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Luis Reuss - One of the best experts on this subject based on the ideXlab platform.
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Regulation of cAMP-Activated Apical Membrane Chloride Conductance in
2013Co-Authors: Gallbladder Epithelium, J Copello, T A Heming, Luis ReussAbstract:A B S T R ACT Regulation of the cAMP-activated Apical Membrane C1- conductance (G~l) in Necturus gallbladder (NGB) epithelial cells was investigated with intracellular-microelectrode techniques. G~. l was increased by exposure to 8-Br-cAMP, theophylline or forskolin. Neither 8-Br-cGMP nor elevation of intracellular [Ca 2+] using ionomycin had effects on G~. 1 or interfered with activation of G~. t by forskolin. N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide (HS), an inhibitor of cAMPdependent protein kinase (PKA), slowed but did not prevent the G~I response to 8-Br-cAMP. Phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), stimulated G~. l but had no effects on intracellular [cAMP]. G~I was unaffected by 4c~-phorbol, a PMA analog which does not activate PKC. Okadaic acid (OA), an inhibitor of protein phosphatases (PP) types 1 and 2A, slowed the activation of G~. l by 8-Br-cAMP, hastened the return of G~E to basal values following removal of 8-Br-cAMP, and significantly reduced the elevation in intracellular [cAMP] produced by forskolin. OA had no effects on the G~. l changes elicited by theophylline. We conclude that: (a) NGB G~. 1 can be activated by PKA-mediated phosphorylation of Apical Membrane C1- channels or a regulatory protein, (b) G~. l can also be activated via PKC, by a cAMP-independent mechanism, (c) OA-sensitive PP are not required for inactivation of G~.I; OA appears to stimulate phosphodiesterase, which lowers intracellular [cAMP] and affects G~I activation, and (d) the Apical Membrane of NGB epithelium lacks a Ca2+-activated C1- conductance
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Cl-/HCO 3 Exchange at the Apical Membrane of Necturus Gallbladder
2013Co-Authors: Luis Reuss, James LAbstract:AB STRACT The hypothesis of Cl-/HCO s exchange across the Apical Membrane of the epithelial cells of Necturus gallbladder was tested by means of measurements of extracellular pH (pH.), intracellular pH (pHi), and Cl- activity (aCli) with ion-sensitive microelectrodes. Luminal pH changes were measured after stopping mucosal superfusion with a solution of low buffering power. Under control conditions, the luminal solution acidifies when superfusion is stopped. Shortly after addition of the Na'/H ' exchange inhibitor amiloride (10-3 M) to the superfusate, alkalinization was observed. During prolonged (10 min) exposure to amiloride, no significant pH. change occurred. Shortly after amiloride removal, luminal acidification increased, returning to control rates in 10 min. The absence of Na ' in the superfusate (TMA ' substitution) caused changes in the same direction, but they were larger than those observed with amiloride. Removal of CI- (cyclamate or sulfate substitution) caused a shortlived increase in the rate of luminal acidification, followed by a return to control values (10-30 min). Upon re-exposure to Cl-, there was a transient reduction of luminal acidification. The initial increase in acidification produced by CIremoval was partially inhibited by SITS (0.5 mM). The pH i increased rapidly and reversibly when the Cl- concentration o £ the mucosal bathing solution was reduced to nominally 0 mM. The pH; changes were larger in 10 mM HC03-Ringer's than in 1 mM HEPES-Ringer's, which suggests that HCO-3 is transported in exchange for CI-. In both HEPES- and HC03-Ringer's, SITS inhibited the pHi changes. Finally, intracellular acidification or alkalinization (partial replacement of NaCl with sodium propionate or ammonium chloride, respectively) caused a reversible decrease or increase of aCl i. These results support the hypothesis of Apical Membrane CI-/HC03 exchange, which can be dissociated from Na''/H+ exchange and operates under control conditions. The coexistence at the Apical Membrane of Na'/H ' and CI-/HCO3 antiports suggests that NaCl entry can occur through these transporters
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Cyclic AMP Inhibits Na'/H' Exchange at the Apical Membrane
2013Co-Authors: Of Necturus Gallbladder Epithelium, Luis Reuss, Karl-uwe PetersenAbstract:ABSTRACT The effects of elevating intracellular cAMP levels on Na ' transport across the Apical Membrane of Necturus gallbladder epithelium were studied by intracellular and extracellular microelectrode techniques. Intracellular cAMP was raised by serosal addition of the phosphodiesterase inhibitor theophylline (3 mM) or mucosal addition of either 8-Br-cAMP (1 mM) or the adenylate cyclase activator forskolin (10 pM). During elevation of intracellular cAMP, intracellular Na ' activity (aNai) and intracellular pH (pHi) decreased significantly. In addition, acidification ofthe mucosal solution, which contained either 100 or 10 mM Na', was inhibited by ^-50 %. The inhibition was independent of the presence of Cl- in the bathing media. The rates of change of aNa i upon rapid alterations of mucosal [Na'] from 100 to 10 mM and from 10 to 100 mM were both decreased, and the rate ofpHi recovery upon acid loading was also reduced by elevated cAMP levels. Inhibition was ^-50 % for all of these processes. These results indicate that cAMP inhibits Apical Membrane Na'/H' exchange. The results of measurements of pH i recovery at 10 and 100 mM mucosal [Na'] and a kinetic analysis of recovery as a function of pHi suggest that the main or sole mechanism of the inhibitory effect of cAMP is a reduction in the maximal rate of acid extrusion. In conjunction with the increase in Apical Membrane electrodiffusional Cl- permeability, produced by cAMP, which causes a decrease in net Cl- entry (Petersen, K.U., and L. Reuss, 1983,J: Gen. Physiol., 81:705), inhibition of Na'/H ' exchange contributes to the reduction of fluid absorption elicited by this agent. Similar mechanisms may account for the effects of cAMP in other epithelia with similar transport properties. It is also possible that inhibition of Na'/H ' exchange by cAMP plays a role in the regulation of pH i in other cell types
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Independence of Apical Membrane Na ' and C1- Entry in
2013Co-Authors: Necturus Gallbladder Epithelium, Luis ReussAbstract:ABSTRACT Transepithelial fluid transport(/,) and intracellular Na ' and CIactivities (aNai, aCl;) were measured in isolated Necturus gallbladders to establish the contribution of different proposed Apical Membrane entry mechanisms to transepithelial salt transport. In 10 mM HC0 3-Ringer's, Jv was 13.5 ± 1.1 ttl-cm-'-h-,'and was significantly reduced by a low bicarbonate medium and by addition of amiloride (10-3 M) or SITS (0.5 X 10-3 M) to the mucosal bathing solution. Bumetanide (10-5 M) was ineffective. Bilateral Na ' removal abolished Jv. The hypothesis of NaCl cotransport was rejected on the basis ofthe following results, all obtained during mucosal bathing solution changes: (a) during Na' removal, aNai fell 4.3 times faster than aCl i; (b) during CI- removal, aCl; fell 7.5 times faster than aNai; (e) amiloride (10-3 M) reduced aNai at a rate of 2.4 ± 0.3 mM/min, whereas aCl i was not changed; (d) bumetanide (10-5 M) had no significant effects on Jv or aCli. The hypothesis of Na-K-CI cotransport was rejected for the same reasons; in addition, K+ removal from the mucosal bathing solution (with concomitant Bat ' addition) did not alter aNai or aCl i. The averag
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Cyclic AMP Inhibits C1-/HCO Exchange at the Apical Membrane of
2013Co-Authors: Necturus Gallbladder Epithelium, Luis ReussAbstract:ABSTRACT Intracellular microelectrode techniques were employed to study the effect of cyclic AMP on Apical Membrane Ci-/HCOi exchange and electrodiffusive HCOi transport in Necturus gallbladder epithelium, lntracellular cAMP levels were raised by addition of either the phosphodiesterase inhibitor theophyiline (3 x 10-s M) or the adenylate cyclase activator forskolin (10-5 M) to the serosal bathing solution. Measurements of pH in a poorly buffered control mucosal solution upon stopping superfusion show acidification, owing to secretion of both H + and HCOL When the same experiment is performed after addition of amiloride or removal of Na + from the mucosal bathing medium, alkalinization is observed since H + transport is either inhibited or reversed, whereas HCOi secretion persists. The changes in pH in both amiloride or Na-free medium were significantly decreased in theophylline-treated tissues. Theophylline had no effect on the initial rates of fall of intracellular CIactivity (aCl 0 upon reducing mucosal solution [CI-] to either 10 or 0 mM, although Membrane voltage and resistance measurements were consistent wit
Philippe Poujeol - One of the best experts on this subject based on the ideXlab platform.
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chloride channels in Apical Membrane of primary cultures of rabbit distal bright convoluted tubule
American Journal of Physiology-renal Physiology, 1994Co-Authors: V Poncet, Michel Bidet, Michel Tauc, Philippe PoujeolAbstract:Using the patch clamp technique on the Apical Membrane of primary cultures of rabbit distal bright convoluted tubule cells (DCTb), two types of Cl- channel were identified. A small channel of 9 pS ...
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a calcium permeable channel in the Apical Membrane of primary cultures of the rabbit distal bright convoluted tubule
Pflügers Archiv: European Journal of Physiology, 1992Co-Authors: V Poncet, Jean Merot, Philippe PoujeolAbstract:Calcium is actively reabsorbed in the distal nephron segments and recent studies have demonstrated the presence of Ca2+ channels in these epithelial cells, which could be involved in transepithelial transport. To test this possibility, single-channel currents were recorded by the patch-clamp technique in the Apical Membrane of primary cultures of the rabbit distal bright convoluted tubule cells (DCTb). In the cell-attached mode with 100 mmol/l BaCl2 in the pipette and 145 mmol/l NaCl in the bath, inward negative currents, consistent with Ba2+ currents, were recorded. In these conditions, the single-channel conductance was 15 pS. In excised insideout patches, the single-channel conductance was 13 pS and the current reversal potential of +60 mV was close to the Nernst equilibrium potential for Ba2+ (>+58 mV). Similar experiments conducted with Ca2+ as the main charge carrier showed that this ion was less permeant through the channel than Ba2+ (PBa/PCa≈1.4). We also showed that the Ca2+-channel blocker, lanthanum (1 μmol/l La3+), added on the cytosolic side of the Membrane, reversibly blocked the channel activity. On the other hand, verapamil (0.1 mmol/l) and nifedipine (10 μmol/l), perfused on the cytosolic side of the Membrane, abolished the channel activity but this effect was not reversible. Another type of channel was also identified in the Apical Membrane of cultured DCTb cells. Ion-substitution experiments showed that this 21-pS conductance channel did not discriminate between Na+ and K+ and did not conduct Ba2+. 4′-Methyl-2-diphenylamine-carboxylic acid (10 μmol/l), added on the cytosolic side of the Membrane, reversibly blocked this cationic channel whereas La3+ (10(μmol/l) had no effect on its activity. We conclude that (a) La3+-sensitive Ca2+ channels are present in the Apical Membrane of DCTb cells and may represent the Apical uptake pathway in the transepithelial calcium transport. (b) These Ca2+ channels are distinct from the non-selective cationic channels that do not show Ba2+ permeability.
Levan Mchedlishvili - One of the best experts on this subject based on the ideXlab platform.
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signaling dependent control of Apical Membrane size and self renewal in rosette stage human neuroepithelial stem cells
Stem cell reports, 2018Co-Authors: Janphilip Medelnik, Kathleen Roensch, Satoshi Okawa, Antonio Del Sol, Osvaldo Chara, Levan MchedlishviliAbstract:In the developing nervous system, neural stem cells are polarized and maintain an Apical domain facing a central lumen. The presence of Apical Membrane is thought to have a profound influence on maintaining the stem cell state. With the onset of neurogenesis, cells lose their polarization, and the concomitant loss of the Apical domain coincides with a loss of the stem cell identity. Little is known about the molecular signals controlling Apical Membrane size. Here, we use two neuroepithelial cell systems, one derived from regenerating axolotl spinal cord and the other from human embryonic stem cells, to identify a molecular signaling pathway initiated by lysophosphatidic acid that controls Apical Membrane size and consequently controls and maintains epithelial organization and lumen size in neuroepithelial rosettes. This Apical domain size increase occurs independently of effects on proliferation and involves a serum response factor-dependent transcriptional induction of junctional and Apical Membrane components.
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signaling dependent control of Apical Membrane size and self renewal in rosette stage human neuroepithelial stem cells
bioRxiv, 2017Co-Authors: Janphilip Medelnik, Kathleen Roensch, Satoshi Okawa, Antonio Del Sol, Osvaldo Chara, Levan MchedlishviliAbstract:Summary In the early developing nervous system, self-renewing neural stem cells are polarized and maintain an Apical domain facing a central lumen. The presence of Apical Membrane is thought to have a profound influence on maintaining the stem cell state. With the onset of neurogenesis cells lose their polarization and the concomitant loss of the Apical domain coincides with a loss of the stem cell identity. Very little is known about the molecular signals controlling Apical Membrane size. Here we use two neuroepithelial cell systems, one derived from regenerating axolotl spinal cord and the other from human ESCs to identify a conserved molecular signalling pathway initiated by lysophosphatidic acid (LPA) that controls Apical Membrane size and consequently controls and maintains epithelial organization and lumen size in neuroepithelial rosettes. This Apical domain size increase occurs independently of effects on proliferation and involves a SRF-dependent transcriptional induction of junctional and Apical Membrane components.
V Poncet - One of the best experts on this subject based on the ideXlab platform.
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chloride channels in Apical Membrane of primary cultures of rabbit distal bright convoluted tubule
American Journal of Physiology-renal Physiology, 1994Co-Authors: V Poncet, Michel Bidet, Michel Tauc, Philippe PoujeolAbstract:Using the patch clamp technique on the Apical Membrane of primary cultures of rabbit distal bright convoluted tubule cells (DCTb), two types of Cl- channel were identified. A small channel of 9 pS ...
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a calcium permeable channel in the Apical Membrane of primary cultures of the rabbit distal bright convoluted tubule
Pflügers Archiv: European Journal of Physiology, 1992Co-Authors: V Poncet, Jean Merot, Philippe PoujeolAbstract:Calcium is actively reabsorbed in the distal nephron segments and recent studies have demonstrated the presence of Ca2+ channels in these epithelial cells, which could be involved in transepithelial transport. To test this possibility, single-channel currents were recorded by the patch-clamp technique in the Apical Membrane of primary cultures of the rabbit distal bright convoluted tubule cells (DCTb). In the cell-attached mode with 100 mmol/l BaCl2 in the pipette and 145 mmol/l NaCl in the bath, inward negative currents, consistent with Ba2+ currents, were recorded. In these conditions, the single-channel conductance was 15 pS. In excised insideout patches, the single-channel conductance was 13 pS and the current reversal potential of +60 mV was close to the Nernst equilibrium potential for Ba2+ (>+58 mV). Similar experiments conducted with Ca2+ as the main charge carrier showed that this ion was less permeant through the channel than Ba2+ (PBa/PCa≈1.4). We also showed that the Ca2+-channel blocker, lanthanum (1 μmol/l La3+), added on the cytosolic side of the Membrane, reversibly blocked the channel activity. On the other hand, verapamil (0.1 mmol/l) and nifedipine (10 μmol/l), perfused on the cytosolic side of the Membrane, abolished the channel activity but this effect was not reversible. Another type of channel was also identified in the Apical Membrane of cultured DCTb cells. Ion-substitution experiments showed that this 21-pS conductance channel did not discriminate between Na+ and K+ and did not conduct Ba2+. 4′-Methyl-2-diphenylamine-carboxylic acid (10 μmol/l), added on the cytosolic side of the Membrane, reversibly blocked this cationic channel whereas La3+ (10(μmol/l) had no effect on its activity. We conclude that (a) La3+-sensitive Ca2+ channels are present in the Apical Membrane of DCTb cells and may represent the Apical uptake pathway in the transepithelial calcium transport. (b) These Ca2+ channels are distinct from the non-selective cationic channels that do not show Ba2+ permeability.
Janphilip Medelnik - One of the best experts on this subject based on the ideXlab platform.
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signaling dependent control of Apical Membrane size and self renewal in rosette stage human neuroepithelial stem cells
Stem cell reports, 2018Co-Authors: Janphilip Medelnik, Kathleen Roensch, Satoshi Okawa, Antonio Del Sol, Osvaldo Chara, Levan MchedlishviliAbstract:In the developing nervous system, neural stem cells are polarized and maintain an Apical domain facing a central lumen. The presence of Apical Membrane is thought to have a profound influence on maintaining the stem cell state. With the onset of neurogenesis, cells lose their polarization, and the concomitant loss of the Apical domain coincides with a loss of the stem cell identity. Little is known about the molecular signals controlling Apical Membrane size. Here, we use two neuroepithelial cell systems, one derived from regenerating axolotl spinal cord and the other from human embryonic stem cells, to identify a molecular signaling pathway initiated by lysophosphatidic acid that controls Apical Membrane size and consequently controls and maintains epithelial organization and lumen size in neuroepithelial rosettes. This Apical domain size increase occurs independently of effects on proliferation and involves a serum response factor-dependent transcriptional induction of junctional and Apical Membrane components.
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signaling dependent control of Apical Membrane size and self renewal in rosette stage human neuroepithelial stem cells
bioRxiv, 2017Co-Authors: Janphilip Medelnik, Kathleen Roensch, Satoshi Okawa, Antonio Del Sol, Osvaldo Chara, Levan MchedlishviliAbstract:Summary In the early developing nervous system, self-renewing neural stem cells are polarized and maintain an Apical domain facing a central lumen. The presence of Apical Membrane is thought to have a profound influence on maintaining the stem cell state. With the onset of neurogenesis cells lose their polarization and the concomitant loss of the Apical domain coincides with a loss of the stem cell identity. Very little is known about the molecular signals controlling Apical Membrane size. Here we use two neuroepithelial cell systems, one derived from regenerating axolotl spinal cord and the other from human ESCs to identify a conserved molecular signalling pathway initiated by lysophosphatidic acid (LPA) that controls Apical Membrane size and consequently controls and maintains epithelial organization and lumen size in neuroepithelial rosettes. This Apical domain size increase occurs independently of effects on proliferation and involves a SRF-dependent transcriptional induction of junctional and Apical Membrane components.