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Tsai YM, Jones F, Mullen P, Porter KE, Steele D, Peers C, Gamper N. Vascular Kv7 channels control intracellular Ca 2+ dynamics in smooth muscle. Cell Calcium 2020; 92:102283. [PMID: 32950876 PMCID: PMC7695684 DOI: 10.1016/j.ceca.2020.102283] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2020] [Revised: 08/21/2020] [Accepted: 08/26/2020] [Indexed: 01/23/2023]
Abstract
Voltage-gated Kv7 (or KCNQ) channels control activity of excitable cells, including vascular smooth muscle cells (VSMCs), by setting their resting membrane potential and controlling other excitability parameters. Excitation-contraction coupling in muscle cells is mediated by Ca2+ but until now, the exact role of Kv7 channels in cytosolic Ca2+ dynamics in VSMCs has not been fully elucidated. We utilised microfluorimetry to investigate the impact of Kv7 channel activity on intracellular Ca2+ levels and electrical activity of rat A7r5 VSMCs and primary human internal mammary artery (IMA) SMCs. Both, direct (XE991) and G protein coupled receptor mediated (vasopressin, AVP) Kv7 channel inhibition induced robust Ca2+ oscillations, which were significantly reduced in the presence of Kv7 channel activator, retigabine, L-type Ca2+ channel inhibitor, nifedipine, or T-type Ca2+ channel inhibitor, NNC 55-0396, in A7r5 cells. Membrane potential measured using FluoVolt exhibited a slow depolarisation followed by a burst of sharp spikes in response to XE991; spikes were temporally correlated with Ca2+ oscillations. Phospholipase C inhibitor (edelfosine) reduced AVP-induced, but not XE991-induced Ca2+ oscillations. AVP and XE991 induced a large increase of [Ca2+]i in human IMA, which was also attenuated with retigabine, nifedipine and NNC 55-0396. RT-PCR, immunohistochemistry and electrophysiology suggested that Kv7.5 was the predominant Kv7 subunit in both rat and human arterial SMCs; CACNA1C (Cav1.2; L-type) and CACNA1 G (Cav3.1; T-type) were the most abundant voltage-gated Ca2+ channel gene transcripts in both types of VSMCs. This study establishes Kv7 channels as key regulators of Ca2+ signalling in VSMCs with Kv7.5 playing a dominant role.
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Affiliation(s)
- Yuan-Ming Tsai
- School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom; Division of Thoracic Surgery, Department of Surgery, Tri-Service General Hospital, National Defence Medical Centre, Taipei 11490, Taiwan.
| | - Frederick Jones
- School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom
| | - Pierce Mullen
- School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom
| | - Karen E Porter
- Leeds Institute of Cardiovascular and Metabolic Medicine, Faculty of Medicine and Health, University of Leeds, Leeds, LS2 9JT, United Kingdom
| | - Derek Steele
- School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom
| | - Chris Peers
- Leeds Institute of Cardiovascular and Metabolic Medicine, Faculty of Medicine and Health, University of Leeds, Leeds, LS2 9JT, United Kingdom
| | - Nikita Gamper
- School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom.
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Zhan B, Kong C, Zhang Z, Dong X, Zhang N. Inhibition of PKCα reduces the ability of migration of kidney cancer cells but has no impact on cell apoptosis. Exp Ther Med 2017; 13:2473-2479. [PMID: 28565866 DOI: 10.3892/etm.2017.4258] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2015] [Accepted: 01/20/2017] [Indexed: 12/29/2022] Open
Abstract
Kidney cancer is among the most important causes of cancer-associated mortality worldwide. The present study aimed to evaluate protein kinase C α (PKCα) expression in kidney cancer tissues and cell lines, and its significance in apoptosis and migration. Expression of PKCα was analyzed using quantitative polymerase chain reaction and western blotting. In addition, the inhibitor of PKCα (calphostin C and GO6976) was used to treat kidney cancer cells. The ACHN cell line was generated with PKCα-small-interfering RNA (siRNA) and a stable expression of PKCα, in order to facilitate the analysis of apoptosis and migration of PKCα during knockdown and inactivation. Flow cytometry was used to determine the rates of apoptosis. Immunohistochemical staining was used to identify the localization of PKCα in renal clear cell carcinoma and normal sections. PKCα expression in normal tissues was found to be greater than in cancerous tissues. Furthermore, apoptosis was not promoted with PKCα inhibitors or PKCα-siRNA treatment, and a decrease of the migration ability was observed following transfection with PKCα-dominant negative. The results indicated that inhibition of PKCα might not contribute to apoptosis progression in kidney carcinoma.
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Affiliation(s)
- Bo Zhan
- Department of Urology, The First Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China
| | - Chuize Kong
- Department of Urology, The First Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China
| | - Zhe Zhang
- Department of Urology, The First Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China
| | - Xiao Dong
- Department of Urology, The First Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China
| | - Naiwen Zhang
- Department of Urology, The First Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China
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Sakakibara K, Feng GG, Li J, Akahori T, Yasuda Y, Nakamura E, Hatakeyama N, Fujiwara Y, Kinoshita H. Kynurenine causes vasodilation and hypotension induced by activation of KCNQ-encoded voltage-dependent K(+) channels. J Pharmacol Sci 2015; 129:31-7. [PMID: 26318674 DOI: 10.1016/j.jphs.2015.07.042] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2015] [Revised: 07/25/2015] [Accepted: 07/31/2015] [Indexed: 12/23/2022] Open
Abstract
Kynurenine is a potential contributor to hypotension in animal and human sepsis. The present study was designed to examine whether the voltage-dependent K(+) channels encoded by the KCNQ gene family (Kv7 channels) mediate vasodilator effects of kynurenine and whether modulation of these channels ameliorates hypotension caused by this compound. Rat aortas and mesenteric arteries or human omental arteries without endothelium were used. Some rings were incubated with the selective Kv7 channel inhibitor linopirdine (10 μM). l-Kynurenine (10 μM-1 mM) induced concentration-dependent relaxation in rat aortas and mesenteric arteries as well as human omental arteries, whereas linopirdine abolished the relaxation. l-Kynurenine (1 mM) produced hyperpolarization of vascular smooth muscle, which was reversed by linopirdine (10 μM). Wistar rats received l-kynurenine (1 mM) iv and subsequent linopirdine (10 μM) iv under 3% sevoflurane inhalation. l-Kynurenine iv caused hypotension, whereas linopirdine iv partially reversed it. In conclusion, kynurenine dilates arteries from rats as well as humans via Kv7 channels in the vascular smooth muscle. In rats, this tryptophan metabolite causes hypotension, which is partly counteracted by Kv7 channel inhibition. These results suggest that modulation of Kv7 channels may be a novel strategy to treat hypotension induced by the kynurenine.
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Affiliation(s)
- Kensuke Sakakibara
- Department of Anesthesiology, Aichi Medical University School of Medicine, 1-1 Yazako Karimata, Nagakute, Aichi 480-1195, Japan
| | - Guo-Gang Feng
- Department of Pharmacology, Aichi Medical University School of Medicine, 1-1 Yazako Karimata, Nagakute, Aichi 480-1195, Japan
| | - Jiazheng Li
- Department of Anesthesiology, Aichi Medical University School of Medicine, 1-1 Yazako Karimata, Nagakute, Aichi 480-1195, Japan
| | - Takahiko Akahori
- Department of Anesthesiology, Aichi Medical University School of Medicine, 1-1 Yazako Karimata, Nagakute, Aichi 480-1195, Japan
| | - Yoshitaka Yasuda
- Department of Anesthesiology, Aichi Medical University School of Medicine, 1-1 Yazako Karimata, Nagakute, Aichi 480-1195, Japan
| | - Emi Nakamura
- Department of Anesthesiology, Aichi Medical University School of Medicine, 1-1 Yazako Karimata, Nagakute, Aichi 480-1195, Japan
| | - Noboru Hatakeyama
- Department of Anesthesiology, Aichi Medical University School of Medicine, 1-1 Yazako Karimata, Nagakute, Aichi 480-1195, Japan
| | - Yoshihiro Fujiwara
- Department of Anesthesiology, Aichi Medical University School of Medicine, 1-1 Yazako Karimata, Nagakute, Aichi 480-1195, Japan
| | - Hiroyuki Kinoshita
- Department of Anesthesiology, Aichi Medical University School of Medicine, 1-1 Yazako Karimata, Nagakute, Aichi 480-1195, Japan.
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Selli C, Erac Y, Kosova B, Erdal ES, Tosun M. Silencing of TRPC1 regulates store-operated calcium entry and proliferation in Huh7 hepatocellular carcinoma cells. Biomed Pharmacother 2015; 71:194-200. [DOI: 10.1016/j.biopha.2015.02.024] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2015] [Accepted: 02/21/2015] [Indexed: 01/23/2023] Open
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Brueggemann LI, Mackie AR, Cribbs LL, Freda J, Tripathi A, Majetschak M, Byron KL. Differential protein kinase C-dependent modulation of Kv7.4 and Kv7.5 subunits of vascular Kv7 channels. J Biol Chem 2013; 289:2099-111. [PMID: 24297175 DOI: 10.1074/jbc.m113.527820] [Citation(s) in RCA: 58] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Abstract
The Kv7 family (Kv7.1-7.5) of voltage-activated potassium channels contributes to the maintenance of resting membrane potential in excitable cells. Previously, we provided pharmacological and electrophysiological evidence that Kv7.4 and Kv7.5 form predominantly heteromeric channels and that Kv7 activity is regulated by protein kinase C (PKC) in response to vasoconstrictors in vascular smooth muscle cells. Direct evidence for Kv7.4/7.5 heteromer formation, however, is lacking. Furthermore, it remains to be determined whether both subunits are regulated by PKC. Utilizing proximity ligation assays to visualize single molecule interactions, we now show that Kv7.4/Kv.7.5 heteromers are endogenously expressed in vascular smooth muscle cells. Introduction of dominant-negative Kv7.4 and Kv7.5 subunits in mesenteric artery myocytes reduced endogenous Kv7 currents by 84 and 76%, respectively. Expression of an inducible protein kinase Cα (PKCα) translocation system revealed that PKCα activation is sufficient to suppress endogenous Kv7 currents in A7r5 rat aortic and mesenteric artery smooth muscle cells. Arginine vasopressin (100 and 500 pm) and the PKC activator phorbol 12-myristate 13-acetate (1 nm) each inhibited human (h) Kv7.5 and hKv7.4/7.5, but not hKv7.4 channels expressed in A7r5 cells. A decrease in hKv7.5 and hKv7.4/7.5 current densities was associated with an increase in PKC-dependent phosphorylation of the channel proteins. These findings provide further evidence for a differential regulation of Kv7.4 and Kv7.5 channel subunits by PKC-dependent phosphorylation and new mechanistic insights into the role of heteromeric subunit assembly for regulation of vascular Kv7 channels.
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Modulation of Ca(2+) release through ryanodine receptors in vascular smooth muscle by protein kinase Calpha. Pflugers Arch 2010; 460:791-802. [PMID: 20571823 DOI: 10.1007/s00424-010-0850-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2009] [Revised: 04/15/2010] [Accepted: 05/11/2010] [Indexed: 01/23/2023]
Abstract
The role of protein kinase C (PKC) in Ca(2+) release through ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (SMCs) is not well understood. Caffeine was used to activate RyRs and the intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured in both freshly isolated and cultured mouse aortic SMCs (ASMCs). Pre-activation of PKC with 1,2-dioctanoyl-sn-glycerol (DOG) prevented caffeine-induced [Ca(2+)](i) transients. Application of the PKC inhibitor calphostin C caused [Ca(2+)](i) transients which were not blocked by nifedipine or by removing extracellular Ca(2+) but were abolished after inhibition of the SR Ca(2+)-ATPase with thapsigargin or after inhibition of RyRs with ryanodine. In addition, chelerythrine and GF109203X also elevated resting [Ca(2+)](i) but no further [Ca(2+)](i) increase was seen with subsequent application of caffeine. Selective inhibition of PKCalpha with safingol blocked caffeine-induced [Ca(2+)](i) transients, but the PKCepsilon inhibitory peptide V1-2 did not. In cells expressing a EGFP-tagged PKCalpha, caffeine-induced [Ca(2+)](i) transients were associated with a rapid focal translocation near the cell periphery, while application of ionomycin and DOG caused translocation to the plasma membrane. Western blot showed that caffeine increased the relative amount of PKCalpha in the particulate fraction in a time-dependent manner. Co-immunoprecipitation of RyRs and PKCalpha indicated that they interact. In conclusion, our studies suggest that PKC activation can inhibit the gating activity of RyRs in the SR of ASMCs, and this regulation is most likely mediated by the Ca(2+)-dependent PKCalpha isoform.
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Brueggemann L, Markun D, Barakat J, Chen H, Byron K. Evidence against reciprocal regulation of Ca2+ entry by vasopressin in A7r5 rat aortic smooth-muscle cells. Biochem J 2009; 388:237-44. [PMID: 15603557 PMCID: PMC1186712 DOI: 10.1042/bj20041360] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
Recent studies by Moneer and Taylor [(2002) Biochem. J. 362, 13-21] have proposed a reciprocal regulation of two Ca2+-entry pathways by AVP ([Arg8]-vasopressin) in A7r5 vascular smooth-amuscle cells. Their model proposes that AVP inhibits CCE (capacitative Ca2+ entry) and predicts a rebound of CCE after the removal of AVP. In the present study, we used whole-cell perforated patch-clamp techniques to measure ISOC (store-operated current) corresponding to CCE in A7r5 cells. When 100 nM AVP is present, it activates ISOC with no apparent rebound on removal of AVP. ISOC activated by thapsigargin or cyclopiazonic acid was not inhibited by 100 nM AVP. We also used fura 2 fluorescence techniques to re-examine the model of Moneer and Taylor, specifically focusing on the proposed inhibition of CCE by AVP. We find that 100 nM AVP activates capacitative Mn2+ entry and does not inhibit thapsigargin- or cyclopiazonic acid-activated Mn2+ entry. Moreover, Ca2+ entry after depletion of intracellular Ca2+ stores is enhanced by AVP and we detect no rebound of Ca2+ or Mn2+ entry after AVP removal. On the basis of these findings, we conclude that AVP does not inhibit CCE in A7r5 cells.
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Affiliation(s)
- Lioubov I. Brueggemann
- *Department of Medicine, Cardiovascular Institute, Loyola University Chicago, Maywood, IL 60153, U.S.A
| | - Daniel R. Markun
- *Department of Medicine, Cardiovascular Institute, Loyola University Chicago, Maywood, IL 60153, U.S.A
| | - John A. Barakat
- *Department of Medicine, Cardiovascular Institute, Loyola University Chicago, Maywood, IL 60153, U.S.A
| | - Haiyan Chen
- †Department of Physiology, Cardiovascular Institute, Loyola University Chicago, Maywood, IL 60153, U.S.A
| | - Kenneth L. Byron
- *Department of Medicine, Cardiovascular Institute, Loyola University Chicago, Maywood, IL 60153, U.S.A
- To whom correspondence should be addressed (email )
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8
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Selli C, Erac Y, Kosova B, Tosun M. Post-transcriptional silencing of TRPC1 ion channel gene by RNA interference upregulates TRPC6 expression and store-operated Ca2+ entry in A7r5 vascular smooth muscle cells. Vascul Pharmacol 2009; 51:96-100. [PMID: 19386284 DOI: 10.1016/j.vph.2009.04.001] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2008] [Revised: 04/06/2009] [Accepted: 04/13/2009] [Indexed: 01/23/2023]
Abstract
This study investigates functional consequences of TRPC1 ion channel downregulation observed in aging rat aorta by employing RNA interference in cultured vascular smooth muscle cells. For this purpose, A7r5 aortic smooth muscle cells were used in quantitative gene and protein expression as well as in functional analyses. According to quantitative RT-PCR results, TRPC3, TRPC4 and TRPC5 mRNAs were not at detectable levels. In siTRPC1-transfected cells, TRPC1 mRNA and protein levels were decreased by 40% and 64%; however, those of TRPC6 were drastically increased by 100% and 200%, respectively. In fura-2-loaded TRPC1 knockdown cells, despite the decreased TRPC1 levels, cyclopiazonic acid-induced Ca2+ entry and store-operated Ca2+ entry following Ca2+ addition were elevated by 77% and 135%, respectively. Results suggest that decrease in TRPC1 may be compensated by upregulated TRPC6 that possibly takes part in store-operated Ca2+ entry in vascular smooth muscle cells.
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Affiliation(s)
- Cigdem Selli
- Department of Pharmacology, Faculty of Pharmacy, Ege University, 35100, Izmir, Turkey
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9
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Mani BK, Brueggemann LI, Cribbs LL, Byron KL. Opposite regulation of KCNQ5 and TRPC6 channels contributes to vasopressin-stimulated calcium spiking responses in A7r5 vascular smooth muscle cells. Cell Calcium 2009; 45:400-11. [PMID: 19246091 DOI: 10.1016/j.ceca.2009.01.004] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2008] [Revised: 01/16/2009] [Accepted: 01/22/2009] [Indexed: 12/12/2022]
Abstract
Physiologically relevant concentrations of [Arg(8)]-vasopressin (AVP) induce repetitive action potential firing and Ca(2+) spiking responses in the A7r5 rat aortic smooth muscle cell line. These responses may be triggered by suppression of KCNQ potassium currents and/or activation of non-selective cation currents. Here we examine the relative contributions of KCNQ5 channels and TRPC6 non-selective cation channels to AVP-stimulated Ca(2+) spiking using patch clamp electrophysiology and fura-2 fluorescence measurements in A7r5 cells. KCNQ5 or TRPC6 channel expression levels were suppressed by short hairpin RNA constructs. KCNQ5 knockdown resulted in more positive resting membrane potentials and induced spontaneous action potential firing and Ca(2+) spiking. However physiological concentrations of AVP induced additional depolarization and increased Ca(2+) spike frequency in KCNQ5 knockdown cells. AVP activated a non-selective cation current that was reduced by TRPC shRNA treatment or removal of external Na(+). Neither resting membrane potential nor the AVP-induced depolarization was altered by knockdown of TRPC6 channel expression. However, both TRPC6 shRNA and removal of external Na(+) delayed the onset of Ca(2+) spiking induced by 25pM AVP. These results suggest that suppression of KCNQ5 currents alone is sufficient to excite A7r5 cells, but AVP-induced activation of TRPC6 contributes to the stimulation of Ca(2+) spiking.
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Affiliation(s)
- Bharath K Mani
- Department of Pharmacology and Experimental Therapeutics, Loyola University, Chicago, Maywood, IL 60153, United States
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Vanderheyden V, Devogelaere B, Missiaen L, De Smedt H, Bultynck G, Parys JB. Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release by reversible phosphorylation and dephosphorylation. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2008; 1793:959-70. [PMID: 19133301 DOI: 10.1016/j.bbamcr.2008.12.003] [Citation(s) in RCA: 155] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/30/2008] [Revised: 11/27/2008] [Accepted: 12/03/2008] [Indexed: 12/12/2022]
Abstract
The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is a universal intracellular Ca2+-release channel. It is activated after cell stimulation and plays a crucial role in the initiation and propagation of the complex spatio-temporal Ca2+ signals that control cellular processes as different as fertilization, cell division, cell migration, differentiation, metabolism, muscle contraction, secretion, neuronal processing, and ultimately cell death. To achieve these various functions, often in a single cell, exquisite control of the Ca2+ release is needed. This review aims to highlight how protein kinases and protein phosphatases can interact with the IP3R or with associated proteins and so provide a large potential for fine tuning the Ca2+-release activity and for creating efficient Ca2+ signals in subcellular microdomains.
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Affiliation(s)
- Veerle Vanderheyden
- Laboratory of Molecular and Cellular Signalling, Department Molecular and Cellular Biology, Campus Gasthuisberg O/N1-K. U. Leuven, Herestraat 49-Bus 802, B-3000 Leuven, Belgium
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Mackie AR, Byron KL. Cardiovascular KCNQ (Kv7) potassium channels: physiological regulators and new targets for therapeutic intervention. Mol Pharmacol 2008; 74:1171-9. [PMID: 18684841 DOI: 10.1124/mol.108.049825] [Citation(s) in RCA: 89] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Potassium channels play an important role in electrical signaling of excitable cells such as neurons, cardiac myocytes, and vascular smooth muscle cells (VSMCs). In particular, the KCNQ (Kv7) family of voltage-activated K(+) channels functions to stabilize negative resting membrane potentials and thereby opposes electrical excitability. Of the five known members of the mammalian Kv7 family, Kv7.1 was originally recognized for its role in cardiac myocytes, where it contributes to repolarization of the cardiac action potential. Kv7.2 to Kv7.5 were first discovered in neurons, in which they play a well characterized role in neurotransmitter-stimulated action potential firing. Over the past 5 years, important new roles for Kv7 channels have been identified. Kv7 channels have been found to be expressed in VSMCs from several vascular beds where they contribute to the regulation of vascular tone. There is evidence that Kv7.5 channels in VSMCs are targeted by the hormone vasopressin to mediate its physiological vasoconstrictor actions and evidence that neuronal Kv7 channels in the baroreceptors of the aortic arch adjust the sensitivity of the mechanosensitive neurons to changes in arterial blood pressure. These newly identified physiological roles for Kv7 channels in the cardiovascular system warrant increased attention because pharmacological modulators of this family of channels are being used clinically to treat a variety of neurological disorders. This raises questions about the cardiovascular side effects associated with existing therapies, but there is also obvious potential to capitalize on the established and evolving pharmacology of these channels to develop new therapies for cardiovascular diseases.
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Affiliation(s)
- Alexander R Mackie
- Loyola University Medical Center, 2160 S. First Avenue, Maywood, IL 60153, USA
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Yang SJ, Hwang JC. Ca++ influx is essential for the hypotensive response to arginine vasopressin-induced neuron activation of the area postrema in the rat. Brain Res 2007; 1163:56-71. [PMID: 17644073 DOI: 10.1016/j.brainres.2007.06.010] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2007] [Revised: 06/04/2007] [Accepted: 06/08/2007] [Indexed: 01/23/2023]
Abstract
We attempted to relate the signal pathway to the hypotension induced by arginine vasopressin (AVP) injection into the area postrema (AP) in urethane-anesthetized and ventilated rats with vagotomy. A femoral artery and vein were catheterized to measure the blood pressure (BP) and administer drugs, respectively. The rat was placed on a stereotaxic apparatus to expose the calamus sriptorius (CS) by craniostomy and maintained at normocapnia in hyperoxia. In protocol 1, hypotension evoked by AVP (3.0 x 10(-5) IU) microinjected into the AP 0.2 mm rostral to the CS of the midline was abolished by V(1A) antagonist, U73122 (phospholipase C blocker), and BAPTA-AM (Ca(++) chelator), suggesting that an increasing intracellular Ca(++) is essential for AVP-induced hypotension. In protocol 2, AVP-induced hypotension was abolished by EGTA (extracellular Ca(++) chelator) and Ca(++) blockers such as nifedipine, nimodipine (L-types), and omega-conotoxin MVIIC (P/Q-type), but not by omega-conotoxin GVIA (N-type). In protocol 3, AVP-induced hypotension was blocked by calphostin C (protein kinase C inhibitor) and mimicked by an increase in intracellular K(+) ions that was reversed by EGTA. Vehicle injections produced no changes in BP. In protocol 4, glutamate-induced hypotension was reversed by BAPTA-AM but not by EGTA or V(1A) antagonist. Our data suggest that AVP-induced hypotension depends on Ca(++) influx through a signal pathway from phospholipase C to protein kinase C which inactivates K(+) channels that may depolarize AP neurons to activate L- and P/Q-type Ca(++) channels. This may provide new insights into establishing a relationship between the signal pathway and physiological functions.
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Affiliation(s)
- Shu-Ju Yang
- Department of Life Science, National Taiwan Normal University, Taipei, Taiwan 106, ROC
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Zhong Y, Gao J, Ye W, Huang P, Cheng Y, Jiao Q. Effect of Latanoprost Acid and Pilocarpine on Cultured Rabbit Ciliary Muscle Cells. Ophthalmic Res 2007; 39:232-40. [PMID: 17622744 DOI: 10.1159/000104832] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2006] [Accepted: 05/09/2007] [Indexed: 12/29/2022]
Abstract
OBJECTIVE To study the interaction between latanoprost and pilocarpine on cultured rabbit ciliary muscle (RCM) cells, and investigate the time courses of the two drugs, when given alone or in combination. METHODS Cultured RCM cells were treated for 24 h with different concentrations of latanoprost acid, pilocarpine and mixtures of latanoprost acid and pilocarpine. RNA was extracted, expressions of matrix metalloproteinase 1 (MMP-1), tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2 were detected by reverse transcription-polymerase chain reaction (RT-PCR), and the optimum concentrations of those drugs were found. Then the cells were treated with the optimum concentrations of those drugs for various periods. RNA was extracted after the treatment and expressions of MMP-1, TIMP-1 and TIMP-2 were detected by RT-PCR again. Changes in [Ca(2+)](i) were estimated by fluorescence measurement using the Ca(2+) indicator Fluo-3 AM with a laser scanning confocal microscope. [Ca(2+)](i) of each cell was monitored continually after administration of the drugs. Gray values at 5 s and 2, 4, 6, 8 and 10 min were chosen for statistical analysis, and the influence and time-effect relationship of those drugs on [Ca(2+)](i) of the cultured cells were evaluated. RESULTS Exposure of the cells to increasing concentrations of latanoprost acid induced increased MMP-1 mRNA and decreased TIMP-1 and TIMP-2 mRNA in a dose-dependent manner. After 24 h of treatment, the optimum concentration of latanoprost acid for maximal changes in MMP-1 and TIMP-2 expression was 2 x 10(-7)M, and for maximal changes in TIMP-1 expression, the optimum concentration was 5 x 10(-7)M. When the optimum concentrations of latanoprost acid were chosen to treat the cells for various periods, the optimum time of the peak MMP-1 expression and trough TIMP-1 expression was 24 h, and of the trough TIMP-2 expression, it was 36 h after initiation of treatment. No significant expression changes of MMP-1, TIMP-1 and TIMP-2 mRNA were found when the cells were treated with pilocarpine at any concentration or at any time. Exposure of the cells to the mixtures of latanoprost acid and pilocarpine induced the same changes and time course of MMP-1, TIMP-1, and TIMP-2 mRNA expression as exposure of the cells to latanoprost acid alone. Exposure of ciliary muscle cells to pilocarpine induced an increase in [Ca(2+)](i), with the peak of increase observed at 5 s after initiation of treatment; then [Ca(2+)](i) gradually decreased near to baseline level within 10 min. Exposure of the cells to latanoprost acid did not significantly change [Ca(2+)](i). Exposure of the cells to the mixtures of latanoprost acid and pilocarpine induced the same [Ca(2+)](i) change as exposure to pilocarpine alone. CONCLUSION Latanoprost and pilocarpine have no interaction in their various effects on the cultured RCM cells.
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Affiliation(s)
- Yisheng Zhong
- Department of Ophthalmology, Ruijin Hospital Affiliated with Shanghai Jiaotong University, Shanghai, China.
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Henderson KK, Byron KL. Vasopressin-induced vasoconstriction: two concentration-dependent signaling pathways. J Appl Physiol (1985) 2007; 102:1402-9. [PMID: 17204577 PMCID: PMC2580829 DOI: 10.1152/japplphysiol.00825.2006] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Abstract
Current scientific literature generally attributes the vasoconstrictor effects of [Arg(8)]vasopressin (AVP) to the activation of phospholipase C (PLC) and consequent release of Ca(2+) from the sarcoplasmic reticulum. However, half-maximal activation of PLC requires nanomolar concentrations of AVP, whereas vasoconstriction occurs when circulating concentrations of AVP are orders of magnitude lower. Using cultured vascular smooth muscle cells, we previously identified a novel Ca(2+) signaling pathway activated by 10-100 pM AVP. This pathway is distinguished from the PLC pathway by its dependence on protein kinase C (PKC) and L-type voltage-sensitive Ca(2+) channels (VSCC). In the present study, we used isolated, pressurized rat mesenteric arteries to examine the contributions of these different Ca(2+) signaling mechanisms to AVP-induced vasoconstriction. AVP (10(-14)-10(-6) M) induced a concentration-dependent constriction of arteries that was reversible with a V(1a) vasopressin receptor antagonist. Half-maximal vasoconstriction at 30 pM AVP was prevented by blockade of VSCC with verapamil (10 microM) or by PKC inhibition with calphostin-C (250 nM) or Ro-31-8220 (1 microM). In contrast, acute vasoconstriction induced by 10 nM AVP (maximal) was insensitive to blockade of VSCC or PKC inhibition. However, after 30 min, the remaining vasoconstriction induced by 10 nM AVP was partially dependent on PKC activation and almost fully dependent on VSCC. These results suggest that different Ca(2+) signaling mechanisms contribute to AVP-induced vasoconstriction over different ranges of AVP concentration. Vasoconstrictor actions of AVP, at concentrations of AVP found within the systemic circulation, utilize a Ca(2+) signaling pathway that is dependent on PKC activation and can be inhibited by Ca(2+) channel blockers.
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Affiliation(s)
- Kyle K Henderson
- Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago, Maywood, Illinois 60153, USA
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15
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Brueggemann LI, Moran CJ, Barakat JA, Yeh JZ, Cribbs LL, Byron KL. Vasopressin stimulates action potential firing by protein kinase C-dependent inhibition of KCNQ5 in A7r5 rat aortic smooth muscle cells. Am J Physiol Heart Circ Physiol 2006; 292:H1352-63. [PMID: 17071736 PMCID: PMC2577603 DOI: 10.1152/ajpheart.00065.2006] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
[Arg(8)]-vasopressin (AVP), at low concentrations (10-500 pM), stimulates oscillations in intracellular Ca(2+) concentration (Ca(2+) spikes) in A7r5 rat aortic smooth muscle cells. Our previous studies provided biochemical evidence that protein kinase C (PKC) activation and phosphorylation of voltage-sensitive K(+) (K(v)) channels are crucial steps in this process. In the present study, K(v) currents (I(Kv)) and membrane potential were measured using patch clamp techniques. Treatment of A7r5 cells with 100 pM AVP resulted in significant inhibition of I(Kv). This effect was associated with gradual membrane depolarization, increased membrane resistance, and action potential (AP) generation in the same cells. The AVP-sensitive I(Kv) was resistant to 4-aminopyridine, iberiotoxin, and glibenclamide but was fully inhibited by the selective KCNQ channel blockers linopirdine (10 microM) and XE-991 (10 microM) and enhanced by the KCNQ channel activator flupirtine (10 microM). BaCl(2) (100 microM) or linopirdine (5 microM) mimicked the effects of AVP on K(+) currents, AP generation, and Ca(2+) spiking. Expression of KCNQ5 was detected by RT-PCR in A7r5 cells and freshly isolated rat aortic smooth muscle. RNA interference directed toward KCNQ5 reduced KCNQ5 protein expression and resulted in a significant decrease in I(Kv) in A7r5 cells. I(Kv) was also inhibited in response to the PKC activator 4beta-phorbol 12-myristate 13-acetate (10 nM), and the inhibition of I(Kv) by AVP was prevented by the PKC inhibitor calphostin C (250 nM). These results suggest that the stimulation of Ca(2+) spiking by physiological concentrations of AVP involves PKC-dependent inhibition of KCNQ5 channels and increased AP firing in A7r5 cells.
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Brueggemann LI, Markun DR, Henderson KK, Cribbs LL, Byron KL. Pharmacological and electrophysiological characterization of store-operated currents and capacitative Ca(2+) entry in vascular smooth muscle cells. J Pharmacol Exp Ther 2006; 317:488-99. [PMID: 16415091 DOI: 10.1124/jpet.105.095067] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Capacitative Ca(2+) entry (CCE) in vascular smooth muscle cells contributes to vasoconstrictor and mitogenic effects of vasoactive hormones. In A7r5 rat aortic smooth muscle cells, measurements of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) have demonstrated that depletion of intracellular Ca(2+) stores activates CCE. However, there is disagreement in published studies regarding the regulation of this mechanism by the vasoconstrictor hormone [Arg(8)]-vasopressin (AVP). We have employed electrophysiological methods to characterize the membrane currents activated by store depletion [store-operated current (I(SOC))]. Because of different recording conditions, it has not been previously determined whether I(SOC) corresponds to CCE measured using fura-2; nor has the channel protein responsible for CCE been identified. In the present study, the pharmacological characteristics of I(SOC), including its sensitivity to blockade by 2-aminoethoxydiphenylborane, diethylstilbestrol, or micromolar Gd(3+), were found to parallel the effects of these drugs on thapsigargin- or AVP-activated CCE measured under identical external ionic conditions using fura-2. Thapsigargin-stimulated I(SOC) was also measured in freshly isolated rat mesenteric artery smooth muscle cells (MASMC). Members of the transient receptor potential (TRP) family of nonselective cation channels, TRPC1, TRPC4, and TRPC6, were detected by reverse transcription-polymerase chain reaction and Western blot in both A7r5 cells and MASMC. TRPC1 expression was reduced in a stable A7r5 cell line expressing a small interfering RNA (siRNA) or by infection of A7r5 cells with an adenovirus expressing a TRPC1 antisense nucleotide sequence. Thapsigargin-stimulated I(SOC) was reduced in both the TRPC1 siRNA- and TRPC1 antisense-expressing cells, suggesting that the TRPC1 channel contributes to the I(SOC)/CCE pathway.
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Affiliation(s)
- Lioubov I Brueggemann
- Department of Pharmacology and Experimental Therapeutics, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153, USA
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17
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Hughes AK, Kohan DE. Mechanism of vasopressin-induced contraction of renal medullary interstitial cells. Nephron Clin Pract 2006; 103:p119-24. [PMID: 16557030 DOI: 10.1159/000092245] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2005] [Accepted: 10/26/2005] [Indexed: 11/19/2022] Open
Abstract
BACKGROUND/AIMS Previous studies have identified a contractile function for renomedullary interstitial cells (RMIC). Such studies focused on the mechanism of endothelin-1-induced RMIC contraction; however, vasopressin (AVP) was also noted to contract RMIC. Since AVP-induced RMIC contraction may be relevant to the medullary effects of AVP on urinary concentration, these initial observations have been extended to examination of the mechanism of AVP-induced RMIC contraction. METHODS Cultured rat RMIC were exposed to AVP and other agents, and examined using video microscopy. RESULTS AVP caused a slowly developing and dose-dependent reduction in RMIC surface area. AVP-induced RMIC contraction was abolished by blockade of V1, but not V2, receptors. Nifedipine and nickel reduced AVP-stimulated RMIC contraction, indicating that this effect is dependent upon dihydropyridine-sensitive calcium channels. H7, a protein kinase C inhibitor, completely abrogated AVP action, while the nitric oxide synthase inhibitor, NMMA, had no effect. Indomethacin enhanced AVP-induced RMIC contraction, and addition of PGE2 together with indomethacin reduced AVP action. CONCLUSION These data indicate that AVP potently contracts RMIC via V1 receptor stimulation of PKC and intracellular calcium accumulation, and that AVP-stimulated prostaglandin production downregulates the contractile effect of AVP on RMIC. AVP modulation of RMIC contraction may be involved in the regulation of urinary concentration.
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Affiliation(s)
- Alisa K Hughes
- Department of Veterans Affairs Medical Center, University of Utah Health Sciences Center, Salt Lake City, Utah 84132, USA
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18
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Zheyu C, Qinghui QI, Lixin L, Tao MA, Xu J, Zhang L, Lunan Y. Effects of emodin on Ca2+ signal transduction of smooth muscle cells in multiple organ dysfunction syndrome. J Surg Res 2005; 131:80-5. [PMID: 16271368 DOI: 10.1016/j.jss.2005.08.031] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2005] [Revised: 08/25/2005] [Accepted: 08/30/2005] [Indexed: 02/05/2023]
Abstract
We have made several reports on the signal transduction mechanism that emodin enhance the calcium concentrations of smooth muscle cells (SMCs) in the physiological condition by inositol [1, 4, 5]-friphosphate (IP3). The observation that IP3 concentrations in SMCs were decreased in multiple organ dysfunction syndrome (MODS) prompted us to ask whether emodin can activate SMCs to contract by way of elevating [Ca2+] and thus modulating the critical Ca2+ signal transduction pathways involved in the contraction of the SMCs in the pathological setting of MODS. To test this hypothesis, we used the rat model of MODS to explore the potential roles of emodin in Ca2+ signal transduction in the SMCs of colon in rats. ML-7 [an inhibitor of myosin light-chain kinase (MLCK)] and Calphostin C [an inhibitor of protein kinase C (PKC)] were used to observe the influence of emodin on the muscle strips and SMCs in rats after MODS. Nifedipine (an antagonist of voltage-gated Ca2+ channel), EGTA (removal of extracellular Ca2+), heparine (a specific IP3 receptor antagonist), and ryanodine were used to probe the potential mechanisms involved in emodin-mediated elevation of the global cytoplasmic Ca2+ in SMCs of colon in the rats after MODS. Our results show that emodin is capable of contract the smooth muscles of colon in rats after MODS by MLCK increasing [Ca2+] of SMCs, and by PKC enhancing the calcium sensitivity of SMCs. The mechanism by which emodin triggers elevated [Ca2+] of smooth muscles of colon in rats after MODS is likely to operate through IP3 and RyR receptors in the sarcoplasm. It is hoped that deeper insights into how emodin modulates the critical calcium signaling in SMCs might lead to the potential development of emodin in the treatment of MODS.
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Affiliation(s)
- Chen Zheyu
- Department of General Surgery of West China Hospital, Sichuan University, Chengdu, China
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19
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Fedoryak OD, Searls Y, Smirnova IV, Burns DM, Stehno-Bittel L. Spontaneous Ca2+ oscillations in subcellular compartments of vascular smooth muscle cells rely on different Ca2+ pools. Cell Res 2005; 14:379-88. [PMID: 15538970 DOI: 10.1038/sj.cr.7290238] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Abstract
Spontaneous Ca2+ oscillations in vascular smooth muscle cells have been modeled using a single Ca2+ pool. This report describes spontaneous Ca2+ oscillations dependent on two separate Ca2+ sources for the nuclear versus cytoplasmic compartments. Changes in free intracellular Ca2+ were monitored with ratiometric Ca2+- fluorophores using confocal microscopy. On average, spontaneous oscillations developed in 79% of rat aortic smooth muscle cells that were synchronous between the cytoplasm and nucleus. Reduction of extracellular Ca2+ (less than 1 microM)decreased the frequency and amplitude of the cytoplasmic oscillations with 48% of the oscillations asynchronous between the nuclear and cytoplasmic compartments. Similar results were obtained with the Ca2+ channel blockers, nimodipine and diltiazem. Arg-vasopressin (AVP) induced a rapid release of intracellular Ca2+ stores that was greater in the nuclear compartment (4.20 +/- 0.23 ratio units, n = 56) than cytoplasm (2.54 +/- 0.28) in cells that had spontaneously developed prior oscillations. Conversely, cells in the same conditions lacking oscillations had a greater AVP-induced Ca2+ transient in the cytoplasm (4.99 +/- 0.66, n = 17) than in the nucleus (2.67 +/- 0.29). Pre-treatment with Ca2+ channel blockers depressed the AVP responses in both compartments with the cytoplasmic Ca2+ most diminished. Depletion of internal Ca2+ stores prior to AVP exposure blunted the nuclear response, mimicking the response of cells that lacked prior oscillations. Spontaneous oscillating cells had a greater sarcoplasmic reticulum network than cells that did not oscillate. We propose that spontaneous nuclear oscillations rely on perinuclear sarcoplasmic reticulum stores, while the cytoplasmic oscillations rely on Ca2+ influx.
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Affiliation(s)
- Olesya D Fedoryak
- Department of Physical Therapy, University of Kansas Medical Center, Kansas City, KS 66160, USA
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Amorim JBO, Musa-Aziz R, Mello-Aires M, Malnic G. Signaling path of the action of AVP on distal K+ secretion. Kidney Int 2004; 66:696-704. [PMID: 15253724 DOI: 10.1111/j.1523-1755.2004.00800.x] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
Abstract
BACKGROUND Previous studies from our laboratory have shown that luminal perfusion with arginine vasopressin (AVP) stimulates distal tubule secretory potassium flux (JK) via V1 receptors (Am J Physiol 278:F809-F816, 2000). In the present work, we investigate the cell signaling mechanism of this process. METHODS In vivo stationary microperfusion was performed in rat cortical distal tubules and luminal K+ was measured using double K+ resin/reference microelectrodes. RESULTS In control conditions, JK was 0.71 +/- 0.05 nmol.cm(-2).second(-1); this process was inhibited (14%) by 10(-5) mol/L 8-bromo-cyclic adenosine monophosphate (cAMP), and increased by 35% with 10(-8) mol/L phorbol ester [phorbol12-myristate 13-acetate (PMA), which activates protein kinase C (PKC)]. During luminal perfusion with 10(-11) mol/L AVP, JK increased to 0.88 +/- 0.08 nmol.cm(-2).seconds(-1). In the presence of 10(-11) mol/L AVP, JK was not affected by 10(-4) mol/L H89, a blocker of protein kinase A (PKA), but was inhibited (45%) by 10(-5) mol/L staurosporine, an inhibitor of PKC, and by 41% during perfusion with 5 x 10(-5) mol/L of the cell Ca2+ chelator bis (2-aminophenoxy) ethane-tetraacetic acid (BAPTA). In order to study the role of Ca(2+)-dependent K channels in the luminal hormonal action, the tubules were perfused with 5 mmol/L tetraethylammonium chloride (TEA) or 10(-7) mol/L iberiotoxin, in the presence of AVP, and JK was significantly reduced by both agents. Iberiotoxin reduced AVP-stimulated JK by 36.4%, and AVP-independent JK (after blocking V1 receptors) by only 16%. CONCLUSION The results suggest that the luminal V1-receptor effect of AVP on JK was mediated by the phospholipase C (PLC)/Ca2+/PKC signaling path and not byadenylate cyclase/cAMP/PKA, therefore probably acting on maxi-potassium channels.
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Affiliation(s)
- José B O Amorim
- Basic Science Deparment, Faculdade de Odontologia de São José dos Campos, UNESP, São Paulo, Brazil
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Ma T, Qi QH, Xu J, Dong ZL, Yang WX. Signal pathways involved in emodin-induced contraction of smooth muscle cells from rat colon. World J Gastroenterol 2004; 10:1476-9. [PMID: 15133857 PMCID: PMC4656288 DOI: 10.3748/wjg.v10.i10.1476] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To investigate the effects induced by emodin on single smooth muscle cells from rat colon in vitro, and to determine the signal pathways involved.
METHODS: Cells were isolated from the muscle layers of Wistar rat colon by enzymatic digestion. Cell length was measured by computerized image micrometry. Intracellular Ca2+ ([Ca2+]i) signals were studied using the fluorescent Ca2+ indicator fluo-3 and confocal microscopy. PKCα distribution at rest state or after stimulation was measured with immunofluorescence confocal microscopy.
RESULTS: (1) Emodin dose-dependently caused colonic smooth muscle cells contraction; (2) emodin induced an increase in intracellular Ca2+ concentration; (3) the contractile responses induced by emodin were respectively inhibited by preincubation of the cells with ML-7 (an inhibitor of MLCK) and calphostin C (an inhibitor of PKC); and (4) Incubation of cells with emodin caused translocation of PKCα from cytosolic area to the membrane.
CONCLUSION: Emodin has a direct contractile effect on colonic smooth muscle cell. This signal cascade induced by emodin is initiated by increased [Ca2+]i and PKCα translocation, which in turn lead to the activation of MLCK and the suppression of MLCP. Both of them contribute to the emodin-induced contraction.
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Affiliation(s)
- Tao Ma
- Department of Surgery, General Hospital of Tianjin Medical University, Tianjin 300052, China.
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Ma T, Qi QH, Jian X, Fei NX. Effects of emodin on intracellular Ca 2+ signaling in the circular smooth muscle cells of rat colon. Shijie Huaren Xiaohua Zazhi 2003; 11:1699-1702. [DOI: 10.11569/wcjd.v11.i11.1699] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate whether emodin has any effects on circular smooth muscle cells of rat colon and to examine the underlying mechanisms.
METHODS Smooth muscle cells were isolated from the circular muscle layers of Wistar rat colon and cell length was measured by computerized image micrometry. Intracellular Ca2+ ([Ca2+]i) signaling was studied in smooth muscle cells using Ca2+ indicator Fluo-3 AM by laser-scanning confocal microscopy.
RESULTS Emodin dose-dependently induced smooth muscle cells contraction, caused a large, transient increase in [Ca2+]i followed by a Sustained elevation in [Ca2+]i. Emodin-induced increase in [Ca2+]i was unaffected by nifedipine, a votage-gated Ca2+-channel antagonist, and the Sustained phase of rising of [Ca2+]i was attenuated by extracellular Ca2+ removal with EGTA solution. Inhibiting Ca2+ release from ryanodine-sensitive intracellular stores by ryanodine reduced the Peak increase in [Ca2+]i. However, the application of heparine, an antagonist of IP3R, nearly abolished the Peak increase in [Ca2+]i induced by emodin.
CONCLUSION Emodin has direct excitatory effect on circular smooth muscle cells from rat colon and its effect is mediated through Ca2+-dependent pathways. Furthermore, emodin-induced Peak [Ca2+]i increase may be attributable to the Ca2+ release from IP3 sensitive stores, which promotes Ca2+ release from ryanodine-sensitive stores through CICR mechanism. Additionally, Ca2+ influx from extracellular medium contributes to the Sustained increase in [Ca2+]i.
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Affiliation(s)
- Tao Ma
- Department of Surgery, General Hospital of Tianjin Medical University, Tianjin 300052, China
| | - Qing-Hui Qi
- Department of Surgery, General Hospital of Tianjin Medical University, Tianjin 300052, China
| | - Xu Jian
- Department of Surgery, General Hospital of Tianjin Medical University, Tianjin 300052, China
| | - Nai-Xin Fei
- Department of Surgery, General Hospital of Tianjin Medical University, Tianjin 300052, China
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Ma T, Qi QH, Yang WX, Xu J, Dong ZL. Contractile effects and intracellular Ca 2+ signalling induced by emodin in circular smooth muscle cells of rat colon. World J Gastroenterol 2003; 9:1804-7. [PMID: 12918125 PMCID: PMC4611548 DOI: 10.3748/wjg.v9.i8.1804] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate whether emodin has any effects on circular smooth muscle cells of rat colon and to examine the mechanism underlying its effect.
METHODS: Smooth muscle cells were isolated from the circular muscle layer of Wistar rat colon and the cell length was measured by computerized image micrometry. Intracellular Ca2+ ([Ca2+]i) signalling was studied in smooth muscle cells using Ca2+ indicator Fluo-3 AM on a laser-scanning confocal microscope.
RESULTS: Emodin dose-dependently induced smooth muscle cells contraction. The contractile responses induced by emodin were inhibited by preincubation of the cells with ML-7 (an inhibitor of MLCK). Emodin caused a large, transient increase in [Ca2+]i followed by a sustained elevation in [Ca2+]i. The emodin –induced increase in [Ca2+]i was unaffected by nifedipine, a voltage-gated Ca2+-channel antagonist, and the sustained phase of the rising of [Ca2+]i was attenuated by extracellular Ca2+ removal with EGTA solution. Inhibiting Ca2+ release from ryanodine-sensitive intracellular stores by ryanodine reduced the peak increase in [Ca2+]i. Using heparin, an antagonist of IP3R, almost abolished the peak increase in [Ca2+]i.
CONCLUSION: Emodin has a direct excitatory effect on circular smooth muscle cells in rat colon mediated via Ca2+/ CaM dependent pathways. Furthermore, emodin-induced peak [Ca2+]i increase may be attributable to the Ca2+ release from IP3 sensitive stores, which further promote Ca2+ release from ryanodine-sensitive stores through CICR mechanism. Additionally, Ca2+ influx from extracellular medium contributes to the sustained increase in [Ca2+]i.
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Affiliation(s)
- Tao Ma
- Department of Surgery, General Hospital of Tianjin Medical University, Tianjin 300052, China.
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Shirasawa Y, Rutland TJ, Young JL, Dean DA, Benoit JN. Modulation of protein kinase C (PKC)-mediated contraction and the possible role of PKC epsilon in rat mesenteric arteries. FRONTIERS IN BIOSCIENCE : A JOURNAL AND VIRTUAL LIBRARY 2003; 8:a133-8. [PMID: 12700091 PMCID: PMC4400799 DOI: 10.2741/1087] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
he involvement of protein kinase C (PKC) in isometric tension development of rat mesenteric arteries was investigated. Non-selective inhibition of PKC and selective inhibition of the epsilon isoform were performed using the PKC inhibitor, chelerythrine, and non-viral gene-transfer of a kinase inactive mutant of PKCepsilon (PKCepsilon-KN), respectively. Chelerythrine (2.5 or 5.0 microM) significantly and equally attenuated phenylephrine-induced but not potassium-induced contractions. Higher concentrations of chelerythrine (10 microM) caused the vessels to lose responsiveness to both phenylephrine and potassium chloride. Transfection of blood vessels with epsilon-KN also resulted in significant attenuation of contractile responses to phenylephrine. Potassium chloride-induced responses were not altered in transfected arteries. In a separate group of vessels, the relationship between [Ca2+]i and isometric tension was evaluated. These studies suggested that calcium sensitivity of the contractile apparatus was decreased in vessels when PKC-epsilon activity was compromised. The results of the study suggest that PKC-epsilon can modulate phenylephrine-induced contraction in mesenteric arteries via calcium-independent pathways.
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Affiliation(s)
- Yuichi Shirasawa
- Department of Pharmacology, Physiology and Therapeutics, University of North Dakota, Grand Forks, North Dakota
| | - Travis J. Rutland
- Department of Physiology, University of South Alabama College of Medicine, Mobile, Alabama
| | - Jennifer L. Young
- Department of Medicine, Northwestern University Medical School, Chicago, Illinois
| | - David A. Dean
- Department of Medicine, Northwestern University Medical School, Chicago, Illinois
| | - Joseph N. Benoit
- Department of Pharmacology, Physiology and Therapeutics, University of North Dakota, Grand Forks, North Dakota
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Feitelson JBA, Rowell PP, Roberts CS, Fleming JT. Two week nicotine treatment selectively increases bone vascular constriction in response to norepinephrine. J Orthop Res 2003; 21:497-502. [PMID: 12706023 DOI: 10.1016/s0736-0266(02)00235-8] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
This study was designed to determine if nicotine treatment alters the constrictor and/or dilator function of the vessels which regulate blood flow to intact bone. Nicotine (1.7 mg/kg/day) or nicotine-free, phosphate-buffered saline was administered subcutaneously to mature male rats for 2 weeks via osmotic mini-pumps. On the 14th day, the rats were anesthetized and in vivo experiments were performed to quantitate the changes in arterial blood pressure and perfusion of the intact tibia (measured by laser Doppler flowmetry) in response to two constrictor agonists (norepinephrine, NE and arginine vasopressin, AVP) and two vasodilator agents (acetylcholine, ACh and sodium nitroprusside, SNP). Dose-response curves were generated by plotting the change in the bone vascular resistance index (mmHg/bone perfusion units) evoked by each dose of agonist. In addition, bone arteriolar expression of endothelial nitric oxide synthase protein was quantitated by Western blot analysis. Nicotine treatment significantly enhanced the constriction of the bone vasculature in response to NE, but not to AVP. Vascular dilation in response to ACh and SNP was not changed by nicotine. These results indicate that nicotine selectively accentuates the constrictor response of the bone vasculature to exogenous NE. This enhanced constriction to NE is not due to impaired endothelial cell release of nitric oxide or diminished smooth muscle response to nitric oxide. Since NE and AVP activate similar cell signaling mechanisms to induce constriction, the selective enhancement of NE-induced constriction suggests that nicotine alters a mechanism unique to NE signaling; possibly the number or binding affinity of alpha adrenergic receptors. Since endogenous NE regulates basal blood flow to bone, the effect of nicotine to augment NE-induced constriction could lead to a chronic reduction in blood flow to bone.
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Affiliation(s)
- J B A Feitelson
- Department of Physiology and Biophysics, 1115A, Health Sciences Center, University of Louisville, KY 40292, USA
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26
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Vermassen E, Van Acker K, Annaert WG, Himpens B, Callewaert G, Missiaen L, De Smedt H, Parys JB. Microtubule-dependent redistribution of the type-1 inositol 1,4,5-trisphosphate receptor in A7r5 smooth muscle cells. J Cell Sci 2003; 116:1269-77. [PMID: 12615969 DOI: 10.1242/jcs.00354] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Abstract
In A7r5 vascular smooth muscle cells, the two expressed inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms were differentially localized. IP(3)R1 was predominantly localized in the perinuclear region, whereas IP(3)R3 was homogeneously distributed over the cytoplasm. Prolonged stimulation (1-5 hours) of cells with 3 microM arginine-vasopressin induced a redistribution of IP(3)R1 from the perinuclear region to the entire cytoplasm, whereas the localization of IP(3)R3 appeared to be unaffected. The redistribution process occurred independently of IP(3)R downregulation. No structural changes of the endoplasmic reticulum were observed, but SERCA-type Ca(2+) pumps redistributed similarly to IP(3)R1. The change in IP(3)R1 localization induced by arginine-vasopressin could be blocked by the simultaneous addition of nocodazole or taxol and depended on Ca(2+) release from intracellular stores since Ca(2+)-mobilizing agents such as thapsigargin and cyclopiazonic acid could induce the redistribution. Furthermore, various protein kinase C inhibitors could inhibit the redistribution of IP(3)R1, whereas the protein kinase C activator 1-oleoyl-2-acetyl-sn-glycerol induced the redistribution. Activation of protein kinase C also induced an outgrowth of the microtubules from the perinuclear region into the cytoplasm, similar to what was seen for the redistribution of IP(3)R1. Finally, blocking vesicular transport at the level of the intermediate compartment inhibited the redistribution. Taken together, these findings suggest a role for protein kinase C and microtubuli in the redistribution of IP(3)R1, which probably occurs via a mechanism of vesicular trafficking.
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MESH Headings
- Animals
- Arginine Vasopressin/pharmacology
- Calcium Channels/drug effects
- Calcium Channels/metabolism
- Calcium Signaling/drug effects
- Calcium Signaling/physiology
- Calcium-Transporting ATPases/drug effects
- Calcium-Transporting ATPases/metabolism
- Cell Compartmentation/drug effects
- Cell Compartmentation/physiology
- Cell Line
- Cell Nucleus/drug effects
- Cell Nucleus/metabolism
- Cytoplasm/drug effects
- Cytoplasm/metabolism
- Down-Regulation/drug effects
- Down-Regulation/physiology
- Enzyme Inhibitors/pharmacology
- Inositol 1,4,5-Trisphosphate Receptors
- Microtubules/drug effects
- Microtubules/metabolism
- Muscle, Smooth, Vascular/drug effects
- Muscle, Smooth, Vascular/metabolism
- Muscle, Smooth, Vascular/ultrastructure
- Protein Kinase C/antagonists & inhibitors
- Protein Kinase C/metabolism
- Protein Transport/drug effects
- Protein Transport/physiology
- Rats
- Receptors, Cytoplasmic and Nuclear/drug effects
- Receptors, Cytoplasmic and Nuclear/metabolism
- Sarcoplasmic Reticulum Calcium-Transporting ATPases
- Transport Vesicles/drug effects
- Transport Vesicles/metabolism
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Affiliation(s)
- Elke Vermassen
- Laboratory of Physiology, CME/VIB04, K.U. Leuven Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium
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27
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Hai CM, Hahne P, Harrington EO, Gimona M. Conventional protein kinase C mediates phorbol-dibutyrate-induced cytoskeletal remodeling in a7r5 smooth muscle cells. Exp Cell Res 2002; 280:64-74. [PMID: 12372340 DOI: 10.1006/excr.2002.5592] [Citation(s) in RCA: 100] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Phorbol dibutyrate (PDBu) induced the formation of podosome-like structures together with partial disassembly of actin stress fibers in A7r5 smooth muscle cells. These podosomes contained alpha-actinin, F-actin, and vinculin and exhibit a tubular, column-like structure arising perpendicularly from the bottom of PDBu-treated cells. The conventional protein kinase C (PKC) antagonist, GO6976, inhibited PDBu-induced cytoskeletal remodeling at 0.1 microM, whereas the novel PKC antagonist, rottlerin, was ineffective at 10 microM. PDBu induced the translocation of the conventional PKC-alpha but not the novel PKC-delta to the sites of podosome formation in A7r5 cells. Although partial disassembly of actin stress fibers was observed in both Y-27632- and PDBu-treated cells, focal adhesions were much reduced in number and size only in Y-27632-treated cells. Furthermore, PDBu restored focal adhesions in Y-27632-treated cells. Live video fluorescence microscopy of alpha-actinin GFP revealed a lag phase of about 20 min prior to the rapid formation and dynamic reorganization of podosomes during PDBu treatment. These findings suggest that conventional PKCs mediate PDBu-induced formation of dynamic podosome-like structures in A7r5 cells, and Rho-kinase is unlikely to be the underlying mechanism. The podosome columns could represent molecular scaffolds where PKC-alpha phosphorylates regulatory proteins necessary for Ca(2+) sensitization in smooth muscle cells.
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Affiliation(s)
- Chi-Ming Hai
- Department of Molecular Pharmacology, Physiology, & Biotechnology, Austrian Academy of Sciences, Salzburg, Austria.
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28
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Endemann D, Touyz RM, Yao G, Schiffrin EL. Tyrosine kinase inhibition attenuates vasopressin-induced contraction of mesenteric resistance arteries: alterations in spontaneously hypertensive rats. J Cardiovasc Pharmacol 2002; 40:123-32. [PMID: 12072585 DOI: 10.1097/00005344-200207000-00015] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
This study examined the role of tyrosine kinase-dependent signaling pathways in arginine vasopressin (AVP)-induced contractile responses in resistance arteries from spontaneously hypertensive rats (SHR). Systolic blood pressure was measured in conscious 6- and 21-week old SHR and Wistar Kyoto rats (WKY) by tail cuff measurements. Segments of third-order mesenteric arteries (about 200 microm in diameter, 2mm in length) were mounted in a pressurized chamber with the intraluminal pressure maintained at 45 mmHg. Contractile effects of AVP (10-12 to 10-7 mol/l) were determined in the absence and presence of the selective tyrosine kinase inhibitor tyrphostin A23 (10-5 mol/l) and the inactive analogue, tyrphostin A1 (10-5 mol/l). Systolic blood pressure was significantly higher in SHR compared with age-matched WKY (p < 0.01). AVP increased contraction in a dose-dependent manner with significantly greater responses in adult SHR (pD2 = 10.3 +/- 0.06) than age-matched WKY (pD2 = 9.4 +/- 0.04). Tyrphostin A23 shifted the AVP dose response curve to the right in 6- and 21-week WKY and 6-week SHR, but had little effect on AVP-induced responses in 21-week-old SHR. Tyrphostin A1 did not influence contraction in any groups. Protein tyrosine phosphorylation in VSMCs and mesenteric arteries was increased 2-3 fold in 21-week SHR compared with WKY counterparts. AVP significantly increased tyrosine phosphorylation in VSMCs, with enhanced effects in SHR compared with WKY (p < 0.05). These effects were inhibited by tyrphostin A23. Our findings demonstrate that protein tyrosine kinases contribute to AVP-induced contraction of resistance arteries from WKY and SHR during the phase of developing hypertension. These processes do not seem to play an important role in AVP-induced hypercontractility in SHR with established hypertension.
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Affiliation(s)
- Dierk Endemann
- Multidisciplinary Research Group on hypertension, Clinical Research Institute of Montreal, University of Montreal, Montreal, Quebec, Canada
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29
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Byron KL, Lucchesi PA. Signal transduction of physiological concentrations of vasopressin in A7r5 vascular smooth muscle cells. A role for PYK2 and tyrosine phosphorylation of K+ channels in the stimulation of Ca2+ spiking. J Biol Chem 2002; 277:7298-307. [PMID: 11739373 DOI: 10.1074/jbc.m104726200] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The signal transduction pathway linking physiological concentrations of [Arg(8)]vasopressin (AVP) to an increase in frequency of Ca(2+) spiking was examined in confluent cultures of A7r5 vascular smooth muscle cells. Immunoprecipitation/Western blot studies revealed a robust increase in tyrosine phosphorylation of the non-receptor tyrosine kinase, PYK2, in A7r5 cells treated with 4beta-phorbol 12-myristate 13-acetate or ionomycin. 100 pm AVP also induced PYK2 tyrosine phosphorylation, and this effect was inhibited by protein kinase C inhibitors Ro-31-8220 (1-10 microm) or chelerythrine chloride (1-20 microm). In fura-2-loaded A7r5 cells, the stimulation of Ca(2+) spiking by 100 pm AVP or 1 nm 4beta-phorbol 12-myristate 13-acetate was completely blocked by PP2 (10 microm, a Src family kinase inhibitor). Salicylate (20 mm, recently identified as a PYK2 inhibitor) and the tyrosine kinase inhibitor, tyrphostin A47 (50 microm), but not its inactive analog, tyrphostin A63, also blocked AVP-stimulated Ca(2+) spiking. PYK2 phosphorylation was inhibited by both PP2 and salicylate, whereas tyrphostin A47 failed to inhibit PYK2 tyrosine phosphorylation. ERK1/2 kinases did not appear to be involved because 1) 100 pm AVP did not appreciably increase ERK1/2 phosphorylation and U-0126 (2.5 microm) did not inhibit AVP-stimulated Ca(2+) spiking; and 2) epidermal growth factor (10 nm) robustly stimulated ERK1/2 phosphorylation but did not induce Ca(2+) spiking. Delayed rectifier K(+) channels may mediate the PYK2 activity because Kv1.2 channel protein co-immunoprecipitated with PYK2 and tyrosine phosphorylation of Kv1.2 was stimulated by AVP and inhibited by Ro-31-8220, PP2, and salicylate but not tyrphostin A47. Our findings are consistent with a role for PYK2 and phosphorylation of K(+) channels in the stimulation of Ca(2+) spiking by physiological concentrations of AVP.
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Affiliation(s)
- Kenneth L Byron
- Department of Medicine, Cardiovascular Institute, Loyola University Chicago, 2160 South First Avenue, Maywood, IL 60153, USA.
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Tanaka S, Saitoh O, Tabata K, Matsuse R, Kojima K, Sugi K, Nakagawa K, Kayazawa M, Teranishi T, Uchida K, Hirata I, Katsu K. Medium-chain fatty acids stimulate interleukin-8 production in Caco-2 cells with different mechanisms from long-chain fatty acids. J Gastroenterol Hepatol 2001; 16:748-54. [PMID: 11446882 DOI: 10.1046/j.1440-1746.2001.02537.x] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
BACKGROUND AND AIM It has been suggested that dietary fat exacerbates intestinal inflammation. We investigated the effect of fatty acids on interleukin (IL)-8 production in a human intestinal epithelial cell line (Caco-2). METHODS The cells were cultured as monolayers on microporous membranes in culture inserts. Oleic acid (OA), capric acid (CA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were applied to the apical compartment of Caco-2 cell monolayers. The concentration of IL-8 in the basolateral medium was measured by using enzyme-linked immunosorbent assay, and the expression of IL-8 mRNA was measured by using competitive reverse transcription--polymerase chain reaction. Protein kinase C inhibitors (GF109203X and calphostin C) and H-7 (a protein kinase inhibitor) were used to study the mechanisms by which IL-8 production is stimulated. RESULTS Both OA and CA enhanced IL-8 production (approximately fivefold), whereas DHA and EPA did not. Both OA and CA also enhanced IL-1-induced IL-8 production. The onset of OA-induced IL-8 production was delayed compared with that of CA-induced IL-8 production. Both OA and CA enhanced IL-8 mRNA expression (approximately fivefold) after 6 and 3 h, respectively. The protein kinase inhibitor (H-7) reduced both OA- and CA-induced IL-8 production by 88.0 and 85.9%, respectively. The protein kinase C inhibitors (GF109203X and calphostin C) reduced OA-induced IL-8 production by 29.3 and 54.5%, respectively, but showed no effect on CA-induced IL-8 production. CONCLUSIONS These findings suggest that not only OA but also CA stimulates IL-8 production in intestinal epithelial cells, and the mechanisms of action differ between OA and CA.
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Affiliation(s)
- S Tanaka
- Second Department of Internal Medicine, Osaka Medical College, Takatsuki, Japan.
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31
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Li Y, Shiels AJ, Maszak G, Byron KL. Vasopressin-stimulated Ca2+ spiking in vascular smooth muscle cells involves phospholipase D. Am J Physiol Heart Circ Physiol 2001; 280:H2658-64. [PMID: 11356622 DOI: 10.1152/ajpheart.2001.280.6.h2658] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Physiological concentrations of [Arg(8)]vasopressin (AVP; 10-500 pM) stimulate oscillations of cytosolic free Ca2+ concentration (Ca2+ spikes) in A7r5 vascular smooth muscle cells. We previously reported that this effect of AVP was blocked by a putative phospholipase A2 (PLA2) inhibitor, ONO-RS-082 (5 microM). In the present study, the products of PLA2, arachidonic acid (AA), and lysophospholipids were found to be ineffective in stimulating Ca2+ spiking, and inhibitors of AA metabolism did not prevent AVP-stimulated Ca2+ spiking. Thin layer chromatography was used to monitor the release of AA and phosphatidic acid (PA), which are the products of PLA2 and phospholipase D (PLD), respectively. AVP (100 pM) stimulated both AA and PA formation, but only PA formation was inhibited by ONO-RS-082 (5 microM). Exogenous PLD (type VII; 2.5 U/ml) stimulated Ca2+ spiking equivalent to the effect of 100 pM AVP. AVP stimulated transphosphatidylation of 1-butanol (a PLD-catalyzed reaction) but not 2-butanol, and 1-butanol (but not 2-butanol) completely prevented AVP-stimulated Ca2+ spiking. Protein kinase C (PKC) inhibition, which completely prevents AVP-stimulated Ca2+ spiking, did not inhibit AVP-stimulated phosphatidylbutanol formation. These results suggest that AVP-stimulated Ca2+ spiking depends on activation of PLD rather than PLA2 and that PKC activation may be downstream of PLD in the signaling cascade.
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Affiliation(s)
- Y Li
- Department of Physiology, Loyola University, Chicago, Illinois 60626, USA
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