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Kurtz I, Schwartz GJ. Base (HCO3-/CO32-) Transport Properties of SLC4 Proteins: New Insights in Acid-Base Kidney Physiology. J Am Soc Nephrol 2023; 34:8-13. [PMID: 36719145 PMCID: PMC10101619 DOI: 10.1681/asn.0000000000000008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2022] [Accepted: 09/30/2022] [Indexed: 01/22/2023] Open
Abstract
H+ or base transporters and channels in the mammalian genome play important roles in the maintenance of numerous cellular biochemical and physiologic processes throughout the body. Among the known base transporters, those within the SLC4 and SLC26 gene families are involved in cell, transepithelial, and whole organ function. Whether the functional properties of these transporters involve HCO3-, CO32-, or HCO3-/CO32- stimulated H+ (or OH-) transport has not received widespread attention in the literature. Accordingly, "bicarbonate" is the term typically used in most textbooks without greater specificity. Moreover, clinicians and physiologists have historically focused on the blood HCO3- concentration as the base term in the Henderson-Hasselbalch equation in the analysis of clinical acid-base abnormalities, thus, bicarbonate has been assumed to be the species reabsorbed along the nephron as required to maintain the blood [HCO3-] at approximately 25 mM. However, accumulating data in the literature suggest that carbonate, rather than bicarbonate, is the species absorbed across the proximal tubule basolateral membrane, whereas in the collecting duct, bicarbonate is indeed transported. Various experimental approaches leading to this new concept are herein reviewed.
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Affiliation(s)
- Ira Kurtz
- Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California
- Brain Research Institute, David Geffen School of Medicine, University of California, Los Angeles, California
| | - George J. Schwartz
- Department of Pediatrics, University of Rochester Medical Center, Rochester, New York
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Revisiting the Role of Ser982 Phosphorylation in Stoichiometry Shift of the Electrogenic Na +/ qHCO 3- Cotransporter NBCe1. Int J Mol Sci 2021; 22:ijms222312817. [PMID: 34884619 PMCID: PMC8657473 DOI: 10.3390/ijms222312817] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2021] [Revised: 11/17/2021] [Accepted: 11/24/2021] [Indexed: 11/16/2022] Open
Abstract
In most cell types and heterologous expression systems, the electrogenic sodium-bicarbonate cotransporter NBCe1 operates with a 1Na+-2HCO3- stoichiometry that, given typical transmembrane electrochemical gradients, promotes Na+ and HCO3- influx. However, NBCe1 in the kidney mediates HCO3- efflux (HCO3- reabsorption), a direction that has been predicted to be favored only if NBCe1 operates with a 1:3 stoichiometry. The phosphorylation state of Ser982 in the cytosolic carboxy-terminal domain of NBCe1 has been reported to be a key determinant of the transporter stoichiometry, with non-phosphorylated Ser982 favoring a 1:3 stoichiometry. Conversely, phosphoproteomic data from renal cortical preparations have revealed the presence of NBCe1 peptides including phosphoserine982 (pSer982) and/or pSer985 although it was not known what proportion of NBCe1 molecules were phosphorylated. In the present study, we report the generation, characterization, and application of a novel phosphospecific antibody raised against NBCe1/pSer982 and show that, contrary to expectations, Ser982 is more prevalently phosphorylated in murine kidneys (in which NBCe1 mediates HCO3- efflux) than in murine colons (in which NBCe1 mediates HCO3- influx). Using phosphomimetic mutants of murine NBCe1 expressed in Xenopus oocytes, we found no evidence that the phosphorylation state of Ser982 or Ser985 alone influences the transport stoichiometry or conductance. Furthermore, we found that the phosphorylation of NBCe1/Ser982 is enhanced in murine kidneys following a 24 h induction of metabolic acidosis. We conclude that the phosphorylation status of Ser982 is not a key determinant of NBCe1 stoichiometry but correlates with presumed NBCe1 activity.
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Myers EJ, Yuan L, Felmlee MA, Lin YY, Jiang Y, Pei Y, Wang O, Li M, Xing XP, Marshall A, Xia WB, Parker MD. A novel mutant Na + /HCO3 - cotransporter NBCe1 in a case of compound-heterozygous inheritance of proximal renal tubular acidosis. J Physiol 2016; 594:6267-6286. [PMID: 27338124 DOI: 10.1113/jp272252] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2016] [Accepted: 06/08/2016] [Indexed: 11/08/2022] Open
Abstract
KEY POINTS The inheritance of two defective alleles of SLC4A4, the gene that encodes the widely-expressed electrogenic sodium bicarbonate cotransporter NBCe1, results in the bicarbonate-wasting disease proximal renal tubular acidosis (pRTA). In the present study, we report the first case of compound-heterozygous inheritance of pRTA (p.Arg510His/p.Gln913Arg) in an individual with low blood pH, blindness and neurological signs that resemble transient ischaemic attacks. We employ fluorescence microscopy on non-polarized (human embryonic kidney) and polarized (Madin-Darby canine kidney) renal cell lines and electrophysiology on Xenopus oocytes to characterize the mutant transporters (R510H and Q913R). Both mutant transporters exhibit enhanced intracellular retention in renal cells, an observation that probably explains the HCO3- transport deficit in the individual. Both mutants retain a close-to-normal per molecule Na+ /HCO3- cotransport activity in Xenopus oocytes, suggesting that they are suitable candidates for folding-correction therapy. However, Q913R expression is uniquely associated with a depolarizing, HCO3- independent, Cl- -conductance in oocytes that could have pathological consequences if expressed in the cells of patients. ABSTRACT Proximal renal tubular acidosis (pRTA) is a rare, recessively-inherited disease characterized by abnormally acidic blood, blindness, as well as below average height and weight. pRTA is typically associated with homozygous mutation of the solute carrier 4 family gene SLC4A4. SLC4A4 encodes the electrogenic sodium bicarbonate cotransporter NBCe1, a membrane protein that acts to maintain intracellular and plasma pH. We present the first description of a case of compound-heterozygous inheritance of pRTA. The individual has inherited two mutations in NBCe1: p.Arg510His (R510H) and p.Gln913Arg (Q913R), one from each parent. In addition to the usual features of pRTA, the patient exhibits unusual signs, such as muscle spasms and fever. We have recreated these mutant transporters for expression in model systems. We find that both of the mutant proteins exhibit substantial intracellular retention when expressed in mammalian renal cell lines. When expressed in Xenopus oocytes, we find that the R510H and Q913R-mutant NBCe1 molecules exhibit apparently normal Na+ /HCO3- cotransport activity but that Q913R is associated with an unusual HCO3- independent anion-leak. We conclude that a reduced accumulation of NBCe1 protein in the basolateral membrane of proximal-tubule epithelia is the most probable cause of pRTA in this case. We further note that the Q913R-associated anion-leak could itself be pathogenic if expressed in the plasma membrane of mammalian cells, compromising the benefit of strategies aiming to enhance mutant NBCe1 accumulation in the plasma membrane.
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Affiliation(s)
- Evan J Myers
- Department of Physiology and Biophysics, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo: The State University of New York, Buffalo, New York, NY, USA
| | - Lu Yuan
- Department of Endocrinology, Key Laboratory of Endocrinology of Ministry of Health, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China
| | - Melanie A Felmlee
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo: The State University of New York, Buffalo, New York, NY, USA.,Department of Pharmaceutics and Medicinal Chemistry, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton, California, USA
| | - Yuan-Yuan Lin
- Department of Endocrinology, Key Laboratory of Endocrinology of Ministry of Health, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China
| | - Yan Jiang
- Department of Endocrinology, Key Laboratory of Endocrinology of Ministry of Health, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China
| | - Yu Pei
- Department of Endocrinology, Chinese People's Army General Hospital, Beijing, China
| | - Ou Wang
- Department of Endocrinology, Key Laboratory of Endocrinology of Ministry of Health, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China
| | - Mei Li
- Department of Endocrinology, Key Laboratory of Endocrinology of Ministry of Health, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China
| | - Xiao-Ping Xing
- Department of Endocrinology, Key Laboratory of Endocrinology of Ministry of Health, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China
| | - Aniko Marshall
- Department of Physiology and Biophysics, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo: The State University of New York, Buffalo, New York, NY, USA
| | - Wei-Bo Xia
- Department of Endocrinology, Key Laboratory of Endocrinology of Ministry of Health, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China.
| | - Mark D Parker
- Department of Physiology and Biophysics, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo: The State University of New York, Buffalo, New York, NY, USA. .,Department of Ophthalmology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo: The State University of New York, Buffalo, New York, NY, USA. .,State University of New York Eye Institutes, University at Buffalo: The State University of New York, Buffalo, New York, NY, USA.
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Abstract
Cation-coupled HCO3(-) transport was initially identified in the mid-1970s when pioneering studies showed that acid extrusion from cells is stimulated by CO2/HCO3(-) and associated with Na(+) and Cl(-) movement. The first Na(+)-coupled bicarbonate transporter (NCBT) was expression-cloned in the late 1990s. There are currently five mammalian NCBTs in the SLC4-family: the electrogenic Na,HCO3-cotransporters NBCe1 and NBCe2 (SLC4A4 and SLC4A5 gene products); the electroneutral Na,HCO3-cotransporter NBCn1 (SLC4A7 gene product); the Na(+)-driven Cl,HCO3-exchanger NDCBE (SLC4A8 gene product); and NBCn2/NCBE (SLC4A10 gene product), which has been characterized as an electroneutral Na,HCO3-cotransporter or a Na(+)-driven Cl,HCO3-exchanger. Despite the similarity in amino acid sequence and predicted structure among the NCBTs of the SLC4-family, they exhibit distinct differences in ion dependency, transport function, pharmacological properties, and interactions with other proteins. In epithelia, NCBTs are involved in transcellular movement of acid-base equivalents and intracellular pH control. In nonepithelial tissues, NCBTs contribute to intracellular pH regulation; and hence, they are crucial for diverse tissue functions including neuronal discharge, sensory neuron development, performance of the heart, and vascular tone regulation. The function and expression levels of the NCBTs are generally sensitive to intracellular and systemic pH. Animal models have revealed pathophysiological roles of the transporters in disease states including metabolic acidosis, hypertension, visual defects, and epileptic seizures. Studies are being conducted to understand the physiological consequences of genetic polymorphisms in the SLC4-members, which are associated with cancer, hypertension, and drug addiction. Here, we describe the current knowledge regarding the function, structure, and regulation of the mammalian cation-coupled HCO3(-) transporters of the SLC4-family.
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Affiliation(s)
- Christian Aalkjaer
- Department of Biomedicine, and the Water and Salt Research Center, Aarhus University, Aarhus, Denmark; Department of Physiology, Emory University School of Medicine, Atlanta, USA
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Seki G, Nakamura M, Suzuki M, Satoh N, Horita S. Species differences in regulation of renal proximal tubule transport by certain molecules. World J Nephrol 2015; 4:307-312. [PMID: 25949945 PMCID: PMC4419141 DOI: 10.5527/wjn.v4.i2.307] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/25/2014] [Revised: 12/24/2014] [Accepted: 01/20/2015] [Indexed: 02/06/2023] Open
Abstract
Renal proximal tubules (PTs) play important roles in the regulation of acid/base, plasma volume and blood pressure. Recent studies suggest that there are substantial species differences in the regulation of PT transport. For example, thiazolidinediones (TZDs) are widely used for the treatment of type 2 diabetes mellitus, but the use of TZDs is associated with fluid overload. In addition to the transcriptional enhancement of sodium transport in distal nephrons, TZDs rapidly stimulate PT sodium transport via a non-genomic mechanism depending on peroxisome proliferator activated receptor γ/Src/epidermal growth factor receptor (EGFR)/MEK/ERK. In mouse PTs, however, TZDs fail to stimulate PT transport probably due to constitutive activation of Src/EGFR/ERK pathway. This unique activation of Src/ERK may also affect the effect of high concentrations of insulin on mouse PT transport. On the other hand, the effect of angiotensin II (Ang II) on PT transport is known to be biphasic in rabbits, rats, and mice. However, Ang II induces a concentration-dependent, monophasic transport stimulation in human PTs. The contrasting responses to nitric oxide/guanosine 3’,5’-cyclic monophosphate pathway may largely explain these different effects of Ang II on PT transport. In this review, we focus on the recent findings on the species differences in the regulation of PT transport, which may help understand the species-specific mechanisms underlying edema formation and/or hypertension occurrence.
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Roles of renal proximal tubule transport in acid/base balance and blood pressure regulation. BIOMED RESEARCH INTERNATIONAL 2014; 2014:504808. [PMID: 24982885 PMCID: PMC4058521 DOI: 10.1155/2014/504808] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/05/2014] [Accepted: 05/16/2014] [Indexed: 02/06/2023]
Abstract
Sodium-coupled bicarbonate absorption from renal proximal tubules (PTs) plays a pivotal role in the maintenance of systemic acid/base balance. Indeed, mutations in the Na+-HCO3− cotransporter NBCe1, which mediates a majority of bicarbonate exit from PTs, cause severe proximal renal tubular acidosis associated with ocular and other extrarenal abnormalities. Sodium transport in PTs also plays an important role in the regulation of blood pressure. For example, PT transport stimulation by insulin may be involved in the pathogenesis of hypertension associated with insulin resistance. Type 1 angiotensin (Ang) II receptors in PT are critical for blood pressure homeostasis. Paradoxically, the effects of Ang II on PT transport are known to be biphasic. Unlike in other species, however, Ang II is recently shown to dose-dependently stimulate human PT transport via nitric oxide/cGMP/ERK pathway, which may represent a novel therapeutic target in human hypertension. In this paper, we will review the physiological and pathophysiological roles of PT transport.
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Kurtz I. NBCe1 as a model carrier for understanding the structure-function properties of Na⁺ -coupled SLC4 transporters in health and disease. Pflugers Arch 2014; 466:1501-16. [PMID: 24515290 DOI: 10.1007/s00424-014-1448-8] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2013] [Revised: 01/07/2014] [Accepted: 01/08/2014] [Indexed: 01/17/2023]
Abstract
SLC4 transporters are membrane proteins that in general mediate the coupled transport of bicarbonate (carbonate) and share amino acid sequence homology. These proteins differ as to whether they also transport Na(+) and/or Cl(-), in addition to their charge transport stoichiometry, membrane targeting, substrate affinities, developmental expression, regulatory motifs, and protein-protein interactions. These differences account in part for the fact that functionally, SLC4 transporters have various physiological roles in mammals including transepithelial bicarbonate transport, intracellular pH regulation, transport of Na(+) and/or Cl(-), and possibly water. Bicarbonate transport is not unique to the SLC4 family since the structurally unrelated SLC26 family has at least three proteins that mediate anion exchange. The present review focuses on the first of the sodium-dependent SLC4 transporters that was identified whose structure has been most extensively studied: the electrogenic Na(+)-base cotransporter NBCe1. Mutations in NBCe1 cause proximal renal tubular acidosis (pRTA) with neurologic and ophthalmologic extrarenal manifestations. Recent studies have characterized the important structure-function properties of the transporter and how they are perturbed as a result of mutations that cause pRTA. It has become increasingly apparent that the structure of NBCe1 differs in several key features from the SLC4 Cl(-)-HCO3 (-) exchanger AE1 whose structural properties have been well-studied. In this review, the structure-function properties and regulation of NBCe1 will be highlighted, and its role in health and disease will be reviewed in detail.
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Affiliation(s)
- Ira Kurtz
- Division of Nephrology, David Geffen School of Medicine, and Brain Research Institute, UCLA, Los Angeles, CA, USA,
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Kurtz I, Zhu Q. Proximal renal tubular acidosis mediated by mutations in NBCe1-A: unraveling the transporter's structure-functional properties. Front Physiol 2013; 4:350. [PMID: 24391589 PMCID: PMC3867943 DOI: 10.3389/fphys.2013.00350] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2013] [Accepted: 11/13/2013] [Indexed: 12/20/2022] Open
Abstract
NBCe1 belongs to the SLC4 family of base transporting membrane proteins that plays a significant role in renal, extrarenal, and systemic acid-base homeostasis. Recent progress has been made in characterizing the structure-function properties of NBCe1 (encoded by the SLC4A4 gene), and those factors that regulate its function. In the kidney, the NBCe1-A variant that is expressed on the basolateral membrane of proximal tubule is the key transporter responsible for overall transepithelial bicarbonate absorption in this nephron segment. NBCe1 mutations impair transepithelial bicarbonate absorption causing the syndrome of proximal renal tubular acidosis (pRTA). Studies of naturally occurring NBCe1 mutant proteins in heterologous expression systems have been very helpful in elucidation the structure-functional properties of the transporter. NBCe1 mutations are now known to cause pRTA by various mechanisms including the alteration of the transporter function (substrate ion interaction, electrogenicity), abnormal processing to the plasma membrane, and a perturbation in its structural properties. The elucidation of how NBCe1 mutations cause pRTA in addition to the recent studies which have provided further insight into the topology of the transporter have played an important role in uncovering its critically important structural-function properties.
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Affiliation(s)
- Ira Kurtz
- Division of Nephrology, David Geffen School of Medicine, UCLA Los Angeles, CA, USA ; Brain Research Institute, UCLA Los Angeles, CA, USA
| | - Quansheng Zhu
- Division of Nephrology, David Geffen School of Medicine, UCLA Los Angeles, CA, USA
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Seki G, Horita S, Suzuki M, Yamazaki O, Usui T, Nakamura M, Yamada H. Molecular mechanisms of renal and extrarenal manifestations caused by inactivation of the electrogenic Na(+)-HCO3 (-) cotransporter NBCe1. Front Physiol 2013; 4:270. [PMID: 24101904 PMCID: PMC3787273 DOI: 10.3389/fphys.2013.00270] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2013] [Accepted: 09/10/2013] [Indexed: 11/13/2022] Open
Abstract
The electrogenic Na(+)-HCO3 (-) cotransporter NBCe1 plays an essential role in bicarbonate absorption from renal proximal tubules, but also mediates the other biological processes in extrarenal tissues such as bicarbonate secretion from pancreatic ducts, maintenance of tissue homeostasis in eye, enamel maturation in teeth, or local pH regulation in synapses. Homozygous mutation in NBCe1 cause proximal renal tubular acidosis (pRTA) associated with extrarenal manifestations such as short stature, ocular abnormalities, enamel abnormalities, and migraine. Functional analyses of NBCe1 mutants using different expression systems suggest that at least a 50% reduction of the transport activity may be required to induce severe pRTA. In addition to functional impairments, some NBCe1 mutants show trafficking defects. Some of the pRTA-related NBCe1 mutants showing the cytoplasmic retention have been shown to exert a dominant negative effect through hetero-oligomer complexes with wild-type NBCe1 that may explain the occurrence of extrarenal manifestations in the heterozygous carries of NBCe1 mutations. Both NBCe1 knockout (KO) and W516X knockin (KI) mice showed very severe pRTA and reproduced most of the clinical manifestations observed in human pRTA patients. Functional analysis on isolated renal proximal tubules from W516X KI mice directly confirmed the indispensable role of NBCe1 in bicarbonate absorption from this nephron segment. In this review, we will focus on the molecular mechanisms underling the renal and extrarenal manifestations caused by NBCe1 inactivation.
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Affiliation(s)
- George Seki
- Department of Internal Medicine, School of Medicine, The University of Tokyo Tokyo, Japan
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Zhu Q, Shao XM, Kao L, Azimov R, Weinstein AM, Newman D, Liu W, Kurtz I. Missense mutation T485S alters NBCe1-A electrogenicity causing proximal renal tubular acidosis. Am J Physiol Cell Physiol 2013; 305:C392-405. [PMID: 23636456 DOI: 10.1152/ajpcell.00044.2013] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Mutations in SLC4A4, the gene encoding the electrogenic Na(+)-HCO3(-) cotransporter NBCe1, cause severe proximal renal tubular acidosis (pRTA), growth retardation, decreased IQ, and eye and teeth abnormalities. Among the known NBCe1 mutations, the disease-causing mechanism of the T485S (NBCe1-A numbering) mutation is intriguing because the substituted amino acid, serine, is structurally and chemically similar to threonine. In this study, we performed intracellular pH and whole cell patch-clamp measurements to investigate the base transport and electrogenic properties of NBCe1-A-T485S in mammalian HEK 293 cells. Our results demonstrated that Ser substitution of Thr485 decreased base transport by ~50%, and importantly, converted NBCe1-A from an electrogenic to an electroneutral transporter. Aqueous accessibility analysis using sulfhydryl reactive reagents indicated that Thr485 likely resides in an NBCe1-A ion interaction site. This critical location is also supported by the finding that G486R (a pRTA causing mutation) alters the position of Thr485 in NBCe1-A thereby impairing its transport function. By using NO3(-) as a surrogate ion for CO3(2-), our result indicated that NBCe1-A mediates electrogenic Na(+)-CO3(2-) cotransport when functioning with a 1:2 charge transport stoichiometry. In contrast, electroneutral NBCe1-T485S is unable to transport NO3(-), compatible with the hypothesis that it mediates Na(+)-HCO3(-) cotransport. In patients, NBCe1-A-T485S is predicted to transport Na(+)-HCO3(-) in the reverse direction from blood into proximal tubule cells thereby impairing transepithelial HCO3(-) absorption, possibly representing a new pathogenic mechanism for generating human pRTA.
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Affiliation(s)
- Quansheng Zhu
- Division of Nephrology, Department of Medicine, University of California, Los Angeles, CA 90095-1689, USA.
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Lee SK, Boron WF, Parker MD. Substrate specificity of the electrogenic sodium/bicarbonate cotransporter NBCe1-A (SLC4A4, variant A) from humans and rabbits. Am J Physiol Renal Physiol 2013; 304:F883-99. [PMID: 23324180 PMCID: PMC3625843 DOI: 10.1152/ajprenal.00612.2012] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2012] [Accepted: 01/10/2013] [Indexed: 11/22/2022] Open
Abstract
In the basolateral membrane of proximal-tubule cells, NBCe1-A (SLC4A4, variant A), operating with an apparent Na(+):HCO(3)(-) stoichiometry of 1:3, contributes to the reclamation of HCO(3)(-) from the glomerular filtrate, thereby preventing whole body acidosis. Others have reported that NBCe1-like activity in human, rabbit, and rat renal preparations is substantially influenced by lithium, sulfite, oxalate, and harmaline. These data were taken as evidence for the presence of distinct Na(+) and CO(3)(2-) binding sites in NBCe1-A, favoring a model of 1 Na(+):1 HCO(3)(-):1 CO(3)(2-). Here, we reexamine these findings by expressing human or rabbit NBCe1-A clones in Xenopus oocytes. In oocytes, NBCe1-A exhibits a 1:2 stoichiometry and could operate in one of five thermodynamically equivalent transport modes: 1) cotransport of Na(+) + 2 HCO(3)(-), 2) cotransport of Na(+) + CO(3)(2-), 3) transport of NaCO(3)(-), 4) exchange of Na(+) + HCO(3)(-) for H(+), or 5) HCO(3)(-)-activated exchange of Na(+) for 2 H(+). In contrast to the behavior of NBCe1-like activity in renal preparations, we find that cloned NBCe1-A is only slightly stimulated by Li(+), not at all influenced by sulfite or oxalate, and only weakly inhibited by harmaline. These negative data do not uniquely support any of the five models above. In addition, we find that NBCe1-A mediates a small amount of Na(+)-independent NO(3)(-) transport and that NBCe1-A is somewhat inhibited by extracellular benzamil. We suggest that the features of NBCe1-like activity in renal preparations are influenced by yet-to-be-identified renal factors. Thus the actual ionic substrates of NBCe1 remain to be identified.
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Affiliation(s)
- Seong-Ki Lee
- Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA
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Parker MD, Boron WF. The divergence, actions, roles, and relatives of sodium-coupled bicarbonate transporters. Physiol Rev 2013; 93:803-959. [PMID: 23589833 PMCID: PMC3768104 DOI: 10.1152/physrev.00023.2012] [Citation(s) in RCA: 208] [Impact Index Per Article: 17.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The mammalian Slc4 (Solute carrier 4) family of transporters is a functionally diverse group of 10 multi-spanning membrane proteins that includes three Cl-HCO3 exchangers (AE1-3), five Na(+)-coupled HCO3(-) transporters (NCBTs), and two other unusual members (AE4, BTR1). In this review, we mainly focus on the five mammalian NCBTs-NBCe1, NBCe2, NBCn1, NDCBE, and NBCn2. Each plays a specialized role in maintaining intracellular pH and, by contributing to the movement of HCO3(-) across epithelia, in maintaining whole-body pH and otherwise contributing to epithelial transport. Disruptions involving NCBT genes are linked to blindness, deafness, proximal renal tubular acidosis, mental retardation, and epilepsy. We also review AE1-3, AE4, and BTR1, addressing their relevance to the study of NCBTs. This review draws together recent advances in our understanding of the phylogenetic origins and physiological relevance of NCBTs and their progenitors. Underlying these advances is progress in such diverse disciplines as physiology, molecular biology, genetics, immunocytochemistry, proteomics, and structural biology. This review highlights the key similarities and differences between individual NCBTs and the genes that encode them and also clarifies the sometimes confusing NCBT nomenclature.
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Affiliation(s)
- Mark D Parker
- Dept. of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4970, USA.
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Lopardo ML, Diaz-Sylvester P, Amorena C. The effect of shear stress on the basolateral membrane potential of proximal convoluted tubule of the rat kidney. Pflugers Arch 2007; 454:289-95. [PMID: 17219192 DOI: 10.1007/s00424-006-0198-7] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2006] [Revised: 11/14/2006] [Accepted: 12/04/2006] [Indexed: 10/23/2022]
Abstract
As consequence of glomerular filtration the viscosity of blood flowing through the efferent arteriole increases. Recently, we found that shear stress modulates proximal bicarbonate reabsorption and nitric oxide (NO.) was the chemical mediator of this effect. In the present work, we found that agonists of NO. production affected basolateral membrane potential (V (blm)) of the proximal convoluted tubule (PCT) epithelium. Using paired micropuncture experiments, we perfused peritubular capillaries with solutions with different viscosity while registering the V (blm). Our results showed that a 50% increment in the viscosity, or the addition of bradykinin (10(-5) M) to the peritubular perfusion solution, induced a significant and similar hyperpolarization of the V (blm) at the PCT epithelium of 6 +/- 0.7 mV (p < 0.05). Both hyperpolarizations were reverted by L-NAME (10(-4) M). Addition of 2,2'-(hydroxynitrosohydrazino) bis-ethanamine (NOC-18) 3 x 10(-4) M to the peritubular perfusion solution induced a hyperpolarization of the same magnitude of that high viscosity or bradykinin. These results strongly suggest the involvement of NO. in the effect of high viscosity solutions. This effect seems to be mediated by activation of K+(ATP) channels as glybenclamide (5 x 10(-5) M) added to peritubular solutions induced a larger depolarization of the V (blm) with high viscosity solutions. Acetazolamide (5 x 10(-5) M) added to high viscosity solutions induced a larger hyperpolarization (8 +/- 1 mV; p < 0.05), suggesting that depolarizing current due to HCO(-)3 exit across the basolateral membrane damps the hyperpolarizing effect of high viscosity. Considering that Na(+) and consequently water reabsorption is highly dependent on electrical gradient, the present data suggest that the endothelium of kidney vascular bed interacts in paracrine fashion with the epithelia, affecting V (blm) and thus modulating PCT reabsorption.
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Affiliation(s)
- Mariano L Lopardo
- CESyMA, Escuela de Ciencia y Tecnología, Universidad Nacional de Gral. San Martín, Avenida Gral Paz 5445, Ed. 23, 1650 San Martín, Argentina
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14
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Horita S, Yamada H, Inatomi J, Moriyama N, Sekine T, Igarashi T, Endo Y, Dasouki M, Ekim M, Al-Gazali L, Shimadzu M, Seki G, Fujita T. Functional analysis of NBC1 mutants associated with proximal renal tubular acidosis and ocular abnormalities. J Am Soc Nephrol 2005; 16:2270-8. [PMID: 15930088 DOI: 10.1681/asn.2004080667] [Citation(s) in RCA: 86] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022] Open
Abstract
Mutations in the Na+-HCO3- co-transporter (NBC1) cause permanent proximal renal tubular acidosis (pRTA) with ocular abnormalities. However, little has been known about the relationship between the degree of NBC1 inactivation and the severity of pRTA. This study identified three new homozygous mutations (T485S, A799V, and R881C) in the common coding regions of NBC1. Functional analysis of these new as well as the known mutants (R298S and R510H) in Xenopus oocytes revealed a considerable variation in their electrogenic activities. Whereas the activities of R298S, A799V, and R881C were 15 to 40% of the wild-type (WT) activity, T485S and R510H, as a result of poor surface expression, showed almost no activities. However, T485S, like R510H, had the transport activity corresponding to approximately 50% of the WT activity in ECV304 cells, indicating that surface expression of T485S and R510H varies between the different in vitro cell systems. Electrophysiologic analysis showed that WT, R298S, and R881C all function with 2HCO3- to 1Na+ stoichiometry and have similar extracellular Na+ affinity, indicating that reduction in Na+ affinity cannot explain the inactivation of R298S and R881C. These results, together with the presence of nonfunctional mutants (Q29X and DeltaA) in other patients, suggest that at least approximately 50% reduction of NBC1 activity would be required to cause severe pRTA.
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MESH Headings
- Acidosis, Renal Tubular/genetics
- Acidosis, Renal Tubular/pathology
- Adolescent
- Animals
- Blotting, Western
- Cell Line
- Cell Membrane/metabolism
- Child, Preschool
- DNA, Complementary/metabolism
- Electrophysiology
- Eye Abnormalities/genetics
- Eye Abnormalities/pathology
- Female
- Gene Expression Regulation
- Genetic Techniques
- Homozygote
- Humans
- Hydrogen-Ion Concentration
- Kidney Tubules, Proximal/metabolism
- Kidney Tubules, Proximal/pathology
- Male
- Membrane Potentials
- Microscopy, Fluorescence
- Models, Statistical
- Mutagenesis
- Mutation
- Mutation, Missense
- Oocytes/cytology
- Oocytes/metabolism
- Sodium/metabolism
- Sodium-Bicarbonate Symporters/genetics
- Xenopus laevis
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Affiliation(s)
- Shoko Horita
- Department of Internal Medicine, Faculty of Medicine, Tokyo University, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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15
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Li HC, Szigligeti P, Worrell RT, Matthews JB, Conforti L, Soleimani M. Missense mutations in Na+:HCO3- cotransporter NBC1 show abnormal trafficking in polarized kidney cells: a basis of proximal renal tubular acidosis. Am J Physiol Renal Physiol 2005; 289:F61-71. [PMID: 15713912 DOI: 10.1152/ajprenal.00032.2005] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
The kidney Na(+):HCO(3)(-) cotransporter NBC1 is located exclusively on the basolateral membrane of kidney proximal tubule cells and is responsible for the reabsorption of majority of filtered bicarbonate. Two well-described missense mutations in NBC1, R510H and S427L, are associated with renal tubular acidosis (RTA). However, the exact relationship between these mutations and NBC1 dysregulation remains largely unknown. To address this question, cDNAs for wild-type kidney NBC1 and its mutants R510H and S427L were generated, fused in frame with NH(2) terminally tagged GFP, and transiently expressed in Madin-Darby canine kidney cells. In parallel studies, oocytes were injected with the wild-type and mutant NBC1 cRNAs and studied for membrane expression and activity. In monolayer cells grown to polarity, the wild-type GFP-NBC1 was exclusively localized on the basolateral membrane domain. However, GFP-NBC1 mutant R510H was detected predominantly in the cytoplasm. GFP-NBC1 mutant S427L, on the other hand, was detected predominantly on the apical membrane with residual cytoplasmic retention and basolateral membrane labeling. In oocytes injected with the wild-type or mutant GFP-NBC1 cRNAs, Western blot analysis showed that wild-type NBC1 is predominantly localized in the membrane fraction, whereas NBC1-R510H mutant was predominantly expressed in the cytoplasm. NBC1-S427L mutant was mostly expressed in the membrane fraction. Functional analysis of NBC1 activity in oocytes by membrane potential recording demonstrated that compared with wild-type GFP-NBC1, the GFP-NBC1 mutants H510R and S427L exhibited significant reduction in activity. These findings suggest that the permanent isolated proximal RTA in patients with H510R or S427L mutation resulted from a combination of inactivation and mistargeting of kidney NBC1, with H510R mutant predominantly retained in the cytoplasm, whereas S427L mutant is mistargeted to the apical membrane.
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Affiliation(s)
- Hong C Li
- Dept. of Medicine, University of Cincinnati, Cincinnati, OH 45267-0585, USA
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16
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Kurtz I, Petrasek D, Tatishchev S. Molecular mechanisms of electrogenic sodium bicarbonate cotransport: structural and equilibrium thermodynamic considerations. J Membr Biol 2004; 197:77-90. [PMID: 15014910 DOI: 10.1007/s00232-003-0643-x] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2003] [Indexed: 12/21/2022]
Abstract
The electrogenic Na(+)-HCO(3)(-) cotransporters play an essential role in regulating intracellular pH and extracellular acid-base homeostasis. Of the known members of the bicarbonate transporter superfamily (BTS), NBC1 and NBC4 proteins have been shown to be electrogenic. The electrogenic nature of these transporters results from the unequal coupling of anionic and cationic fluxes during each transport cycle. This unique property distinguishes NBC1 and NBC4 proteins from other sodium bicarbonate cotransporters and members of the bicarbonate transporter superfamily that are known to be electroneutral. Structure-function studies have played an essential role in revealing the basis for the modulation of the coupling ratio of NBC1 proteins. In addition, the recent transmembrane topographic analysis of pNBC1 has shed light on the potential structural determinants that are responsible for ion permeation through the cotransporter. The experimentally difficult problem of determining the nature of anionic species being transported by these proteins (HCO(3)(-) versus CO(3)(2-)) is analyzed using a theoretical equilibrium thermodynamics approach. Finally, our current understanding of the molecular mechanisms responsible for the regulation of ion coupling and flux through electrogenic sodium bicarbonate cotransporters is reviewed in detail.
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Affiliation(s)
- I Kurtz
- Division of Nephrology, David Geffen School of Medicine at UCLA, Los Angeles, California 90095-1689, USA.
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17
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Satoh H, Moriyama N, Hara C, Yamada H, Horita S, Kunimi M, Tsukamoto K, Iso-O N, Inatomi J, Kawakami H, Kudo A, Endou H, Igarashi T, Goto A, Fujita T, Seki G. Localization of Na+-HCO-3 cotransporter (NBC-1) variants in rat and human pancreas. Am J Physiol Cell Physiol 2003; 284:C729-37. [PMID: 12444017 DOI: 10.1152/ajpcell.00166.2002] [Citation(s) in RCA: 57] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Mutations in Na(+)-HCO(3)(-) cotransporter (NBC-1) cause proximal renal tubular acidosis (pRTA) associated with ocular abnormalities. One pRTA patient had increased serum amylase, suggesting possible evidence of pancreatitis. To further delineate a link between NBC-1 inactivation and pancreatic dysfunction, immunohistochemical analysis was performed on rat and human pancreas using antibodies against kidney-type (kNBC-1) and pancreatic-type (pNBC-1) transporters. In rat pancreas, the anti-pNBC-1 antibody labeled acinar cells and both apical and basolateral membranes of medium and large duct cells. In human pancreas, on the other hand, the anti-pNBC-1 antibody did not label acinar cells, although it did label the basolateral membranes of the entire duct system. The labeling by anti-kNBC-1 antibody was detected in only a limited number of rat pancreatic duct cells. To examine the effects of pRTA-related mutations, R342S and R554H, on pNBC-1 function, we performed functional analysis and found that both mutants had reduced transport activities compared with the wild-type pNBC-1. These results indicate that pNBC-1 is the predominant variant that mediates basolateral HCO(3)(-) uptake into duct cells in both rat and human pancreas. The loss of pNBC-1 function is predicted to have significant impact on overall ductal HCO(3)(-) secretion, which could potentially lead to pancreatic dysfunction.
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Affiliation(s)
- Hiroaki Satoh
- Department of Internal Medicine, Faculty of Medicine, Tokyo University, Japan
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18
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Horita S, Zheng Y, Hara C, Yamada H, Kunimi M, Taniguchi S, Uwatoko S, Sugaya T, Goto A, Fujita T, Seki G. Biphasic regulation of Na+-HCO3- cotransporter by angiotensin II type 1A receptor. Hypertension 2002; 40:707-12. [PMID: 12411466 DOI: 10.1161/01.hyp.0000036449.70110.de] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Although angiotensin (Ang) II is known to regulate renal proximal transport in a biphasic way, the receptor subtype(s) mediating these Ang II effects remained to be established. To clarify this issue, we compared the effects of Ang II in wild-type mice (WT) and Ang II type 1A receptor-deficient mice (AT(1A) KO). The Na+-HCO3- cotransporter (NBC) activity, analyzed in isolated nonperfused tubules with a fluorescent probe, was stimulated by 10(-10) mol/L Ang II but was inhibited by 10(-6) mol/L Ang II in WT. Although valsartan (AT1 antagonist) blocked both stimulation and inhibition by Ang II, PD 123,319 (AT2 antagonist) did not modify these effects of Ang II. In AT1A KO, in contrast, this biphasic regulation was lost, and only stimulation of NBC activity by 10(-6) mol/L Ang II was observed. This stimulation was blocked by valsartan but not by PD 123,319. More than 10(-8) mol/L Ang II induced a transient increase in cell Ca2+ concentrations in WT, which was again blocked by valsartan but not by PD 123,319. However, up to 10(-5) mol/L Ang II did not increase cell Ca2+ concentrations in AT1A KO. Finally, the addition of arachidonic acid inhibited the NBC activity similarly in WT and AT(1A) KO, suggesting that the inhibitory pathway involving P-450 metabolites is preserved in AT(1A) KO. These results indicate that AT(1A) mediates the biphasic regulation of NBC. Although low-level expression of AT(1B) could be responsible for the stimulation by 10(-6) mol/L Ang II in AT1A KO, no evidence was obtained for AT2 involvement.
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MESH Headings
- Angiotensin II/pharmacology
- Angiotensin Receptor Antagonists
- Animals
- Arachidonic Acid/pharmacology
- Bicarbonates/metabolism
- Calcium/metabolism
- Dose-Response Relationship, Drug
- Enzyme Activators/pharmacology
- Fluorescent Dyes
- Imidazoles/pharmacology
- In Vitro Techniques
- Intracellular Fluid/metabolism
- Kidney Tubules, Proximal/drug effects
- Kidney Tubules, Proximal/metabolism
- Male
- Mice
- Mice, Inbred C57BL
- Mice, Knockout
- Pyridines/pharmacology
- Receptor, Angiotensin, Type 1
- Receptor, Angiotensin, Type 2
- Receptors, Angiotensin/drug effects
- Receptors, Angiotensin/genetics
- Receptors, Angiotensin/metabolism
- Sodium/metabolism
- Sodium-Bicarbonate Symporters/antagonists & inhibitors
- Sodium-Bicarbonate Symporters/metabolism
- Tetrazoles/pharmacology
- Valine/analogs & derivatives
- Valine/pharmacology
- Valsartan
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Affiliation(s)
- Shoko Horita
- Department of Internal Medicine, Faculty of Medicine, Tokyo University, Tokyo, Japan
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19
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Kottra G, Stamfort A, Daniel H. PEPT1 as a paradigm for membrane carriers that mediate electrogenic bidirectional transport of anionic, cationic, and neutral substrates. J Biol Chem 2002; 277:32683-91. [PMID: 12082113 DOI: 10.1074/jbc.m204192200] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The capability for electrogenic inward transport of substrates that carry different net charge is a phenomenon observed in a variety of membrane-solute transporters but is not yet understood. We employed the two-electrode voltage clamp technique combined with intracellular pH recordings and the giant patch technique to assess the selectivity for bidirectional transport and the underlying stoichiometries in proton to substrate flux coupling for electrogenic transfer of selected anionic, cationic, and neutral dipeptides by the intestinal peptide transporter PEPT1. Anionic dipeptides such as Gly-Asp and Asp-Gly are transported in their neutral and negatively charged forms with high and low affinities, respectively. The positive transport current obtained with monoanionic substrates results from the cotransport of two protons. Cationic dipeptides can be transported in neutral and positively charged form, resulting in an excess transport current as compared with neutral substrates. However, binding and transport of cationic dipeptides shows a pronounced selectivity for the position of charged side chains demonstrating that the binding domain of PEPT1 is asymmetric, both in its inward and outward facing conformation. The simultaneous presence of identically charged substrates on both membrane surfaces generates outward and, unexpectedly, enhanced inward transport currents probably by increasing the turnover rate.
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Affiliation(s)
- Gabor Kottra
- Molecular Nutrition Unit, Technical University of Munich, Hochfeldweg 2, D-85350 Freising-Weihenstephan, Germany.
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20
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Usui T, Hara M, Satoh H, Moriyama N, Kagaya H, Amano S, Oshika T, Ishii Y, Ibaraki N, Hara C, Kunimi M, Noiri E, Tsukamoto K, Inatomi J, Kawakami H, Endou H, Igarashi T, Goto A, Fujita T, Araie M, Seki G. Molecular basis of ocular abnormalities associated with proximal renal tubular acidosis. J Clin Invest 2001; 108:107-15. [PMID: 11435462 PMCID: PMC209339 DOI: 10.1172/jci11869] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Proximal renal tubular acidosis associated with ocular abnormalities such as band keratopathy, glaucoma, and cataracts is caused by mutations in the Na(+)-HCO(3)(-) cotransporter (NBC-1). However, the mechanism by which NBC-1 inactivation leads to such ocular abnormalities remains to be elucidated. By immunological analysis of human and rat eyes, we demonstrate that both kidney type (kNBC-1) and pancreatic type (pNBC-1) transporters are present in the corneal endothelium, trabecular meshwork, ciliary epithelium, and lens epithelium. In the human lens epithelial (HLE) cells, RT-PCR detected mRNAs of both kNBC-1 and pNBC-1. Although a Na(+)-HCO(3)-cotransport activity has not been detected in mammalian lens epithelia, cell pH (pH(i)) measurements revealed the presence of Cl(-)-independent, electrogenic Na(+)-HCO(3)-cotransport activity in HLE cells. In addition, up to 80% of amiloride-insensitive pH(i) recovery from acid load in the presence of HCO(3)(-)/CO(2) was inhibited by adenovirus-mediated transfer of a specific hammerhead ribozyme against NBC-1, consistent with a major role of NBC-1 in overall HCO(3)-transport by the lens epithelium. These results indicate that the normal transport activity of NBC-1 is indispensable not only for the maintenance of corneal and lenticular transparency but also for the regulation of aqueous humor outflow.
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MESH Headings
- 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology
- Acidosis, Renal Tubular/complications
- Acidosis, Renal Tubular/genetics
- Amiloride/pharmacology
- Animals
- Bicarbonates/metabolism
- Blotting, Western
- Carrier Proteins/genetics
- Carrier Proteins/metabolism
- Cataract/etiology
- Cataract/genetics
- Cells, Cultured
- Chlorides/metabolism
- Cornea/metabolism
- Cornea/pathology
- Corneal Opacity/etiology
- Corneal Opacity/genetics
- Epithelial Cells/drug effects
- Epithelial Cells/metabolism
- Eye Proteins/genetics
- Eye Proteins/metabolism
- Glaucoma/etiology
- Glaucoma/genetics
- Humans
- Ion Transport/genetics
- Kidney Tubules, Proximal/metabolism
- Lens, Crystalline/metabolism
- Lens, Crystalline/pathology
- Pancreas/metabolism
- Protein Isoforms/deficiency
- Protein Isoforms/genetics
- Protein Isoforms/metabolism
- RNA, Catalytic/chemistry
- RNA, Catalytic/pharmacology
- RNA, Messenger/biosynthesis
- Rats
- Reverse Transcriptase Polymerase Chain Reaction
- Sodium/metabolism
- Sodium-Bicarbonate Symporters
- Valinomycin/pharmacology
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Affiliation(s)
- T Usui
- Department of Ophthalmology, Faculty of Medicine, Tokyo University, Bunkyo-ku, Tokyo, Japan
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21
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Sun XC, Bonanno JA, Jelamskii S, Xie Q. Expression and localization of Na(+)-HCO(3)(-) cotransporter in bovine corneal endothelium. Am J Physiol Cell Physiol 2000; 279:C1648-55. [PMID: 11029313 DOI: 10.1152/ajpcell.2000.279.5.c1648] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Functional studies support the presence of the Na(+)-HCO(3)(-) cotransporter (NBC) in corneal endothelium and possibly corneal epithelium; however, molecular identification and membrane localization have not been reported. To test whether NBC is expressed in bovine cornea, Western blotting was performed, which showed a single band at approximately 130 kDa for freshly isolated and cultured endothelial cells, but no band for epithelium. Two isoforms of NBC have recently been cloned in kidney (kNBC) and pancreas (pNBC). RT-PCR was run using cultured and fresh bovine corneal endothelial and fresh corneal epithelial total RNA and specific primers for kNBC and pNBC. RT-PCR analysis for pNBC was positive in endothelium and weak in epithelium. The RT-PCR product was subcloned and confirmed as pNBC by sequencing. No specific bands for kNBC were obtained from corneal cells. Indirect immunofluorescence and confocal microscopy indicated that NBC locates predominantly to the basolateral membrane in corneal endothelial cells. Furthermore, Na(+)-dependent HCO(3)(-) fluxes and HCO(3)(-)-dependent cotransport with Na(+) were elicited only from the basolateral side of corneal endothelial cells. Therefore, we conclude that pNBC is present in the basolateral membrane of both fresh and cultured bovine corneal endothelium and weakly expressed in the corneal epithelium.
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Affiliation(s)
- X C Sun
- School of Optometry, Indiana University, Bloomington, Indiana 47401, USA
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22
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Lane J, Wigham CG, Hodson SA. A chloride-activated Na(+)/HCO(3)(-)-coupled transport activity in corneal endothelial membranes. Biophys J 2000; 78:2493-8. [PMID: 10777746 PMCID: PMC1300839 DOI: 10.1016/s0006-3495(00)76794-4] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Investigations of corneal endothelium were made to resolve the apparent contradiction of the presence of sodium/bicarbonate cotransporter (NBC) in fresh and cultured cells and NBC's reported absence in isolated plasma membrane vesicles. Gradient-driven ion fluxes into the vesicles were measured. Short-term incubations (0-30 s) showed the presence of a bicarbonate-dependent inward sodium flux (BDSF), which was active when the insides of the vesicles were preloaded with chloride ions. The BDSF was absent if chloride was present only externally to the vesicles. Chloride at concentrations between 30 and 40 mM inside the vesicle had its maximum effect on BDSF. Other anions (acetate, thiocyanate, or gluconate) inside the vesicles did not mimic the chloride effect. Associated with the net inward sodium flux was a net inward bicarbonate flux. Hill plots of sodium influx with respect to external bicarbonate concentrations indicated that the stoichiometry of the net transfer was 1.7 +/- 0.2 (mean +/- standard error, n = 5) bicarbonate ions for each sodium ion transported. There was no net chloride flux found across the membrane vesicles. The finding of a novel chloride-activated NBC activity fully resolves the apparent contradiction between whole-cell and membrane vesicle preparations.
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Affiliation(s)
- J Lane
- Laboratory of Ocular Biophysics, Department of Optometry and Vision Sciences, Cardiff University, Cardiff CF1 3XF, Wales
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23
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Kunimi M, Seki G, Hara C, Taniguchi S, Uwatoko S, Goto A, Kimura S, Fujita T. Dopamine inhibits renal Na+:HCO3- cotransporter in rabbits and normotensive rats but not in spontaneously hypertensive rats. Kidney Int 2000; 57:534-43. [PMID: 10652030 DOI: 10.1046/j.1523-1755.2000.00873.x] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
BACKGROUND Dopamine (DA) is thought to regulate renal proximal transport through the inhibition of the Na+,K+-ATPase and/or Na+/H+ exchanger. Defects in this dopaminergic system are proposed to be a pathogenic factor of genetic hypertension. However, microperfusion studies have not consistently confirmed direct tubular effects of DA. METHODS Isolated proximal straight tubules were perfused peritubularly with Dulbecco's modified Eagle's tissue culture medium (DMEM) containing norepinephrine (NE) to improve incubation conditions. Intracellular Na+ concentrations ([Na+]i) and cell pH (pHi) were measured with fluorescence probes. RESULTS When incubated in DMEM plus NE, DA increased [Na+]i in rabbit tubules. Inhibition of Na+,K+-ATPase could not explain this response, as it was not suppressed by ouabain. An analysis of pHi responses to bath HCO3- reduction revealed that DA, SKF 38393 (a DA1 agonist), and adenosine 3',5'-cyclic monophosphate (cAMP) inhibited the basolateral Na+:HCO3- cotransporter in rabbit and Wistar-Kyoto rat (WKY), if its transport stoichiometry was converted to 3 HCO3-:1 Na+ by DMEM plus NE incubation. The inhibitory effect of DA was abolished by SCH 23390, a DA1 antagonist, but not by (-)-sulpiride, a DA2 antagonist. In spontaneously hypertensive rats (SHRs), however, DA and SKF 38393 failed to inhibit the cotransporter, although the inhibitory effects of cAMP and parathyroid hormone were comparable to those in WKY. CONCLUSION These results indicate that DA inhibits the Na+:HCO3- cotransporter in renal proximal tubules and also suggest that dysregulation of the cotransporter, possibly through the defect in DA1 receptor signaling, could play an important role in development of hypertension in SHRs.
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MESH Headings
- 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine/pharmacology
- Acetazolamide/pharmacology
- Adenylyl Cyclases/metabolism
- Animals
- Bicarbonates/pharmacokinetics
- Biological Transport/drug effects
- Carrier Proteins/antagonists & inhibitors
- Carrier Proteins/metabolism
- Cyclic AMP/analogs & derivatives
- Cyclic AMP/pharmacology
- Diuretics/pharmacology
- Dopamine/pharmacology
- Dopamine Agonists/pharmacology
- Enzyme Inhibitors/pharmacology
- Female
- Hydrogen-Ion Concentration
- Hypertension, Renal/genetics
- Hypertension, Renal/metabolism
- Kidney Tubules, Proximal/chemistry
- Kidney Tubules, Proximal/cytology
- Kidney Tubules, Proximal/enzymology
- Male
- Parathyroid Hormone/pharmacology
- Rabbits
- Rats
- Rats, Inbred SHR
- Rats, Inbred WKY
- Receptors, Dopamine D1/physiology
- Second Messenger Systems/physiology
- Sodium/pharmacokinetics
- Sodium-Bicarbonate Symporters
- Sodium-Potassium-Exchanging ATPase/metabolism
- Thionucleotides/pharmacology
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Affiliation(s)
- M Kunimi
- Department of Nephrology and Endocrinology, and Department of Infectious Diseases, Faculty of Medicine, University of Tokyo, Japan
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24
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Abstract
Bicarbonate and CO2 comprise the major pH buffer of biological fluids. In the renal proximal tubule most of the filtered HCO3- is reabsorbed by an electrogenic Na/HCO3 cotransporter located at the basolateral membrane. This Na+ bicarbonate cotransporter (NBC) was recently cloned. This review highlights the recent developments leading to and since the cloning of NBC: NBC expression cloning, protein features, clone physiology, isoforms and genes, mRNA distribution, and protein distribution. With the NBC amino acid sequence 30-35% identical to the anion exchangers (AE1-3), a superfamily of HCO3- transporters is emerging. Physiologically, NBC is electrogenic, Na+ dependent, HCO3- dependent, Cl- independent, and inhibited by stilbenes (DIDS and SITS). NBC clones and proteins have been isolated from several tissues (other than kidney) thought to have physiologically distinct HCO3- transporters. For example, NBC occurs in pancreas, prostate, brain, heart, small and large intestine, stomach, and epididymis. Finally, there are at least two genes that encode NBC proteins. Possible future directions of research are discussed.
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Affiliation(s)
- M F Romero
- Department of Physiology and Biophysics and Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4790, USA.
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25
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Abuladze N, Lee I, Newman D, Hwang J, Pushkin A, Kurtz I. Axial heterogeneity of sodium-bicarbonate cotransporter expression in the rabbit proximal tubule. THE AMERICAN JOURNAL OF PHYSIOLOGY 1998; 274:F628-33. [PMID: 9530281 DOI: 10.1152/ajprenal.1998.274.3.f628] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
It is generally accepted that Na(HCO3)n cotransport is the most important mechanism mediating basolateral bicarbonate efflux in the early proximal tubule. The presence of basolateral Na(HCO3)n cotransport in the late proximal tubule (S3 segment) and in the juxtamedullary S1 and S2 segments has been controversial. The renal sodium-bicarbonate cotransporter (NBC) has been recently cloned from rat (M. F. Romero, M. A. Hediger, E. L. Boulpaep, and W. F. Boron. J. Am. Soc. Nephrol. 7: 1259, 1996), salamander (M. F. Romero, M. A. Hediger, E. L. Boulpaep, and W. F. Boron. Nature 387: 409-413, 1997), and human (C. E. Burnham, H. Amlal, Z. Wang, G. E. Shull, and M. Soleimani. J. Biol. Chem. 272: 19111-19114, 1997). The localization of NBC in the kidney is unknown. The present study was designed to localize NBC mRNA expression in the rabbit proximal tubule. In situ hybridization studies were combined with functional studies of basolateral Na(HCO3)n cotransport in superficial and juxtamedullary S1, S2, and S3 segments of the rabbit proximal tubule. The results demonstrate that NBC mRNA is localized predominantly to the cortex, with less expression in the outer medulla. NBC expression was not detected in the inner medulla. The highest level of NBC mRNA is in the S1 proximal tubule. NBC is expressed at a low levels in the S3 segment, with intermediate expression in the S2 segment. In bicarbonate-buffered solutions, the rate of base efflux mediated by Na(HCO3)n cotransport followed a similar pattern in superficial and juxtamedullary proximal tubule segments, i.e., S1 > S2 > S3. The juxtamedullary S1 segment had the greatest rate of basolateral Na(HCO3)n cotransport and the highest level of NBC expression in the proximal tubule.
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Affiliation(s)
- N Abuladze
- Division of Nephrology, University of California Los Angeles School of Medicine 90095-1698, USA
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26
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Ishiguro H, Steward MC, Lindsay AR, Case RM. Accumulation of intracellular HCO3- by Na(+)-HCO3- cotransport in interlobular ducts from guinea-pig pancreas. J Physiol 1996; 495 ( Pt 1):169-78. [PMID: 8866360 PMCID: PMC1160733 DOI: 10.1113/jphysiol.1996.sp021582] [Citation(s) in RCA: 136] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
1. Short segments of interlobular duct were microdissected from guinea-pig pancreas following enzymatic digestion. After overnight culture, intracellular pH (pH1) and Na+ concentration ([Na+]i) were measured by microfluorometry in duct cells loaded with either the pH-sensitive fluoroprobe 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) or the sodium-binding benzofuran isophthalate (SBFI). 2. The transporters responsible for maintaining pHi above equilibrium were investigated by using the NH4Cl pulse technique to acid load the cells. In the absence of HCO3-/CO2, the recovery of pH1 was Na+ dependent, abolished by 0.2 mM amiloride and by 10 microM N-methyl-N-isobutylamiloride and was therefore attributed to Na(+)-H+ exchange. 3. In the presence of HCO3-/CO2, amiloride only partially inhibited the recovery from acid loading. The amiloride-insensitive component was abolished by 0.5 mM H2DIDS and unaffected by depletion of intracellular Cl- and was therefore attributed to Na(+)-HCO3- cotransport. 4. Stimulation with 10 nM secretin did not cause a significant change in pH1 despite a significant increase in HCO3- efflux. However, in the presence of secretin, addition of 0.5 mM H2DIDS caused a decline in pH1 that was three times more rapid than that obtained with 0.2 mM amiloride. 5. In secretin-stimulated ducts, Na+ uptake increased when HCO3-/CO2 was added to the bath and this increase was strongly inhibited by 0.5 mM H2DIDS. 6. We conclude that Na(+)-HCO3- cotransport contributes approximately 75% of the HCO3- taken up by guinea-pig pancreatic duct cells during stimulation with secretin. It is proposed that electrical coupling between HCO3- efflux at the luminal membrane and electrogenic Na(+)-HCO3- cotransport at the basolateral membrane explains why secretin causes little change in pH1.
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Affiliation(s)
- H Ishiguro
- School of Biological Sciences, University of Manchester, UK
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27
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Seki G, Coppola S, Yoshitomi K, Burckhardt BC, Samarzija I, Müller-Berger S, Frömter E. On the mechanism of bicarbonate exit from renal proximal tubular cells. Kidney Int 1996; 49:1671-7. [PMID: 8743474 DOI: 10.1038/ki.1996.244] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
We compare here the results of electrophysiological measurements on proximal tubular cells performed on rat kidney in vivo and on isolated rabbit and rat tubules in vitro. Based on different effects of carbonic anhydrase inhibitors in the in vivo and in vitro preparation, we conclude that NaHCO3 cotransport across the basolateral cell membrane functions as Na(+)-CO3(2-)-HCO3- cotransport in vivo, but as Na(+)-HCO3(-)-HCO3- cotransport in the classical in vitro preparation. The former, but not the latter, transport mode is characterized by generation of local disequilibrium pH/CO3(2-) concentrations that oppose fluxes if membrane-bound carbonic anhydrase is inhibited. In support of this conclusion, we find that overall transport functions with a HCO3- to Na+ stoichiometry of 3:1 in vivo (since each transported CO3(2-) eventually generates 2 HCO3- ions), but 2:1 in vitro. This has been deduced from various measurements, among them super-Nernstian and reverse nernstian, potential responses to changing ion concentrations which are characteristic of obligatorily coupled cation-anion cotransporters, but are not known in classical electrochemistry. The different transport modes in vivo and in vitro suggest that isolated proximal tubules have functional deficits compared to proximal tubules in vivo.
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Affiliation(s)
- G Seki
- Zentrum der Physiologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt/Main, Germany
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28
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Curci S, Debellis L, Caroppo R, Frömter E. Model of bicarbonate secretion by resting frog stomach fundus mucosa. I. Transepithelial measurements. Pflugers Arch 1994; 428:648-54. [PMID: 7838688 DOI: 10.1007/bf00374589] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
In the present in vitro experiments on gastric fundus mucosa of Rana esculenta we try to define the mechanism of alkaline secretion that is observed in summer frogs in the resting stomach (blockage of HCl secretion by ranitidine, 10(-5) mol/l). The transepithelial voltage and the rate of alkalinization (ASR) of an unbuffered gastric lumen perfusate was measured as a function of serosal (and mucosal) fluid composition. ASR was high (0.88 +/- S.E. 0.09 microEq.cm-2.h-1, n = 11) during serosal bath perfusion with HCO(3-)-Ringer solution, decreased slightly to 0.50 +/- 0.07 microEq.cm-2.h-1 (n = 6) in HCO(3-)-free HEPES-buffered Ringer solution of the same pH, and decreased to approximately 20% when carbonic anhydrase was inhibited by acetazolamide. While replacement of mucosal or serosal Cl- did not--within 1 h--significantly alter ASR, replacement of serosal Na+ in the presence or absence of HCO3- strongly reduced ASR, and a similar reduction was observed after serosal application of the anion transport inhibitor DIDS (4,4-diisothiocyanatostilbene-2,2-disulphonate, 2.10(-4) mol/l), the metabolic poison rotenone (10(-5) mol/l), the uncoupler dinitrophenol (10(-4) mol/l), and the Na+ pump inhibitor ouabain (10(-4) mol/l), while serosal amiloride (10(-4) mol/l) had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)
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Affiliation(s)
- S Curci
- Istituto di Fisiologia Generale, Università di Bari, Italy
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29
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Köttgen M, Leipziger J, Fischer KG, Nitschke R, Greger R. pH regulation in HT29 colon carcinoma cells. Pflugers Arch 1994; 428:179-85. [PMID: 7971175 DOI: 10.1007/bf00374856] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
The pH regulation in HT29 colon carcinoma cells has been investigated using the pH-sensitive fluorescent indicator 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under control conditions, intracellular pH (pHi) was 7.21 +/- 0.07 (n = 22) in HCO3(-)-containing and 7.21 +/- 0.09 (n = 12) in HCO3(-)-free solution. HOE-694 (10 mumol/l), a potent inhibitor of the Na+/H+ exchanger, did not affect control pHi. As a means to acidify cells we used the NH4+/NH3 (20 mmol/l) prepulse technique. The mean peak acidification was 0.37 +/- 0.07 pH units (n = 6). In HCO3(-)-free solutions recovery from acid load was completely blocked by HOE-694 (1 mumol/l), whereas in HCO3(-)-containing solutions a combination of HOE-694 and 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS, 0.5 mmol/l) was necessary to show the same effect. Recovery from acid load was Na(+)-dependent in HCO3(-)-containing and HCO3(-)-free solutions. Removal of external Cl- caused a rapid, DIDS-blockable alkalinization of 0.33 +/- 0.03 pH units (n = 15) and of 0.20 +/- 0.006 pH units (n = 5), when external Na+ was removed together with Cl-. This alkalinization was faster in HCO3(-)-containing than in HCO3(-)-free solutions. The present observations demonstrate three distinct mechanisms of pHi regulation in HT29 cells: (a) a Na+/H+ exchanger, (b) a HCO3-/Cl- exchanger and (c) a Na(+)-dependent HCO3- transporter, probably the Na(+)-HCO3-/Cl- antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)
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Affiliation(s)
- M Köttgen
- Physiologisches Institut, Albert-Ludwigs-Universität, Freiburg, Germany
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30
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Coppola S, Frömter E. An electrophysiological study of angiotensin II regulation of Na-HCO3 cotransport and K conductance in renal proximal tubules. II. Effect of micromolar concentrations. Pflugers Arch 1994; 427:151-6. [PMID: 8058464 DOI: 10.1007/bf00585954] [Citation(s) in RCA: 27] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
In the first part of our study, we described the effect of picomolar concentrations of angiotensin II (AII) on cell membrane potential (Vb) and cell pH (pHi) of isolated perfused rabbit renal proximal tubules. In the present publication we summarize respective observations with micromolar concentrations of AII. With a few exceptions nearly all experiments showed mirror-image-like results. In the majority of the experiments 10(-6) mol/l AII, when applied from the bath (but not when applied from the lumen), slightly hyperpolarized the cells by -3.4 +/- 0.3 mV (mean +/- SEM, n = 20) and alkalinized them by up to 0.06 pH units, while the lower AII concentrations, which were applied in the previous study, depolarized and acidified. The present observations suggest that micromolar concentrations of AII inhibit basolateral Na-HCO3 cotransport. This conclusion was confirmed by a decreasing Vb response to step changes of basolateral HCO3 concentration. In addition, there was a tendency of the Vb response to K concentration steps to decrease, but measurements of the voltage divider ratio did not point to a significant inhibition of a basolateral K conductance. In spite of the almost perfect reciprocity of the results with 10(-6) and 10(-11) mol/l AII, some specific observations suggest that micromolar concentrations of AII do not simply cause mirror-image-like effects, but influence still further transport systems compared to picomolar concentrations.
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Affiliation(s)
- S Coppola
- Zentrum der Physiologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt/Main, Germany
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31
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Coppola S, Frömter E. An electrophysiological study of angiotensin II regulation of Na-HCO3 cotransport and K conductance in renal proximal tubules. I. Effect of picomolar concentrations. Pflugers Arch 1994; 427:143-50. [PMID: 8058463 DOI: 10.1007/bf00585953] [Citation(s) in RCA: 25] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
The effect of picomolar concentrations of angiotensin II (AII) was investigated in isolated perfused rabbit renal proximal tubules using conventional or pH-sensitive intracellular microelectrodes. Under control conditions cell membrane potential (Vb) and cell pH (pHi) averaged -53.8 +/- 1.9 mV (mean +/- SEM, n = 49) and 7.24 +/- 0.01 (n = 10), respectively. AII (at 10(-11) mol/l), when applied from the bath (but not when applied from the lumen perfusate), produced the following effects: approximately 85% of the viable tubules responded with a small depolarization (+5.5 +/- 0.4 mV, n = 43) which was accompanied in half of the pHi measurements by a slow acidification (delta pHi = -0.03 +/- 0.01, n = 5). The remaining 15% responded with a small hyperpolarization (delta Vb = -3.1 +/- 0.4 mV, n = 6). All changes were fully reversible and repeatable. Experiments with fast changes in bath HCO3 or K concentrations, as well as measurements of the basolateral voltage divider fraction in response to transepithelial current flow, explain these observations as stimulation of a basolateral Na-HCO3 cotransporter and of a basolateral K conductance. Both counteract in their effect on Vb, but can be individuated by blocker experiments with 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and barium. Both the stimulation of Na-HCO3 cotransport and the stimulation of the K conductance may result from down-regulation of the level of cyclic adenosine monophosphate in the cell.
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Affiliation(s)
- S Coppola
- Zentrum der Physiologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt/Main, Germany
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