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Br-exchange in the epithelium of toad skin (bufo viridis)
Abstracts D5 APICAL CELLULAR MATERIAL PREPAREDFHOM THE AMPHIBIANBLADDEREWIWELIUM. G. Calamita, V. Ripoche, G. Vale&i, J. Bourquet and J.S. C.E.N.de Saclay, HugOn, Dept. de Biologie, Gif-sur-Yvette, France and Istituto di Fisiologia Generale, Univ. degli Studi, Sari, Italia. Using a modification of the freeze fracture (Ann. Rev. Fhysiol. method of K. Fischer 261) we have obtained apical cel1980, 42, lular material derived from the ADH-responsive epithelial cells of the amphibian bladder. In our procedure the apical surface of pushed against a the epithelium is gently nuclepore filter (pore size 3 ,u) by a low hydrostatic pressure (20 cm H 0) exerted on . the serosal side of the prepara t ion. At times ranging from 60 to 165 -min. a polylysine coated coverslio was aonlied to the filter, and the sandwich of-coverslip, filter, and bladder was frozen with liquid nitrogen or dry ice. The coverslip was then quickly pulled away from the filter, and the apical material fractured from the cells recovered in SDS buffer. Samples were then compared to crude homogenate of the whole epithelium by electrophoresis. After silver-staining, apical material recovered in these conditions exhibited several differences from the crude some bands being enriched while homogenate, others were reduced.
D7 CELLULAR Cl,&?EKCHANGEIN THE EPITHELIUN OF TOADSKIN (BUFGVIRIDIS). A. Diirge, W. Nagel, and F. Beck, Dept. Physiol., Univ. R. Rick, Nunich, D-8000 Munich 2, FRG. To further characterize the transepithelial anion pathway in the toad skin, cellular electrolyte concentrations were measured by electron microprobe analysis after replacing the extracellular Cl on the outside or the inside with Br. In control, the Cl concentration was 2024 in the mitochondria rich cells (NRC) and 35+5 mmol/kg wet weight in the epithelial cell layers. Replacing Cl by Br on the outside resulted in a partial exchange of Cl with Br in some MRCs. Replacing Cl by Br on the inside led to a partial exchanqe of Cl with Br in all epithelial cells analysed. Activating anion-influx by clamping the transepithelial potential to 100 mV (inside positive) resulted in increases in the cellular Cl and Na concentrations in some but not all MRCs, when the outside solution was normal Ringer’s solution, and in increases in Br and Na concentrations, when the outside Cl was replaced with Br. Since under the latter condition almost no Br could be detected in the different epithelial cell layers it might be concluded that MRCs are involved in transepithelial anion transport.
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D6
EDENCB FOR THE ROLE OF THB Ns PROTEIN IN THE FORSKOLINACTIONSON OSMOTICWATEX FLOW.
V. Casavola, G. Valenti, G. Calamita, J. Bourquet and M. Svelto. Inst. of General Physiology, Univ. of Bari, Italy.
We have previously reported that forskolin (FSK) in frog urinary bladders at concentrations higher than 0.1 PM induces an hydrosmotic effect whereas at concentrations lower than 0.05 PM it markedly potentiates the hydrosmotic response induced by submaximal doses (2mU/ml) of vasopressin (VP). Preincubation of the bladder with 0.7 mMcycloheximide significantly reduces both the FSK-induced osmotic water flow and the FSK-synergic effect with VP. These results indicate that both actions of FSK on osmotic water-flow may reside on a protein with a relatively short half life. Serosal addition of 100 ,uM Gpp(NH)p an activator of the guanine nucleotide regulatory proteins(Ns) significantly enhances both FSK-effects leaving the basal osmotic water flow unchanged. These results indicate that the activity state of Ns modulates both the hydrosmotic and the synergic effect of FSK.
D8 K THB WATER CHANNEL ACHGSSCELLMEMBRANES N0N.EOTHERTHANTHEGLUCOSE TRANSPORTER CHANNEL? J. Fischbarg, L.S. Liebovitch and J.P. Koniarek, Dept. of Physiology, Columbia Univ., New York, Specific inhibitors of either anion or glucose transport have not been found to block water transport until now, and hence a precise identification of the water channel has not been possible. However, we have reinvestigated the effects of inhibitors on the osmotic water flows across rabbit cornea1 endothelium. We have confirmed prior findings of a) inhibition by PCMBS, and b) lack of inhibition by DTNB and DIDS. On the other hand, cf we have found for the first time that several blockers of glucose facilitated diffusion, namely, phloretin (2 m&Q, phloridzin (2 m&I) dia~lyldie~vlstil~strol (0.1 mMf , cytochalasin B- (20 pg;?nl) , and ethylidene-D-)-glucose (200 mM). all clearlv inhibit osmotic-flow. We conclude that water-and glucose may traverse these cell membranes through the same channel-like pathway contained in the glucose transporter membranespanning protein.
D7 CELLULAR Cl,&?EKCHANGEIN THE EPITHELIUN OF TOADSKIN (BUFGVIRIDIS). A. Diirge, W. Nagel, and F. Beck, Dept. Physiol., Univ. R. Rick, Nunich, D-8000 Munich 2, FRG. To further characterize the transepithelial anion pathway in the toad skin, cellular electrolyte concentrations were measured by electron microprobe analysis after replacing the extracellular Cl on the outside or the inside with Br. In control, the Cl concentration was 2024 in the mitochondria rich cells (NRC) and 35+5 mmol/kg wet weight in the epithelial cell layers. Replacing Cl by Br on the outside resulted in a partial exchange of Cl with Br in some MRCs. Replacing Cl by Br on the inside led to a partial exchanqe of Cl with Br in all epithelial cells analysed. Activating anion-influx by clamping the transepithelial potential to 100 mV (inside positive) resulted in increases in the cellular Cl and Na concentrations in some but not all MRCs, when the outside solution was normal Ringer’s solution, and in increases in Br and Na concentrations, when the outside Cl was replaced with Br. Since under the latter condition almost no Br could be detected in the different epithelial cell layers it might be concluded that MRCs are involved in transepithelial anion transport.
825
D6
EDENCB FOR THE ROLE OF THB Ns PROTEIN IN THE FORSKOLINACTIONSON OSMOTICWATEX FLOW.
V. Casavola, G. Valenti, G. Calamita, J. Bourquet and M. Svelto. Inst. of General Physiology, Univ. of Bari, Italy.
We have previously reported that forskolin (FSK) in frog urinary bladders at concentrations higher than 0.1 PM induces an hydrosmotic effect whereas at concentrations lower than 0.05 PM it markedly potentiates the hydrosmotic response induced by submaximal doses (2mU/ml) of vasopressin (VP). Preincubation of the bladder with 0.7 mMcycloheximide significantly reduces both the FSK-induced osmotic water flow and the FSK-synergic effect with VP. These results indicate that both actions of FSK on osmotic water-flow may reside on a protein with a relatively short half life. Serosal addition of 100 ,uM Gpp(NH)p an activator of the guanine nucleotide regulatory proteins(Ns) significantly enhances both FSK-effects leaving the basal osmotic water flow unchanged. These results indicate that the activity state of Ns modulates both the hydrosmotic and the synergic effect of FSK.
D8 K THB WATER CHANNEL ACHGSSCELLMEMBRANES N0N.EOTHERTHANTHEGLUCOSE TRANSPORTER CHANNEL? J. Fischbarg, L.S. Liebovitch and J.P. Koniarek, Dept. of Physiology, Columbia Univ., New York, Specific inhibitors of either anion or glucose transport have not been found to block water transport until now, and hence a precise identification of the water channel has not been possible. However, we have reinvestigated the effects of inhibitors on the osmotic water flows across rabbit cornea1 endothelium. We have confirmed prior findings of a) inhibition by PCMBS, and b) lack of inhibition by DTNB and DIDS. On the other hand, cf we have found for the first time that several blockers of glucose facilitated diffusion, namely, phloretin (2 m&Q, phloridzin (2 m&I) dia~lyldie~vlstil~strol (0.1 mMf , cytochalasin B- (20 pg;?nl) , and ethylidene-D-)-glucose (200 mM). all clearlv inhibit osmotic-flow. We conclude that water-and glucose may traverse these cell membranes through the same channel-like pathway contained in the glucose transporter membranespanning protein.