Reversed short-circuit current across isolated skin of the toadBufo arenarum

Short-circuit current (SCC) aross isolated pelvic skin of the toadBufo arenarum has been shown to be reflected by the algebraic sum of net sodium and chloride transport. After the animals had been maintained in tap water, amiloride — an apical sodium channel blockerled to a reversal of potential difference (rPD) across this preparation, to which corresponded a reversed short-circuit current (rSCC). Both rSCC and rPD were abolished by dermal treatment of skins with the metabolic inhibitor dinitrophenol, or by omission of chloride ion from the Ringer solution bathing both sides of the skin. There was a significant positive correlation between rSCC and isotopically determined net chloride transport after amiloride. An inhibitory action of amiloride on unidirectional chloride fluxes was detected, but only early after drug addition. rSCC was absent in skins of toads exposed to 110 mmol/l NaCl in tap water during 10 days. Together, our results suggest that amiloride addition —by inhibiting active sodium movement — can in certain conditions reveal the existence of an inward active chloride transport.     

Passive transepithelial cationic fluxes across the frog skin under short-circuit conditions

The short-circuit current (Isc) across the frog skin in the steady state reflects the active Na+ transport. Inhibition of the Na+-K+ pump by ouabain causes slow decay of Isc. It has been suggested that this slow ouabain effect on Isc could be due to the asymmetric ionic permeations of frog skin. That is, Na+ flux at the apical membrane and K+ flux at the basolateral membrane transiently generate the transepithelial cationic fluxes and are measured as the Isc even under the condition of active Na+ transport arrest. However, this hypothesis on the transient Isc has not been studied experimentally. In the present study, transient inward and outward Isc were observed alternately even after pump arrest by changing the ionic compositions of the bathing solutions in a Ussing's chamber. The time constant of Isc decay was 20-30 min. The Isc responses were quicker and stronger on the isolated epithelia than on the whole skin. Both Isc responses were blocked by amiloride, a Na+ channel blocker. Measurements of the ionic composition of isolated epithelia under experimental conditions indicate that the passive Na+ flux across the apical membrane and the passive K+ flux across the basolateral membrane cause both transient inward and outward Isc under the ouabain-treated condition of frog skin.     

Endogenous prostaglandins, adenosine 3':5'-monophosphate and sodium transport across isolated frog skin.

1. Sodium transport across isolated frog skin, as measured by the short-circuit current, was decreased by acetylsalicylic acid, mefenamic acid, paracetamol and phenylbutazone. Indomethacin (6 X 10(-6) M) had a biphasic effect on the short-circuit current: a transient increase followed by a sustained decrease. 2. The release of prostaglandin-like material from the skin was reduced by acetylsalicylic acid and indomethacin. Paracetamol caused a significant reduction in the short-circuit current response of the skin to low doses of arachidonic acid, but the response to the highest dose tested was not significantly altered. 3. Indomethacin (6 X 10(-6) M) increased the sensitivity of the skin to applied prostaglandin E1. The other prostaglandin synthetase inhibitors did not have this effect. Indomethacin (6 X 10(-6) M) also enhanced the effect of antidiuretic hormone on the short-circuit current. 4. Indomethacin (30 X 10(-6) M) increased the short-circuit current and diminished the response to applied prostaglandin E1. 5. In sulphate Ringer, theophylline increased the short-circuit current and diminished the response to prostaglandin E1. 6. Prostaglandin E1 increased the levels of cyclic AMP in frog skin and these increases preceded the increases in short-circuit current. There was a seasonal variation in the level of cyclic AMP in the skin: the levels in winter exceeded those in summer. There was also a seasonal variation in the cyclic AMP response to prostaglandin E1: the winter response was greater than that in summer. 7. Indomethacin (6 X 10(-6) M) had a biphasic effect on cyclic AMP levels in the skin, an initial increase followed by a decrease. Indomethacin also potentiated prostaglandin E1 stimulated cyclic AMP accumulation. 8. Theophylline increased cyclic AMP levels in the skin and potentiated prostaglandin E1 stimulated cyclic AMP accumulation. 9. Pre-treatment of the skin with theophylline reversed the effects of cyclic AMP on the short-circuit current and open-circuit potential. 10. It is concluded that endogenous prostaglandins help to maintain sodium transport across isolated frog skin and that the effects of E-type prostaglandins on the short-circuit current are mediated by increased cyclic AMP levels. The transient increase in short-circuit current and the increased skin sensitivity caused by indomethacin (6 X 10(-6) M) are attributed to inhibition of phosphodiesterase activity. The failure of theophylline to potentiate the short-circuit current response of the skin to prostaglandin E1 is attributed to alteration of cyclic AMP action on the skin by theophylline.     

Inhibition of short-circuit current by triaminopyrimidine in isolated toad urinary bladder.

The organic cation 2,4,6-triaminopyrimidine (TAP) produced inhibition of short-circuit current (SCC) when added to either the mucosal or serosal surface of the isolated urinary bladder of the toad. Fifty percent inhibition was produced by 10(-3) M TAP in the mucosal solution at pH 6.8 when the mucosal [Na+] was 113 mM. The actions of TAP resemble those produced by amiloride in several ways: a) inhibition of SCC by mucosal application is rapid; b) the mucosal inhibition is fully reversible; c) high concentrations in the serosal solutions produce irreversible inhibition; and d) the concentration required to produce 50% inhibition from the mucosal side is reduced when mucosal [Na+] is reduced. It is postulated that mucosal application of TAP and amiloride inhibit short-circuit current in high-resistance epithelia via action at a common locus.     

Inotropic responses of the isolated frog ventricle to serum

A study has been made of the effects of newborn calf-serum on the isolated frog ventricle. Preparations were superfused with solutions containing different concentrations of serum and the changes in contractility measured. Ordinary calf-serum evoked a dose-dependent increase in contractile force which was unaffected by adrenergic receptor antagonists (propranolol and phentolamine). Serum which was either used in growing HeLa cells or incubated with chymotrypsin failed to elicit a positive inotropic response. However, if prostaglandin-like materials were extracted from the serum then its ability to potentiate the twitch remained unaltered. These results suggest that the cardioactive-factor present in calf-serum is most probably related to a polypeptide, and possibly a growth factor.     

The potential and short-circuit current across isolated rumen epithelium of the sheep.

1. A technique is described for working with isolated sheets of rumen epithelium. Epithelium was obtained at operations for rumen fistulation or in acute experiments and was rapidly mounted between two cone-shaped chambers each of which held 40 ml. of bathing fluid. The fluids were gassed with 95% O2/5% CO2 and 20 cm2 of epithelium was exposed to the fluids. 2. Electrical measurements were made of (a) the potential generated across the epithelium, (b) the short-circuit current which flowed and (c) the resistance of the epithelium. 3. The results are grouped according to the composition of the sheep Ringer solution used to bathe the epithelium. The most stable preparation was obtained when the solutions contained 5 mM each of glucose, acetate, propionate and butyrate as well as bicarbonate and phosphate buffers. 4. In Cl- media, the average initial p.d., short-circuit current (s.-c.c.) and resistance were 9.3 mV, 11.6 microA/cm2 and 1156 omega x cm2 respectively. Higher values of 17.6 mV, 17.3 microA/cm2 and 1501omega x cm2 were recorded in SO4(2-) media. In both media, the s.-c.c. declined at approximately 1.5 microA/cm2. hr and preparations were suitable for study over periods from 4 to 6 hr. 5. When the concentration of potassium was varied on either side of the epithelium at constant [Na+], the potential showed a linear relation with log [K+] for both sides of the epithelium. 6. Changing the sodium concentrations at constant [K+] on either side of the epithelium caused negligible alterations in the potential. 7. Exclusion of sodium from the media bathing both sides of the epithelium abolished the potential and s.-c.c. 8. The addition of ouabain to the blood side of the preparation abolished the potential and s.-c.c. whereas on the rumen side these parameters were unaffected.     

Tizolemide-induced changes of passive transport components across the basolateral membrane of isolated frog skin

The effect on transepithelial Na transport of tizolemide was investigated in isolated frog skin (Rana temporaria). It was found that tizolemide (2–5 mM, serosal side) decreased transepithelial Na transport (measured as short circuit current and as net sodium flux) within 60 min to 25–40% of the control level resulting from reduction of the unidirectional sodium influx. Intracellular recording with microelectrodes revealed that these changes were associated with depolarization of the intracellular space to less than 40% of the control values (averaging –71.7±5.1 mV) which is a consequence of a decrease in conductance of the basolateral border to about 25% of the control values. The conductance of the apical border was only slightly reduced. It is suggested that tizolemde blocks the partial conductance of potassium at the basolateral border which secondarily diminishes trans-epithelial Na transport due to a decrease of the driving force for apical border Na entry. A certain degree of inhibition of the Na-K-ATPase by tizolemide cannot be excluded. When vasopressin (ADH) was added to frog skin after treatment with tizolemide, the response was markedly reduced compared to that of untreated control preparations. Under these conditions, the conductance of the basolateral border increased while the apical border remained little influenced by the hormone — opposite to the response of frog skins under control conditions. It is concluded that the mode of action of ADH is more complex than has been recognized hitherto and includes effects at the basolateral border.Dedicated to Professor H. Schwiegk on the occassion of his 75th birthday.Some of these results have been presented at the 8. Int. Congr. Néphrology, Athens 1981.     

A comparison of the responses of frog skin receptors to mechanical and electrical stimulation

1. The latencies of spike responses evoked alternatively by brief mechanical (M) and electrical (E) pulses applied to single mechanoreceptive terminals in frog skin were compared on the same receptor.2. Latency was found to be a maximum at threshold and to decrease with increased stimulus strength for both modes of excitation, but at all strengths M latency exceeded E latency. Mean maximum and minimum values for M latency were 4.8 and 2.85 msec; for E latency the maximum was 2.8 and minimum 2.3 msec.3. At high frequency and strength of E stimulation there was an abrupt and marked shortening of latency to a fixed minimum value which ranged from 0.5 to 1.2 msec (mean 0.8). This was taken to be the response of the parent myelinated axon excited directly. The gap (1.5 msec) between the minimum value for the receptor response (2.3 msec) and the axonal response (0.8 msec) was taken to represent conduction time in the terminal branches of the sensory axon.4. The response latency for excitation of the sensory terminal was also dependent on the duration of the stimulus pulse, but whereas the latency range for the M stimulus could be greatly extended that for the E stimulus was only slightly affected by increase in pulse duration.5. The responses evoked by direct currents were complex, and consisted of an early brief discharge at the start of a cathodal current followed after a delay of 5-30 sec by a prolonged multi-fibre discharge which out-lasted the stimulus. It is proposed that the sensory terminal is rapidly accommodating to current flow and that the delayed discharge is due to release of chemical material.6. It is suggested that delay in mechanical excitation may be due to non-rigid coupling of the receptor terminal to the skin tissues.     

Volume regulatory responses in frog isolated proximal cells

Cells respond to increases in volume by activating solute efflux pathways, resulting in water loss and restoration of the original cell volume. The solute efflux pathways underlying these volume regulatory decrease (VRD) responses have been relatively well studied. However, the transduction pathways whereby the change in cell volume is converted into an intracellular signal resulting in VRD are much less well understood. We have examined VRD in isolated proximal tubule cells from the frog, with particular attention to the roles of stretch-activated channels, Ca²⁺ and protein kinases. Cell length was taken as an index of cell volume, and was measured continuously using a photodiode array. VRD was observed in approximately 50% of cells, and was inhibited by Ba²⁺, Gd³⁺ and 4,4-diisothiocyanatostilbene 2,2-disulphonic acid (DIDS), and removal of extracellular Ca²⁺. VRD was accelerated by the active phorbol ester, phorbol 12-myristate 13-acetate (PMA), and the phosphatase inhibitor F^–; on the other hand, VRD was prolonged by 4-phorbol 12,13-didecanoate (PDC), an inactive phorbol ester), and inhibited by PMA and Gd³⁺, PMA and 0 Ca²⁺, and staurosporine. Volume regulation was unaffected by di-butyryl cAMP and 3-isobutyl-1-methyl-xanthene (IBMX). These data suggest that Ca²⁺ and PKC, via protein phosphorylation, play a stimulatory role in VRD.     

Force responses to rapid length changes in single intact cells from frog heart.

1. Force transients in response to step perturbations in length were recorded in intact atrial cells from frog heart at various temperatures (6-15 degrees C). Length changes of various sizes and in either direction, complete in 0.5 ms, were applied to single myocytes near slack length (initial sarcomere length 2.1-2.2 microns) just before the peak of an isometric twitch. The frequency response of the force transducers used was 2-4 kHz in Ringer solution. 2. An early quick force recovery phase was clearly observed after the elastic force response to the length step and before the start of much slower recovery processes. The quick recovery phase became progressively faster with larger shortening steps and was almost as fast as that originally described in intact frog skeletal muscle fibres (rate constants above 1000 s-1 in large releases at 10 degrees C). 3. The force-extension relation determined at the end of the length change (T1 curve) indicates that an instantaneous shortening of 0.5-0.6% of the initial cell length (L0) brings the force to zero. The force--extension relation determined at the end of the quick recovery phase (T2 curve) showed that the early recovery leads to an almost complete restoration of the original force with small stretches and releases (up to 0.3% L0) and that it becomes negligible in shortening steps of about 1.4% L0. 4. The results suggest that the mechanical properties of attached cross-bridges and the rate of transitions between attached cross-bridge states are approximately the same in frog atrial cells and fast skeletal muscle fibres.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na and K movements across the membranes of frog skin epithelia associated with transient current changes

Summary The ionic composition of the current crossing each membrane of the frog skin epithelia during a) the positive and transient current responses elicited by sudden addition of Na ions to the outer Na free medium b) the negative and transient current responses observed when Na loaded preparations are suddenly exposed to outer Na free solution was determined using isotopic techniques. It is shown that Na ions carry the current across the outer membrane while K ions are mainly involved in the transfert of charges across the inner membrane. The amount of Na accumulated by the epithelial cells during the responses is correlated to the area under the transient part of the current responses. Determinations of²⁴Na uptake at different time of these transient positive responses show that the unidirectional Na influx across the outer membrane decreases as a function of time. It is suggested that the intracellular Na concentration might control the Na uptake mechanism across the outer membrane. During the negative responses, the Na efflux into the outer medium is highly correlated, either in time course or magnitude, to the current response. Both Na efflux and negative current are sensitive to amiloride, suggesting that the mechanism of Na uptake by the frog skin also is able to promote Na movement out of the epithelial cells.     

Sodium transport across the isolated epithelium of the frog skin

1. A method to separate the epithelium from the underlying layers of the frog skin is described. The method is based on the combined use of collagenase and hydrostatic pressures.2. The potential difference and the short-circuit current values of isolated epithelia and whole skins are similar. Na net flux and short-circuit current are equivalent.3. The time course of changes in potential following rapid changes in composition of the bathing solutions shows that the barrier to K diffusion at the internal surface of the isolated epithelium is larger than the barrier to Na diffusion at the external surface.4. In the isolated epithelium there are 133 m-mole K(+) and 24.7 m-mole Na/l. cellular water. The amount of extracellular water was considered to be equal to the inulin space.5. Arginine vasopressin (0.1 u./ml.) markedly increased short-circuit current and potential difference in isolated epithelia. The amount of Na in the epithelium that equilibrated with Na in the external solution was not increased by the hormone.6. Ouabain (10(-4)M) reduced short circuit current and potential difference to values close to zero. The ouabain treated epithelia contained an increased amount of Na originating in the internal solution. On the other hand the amount of Na that originated from the external solution was not increased.7. The amount of epithelial Na that equilibrated with Na in the external solution was 0.009 mu-equiv/cm(2). This figure is about ten times smaller than the values found in whole skins.     

Tension responses to rapid length changes in skinned muscle fibres of the frog

Tension responses due to rapid length changes completed in 50 and 150 s were obtained from activated skinned single fibres of the ileofibularis muscle of the frog. The natural frequency of the force transducer was about 50 kHz. The length changes ranged between –1% and +0.5% of the fibre segment length. The sarcomere length was adjusted to 2.15 m. The temperature was maintained at 2.5° C. The transmission velocity estimated from these recordings obtained on fibre segments with different length was 230 m/s in fully activated segments and 112 m/s in relaxed segments. The initial part of the responses during the length changes consisted of an abrupt change in tension reaching an extreme value T₁, which depended on the amplitude as well as the duration of the length change. A partial rapid recovery towards a plateau occurred after the length change. The reciprocal half-time of this recovery increased with the amplitude of the displacement both for stretches as well as releases up to about 5 nm/half sarcomere.     

The mechanism of lithium accumulation in the isolated frog skin epithelium

Summary The intracellular concentration of Li ([Li]c) of epithelia isolated from frog skins was determined after a one hour exposure to external Na free Choline Ringer containing from 1 to 25 mM of LiCl. In both open and short circuit conditions, [Li]c was found to have values up to ten times that of the outside Li concentration, indicating accumulation of Li in the epithelia. A loss of an approximately equivalent amount of cellular K was associated with this Li accumulation, whereas no significant change in epithelial content of Na was observed. This accumulation of Li is reduced if Na is present together with Li in the outer solution. Preincubation of the epithelia with Amiloride (10^–4 M) suppressed short circuit current and potential difference changes consecutive to externali solutions exposure, prevented Li accumulation and K loss and reduced transepithelial movements of Li. The entry of Li into the cell was speeded up by oxytocin (20 mU/ml). In cyanide (10^–4 M) pretreated epithelia, Li uptake was reduced and accumulation failed to occur. 2,4 dinitrophenol (5×10^–4 M) also lowered the Li uptake. It is concluded that the main mechanism for monovalent cations entry into the epithelium is a process for which Na and Li compete. The existence of an active transport step at the level of the outward facing membrane of the frog skin epithelium is proposed.     

Ba2+-induced changes in the Na+- and K+-permeability of the isolated frog skin

Addition of the K+-channel blocking agent Ba2+ to the basolateral solution (in a concentration which is assumed to block the K+-flux via the K+-channels completely) resulted initially in a two-thirds reduction in the short-circuit current (SCC), followed by a complete recovery of the SCC. To examine the reason for this recovery, experiments were carried out which made it possible to calculate the Na+-permeability of the apical membrane (PaNa) and the K+-permeability of the basolateral membrane (PbK). The presence of Ba2+ had no significant effect on the cell volume and the cellular Na+- and K+-concentration. Addition of Ba2+ resulted in a depolarization of the intracellular potential (VSCC) from a control value of -76.3 +/- 2.8 mV to -15.1 +/- 1.7 mV. Although a complete recovery in the SCC was observed, VSCC did not recover. The K+-flux across the basolateral membrane was estimated from washout experiments. The washout of 42K+ (the K+-efflux) could be described by a single exponential component with a half time of 30-70 min. The addition of Ba2+ during the washout resulted in a transient decrease in 42K+-efflux from the epithelium. From VSCC and the cellular K+ and Na+-concentration and the coupling ratio of the Na-K pump, it was found that Na+-permeability of the apical membrane was 6.5 X 10(-7) cm X s-1 before the addition of Ba2+ and 1.7 X 10(-6) cm X s-1 when the SCC had recovered after the addition of Ba2+ and PbK changed from 8.8 X 10(-6) cm X s-1 to 1.5 X 10(-6) cm X s-1. Thus, the observed recovery in SCC was due to a considerable increase in Na+-permeability of the apical membrane and the presence or appearance of a small Ba2+-insensitive K+-permeability in the basolateral membrane.