5-Hydroxytryptamine2 and 5-hydroxytryptamine3 receptors mediate serotonin-induced short-circuit current in pig jejunum

The purpose of this study was to determine the effect of methysergide, ketanserin, granisetron, cisapride, and renzapride on serotonin 5-hydroxytryptamine-evoked short-circuit current in muscle and myenteric plexus-stripped pig jejunum using the Ussing chamber technique. Ketanserin, granisetron, cisapride, and renzapride all reduced the 5-hydroxytryptamine-induced increase in short-circuit current by about 50%. Combination of ketanserin and granisetron only reduced the 5-hydroxytryptamine-induced peak increase in short-circuit current by 25%. Cisapride caused a small concentration-dependent increase in short-circuit current. Atropine and hexamethonium both almost completely suppressed the cisapride-induced peak increase in short-circuit current. Ketanserin, granisetron, methysergide, and renzapride did not alter the basal short-circuit current. These results suggest that 5-hydroxytryptamine elicits an increase in short-circuit current by activating epithelial and submucosal 5-hydroxytryptamine₂ and 5-hydroxytryptamine₃ receptor subtypes. Furthermore, the short-circuit current-increasing effect of cisapride, is due to activation of at least muscarinic and nicotinic receptors.Abbreviations 5-HT 5-hydroxytryptamine, serotonin - AUC area under the curve - EC enterochromaffin - ENS enteric nervous system - GI gastrointestinal - MW molecular weight - 5-HTP-DP N-acetyl-5-hydroxytryptophyl-5-hydroxytrytophan amide - SSC short-circuit current - TTX tetrodotoxin     

Branchial versus intestinal silver toxicity and uptake in the marine teleost Parophrys vetulus

Exposure to elevated waterborne silver as AgNO3 (4.07 microM=448 microg l(-1)) in seawater resulted in osmoregulatory disturbance in the lemon sole (Parophrys vetulus). The main effects were increased plasma Na+ and Cl- concentrations which translated into increased plasma osmolality. Plasma Mg2+ levels were also slightly increased after 96 h exposure. Using radioisotopic flux measurements, a 50% reduction in branchial unidirectional Na+ extrusion was observed after 48 h silver exposure. By applying an intestinal perfusion approach, we were able to separate and thus quantify the intestinal contribution to the observed silver-induced physiological disturbance and internal silver accumulation. This analysis revealed that the intestinal contribution to silver-induced ionoregulatory toxicity was as high as 50-60%. In marked contrast, internal silver accumulation (in liver and kidney) was found to be derived exclusively from uptake across the gills. Drinking of silver-contaminated seawater resulted in substantial silver accumulation in the intestinal tissue (but apparently not silver uptake across the intestine), which probably explains the intestinal contribution to silver-induced physiological disturbance.     

The Coupling of the Short-Circuit Current to Metabolism in the Urinary Bladder of the Toad

The relationship of the short-circuit current to metabolism was studied in the toad bladder in vitro. Substrates and inhibitors were added to the bathing medium and the effect on the short-circuit current was determined. The spontaneous decline in the short-circuit current that occurred in substrate-free media was prevented or reversed by the addition of glucose, pyruvate, lactate, or β-hydroxybutyrate, whereas acetate and tricarboxylic acid cycle intermediates had no effect. A variety of metabolic inhibitors depressed the short-circuit current; depression by iodoacetate and by malonate was delayed by prior addition of pyruvate or lactate but not by glucose. The ability of a substrate to stimulate the current did not correlate with its rate of oxidation to CO₂. On the basis of earlier studies, the metabolic effects on the short-circuit current were assumed to reflect equivalent effects on the rate of active Na transport. It is suggested that the energy for Na transport is provided not by a general cellular metabolic pool but by a specific metabolic pathway or pathways spatially linked to the transport mechanism.     

Alterations in electrophysiology of isolated amphibian small intestine produced by removing the muscle layers

Isolated segments of Amphiuma small intestine bathed in chloride or sulfate buffer generate a greater short-circuit current and a larger change in current in response to galactose when the serosal muscle layers are stripped from the mucosa. Intact (unstripped) segments are not apparently anoxic since stripped segments exposed to serosal N₂ for 3 h display normal short-circuit currents but a reduced potential response to galactose, while the presence of muscle layers tends to reduce the short-circuit current but does not alter the potential response to galactose. Bullfrog small intestine also generates greater short-circuit current following removal of the muscle layers. The enhancing effect of stripping appears to be related to removal of a resistance to ion flow across the tissue.     

Ion transport across the frog olfactory mucosa: the basal and odorant-stimulated states

The Ussing method was adapted to study the basal electrolyte transfer as well as the events that occur upon odorant stimulation in frog olfactory mucosa. The unstimulated short-circuit current was due mainly to a furosemide-sensitive ion transport system on the apical side of the olfactory mucosa. This current was not amiloride sensitive. The current-voltage relationship of the unstimulated state was linear. That of the odorant-evoked current was non-linear and amiloride-sensitive. Ouabain caused collapse of both the unstimulated and odorant-stimulated short-circuit current. In this case, voltage-clamping the tissue to non-zero values restored the odorant-evoked current with polarity depending on that of the clamping voltage. This suggested that the direction of the current is determined by that of the sodium electrochemical potential difference. Our results indicate that the unstimulated short-circuit current occurs through an apical sodium cotransport system, while the odorant-evoked current is due to odorant-activated, passive sodium channels that are amiloride sensitive.     

Sodium transport by isolated bullfrog small intestine. Effect of prostaglandin E1.

Addition of 446 micron prostaglandin E1 (PGE1) to the serosal medium of isolated short-circuited bullfrog small intestine elicited small increases transmural potential difference and short-circuit current while addition of PGE1 to the mucosal medium caused no change in the electrical parameters. Addition of 100 micron indomethacin to the mucosal medium inhibited both potential difference and short-circuit current with a resultant increase in steady-state tissue resistance. In the presence of mucosal 100 micron indomethacin, serosal 60 micron PGE1 markedly stimulated transmural potential difference and short-circuit current with a resultant decrease in steady-state tissue resistance. Serosal arachidonic acid (330 micron) stimulated transmural potential difference and short-circuit current and this effect was abolished by the addition of 100 micron indomethacin to the mucosal medium. Serosal 60 micron PGE1 only stimulated the M (mucosa) leads to S (serosa) unidirectional flux of sodium. These results strongly suggest that the PGE1 action is mediated either via a series of metabolic reactions which possibly increase the permeability of the mucosal membrane to sodium or via direct stimulation of rheogenic sodium pump activity.     

Different sensitivity to amiloride of body and tail skins of Rana catesbeiana tadpoles during metamorphosis

Regional differences in potential difference and short-circuit current between the body (dorsal) and the tail skin during metamorphosis of Rana catesbeiana tadpoles were investigated. In body skin, the potential difference and the short-circuit current across the skin develop in two successive steps. At stage XX, the potential difference and the short-circuit current across the body skins were amiloride-insensitive (1st step). At stage XXII, however, amiloride-sensitive potential difference and the short circuit current appeared (2nd step). By contrast, in tail skin the potential difference and the short-circuit current remained amiloride-insensitive (1st step) even at stage XXIII. Since the tail regresses after stage XXIII, the appearance of the second step could not be followed in vivo. To determine whether or not the second step can be induced in the tail, tail skin was cultured under conditions where the skin survives for a much longer period than it does in normally developing tadpoles. Such cultured tail skin generated the amiloride-sensitive potential difference and the short-circuit current and cultured body skin also generated them. Therefore, development of the 2nd step in the tail skin may be delayed in vivo. To characterize the differences between body and tail skin, skins were mutally grafted between body and tail at stage XIII–XV. The body skin grafted on the tail underwent both the 1st and 2nd steps by stage XXII, whereas the tail skin grafted on the body only showed the 1st step by the same stage. These results suggest that the regional specificity of the skin is already established before the prometamorphic stage.Abbreviations CMFS Ca²⁺- and Mg²⁺-free saline - CTS charcoal-treated serum - EDTA ethylene diamine tetra-acetate - I current - PD potential difference - R skin resistance - SCC short-circuit current     

Active sodium transport by the isolated toad bladder

Studies were made of the active ion transport by the isolated urinary bladder of the European toad, Bufo bufo, and the large American toad, Bufo marinus. The urinary bladder of the toad is a thin membrane consisting of a single layer of mucosal cells supported on a small amount of connective tissue. The bladder exhibits a characteristic transmembrane potential with the serosal surface electrically positive to the mucosal surface. Active sodium transport was demonstrated by the isolated bladder under both aerobic and anaerobic conditions. Aerobically the mean net sodium flux across the bladder wall measured with radioactive isotopes, Na²⁴ and Na²², just equalled the simultaneous short-circuit current in 42 periods each of 1 hour's duration. The electrical phenomenon exhibited by the isolated membrane was thus quantitatively accounted for solely by active transport of sodium. Anaerobically the mean net sodium flux was found to be slightly less than the short-circuit current in 21 periods of observation. The cause of this discrepancy is not known. The short-circuit current of the isolated toad bladder was regularly stimulated with pure oxytocin and vasopressin when applied to the serosal surface under aerobic and anaerobic conditions. Adrenaline failed to stimulate the short-circuit current of the toad bladder.     

Sodium-dependent short-circuit current across the yolk sac membrane during embryonic development in normal and shell-less cultured chicks

Summary The transepithelial electrical characteristics of the isolated yolk sac membrane of normal in ovo or shell-less cultured chick embryos were investigated. In normal chicks the potential difference (blood side positive relative to yolk side) and short-circuit current of the membrane increased during development. Ouabain (10^-4 M) on the blood side (basolateral side, serosal side) significantly decreased potential difference and short-circuit current but was without effect on the yolk side (brush border side, mucosal side). Substitution of choline for Na⁺ in the bathing solutions abolished the potential difference and the short-circuit current; when Na⁺ replaced choline this effect was reversed. Amiloride added to both sides of the yolk sac membrane had no effect on potential difference or short-circuit current. Injection of aldosterone (50 g) and T₃ (10 M) into yolk did not induce amiloride sensitivity. The short-circuit current was not altered by addition of either glucose or alanine to the bath. The short-circuit current of the yolk sac membrane of shell-less cultured embryos was significantly lower than that of normal controls. Addition of Ca²⁺ to the serosal bathing medium did not reverse the foregoing condition, but decreased the short-circuit current. It is concluded that the yolk sac short-circuit current is Na⁺ dependent and increases with developmental age in the chick embryo.Abbreviations Hepes N-2-hydroxyethylpiperazine-N-2-ethaneoulphonic acid - PD potential difference - R resistance - SCC short-circuit current - TRIS tris-hydroxymethyl aminomethane - T₃ 3,3-5-triiodo-l-thyronine     

Conductive chloride flux across amphibian skin: inhibition by indacrinone and cobalt ion.

When amphibian skin was incubated under conditions in which transepithelial sodium transport was abolished, a conductive transepithelial Cl- flux arose when Cl- was removed from one of the compartments. This flux was matched by short-circuit current and it accounted entirely for transepithelial conductance. Cl- influx was larger than efflux; it was linearly related to the magnitude of transepithelial Cl- concentration difference. When applied to the epithelial surface of the tissue, divalent metal cations such as Co2+, and the ethacrynic acid derivative, indacrinone, reduced rapidly and reversibly both transepithelial Cl- (in)flux and short-circuit current. Frog skin proved to be more sensitive to these inhibitors than toad skin. Further characterization of transepithelial Cl- pathway(s) should benefit from the fact that Cl- across amphibian skin can easily be monitored by the short-circuit current method, and from the availability of agents which inhibit this passive flux rapidly and reversibly.     

Sodium transport inhibition by selective mitochondrial inhibitors in the urinary bladder of the toad

The effects of selective mitochondrial inhibitors on the short-circuit current and oxygen consumption displayed by the isolated urinary bladder of the toad was studied. Three types of compounds were used: (a) electron transfer inhibitors, Amytal, Cyanide and Antimycin A; (b) energy transfer inhibitors Guanidine, Oligomycin and Rutamycin; and (c) uncoupling agents, Carbonyl cyanide m-chlorophenylhydrazone and 2–4 dinitrophenol. The kinetics of inhibition of oxygen consumption indicated that the inhibitors tested were effectively reaching the mitochondria of the bladder cells. Different kinetics of inhibition of short-circuit current were obtained with the various inhibitors tested. Uncouplers and electron transfer inhibitors rapidly blocked the short-circuit current; energy transfer inhibitors only produced a slow and partial inhibition. A site of energy-coupling, tentatively identified with the intermediate formed in the energy transfer reactions closest to the electron transfer chain, is proposed.     

Ethanol and silver effects on ion transport across toad skin

Silver stimulated short-circuit current and transepithelial potential difference. Ethanol inhibited transpithelial potential difference. Ethanol had no effect on short-circuit current. Ethanol stimulated unidirectional movements of chloride from outside to inside and from inside to outside.     

Thiocyanate inhibition of active chloride absortion in Aplysia intestine

This investigation was principally undertaken to examine the mechanism of active chloride absorption across the Aplysia californica intestine by using various inhibitors of ion transport. Isolated intestine, mounted between identical oxygenated sodium-free seawater solutions, maintained stable transmural potential differences (serosa negative) and short-circuit currents for several hours at 25°C. The metabolic inhibitors, 2,4-dinitrophenol and flouride, reduced both transmural potential difference and short-circuit current; however, the electrical characteristics were predominantly dependent upon glycolytic energy. The addition of thiocyanate to the mucosal solution inhibited both electrical characteristics in parallel, and this inhibition could be titrated according to the thiocyanate concentration. The short-circuit current was carried wholly by a net active chloride transfer from mucosa to serosa as determined by flux measurements. These results suggest that active chloride absorption may be mediated by a primary active transport process.     

Effects of exogenous ATP on short-circuit current and potential difference of the isolated frog skin.

The addition of ATP (10(-3) M = final concentration) to the bathing medium of either side of the isolated frog skin resulted in parallel increases in potential difference and short-circuit current. Reductions in these electrical parameters induced by anaerobic conditions and sodium azide could be partially reversed by exogenous ATP. The response is apparently not mediated by cyclic adenylic acid, as it was not enhanced by theophylline. Ouabain failed to reduce rates of phosphate liberation induced by ATP, although potential difference and short-circuit current were reduced.     

Thiocyanate inhibition of active chloride absorption in Aplysia intestine

This investigation was principally undertaken to examine the mechanism of active chloride absorption across the Aplysia californica intestine by using various inhibitors of ion transport. Isolated intestine, mounted between identical oxygenated sodium-free seawater solutions, maintained stable transmural potential differences (serosa negative) and short-circuit currents for several hours at 25 degrees C. The metabolic inhibitors, 2,4-dinitrophenol and fluoride, reduced both transmural potential difference and short-circuit current; however, the electrical characteristics were predominantly dependent upon glycolytic energy. The addition of thiocyanate to the mucosal solution inhibited both electrical characteristics in parallel, and this inhibition could be titrated according to the thiocyanate concentration. The short-circuit current was carried wholly by a net active chloride transfer from mucosa to serosa as determined by flux measurements. These results suggest that active chloride absorption may be mediated by a primary active transport process.     

Sodium transport by isolated bullfrog small intestine. Effect of Prostaglandin E1

Addition of 446 μM prostaglandin E₁ (PGE₁) to the serosal medium of isolated short-circuited bullfrog small intestine elicited small increases in transmural potential difference and short-circuit current while addition of PGE₁ to the mucosal medium caused no change in the electrical parameters. Addition of 100 μM indomethacin to the mucosal medium inhibited both potential difference and short-circuit current with a resultant increase in steady-state tissue resistance. In the presence of mucosal 100 μM indomethacin, serosal 60 μM PGE₁ markedly stimulated transmural potential difference and short-circuit current with a resultant decrease in steady-state tissue resistance. Serosal arachidonic acid (330μM) stimulated transmural potential difference and short-circuit current and this effect was abolished by the addition of 100 μM indomethacin to the mucosal medium. Serosal 60 μM PGE₁ only stimulated the M (mucosa) → S (serosa) unidirectional flux of sodium. These results strongly suggest that the PGE₁ action is mediated either via a series of metabolic reactions which possibly increase the permeability of the mucosal membrane to sodium or via direct stimulation of rheogenic sodium pump activity.     

Effect of 5-hydroxytryptamine on sodium transport across bullfrog skin

Summary 5-hydroxytryptamine, when present in the solution bathing the inside surface of bullfrog skin at concentrations of 0.25–25.0 mM, reduced both electrical potential difference and short-circuit current across the skin. The magnitude of reduction in potential difference and short-circuit current was dependent on 5 HT concentration. Reduction in sodium influx entirely accounted for the reduction in short-circuit current. Preliminary evidence suggested a competition between 5 HT and vasopressin in the production of their effects on sodium transport across the skin, while high Ca⁺⁺ concentrations and 5 HT seemed to act independently of each other.Dr. Henry C. and Bertha H. Buswell Fellow.     

Exploration of apical sodium transport mechanisms in an epithelial model by network thermodynamic simulation of the effect of mucosal sodium depletion: I. Comparison of three different apical sodium permeability expressions

A model based on that of Koefoed-Johnsen & Ussing (1958) and elaborated by Hviid Larsen (1978) and Lew et al. (1979), is designed using network thermodynamic theory and used to simulate experiments performed on epithelia. Three different expressions for the apical sodium permeability are tested for their ability to reproduce the saturation of the short-circuit current with increasing mucosal sodium concentration. Using the parameters from the previous models, the sodium entry step is shown to be the rate limiting step. If the apical sodium permeability is constant, there is no saturation of the short-circuit current with increasing mucosal sodium. The saturation of the short-circuit current is simulated with versions of the model which include a variable apical sodium permeability. The phenomenological expressions used for the variable permeabilities are those proposed by Fuchs et al. (1977) and Civan & Bookman (1982). They describe the so-called feedback effect of the mucosal and intracellular sodium concentrations.     

Actions of carbaryl on the ionic transport across the isolated skin of Rana esculenta

1. Carbaryl, a carbamate used as a pesticide, increases the short-circuit current (SCC) across the isolated frog skin in a dose-dependent manner. 2. This effect is due to the stimulation of sodium absorption and chloride secretion. 3. Carbaryl action on short-circuit current is unrelated to its inhibitory power on cholinesterase; this statement is supported by two experimental results: (a) carbaryl is equally active on both sides of the skin, (b) atropine pretreatment does not inhibit the carbaryl action on SCC.     

Mechanism of inhibition by lithium of sodium transport in the toad bladder

Lithium transport across the urinary bladder of Bufo marinus has been studied by means of the short-circuit current technique, as well as unidirectional ion flux measurements. Exposure to lithium of the epithelial (mucosal) surface of this preparation led to a slow, progressive decrease of ion transport, with increasing discrepancy between short-circuit current and lithium influx; in fact there was still an appreciable lithium influx across bladder exposed to amiloride even though short-circuit current was suppressed. Ohmic conductance and sodium efflux barely increased under these circumstances. Upon replacement of lithium by sodium on the epithelial side, the preparations recovered slowly indeed, and residual lithium could be detected in bladder tissue for more than 2 hr while the rate of sodium extrusion at the basal-lateral cell border was slowed down. Recovery from exposure to lithium was accelerated by vasopressin and amphotericin, both of which facilitate sodium entry at the apical border of the epithelium. Thus the lasting deleterious influence of lithium on sodium transport might result from the fact that this ion, once trapped in the cytoplasm, closes the sodium channels.