KCl Ringer's Solution Research Articles

Stretch-activated cation channel from larval bullfrog skin

Cell-attached patches from isolated epithelial cells from larval bullfrog skin revealed a cation channel that was activated by applying suction (-1 kPa to -4.5 kPa) to the pipette. Activation was characterized by an initial large current spike that rapidly attenuated to a stable value and showed a variable pattern of opening and closing with continuing suction. Current-voltage plots demonstrated linear or inward rectification and single channel conductances of 44-56 pS with NaCl or KCl Ringer's solution as the pipette solution, and a reversal potential (-V(p)) of 20-40 mV. The conductance was markedly reduced with N-methyl-D-glucamide (NMDG)-Cl Ringer's solution in the pipette. Neither amiloride nor ATP, which are known to stimulate an apical cation channel in Ussing chamber preparations of larval frog skin, produced channel activation nor did these compounds affect the response to suction. Stretch activation was not affected by varying the pipette concentrations of Ca(2+) between 0 mmol l(-1) and 4 mmol l(-1) or by varying pH between 6.8 and 8.0. However, conductance was reduced with 4 mmol l(-1) Ca(2+). Western blot analysis of membrane homogenates from larval bullfrog and larval toad skin identified proteins that were immunoreactive with mammalian TRPC1 and TRPC5 (TRPC, canonical transient receptor potential channel) antibodies while homogenates of skin from newly metamorphosed bullfrogs were positive for TRPC1 and TRPC3/6/7 antibodies. The electrophysiological response of larval bullfrog skin resembles that of a stretch-activated cation channel characterized in Xenopus oocytes and proposed to be TRPC1. These results indicate this channel persists in all life stages of anurans and that TRP isoforms may be important for sensory functions of their skin.

Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium.

Inward-rectifier K channel: using macroscopic voltage clamp and single-channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current-voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell-attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. under physiological ionic gradients, a 15-pS inward-rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.

Differential modulation of Ca2(+)-activated K+ channels by substance P

Substance P released from motoneurons or primary afferent nerves innervating the gut is an excitatory noncholinergic regulator of gastrointestinal motility and has been shown to stimulate contractions in longitudinal colonic muscle. By using the patch-clamp technique on single myocytes from the longitudinal muscle of the rabbit colon, we have studied the action of the peptide on membrane conductances. In cell-attached patches, addition of the peptide to the bath activates a large conductance Ca2(+)-activated K+ channel. At peptide concentrations (10(-12) M) that did not result in cell contraction, K+ channels were activated in a synchronized, cyclical fashion. The activation did not occur in the presence of nifedipine (10(-6) M), a blocker of dihydropyridine-sensitive Ca2+ channels. The activation was also absent when the cells were depolarized in 126 mM KCl-Ringer solution. In contrast, at a concentration of the peptide (10(-7) M) that resulted in cell contraction, there was a transient activation of K+ channels, followed by a prolonged inhibition. Nifedipine (10(-6) M) did not block the K+ channel activation by this concentration of the peptide. Removal of bath Ca2+ abolished activation of the K+ channels by both the high and low concentrations of substance P. These results indicate that substance P exerts its effect via two different membrane pathways for extracellular Ca2+.

Effect of amiloride on the poorly selective cation channel of larval bullfrog skin

A small, inward-directed, short-circuit current (SCC) was measured across the isolated skin of larval bullfrogs (Rana catesbeiana) when either NaCl or KCl Ringer solution bathed the mucosal surface. The addition of amiloride, in concentrations of 1-100 microM, produced a stepwise increase in SCC. As SCC values became maximally elevated by amiloride, the plateau value (So) of the Lorentzian component in the power-density spectrum increased, whereas the corner frequency (fc) decreased. This agonist effect of amiloride can be explained by an increase in the open probability and possibly the single-channel current of the larval channel. When the amiloride concentration was increased above 100 microM, the SCC values declined progressively but usually remained above pretreatment values. This suggests an antagonist effect of amiloride that is concurrent with the agonist effect. The removal of Ca2+ from the mucosal Ringers increased SCC in conjunction with an increase in So and a decrease in fc. Under these conditions, the maximal agonist effect of amiloride was observed at concentrations of 10-20 microM. Ca2+ thus exerts an inhibitory effect on the larval cation channel that interferes with the agonist effect of amiloride. The addition of Ba2+ to Ca2+-free preparations lowered SCC and reduced the agonist effect of amiloride.

Physiological role of apical potassium ion channels in frog skin.

The K+ permeability of the apical membrane of frog skin (Rana temporaria) was analysed by recording the short-circuit current and its fluctuations in the presence of a mucosa-to-serosa-oriented K+ concentration gradient. Loading of the animals with KCl resulted in an augmentation of the Ba2+-blockade component of the short-circuit current and the plateau value of the K+-dependent relaxation noise. Poisoning of active transport and exposing both sides of the epithelium to KCl Ringer solution caused an increase of the K+ current and its fluctuations recorded after restoring the inward-oriented K+ gradient. Serosal quinidine (5 X 10(-4) M), which is thought to increase intracellular Ca2+ activity, depressed the K+ current and the relaxation noise. This effect was completely reversible. Removal of Na+ from the serosal solution, which is known to result in an elevation of intracellular Ca2+ by abolishing the driving force for the Na+/Ca2+ exchanger, also reduced the K+ current and the Lorentzian plateau. Both parameters returned to their control values after restoring the Na+ gradient across the basolateral membranes. It is concluded from these experiments that the apical K+ permeability is controlled by factors which depend on the intracellular K+ and Ca2+ concentration and that the apical K+ channels may constitute a pathway for K+ secretion.

Potassium-dependent chloride and water transport across the seawater eel intestine

Simultaneous measurements of net ion and water fluxes and transepithelial potential difference (PD) were made in the stripped intestine of the seawater eel, and it was examined whether Cl- was driven following electrochemical gradient for Na+ across the brush border membrane of the epithelium or not. When mucosal Na+ was completely replaced with K+, while the serosa was being bathed with normal Ringer's solution, net Cl- and water fluxes were maintained as high as those in normal Ringer's solution. After serosal Na+ was completely replaced with choline+ while the mucosa was being bathed with Na+-free KCl Ringer's solution, 80% of the original Cl- and water fluxes still persisted, indicating significant Na+-independent Cl- and water transport. These results are against a hypothesis that Cl- is driven by electrochemical gradient of Na+ across the brush border membrane. The Na+-independent Cl- and water fluxes were a saturable function of mucosal K+ concentration, suggesting K+-dependent Cl- and water transport. A possible mechanism of Cl- transport is discussed in relation to K+ transport. On the other hand, a good correlation was observed between the net Cl- and water fluxes. This suggests that water transport depends on Cl- transport system; NaCl and/or KCl cotransport.

Electrical properties of the rabbit urinary bladder assessed using gramicidin D.

Recently, antibiotics have enjoyed widespread usage as tools in studies of epithelial transport. In the present study we assess the usefulness of the pore-forming antibiotic gramicidin D as a means for probing the electrical properties of the tight epithelium rabbit urinary bladder. Addition of 50 micron gramicidin to the mucosal bath (either a NaCl or KCl Ringer's solution) led to a large irreversible increase in the transepithelial conductance (GT) with 800 sec. GT increased by approximately 1200% and 500% in KCl and NaCl Ringer's solutions, respectively. Microelectrode measurements of the resistance ratio (the ratio of apical membrane resistance to basolateral membrane resistance) showed that apical membrane resistance is decreased by the drug. Measurements of the basolateral membrane resistance (Rbl) and tight junctional resistance (Rj) using a new and independent method (based on the perturbation of basolateral membrane electrogenic Na+ pump) demonstrated that Rbl and Rj were unaffected, suggesting that the effects of gramicidin are restricted to the apical membrane for periods of at least 2 hours after drug addition. The selectivity of the gramicidin-induced permeability in the apical membrane was calculated from measurements of the apical membrane potential after ion substitutions using a modified version of the constant field equation. The selectivity sequence for cations was Cs+ greater than K+ greater than Na+ greater than Li+ greater than choline. Unlike the commonly used polyene antibiotics nystatin and amphotericin B, gramicidin did not induce a significant Cl- permeability. In addition, the dose-response curve had a slope of 1. A method is described for calculating membrane resistances directly from transepithelial measurements under some conditions of gramicidin use, without requiring the use of microelectrode measurements.

Stabilizing-labilizing effects of reserpine, chlorpromazine, tetracaine, and quinidine on frog skeletal muscle fibers

In order to characterize the interaction of reserpine, chlorpromazine, tetracaine, and quinidine with membrane phenomena, electrophysiological studies were performed on frog skeletal muscle fibers under various ionic environments. In addition the influence of these drugs on the contractility of the frog skeletal muscle was investigated. 1. Measurements of intracellular membrane potentials of the sartorius muscle fibers revealed that tetracaine, quinidine, chlorpromazine, and reserpine diminished the depolarizing action of high [K+]0: while the resting potential in normal Ringer's solution fell from −90 mV to −9.3 mV when 100 mM NaCl was replaced by the same amount of KCl, a time and dose-dependent increase of the membrane potential depolarized by potassium was observed after addition of these drugs. The order of the polarizing potency was reserpine > chlorpromazine > tetracaine > quinidine. 2. Maximal stabilizing effects (i. e. increase of the membrane potential in 100 mM KCl Ringer's solution to more negative values) were observed with the following drug concentrations (20 min incubation time): reserpine 10−4 M (13 mV increase); chlorpromazine 3 X104 M (21 mV increase); quinidine 3 × 10−3 M (15 mV increase); tetracaine 10−2M (28 mV increase). At higher concentrations, reversion of the polarizing effects occurred as a consequence of labilization of the muscle membrane. 3. Evidence for such a biphasic effect, i.e. stabilizing-labilizing effect of the drugs came also from measurements of the relative membrane resistanceRr. In 100 mM KCl Ringer's solution tetracaine in concentrations up to 10−3 M caused an absolute increase. Higher concentrations produced again a subsequent fall ofRr. Quinidine, chlorpromazine and reserpine inhibited only the time-dependent decrease ofRr normally found in control preparations. 4. In chloride-deficient 112 mM KCH3S04 Ringer's solution, tetracaine produced a several fold higher increase ofRr. Under these experimental conditions also quinidine and chlorpromazine caused an absolute enhancement ofRr, but not reserpine. These findings indicate that chiefly the potassium conductancegK of the muscle fiber membrane was affected by tetracaine, chlorpromazine, and quinidine. 5. Tetracaine, quinidine, chlorpromazine, and reserpine inhibited the KCl-induced contractions of the sartorius muscle as well as the acetylcholine-induced mechanical activity of the frog rectus abdominis muscle. 6. From the results it is concluded that the different drugs used in this investigation may exert their effects by a rather common mechanism, i.e. via membrane stabilization; this is indicated by the observed impairment of the membrane permeability for cations.

Sodium azide stimulated potassium efflux from striated muscles.

Sodium azide (NaN3) in concentrations as low as 0.25 mM was capable of altering potassium fluxes in frog sartorius muscle fibers. Influx of potassium was maximally reduced in the presence of 2.0 mM NaN3 to between 32 and 39% of control. All concentrations of azide tested increased potassium efflux, but above 2.0 mM NaN3 the rate constant for potassium (K-42) efflux increased continuously. After the addition of 3.0 mM NaN3 to the normal Ringer's solution the rate constant for potassium efflux increased from 267% to 358% over a 2 h period. The stimulation of potassium efflux by sodium azide was always greater than the reduction of potassium influx. Net intracellular sodium was unaltered by axide concentrations below 2.0 mM, but markedly increased in the presence of higher azide concentrations. Muscles were rapidly depolarized by sodium azide in both 2.5 mM KCl and 5.0 mM KCl Ringer's solution. At higher azide concentrations (>2.0 mM) the depolarization was constant and did not reflect the continuous loss of potassium ions. The loss of intracellular potassium which was not completely compensated by the gain of sodium was probably due to an increase in potassium permeability.