Evaluation by capacitance measurements of antidiuretic hormone induced membrane area changes in toad bladder (original) (raw)
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Effects of ADH on the apical and basolateral membranes of toad urinary bladder epithelial cells
Pflugers Archiv-european Journal of Physiology, 1993
Short-circuited urinary bladders from Bufo marinus were supported on their apical surface by an agar mounting method and impaled with microelectrodes via their basolateral membrane. This arrangement provided stable and long-lasting impalements of epithelial cells and yielded reliable membrane potentials and voltage divider ratios (R a/R b), where R a and R b are apical and basolateral membrane resistances respectively. The membrane potential under short-circuit conditions (V sc) was −51.4±2.2 mV (n=59), while under open-circuit conditions apical membrane potential (V a) and basolateral membrane potential (V b) were −31.0±2.4 and 59.5±2.4 mV, respectively. This yields a “well-shaped” potential profile across the toad urinary bladder, where V a is inversely related to the rate of transport, I sc. Antidiuretic hormone (ADH) produced a hyperpolarisation of V sc and V b but had no significant effect on V a. In addition, R a/R b was significantly increased by ADH (4.6±0.5 to 10.2±3.6). Calculation of individual membrane resistances following the addition of amiloride showed that ADH produced a parallel decrease in R a and R b membrane resistance, with the observed increase in R a/R b being due to a greater percentage decrease in R b than in R a. The ability of ADH to effect parallel changes in apical and basolateral membrane conductance helps to maintain a constant cellular volume despite an increase in transepithelial transport.
Pflügers Archiv, 1990
The role of Ca 2 + in the regulation of antidiuretic hormone(ADH)-induced water permeability of the apical membrane of the toad urinary bladder was examined. The effects of modifying Ca 2 + entry through the apical membrane of toad urinary bladders on the hydroosmotic water flow (~bH2o) and short circuit current (Iso) were measured. In most experiments the bladders were treated with small amounts of Ag + (10-7 mol/1) on the apical side. This treatment was used because previous experiments indicate that it markedly increases alkaliearth cation fluxes through an amiloride-insensitive cation channel in the apical membrane of the urinary bladder. Moreover, when Ca 2+ is the major cation in the apical solution of these Ag+-treated bladders, Isc is mostly due to Ca 2 + entry through the apical membrane. Ag + increased Iso and simultaneously inhibited ~H2o in bladders perfused with Ca 2 + solutions on the apical side. Addition of La 3+ to the apical solution reversed the stimulation of Isc and the inhibition of qsi~o produced by Ag +. When bladders were perfused with Ca 2 +-free solutions on the apical side, addition of Ag + did not inhibit ~bH~ o while the stimulation of cation movements through the amiloride-insensitive cation channel persisted. In bladders perfused with apical Ca 2 + solutions and treated with chlorophenyl thio-cyclic adenosine monophosphate (C1PheS-cAMP) the addition of Ag + did not inhibit ~H~o while it still increased Iso. Finally, addition of Ca 2 § to the apical solution of bladders not treated with Ag § reduced ~bn2o. These results taken together with other findings in the literature suggest' (1) Ca 2 + entry through the Ag+-treated amiloride-insensitive cation channel of the apical membrane inhibits ~H2o; (2) the effects of Ca 2 + entry are at a regulatory site that precedes the interaction of cAMP with the water channels; (3) it is also possible that Ca z + entry through the unmodified amiloride-insensitive cation channel may have some inhibitory effect on (~H20-Offprint requests to.
Annals of the New York Academy of Sciences, 1981
Previous studies have shown that in addition to the action of antidiuretic hormone (ADH) , elicited via cyclic AMP accumulation, the modification of the local cellular environment can also regulate water permeability in frog urinary bladder. For instance, serosal hypertonicity can increase water permeability by interacting with some biochemical step, probably located at a "post cyclic AMP" level and also involved in the ADH mechanism of acti0n.l * M. P. is a career investigator from the Centre National de la Recherche Scientifique (CNRS), France.
Tissue and Cell, 1983
The calcium antagonist verapamil and the calcium ionophore A23187 have been shown to inhibit the hydro-osmotic actions of antidiuretic hormone (ADH) presumably by different mechanisms. Presently, urinary bladders of the frog (Ram pipiem) were examined under SEM following exposure to calcium ionophore A23187, verapamil and ADH in the presence and absence of an osmotic gradient. Cells exposed to ADH show marked changes in surface substructure which is accompanied by an expansion of microridges, cell borders and the appearance of microvilli in the granular cells. The microvilli are pronounced and appear at the junction of microridges. In the presence of an osmotic gradient, ADH induces granular cell swelling and some cells show a blistering effect. Calcium ionophore, in the absence of an osmotic gradient, induced pronounced morphological changes in the granular cells, where the microvilli become prominently visible as 'finger-like' projections. This effect may be due to the action of calcium in promoting elongation of microtubules. Cells exposed to ionophore plus ADH are indistinguishable from ionophore alone. The most apparent effect of verapamil on surface substructure was on the elevation of the mitochondrial-rich cells above the surrounding granular cells. These cells show some degree of separation from the granular cells and are accentuated in tissues exposed to verapamil plus ADH. The present observations suggest that these agents, verapamil and calcium ionophore, have marked effects on cellular morphology. These actions are mediated through alterations in calcium movements and reflect the relative importance of cellular calcium in transepithelial water flow and the actions of antidiuretic hormone.
The single file hypothesis and the water channels induced by antidiuretic hormone
The Journal of Membrane Biology, 1983
Unidirectional and net water movements were determined at minute intervals in frog urinary bladders. The changes in both parameters were followed, during the action of antidiuretic hormone (ADH), at different temperatures and stirring conditions. After correction for external unstirred layer effects, the ratio of the osmotic (P f) and diffusional (Pd) permeability coefficients was remarkably constant, at different times and in different experimental conditions, In the presence of ADH the A Pf/A P d ratio in the mucosal border was probably greater than 9. On the other hand, in nonstimulated preparations the ratio was smaller, and probably not different from 1. These results, together with previous observations indicating that other small molecules (like urea) are excluded from the ADHinduced channel, might indicate that single-file water movement can occur through this structure. Alternatively, the APf/APd ratio could result from a complex geometric arrangement in series with the aqueous pore.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1993
Fluorescence is transferred across the toad urinary bladder when fura-2/AM is added to the mucosal or serosal sides of the epithelium. It was now observed that: (1) Oxytocin (20 nM, serosal) increased fluorescence transfer from the mucosal to the serosal but not from the serosal to the mucosal baths. The ratio between the fluorescence intensities recorded with excitation wavelengths of 340 and 380 nm indicates that the calcium sensitive probe (free fura-2) was transferred to the serosal but not to the mucosal compartment by an oxytocin sensitive transport. (2) Preincubation with probenecid did not change fluorescence transfer in basal conditions but significantly reduced the oxytocin induced increase in free fura-2 transport. (3) Fluorescence accumulation inside the tissue was strongly reduced by oxytocin, but only when fura-2/AM was added to the mucosal side. (4) An osmotic gradient, in the presence of oxytocin, further increased the transfer of fluorescence at 380 nm but not at 340 nm. This indicated that the transfer of a calcium-insensitive fraction was being stimulated. (5) Preincubation with colchicine strongly inhibited fluorescence transfer across the tissue, at both 340 and 380 nm (the 340/380 ratio did not change). (6) Tissue accumulation was increased by colchicine. (7) Vanadate did not inhibit fura-2 transfer in the toad urinary bladder. We conclude that intracellularly-generated free fura-2 is only transported across the basolateral border, and that this transfer is stimulated by ADH. The calcium-insensitive fraction is transferred by a temperature-dependent process, sensitive to an osmotic gradient and colchicine.