Deviating flux rations for Na+ in ouabain-treated frog skin

Summary Unidirectional Na⁺ fluxes across ouabain-treated frog skins were measured at different applied voltages. The calculated influx/efflux ratios, appear to deviate markedly from Ussing's flux-ratio equation. This means that interactions of Na⁺ ions with some component in the system occur. Possible mechanisms, responsible for this phenomenon, are indicated.with technical assistance of Anneke Adams-Schrijen     

Oxytocin stimulates the apical K+ conductance in frog skin

We measured the effects of oxytocin (0.1 U/ml) on the current (I _sc) recorded through skins ofRana temporaria incubated with an isotonic K⁺ solution on the api al side while the transepithelial potential was clamped to zero. Under these conditions,I _sc is carried by inward K⁺ movements. Oxytocin markedly stimulated this inward K⁺ current. When the spontaneous fluctuations were analyzed we found that oxytocin increased the plateau (S _o ⁽¹⁾) of the spontaneous Lorentzian component without modifying the corner frequency (f _c ⁽¹⁾). Addition of Ba²⁺ to the mucosal solution blockedI _sc both in the presence and absence of oxytocin. Moreover, with mucosal Ba²⁺ a characteristic blocker-induced Lorentzian component appeared in the power spectrum. Analysis of this blocker-induced noise showed that oxytocin increased the number of active K⁺ channels in the apical membrane, while the changes in single channel current were in agreement with expected alterations of the electrochemical driving force.     

Intracellular calcium modulates basolateral K+-permeability in frog skin epithelium

Cytosolic calcium ([Ca²⁺]_i) has been suggested as a key modulator in the regulation of active sodium transport across electrically tight (high resistance) epithelia. In this study we investigated the effects of calcium on cellular electrophysiological parameters in a classical model tissue, the frog skin. [Ca²⁺]_i was measured with fura-2 in an epifluorescence microscope setup. An inhibition of basolateral potassium permeability was observed when cytosolic calcium was increased. This inhibition was reversible upon removal of calcium from the serosal solution.     

Effect of caffeine on K+ efflux in frog skeletal muscle

 The exposure of frog skeletal muscle to caffeine (3–4 mM) generates an increase of the K⁺ (⁴²K⁺) efflux rate coefficient (k _K,o) which exhibits the following characteristics. First it is promoted by the rise in cytosolic Ca²⁺ ([Ca²⁺]_i), because the effect is mimicked by ionomycin (1.25 μM), a Ca²⁺ ionophore. Second, the inhibition of caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum (SR) by 40 μM tetracaine significantly reduced the increase in k _K,o (Δk _K,o). Third, charybdotoxin (23 nM), a blocker of the large-conductance Ca²⁺-dependent K⁺ channels (BK_Ca channels) reduced Δk _K,o by 22%. Fourth, apamin (10 nM), a blocker of the small-conductance Ca²⁺-dependent K⁺ channels (SK_Ca channels), did not affect Δk _K,o. Fifth, tolbutamide (800 μM), an inhibitor of K_ATP channels, reduced Δk _K,o by about 23%. Sixth, Ba²⁺, a blocker of most K⁺ channels, did not preclude the caffeine-induced Δk _K,o. Seventh, omitting Na⁺ from the external medium reduced Δk _K,o by about 40%. Eight, amiloride (5 mM) decreased Δk _K,o by 65%. It is concluded that the caffeine-induced rise of [Ca²⁺]_i increases K⁺ efflux, through the activation of: (1) two channels (BK_Ca and K_ATP) and (2) an external Na⁺-dependent amiloride-sensitive process. Received: 13 March 1998 / Received after revision: 17 June 1998 / Accepted: 14 September 1998     

A voltage-dependent K+ channel controlling the membrane potential in frog oocytes

Full-grown frog ovarian oocytes (Rana esculenta), were voltage clamped with a conventional two-microelectrode system. Depolarizations from a holding potential of –60 mV produced slowly developing outward currents. Two-step clamp experiments showed that, in Ringer's solution, this current has a reversal potential at about –84 mV. Substitution of either sodium or chloride with impermeant ions in the external solution did not alter significantly the activation of the current nor its reversal potential. Increasing the potassium ions concentration caused a shift on the reversal potential in the positive direction with a slope of about 48 mV per decade. The presence of TEA ions (50 mM) in the external solution partially reduced the current. It is concluded that the membrane of full-grown frog ovarian oocytes possesses voltage-dependent ionic channels permeated mainly by potassium. They appear to play an important role in the control of membrane potential.     

Trans- and paracellular K+ transport in diluting segment of frog kidney

In frog diluting segment transepithelial K⁺ net flux (J _te ^K ) occurs via trans- and paracellular transport routes. Inhibition of transcellular K⁺ transport disclosesJ _te ^K across the shunt-pathway. By means of K⁺-sensitive microelectrodes we have measured secretoryJ _te ^K induced by an acute K⁺ load, in the diluting segment of the isolated and doublyperfused frog kidney. Transcellular K⁺ transport was inhibited by blocking the luminal K⁺ permeability either directly by barium or indirectly by the diuretic drug amiloride (via intracellular acidification induced by inhibition of Na⁺/H⁺ exchange), by the the Na⁺/K⁺ pump inhibitor ouabain or by inducing an acute acid load. All experimental maneouvers led to a reduction of secretoryJ _te ^K to about 50% of the controlJ _te ^K . The apparent permeability coefficient for K⁺ of this nephron portion after inhibition of transcellular secretoryJ _te ^K was reduced to a similar extent. We conclude: In frog diluting segment the ratio of trans- over paracellularJ _te ^K is close to unity. This ratio represents a minimum estimate because inhibition of the transcellular K⁺ pathway by barium, amiloride or an acute acid load may have been incomplete. Acidosis and/or amiloride exert large antikaliuretic effects due to the inhibition of the luminal K⁺ permeability.     

Detection of bimodal stimuli in the frog retina

The present report addresses the electrical activity of the frog retina assessed using electroretinogram (ERG) recordings of responses to instantaneous changes from a reference line stimulus with fixed luminance and orientation to a test stimulus consisting of a line of different luminance and orientation. The analysis revealed two types of retinal responses. Responses to onset and offset of the line stimulus were analogous to responses obtained by homogeneous illumination of the retina and were characterized by a high-amplitude b wave (hundreds of microvolts) and significant asymmetry between the b and d waves. At the same time, interaction of these two stimuli yielded more symmetrical b and d waves, with amplitudes an order of magnitude lower, such that this frog retinal activity approached the ERG pattern recorded in response to formed stimuli in higher vertebrates. Analysis of the interaction of the mechanisms detecting the luminance and configurative characteristics of the stimuli, based on construction of V-shaped discrimination functions, showed that when the luminance of the stimulus line was only slightly different from the luminance of the reference line (i.e., discrimination occurred in the retinal stimulus adaptation zone), the activities of the luminance and orientation channels were summed in the ERG. This indicates that these mechanisms function independently and in parallel. From the moment at which the test stimulus luminance became twice that of the reference stimulus, the increase in ERG amplitude grew in a non-linear manner. This two-stage change in amplitude is explained by the existence of two luminance encoding mechanisms in the frog retina, acting simultaneously with the orientation encoding mechanism. One luminance mechanism encodes the action of the stimulus as the discharge power, generating information encoding the absolute level of environmental illumination. The activity of this mechanism is determined mainly by receptors and cells in the outer plexiform layer of the retina. The other mechanism is based on vector encoding of stimulus actions, generating information encoding the spatial and temporal differentiation of light in the visual field and is mainly associated with cells in the inner plexiform layer of the retina. __________ Translated from Zhurnal Vysshei Nervnoi Deyatel’nosti imeni I. P. Pavlova, Vol. 57, No. 1, pp. 65–79, January–February, 2007.     

Single-channel analysis of fast transient outward K+ currents in frog skeletal muscle

 The patch-clamp method was employed to examine the voltage-dependent gating mechanism of A-type K⁺ channels, which generate the transient outward K⁺ currents described previously in a study of vesicles derived from the sarcolemma of frog skeletal muscle. Channels were activated by depolarizing pulses. There is evidence for non-random grouping of records with channel openings and blank records when depolarizations were repeated at brief intervals, suggesting a slow process similar to slow inactivation. Binomial analysis was consistent with independent behaviour of the channels. Ensemble average currents obtained from multichannel patches had kinetics similar to those of the macroscopic A-type K⁺ current, I _A. The rate of activation, fitted to n ⁴ kinetics, was fast and voltage dependent. The rate of inactivation had an exponential time course with a voltage-independent time constant. The mean open time and the probability of a channel being open increased with depolarization. The histograms of latency to first opening revealed the presence of more than two voltage-dependent closed states. Channel openings occurred in bursts and the closed-time histograms could be fitted by the sum of two or three exponentials. These results suggest a gating scheme with at least three closed states, probably two open states, and two inactivated states. Received: 4 November 1997 / Received after revision: 9 January 1998 / Accepted: 12 January 1998     

Modulation of Ca2+-activated K+ channels by Mg2+ and ATP in frog oxyntic cells

Ca²⁺-activated K⁺ channels in the basolateral plasma membrane of bullfrog oxynticopeptic cells are intimately involved in the regulation of acid secretion. Patch-clamp techniques were applied to study the regulating mechanism of these channels. In the excised inside-out configuration, intracellular Mg²⁺ decreased channel activity in a dose-dependent manner. In the absence of Mg²⁺, administration of adenosine 5 triphosphate (ATP) to the cytoplasmic side also inhibited channel activity. On the other hand, in the presence of Mg²⁺, addition of ATP markedly increased channel activity. At a fixed concentration of free Mg²⁺ the Mg-ATP complex caused channel activation and shifted the dose response relationship between channel activity and the intracellular Ca²⁺ concentration to the left. A nonhydrolysable ATP analogue, adenosine 5-[,-imido]triphosphate (AMP-PNP) adenylyl [,-methylene]diphosphate (AMP-PCP), could not substitute for ATP in channel activation, but a hydrolysable ATP analogue, adenosine 5-O-(3-thiotriphosphate) (ATP[S]) could do so. Furthermore, application of alkaline phosphatase to the cytoplasmic side inhibited channel activity. These results demonstrate that Ca²⁺-activated K⁺ channels are regulated by Mg²⁺ and ATP, and suggest that a phosphorylation reaction may be involved in the regulation mechanism of these channels.     

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.     

Passive sugar flux across frog jejunum in vitro

Summary Transmural diffusion of 3-O-methylglucose (3-MG) shows symmetry in bullfrog jejunum perfused in vitro. In both the mucosal to serosal (M to S) and serosal to mucosal (S to M) direction, the permeability coefficient is 1–2×10^–6 cm/sec as measured in isosmotic Na₂SO₄ Ringer's solution containing phlorizin, or K₂SO₄ Ringer's. The coefficient—both from M to S and S to M—increases several fold when osmotic water flow is induced from M to S. This is believed to be due to the opening of intercellular spaces. Furthermore, M to S osmotic flow exerts a solvent drag effect on 3-MG. With S to M osmotic flow which is smaller for the same osmotic driving force and induces no space opening, no permeability change is observed and no significant solvent drag effect. The data, along with measurements of mucosal cell and submucosal tissue concentrations during stationary 3-MG diffusion, indicate that the main resistance to sugar diffusion resides in the mucosal cells, especially in their luminal border. By some isosmotic non-electrolyte Ringer's solutions and moderately hyperosmotic media, the epithelium is progressively destroyed, leading to a time-dependant increase of sugar permeability and a rapid loss of active transport capacity.This work was presented, in part, at the 38. Tagung der Deutschen Physiologischen Gesellschaft, Erlangen, September 1970 [Pflügers Arch.319, R 95 (1970)].     

Apical K+ channels of frog diluting segment: inhibition by acidification

The apical potassium conductance of the amphibian diluting segment is regulated by the intracellular pH. Alkalinisation of the cytosol, whether directly by bathing the cell in an alkaline medium, or following activation of an apical Na/H exchanger by aldosterone, results in an increase in the K conductance. Early distal tubules were isolated from slices of Frog kidneys and the apical membranes exposed by everting the tubule with the aid of microperfusion pipettes. Single channels in the apical membrane were studied in the cell-attached configuration while the tubules were bathed in Ringer with a pH of either 7.4 (control) or 6.6 (acid). Under control conditions single channel currents were readily seen at the resting membrane potential. Upon acidification of the superfusion solution the open probability of the channels was decreased from 0.76 to 0.15. Thus the reduction in apical conductance is brought about, at least in part, by a reduction in the open probability of the channels upon cellular acidification.     

Effect of Ba2+ and furosemide on K+ and Rb+ secretion and absorption in isolated frog skin

The aim of the present investigation was to explore whether in isolated frog skin there should be different pathways for K+ absorption and secretion. Therefore, the unidirectional fluxes of 42K+ and the K+-like isotope 86Rb+ were measured. By using various transport inhibitors, separate pathways for active K+ absorption and secretion were detected. The data obtained indicate that the transepithelial K+ and Rb+ transport across isolated frog skin is made up of four different components: one passive and three active. One of the active components is directed from the apical to the basolateral solution, whereas the other two are in the opposite direction. The direction of the net K+ transport depends on the activities of these three active transport components. The active uptake mechanism, which is present in the epithelial cells, discriminates between K+ and Rb+. The ratio between the K+ and Rb+ influxes, K/Rb, is about 3. The presence of Ba2+, furosemide or ouabain in the apical solution had no effect on the K+ influx. The active secretion of K+ takes place via two different pathways, namely the skin glands and the epithelial cells. The K+ secretion via the glands is inhibited by furosemide (basolateral), but is unaffected by Ba2+ (apical) and does not discriminate between K+ and Rb+. The active K+ secretion via the epithelial cells is blocked by apical Ba2+, and it discriminates between K+ and Rb+. The ratio between the K+ and Rb+ effluxes is about 2. The data presented show that the K+ channels in the apical and the basolateral membrane of the epithelial cells discriminate between K+ and Rb+.(ABSTRACT TRUNCATED AT 250 WORDS)     

β-Adrenergic modulation of the Na+−K+ pump in frog skeletal muscles

Adrenaline markedly increased the ouabain-sensitive 22Na+-efflux by stimulating the Na+-K+ pump in frog skeletal muscle. The facilitatory effects of adrenaline had the following properties. The effects of adrenaline on the ouabain-sensitive Na+-efflux were observed at concentrations greater than 0.1 microM and the magnitude increased with concentration up to 10 microM. At a concentration of 30 microM, adrenaline markedly augmented the ouabain-sensitive Na+-efflux, but other biogenic amines were less effective (noradrenaline and dopamine) or ineffective (histamine and serotonin). The increase of Na+-efflux induced by 1 microM adrenaline was blocked by 3 microM propranolol, but not by 3 microM phenoxybenzamine. The properties of the facilitatory action of adrenaline on the ouabain-sensitive Na+-efflux suggest that beta-adrenoceptors have an important role in modulating the Na+-K+ pump activity in the skeletal muscle membrane. The protein complex localized in excitable membranes, namely the Na+-K+ ATPase-beta-adrenoceptor complex, may be the functional unit which operates the membrane machinery driving the Na+-K+ pump.     

Current-voltage relations of Cs+-inhibited K+ currents through the apical membrane of frog skin

The voltage-dependence of the inhibitory effect of mucosal Cs⁺ on the inward K⁺ current through the apical membrane of frog skin (Rana temporaria) was studied by recording transepithelial current-voltage relations. Experiments were performed with skins exposed to NaCl and KCl Ringer solutions on the serosal and mucosal side respectively (contron skins), as well as with tissues incubated with K₂SO₄ Ringer solutions on both sides (depolarized skins). Studies of the dose-depedence of the Cs⁺ block showed that under both experimental conditions the apparent affinity of Cs⁺ increased as the transepithelial potential was clamped at higher mucosal positive voltages. Under control conditions, the concentration of Cs⁺ required to block 50% of the K⁺ current (K_Cs) recorded while the transepithelial voltage was clamped at zero mV was 16 mmol/1. K_Cs decreased exponentially with muscosal positive voltages. The dependence of K_Cs on the membrane potential was analyzed with Eyring rate theory in which Cs⁺ was assumed to block the K⁺ transport by binding to a site within the channel. The analysis showed that this site is located at a relative electrical distance =0.32 of the voltage drop across the apical membrane, measured from the cytosolic side. The Hill coefficient obtained from this analysis wasn=3.1. Experiments with K⁺-depolarized tissues showed that only inward K⁺ currents recorded with positive transepithelial voltages were depressed by external Cs⁺. Also under these conditions K_Cs showed an exponential dependence on the transepithelial potential. The analysis of these data with the rate theory revealed =0.09 andn=1.7. The difference in found in control and depolarized tissues can be explained by the influence of the basolateral membrane resistance on theI–V relations.     

Non-NMDA receptors in frog retina: an immunocytochemical study

Glutamate is one of the main neurotransmitters in the retina. Its effects are mediated by a large number of ionotropic and metabotropic membrane receptors. The distribution of ionotropic AMPA receptor subunits GluR1-4, kainate receptor subunits GluR5-7 and KA2, delta receptors 1-2, as well as the metabotropic receptor mGluR6 were studied in the frog retina. Indirect immunofluorescence was used to localize the different receptor subunits. Results showed that all subunits, with the exception of GluR1 and GluR5, are widely distributed in the retina. They are mainly located in both plexiform layers: the outer (OPL) and the inner one (IPL), where punctate labelling, a sign of synaptic localization, is observed. The metabotropic receptor mGluR6 is localised only in the OPL. The AMPA receptor subunit GluR4 is localised on the glial Müller cells of the retina. The vast majority of the subunits possess specific patterns of labelling that indicate that they are involved with different retinal functions. The significance of the AMPA receptors and involvement of glia in modulation of synaptic transmission are discussed.     

A local anesthetic, tetracaine, similarly inhibits Ag+ and K+ contractures in frog skeletal muscle

To evaluate usefulness of Ag+ contracture as a tool for elucidating the mechanism underlying the excitation-contraction coupling, the effects of tetracaine on Ag+ contracture were compared with those on K+ and caffeine contractures in frog skeletal muscle. Tetracaine less than 100 microM shortened the duration of 120 mM K+ contracture, without affecting tension amplitude. At higher concentrations of tetracaine, K+ contracture was inhibited dose-dependently and the duration shortened. Treatment of the fibers with 20-500 microM tetracaine for 3 min did not block the contracture induced by 25 mM caffeine. Effects of tetracaine on Ag+ contracture were similar to those on K+ contracture. In the presence of 200 microM tetracaine, 41% inhibition was observed in 120 mM K+ contracture, while 43% in 100 microM Ag+ contracture. Also, 200 microM tetracaine completely inhibited the contractures induced by 40 mM K+ or 5 microM Ag+. These findings suggest that the Ag+ may induce contractures via its action on the T/SR junction, not a direct action on the SR. Therefore, understanding the mechanism involved in the development of Ag+ contracture would be helpful to elucidate the mechanism of excitation-contraction coupling.