Active transport of chloride across skin isolated from Rana esculenta]

The effect of SITS, ouabain and acetazolamide on potential difference (E) and short-circuit current (ccc) across isolated frog skin after Amiloride treatment have been investigated. It has been found that Amiloride (3.5 . 10(-5)M) reverses the E and ccc values. After Amiloride treatment, with the use of SITS (6 . 10(-4)M) positive values of E and ccc have been measured while Ouabain (10(-4)M) only slightly reduces the reversed E and ccc values. On the other hand the effect of Acetazolamide (6 . 10(-4)M) is additional to the effect of SITS. It is suggested that the frog skin possesses an active transport for Cl-, as demonstrated by the use of Amiloride, and that it is carried out by two distinct mechanism: the first SITS and Acetazolamide sensible, the second one sensible to Strofantine.     

Effect of hibernation on sodium and chloride ion transport in isolated frog skin

The aim of the study was to evaluate the effect of hibernation on electrophysiological parameters of isolated frog skin under control incubation (Ringer solution) and after inhibition of Na+ and CI- transepithelial transport by application of amiloride and bumetanide. The transepithelial electrical potential difference (PD in mV) was measured before and after mechanical stimulation of isolated frog skin. The tissues were mounted in a modified Ussing chamber. The results revealed a reduced PD of frog skin during hibernation. In February, as compared with November, PD of frog skin incubated in Ringer solution decreased by about 50%. Hibernation also affected hyperpolarization (dPD) of frog skin after mechanical stimulation. In November and December, dPD was about 50% and 30% lower, respectively, compared with the subsequent two months of the experiment. The incubation of frog skin with amiloride, a sodium ion channel blocker, resulted in reduced values of all measured electrophysiological parameters irrespective of the phase of hibernation. After application of chloride ion transport inhibitor (bumetanide), the PD in November and December decreased compared with the control incubation by about 80% and 75%, while in January and February by about 40% and 25%, respectively. In January and February dPD increased by four times and three times as compared with November and December. Hibernation reduces net ion flow in isolated frog skin. During the initial period of hibernation the sensitivity of the skin to mechanical stimulation also decreases. Towards the end of hibernation, on the other hand, excitation of mechanosensitive ion channels takes place.     

Prostaglandin E2 enhances the sodium conductance of exocrine glands in isolated frog skin (Rana esculenta)

Summary Prostaglandins are known to stimulate the active transepithelial Na⁺ uptake and the active secretion of Cl^– from the glands of isolated frog skin. In the present work the effect of prostaglandin E₂ (PGE₂) on the glandular Na⁺ conductance was examined. In order to avoid interference from the Na⁺ uptake and the glandular Cl^– secretion the experiments were carried out on skins where the Cl^– secretion was inhibited (the skins were bathed in Cl^– Ringer's solution in the presence of furosemide, or in NO ₃ ^– Ringer's solution), and the active Na⁺ uptake was blocked by the addition of amiloride. Transepithelial current, water flow and ion fluxes were measured. A negative current was passed across the skins (the skins were clamped at –100 mV, basolateral solution was taken as reference). When PGE₂, was added to the skins under these experimental conditions, the current became more negative; this was mainly due to an increase in the Na⁺ efflux. Together with the increase in Na⁺ efflux a significant increase of the water secretion was observed. The water secretion was coupled to the efflux of Na⁺, and when one Na⁺ was pulled from the basolateral to the apical solution via this pathway 230 molecules of water follwed. From the data presented it is suggested that this pathway for Na⁺ is confined to the exocrine glands.     

Interdependence between sodium transport, external chloride, and sodium/calcium exchanger in the isolated skin of the Rana pipiens

The aim of this study was to analyze the relationship of the Na+/Ca2+ exchanger, cytosolic calcium, and chloride to the transepithelial transport of sodium in isolated frog skin. Sodium transport was measured as amiloride-inhibitable short circuit current (SCC). We studied the effect of variations in the concentrations of external chloride and of the manipulation of calcium on sensitive amiloride SCC. Modifications in the movement of Ca2+ were induced by an ionophore, A23187, and a Ca2+ channel blocker, nifedipine. Calcium ionophore A23187 (5 and 20 microM), in a normal Ringer's solution, increased SCC and transepithelial potential difference (PD). In contrast, nifedipine (20 microM) reduced SCC and PD. The role of the Na+/Ca2+ exchanger was studied using dichlorobenzamil (DCB, 50 microM) and quinacrine (1 mM), inhibitors of this exchanger. They selectively increased SCC and PD on the mucosal side of the skin, with no effect on the serosal side. This response occurred only in the presence of extracellular calcium. Replacement of NaCl by sodium methanesulfonate or the addition of furosemide (1 mM) at the serosal compartment, decreased basal SCC and PD and blocked the response to A23187 and the mucosal effect of DCB and quinacrine. These results suggest the presence of an Na+/Ca2+ exchanger located on the mucosal side of the frog skin, which participates in the transepithelial sodium transport. The action of this exchanger may be modulated by external chloride and calcium. J. Exp. Zool. 289:23-32, 2001.     

Effect of capsaicin and dimethyl sulphoxide (DMSO) on ion transport in isolated skin of frog (Rana esculenta L.)

The effects ofcapsaicin, dimethyl sulphoxide and pH changes on transport of sodium and/or chlorine ions in an isolated frog skin, were studied using electrophysiological methods, adapted to evaluation of ionic currents occurring in the epithelial tissues and organs. The experiment consisted in measuring potential difference (PD in mV) of an isolated skin of the aquatic frog, Rana esculenta L., placed in a Ussing apparatus. The ionic transport processes were modified through incubation of the tissue in Ringer solution and in Ringer solution supplemented with amiloride, bumetanide, and also with dimethyl sulphoxide. The direct effect of capsaicin and dimethyl sulphoxide (DMSO) on frog skin was assessed while these compounds were added to the Ussing chamber with a pipette and a peristaltic pump. Adaptive reactions of the tissue were assessed following at least 60-min exposure to those compounds. It has been demonstrated that amiloride-inhibited sodium ion transport and acidification of the incubation medium (pH 6.4) inhibited mechanically induced epithelium reactions. Both compounds, capsaicin and DMSO modified ionic transport processes depending on the mechanical stimulation.     

Calcium-dependent chloride transient currents in the immature oocyte of the frog, Rana esculenta

Transient currents of chloride were studied in the plasma membrane of immature frog oocyte in voltage clamp conditions. The transients appeared to be activated by an influx of Ca2+ from the external medium. The mechanism leading to a surge of intracellular Ca2+ concentration needed at least 30 sec before full recovery. It was inhibited by substituting Ba2+ for Ca2+ in the external medium, or in the presence of La3+, Co2+ and Cd2+, or when external Na+ was replaced by Li+. Verapamil proved ineffective. The data suggest that an intracellular system of Ca-activated Ca-release is present in the frog oocyte, which can be primarily activated by membrane hyperpolarization via an influx of Ca2+ through non-selective channels.     

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.     

Electrophysiological effect of ruthenium red and pH on the skin of the edible frog, Rana esculenta L

Amphibian skin is a sensitive interface between the organism and the environment. Metal ions from the external environment, some of them being trace elements, act on the amphibian skin. It had been shown that stimulation of tactile receptors affected Na+ transport in the frog skin and changed the potential difference, therefore the aim of this project was to study the effect of ruthenium complex, known as ruthenium red (RR), on the ion transport in this organ in vitro under control conditions, after mechanical stimulation and also in the presence of the Na+ transport inhibitor-amiloride. Three different concentrations of RR (0.12, 1.2, and 12.0 mM) in two different pH values (6.4 and 7.4) were studied in vitro in the Ussing apparatus. The measured electrophysiological parameters were the transepithelial electrical potential difference (PD) and the changes in PD after mechanical stimulation (dPD). The gentle mechanical stimulus was a jet of bath fluid from a peristaltic pump directed on the mucosal surface of isolated frog skin. After mechanical stimulation, transient hyperpolarization invariably occurred (dPD = 1.5 +/- 0.2 mV). In the presence of RR the hyperpolarization was smaller and this diminution was concentration dependent: 0.5 +/- 0.1 mV for 1.2 mM of RR and 0.1 +/- 0.1 mV for 1.2 mM of RR. At pH 6.4 the reactions of the skins on the mechanical and chemical stimuli were smaller, in the presence of amiloride disappearing completely, but after the washing away of amiloride from the experimental organ in pH 6.4 the action of RR was stimulatory. The natural defensive reactions of frog skin related to the ion transport and electrical potential difference are affected or disappear in the presence of ruthenium complex.     

Effect of thallium ions on sodium and potassium transport in frog skin

It has been shown that Tl+ accumulated in the frog skin cells (Rana temporaria) inhibits irreversibly the unidirectional transport of Na+ estimated by the short circuit current (SCC). The inhibiting effect of Tl+ cannot be attributed to a decrease of Na+ penetration through the apical membranes. The influx of 22Na+ from mucosal bathing solution into the skin poisoned with Tl+ was about 50% of that observed in the intact skin, while the SCC was completely inhibited. The activity of the ouabain-sensitive Na+/K+ pumps located in the basolateral cell membranes was estimated by studying the uptake of 86Rb+ as a tracer for K+. This activity was high enough to maintain the ion composition of epithelial cells in spite of their ability to accomplish the undirectional transport of Na+. Tl+ seems to inhibit the production of respiration energy utilized in the undirectional Na+ transport, while the ion homeostasis of epithelial cells may be supported by the Na+/K+ pumps consuming energy of glycolytic reactions.     

Prostaglandin E2-stimulated glandular ion and water secretion in isolated frog skin (Rana esculenta)

Summary Prostaglandins are known to stimulate the active sodium absorption in frog skin. In this paper it is shown that prostaglandin E₂ (PGE₂) stimulates an active secretion of Cl^–, Na⁺, and K⁺ from the skin glands inRana esculenta. The active Cl secretion is enhanced more than the Na and K secretion. Therefore, in skins where the Na absorption is inhibited by amiloride, the addition of PGE₂ results in an increase in the short-circuit current (SCC). The PGE₂-stimulated Cl secretion could be inhibited by the presence of ouabain or furosemide in the basolateral solution or diphenylamine-2-carboxylate in the apical solution. The PGE₂-stimulated Cl secretion was enhanced by the phosphodiesterase inhibitor, theophylline, indicating that the effect of PGE₂ was caused by an increase in the intracellular cAMP level in the gland cells. The calcium ionophore A23187, which increases the PGE₂ synthesis in frog skin, stimulated the glandular Cl secretion. This secretion could be blocked by the prostaglandin synthesis inhibitor indomethacin, indicating that A23187 acts by increasing the prostaglandin synthesis and not by a direct action of Ca²⁺ ionsper se. The net water flow (J _w) and the Cl secretion were measured simultaneously under the conditions outlined above. The stimulation, inhibition, and the time-course of the outward-directedJ _w were similar to the change observed for the Cl secretion. These results show that PGE₂ stimulates a glandular secretion of Cl and water in frog skin, probably by increasing the cAMP level in the gland cells.     

Ovarian activity and reproduction in the frog, Rana esculenta

Rana esculenta specimens were collected, during the last 13 years, in well-defined areas around Naples. The annual ovarian cycle shows distinct phases of recrudescence (starting September; vitellogenesis), breeding (late March-early July; egg deposition and active oogenesis) and quiescence (July-August; no follicular growth). Previtellogenic follicles are recruited for vitellogenesis in early September and in between two successive ovulatory waves. Breeding congregations are generally formed after a heavy rain fall and eggs are laid in standing waters, temporary or permanent. A maximum of three clutches of eggs is produced during the breeding season, at roughly monthly intervals. All mature females reproduce to some extent. Ovarian weight and clutch size are positively correlated to body weight. Depending upon the body size, the potential clutch size ranges from 1000 to 3500 eggs during the first wave of ovulation and it is notably smaller in the successive wave(s) of ovulation. Egg masses and tadpoles are left unprotected and mortality is high. The life cycle from the fertilized egg to completion of metamorphosis is 2 months and oogenesis in the ovary starts in the larva before the onset of metamorphic climax. Young females hatching from the first clutch of eggs may reach sexual maturity and breed in May the following year; those hatching from the last clutch require nearly 20 months to reach sexual maturity. The importance of some endocrine and exocrine factors for the regulation of ovarian activity and reproduction is discussed.