Stimulation by caveolin-1 of the hypotonicity-induced release of taurine and ATP at basolateral, but not apical, membrane of Caco-2 cells

2006 ◽  
Vol 290 (5) ◽  
pp. C1287-C1296 ◽  
Author(s):  
Nina Ullrich ◽  
Adrian Caplanusi ◽  
Bert Brône ◽  
Diane Hermans ◽  
Els Larivière ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Regulatory Volume Decrease ◽  
Protective Mechanism ◽  
Organic Osmolytes ◽  
Wild Type ◽  
Caveolin 1 ◽  
Water Efflux ◽  
Electrophysiological Characterization

Regulatory volume decrease (RVD) is a protective mechanism that allows mammalian cells to restore their volume when exposed to a hypotonic environment. A key component of RVD is the release of K+, Cl−, and organic osmolytes, such as taurine, which then drives osmotic water efflux. Previous experiments have indicated that caveolin-1, a coat protein of caveolae microdomains in the plasma membrane, promotes the swelling-induced Cl− current ( ICl,swell) through volume-regulated anion channels. However, it is not known whether the stimulation by caveolin-1 is restricted to the release of Cl− or whether it also affects the swelling-induced release of other components, such as organic osmolytes. To address this problem, we have studied ICl,swell and the hypotonicity-induced release of taurine and ATP in wild-type Caco-2 cells that are caveolin-1 deficient and in stably transfected Caco-2 cells that express caveolin-1. Electrophysiological characterization of wild-type and stably transfected Caco-2 showed that caveolin-1 promoted ICl,swell, but not cystic fibrosis transmembrane conductance regulator currents. Furthermore, caveolin-1 expression stimulated the hypotonicity-induced release of taurine and ATP in stably transfected Caco-2 cells grown as a monolayer. Interestingly, the effect of caveolin-1 was polarized because only the release at the basolateral membrane, but not at the apical membrane, was increased. It is therefore concluded that caveolin-1 facilitates the hypotonicity-induced release of Cl−, taurine, and ATP, and that in polarized epithelial cells, the effect of caveolin-1 is compartmentalized to the basolateral membrane.

Electrophysiological characterization of upper portion of descending limb of long-looped nephron

1991 ◽  
Vol 260 (3) ◽  
pp. F311-F316 ◽  
Author(s):  
K. Yoshitomi ◽  
M. Imai
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
High Water ◽  
Membrane Resistance ◽  
Membrane Voltage ◽  
Major Pathway ◽  
K Concentration ◽  
Electrophysiological Characterization

The upper portion of the descending limb of long-looped nephron (LDLu) of the hamster is characterized by high water and ion permeabilities. Although the paracellular route is considered to be the major pathway representing cation permselectivity of this segment, ion transport mechanisms through the transcellular pathway are unknown. To study this issue; we applied cable analysis and conventional microelectrode technique to the hamster LDLu perfused in vitro. The transmural voltage (VT) was not different from zero, and transmural resistance (RT) was very low, 18.3 +/- 2.0 omega.cm2 (n = 12). The basolateral membrane voltage was -80 +/- 2 mV (n = 55), and fractional apical membrane resistance was 0.92 +/- 0.23 (n = 5). Ouabain (0.1 mM) in the bath decreased basolateral membrane voltage (VB) by 23 +/- 3 mV (n = 6, P less than 0.001). Increase in K+ concentration in bath and in lumen from 5 to 50 mM decreased VB by 39 +/- 2 (n = 7, P less than 0.01) and apical membrane voltage (VA) by 10 +/- 1 mV (n = 7, P less than 0.001), respectively. Addition of 2 mM Ba2+ to bath and to lumen decreased VB by -47 +/- 2 (n = 11, P less than 0.001) and decreased VA by 8 +/- 1 mV, respectively. Reduction of HCO3- in bath from 25 to 2.5 mM decreased VB by 4 +/- 1 mV (n = 7, P less than 0.005). Reduction of bath Cl- did not cause any rapid deflection of VB. No appreciable Na+ conductance was detected in the apical membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

PAT-1 (Slc26a6) is the predominant apical membrane Cl−/HCO3− exchanger in the upper villous epithelium of the murine duodenum

2007 ◽  
Vol 292 (4) ◽  
pp. G1079-G1088 ◽  
Author(s):  
Janet E. Simpson ◽  
Clifford W. Schweinfest ◽  
Gary E. Shull ◽  
Lara R. Gawenis ◽  
Nancy M. Walker ◽  
...  
Keyword(s):  
Anion Exchanger ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Duodenal Mucosa ◽  
Transport Processes ◽  
Wild Type ◽  
Rt Pcr ◽  
Targeted Deletion ◽  
Anion Transporter ◽  
Dependent Transport

Basal HCO3− secretion across the duodenum has been shown in several species to principally involve the activity of apical membrane Cl−/HCO3− exchanger(s). To investigate the identity of relevant anion exchanger(s), experiments were performed using wild-type (WT) mice and mice with gene-targeted deletion of the following Cl−/HCO3− exchangers localized to the apical membrane of murine duodenal villi: Slc26a3 [down-regulated in adenoma (DRA)], Slc26a6 [putative anion transporter 1 (PAT-1)], and Slc4a9 [anion exchanger 4 (AE4)]. RT-PCR of the isolated villous epithelium demonstrated PAT-1, DRA, and AE4 mRNA expression. Using the pH-sensitive dye BCECF, anion exchange rates were measured across the apical membrane of epithelial cells in the upper villus of the intact duodenal mucosa. Under basal conditions, Cl−/HCO3− exchange activity was reduced by 65–80% in the PAT-1(−) duodenum, 30–40% in the DRA(−) duodenum, and <5% in the AE4(−) duodenum compared with the WT duodenum. SO42−/HCO3− exchange was eliminated in the PAT-1(−) duodenum but was not affected in the DRA(−) and AE4(−) duodenum relative to the WT duodenum. Intracellular pH (pHi) was reduced in the PAT-1(−) villous epithelium but increased to WT levels in the absence of CO2/HCO3− or during methazolamide treatment. Further experiments under physiological conditions indicated active pHi compensation in the PAT-1(−) villous epithelium by combined activities of Na+/H+ exchanger 1 and Cl−-dependent transport processes at the basolateral membrane. We conclude that 1) PAT-1 is the major contributor to basal Cl−/HCO3− and SO42−/HCO3− exchange across the apical membrane and 2) PAT-1 plays a role in pHi regulation in the upper villous epithelium of the murine duodenum.

Basolateral K+, Cl-, and HCO3- conductances and cell volume regulation in rabbit PCT

1993 ◽  
Vol 264 (2) ◽  
pp. F365-F376 ◽  
Author(s):  
P. Macri ◽  
S. Breton ◽  
J. S. Beck ◽  
J. Cardinal ◽  
R. Laprade
Keyword(s):  
Volume Regulation ◽  
Basolateral Membrane ◽  
Regulatory Volume Decrease ◽  
Cell Volume Regulation ◽  
Temporal Variations ◽  
Hypotonic Shock ◽  
Control Value ◽  
Water Efflux ◽  
To Come ◽  
Cellular Water

The relationship between changes in cellular volume, intracellular pH (pHi), basolateral membrane potential (VBL), and membrane partial basolateral conductances to K+ (tK) and Cl- (tCl) and mediated by the Na-HCO3 cotransporter (tNaHCO3) was determined in the collapsed proximal convoluted tubule (PCT) submitted to a 125-mosmol/kg hypotonic shock. The shock that produces a rapid swelling followed by partial volume regulation was accompanied by a rapid and transient VBL hyperpolarization of 10.0 +/- 1.5 mV and a second gradual hyperpolarization of 5.0 +/- 0.7 mV with respect to a control value of -44.0 +/- 4.6 mV.tK was 0.12 +/- 0.03 in control, increased transiently to 0.15 +/- 0.03, and then gradually increased to reach 0.32 +/- 0.06 at the end of hypotonic shock. In contrast, tCl was 0.03 +/- 0.01 in control, increased rapidly to a maximum of 0.16 +/- 0.01, and then decreased slowly to 0.08 +/- 0.02. During the same period, tNaHCO3 decreased rapidly from 0.41 +/- 0.04 to a minimum of 0.11 +/- 0.02 and slowly reincreased to reach 0.16 +/- 0.01.pHi increased transiently from 7.09 +/- 0.03 in control to 7.24 +/- 0.05 to come back gradually to 7.15 +/- 0.05 at the end of the hypotonic period. The membrane absolute conductance mediated by the Na-HCO3 cotransporter was found to increase only slightly in hypotonic conditions, whereas that to K+ and Cl-, GK and GCl, increased by at least factors of 8 and 17, respectively, with the increase of GCl being much faster than that of GK. In addition, the temporal variations in GCl followed closely those of the cellular water efflux. We conclude that the hypotonic swelling leads to important increases in the conductive pathways for K+ and Cl- and that the Cl- conductance pathway appears to be the rate limiting step in triggering and supporting regulatory volume decrease.

scholarly journals Purinergic activation of anion conductance and osmolyte efflux in cultured rat hippocampal neurons

2008 ◽  
Vol 295 (6) ◽  
pp. C1550-C1560 ◽  
Author(s):  
Guangze Li ◽  
James E. Olson
Keyword(s):  
Mammalian Cells ◽  
Hippocampal Neurons ◽  
Regulatory Volume Decrease ◽  
Extracellular Atp ◽  
Cell Swelling ◽  
Nucleotide Receptors ◽  
Organic Osmolytes ◽  
Anion Channels ◽  
Critical Signal ◽  
Current Activation

The majority of mammalian cells demonstrate regulatory volume decrease (RVD) following swelling caused by hyposmotic exposure. A critical signal initiating RVD is activation of nucleotide receptors by ATP. Elevated extracellular ATP in response to cytotoxic cell swelling during pathological conditions also may initiate loss of taurine and other intracellular osmolytes via anion channels. This study characterizes neuronal ATP-activated anion current and explores its role in net loss of amino acid osmolytes. To isolate anion currents, we used CsCl as the major electrolyte in patch electrode and bath solutions and blocked residual cation currents with NiCl2 and tetraethylammonium. Anion currents were activated by extracellular ATP with a Km of 70 μM and increased over fourfold during several minutes of ATP exposure, reaching a maximum after 9.0 min (SD 4.2). The currents were blocked by inhibitors of nucleotide receptors and volume-regulated anion channels (VRAC). Currents showed outward rectification and inactivation at highly depolarizing membrane potentials, characteristics of swelling-activated anion currents. P2X agonists failed to activate the anion current, and an inhibitor of P2X receptors did not block the effect of ATP. Furthermore, current activation was observed with extracellular ADP and 2-(methylthio)adenosine 5′-diphosphate, a P2Y1 receptor-specific agonist. Much less current activation was observed with extracellular UTP, suggesting the response is mediated predominantly by P2Y1 receptors. ATP caused a dose-dependent loss of taurine and alanine that could be blocked by inhibitors of VRAC. ATP did not inhibit the taurine uptake transporter. Thus extracellular ATP triggers a loss of intracellular organic osmolytes via activation of anion channels. This mechanism may facilitate neuronal volume homeostasis during cytotoxic edema.

Missense mutations in Na+:HCO3− cotransporter NBC1 show abnormal trafficking in polarized kidney cells: a basis of proximal renal tubular acidosis

2005 ◽  
Vol 289 (1) ◽  
pp. F61-F71 ◽  
Author(s):  
Hong C. Li ◽  
Peter Szigligeti ◽  
Roger T. Worrell ◽  
Jeffrey B. Matthews ◽  
Laura Conforti ◽  
...  
Keyword(s):  
Renal Tubular Acidosis ◽  
Membrane Fraction ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Kidney Cells ◽  
Missense Mutations ◽  
Wild Type ◽  
Membrane Expression ◽  
Potential Recording ◽  
Renal Tubular

The kidney Na+:HCO3− 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 NH2 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.

scholarly journals Characterization of Na+/H+ exchanger NHE8 in cultured renal epithelial cells

2007 ◽  
Vol 293 (3) ◽  
pp. F761-F766 ◽  
Author(s):  
Jianning Zhang ◽  
Ion Alexandru Bobulescu ◽  
Sunita Goyal ◽  
Peter S. Aronson ◽  
Michel G. Baum ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Functional Characterization ◽  
Protein Levels ◽  
Primary Amino ◽  
Interfering Rna

NHE8 is expressed in the apical membrane of the proximal tubule and is predicted to be a Na+/H+ exchanger on the basis of its primary amino acid sequence. Functional characterization of native NHE8 in mammalian cells has not been possible to date. We screened a number of polarized renal cell lines for the plasma membrane Na+/H+ exchangers (NHE1, 2, 3, 4, and 8) and found only NHE1 and NHE8 transcripts in NRK cells by RT-PCR. NHE8 protein is expressed in the apical membrane of NRK cells as demonstrated by immunoblots, confocal fluorescent immunocytochemistry, and immunoelectron microscopy. NHE1, on the other hand, is expressed primarily in the basolateral membrane. Bilateral perfusion of NRK cells grown on permeable supports shows Na+/H+ exchange activity on both the apical and basolateral membranes. NHE8-specific small interfering RNA knocks down NHE8 protein expression but does not affect NHE1 protein levels. Knockdown of NHE8 protein is accompanied by a commensurate reduction in apical NHE activity, without altered basolateral NHE activity. Conversely, transfection of NHE1-specific small interfering RNA knocks down NHE1 protein expression without affecting NHE8 protein levels and reduces basolateral NHE activity without affecting apical NHE activity. NHE8 is the only apical membrane Na+/H+ exchanger in NRK cells. NHE8 activity is Na+ dependent, displaying a cooperative sigmoidal relationship, and is highly sensitive to 5-( N-ethyl- n-isopropyl)-amiloride (EIPA). NRK cells provide a useful system where NHE8 can be studied in its native environment.

Expression of caveolin-1 and polarized formation of invaginated caveolae in Caco-2 and MDCK II cells

1998 ◽  
Vol 111 (6) ◽  
pp. 825-832 ◽  
Author(s):  
U. Vogel ◽  
K. Sandvig ◽  
B. van Deurs
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Epithelial Cell ◽  
Basolateral Membrane ◽  
Apical Surface ◽  
Wild Type ◽  
Caveolin 1 ◽  
Endogenous Level ◽  
Epithelial Cell Lines ◽  
Different Levels

We have studied caveolin-1 expression and the frequency and distribution of typical invaginated caveolae as they are identified by electron microscopy in the polarized epithelial cell lines MDCK II and Caco-2. In wild-type MDCK II cells caveolin expression is high and more than 400 caveolae/mm filter were observed at the basolateral membrane. No caveolae were found at the apical surface. By contrast, wild-type Caco-2 cells do not express caveolin-1 and have extremely few, if any caveolae. Caco-2 cells were stably transfected with the gene for caveolin-1 in order to investigate if the formation of caveolae is polarized also in these cells. We have isolated Caco-2 clones expressing different levels of caveolin-1, where the level of expression varies from 10–100% of the endogenous level in MDCK II cells. Caveolin-1 expression in Caco-2 cells gives rise to a marked immunofluorescense labeling mainly at the lateral plasma membrane. By electron microscopy an increase from less than 4 caveolae/mm filter in wild-type Caco-2 cells to 21–76 caveolae/mm filter in Caco-2 clones transfected with caveolin-1 was revealed and these caveolae were exclusively localized to the basolateral membrane. Thus expression of heterologous caveolin-1 in Caco-2 cells leads to polarized formation of caveolae, but there is a lack of correlation between the amount of caveolin expressed in the cells and the number of caveolae, suggesting that factors in addition to caveolin are required for generation of caveolae.

scholarly journals Identification and Characterization of a Putative Human Iodide Transporter Located at the Apical Membrane of Thyrocytes

2002 ◽  
Vol 87 (7) ◽  
pp. 3500-3503 ◽  
Author(s):  
Anne-Marie Rodriguez ◽  
Barbara Perron ◽  
Ludovic Lacroix ◽  
Bernard Caillou ◽  
Gérard Leblanc ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Human Kidney ◽  
Protein Product ◽  
Sodium Iodide Symporter ◽  
Pcr Cloning ◽  
Thyroid Cells ◽  
Iodide Transport ◽  
Acid Protein

Iodide transport by thyrocytes is a two step process involving transporters located either in the basal or in the apical membranes of the cell. The sodium iodide symporter (NIS) is localized in the basolateral membrane facing the bloodstream and mediates iodide accumulation into thyrocytes. Pendrin has been proposed as an apical transporter. In order to identify new iodide transporters, we developed a PCR cloning strategy based on NIS sequence homologies. From a human kidney cDNA library, we characterized a gene, located on chromosome 12q23, that encodes a 610 amino acid protein sharing 46% identity (70% similarity) with the human NIS. Functional analysis of the protein expressed in mammalian cells indicates that it catalyzes a passive iodide transport. The protein product was immunohistochemically localized at the apical pole of the thyroid cells facing the colloid lumen. These results suggest that this new identified protein mediates iodide transport from the thyrocyte into the colloid lumen through the apical membrane. It was designated hAIT for human Apical Iodide Transporter.

Ontogeny of rabbit renal cortical NHE3 and NHE1: effect of glucocorticoids

1995 ◽  
Vol 268 (5) ◽  
pp. F815-F820 ◽  
Author(s):  
M. Baum ◽  
D. Biemesderfer ◽  
D. Gentry ◽  
P. S. Aronson
Keyword(s):  
Proximal Tubule ◽  
Mammalian Cells ◽  
Renal Cortex ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Subcutaneous Administration ◽  
Mrna Abundance ◽  
Protein Abundance ◽  
Twofold Increase ◽  
Threefold Increase

The neonatal proximal tubule has a lower rate of bicarbonate absorption and Na+/H+ antiporter activity than the proximal tubule of adult animals. Two isoforms of the Na+/H+ antiporter have been localized to the proximal tubule. NHE3 is located on the apical membrane, whereas NHE1, the isoform found on most mammalian cells, is present on the basolateral membrane. The Na+/H+ antiporter isoforms that increase with renal maturation are unknown. The purpose of the present study was to examine the maturation of rabbit renal cortical NHE3 and NHE1 mRNA and protein abundance and to determine whether the rate of maturation of these isoforms was affected by glucocorticoids. Renal cortex from neonatal rabbits (1 wk) had approximately one-fourth the NHE3 mRNA and protein abundance as that from adult animals. Renal cortical NHE1 mRNA and protein abundance did not change significantly during maturation. Glucocorticoids have been shown to accelerate the maturation of neonatal bicarbonate absorption and apical membrane Na+/H+ antiporter activity. Daily subcutaneous administration of dexamethasone starting at 4 days of age (10 micrograms/100 g body wt) for 3 days and 2 h before being killed resulted in a twofold increase in NHE3 mRNA abundance and a threefold increase in NHE3 protein abundance. NHE1 mRNA and protein abundance were unaffected. These data show that there is selective maturation of NHE3 during renal cortical development, which can be accelerated by administration of glucocorticoids.

scholarly journals Mistargeting of a truncated Na-K-2Cl cotransporter in epithelial cells

2018 ◽  
Vol 315 (2) ◽  
pp. C258-C276 ◽  
Author(s):  
Rainelli Koumangoye ◽  
Salma Omer ◽  
Eric Delpire
Keyword(s):  
Epithelial Cells ◽  
De Novo ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Dominant Negative ◽  
De Novo Mutation ◽  
Wild Type ◽  
Saliva Secretion

We recently reported the case of a young patient with multisystem failure carrying a de novo mutation in SLC12A2, the gene encoding the Na-K-2Cl cotransporter-1 (NKCC1). Heterologous expression studies in nonepithelial cells failed to demonstrate dominant-negative effects. In this study, we examined expression of the mutant cotransporter in epithelial cells. Using Madin-Darby canine kidney (MDCK) cells grown on glass coverslips, permeabilized support, and Matrigel, we show that the fluorescently tagged mutant cotransporter is expressed in cytoplasm and at the apical membrane and affects epithelium integrity. Expression of the mutant transporter at the apical membrane also results in the mislocalization of some of the wild-type transporter to the apical membrane. This mistargeting is specific to NKCC1 as the Na+-K+-ATPase remains localized on the basolateral membrane. To assess transporter localization in vivo, we created a mouse model using CRISPR/cas9 that reproduces the 11 bp deletion in exon 22 of Slc12a2. Although the mice do not display an overt phenotype, we show that the colon and salivary gland expresses wild-type NKCC1 abundantly at the apical pole, confirming the data obtained in cultured epithelial cells. Enough cotransporter must remain, however, on the basolateral membrane to participate in saliva secretion, as no significant decrease in saliva production was observed in the mutant mice.

Sign in / Sign up

Export Citation Format

Share Document