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ABSTRACT: Sulfhydryl (SH) reactive reagents, such as eosin derivatives, have been found to be useful in labeling water pathways in red cells. In the present study we used an impermeable SH-reagent, a fluorescent maleimide analogue EMA (eosin-5'-maleimide), in order to identify proteins involved in water permeability response to antidiuretic hormone (ADH). We observed that: 1) EMA (1 mM) mucosal pretreatment did not modify either the basal water flux or the subsequent ADH-induced hydrosmotic response; 2) EMA added to the mucosal bath at the maximum response to ADH, significantly decreased net water flux by about 40%; similar results were obtained when 10(-5) M forskolin was used as a hydrosmotic agent. These results suggest that the inhibitory effect of EMA occurs at a post cAMP step, possibly at the level of the sulfhydryl groups of the water channels themselves. Fluorescence distribution in SDS-PAGE of Triton X-100 extracted proteins from bladder labeled with EMA in both control conditions and under ADH stimulation allowed us to identify apical membrane proteins, labeled during ADH stimulation and not labeled in water impermeable controls. Of particular importance are four proteins of 52, 32-35, 26, 17, kDa. These polypeptides are probably involved in ADH-stimulated water transport and may be components of the water channels.
Biology of the Cell 02/1989; 67(2):115-21. · 3.60 Impact Factor
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ABSTRACT: It is now generally accepted that the increase in water permeability induced by antidiuretic hormone (ADH) in responsive epithelia is accompanied by the insertion of specific structures in the apical membrane of epithelial cells. There are strong indications that these particles, probably proteic in nature, represent water channels. In order to evaluate the nature and role of such proteins, plasma membranes were isolated by the affinity chromatography technique. The method is based on the firm attachment of the external face of the membrane to polycations covalently bound to the surface of polyacrylamide beads, followed by shearing of the rest of the cells. Maximal binding of epithelial cells to beads was achieved in a medium of low ionic strength and pH 5.2 (i.e. sucrose-MES buffer). By this procedure plasma membranes were obtained from both cAMP-stimulated cells and control cells. Membranes isolated on beads were enriched in the activity of typical membrane marker enzymes (LAP; H+ ATPase; Na+, K+ ATPase) with respect to a whole cell homogenate, whereas contamination of plasma membrane fraction by endoplasmic reticulum, lysosomes, and mitochondria was relatively low. Analysis by SDS polyacrylamide gel electrophoresis showed an interesting difference between cAMP-treated and control samples.
Biology of the Cell 02/1989; 66(1-2):85-9. · 3.60 Impact Factor
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ABSTRACT: In the present study several aspects of the osmotic water-flow-regulation mechanism in frog urinary bladder were examined, utilizing forskolin either as a direct hydrosmotic agent or in association with vasopressin. It was found that forskolin induces a hydrosmotic effect similar to the one induced by vasopressin. This effect is rapid, reversible and dose-dependent. The half-maximally effective concentration (Ec50FSK) is 1.37 microM forskolin. No additional effect on the osmotic water flow was observed when maximal concentrations of forskolin and vasopressin were given simultaneously. Moreover, forskolin can also markedly potentiate vasopressin-induced hydrosmotic response. This potentiation was maximal with submaximal doses of vasopressin, whereas it disappeared when the hormonal concentration was increased to very high levels. Therefore, forskolin increases vasopressin potency without affecting vasopressin efficacy. The Ec50FSK for the forskolin-induced increase in vasopressin potency was 11 nmol, about two orders of magnitude lower than the Ec50FSK for the direct effect of forskolin on the osmotic water transport. On the whole, our results are compatible with a two-site model of forskolin action in frog urinary bladder: a low affinity site (Ec50FSK = 1.37 microM) that mediates the direct action of forskolin on the osmotic water flow and a high affinity site (Ec50FSK = 11 nmol), which mediates the synergic effect of forskolin with the antidiuretic hormone.
Pflügers Archiv - European Journal of Physiology 08/1987; 409(4-5):486-91. · 4.46 Impact Factor
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ABSTRACT: Serosal preincubation of frog skin with tetradecanoyl phorbol acetate, TPA, an activator of protein kinase C, inhibits the hydrosmotic response elicited by vasopressin (AVP) but not that induced by 8br-cAMP. This proves that serosal TPA primarily influences a pre-cAMP step. The TPA-induced inhibition of AVP response appears to be related to TPA-induced prostaglandin synthesis. The pretreatment with naproxen, in fact, prevents the inhibition induced by serosal TPA on the AVP response. On the contrary, mucosal TPA produces a more marked inhibition of the response to AVP and significantly diminishes the water flow induced by 8br-cAMP; this suggests that mucosal TPA interferes mainly with a post-cAMP step. Furthermore, naproxen is unable to completely prevent the inhibition induced by mucosal TPA on AVP response thus indicating that mucosal TPA may also activate a prostaglandin-independent mechanism able to inhibit one of the last steps of the hydrosmotic response to AVP.
Pflügers Archiv - European Journal of Physiology 04/1987; 408(3):318-20. · 4.46 Impact Factor
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Bollettino della Società italiana di biologia sperimentale 08/1985; 61(7):989-93.
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ABSTRACT: Treatment of ventral frog skin with serosal A23187 calcium ionophore caused an initially transient increase in transepithelial sodium transport. After 60 min of treatment with A23187, a steady-state transport value was reached which was significantly lower than the initial one. Furthermore, it was found that ionophore treatment greatly inhibited the natriferic response to ADH and to 8br-cAMP. A further analysis on the possible ionophore action mechanism was carried out through pretreatment of the skin with indomethacin, very powerful prostaglandin synthesis inhibitor. In the experimental conditions reported, A23187 seems no longer capable of inducing a transient increase in sodium transport, although it does inhibit the natriferic response to ADH.
Comparative Biochemistry and Physiology Part A Physiology 02/1985; 80(3):329-32.
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ABSTRACT: The A23187 calcium ionophore strongly inhibits the hydrosmotic response to vasopressin. On the contrary neither exogenous cAMP nor theophylline-induced hydrosmotic response are affected by Ca++-ionophore. The effects obtained with A23187 suggest that calcium ions modulate vasopressin-induced osmotic water flow by interfering with a pre-cAMP step. The increase in intracellular calcium concentration induced by A23187, in fact, may inhibit the rate of cAMP formation by interfering with the vasopressin-stimulated adenylate cyclase system. This inhibition seems to be restricted to a locus proximal to the catalytic subunit of adenylate cyclase, whereas the hydrosmotic response to forskolin, a non-hormonal activator, is not affected by calcium ionophore addition. In order to clarify whether calcium ions act directly on the adenylate cyclase system or activate a more complex pathway, we studied the interaction between calcium and prostaglandins in modulating the hydrosmotic response to vasopressin. Direct measurements by radioimmunoassay of PGE2 release in the serosal medium show that A23187 notably increases PGE2 release. Furthermore, the inhibition of prostaglandin biosynthesis by hydrocortisone (a phospholipase inhibitor) or by indomethacin and naproxen (agents that inhibit arachidonic acid oxygenase) results in augmented vasopressin-stimulated water flow and prevents the inhibitory effect of the ionophore. Collectively these results strongly suggest that the effects obtained with A23187 on hydrosmotic response to ADH, are closely linked to calcium-stimulated PGE2 biosynthesis.
Biology of the Cell 02/1985; 55(3):199-205. · 3.60 Impact Factor
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ABSTRACT: Treatment with the calcium ionophore A23187 on either the serosal or mucosal sides of frog skin, strongly inhibits the hydrosmotic response to vasopressin. On the contrary, the hydrosmotic response to 8-br-cAMP is not affected by treatment with the A23187. Trifluoperazine, a drug which inhibits the Ca2+-calmodulin complex, selectively inhibits vasopressin-induced water transport. Collectively, our results suggest that an increase in the intracellular concentration of Ca2+, obtained by treatment with the ionophore A23187, interferes with a pre-cAMP step of the hydrosmotic response to the antidiuretic hormone. Calcium ions could regulate adenyl-cyclase activity and consequently intracellular levels of cAMP. This effect may probably involve calmodulin.
Pflügers Archiv - European Journal of Physiology 11/1984; 402(2):166-70. · 4.46 Impact Factor
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Bollettino della Società italiana di biologia sperimentale 06/1984; 60 Suppl 4:201-5.
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ABSTRACT: The in vivo gastric mucosa actively transport Cl- (serosa to mucosa) and it has been shown that the e.m.f. generated by the epithelium, as well as the short circuit current are both manifestations of the same phenomenon: the Cl- movement. Also, it has been postulated the presence of a Cl- - HCO-3 exchange in the gastric epithelium probably located on different cell types. The addition of niflumic acid (10(-4) M in the serosal solution) resulted in a decline towards zero of both transepithelial p.d. and Isc. The mechanism of the niflumic acid action is postulated to be a blockage of the Cl- - HCO-3 exchange, similarly to the SITS action mechanism.
Bollettino della Società italiana di biologia sperimentale 08/1983; 59(7):928-34.
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ABSTRACT: The addition of the Ca++ ionophore A23187 (10 microM) to the inside solution of the frog skin induced a transient increase in the active Na+ transport in frog skin (Rana esculenta) which decayed to the control values 60 minutes after the addition. At the same time the skin resistance failed significantly; antidiuretic hormone addition resulted in no-more increase of the Na+ active transport; the skin resistance remained unchanged. To further investigate the role of intracellular calcium on the skin transepithelial permeability, the effect of A23187 ionophore on thiourea permeability has been tested. Increase in intracellular Ca++ concentration brought about by calcium ionophores have been shown to modify both basal and ADH-stimulated thiourea transport.
Bollettino della Società italiana di biologia sperimentale 08/1983; 59(7):935-41.
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ABSTRACT: The effect of noradrenaline on intracellular levels of cAMP in epithelial cells isolated from frog skin, has been studied both in the presence and in the absence of alpha (phentolamine) or beta (propranolol) adrenergic blocking agents. Such levels are increased by hormonal treatment and are linked to the stimulation of beta-adrenergic receptors, while alpha-stimulation has an inhibitory effect. Higher levels of cAMP are reached in the presence of both Ca2+ and Mg2+, confirming the existence of a Ca2+ and Mg2+-dependent adenyl-cyclase, stimulated by noradrenaline. The experimental conditions necessary to induce the highest intracellular cAMP levels, are those in which the permeabilizing effect reaches its maximum.
General Pharmacology 02/1983; 14(6):673-6.
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ABSTRACT: This report presents evidence for urea active absorption by isolated skin of Rana esculenta. One of the supporting factors of such evidence is that at a low concentration the urea influx is five times greater than the outflux, in the absence of a chemical gradient. The transport shows a saturation kinetics with an apparent Km = 1.33 mM and is inhibited by un uncoupling agent (FCCP). 5 x 10(-4) M Phloretin, added to the external side, markedly inhibits inward urea transport, whereas it is ineffective when added to the serosal fluid. This provides evidence for a phloretin-sensitive mechanism located at the external side of the epithelium. Phloretin stimulates the sodium active transport; the possible coupling of urea and sodium movement is analysed.
Pflügers Archiv - European Journal of Physiology 10/1982; 394(3):226-9. · 4.46 Impact Factor
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ABSTRACT: In several epithelial tissues such as toad bladder, gallbladder and human red cells, it has been established that urea movement implies a phloretin sensitive mediated transport. In the skin of the toad Bufo viridis also it has been described an active transport of urea. Our data, obtained on the frog skin seem to demonstrate the existence of some specific mechanism for urea transport towards the inside solution. In fact, two molecules having the some molecular diameter, such as urea and thiourea, show a large difference in permeability at low concentration. In addition 0.1 mM urea influxes and outfluxes, measured on paired skin halves in the absence of concentration gradient, exhibit an evident asymmetry. Further approaches with phloretin experiments were made in order to characterize the urea transport system. Phloretin (5.10(-4)M) added to the external solution significantly inhibits the urea influx. Little can be said at this time about the composition or kinetics of the carrier involved in the transport.
Bollettino della Società italiana di biologia sperimentale 07/1982; 58(12):752-5.
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ABSTRACT: The effect of microtubular-poisons, such as colchicine and vincristine, on frog skin permeability has been investigated. Three-hour treatment with the drugs has no effect on nonelectrolyte basal transepithelial permeability, but completely suppresses the effect of ADH. Colchicine and vincristine, in addition, affect both basal sodium transport and the rise in short circuit current induced by vasopressin. The inhibition produced by microtubular-poisons disappears, however, when hydrocortisone, a glucocorticoid known to preserve junctional communications is used. Together with the results previously obtained with isolated epithelial cells (Svelto et al. 1979), these findings provide further support for our hypothesis that the microtubular-microfilament-system, is involved in cell-to-cell exchange.
Pflügers Archiv - European Journal of Physiology 01/1982; 392(2):152-6. · 4.46 Impact Factor
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ABSTRACT: Transepithelial urea outfluxes across toad gallbladder were determined before and after the addition of cycloheximide. The drug inhibits the movement of urea but has no effect on thiourea and antipyrine outfluxes. The inhibition of amide transport is time dependent as also shown in counterflow experiments. These results are consistent with the hypothesis that cycloheximide inhibits the synthesis of membrane proteic sites involved in urea mediated transport.
Pflügers Archiv - European Journal of Physiology 04/1980; 384(2):155-8. · 4.46 Impact Factor
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ABSTRACT: Counterflow experiments demonstrate the existence of urea counter-transport on the epithelium luminal surface. This phenomenon disappears when 10(-4) M phloretin is added to the perfusion fluid. Moreover counterflow experiments made using thiourea as elicitor, demonstrate that the phenomenon is specific for the urea.
Archives internationales de physiologie et de biochimie 06/1978; 86(2):243-50.
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ABSTRACT: The toad gallbladder epithelium is much more selective than that of the rabbit especially as to the permeability of two molecules like urea and thiourea. These observations can probably be attributed to different permeation mechanisms of the 2 molecules. Neither active transport nor solvent drag can explain these phenomena. 10(-4) M phloretin strongly inhibits urea movement, but does not alter either thiourea fluxes or isotonic net water transport: these results suggest that a specific mechanism is involved in urea movement. The urea transport shows saturation kinetic which is consistent with the presence of a facilitated mechanism.
Pflügers Archiv - European Journal of Physiology 04/1976; 362(2):109-12. · 4.46 Impact Factor
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ABSTRACT: Amphotericin B treatment increases the thiourea, D-xylose and mannitol fluxes and lowers those of urea, N-methyl-urea, acetamide, formamide, and N-N'-dimethyl-thiourea. The degree of flux inhibition is related to the cellular permeability of these compounds. Most probably Amphotericin B increases the permeability of all those molecules across the luminal plasma membrane, but simultaneously elicits a cellular swelling, which reduces the diffusion across the lateral plasma membranes. This effect masks the polyene effect especially for molecules showing a mainly cellular permeation pathway such as amides and lipid soluble molecules.
Pflügers Archiv - European Journal of Physiology 04/1975; 355(3):267-71. · 4.46 Impact Factor
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Bollettino della Società italiana di biologia sperimentale 09/1974; 50(16-2):1311-4.
