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Gupta N, Baker SA, Sanders KM, Rabab KE, Thean DK, Alkawadri T, Griffin CS, Sergeant GP, Hollywood MA, Thornbury KD, Drumm BT. ANO1 channels are expressed in mouse urethral smooth muscle but do not contribute to agonist or neurally evoked contractions. Sci Rep 2025; 15:17365. [PMID: 40389459 PMCID: PMC12089485 DOI: 10.1038/s41598-025-00953-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2025] [Accepted: 05/02/2025] [Indexed: 05/21/2025] Open
Abstract
Anoctamin-1 Ca2+-activated Cl- channels (ANO1) are proposed to modulate contractility of urethra smooth muscle cells (USMC), but their cellular expression and contribution to agonist/neural evoked activity is unclear. ANO1 is implicated as a potential target to treat incontinence, thus this is an important issue to resolve. We sought to clarify roles of ANO1 in contractility of mouse USMC. We found expression of Ano1 transcripts in murine urethra, with no difference between male and females. Immunolabelling revealed ANO1 was expressed in USMC and not in specialized populations of interstitial cells (c-kit+ interstitial of Cajal-like cells (ICC-LC) and PDGFRα+ cells). However, a specific ANO1 channel inhibitor, Ani9, failed to affect urethral contractions elicited by phenylephrine, arginine vasopressin or electrical field stimulation of intrinsic nerves. CaCCinhA01 also failed to affect urethral contractions. In addition, Ani9 had no effect on Ca2+ signals generated by USMC in situ. In contrast, Ani9 effectively reduced spontaneous contractions and Ca2+ signals of mouse proximal colon. In addition, Ani9 inhibited ANO1 currents recorded in HEK 293 cells, at concentrations 30 times less than those used in organ bath experiments. Our data suggest that despite expression of ANO1 in USMC, these channels do not contribute to basal Ca2+ signalling, or agonist and neurally-evoked contractions in murine urethra.
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Affiliation(s)
- Neha Gupta
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Salah A Baker
- Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
| | - Kenton M Sanders
- Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
| | - Kaneez E Rabab
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Denzel Kf Thean
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Tuleen Alkawadri
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Caoimhin S Griffin
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Gerard P Sergeant
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Mark A Hollywood
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Keith D Thornbury
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Bernard T Drumm
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland.
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Gupta N, Baker SA, Sanders KM, Griffin CS, Sergeant GP, Hollywood MA, Thornbury KD, Drumm BT. Interstitial cell of Cajal-like cells (ICC-LC) exhibit dynamic spontaneous activity but are not functionally innervated in mouse urethra. Cell Calcium 2024; 123:102931. [PMID: 39068674 DOI: 10.1016/j.ceca.2024.102931] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2024] [Revised: 07/09/2024] [Accepted: 07/20/2024] [Indexed: 07/30/2024]
Abstract
Urethral smooth muscle cells (USMC) contract to occlude the internal urethral sphincter during bladder filling. Interstitial cells also exist in urethral smooth muscles and are hypothesized to influence USMC behaviours and neural responses. These cells are similar to Kit+ interstitial cells of Cajal (ICC), which are gastrointestinal pacemakers and neuroeffectors. Isolated urethral ICC-like cells (ICC-LC) exhibit spontaneous intracellular Ca2+ signalling behaviours that suggest these cells may serve as pacemakers or neuromodulators similar to ICC in the gut, although observation and direct stimulation of ICC-LC within intact urethral tissues is lacking. We used mice with cell-specific expression of the Ca2+ indicator, GCaMP6f, driven off the endogenous promoter for Kit (Kit-GCaMP6f mice) to identify ICC-LC in situ within urethra muscles and to characterize spontaneous and nerve-evoked Ca2+ signalling. ICC-LC generated Ca2+ waves spontaneously that propagated on average 40.1 ± 0.7 μm, with varying amplitudes, durations, and spatial spread. These events originated from multiple firing sites in cells and the activity between sites was not coordinated. ICC-LC in urethra formed clusters but not interconnected networks. No evidence for entrainment of Ca2+ signalling between ICC-LC was obtained. Ca2+ events in ICC-LC were unaffected by nifedipine but were abolished by cyclopiazonic acid and decreased by an antagonist of Orai Ca2+ channels (GSK-7975A). Phenylephrine increased Ca2+ event frequency but a nitric oxide donor (DEA-NONOate) had no effect. Electrical field stimulation (EFS, 10 Hz) of intrinsic nerves, which evoked contractions of urethral rings and increased Ca2+ event firing in USMC, failed to evoke responses in ICC-LC. Our data suggest that urethral ICC-LC are spontaneously active but are not regulated by autonomic neurons.
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Affiliation(s)
- Neha Gupta
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Salah A Baker
- Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
| | - Kenton M Sanders
- Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
| | - Caoimhin S Griffin
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Gerard P Sergeant
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Mark A Hollywood
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Keith D Thornbury
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Bernard T Drumm
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland; Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA.
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Drumm BT, Gupta N, Mircea A, Griffin CS. Cells and ionic conductances contributing to spontaneous activity in bladder and urethral smooth muscle. J Physiol 2024. [PMID: 39323077 DOI: 10.1113/jp284744] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2023] [Accepted: 09/02/2024] [Indexed: 09/27/2024] Open
Abstract
Smooth muscle organs of the lower urinary tract comprise the bladder detrusor and urethral wall, which have a reciprocal contractile relationship during urine storage and micturition. As the bladder fills with urine, detrusor smooth muscle cells (DSMCs) remain relaxed to accommodate increases in intravesical pressure while urethral smooth muscle cells (USMCs) sustain tone to occlude the urethral orifice, preventing leakage. While neither organ displays coordinated regular contractions as occurs in small intestine, lymphatics or renal pelvis, they do exhibit patterns of rhythmicity at cellular and tissue levels. In rabbit and guinea-pig urethra, electrical slow waves are recorded from USMCs. This activity is linked to cells expressing vimentin, c-kit and Ca2+-activated Cl- channels, like interstitial cells of Cajal in the gastrointestinal tract. In mouse, USMCs are rhythmically active (firing propagating Ca2+ waves linked to contraction), and this cellular rhythmicity is asynchronous across tissues and summates to form tone. Experiments in mice have failed to demonstrate a voltage-dependent mechanism for regulating this rhythmicity or contractions in vitro, suggesting that urethral tone results from an intrinsic ability of USMCs to 'pace' their own Ca2+ mobilization pathways required for contraction. DSMCs exhibit spontaneous transient contractions, increases in intracellular Ca2+ and action potentials. Consistent across numerous species, including humans, this activity relies on voltage-dependent Ca2+ influx in DSMCs. While interstitial cells are present in the bladder, they do not 'pace' the organ in an excitatory manner. Instead, specialized cells (PDGFRα+ interstitial cells) may 'negatively pace' DSMCs to prevent bladder overexcitability.
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Affiliation(s)
- Bernard T Drumm
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Ireland
| | - Neha Gupta
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Ireland
| | - Alexandru Mircea
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Ireland
| | - Caoimhin S Griffin
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Ireland
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Xu D, Ma SL, Huang ML, Zhang H. Expression and functional study of cholecystokinin-A receptors on the interstitial Cajal-like cells of the guinea pig common bile duct. World J Gastroenterol 2023; 29:5374-5382. [PMID: 37900582 PMCID: PMC10600798 DOI: 10.3748/wjg.v29.i38.5374] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/27/2023] [Revised: 09/16/2023] [Accepted: 09/26/2023] [Indexed: 10/12/2023] Open
Abstract
BACKGROUND Many studies have shown that interstitial Cajal-like cell (ICLC) abnormalities are closely related to a variety of dynamic gastrointestinal disorders. ICLCs are pacemaker cells for gastrointestinal movement and are involved in the transmission of nerve impulses. AIM To elucidate the expression profile and significance of cholecystokinin-A (CCK-A) receptors in ICLCs in the common bile duct (CBD), as well as the role of CCK in regulating CBD motility through CCK-A receptors on CBD ICLCs. METHODS The levels of tyrosine kinase receptor (c-kit) and CCK-A receptors in CBD tissues and isolated CBD cells were quantified using the double immunofluorescence labeling technique. The CCK-mediated enhancement of the movement of CBD muscle strips through CBD ICLCs was observed by a muscle strip contraction test. RESULTS Immunofluorescence showed co-expression of c-kit and CCK-A receptors in the CBD muscularis layer. Observations of isolated CBD cells showed that c-kit was expressed on the surface of ICLCs, the cell body and synapse were colored and polygonal, and some cells presented protrusions and formed networks adjacent to the CBD while others formed filaments at the synaptic terminals of local cells. CCK-A receptors were also expressed on CBD ICLCs. At concentrations ranging from 10-6 mol/L to 10-10 mol/L, CCK promoted CBD smooth muscle contractility in a dose-dependent manner. In contrast, after ICLC removal, the contractility mediated by CCK in CBD smooth muscle decreased. CONCLUSION CCK-A receptors are highly expressed on CBD ICLCs, and CCK may regulate CBD motility through the CCK-A receptors on ICLCs.
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Affiliation(s)
- Dan Xu
- Department of Gastroenterology, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, Hubei Province, China
- Key Laboratory for Molecular Diagnosis of Hubei Province, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, Hubei Province, China
| | - Song-Lin Ma
- Department of Gastroenterology, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, Hubei Province, China
- Key Laboratory for Molecular Diagnosis of Hubei Province, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, Hubei Province, China
| | - Man-Lin Huang
- Department of Gastroenterology, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, Hubei Province, China
- Key Laboratory for Molecular Diagnosis of Hubei Province, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, Hubei Province, China
| | - Heng Zhang
- Department of Gastroenterology, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, Hubei Province, China
- Key Laboratory for Molecular Diagnosis of Hubei Province, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, Hubei Province, China
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Spencer NJ, Costa M. Rhythmicity in the Enteric Nervous System of Mice. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2022; 1383:295-306. [PMID: 36587167 DOI: 10.1007/978-3-031-05843-1_27] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
The enteric nervous system (ENS) is required for many cyclical patterns of motor activity along different regions of the gastrointestinal (GI) tract. What has remained mysterious is precisely how many thousands of neurons within the ENS are temporally activated to generate cyclical neurogenic contractions of GI-smooth muscle layers. This has been an especially puzzling conundrum, since the ENS consists of an extensive network of small ganglia, with each ganglion consisting of a heterogeneous population of neurons, with diverse cell soma morphologies, neurochemical and biophysical characteristics, and neural connectivity. Neuronal imaging studies of the mouse large intestine have provided major new insights into how the different classes of myenteric neurons are activated during cyclical neurogenic motor patterns, such as the colonic motor complex (CMC). It has been revealed that during CMCs (in the isolated mouse whole colon), large populations of myenteric neurons, across large spatial fields, coordinate their firing, via bursts of fast synaptic inputs at ~2 Hz. This coordinated firing of many thousands of myenteric neurons synchronously over many rows of interconnected ganglia occurs irrespective of the functional class of neuron. Aborally directed propulsion of content along the mouse colon is due, in large part, to polarity of the enteric circuits including the projections of the intrinsic excitatory and inhibitory motor neurons but still involves the fundamental ~2 Hz rhythmic activity of specific classes of enteric neurons. What remains to be determined are the mechanisms that initiate and terminate the patterned firing of large ensembles of enteric neurons during cyclic activity. This remains an exciting challenge for future studies.
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Affiliation(s)
- Nick J Spencer
- Visceral Neurophysiology Laboratory, Department of Physiology, College of Medicine and Public Health & Centre for Neuroscience, Flinders University, Bedford Park, SA, Australia.
| | - Marcello Costa
- Visceral Neurophysiology Laboratory, Department of Physiology, College of Medicine and Public Health, Flinders University, Bedford Park, SA, Australia
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Spencer NJ, Travis L, Wiklendt L, Costa M, Hibberd TJ, Brookes SJ, Dinning P, Hu H, Wattchow DA, Sorensen J. Long range synchronization within the enteric nervous system underlies propulsion along the large intestine in mice. Commun Biol 2021; 4:955. [PMID: 34376798 PMCID: PMC8355373 DOI: 10.1038/s42003-021-02485-4] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2020] [Accepted: 07/15/2021] [Indexed: 02/07/2023] Open
Abstract
How the Enteric Nervous System (ENS) coordinates propulsion of content along the gastrointestinal (GI)-tract has been a major unresolved issue. We reveal a mechanism that explains how ENS activity underlies propulsion of content along the colon. We used a recently developed high-resolution video imaging approach with concurrent electrophysiological recordings from smooth muscle, during fluid propulsion. Recordings showed pulsatile firing of excitatory and inhibitory neuromuscular inputs not only in proximal colon, but also distal colon, long before the propagating contraction invades the distal region. During propulsion, wavelet analysis revealed increased coherence at ~2 Hz over large distances between the proximal and distal regions. Therefore, during propulsion, synchronous firing of descending inhibitory nerve pathways over long ranges aborally acts to suppress smooth muscle from contracting, counteracting the excitatory nerve pathways over this same region of colon. This delays muscle contraction downstream, ahead of the advancing contraction. The mechanism identified is more complex than expected and vastly different from fluid propulsion along other hollow smooth muscle organs; like lymphatic vessels, portal vein, or ureters, that evolved without intrinsic neurons.
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Affiliation(s)
- Nick J Spencer
- Visceral Neurophysiology Laboratory, College of Medicine and Public Health, Centre for Neuroscience, Flinders University, Bedford Park, SA, Australia.
| | - Lee Travis
- Visceral Neurophysiology Laboratory, College of Medicine and Public Health, Centre for Neuroscience, Flinders University, Bedford Park, SA, Australia
| | - Lukasz Wiklendt
- Discipline of Gastroenterology, College of Medicine and Public Health, Flinders Medical Centre, Bedford Park, SA, Australia
| | - Marcello Costa
- Visceral Neurophysiology Laboratory, College of Medicine and Public Health, Centre for Neuroscience, Flinders University, Bedford Park, SA, Australia
| | - Timothy J Hibberd
- Visceral Neurophysiology Laboratory, College of Medicine and Public Health, Centre for Neuroscience, Flinders University, Bedford Park, SA, Australia
| | - Simon J Brookes
- Visceral Neurophysiology Laboratory, College of Medicine and Public Health, Centre for Neuroscience, Flinders University, Bedford Park, SA, Australia
| | - Phil Dinning
- Discipline of Gastroenterology, College of Medicine and Public Health, Flinders Medical Centre, Bedford Park, SA, Australia
| | - Hongzhen Hu
- Department of Anesthesiology, The Center for the Study of Itch, Washington University, St Louis, MO, USA
| | - David A Wattchow
- Discipline of Surgery, College of Medicine and Public Health, Flinders Medical Centre, Bedford Park, SA, Australia
| | - Julian Sorensen
- Visceral Neurophysiology Laboratory, College of Medicine and Public Health, Centre for Neuroscience, Flinders University, Bedford Park, SA, Australia
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Drumm BT, Thornbury KD, Hollywood MA, Sergeant GP. Role of Ano1 Ca 2+-activated Cl - channels in generating urethral tone. Am J Physiol Renal Physiol 2021; 320:F525-F536. [PMID: 33554780 DOI: 10.1152/ajprenal.00520.2020] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Urinary continence is maintained in the lower urinary tract by the contracture of urethral sphincters, including smooth muscle of the internal urethral sphincter. These contractions occlude the urethral lumen, preventing urine leakage from the bladder to the exterior. Over the past 20 years, research on the ionic conductances that contribute to urethral smooth muscle contractility has greatly accelerated. A debate has emerged over the role of interstitial cell of Cajal (ICC)-like cells in the urethra and their expression of Ca2+-activated Cl- channels encoded by anoctamin-1 [Ano1; transmembrane member 16 A (Tmem16a) gene]. It has been proposed that Ano1 channels expressed in urethral ICC serve as a source of depolarization for smooth muscle cells, increasing their excitability and contributing to tone. Although a clear role for Ano1 channels expressed in ICC is evident in other smooth muscle organs, such as the gastrointestinal tract, the role of these channels in the urethra is unclear, owing to differences in the species (rabbit, rat, guinea pig, sheep, and mouse) examined and experimental approaches by different groups. The importance of clarifying this situation is evident as effective targeting of Ano1 channels may lead to new treatments for urinary incontinence. In this review, we summarize the key findings from different species on the role of ICC and Ano1 channels in urethral contractility. Finally, we outline proposals for clarifying this controversial and important topic by addressing how cell-specific optogenetic and inducible cell-specific genetic deletion strategies coupled with advances in Ano1 channel pharmacology may clarify this area in future studies.NEW & NOTEWORTHY Studies from the rabbit have shown that anoctamin-1 (Ano1) channels expressed in urethral interstitial cells of Cajal (ICC) serve as a source of depolarization for smooth muscle cells, increasing excitability and tone. However, the role of urethral Ano1 channels is unclear, owing to differences in the species examined and experimental approaches. We summarize findings from different species on the role of urethral ICC and Ano1 channels in urethral contractility and outline proposals for clarifying this topic using cell-specific optogenetic approaches.
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Affiliation(s)
- Bernard T Drumm
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Ireland
| | - Keith D Thornbury
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Ireland
| | - Mark A Hollywood
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Ireland
| | - Gerard P Sergeant
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Ireland
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The intracellular Ca 2+ release channel TRPML1 regulates lower urinary tract smooth muscle contractility. Proc Natl Acad Sci U S A 2020; 117:30775-30786. [PMID: 33199609 PMCID: PMC7720193 DOI: 10.1073/pnas.2016959117] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel that is localized to late endosomes and lysosomes. Here, we investigated the function of TRPML1 channels in regulating lower urinary tract (LUT) smooth muscle cell (SMC) contractility. We found that TRPML1 forms a stable signaling complex with ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR). We further showed that TRPML1 channels are important for initiating an essential Ca2+-signaling negative feedback mechanism between RyRs on SR membranes and K+ channels on the plasma membrane. Knockout of TRPML1 channels in mice impaired this pathway, resulting in LUT smooth muscle hypercontractility and symptoms of overactive bladder. Our findings demonstrate a critical role for TRPML1 in LUT function. TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel that is predominantly localized to the membranes of late endosomes and lysosomes (LELs). Intracellular release of Ca2+ through TRPML1 is thought to be pivotal for maintenance of intravesicular acidic pH as well as the maturation, fusion, and trafficking of LELs. Interestingly, genetic ablation of TRPML1 in mice (Mcoln1−/−) induces a hyperdistended/hypertrophic bladder phenotype. Here, we investigated this phenomenon further by exploring an unconventional role for TRPML1 channels in the regulation of Ca2+-signaling activity and contractility in bladder and urethral smooth muscle cells (SMCs). Four-dimensional (4D) lattice light-sheet live-cell imaging showed that the majority of LELs in freshly isolated bladder SMCs were essentially immobile. Superresolution microscopy revealed distinct nanoscale colocalization of LEL-expressing TRPML1 channels with ryanodine type 2 receptors (RyR2) in bladder SMCs. Spontaneous intracellular release of Ca2+ from the sarcoplasmic reticulum (SR) through RyR2 generates localized elevations of Ca2+ (“Ca2+ sparks”) that activate plasmalemmal large-conductance Ca2+-activated K+ (BK) channels, a critical negative feedback mechanism that regulates smooth muscle contractility. This mechanism was impaired in Mcoln1−/− mice, which showed diminished spontaneous Ca2+ sparks and BK channel activity in bladder and urethra SMCs. Additionally, ex vivo contractility experiments showed that loss of Ca2+ spark–BK channel signaling in Mcoln1−/− mice rendered both bladder and urethra smooth muscle hypercontractile. Voiding activity analyses revealed bladder overactivity in Mcoln1−/− mice. We conclude that TRPML1 is critically important for Ca2+ spark signaling, and thus regulation of contractility and function, in lower urinary tract SMCs.
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Mah SA, Avci R, Cheng LK, Du P. Current applications of mathematical models of the interstitial cells of Cajal in the gastrointestinal tract. WIREs Mech Dis 2020; 13:e1507. [PMID: 33026190 DOI: 10.1002/wsbm.1507] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2020] [Revised: 08/31/2020] [Accepted: 09/04/2020] [Indexed: 12/25/2022]
Abstract
The interstitial cells of Cajal (ICC) form interconnected networks throughout the gastrointestinal (GI) tract. ICC act as the pacemaker cells that initiate the rhythmic bioelectrical slow waves and intermediary between the GI musculature and nerves, both of which are critical to GI motility. Disruptions to the number of ICC and the integrity of ICC networks have been identified as a key pathophysiological mechanism in a number of clinically challenging GI disorders. The current analyses of ICC generally rely on either functional recordings taken directly from excised tissue or morphological analysis based on images of labeled ICC, where the structural-functional relationship is investigated in an associative manner rather than mechanistically. On the other hand, computational physiology has played a significant role in facilitating our understanding of a number of physiological systems in both health and disease, and investigations in the GI field are beginning to incorporate several mathematical models of the ICC. The main aim of this review is to present the major modeling advances in GI electrophysiology, in order to introduce a multi-scale framework for mathematically quantifying the functional consequences of ICC degradation at both cellular and tissue scales. The outcomes will inform future investigators utilizing modeling techniques in their studies. This article is categorized under: Metabolic Diseases > Computational Models.
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Affiliation(s)
- Sue Ann Mah
- Auckland Bioengineering Institute, University of Auckland, Auckland, New Zealand
| | - Recep Avci
- Auckland Bioengineering Institute, University of Auckland, Auckland, New Zealand
| | - Leo K Cheng
- Auckland Bioengineering Institute, University of Auckland, Auckland, New Zealand
| | - Peng Du
- Auckland Bioengineering Institute, University of Auckland, Auckland, New Zealand.,Department of Engineering Science, University of Auckland, Auckland, New Zealand
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Dalghi MG, Montalbetti N, Carattino MD, Apodaca G. The Urothelium: Life in a Liquid Environment. Physiol Rev 2020; 100:1621-1705. [PMID: 32191559 PMCID: PMC7717127 DOI: 10.1152/physrev.00041.2019] [Citation(s) in RCA: 112] [Impact Index Per Article: 22.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2019] [Revised: 03/02/2020] [Accepted: 03/14/2020] [Indexed: 02/08/2023] Open
Abstract
The urothelium, which lines the renal pelvis, ureters, urinary bladder, and proximal urethra, forms a high-resistance but adaptable barrier that surveils its mechanochemical environment and communicates changes to underlying tissues including afferent nerve fibers and the smooth muscle. The goal of this review is to summarize new insights into urothelial biology and function that have occurred in the past decade. After familiarizing the reader with key aspects of urothelial histology, we describe new insights into urothelial development and regeneration. This is followed by an extended discussion of urothelial barrier function, including information about the roles of the glycocalyx, ion and water transport, tight junctions, and the cellular and tissue shape changes and other adaptations that accompany expansion and contraction of the lower urinary tract. We also explore evidence that the urothelium can alter the water and solute composition of urine during normal physiology and in response to overdistension. We complete the review by providing an overview of our current knowledge about the urothelial environment, discussing the sensor and transducer functions of the urothelium, exploring the role of circadian rhythms in urothelial gene expression, and describing novel research tools that are likely to further advance our understanding of urothelial biology.
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Affiliation(s)
- Marianela G Dalghi
- Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Nicolas Montalbetti
- Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Marcelo D Carattino
- Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Gerard Apodaca
- Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
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11
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Chen D, Meng W, Shu L, Liu S, Gu Y, Wang X, Feng M. ANO1 in urethral SMCs contributes to sex differences in urethral spontaneous tone. Am J Physiol Renal Physiol 2020; 319:F394-F402. [PMID: 32686521 DOI: 10.1152/ajprenal.00174.2020] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Stress urinary incontinence (SUI) is more common in women than in men, and sex differences in anatomic structure and physiology have been suggested as causes; however, the underlying cellular and molecular mechanisms remain unclear. The spontaneous tone (STT) of the urethra has been shown to have a fundamental effect on preventing the occurrence of SUI. Here, we investigated whether the urethral STT exhibited sex differences. First, we isolated urethral smooth muscle (USM) and detected STT in female mice and women. No STT was found in male mice or men. Furthermore, caffeine induced increased contractility and intracellular Ca2+ concentration in urethrae from female mice compared with male mice. EACT [an N-aroylaminothiazole, anoctamin-1 (ANO1) activator] elicited increased intracellular Ca2+ concentration and stronger currents in female mice than in male mice. Moreover, ANO1 expression in single USM cells from women and female mice was almost twofold higher than that found in cells from men and male mice. In summary, ANO1 in USM contributes to sex differences in urethral spontaneous tone. This finding may provide new guidance for the treatment of SUI in women and men.
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Affiliation(s)
- Defang Chen
- Department of Outpatient, Central Hospital Affiliated to Shandong First Medical University, Jinan, China
| | - Wen Meng
- Pharmacy Intravenous Admixture Services, Central Hospital Affiliated to Shandong First Medical University, Jinan, China
| | - Ling Shu
- Operating Room, Central Hospital Affiliated to Shandong First Medical University, Jinan, China
| | - Shuang Liu
- Central Laboratory, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
| | - Yongzhong Gu
- Department of Obstetrics and Gynecology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
| | - Xiaoyan Wang
- General Practice Department, College of Traditional Chinese Medicine, Shandong University of Traditional Chinese Medicine, Jinan, China
| | - Mei Feng
- Central Laboratory, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China,Central Laboratory, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China
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Rembetski BE, Sanders KM, Drumm BT. Contribution of Ca v1.2 Ca 2+ channels and store-operated Ca 2+ entry to pig urethral smooth muscle contraction. Am J Physiol Renal Physiol 2020; 318:F496-F505. [PMID: 31904286 DOI: 10.1152/ajprenal.00514.2019] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Urethral smooth muscle (USM) generates tone to prevent urine leakage from the bladder during filling. USM tone has been thought to be a voltage-dependent process, relying on Ca2+ influx via voltage-dependent Ca2+ channels in USM cells, modulated by the activation of Ca2+-activated Cl- channels encoded by Ano1. However, recent findings in the mouse have suggested that USM tone is voltage independent, relying on Ca2+ influx through Orai channels via store-operated Ca2+ entry (SOCE). We explored if this pathway also occurred in the pig using isometric tension recordings of USM tone. Pig USM strips generated myogenic tone, which was nearly abolished by the Cav1.2 channel antagonist nifedipine and the ATP-dependent K+ channel agonist pinacidil. Pig USM tone was reduced by the Orai channel blocker GSK-7975A. Electrical field stimulation (EFS) led to phentolamine-sensitive contractions of USM strips. Contractions of pig USM were also induced by phenylephrine. Phenylephrine-evoked and EFS-evoked contractions of pig USM were reduced by ~50-75% by nifedipine and ~30% by GSK-7975A. Inhibition of Ano1 channels had no effect on tone or EFS-evoked contractions of pig USM. In conclusion, unlike the mouse, pig USM exhibited voltage-dependent tone and agonist/EFS-evoked contractions. Whereas SOCE plays a role in generating tone and agonist/neural-evoked contractions in both species, this dominates in the mouse. Tone and agonist/EFS-evoked contractions of pig USM are the result of Ca2+ influx primarily through Cav1.2 channels, and no evidence was found supporting a role of Ano1 channels in modulating these mechanisms.
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Affiliation(s)
- Benjamin E Rembetski
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno Nevada
| | - Kenton M Sanders
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno Nevada
| | - Bernard T Drumm
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno Nevada
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13
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Mucosa-Dependent, Stretch-Sensitive Spontaneous Activity in Seminal Vesicle. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019. [PMID: 31183829 DOI: 10.1007/978-981-13-5895-1_9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register]
Abstract
Seminal vesicles (SVs), a pair of male accessory glands, contract upon sympathetic nerve excitation during ejaculation while developing spontaneous phasic constrictions in the inter-ejaculatory storage phase. Recently, the fundamental role of the mucosa in generating spontaneous activity in SV of the guinea pig has been revealed. Stretching the mucosa-intact but not mucosa-denuded SV smooth muscle evokes spontaneous phasic contractions arising from action potential firing triggered by electrical slow waves and associated Ca2+ flashes. These spontaneous events primarily depend on sarco-endoplasmic reticulum (SR/ER) Ca2+ handling linked with the opening of Ca2+-activated chloride channels (CaCCs) resulting in the generation of slow waves. Slow waves in mucosa-intact SV smooth muscle are abolished upon blockade of gap junctions, suggesting that seminal smooth muscle cells are driven by cells distributed in the mucosa. In the SV mucosal preparations dissected free from the smooth muscle layer, a population of cells located just beneath the epithelium develop spontaneous Ca2+ transients relying on SR/ER Ca2+ handling. In the lamina propria of the SV mucosa, vimentin-immunoreactive interstitial cells including platelet-derived growth factor receptor α (PDGFRα)-immunoreactive cells are distributed, while known pacemaker cells in other smooth muscle tissues, e.g. c-Kit-positive interstitial cells or α-smooth muscle actin-positive atypical smooth muscle cells, are absent. The spontaneously-active subepithelial cells appear to drive spontaneous activity in SV smooth muscle either by sending depolarizing signals or by releasing humoral substances. Interstitial cells in the lamina propria may act as intermediaries of signal transmission from the subepithelial cells to the smooth muscle cells.
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Sergeant GP, Hollywood MA, Thornbury KD. Spontaneous Activity in Urethral Smooth Muscle. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1124:149-167. [DOI: 10.1007/978-981-13-5895-1_6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
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Abstract
Veins exhibit spontaneous contractile activity, a phenomenon generally termed vasomotion. This is mediated by spontaneous rhythmical contractions of mural cells (i.e. smooth muscle cells (SMCs) or pericytes) in the wall of the vessel. Vasomotion occurs through interconnected oscillators within and between mural cells, entraining their cycles. Pharmacological studies indicate that a key oscillator underlying vasomotion is the rhythmical calcium ion (Ca2+) release-refill cycle of Ca2+ stores. This occurs through opening of inositol 1,4,5-trisphosphate receptor (IP3R)- and/or ryanodine receptor (RyR)-operated Ca2+ release channels in the sarcoplasmic/endoplasmic (SR/ER) reticulum and refilling by the SR/ER reticulum Ca2+ATPase (SERCA). Released Ca2+ from stores near the plasma membrane diffuse through the cytosol to open Ca2+-activated chloride (Cl-) channels, this generating inward current through an efflux of Cl-. The resultant depolarisation leads to the opening of voltage-dependent Ca2+ channels and possibly increased production of IP3, which through Ca2+-induced Ca2+ release (CICR) of IP3Rs and/or RyRs and IP3R-mediated Ca2+ release provide a means by which store oscillators entrain their activity. Intercellular entrainment normally involves current flow through gap junctions that interconnect mural cells and in many cases this is aided by additional connectivity through the endothelium. Once entrainment has occurred the substantial Ca2+ entry that results from the near-synchronous depolarisations leads to rhythmical contractions of the mural cells, this often leading to vessel constriction. The basis for venous/venular vasomotion has yet to be fully delineated but could improve both venous drainage and capillary/venular absorption of blood plasma-associated fluids.
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Eggermont M, De Wachter S, Eastham J, Gillespie J. Innervation of the Epithelium and Lamina Propria of the Urethra of the Female Rat. Anat Rec (Hoboken) 2018; 302:201-214. [DOI: 10.1002/ar.23937] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2017] [Revised: 01/19/2018] [Accepted: 01/26/2018] [Indexed: 02/02/2023]
Affiliation(s)
- Monica Eggermont
- Department of Urology, Faculty of Medicine and Health SciencesUniversity of Antwerp Antwerp Belgium
| | - Stefan De Wachter
- Department of Urology, Faculty of Medicine and Health SciencesUniversity of Antwerp Antwerp Belgium
| | - Jane Eastham
- Uro‐physiology Research Group, The Dental and Medical SchoolNewcastle University Newcastle upon Tyne UK
| | - James Gillespie
- Department of Urology, Faculty of Medicine and Health SciencesUniversity of Antwerp Antwerp Belgium
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Drumm BT, Rembetski BE, Cobine CA, Baker SA, Sergeant GP, Hollywood MA, Thornbury KD, Sanders KM. Ca 2+ signalling in mouse urethral smooth muscle in situ: role of Ca 2+ stores and Ca 2+ influx mechanisms. J Physiol 2018; 596:1433-1466. [PMID: 29383731 PMCID: PMC5899989 DOI: 10.1113/jp275719] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2017] [Accepted: 01/17/2018] [Indexed: 11/08/2022] Open
Abstract
KEY POINTS Contraction of urethral smooth muscle cells (USMCs) contributes to urinary continence. Ca2+ signalling in USMCs was investigated in intact urethral muscles using a genetically encoded Ca2+ sensor, GCaMP3, expressed selectively in USMCs. USMCs were spontaneously active in situ, firing intracellular Ca2+ waves that were asynchronous at different sites within cells and between adjacent cells. Spontaneous Ca2+ waves in USMCs were myogenic but enhanced by adrenergic or purinergic agonists and decreased by nitric oxide. Ca2+ waves arose from inositol trisphosphate type 1 receptors and ryanodine receptors, and Ca2+ influx by store-operated calcium entry was required to maintain Ca2+ release events. Ca2+ release and development of Ca2+ waves appear to be the primary source of Ca2+ for excitation-contraction coupling in the mouse urethra, and no evidence was found that voltage-dependent Ca2+ entry via L-type or T-type channels was required for responses to α adrenergic responses. ABSTRACT Urethral smooth muscle cells (USMCs) generate myogenic tone and contribute to urinary continence. Currently, little is known about Ca2+ signalling in USMCs in situ, and therefore little is known about the source(s) of Ca2+ required for excitation-contraction coupling. We characterized Ca2+ signalling in USMCs within intact urethral muscles using a genetically encoded Ca2+ sensor, GCaMP3, expressed selectively in USMCs. USMCs fired spontaneous intracellular Ca2+ waves that did not propagate cell-to-cell across muscle bundles. Ca2+ waves increased dramatically in response to the α1 adrenoceptor agonist phenylephrine (10 μm) and to ATP (10 μm). Ca2+ waves were inhibited by the nitric oxide donor DEA NONOate (10 μm). Ca2+ influx and release from sarcoplasmic reticulum stores contributed to Ca2+ waves, as Ca2+ free bathing solution and blocking the sarcoplasmic Ca2+ -ATPase abolished activity. Intracellular Ca2+ release involved cooperation between ryanadine receptors and inositol trisphosphate receptors, as tetracaine and ryanodine (100 μm) and xestospongin C (1 μm) reduced Ca2+ waves. Ca2+ waves were insensitive to L-type Ca2+ channel modulators nifedipine (1 μm), nicardipine (1 μm), isradipine (1 μm) and FPL 64176 (1 μm), and were unaffected by the T-type Ca2+ channel antagonists NNC-550396 (1 μm) and TTA-A2 (1 μm). Ca2+ waves were reduced by the store operated Ca2+ entry blocker SKF 96365 (10 μm) and by an Orai antagonist, GSK-7975A (1 μm). The latter also reduced urethral contractions induced by phenylephrine, suggesting that Orai can function effectively as a receptor-operated channel. In conclusion, Ca2+ waves in mouse USMCs are a source of Ca2+ for excitation-contraction coupling in urethral muscles.
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Affiliation(s)
- Bernard T. Drumm
- Department of Physiology and Cell BiologyUniversity of Nevada, Reno School of MedicineRenoNV89557USA
| | - Benjamin E. Rembetski
- Department of Physiology and Cell BiologyUniversity of Nevada, Reno School of MedicineRenoNV89557USA
| | - Caroline A. Cobine
- Department of Physiology and Cell BiologyUniversity of Nevada, Reno School of MedicineRenoNV89557USA
| | - Salah A. Baker
- Department of Physiology and Cell BiologyUniversity of Nevada, Reno School of MedicineRenoNV89557USA
| | - Gerard P. Sergeant
- Smooth Muscle Research CentreDundalk Institute of TechnologyCo. LouthDundalkRepublic of Ireland
| | - Mark A. Hollywood
- Smooth Muscle Research CentreDundalk Institute of TechnologyCo. LouthDundalkRepublic of Ireland
| | - Keith D. Thornbury
- Smooth Muscle Research CentreDundalk Institute of TechnologyCo. LouthDundalkRepublic of Ireland
| | - Kenton M. Sanders
- Department of Physiology and Cell BiologyUniversity of Nevada, Reno School of MedicineRenoNV89557USA
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18
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Drake MJ, Fry CH, Hashitani H, Kirschner-Hermanns R, Rahnama'i MS, Speich JE, Tomoe H, Kanai AJ, McCloskey KD. What are the origins and relevance of spontaneous bladder contractions? ICI-RS 2017. Neurourol Urodyn 2018; 37:S13-S19. [PMID: 29360173 DOI: 10.1002/nau.23485] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2017] [Accepted: 12/14/2017] [Indexed: 12/16/2022]
Abstract
INTRODUCTION Storage phase bladder activity is a counter-intuitive observation of spontaneous contractions. They are potentially an intrinsic feature of the smooth muscle, but interstitial cells in the mucosa and the detrusor itself, as well as other muscular elements in the mucosa may substantially influence them. They are identified in several models explaining lower urinary tract dysfunction. METHODS A consensus meeting at the International Consultation on Incontinence Research Society (ICI-RS) 2017 congress considered the origins and relevance of spontaneous bladder contractions by debating which cell type(s) modulate bladder spontaneous activity, whether the methodologies are sufficiently robust, and implications for healthy and abnormal lower urinary tract function. RESULTS The identified research priorities reflect a wide range of unknown aspects. Cellular contributions to spontaneous contractions in detrusor smooth muscle are still uncertain. Accordingly, insight into the cellular physiology of the bladder wall, particularly smooth muscle cells, interstitial cells, and urothelium, remains important. Upstream influences, such as innervation, endocrine, and paracrine factors, are particularly important. The cellular interactions represent the key understanding to derive the integrative physiology of organ function, notably the nature of signalling between mucosa and detrusor layers. Indeed, it is still not clear to what extent spontaneous contractions generated in isolated preparations mirror their normal and pathological counterparts in the intact bladder. Improved models of how spontaneous contractions influence pressure generation and sensory nerve function are also needed. CONCLUSIONS Deriving approaches to robust evaluation of spontaneous contractions and their influences for experimental and clinical use could yield considerable progress in functional urology.
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Affiliation(s)
- Marcus J Drake
- Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom.,Bristol Urological Institute, Southmead Hospital, Bristol, United Kingdom
| | - Christopher H Fry
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom
| | - Hikaru Hashitani
- Department of Cell Physiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan
| | - Ruth Kirschner-Hermanns
- Neuro-Urology/Urology, University Clinic, Rheinische Friedrich Wilhelms University Bonn and Neurological Rehabilitation Center Godeshöhe, Bonn, Germany
| | | | - John E Speich
- Department of Mechanical and Nuclear Engineering, Virginia Commonwealth University, Richmond, Virginia
| | - Hikaru Tomoe
- Department of Urology and Pelvic Reconstructive Surgery, Tokyo Women's Medical University Medical Center East, Tokyo, Japan
| | - Anthony J Kanai
- Department of Medicine, Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Karen D McCloskey
- Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, Belfast, United Kingdom
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Sancho M, Bradley E, Garcia-Pascual A, Triguero D, Thornbury KD, Hollywood MA, Sergeant GP. Involvement of cyclic nucleotide-gated channels in spontaneous activity generated in isolated interstitial cells of Cajal from the rabbit urethra. Eur J Pharmacol 2017; 814:216-225. [DOI: 10.1016/j.ejphar.2017.08.020] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2016] [Revised: 08/15/2017] [Accepted: 08/15/2017] [Indexed: 11/16/2022]
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Fedigan S, Bradley E, Webb T, Large RJ, Hollywood MA, Thornbury KD, McHale NG, Sergeant GP. Effects of new-generation TMEM16A inhibitors on calcium-activated chloride currents in rabbit urethral interstitial cells of Cajal. Pflugers Arch 2017; 469:1443-1455. [PMID: 28733893 DOI: 10.1007/s00424-017-2028-5] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2016] [Revised: 06/21/2017] [Accepted: 07/03/2017] [Indexed: 12/18/2022]
Abstract
Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit Ca2+-activated Cl- currents (I ClCa) that are important for the development of urethral tone. Here, we examined if TMEM16A (ANO1) contributed to this activity by examining the effect of "new-generation" TMEM16A inhibitors, CACCinh-A01 and T16Ainh-A01, on I ClCa recorded from freshly isolated rabbit urethral ICC (RUICC) and on contractions of intact strips of rabbit urethra smooth muscle. Real-time quantitative PCR experiments demonstrated that TMEM16A was highly expressed in rabbit urethra smooth muscle, in comparison to TMEM16B and TMEM16F. Single-cell RT-PCR experiments revealed that only TMEM16A was expressed in freshly isolated RUICC. Depolarization-evoked I ClCa in isolated RUICC, recorded using voltage clamp, were inhibited by CACCinh-A01 and T16Ainh-A01 with IC50 values of 1.2 and 3.4 μM, respectively. Similarly, spontaneous transient inward currents (STICs) recorded from RUICC voltage clamped at -60 mV and spontaneous transient depolarizations (STDs), recorded in current clamp, were also inhibited by CACCinh-A01 and T16Ainh-A01. In contrast, spontaneous Ca2+ waves in isolated RUICC were only partially reduced by CACCinh-A01 and T16Ainh-A01. Finally, neurogenic contractions of strips of rabbit urethra smooth muscle (RUSM), evoked by electric field stimulation (EFS), were also significantly reduced by CACCinh-A01 and T16Ainh-A01. These data are consistent with the idea that TMEM16A is involved with CACCs in RUICC and in contraction of rabbit urethral smooth muscle.
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Affiliation(s)
- Stephen Fedigan
- The Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Eamonn Bradley
- The Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Timothy Webb
- The Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Roddy J Large
- The Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Mark A Hollywood
- The Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Keith D Thornbury
- The Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Noel G McHale
- The Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Gerard P Sergeant
- The Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland.
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Hollywood MA, Thornbury KD, Sergeant GP. A seminal study on the mechanisms underlying spontaneous activity of the seminal vesicles? J Physiol 2017; 595:4567. [PMID: 28488278 DOI: 10.1113/jp274499] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Affiliation(s)
- M A Hollywood
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dublin Road, Dundalk, Co. Louth, Ireland
| | - K D Thornbury
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dublin Road, Dundalk, Co. Louth, Ireland
| | - G P Sergeant
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dublin Road, Dundalk, Co. Louth, Ireland
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Proliferation of Interstitial Cells in the Cyclophosphamide-Induced Cystitis and the Preventive Effect of Imatinib. BIOMED RESEARCH INTERNATIONAL 2017; 2017:3457093. [PMID: 28698872 PMCID: PMC5494099 DOI: 10.1155/2017/3457093] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/22/2017] [Revised: 04/21/2017] [Accepted: 05/10/2017] [Indexed: 11/25/2022]
Abstract
Cyclophosphamide- (CYP-) induced cystitis in the rat is a well-known model of bladder inflammation that leads to an overactive bladder, a process that appears to involve enhanced nitric oxide (NO) production. We investigated the changes in the number and distribution of interstitial cells (ICs) and in the expression of endothelial NO synthase (eNOS) in the bladder and urethra of rats subjected to either intermediate or chronic CYP treatment. Pronounced hyperplasia and hypertrophy of ICs were evident within the lamina propria and in the muscle layer. IC immunolabeling with CD34, PDGFRα, and vimentin was enhanced, as reflected by higher colocalization indexes of the distinct pairs of markers. Moreover, de novo expression of eNOS was evident in vimentin and CD34 positive ICs. Pretreatment with the receptor tyrosine kinase inhibitor Imatinib prevented eNOS expression and ICs proliferation, as well as the increased voiding frequency and urinary tract weight provoked by CYP. As similar results were obtained in the urethra, urethritis may contribute to the uropathology of CYP-induced cystitis.
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Yu Y, Jiang J, He Y, Wang W, Shen C, Yang B. Resveratrol improves urinary dysfunction in rats with chronic prostatitis and suppresses the activity of the stem cell factor/c-Kit signaling pathway. Mol Med Rep 2017; 16:1395-1400. [PMID: 29067468 DOI: 10.3892/mmr.2017.6721] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2016] [Accepted: 03/10/2017] [Indexed: 11/06/2022] Open
Abstract
Chronic prostatitis (CP) is a common urological disorder, with bladder voiding dysfunction being the primary clinical manifestation. Resveratrol is polyphenolic compound isolated from numerous plants, with widely‑reported anti-inflammatory properties. The present study aimed to investigate whether resveratrol may improve overactive bladder in rats with CP and to investigate the underlying molecular mechanisms. Furthermore, the potential pharmacological synergy between resveratrol and solifenacin was also investigated as a potential treatment for CP. Following the successful establishment of a rat model of CP by subcutaneously injecting DPT vaccine, rats were treated with resveratrol or a combination of resveratrol + solifenacin. Bladder pressure and volume tests were performed to investigate the effect of resveratrol and solifenacin on urinary dysfunction in rats with chronic prostatitis. Western blot analysis and immunohistochemical staining were used to examine the expression of c‑Kit receptor, stem cell factor (SCF), AKT and phosphorylated‑AKT (p‑AKT) in the bladder tissue. The results of the bladder pressure and volume test indicated that the maximum capacity of the bladder, residual urine volume and maximum voiding pressure in the control group were 0.57 ml, 0.17 ml and 29.62 cm H2O, respectively. These values were increased by 71, 27 and 206% in rats in the CP group compared with the control group. Following treatment with resveratrol, the results in the resveratrol group were reduced by 25.77, 44.23 and 13.32% compared with the CP group. The results of western blot analysis, immunohistochemical staining and immunofluorescence labeling demonstrate that the protein expression of SCF, c‑Kit and p‑AKT in the bladder of rats in the CP group was 4.32, 6.13 and 6.31 times higher compared with the control group, respectively. Following treatment with resveratrol, protein expression was significantly reduced. However, no significant differences were observed between the protein expression of the SCF, c‑Kit and p‑AKT in the bladder between the resveratrol and combination groups. In conclusion, resveratrol may improve overactive bladder by downregulating the protein expression of SCF, c‑Kit and p‑AKT in the bladder of rats with CP. Furthermore, a combination of resveratrol and solifenacin may have potential pharmacological synergy as a treatment for patients with CP.
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Affiliation(s)
- Yang Yu
- Department of Urology, The Second Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116044, P.R. China
| | - Jiang Jiang
- Dalian Municipal Food and Drug Administration, Dalian, Liaoning 116000, P.R. China
| | - Yi He
- Department of Urology, The Second Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116044, P.R. China
| | - Wei Wang
- Department of Urology, The Second Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116044, P.R. China
| | - Chen Shen
- Department of Urology, The Second Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116044, P.R. China
| | - Bo Yang
- Department of Urology, The Second Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116044, P.R. China
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Takeya M, Hashitani H, Hayashi T, Higashi R, Nakamura KI, Takano M. Role of mucosa in generating spontaneous activity in the guinea pig seminal vesicle. J Physiol 2017; 595:4803-4821. [PMID: 28421606 DOI: 10.1113/jp273872] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2017] [Accepted: 04/11/2017] [Indexed: 01/08/2023] Open
Abstract
KEY POINTS The mucosa may have neuron-like functions as urinary bladder mucosa releases bioactive substances that modulate sensory nerve activity as well as detrusor muscle contractility. However, such mucosal function in other visceral organs remains to be established. The role of mucosa in generating spontaneous contractions in seminal vesicles (SVs), a paired organ in the male reproductive tract, was investigated. The intact mucosa is essential for the generation of spontaneous phasic contractions of SV smooth muscle arising from electrical slow waves and corresponding increases in intracellular Ca2+ . These spontaneous events primarily depend on Ca2+ handling by sarco-endoplasmic reticulum Ca2+ stores. A population of mucosal cells developed spontaneous rises in intracellular Ca2+ relying on sarco-endoplasmic reticulum Ca2+ handling. The spontaneously active cells in the SV mucosa appear to drive spontaneous activity in smooth muscle either by sending depolarizing signals and/or by releasing humoral substances. ABSTRACT The role of the mucosa in generating the spontaneous activity of guinea-pig seminal vesicle (SV) was explored. Changes in contractility, membrane potential and intracellular Ca2+ dynamics of SV smooth muscle cells (SMCs) were recorded using isometric tension recording, intracellular microelectrode recording and epi-fluorescence Ca2+ imaging, respectively. Mucosa-intact but not mucosa-denuded SV preparations generated TTX- (1 μm) resistant spontaneous phasic contractions that were abolished by nifedipine (3 μm). Consistently, SMCs developed mucosa-dependent slow waves (SWs) that triggered action potentials and corresponding Ca2+ flashes. Nifedipine (10 μm) abolished the action potentials and spontaneous contractions, while suppressing the SWs and Ca2+ flashes. Both the residual SWs and spontaneous Ca2+ transients were abolished by cyclopiazonic acid (CPA, 10 μm), a sarco-endoplasmic reticulum Ca2+ -ATPase (SERCA) inhibitor. DIDS (300 μm) and niflumic acid (100 μm), blockers for Ca2+ -activated Cl- channels (CACCs), or low Cl- solution also slowed or prevented the generation of SWs. In SV mucosal preparations detached from the muscle layer, a population of mucosal cells generated spontaneous Ca2+ transients that were blocked by CPA but not nifedipine. These results suggested that spontaneous contractions and corresponding Ca2+ flashes in SV SMCs arise from action potential generation due to the opening of L-type voltage-dependent Ca2+ channels. Spontaneous Ca2+ transients appear to primarily result from Ca2+ release from sarco-endoplasmic reticulum Ca2+ stores to activate CACCs to develop SWs. The mucosal cells firing spontaneous Ca2+ transients may play a critical role in driving spontaneous activity of SV smooth muscle either by sending depolarizing signals or by releasing humoral substances.
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Affiliation(s)
- Mitsue Takeya
- Division of Integrated Autonomic Function, Department of Physiology, Kurume University School of Medicine, Kurume, Japan
| | - Hikaru Hashitani
- Department of Cell Physiology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Tokumasa Hayashi
- Department of Urology, Kurume University School of Medicine, Kurume, Japan
| | - Ryuhei Higashi
- Electron Microscopic Laboratory, Central Research Unit of Kurume University School of Medicine, Kurume, Japan
| | - Kei-Ichiro Nakamura
- Division of Microscopic and Developmental Anatomy, Department of Anatomy, Kurume University School of Medicine, Kurume, Japan
| | - Makoto Takano
- Division of Integrated Autonomic Function, Department of Physiology, Kurume University School of Medicine, Kurume, Japan
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C-kit receptor immunopositive interstitial cells (Cajal-type) in the porcine reproductive tract. Acta Vet Scand 2017; 59:32. [PMID: 28526042 PMCID: PMC5438557 DOI: 10.1186/s13028-017-0300-5] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2016] [Accepted: 05/11/2017] [Indexed: 11/10/2022] Open
Abstract
Background Interstitial Cajal cells have been suspected as being the pacemaker cells of smooth muscle motor activity and discharging slow triggering waves in the gut as well as in other organs containing smooth muscles where they are known as interstitial Cajal-like cells (ICLC). The present study describes ICLC localization and density in the porcine oviduct and uterus. Differences in ICLC density were examined using histological, immunohistochemical and immunofluorescent methods and c-kit expression was determined. Results interstitial Cajal-like cells with characteristic morphological and immunological phenotypes were found. Star-like or spindle-shaped cells with very long, moniliform processes were localized in the muscle layers of the oviduct and uterine walls at variable densities that decreased progressively from high in the oviduct to low in the uterus. Conclusions The detailed description of ICLC in the porcine reproductive tract may lead to a better understanding of reproductive tract motility. Our approach is inexpensive and effective for ICLC evaluation and may in the future be applied to clinical diagnosis.
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Gajewski JB, Rosier PF, Rahnama'i S, Abrams P. Do we assess urethral function adequately in LUTD and NLUTD? ICI-RS 2015. Neurourol Urodyn 2017; 36:935-942. [DOI: 10.1002/nau.23100] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2016] [Accepted: 07/29/2016] [Indexed: 11/09/2022]
Affiliation(s)
| | | | - Sajjad Rahnama'i
- Departemnt of Urology; Maastricht University; Maastricht The Netherlands
| | - Paul Abrams
- Department of Urology; Bristol Urological Institute; Bristol United Kingdom
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Hasirci E, Turunc T, Bal N, Goren MR, Celik H, Kervancioglu E, Dirim A, Tekindal MA, Ozkardes H. Distribution and number of Cajal-like cells in testis tissue with azoospermia. Kaohsiung J Med Sci 2017; 33:181-186. [DOI: 10.1016/j.kjms.2017.01.006] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2016] [Revised: 01/13/2017] [Accepted: 01/19/2017] [Indexed: 10/20/2022] Open
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Drumm BT, Baker SA. Teaching a changing paradigm in physiology: a historical perspective on gut interstitial cells. ADVANCES IN PHYSIOLOGY EDUCATION 2017; 41:100-109. [PMID: 28188197 DOI: 10.1152/advan.00154.2016] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/29/2016] [Revised: 11/11/2016] [Accepted: 12/27/2016] [Indexed: 06/06/2023]
Abstract
The study and teaching of gastrointestinal (GI) physiology necessitates an understanding of the cellular basis of contractile and electrical coupling behaviors in the muscle layers that comprise the gut wall. Our knowledge of the cellular origin of GI motility has drastically changed over the last 100 yr. While the pacing and coordination of GI contraction was once thought to be solely attributable to smooth muscle cells, it is now widely accepted that the motility patterns observed in the GI tract exist as a result of a multicellular system, consisting of not only smooth muscle cells but also enteric neurons and distinct populations of specialized interstitial cells that all work in concert to ensure proper GI functions. In this historical perspective, we focus on the emerging role of interstitial cells in GI motility and examine the key discoveries and experiments that led to a major shift in a paradigm of GI physiology regarding the role of interstitial cells in modulating GI contractile patterns. A review of these now classic experiments and papers will enable students and educators to fully appreciate the complex, multicellular nature of GI muscles as well as impart lessons on how shifting paradigms in physiology are fueled by new technologies that lead to new emerging discoveries.
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Affiliation(s)
- Bernard T Drumm
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - Salah A Baker
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
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Balikci O, Turunç T, Bal N, Çelik H, Özkardeş H. Comparison of Cajal-like cells in pelvis and proximal ureter of kidney with and without hydronephrosis. Int Braz J Urol 2016; 41:1178-84. [PMID: 26742978 PMCID: PMC4756946 DOI: 10.1590/s1677-5538.ibju.2014.0427] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2014] [Accepted: 03/22/2015] [Indexed: 11/21/2022] Open
Abstract
OBJECTIVES To evaluate effects of Cajal-like cells on human renal pelvis and proximal ureter on peristalsis. MATERIALS AND METHODS 63 patients submitted to nephrectomy due to atrophic non-functional kidney associated with hydroureteronephrosis were included as study group and 30 cases with nephrectomy due to other reasons were included as control group. Samples from renal pelvis and proximal ureters were obtained and sections of 5µ form paraffin blocks of these samples were prepared; layers of lamina propria and muscularis mucosa were examined by immune-histochemistry using CD117 in order to determine count and distribution of Cajal-like cells. RESULTS During immune-histochemical examinations of sections, obtained from renal pelvis and proximal ureter of hydronephrotic kidneys by CD117, Cajal-like cells number determined in lamina propria and muscularis propria was statistically significantly lower compared to control group (p<0.001). Distribution of Cajal-like cells in renal pelvis and proximal tubulus was similar under examination by light microscope, and also both groups were not different from each other regarding staining intensity of Cajal-like cells by c-kit. CONCLUSION Significantly reduced number of Cajal-like cells in study group compared to control group, shows that these cells may have a key role in regulation of peristalsis at level of renal pelvis and proximal ureter in urinary system.
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Affiliation(s)
- Omer Balikci
- Department of Urology, Manisa Alaşehir State Hospital, Manisa, Turkey
| | - Tahsin Turunç
- Department of Urology, School of Medicine, Başkent University, Adana, Turkey
| | - Nebil Bal
- Department of Patology, School of Medicine, Başkent University, Adana, Turkey
| | - Hüseyin Çelik
- Department of Urology, School of Medicine, Inonu University, Malatya, Turkey
| | - Hakan Özkardeş
- Department of Urology, School of Medicine, Başkent University, Adana, Turkey
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Abstract
The mammalian urethra is a muscular tube responsible for ensuring that urine remains in the urinary bladder until urination. In order to prevent involuntary urine leakage, the urethral musculature must be capable of constricting the urethral lumen to an extent that exceeds bladder intravesicular pressure during the urine-filling phase. The main challenge in anti-incontinence treatments involves selectively-controlling the excitability of the smooth muscles in the lower urinary tract. Almost all strategies to battle urinary incontinence involve targeting the bladder and as a result, this tissue has been the focus for the majority of research and development efforts. There is now increasing recognition of the value of targeting the urethral musculature in the treatment and management of urinary incontinence. Newly-identified and characterized ion channels and pathways in the smooth muscle of the urethra provides a range of potential therapeutic targets for the treatment of urinary incontinence. This review provides a summary of the current state of knowledge of the ion channels discovered in urethral smooth muscle cells that regulate their excitability.
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Affiliation(s)
- Barry D Kyle
- a Department of Physiology & Pharmacology; Libin Cardiovascular Institute and The Smooth Muscle Research Group ; University of Calgary ; Calgary , AB Canada
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Drumm BT, Large RJ, Hollywood MA, Thornbury KD, Baker SA, Harvey BJ, McHale NG, Sergeant GP. The role of Ca(2+) influx in spontaneous Ca(2+) wave propagation in interstitial cells of Cajal from the rabbit urethra. J Physiol 2015; 593:3333-50. [PMID: 26046824 DOI: 10.1113/jp270883] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2015] [Accepted: 06/01/2015] [Indexed: 12/25/2022] Open
Abstract
KEY POINTS Tonic contractions of rabbit urethra are associated with spontaneous electrical slow waves that are thought to originate in pacemaker cells termed interstitial cells of Cajal (ICC). ICC pacemaker activity results from their ability to generate propagating Ca(2+) waves, although the exact mechanisms of propagation are not understood. In this study, we have identified spontaneous localised Ca(2+) events for the first time in urethral ICC; these were due to Ca(2+) release from the endoplasmic reticulum (ER) via ryanodine receptors (RyRs) and, while they often remained localised, they sometimes initiated propagating Ca(2+) waves. We show that propagation of Ca(2+) waves in urethral ICC is critically dependent upon Ca(2+) influx via reverse mode NCX. Our data provide a clearer understanding of the intracellular mechanisms involved in the generation of ICC pacemaker activity. Interstitial cells of Cajal (ICC) are putative pacemaker cells in the rabbit urethra. Pacemaker activity in ICC results from spontaneous propagating Ca(2+) waves that are modulated by [Ca(2+)]o and whose propagation is inhibited by inositol tri-phosphate receptor (IP3 R) blockers. The purpose of this study was to further examine the role of Ca(2+) influx and Ca(2+) release in the propagation of Ca(2+) waves. Intracellular Ca(2+) was measured in Fluo-4-loaded ICC using a Nipkow spinning disc confocal microscope at fast acquisition rates (50 fps). We identified previously undetected localised Ca(2+) events originating from ryanodine receptors (RyRs). Inhibiting Ca(2+) influx by removing [Ca(2+)]o or blocking reverse mode sodium-calcium exchange (NCX) with KB-R 7943 or SEA-0400 abolished Ca(2+) waves, while localised Ca(2+) events persisted. Stimulating RyRs with 1 mm caffeine restored propagation. Propagation was also inhibited when Ca(2+) release sites were uncoupled by buffering intracellular Ca(2+) with EGTA-AM. This was reversed when Ca(2+) influx via NCX was increased by reducing [Na(+)]o to 13 mm. Low [Na(+)]o also increased the frequency of Ca(2+) waves and this effect was blocked by tetracaine and ryanodine but not 2-aminoethoxydiphenyl borate (2-APB). RT-PCR revealed that isolated ICC expressed both RyR2 and RyR3 subtypes. We conclude: (i) RyRs are required for the initiation of Ca(2+) waves, but wave propagation normally depends on activation of IP3 Rs; (ii) under resting conditions, propagation by IP3 Rs requires sensitisation by influx of Ca(2+) via reverse mode NCX; (iii) propagation can be maintained by RyRs if they have been sensitised to Ca(2+).
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Affiliation(s)
- Bernard T Drumm
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland.,Department of Molecular Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland.,Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada, 89557, USA
| | - Roddy J Large
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Mark A Hollywood
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Keith D Thornbury
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Salah A Baker
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada, 89557, USA
| | - Brian J Harvey
- Department of Molecular Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland
| | - Noel G McHale
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
| | - Gerard P Sergeant
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
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Bradley E, Fedigan S, Webb T, Hollywood MA, Thornbury KD, McHale NG, Sergeant GP. Pharmacological characterization of TMEM16A currents. Channels (Austin) 2015; 8:308-20. [PMID: 24642630 DOI: 10.4161/chan.28065] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Recent studies have shown that transmembrane protein 16 A (TMEM16A) is a subunit of calcium-activated chloride channels (CACCs). Pharmacological agents have been used to probe the functional role of CACCs, however their effect on TMEM16A currents has not been systematically investigated. In the present study, we characterized the voltage and concentration-dependent effects of 2 traditional CACC inhibitors (niflumic acid and anthracene-9-carboxcylic acid) and 2 novel CACC / TMEM16A inhibitors (CACC(inh)A01 and T16A(inh)A01) on TMEM16A currents. The whole cell patch clamp technique was used to record TMEM16A currents from HE K 293 cells that stably expressed human TMEM16A. Niflumic acid, A-9-C, CACC(inh)A01 and T16A(inh)A01 inhibited TMEM16A currents with IC50 values of 12, 58, 1.7 and 1.5 μM, respectively, however, A-9-C and niflumic acid were less efficacious at negative membrane potentials. A-9-C and niflumic acid reduced the rate of TMEM16A tail current deactivation at negative membrane potentials and A-9-C (1 mM) enhanced peak TMEM16A tail current amplitude. In contrast, the inhibitory effects of CACC(inh)A01 and T16A(inh)A01 were independent of voltage and they did not prolong the rate of TMEM16A tail current deactivation. The effects of niflumic acid and A-9-C on TMEM16A currents were similar to previous observations on CACCs in vascular smooth muscle, strengthening the hypothesis that they are encoded by TMEM16A. However, CACC(inh)A01 and T16A(inh)A01 were more potent inhibitors of TMEM16A channels and their effects were not diminished at negative membrane potentials making them attractive candidates to interrogate the functional role of TMEM16A channels in future studies.
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The role of cellular coupling in the spontaneous generation of electrical activity in uterine tissue. PLoS One 2015; 10:e0118443. [PMID: 25793276 PMCID: PMC4368634 DOI: 10.1371/journal.pone.0118443] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2014] [Accepted: 01/16/2015] [Indexed: 11/19/2022] Open
Abstract
The spontaneous emergence of contraction-inducing electrical activity in the uterus at the beginning of labor remains poorly understood, partly due to the seemingly contradictory observation that isolated uterine cells are not spontaneously active. It is known, however, that the expression of gap junctions increases dramatically in the approach to parturition, by more than one order of magnitude, which results in a significant increase in inter-cellular electrical coupling. In this paper, we build upon previous studies of the activity of electrically excitable smooth muscle cells (myocytes) and investigate the mechanism through which the coupling of these cells to electrically passive cells results in the generation of spontaneous activity in the uterus. Using a recently developed, realistic model of uterine muscle cell dynamics, we investigate a system consisting of a myocyte coupled to passive cells. We then extend our analysis to a simple two-dimensional lattice model of the tissue, with each myocyte being coupled to its neighbors, as well as to a random number of passive cells. We observe that different dynamical regimes can be observed over a range of gap junction conductances: at low coupling strength, corresponding to values measured long before delivery, the activity is confined to cell clusters, while the activity for high coupling, compatible with values measured shortly before delivery, may spread across the entire tissue. Additionally, we find that the system supports the spontaneous generation of spiral wave activity. Our results are both qualitatively and quantitatively consistent with observations from in vitro experiments. In particular, we demonstrate that the increase in inter-cellular electrical coupling observed experimentally strongly facilitates the appearance of spontaneous action potentials that may eventually lead to parturition.
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Rabotti C, Mischi M. Propagation of electrical activity in uterine muscle during pregnancy: a review. Acta Physiol (Oxf) 2015; 213:406-16. [PMID: 25393600 DOI: 10.1111/apha.12424] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2014] [Revised: 08/13/2014] [Accepted: 11/07/2014] [Indexed: 11/29/2022]
Abstract
The uterine muscle (the myometrium) plays its most evident role during pregnancy, when quiescence is required for adequate nourishment and development of the foetus, and during labour, when forceful contractions are needed to expel the foetus and the other products of conception. The myometrium is composed of smooth muscle cells. Contraction is initiated by the spontaneous generation of electrical activity at the cell level in the form of action potentials. The mechanisms underlying uterine quiescence during pregnancy and electrical activation during labour remain largely unknown; as a consequence, the clinical management of preterm contractions during pregnancy and inefficient uterine contractility during labour remains suboptimal. In an effort to improve clinical management of uterine contractions, research has focused on understanding the propagation properties of the electrical activity of the uterus. Different perspectives have been undertaken, from animal and in vitro experiments up to clinical studies and dedicated methods for non-invasive parameter estimation. A comparison of the results is not straightforward due to the wide range of different approaches reported in the literature. However, previous studies unanimously reveal a unique complexity as compared to other organs in the pattern of uterine electrical activity propagation, which necessarily needs to be taken into consideration for future studies to be conclusive. The aim of this review is to structure current variegated knowledge on the properties of the uterus in terms of pacemaker position, pattern, direction and speed of the electrical activity during pregnancy and labour.
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Affiliation(s)
- C. Rabotti
- Electrical Engineering Department; Eindhoven University of Technology; Eindhoven the Netherlands
| | - M. Mischi
- Electrical Engineering Department; Eindhoven University of Technology; Eindhoven the Netherlands
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Canda AE, Dogan H, Kandemir O, Atmaca AF, Akbulut Z, Balbay MD. Does diabetes affect the distribution and number of interstitial cells and neuronal tissue in the ureter, bladder, prostate, and urethra of humans? Cent European J Urol 2014; 67:366-74. [PMID: 25667756 PMCID: PMC4310884 DOI: 10.5173/ceju.2014.04.art10] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2014] [Revised: 09/16/2014] [Accepted: 09/19/2014] [Indexed: 11/22/2022] Open
Abstract
INTRODUCTION The aim of this study was to investigate and compare the distribution and number of interstitial cells (ICs) and neuronal tissue in the ureter, bladder, prostate, and urethra of human patients with and without diabetes. MATERIAL AND METHODS Human tissue was obtained from patients who had undergone radical cystectomy for bladder cancer (10 diabetic and 11 non-diabetic males). Interstitial cells were stained immunohistochemically with anti-human CD117 (c-kit) rabbit polyclonal antibody, Vimentin, and Connexin-43. Neural tissue was stained with synaptophysin. The number of ICs and neurons was evaluated and compared between the groups (diabetic versus non-diabetic). RESULTS The mean number of c-kit (+) ICs in bladder lamina propria was significantly decreased in diabetics (32.40 ±12.96 versus 57.18 ±25.37, p = 0.036). The mean number of ICs in the detrusor muscle was significantly decreased in diabetics (40.50 ±16.79 versus 64.55 ±22.08, p = 0.013). Between the groups, no significant differences were detected regarding the number of ICs at the level of the ureter, urethra, and prostate. No significant differences were detected regarding the number of nerves in the ureter, bladder, prostate, and urethra of both groups. CONCLUSIONS The number of ICs may be decreased in the lamina propria and detrusor muscle of the human bladder in diabetes. This can be an underlying cause of lower urinary tract (LUT) dysfunction in diabetics. Research into the development of drugs targeting or stimulating IC function in order to prevent diabetic LUT dysfunction is warranted.
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Affiliation(s)
- Abdullah Erdem Canda
- Yildirim Beyazit University, School of Medicine, Ankara Ataturk Training & Research Hospital, Department of Urology, Ankara, Turkey
| | - Hayriye Dogan
- Ankara Ataturk Training & Research Hospital, Department of Pathology, Ankara, Turkey
| | - Olcay Kandemir
- Ankara Oncology Training & Research Hospital, Department of Pathology, Ankara, Turkey
| | - Ali Fuat Atmaca
- Yildirim Beyazit University, School of Medicine, Ankara Ataturk Training & Research Hospital, Department of Urology, Ankara, Turkey
| | - Ziya Akbulut
- Yildirim Beyazit University, School of Medicine, Ankara Ataturk Training & Research Hospital, Department of Urology, Ankara, Turkey
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Triguero D, Lafuente-Sanchis A, Garcia-Pascual A. Changes in nerve-mediated contractility of the lower urinary tract in a mouse model of premature ageing. Br J Pharmacol 2014; 171:1687-705. [PMID: 24372152 DOI: 10.1111/bph.12567] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2013] [Revised: 12/04/2013] [Accepted: 12/12/2013] [Indexed: 01/10/2023] Open
Abstract
BACKGROUND AND PURPOSE A high incidence of lower urinary tract disorders is associated with ageing. In the senescent-accelerated prone (SAMP8) mouse strain and the senescent-accelerated resistant (SAMR1) strain, we compared smooth muscle contractility in responses to intrinsic neurotransmitters, both in the bladder and urethra. EXPERIMENTAL APPROACH We analysed micturition frequency, the changes in muscle tension induced by electrical field stimulation or agonist administration, the density of nerves (adrenergic, cholinergic and nitrergic) and interstitial cells (ICs), as well as cGMP accumulation in bladder and urethral preparations. KEY RESULTS Senescent mice of the SAMP8 strain displayed increased micturition frequency and excitatory contractility of neurogenic origin in the bladder. While cholinergic nerve density remained unchanged, there was a mild sensitization to ACh in male mice. Potentiation in the detrusor may be also provoked by the stronger contribution of ATP, together with reduced adrenergic innervation in males and COX-derived prostanoid production in females. The greater excitatory contractility in the urethra was probably due to the sensitization to noradrenaline, in conjunction with attenuated nitrergic relaxation. There were also fewer neuronal NOS immunoreactive (ir) nerves and vimentin-positive ICs, although the sildenafil- and diethylamine-NONOate-induced relaxations and cGMP-ir remained unchanged. CONCLUSIONS AND IMPLICATIONS Premature senescent mice exhibit bladder and urethral hyperexcitability, coupled with reduced urethral relaxation of neurogenic origin, which could model the impaired urinary function in elderly humans. We propose that senescence-accelerated mice provide a useful tool to analyse the basic mechanisms of age-related changes in bladder and urethral function.
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Affiliation(s)
- D Triguero
- Department of Physiology, Veterinary School, Complutense University, Madrid, Spain
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Calcium signalling in Cajal-like interstitial cells of the lower urinary tract. Nat Rev Urol 2014; 11:555-64. [PMID: 25224445 DOI: 10.1038/nrurol.2014.241] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Interstitial cells of Cajal (ICC) serve several critical physiological roles in visceral smooth muscle organs, including acting as electrical pacemakers to modulate phasic contractile activity and as intermediaries in motor neurotransmission. The major roles of ICC have been described in the gastrointestinal tract, however, ICC-like cells (ICC-LC) can also be found in other visceral organs, including those of the lower urinary tract (LUT), where they provide similar functions, acting as electrical pacemakers and as intermediary cells involved in the modulation of neurotransmission to adjacent smooth muscle cells. The physiological functions of ICC-LC, in particular their role as pacemakers, relies on their ability to generate transient and propagating intracellular Ca(2+) events. The role of ICC-LC as pacemakers and neuromodulators in the LUT is increasingly apparent and the study of their intracellular Ca(2+) dynamics will provide a better understanding of their role in LUT excitability.
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Sanders KM, Ward SM, Koh SD. Interstitial cells: regulators of smooth muscle function. Physiol Rev 2014; 94:859-907. [PMID: 24987007 DOI: 10.1152/physrev.00037.2013] [Citation(s) in RCA: 347] [Impact Index Per Article: 31.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Smooth muscles are complex tissues containing a variety of cells in addition to muscle cells. Interstitial cells of mesenchymal origin interact with and form electrical connectivity with smooth muscle cells in many organs, and these cells provide important regulatory functions. For example, in the gastrointestinal tract, interstitial cells of Cajal (ICC) and PDGFRα(+) cells have been described, in detail, and represent distinct classes of cells with unique ultrastructure, molecular phenotypes, and functions. Smooth muscle cells are electrically coupled to ICC and PDGFRα(+) cells, forming an integrated unit called the SIP syncytium. SIP cells express a variety of receptors and ion channels, and conductance changes in any type of SIP cell affect the excitability and responses of the syncytium. SIP cells are known to provide pacemaker activity, propagation pathways for slow waves, transduction of inputs from motor neurons, and mechanosensitivity. Loss of interstitial cells has been associated with motor disorders of the gut. Interstitial cells are also found in a variety of other smooth muscles; however, in most cases, the physiological and pathophysiological roles for these cells have not been clearly defined. This review describes structural, functional, and molecular features of interstitial cells and discusses their contributions in determining the behaviors of smooth muscle tissues.
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Affiliation(s)
- Kenton M Sanders
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - Sean M Ward
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - Sang Don Koh
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
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Drumm BT, Sergeant GP, Hollywood MA, Thornbury KD, McHale NG, Harvey BJ. The role of cAMP dependent protein kinase in modulating spontaneous intracellular Ca²⁺ waves in interstitial cells of Cajal from the rabbit urethra. Cell Calcium 2014; 56:181-7. [PMID: 25063367 DOI: 10.1016/j.ceca.2014.07.002] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2014] [Revised: 06/17/2014] [Accepted: 07/02/2014] [Indexed: 11/19/2022]
Abstract
Interstitial cells of Cajal (ICC) serve as electrical pacemakers in the rabbit urethra. Pacemaking activity in ICC results from spontaneous intracellular Ca(2+) waves that rely on Ca(2+) release from endoplasmic reticulum (ER) stores. The purpose of this study was to investigate if the action of protein kinase A (PKA) affected the generation of Ca(2+) waves in ICC. Intracellular [Ca(2+)] was measured in fluo-4 loaded ICC, freshly isolated from the rabbit urethra using a Nipkow spinning disc confocal microscope. Application of the PKA inhibitor H-89 (10 μM) significantly inhibited the generation of spontaneous Ca(2+) waves in ICC and this was associated with a significant decrease in the ER Ca(2+) load, measured with 10mM caffeine responses. Ca(2+) waves could be rescued in the presence of H-89 by stimulating ryanodine receptors (RyRs) with 1mM caffeine but not by activation of inositol 1,4,5 tri-phosphate receptors (IP3Rs) with 10 μM phenylephrine. Increasing intracellular PKA with the cAMP agonists forskolin and 8-bromo-cAMP failed to yield an increase in Ca(2+) wave activity. We conclude that PKA may be maximally active under basal conditions in ICC and that inhibition of PKA with H-89 leads to a decreased ER Ca(2+) load sufficient to inactivate IP3Rs but not RyRs.
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Affiliation(s)
- Bernard T Drumm
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dublin Road, Co. Louth, Ireland; Molecular Medicine Laboratories, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Co. Dublin, Ireland.
| | - Gerard P Sergeant
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dublin Road, Co. Louth, Ireland
| | - Mark A Hollywood
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dublin Road, Co. Louth, Ireland
| | - Keith D Thornbury
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dublin Road, Co. Louth, Ireland
| | - Noel G McHale
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dublin Road, Co. Louth, Ireland
| | - Brian J Harvey
- Molecular Medicine Laboratories, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Co. Dublin, Ireland
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Hall KA, Ward SM, Cobine CA, Keef KD. Spatial organization and coordination of slow waves in the mouse anorectum. J Physiol 2014; 592:3813-29. [PMID: 24951622 DOI: 10.1113/jphysiol.2014.272542] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
The internal anal sphincter (IAS) develops tone and is important for maintaining a high anal pressure while tone in the rectum is less. The mechanisms responsible for tone generation in the IAS are still uncertain. The present study addressed this question by comparing the electrical properties and morphology of the mouse IAS and distal rectum. The amplitude of tone and the frequency of phasic contractions was greater in the IAS than in rectum while membrane potential (Em) was less negative in the IAS than in rectum. Slow waves (SWs) were of greatest amplitude and frequency at the distal end of the IAS, declining in the oral direction. Dual microelectrode recordings revealed that SWs were coordinated over a much greater distance in the circumferential direction than in the oral direction. The circular muscle layer of the IAS was divided into five to eight 'minibundles' separated by connective tissue septa whereas few septa were present in the rectum. The limited coordination of SWs in the oral direction suggests that the activity in adjacent minibundles is not coordinated. Intramuscular interstitial cells of Cajal and platelet-derived growth factor receptor alpha-positive cells were present in each minibundle suggesting a role for one or both of these cells in SW generation. In summary, three important properties distinguish the IAS from the distal rectum: (1) a more depolarized Em; (2) larger and higher frequency SWs; and (3) the multiunit configuration of the muscle. All of these characteristics may contribute to greater tone generation in the IAS than in the distal rectum.
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Affiliation(s)
- K A Hall
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA
| | - S M Ward
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA
| | - C A Cobine
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA
| | - K D Keef
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA
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Fry CH, Jabr RI. T-type Ca2+ channels and the urinary and male genital tracts. Pflugers Arch 2014; 466:781-9. [PMID: 24463704 DOI: 10.1007/s00424-014-1446-x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2013] [Revised: 01/06/2014] [Accepted: 01/07/2014] [Indexed: 11/26/2022]
Abstract
T-type Ca(2+) channels are widely expressed throughout the urinary and male genital tracts, generally alongside L-type Ca(2+) channels. The use of pharmacological blockers of these channels has suggested functional roles in all regions, with the possible exception of the ureter. Their functional expression is apparent not just in smooth muscle cells but also in interstitial cells that lie in close proximity to muscle, nerve and epithelial components of these tissues. Thus, T-type Ca(2+) channels can contribute directly to modulation of muscle function and indirectly to changes of epithelial and nerve function. T-type Ca(2+) channel activity modulates phasic contractile activity, especially in conjunction with Ca(2+)-activated K(+) channels, and also to agonist-dependent responses in different tissues. Upregulation of channel density occurs in pathological conditions associated with enhanced contractile responses, e.g. overactive bladder, but it is unclear if this is causal or a response to the pathological state. Moreover, T-type Ca(2+) channels may have a role in the development of prostate tumours regulating the secretion of mitogens from neuroendocrine cells. Although a number of selective channel blockers exist, their relative selectivity over L-type Ca(2+) channels is often low and makes evaluation of T-type Ca(2+) channel function in the whole organism difficult.
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Affiliation(s)
- C H Fry
- Department of Biochemistry and Physiology, University of Surrey, Guildford, GU2 7XH, UK,
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Drumm BT, Sergeant GP, Hollywood MA, Thornbury KT, Matsuda TT, Baba A, Harvey BJ, McHale NG. The effect of high [K(+)]o on spontaneous Ca(2+) waves in freshly isolated interstitial cells of Cajal from the rabbit urethra. Physiol Rep 2014; 2:e00203. [PMID: 24744882 PMCID: PMC3967686 DOI: 10.1002/phy2.203] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2013] [Revised: 12/19/2013] [Accepted: 12/23/2013] [Indexed: 11/20/2022] Open
Abstract
Interstitial cells of Cajal (ICC) act as putative pacemaker cells in the rabbit urethra. Pacemaker activity in ICC results from spontaneous global Ca2+ waves that can be increased in frequency by raising external [K+]. The purpose of this study was to elucidate the mechanism of this response. Intracellular [Ca2+] was measured in fluo‐4‐loaded smooth muscle cells (SMCs) and ICC using a Nipkow spinning disk confocal microscope. Increasing [K+]o to 60 mmol/L caused an increase in [Ca2+]i accompanied by contraction in SMCs. Raising [K+]o did not cause contraction in ICC, but the frequency of firing of spontaneous calcium waves increased. Reducing [Ca2+]o to 0 mmol/L abolished the response in both cell types. Nifedipine of 1 μmol/L blocked the response of SMC to high [K+]o, but did not affect the increase in firing in ICC. This latter effect was blocked by 30 μmol/L NiCl2 but not by the T‐type Ca2+ channel blocker mibefradil (300 nmol/L). However, inhibition of Ca2+ influx via reverse‐mode sodium/calcium exchange (NCX) using either 1 μmol/L SEA0400 or 5 μmol/L KB‐R7943 did block the effect of high [K+]o on ICC. These data suggest that high K+ solution increases the frequency of calcium waves in ICC by increasing Ca2+ influx through reverse‐mode NCX. Pacemaker activity in ICC results from spontaneous global Ca2+ waves that can be increased in frequency by raising external [K+]. The experiments described support the hypothesis that high K+ solution increases the frequency of calcium waves in ICC by increasing Ca2+ influx through reverse‐mode NCX.
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Affiliation(s)
- Bernard T Drumm
- Smooth Muscle Research Centre, Dundalk Institute of Technology, DundalkCo. Louth, Ireland ; Department of Molecular Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland
| | - Gerard P Sergeant
- Smooth Muscle Research Centre, Dundalk Institute of Technology, DundalkCo. Louth, Ireland
| | - Mark A Hollywood
- Smooth Muscle Research Centre, Dundalk Institute of Technology, DundalkCo. Louth, Ireland
| | - Keith T Thornbury
- Smooth Muscle Research Centre, Dundalk Institute of Technology, DundalkCo. Louth, Ireland
| | - Toshio T Matsuda
- Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
| | - Akemichi Baba
- Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
| | - Brian J Harvey
- Department of Molecular Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland
| | - Noel G McHale
- Smooth Muscle Research Centre, Dundalk Institute of Technology, DundalkCo. Louth, Ireland
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Lam M, Dey A, Lang RJ, Exintaris B. Effects of imatinib mesylate on the spontaneous activity generated by the guinea-pig prostate. BJU Int 2013; 112:E398-405. [PMID: 23879919 DOI: 10.1111/j.1464-410x.2012.11660.x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
UNLABELLED What's known on the subject? and what does the study add?: Several studies have examined the functional role of tyrosine kinase receptors in the generation of spontaneous activity in various segments of the gastrointestinal and urogenital tracts through the application of its inhibitor, imatinib mesylate (Glivec®), but results are fairly inconsistent. This is the first study detailing the effects of imatinib mesylate on the spontaneous activity in the young and ageing prostate gland. As spontaneous electrical activity underlies the spontaneous rhythmic prostatic contractions that occur at rest, elucidating the mechanisms involved in the regulation of the spontaneous electrical activity and the resultant phasic contractions could conceivably lead to the identification of better targets and the development of more specific therapeutic agents to treat prostate conditions. OBJECTIVE To investigate the effect of imatinib mesylate, a tyrosine kinase receptor inhibitor, in the generation of spontaneous electrical and contractile activity in the young and ageing guinea-pig prostate. MATERIALS AND METHODS Standard tension and intracellular recording were used to measure spontaneous contractions and slow waves, respectively from the guinea-pig prostate at varying concentrations of imatinib mesylate (1-50 μm). RESULTS Imatinib mesylate (1-10 μm), did not significantly affect slow waves recorded in the prostate of both age groups but at 50 μm, the amplitude of slow waves from the ageing guinea-pig prostate was significantly reduced (P < 0.05, n = 5). In contrast, the amplitude of contractions across all concentrations in the young guinea-pig prostate was reduced to between 35% and 41% of control, while the frequency was reduced to 15.7% at 1 μm (n = 7), 49.8% at 5 μm (n = 10), 46.2% at 10 μm (n = 7) and 53.1% at 50 μm (n = 5). Similarly, imatinib mesylate attenuated the amplitude and slowed the frequency of contractions in ageing guinea-pigs to 5.15% and 3.3% at 1 μm (n = 6); 21.1% and 20.8% at 5 μm (n = 8); 58.4% and 8.8% at 10 μm (n = 11); 72.7% and 60% at 50 μm (n = 5). CONCLUSIONS A significant reduction in contractions but persistence of slow waves suggests imatinib mesylate may affect the smooth muscle contractile mechanism. Imatinib mesylate also significantly reduced contractions in the prostates of younger guinea pigs more than older ones, which is consistent with the notion that the younger guinea-pig prostate is more reliant on the tyrosine-dependent pacemaker ability of interstitial cells of Cajal-like prostatic interstitial cells.
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Affiliation(s)
- Michelle Lam
- Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Australia
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Lamina propria: The functional center of the bladder? Neurourol Urodyn 2013; 33:9-16. [DOI: 10.1002/nau.22465] [Citation(s) in RCA: 98] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2013] [Accepted: 06/17/2013] [Indexed: 11/07/2022]
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Kyle BD, Bradley E, Large R, Sergeant GP, McHale NG, Thornbury KD, Hollywood MA. Mechanisms underlying activation of transient BK current in rabbit urethral smooth muscle cells and its modulation by IP3-generating agonists. Am J Physiol Cell Physiol 2013; 305:C609-22. [PMID: 23804200 DOI: 10.1152/ajpcell.00025.2013] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
We used the perforated patch-clamp technique at 37°C to investigate the mechanisms underlying the activation of a transient large-conductance K(+) (tBK) current in rabbit urethral smooth muscle cells. The tBK current required an elevation of intracellular Ca(2+), resulting from ryanodine receptor (RyR) activation via Ca(2+)-induced Ca(2+) release, triggered by Ca(2+) influx through L-type Ca(2+) (CaV) channels. Carbachol inhibited tBK current by reducing Ca(2+) influx and Ca(2+) release and altered the shape of spike complexes recorded under current-clamp conditions. The tBK currents were blocked by iberiotoxin and penitrem A (300 and 100 nM, respectively) and were also inhibited when external Ca(2+) was removed or the CaV channel inhibitors nifedipine (10 μM) and Cd(2+) (100 μM) were applied. The tBK current was inhibited by caffeine (10 mM), ryanodine (30 μM), and tetracaine (100 μM), suggesting that RyR-mediated Ca(2+) release contributed to the activation of the tBK current. When IP3 receptors (IP3Rs) were blocked with 2-aminoethoxydiphenyl borate (2-APB, 100 μM), the amplitude of the tBK current was not reduced. However, when Ca(2+) release via IP3Rs was evoked with phenylephrine (1 μM) or carbachol (1 μM), the tBK current was inhibited. The effect of carbachol was abolished when IP3Rs were blocked with 2-APB or by inhibition of muscarinic receptors with the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (1 μM). Under current-clamp conditions, bursts of action potentials could be evoked with depolarizing current injection. Carbachol reduced the number and amplitude of spikes in each burst, and these effects were reduced in the presence of 2-APB. In the presence of ryanodine, the number and amplitude of spikes were also reduced, and carbachol was without further effect. These data suggest that IP3-generating agonists can modulate the electrical activity of rabbit urethral smooth muscle cells and may contribute to the effects of neurotransmitters on urethral tone.
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Affiliation(s)
- Barry D Kyle
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Ireland
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Wang JP, Ding GF, Wang QZ. Interstitial cells of Cajal mediate excitatory sympathetic neurotransmission in guinea pig prostate. Cell Tissue Res 2013; 352:479-86. [PMID: 23411811 DOI: 10.1007/s00441-013-1572-3] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2012] [Accepted: 01/17/2013] [Indexed: 01/26/2023]
Abstract
Morphological and functional studies have confirmed that interstitial cells of Cajal (ICCs) are involved in many enteric motor neurotransmission pathways. Recent investigations have demonstrated that human and guinea pig prostate glands possess a distinct cell type with morphological and immunological similarities to ICCs. These prostate ICCs have a close relationship with nerve bundles and smooth muscle cells. Prostate smooth muscle tone is largely induced by stimulation from the sympathetic nervous system, which releases excitatory norepinephrine (NE) to act on the α1-adrenoceptor. We have performed morphological and functional experiments to determine the role of ICCs in sympathetic neurotransmission in the guinea pig prostate based on the hypothesis that prostate ICCs act as mediators of sympathetic neurotransmission. Immunohistochemistry revealed many close points of contact between ICCs and sympathetic nerve bundles and smooth muscle cells. Double-labeled sections revealed that α1-adrenoceptor and the gap junction protein connexin 43 were expressed in prostate ICCs. Surprisingly, prostate ICCs co-expressed tyrosine hydroxylase and dopamine β-hydroxylase, two markers of sympathetic neurons. Functionally, the application of NE evoked a large single inward current in isolated prostate ICCs in a dose-dependent manner. The inward current evoked by NE was mediated via the activation of α1-adrenoceptors, because it was abolished by the non-specific α-adrenoceptor antagonist, phentolamine and the specific α1-adrenoceptor antagonist, prazosin. Thus, ICCs in the guinea pig prostate are target cells for prostate sympathetic nerves and possess the morphological and functional characteristics required to mediate sympathetic signals.
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Affiliation(s)
- Jiang-ping Wang
- Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
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McCloskey KD. Bladder interstitial cells: an updated review of current knowledge. Acta Physiol (Oxf) 2013; 207:7-15. [PMID: 23034074 DOI: 10.1111/apha.12009] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2011] [Revised: 01/22/2012] [Accepted: 09/10/2012] [Indexed: 01/12/2023]
Abstract
The field of bladder research has been energized by the study of novel interstitial cells (IC) over the last decade. Several subgroups of IC are located within the bladder wall and make structural interactions with nerves and smooth muscle, indicating integration with intercellular communication and key physiological functions. Significant progress has been made in the study of bladder ICs' cellular markers, ion channels and receptor expression, electrical and calcium signalling, yet their specific functions in normal bladder filling and emptying remain elusive. There is increasing evidence that the distribution of IC is altered in bladder pathophysiologies suggesting that changes in IC may be linked with the development of bladder dysfunction. This article summarizes the current state of the art of our knowledge of IC in normal bladder and reviews the literature on IC in dysfunctional bladder.
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Affiliation(s)
- K. D. McCloskey
- Centre for Cancer Research and Cell Biology; Queen's University Belfast; Belfast; Northern Ireland; UK
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Harhun MI, Huggins CL, Ratnasingham K, Raje D, Moss RF, Szewczyk K, Vasilikostas G, Greenwood IA, Khong TK, Wan A, Reddy M. Resident phenotypically modulated vascular smooth muscle cells in healthy human arteries. J Cell Mol Med 2012; 16:2802-12. [PMID: 22862785 PMCID: PMC3492755 DOI: 10.1111/j.1582-4934.2012.01609.x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2012] [Accepted: 07/13/2012] [Indexed: 12/24/2022] Open
Abstract
Vascular interstitial cells (VICs) are non-contractile cells with filopodia previously described in healthy blood vessels of rodents and their function remains unknown. The objective of this study was to identify VICs in human arteries and to ascertain their role. VICs were identified in the wall of human gastro-omental arteries using transmission electron microscopy. Isolated VICs showed ability to form new and elongate existing filopodia and actively change body shape. Most importantly sprouting VICs were also observed in cell dispersal. RT-PCR performed on separately collected contractile vascular smooth muscle cells (VSMCs) and VICs showed that both cell types expressed the gene for smooth muscle myosin heavy chain (SM-MHC). Immunofluorescent labelling showed that both VSMCs and VICs had similar fluorescence for SM-MHC and αSM-actin, VICs, however, had significantly lower fluorescence for smoothelin, myosin light chain kinase, h-calponin and SM22α. It was also found that VICs do not have cytoskeleton as rigid as in contractile VSMCs. VICs express number of VSMC-specific proteins and display features of phenotypically modulated VSMCs with increased migratory abilities. VICs, therefore represent resident phenotypically modulated VSMCs that are present in human arteries under normal physiological conditions.
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Affiliation(s)
- Maksym I Harhun
- Pharmacology and Cell Physiology Research Group, Division of Biomedical Sciences, St. George's, University of London, London, United Kingdom.
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Canda AE, Isgoren AE. Re: Increased risk of diabetes in patients with urinary calculi: a 5-year followup study: S.-D. Chung, Y.-K. Chen and H.-C. Lin J Urol 2011; 186: 1888-1893. J Urol 2012; 187:2279-80. [PMID: 22503233 DOI: 10.1016/j.juro.2012.01.132] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2011] [Indexed: 11/26/2022]
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Dixon RE, Hennig GW, Baker SA, Britton FC, Harfe BD, Rock JR, Sanders KM, Ward SM. Electrical slow waves in the mouse oviduct are dependent upon a calcium activated chloride conductance encoded by Tmem16a. Biol Reprod 2012; 86:1-7. [PMID: 21976594 DOI: 10.1095/biolreprod.111.095554] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2022] Open
Abstract
Myosalpinx contractions are critical for oocyte transport along the oviduct. A specialized population of pacemaker cells-oviduct interstitial cells of Cajal-generate slow waves, the electrical events underlying myosalpinx contractions. The ionic basis of oviduct pacemaker activity is unknown. We examined the role of a new class of Ca(2+)-activated Cl(-) channels (CaCCs)-anoctamin 1, encoded by Tmem16a-in oviduct slow wave generation. RT-PCR revealed the transcriptional expression of Tmem16a-encoded CaCCs in the myosalpinx. Intracellular microelectrode recordings were performed in the presence of two pharmacologically distinct Cl(-) channel antagonists, anthracene-9-carboxylic acid and niflumic acid. Both of these inhibitors caused membrane hyperpolarization, reduced the duration of slow waves, and ultimately inhibited pacemaker activity. Niflumic acid also inhibited propagating calcium waves within the myosalpinx. Slow waves were present at birth in wild-type and heterozygous oviducts but failed to develop by birth in mice homozygous for a null allele of Tmem16a (Tmem16a(tm1Bdh/tm1Bdh)). These data suggest that Tmem16a-encoded CaCCs contribute to membrane potential and are responsible for the upstroke and plateau phases of oviduct slow waves.
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Affiliation(s)
- Rose Ellen Dixon
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, 89557, USA
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