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Biomedical subjects

E Brochiero

Publications and source records attributed to E Brochiero.

7 recordsLinked to original sources
PubMed1997

Characterisation of Ca2+ membrane permeability of renal A6 cells upon different osmotic conditions.

The nature of the calcium-transporting mechanisms involved in A6 cell calcium homeostasis under iso- and hypo-osmotic conditions was investigated using fura-2 (AM) as a cell calcium indicator. Under steady-state conditions, intracellular calcium (Ca2+i) was increased by Bay K8644 or by gramicidin, an ionophore which depolarises A6 cell membranes. The Ca2+i increase following calcium addition (to calcium-depleted cells) or membrane depolarisation was blocked by nifedipine but not by verapamil or omega-conotoxin, indicating that the membrane calcium permeability may be mediated by voltage-dependent and dihydropyridine-sensitive calcium channels. Ca2+i could also be increased by a hypo-osmotic shock having a linear relationship with the osmolarity change. This osmotically induced Ca2+i increase had an extracellular origin since it was absent when cells were suspended in a calcium-free medium and it was not affected by thapsigargin or TMB-8 application. In addition, it was inhibited by the calcium channel inhibitor, nifedipine. Furthermore, under hypo-osmotic conditions, an additional Ca2+i increase, sensitive to nifedipine, was measured when cells were depolarised by gramicidin or K-gluconate addition. It is proposed that the hypo-osmotically induced cell calcium increase implies the activation of voltage-dependent and nifedipine-sensitive calcium channels, presenting the same pharmacological characteristics as those involved in cell calcium homeostasis under iso-osmotic conditions. The initial Ca2+i increase was transient and stabilised to a value nevertheless higher than the iso-osmotic level; this secondary and incomplete regulatory phase did not occur in the presence of thapsigargin or TMB-8, thus providing evidence of intracellular calcium storage.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Apical membrane sodium and chloride entry during osmotic swelling of renal (A6) epithelial cells.

To assess the role of chloride in cell volume and sodium transport regulation, we measured cell height changes (CH), transepithelial chloride and sodium fluxes, and intracellular chloride content during challenge with hyposmotic solutions under open circuit (OC) conditions. CH maximally increased following hyposmotic challenge within approximately 5 minutes. The change in CH was smaller under short circuit (SC) conditions or following replacement of chloride in the mucosal solution by gluconate or cyclamate (Cl(-)-freem). When corrected for the osmotically inactive cell volume (30 +/- 2%), delta CH for controls (OC) were greater than predicted for an ideal osmometer. In contrast, delta CH for Cl(-)-freem or SC conditions were similar to that predicted for an ideal osmometer. Na+ and Cl- mucosa-to-serosa fluxes increased following hyposmotic challenge. Chloride fluxes increased maximally within 5 min, then decreased. In contrast, the Na+ flux increased slowly and reached a steady state after approximately 25 min. Under isosmotic conditions, exposure to Cl(-)-freem solutions led to decreases in the transepithelial conductance, Na+ flux, and CH. Chloride permeabilities in the apical and basolateral membranes were detected using the fluorescent intracellular chloride indicator MQAE. The results indicate that during osmotic swelling, the entry of both sodium and chloride is increased. The time courses of these increases differ, suggesting distinct mechanisms for the osmotic regulation of these apical membrane transport processes.

Animals↗
PubMed1995

Na/Ca exchange in the basolateral membrane of the A6 cell monolayer: role in Cai homeostasis.

The presence of a Na/Ca exchanger in A6 cells was investigated by measuring intracellular calcium (Cai) fluctuations and the 45Ca fluxes through the basolateral membranes (blm) of the cell monolayer. Removal of Na+ from the medium produced a transient increase in Cai followed by a regulatory phase returning Cai to control levels in 3-4 min, this phase being greatly accelerated (< 60 s) by NaCl addition (apparent Km of approximately 5 mM Na+). The Cai increase was only found with the Na(+)-free medium on the basolateral side of the cell monolayer. A twofold increase in the 45Ca influx was observed under these conditions. In Ca(2+)- depleted cells, the initial Cai increase after Ca2+ addition to the medium was greater when the putative Na/Ca exchanger was not functioning (i.e. in a Na(+)-free medium). 45Ca effluxes through the blm of the monolayer were greatly and transiently increased by a Na(+)-free medium on the serosal side and blocked by orthovanadate (1 mM). The Cai increased induced by a hypo-osmotic shock was greater in cells bathed in a Na(+)-medium, conditions expected to block the activity of the Na/Ca exchanger. These findings support the hypothesis that a Na/Ca exchanger is present on the blm of A6 cells and affirm its role in Cai homeostasis in steady-state conditions and following osmotic shock. In addition, a Ca2+ pump also located on the blm and Ca2+ stores sensitive to inositol 1,4,5-trisphosphate were found to be implicated in Cai homeostasis.

Animals↗
PubMed1995

Basolateral membrane chloride permeability of A6 cells: implication in cell volume regulation.

The permeability to Cl- of the basolateral membrane (blm) was investigated in renal (A6) epithelial cells, assessing their role in transepithelial ion transport under steady-state conditions (isoosmotic) and following a hypoosmotic shock (i.e. in a regulatory volume decrease, RVD). Three different complementary studies were made by measuring: (1) the Cl- transport rates (delta F/Fo s-1 (x10(-3))), where F is the fluorescence of N-(6-methoxyquinoyl) acetoethyl ester, MQAE, and Fo the maximal fluorescence (x10(-3)) of both membranes by following the intracellular Cl- activities (ai Cl-, measured with MQAE) after extracellular Cl- substitution (2) the blm 86Rb and 36Cl uptakes and (3) the cellular potential and Cl- current using the whole-cell patch-clamp technique to differentiate between the different Cl- transport mechanisms. The permeability of the blm to Cl- was found to be much greater than that of the apical membranes under resting conditions: aiCl- changes were 5.3 +/- 0.7 mM and 25.5 +/- 1.05 mM (n = 79) when Cl- was substituted by NO3(-) in the media bathing apical and basolateral membranes. The Cl- transport rate of the blm was blocked by bumetanide (100 microM) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 50 microM) but not by N-phenylanthranilic acid (DPC, 100 microM). 86Rb and 36Cl uptake experiments confirmed the presence of a bumetanide- and a NPPB-sensitive Cl- pathway, the latter being approximately three times more important than the former (Na/K/2Cl cotransporter). Appli-cation of a hypoosmotic medium to the serosal side of the cell increased delta F/Fo s-1 (x10(-3)) after extracellular Cl- substitution (1.03 +/- 0.10 and 2.45 +/- 0.17 arbitrary fluorescent units s-1 for isoosmotic and hypoosmotic conditions respectively, n = 11); this delta F/Fo s-1 (x10(-3)) increase was totally blocked by serosal NPPB application; on the other hand, cotransporter activity was decreased by the hypoosmotic shock. Cellular Ca2+ depletion had no effect on delta F/Fo s-1 (x10(-3)) under isoosmotic conditions, but blocked the delta F/Fo s-1 (x10(-3)) increase induced by a hypoosmotic stress. Under isotonic conditions the measured cellular potential at rest was -37.2 +/- 4.0 mV but reached a maximal and transient depolarization of -25.1 +/- 3.7 mV (n = 9) under hypoosmotic conditions. The cellular current at a patch-clamping cellular potential of -85 mV (close to the Nernst equilibrium potential for K+) was blocked by NPPB and transiently increased by hypoosmotic shock (&ap;50% maximum increase). This study demonstrates that the major component of Cl- transport through the blm of the A6 monolayer is a conductive pathway (NPPB-sensitive Cl- channels) and not a Na/K/2Cl cotransporter. These channels could play a role in transepithelial Cl- absorption and cell volume regulation. The increase in the blm Cl- conductance, inducing a depolarization of these membranes, is proposed as one of the early events responsible for the stimulation of the 86Rb efflux involved in cell volume regulation.

Animals↗
PubMed1994

Basolateral potassium membrane permeability of A6 cells and cell volume regulation.

The K+ permeabilities (86Rb(K) transport) of the basolateral membranes (JbK) of a renal cell line (A6) were compared under isosmotic and hypo-osmotic conditions (serosal side) to identify the various components involved in cell volume regulation. Changing the serosal solution to a hypo-osmotic one (165 mOsm) induced a fast transient increase in Cai (max < 1 min) and cell swelling (max at 3-5 min) followed by a regulatory volume decrease (5-30 min) and rise in the SCC (stabilization at 30 min). In isosmotic conditions (247 mOsm), the 86Rb(K) transport and the SCC were partially blocked by Ba2+, quinidine, TEA and glibenclamide, the latter being the least effective. Changing the osmolarity from isosmotic to hypo-osmotic resulted in an immediate (within the first 3-6 min) stimulation of the 86Rb(K) transport followed by a progressive decline to a stable value higher than that found in isosmotic conditions. A serosal Ca(2+)-free media or quinidine addition did not affect the initial osmotic stimulation of JbK but prevented its "secondary regulation", whereas TEA, glibenclamide and DIDS completely blocked the initial JbK increase. Under hypo-osmotic conditions, the initial JbK increase was enhanced by the presence of 1 mM of barium and delayed with higher concentrations (5 mM). In addition, cell volume regulation was fully blocked by quinidine, DIDS, NPPB and glibenclamide, while partly inhibited by TEA and calcium-free media. We propose that a TEA- and glibenclamide-sensitive but quinidine-insensitive increase in K+ permeability is involved in the very first phase of volume regulation of A6 cells submitted to hypo-osmotic media. In achieving cell volume regulation, it would play a complementary role to the quinidine-sensitive K+ permeability mediated by the observed calcium rise.

Animals↗
PubMed1993

Control of Na+ and H+ transports by exocytosis/endocytosis phenomena in a tight epithelium.

The relationship linking Na+ and H+ transports and exocytosis/endocytosis located in the apical membranes of the frog skin epithelium was investigated under various conditions of ion transport stimulation. The exocytosis process, indicating insertion of intracellular vesicles, which were preloaded with fluorescent FITC-dextran (FD), was measured by following the FD efflux in the apical bathing solution. Na+ transport stimulators such as serosal hypotonic shock (replacement of serosal Ringer solution by half-Ringer or 4/5-Ringer), apical PCMPS (10(-3) M) and amphotericin-B (20 micrograms/ml), were also found to stimulate the exocytotic rates of FD. Acidification of the epithelium by CO2 or post NH4 load, conditions which increase the proton secretion also stimulated the FD release in the apical bathing solution. On the other hand, alkalization of the epithelial cells increased the endocytosis rate. Hypotonic shock, acid load and PCMPS induced an increase in cell calcium which is probably the signal within the cell for exocytosis. In addition, quantitative spectrofluorimetric measurements of F-actin content after rhodamine-phalloidin staining, indicated a decrease in the F-actin content as a result of cell acidosis, hypotonic conditions and amphotericin additions. It is proposed that the insertion/retrieval of intracytoplasmic vesicles containing H+ pumps plays a key role in the regulation of proton secretion in tight epithelia. In addition, it is suggested that cytoskeleton depolymerization of F-actin filaments facilitates H+ pump insertion. A comparable working hypothesis for the control of Na+ transport is proposed.

4-Chloromercuribenzenesulfonate↗