Determination of intracellular K+ activity in rat kidney proximal tubular cells

The intracellular K⁺ activity of rat kidney proximal tubular cells was determined in vivo, using intracellular microelectrodes. In order to minimize damage from the impaling electrodes, separate measurements on separate cells, were performed with single-barrelled KCl-filled non-selective electrodes and single-barrelled, K⁺-sensitive microelectrodes, which were filled with a liquid K⁺-exchanger resin that has also a small sensitivity to Na⁺. Both electrodes had tip diameters of 0.2 m or below. The proper intracellular localization of the electrodes was ascertained by recording the cell potential response to intermittent luminal perfusions with glucose. The membrane potential measured with the non-selective microelectrodes was –76.3±8.1 mV (n=81) and the potential difference measured with the K⁺-sensitive microelectrode was –7.2±5.8 mV (n=32). Based on the activity of K⁺ in the extracellular fluid of 3 mmol/l the intracellular K⁺ activity was estimated to be 82 mmol/l. Assuming equal K⁺-activity coefficients to prevail inside and outside the cell, this figure suggests that the intracellular K⁺ concentration is 113 mmol/l which must be considered as a lower estimate, however. The data indicate that the K⁺-ion distribution between cytoplasm and extracellular fluid is not in equilibrium with the membrane potential, but that K⁺ is actively accumulated inside the cell. This result provides direct evidence for the presence of an active K⁺ pump in the tubular cell membranes, which in view of other observations, must be envisaged as a (not necessarily electroneutral) Na⁺/K⁺-exchange pump which operates in the peritubular cell membrane and is eventually responsible for the major part of the tubular solute and water absorption.     

Short-term desensitization of muscarinic K+ channel current in isolated atrial myocytes and possible role of GTP-binding proteins

The short-term desensitization of the acetylcholine (ACh)-induced K⁺ channel current was examined in single atrial cells of guinea-pig heart. The tight-seal whole cell voltage clamp technique was used. The solution in the pipettes contained GTP or guanosine-5-O-(3-thiotriphosphate) (GTP-S, a non-hydrolyzable GTP analogue). In GTP-loaded cells, ACh evoked a specific K⁺ channel current via GTP-binding proteins (G) in a dose-dependent manner. The K⁺ current showed agonist-dependent desensitization similar to those reported in other cardiac tissues (Nilius 1983; Carmeliet and Mubagwa 1986). The cellular response to ACh was also desensitized by activation of P1-purinergic receptors with adenosine (Ado). In GTP-S-loaded cells, the K⁺ current was gradually induced even in the absence of agonists, probably due to direct activation of G proteins by GTP-S. In the early phase of the spontaneous current increase, ACh evoked a large current transiently. As the GTP-S-induced activation of the current progressed, the magnitude of the ACh-evoked current transient became smaller and finally negligible. Similar results were obtained when Ado was used as an agonist instead of ACh to induce the K⁺ current. Therefore, it is indicated that the agonistreceptor interaction may not be essential for the desensitization of ACh-induced K⁺ current in atrial myocytes.     

A new class of inhibitors of cAMP-mediated Cl− secretion in rabbit colon,acting by the reduction of cAMP-activated K+ conductance

Previously we have shown that arylamino-benzoates like 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which are very potent inhibitors of NaCl absorption in the thick ascending limb of the loop of Henle, are only poor inhibitors of the cAMP-mediated secretion of NaCl in rat colon. This has prompted our search for more potent inhibitors of NaCl secretion in the latter system. The chromanole compound 293 B inhibited the equivalent short-circuit current (I _sc) induced by prostaglandin E₂ (n=7), vasoactive intestinal polypeptide (VIP,n=5), adenosine (n=3), cholera toxin (n=4) and cAMP (n=6), but not by ionomycin (n=5) in distal rabbit colon half maximally (IC₅₀) at 2 mol/l from the mucosal and at 0.7 mol/l from the serosal side. The inhibition was reversible and paralleled by a significant increase in transepithelial membrane resistance [e.g. in the VIP series from 116±16 ·cm² to 136±21 ·cm² (n=5)]. A total of 25 derivatives of 293 B were examined and structure activity relations were obtained. It was shown that the racemate 293 B was the most potent compound with-in this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 mol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 mol/l 293 B had no effect on the membrane voltage across the basolateral membrane (V _bl) in non-stimulated crypt cells: –69±3 mV versus –67±3 mV (n=10), whilst in the same cells 1 mmol/l Ba²⁺ depolarised (V _bl) significantly. However, 293 B depolarised (V _bl) significantly in the presence of 1 mol/l forskolin: –45±4mV versus –39±5 mV (n=7). Similar results were obtained with 0.1 mmol/l adenosine. 293 B depolarised (V _bl) from –40±5 mV to –30±4 mV (n=19). This was paralleled by an increase in the fractional resistance of the basolateral membrane. VIP had a comparable effect. The hyperpolarisation induced by 0.1 mmol ATP was not influenced by 10 mol/l 293 B: –75±6 mV versus –75±6 mV (n=6). Also 293 B had no effect on basal K⁺ conductance (n=4). Hence, we conclude that 293 B inhibits the K⁺ conductance induced by cAMP. This conductance is apparently relevant for Cl^– secretion and the basal K⁺ conductance is insufficient to support secretion.     

Positive cooperativity in activation of the cardiac muscarinic K+ channel by intracellular GTP

Concentration-dependent effects of intracellular GTP on activation of the muscarinic K⁺ channel were examined in inside-out patches of cardiac atrial myocytes. The pipette solution contained 0.1 M ACh. GTP (0.01–30 M) and 0.5 mM MgCl₂ were applied to the inside side of the patch membrane. K⁺ channels were activated with GTP concentration above 0.1 M. Channel activation reached a maximal value with 1–3 M GTP. It decreased at GTP concentrations larger than 3 M, probably due to desensitization. The dependence of the open probability of the channel on intracellular GTP showed a sigmoidal relationship with a Hill coefficient of around 3. A positive cooperative effect of intracellular GTP on the K⁺ channel may play an important role in amplifying the signal from the membrane receptor to the K⁺ channel.     

Potassium conductance of smooth muscle cells from rabbit aorta in primary culture

Vascular smooth muscle cells were obtained from rabbit aorta and were studied in primary culture on days 1–7 after seeding with electrophysiological techniques. In impalement experiments a mean membrane potential difference (PD) of –50±0.3 mV (n=387) was obtained with Ringer-type solution in the bath. PD was depolarized by 6±0.3 mV (n=45) and 16±2 mV (n= 5) when the bath K⁺ concentration was increased from the control value of 3.6 mmol/l to 13.6 and 23.6 mmol/l, respectively. Ba²⁺ (0.1–1 mmol/l) depolarized PD. Tetraethylammonium (TEA, 10 mmol/l) depolarized PD only slightly but significantly. Verapamil (0.1 mmol/l) and charybdotoxin (10 nmol/l) had no effect on PD. The conductance properties of these cells were further examined with the patch-clamp technique. K⁺ channels were spontaneously present in cell-attached patches. When the pipette was filled with 145 mmol/l KCl, a mean conductance (g _K) of 209.6±4.6 mV (n=17) was read from the current/voltage curves at a clamp voltage (V _c) of 0 mV. After excision K⁺ channels were found in 129 patches with inside-out and in 50 with outside-out configuration. With KCl on one and NaCl on the other side the mean g _K at a V _c of 0 mV was 134.6±3.9 pS (n=179). The mean permeability was 0.89±0.03×10^–12 cm³/s. With symmetrical KCl solution the mean g _K was 227±6 pS (n=17). The conductance sequence was g _K g _Rb= g _Cs=g _Na=0. TEA blocked dose-dependently only from the outside.(1–10 mmol/l). Lidocaine (5 mmol/l) quinidine (0.01–1 mmol/l) and quinine (0.01–1 mmol/l) blocked from both sides. Charybdotoxin (0.5–5 nmol/l) blocked only from the extracellular side. Ba²⁺ blocked from the cytosolic side and the inhibition was increased by depolarization and reduced by hyperpolarization. At a V _c of 0 mV a half-maximal inhibition (IC₅₀) of 2 mol/l was obtained. Verapamil and diltiazem blocked from both sides, verapamil with an IC₅₀ of 2 mol/l and diltiazem with an IC₅₀ of 10 mol/l. The open probability of this channel was increased by Ca²⁺ on the cytosolic side at activities > 0.1 mol/l. Half-maximal activation occurred at Ca²⁺ activities exceeding 1 mol/l. The present data indicate that the vascular smooth muscle cells of rabbit aorta in primary culture possess a K⁺ conductance. In excised patches only a maxi K⁺ channel was detected. This channel has properties different from the macroscopic K⁺ conductance. Hence, it is likely that the K⁺ conductance of the intact cell is dominated by yet another and thus far not detected K⁺ channel.Supported by DFG Gr 480/10     

Potassium channels in the basolateral membrane of the rectal gland ofSqualus acanthias

The present study examines the influences of pH and Ca²⁺ and several putative inhibitors on the basolateral K⁺ channel of the rectal gland ofSqualus acanthias. Excised membrane patches were examined using the patch clamp technique. It is shown that reduction of the calcium activity on the cytosolic side to less than 10^–9 mol/l has no detectable inhibitory effect on this channel. Conversely, increase in calcium activity to some 10^–3 mol/l reduced the activity of this channel. Variations in cytosolic pH had only a moderate effect on the current amplitude: alkalosis by one pH unit increased and acidosis reduced the single current amplitude by some 15%. Several inhibitors were tested in excised patches when added to the cytosolic side. Ba²⁺ (5·10^–3 mol/l), quinine (10^–3 mol/l), quinidine (10^–4 mol/l), lidocaine (1 mmol/l), tetraethylammonium (10 mmol/l), Cs⁺ (10 mmol/l), and Rb⁺ (20 mmol/l) all blocked this K⁺ channel reversibly. We conclude that the basolateral K⁺ channel of the rectal gland is distinct from other epithelial K⁺ channels inasmuch as it is not stimulated by Ca²⁺ directly, but that it is qualitatively similar to many other known K⁺ channels with respect to its sensitivity towards blockers.This study was supported by Deutsche Forschungsgemeinschaft Gr 480/8 and by NSF and NIH grants to the Mount Desert Island Biological Laboratory     

Different inhibitions of the voltage-dependent K+ current by Ca2+ antagonists in the smooth muscle cell membrane of rabbit small intestine

Actions of Ca²⁺ antagonists (verapamil, nicardipine and diltiazem) on the voltage-dependent K⁺ current, obtained from the fragmented smooth muscle cell membrane (smooth muscle ball; SMB) of the rabbit small intestine, were investigated using voltage clamp techniques. To eliminate the influence of the Ca²⁺-dependent K⁺ current, the voltage-dependent K⁺ current was recorded in 2.5 mM Mn²⁺ (Ca²⁺ omitted) solution. These three Ca²⁺ antagonists inhibited the peak amplitude of the K⁺ current, in a dose-dependent manner. During application of a long command pulse (duration, 3 s), the amplitude of the voltage-dependent K⁺ current decreased slowly with time. Diltiazem inhibited the K⁺ current with a slight prolongation of the 20% decay time, while TEA (tetraethyl-ammonium), a K⁺ channel blocker, inhibited the current, without affecting the decay. By contrast, verapamil and nicardipine accelerated inactivation. In the control, the voltage-dependent inactivation was also seen in the K⁺ current. This inactivation curve below 0 mV was not modified by 10 M diltiazem, 5 M verapamil nor 3 M nicardipine. These results indicate that inhibition of the voltage-dependent K⁺ current by verapamil or nicardipine differed from that by diltiazem.An abstract of this work was reported at the 59th general meeting of Japan Pharmacological Society (Terada et al. 1986).     

G-protein-mediated regulation of a Ca2+-dependent K+ channel in cultured vascular endothelial cells

The purpose of the present study was to determine the mechanism by which bradykinin activates the small conductance, inwardly rectifying, Ca²⁺-activated K⁺ channel (K_Ca) found in cultured bovine aortic endothelial cells. Channel activity was studied using the patch-clamp technique in whole-cell, cell-attached, inside-out and outside-out configurations. Channel conductance at potentials positive to 0 mV was 10±2 pS and at potentials negative to 0 mV 30±3 pS (n=7) when examined in symmetrical K⁺ (150 mmol/l) solutions. The channel open probability (P _o) was only weakly voltage dependent changing approximately 0.2 units over 160 mV. In contrast, raising the intracellular Ca²⁺ concentration from 100 nmol/l to 10 mol/l at –60 mV produced a graded increase in channel P _o from 0.15 to 0.96; the concentration required for half-maximum response (apparent K_0.5) was 719 nmol/l. At a constant Ca²⁺ concentration, application of guanosine triphosphate (GTP) to the cytoplasmic surface of the patch increased channel P _o. This effect was dependent upon the simultaneous presence of both GTP and Mg²⁺, and was reversed by the subsequent application of the guanosine diphosphate (GDP) analogue, guanosine-5-O-(2-thiodiphosphate) (GDPS). The hydrolysis-resistant GTP analogue, guanosine-5-O-(3-thiotriphosphate) (GTPS), induced a long-lasting increase in channel P _o. In the presence of Mg²⁺-GTP, the apparent K_0.5 for Ca²⁺ decreased from a control value of 722 nmol/l to 231 nmol/l. Addition of bradykinin to outside-out patches previously exposed to intracellular Mg²⁺-GTP further enhanced K_Ca activity, shifting the apparent K_0.5 for Ca²⁺ from 228 nmol/l to 107 nmol/l. This activation by bradykinin was not observed in patches following prior exposure to GDPS. These results suggest that bradykinin can activate the K_Ca channel of vascular endothelial cells via a G-protein-mediated change in the sensitivity of the channel for Ca²⁺. We postulate that vasoactive agonists may use this mechanism to maintain an elevated K⁺ permeability as the intracellular Ca²⁺ concentration returns towards normal resting levels.     

Demonstration of a novel apamin-insensitive calcium-activated K+ channel in mouse pancreatic B cells

The whole-cell configuration of the patchclamp technique was used to characterize the biophysical and pharmacological properties of an oscillating K⁺-current that can be induced by intracellular application of GTP[S] in mouse pancreatic B cells (Ämmälä et al. 1991). These K⁺ conductance changes are evoked by periodic increases in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and transiently repolarize the B cell, thus inhibiting action-potential firing and giving rise to a bursting pattern. GTP[S]-evoked oscillations in K⁺ conductance were reversibly suppressed by a high (300 M) concentration of carbamylcholine. By contrast, ₂-adrenoreceptor stimulation by 20 M clonidine did not interfere with the oscillatory behaviour but evoked a small sustained outward current. At 0 mV membrane potential, the oscillating K⁺-current elicited by GTP[S] was highly sensitive to extracellular tetraethylammonium (TEA; 70% block by 1 mM). The TEA-resistant component, which carried approximately 80% of the current at –40 mV, was affected neither by apamin (1 M) nor by tolbutamide (500 M). The current evoked by internal GTP[S] was highly selective for K⁺, as demonstrated by a 51-mV change in the reversal potential for a sevenfold change in [K⁺]_o. Stationary fluctuation analysis indicated a unitary conductance of 0.5 pS when measured with symmetric ( 140mM) KCl solutions. The estimated singlechannel conductance with physiological ionic gradients is 0.1 pS. The results indicate the existence of a novel Ca²⁺-gated K⁺ conductance in pancreatic B cells. Activation of this K⁺ current may contribute to the generation of the oscillatory electrical activity characterizing the B cell at intermediate glucose concentrations.     

Inhibition of voltage-dependent Na+ and K+ currents by forskolin in nodes of Ranvier

The effect of forskolin on voltage-activated Na⁺ and K⁺ currents in nodes of Ranvier from the toad, Bufo marinus, has been examined using the vaseline-gap voltageclamp technique. Peak Na⁺ currents (I _Na) were reduced by 35% and the rate of decline of Na⁺ current during continuous depolarization was accelerated following treatment with 450 M forskolin. However, the voltage-dependence of steady-state inactivation as well as the rate of recovery from fast inactivation remained unchanged. Upon repetitive depolarization at 1–10 Hz, a further inhibition of I _Na (60%) was observed. This use-dependent or phasic inhibition recovers slowly at -80 mV ( 13 s) and had a voltage-dependence like that of activation of the Na conductance. Near maximal steady-state phasic inhibition occurred with depolarizing pulse durations of only 4 ms, consistent with a direct involvement of the open Na⁺ channel in the blocking process. Inhibition of the delayed K⁺ current (I _K) was characterized by a concentration-dependent reduction in steady-state current amplitude (IC₅₀ 80 M) and a concentration-independent acceleration of current inactivation. A similar inhibition of I _K was obtained with 1,9-dideoxyforskolin, a homolog which does not activate adenylate cyclase (AC). The results suggest that the inhibition of I _K and perhaps I _Na follows directly from drug binding and is not a consequence of AC activation.     

Electrogenic Na+/K+-transport in human endothelial cells

Na⁺/K⁺ pump currents were measured in endothelial cells from human umbilical cord vein using the whole-cell or nystatin-perforated-patch-clamp technique combined with intracellular calcium concentration ([Ca²⁺]_i) measurements with Fura-2/AM. Loading endothelial cells through the patch pipette with 40 mmol/l [Na⁺] did not induce significant changes of [Ca²⁺]_i. Superfusing the cells with K⁺-free solutions also did not significantly affect [Ca²⁺]_i. Reapplication of K⁺ after superfusion of the cells with K⁺-free solution induced an outward current at a holding potential of 0 mV. This current was nearly completely blocked by 100 mol/l dihydroouabain (DHO) and was therefore identified as a Na⁺/K⁺ pump current. During block and reactivation of the Na⁺/K⁺ pump no changes in [Ca²⁺]_i could be observed. Pump currents were blocked concentration dependently by DHO. The concentration for half-maximal inhibition was 21 mol/l. This value is larger than that reported for other tissues and the block was practically irreversible. Insulin (10–1000 U/l) did not affect the pump currents. An increase of the intracellular Na⁺ concentration ([Na⁺]_i) enhanced the amplitude of the pump current. Half-maximal activation of the pump current by [Na⁺]_i occurred at about 60 mmol/l. The concentration for half-maximal activation by extracellular K⁺ was 2.4±1.2 mmol/l, and 0.4±0.1 and 8.7±0.7 mmol/l for Tl⁺ and NH₄ ⁺ respectively. The voltage dependence of the DHO-sensitive current was obtained by applying linear voltage ramps. Its reversal potential was more negative than –150 mV. Pump currents measured with the conventional whole-cell technique were about four times smaller than pump currents recorded with the nystatin-perforated-patch method. If however 100 mol/l guanosine 5-O-(3-thiotriphosphate) (GTPS) were added to the pipette solution, the currents measured in the ruptured-whole-cell-mode were not significantly different from the currents measured with the perforated-patch technique. We suppose that the use of the perforated-patch technique prevents wash out of a guanine nucleotide-binding protein (G-protein)-connected intracellular regulator that is necessary for pump activation.     

Activation of atrial muscarinic K+ channels by low concentrations ofβγ subunits of rat brain G protein

Effects of G protein subunits from rat brain on cardiac K⁺ channel was examined in single atrial cells of guinea-pig, using patch clamp techniques. We found that 10 pM concentration of rat brain subunits preparation could activate the atrial muscarine receptor-gated K⁺ channel (I_K.ACh). Neither the detergent, CHAPS, used to suspend nor the boiled preparation activated I_K.ACh. Furthermore, preincubation of subunits preparation in Mg²⁺-free solution, which easily inactivated -GTP-S, did not affect -activation of I_K.ACh. We concluded, therefore, that subunits themselves can activate I_K.ACh.Supported by the grants from the Ministry of Education, Culture and Science of Japan and from the Calcium Signal Workshop on Cardiovascular Systems     

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.     

Quinidine blockade of calcium-activated potassium channels in dissociated gastric smooth muscle cells

The effects of quinidine, an antiarrhythmic alkaloid, on potassium-selective channels in enzymatically dissociated gastric smooth muscle cells fromRana pipiens andBufo marinus were investigated using excised patches and the patch-clamp technique. The predominant potassium channel in these cells is the calcium- and voltage-activated maxi-K channel with a single-channel conductance > 100 pS. Applications of quinidine (100–600 M) resulted in resolvable rapid flickerings between the open and blocked states with a corresponding reduction in open channel amplitude and an increase in open channel noise. The currentvoltage curves in the presence of internal quinidine and symmetrical potassium gradients displayed inward rectification. The time-constant of open-time distributions was found to decrease with increasing quinidine concentrations and membrane depolarization. The power-density spectrum of the channel current noise induced by internal quinidine showed a second Lorentzian component with a corner frequency larger than 300 Hz, suggesting that the noise is caused by rapid fluctuations between the open and blocked states. Apparent dissociation constants of 253 M and 209 M for membrane potentials of +20 mV and –60 mV, respectively, were obtained for the quinidine-induced blockade of Ca²⁺-activated K⁺ channels in these smooth muscle cells. Another potassium-selective channel with a single-channel conductance of 40 pS was completely blocked in the presence of 100 M qunidine. However, a 15 pS potassium channel was not affected by quinidine but was reversibly blocked by tetraethylammonium. Quinidine (500 M) was also observed to decrease the opening probability of a 40 pS potassium channel fromBufo marinus without affecting its channel amplitude. Thus, quinidine appears to have diverse mechanisms of action on potassium-selective channels in smooth muscle cells, ranging from totally ineffective to highly selective, as a slow blocker for some channels and as intermediate and fast blockers for others.     

Polarized ion transport during migration of transformed Madin-Darby canine kidney cells

Epithelial cells lose their usual polarization during carcinogenesis. Although most malignant tumours are of epithelial origin little is known about ion channels in carcinoma cells. Previously, we observed that migration of transformed Madin-Darby canine kidney (MDCK-F) cells depended on oscillating K⁺ channel activity. In the present study we examined whether periodic K⁺ channel activity may cause changes of cell volume, and whether K⁺ channel activity is distributed in a uniform way in MDCK-F cells. After determining the average volume of MDCK-F cells (2013±270 m³; n=8) by means of atomic force microscopy we deduced volume changes by calculating the K⁺ efflux during bursts of K⁺ channel activity. Therefore, we measured the membrane conductance of MDCK-F cells which periodically rose by 22.3±2.5 nS from a resting level of 6.5±1.4 nS (n=12), and we measured the membrane potential which hyperpolarized in parallel from –35.4±1.2 mV to –71.6±1.8 mV (n=11). The distribution of K⁺ channel activity was assessed by locally superfusing the front or rear end of migrating MDCK-F cells with the K⁺ channel blocker charybdotoxin (CTX). Only exposure of the rear end to CTX inhibited migration providing evidence for horizontal polarization of K⁺ channel activity in transformed MDCK-F cells. This is in contrast to the vertical polarization in parent MDCK cells. We propose that the asymmetrical distribution of K⁺ channel activity is a prerequisite for migration of MDCK-F cells.     

The effect of known K+-channel blockers on the electrical activity of bovine lymphatic smooth muscle

The effects of known K⁺-channel blockers on the electrical properties of bovine lymphatic smooth muscle were investigated using the double sucrose-gap technique. Constant current anodal pulses elicited hyperpolarizing electrotonic potentials (EP's) which were characterised by a sag in the potential record. Current/voltage relationship (I/V), which were examined by measuring EP amplitude at the end of 5 s anodal pulses (<30 A), showed an apparent increase in conductance with increasing hyperpolarization. In the presence of caesium (10 mM), 4-aminopyridine (10 mM) or in the absence of external K⁺ the sag in the EP was lost and the inward rectification characteristic of the control I/V relationship was abolished. Barium (2.5 mM) also abolished in sag in the EP although TEA (10 mM) had no effect on either EP shape or I/V relationship. Thus it would appear that lymphatic smooth muscle shows inward rectification which is slowly activating and is blocked by some of the known K⁺-channel blockers or by the removal of external K⁺.     

Tubular transport processes in proximal tubules of hypothyroid rats. Lack of relationship between thyroidal dependent rise of isotonic fluid reabsorption and Na+−K+-ATPase activity

Hypothyroid rats reconstituted with 10 g/kg b.w. per day of tri-iodothironine (T₃) for 4 days resulted in normal free T₃ and TSH levels. FT₃ levels were: 0.53±0.3 pg/ml in hypothyroid rats; 2.78±1.21 pg/ml in hormone reconstituted rats and 2.90±0.90 pg/ml in euthyroid rats. TSH levels were 3,508±513 g/ml in hypothyroid rats; 1,008±204 g/ml in reconstituted rats and 270±184 ng/ml in euthyroid rats.When hypothyroid rats were reconstituted with 50 g T₃/kg b.w. per day, TSH levels were nearly normal after 4 days (1,157±621 ng/ml). However FT₃ levels after 1–4 days were always higher than in euthyroid rats.Hypothyroid rats show a decrease in isotonic fluid reabsorption (J _v) in the proximal tubule (1.50±0.08 versus 4.96±0.23 10^–2 nl·mm^–1·s^–1 in euthyroid animals). 1 day after T₃ (10 g/kg b.w./day) injectionJ _v was increased significantly to 2.05±0.20 10^–2 nl·mm^–1·s^–1 and continued to increase during 4 days of T₃ reconstitution.When 50 g T₃/kg b.w./day was used,J _v increased to 2.75±0.07 after 1 day and to 3.10±0.42 10^–2 nl·mm^–1·s^–1 after 4 days.J _v was never reaching a value close to that of euthyroid rats because the tubular radius in hypothyroid rats (14.7±1.8 m) is less than that of euthyroid rats (19.2±0.5 m). The radius in hypothyroid rats treated with T₃ was unchanged over a 4 day course with either high or low doses of T₃.Na⁺–K⁺-ATPase activity was found to be 2.91±0.16 M P_i/h×mg protein in homogenates of kidney cortex from hypothyroid rats. Treatment of hypothyroid rats with 10 g or 50 g of T₃ resulted in an initial decrease in ATPase activity, followed by an increase to base level in hypothyroid rats with 10 g and a significantly higher level with 50 g. This decrease in ATPase activity was contrasted to the increase inJ _v.These data indicate that there is a dissociation between the effects of physiological doses of thyroid hormones on proximal tubular reabsorption and the effects of T₃ on Na⁺–K⁺-ATPase activity of kidney cortex. This leads to question the relationship between sodium transport and ATPase activity under physiological doses of thyroid hormones. An early effect of physiological doses of thyroid hormones on brush border Na⁺ permeability is suggested.     

Kinetic studies on the effects of ouabain on Na+ fluxes in frog skin

Among 48 pieces of paired frog skins ofRana pipiens in Ringer's solution, 10 pieces showed a strictly monotone decrease in the short circuit current (SCC) following ouabain treatment (10^–4 M). In 9 cases a transient attenuation, and in 27 cases a distinct wave in the ebb of the SCC, was seen. In 2 instances, two waves were seen. Associated with the not-monotone events was a transient rise in electrical skin conductance. The reasons for these mixed skin responses are unknown. One possible reason is considered here: Early during the ouabain action, some of the Na⁺ entering from the mucosal side is trapped in the skin by electroneutral processes, in keeping with the already known fact that ultimately cellular KCl is partly replaced by NaCl. Computer assisted model studies show how monotone, and not-monotone transepithelial net Na⁺ flux curves can be generated. Essential conditions for the generation of notmonotone Na⁺ flux curves are: 1. Presence of two distinct cellular, active Na⁺ pools in the model. 2. Presence of a loop pathway in which a principal transepithelial Na⁺ transport compartment, and a constitutent Na⁺/K⁺ maintenance compartment, are connected to each other and to the extracellular compartment. The model, then, predicts under which kinetic conditions monotone and not-monotone transepithelial Na⁺ flux curves will be seen.     

Microelectrode study of voltage-dependent Ba2+ and Cs+ block of apical K+ channels in the skin of Rana temporaria

The blockage of the apical K⁺ channels in frog species Rana temporaria by Ba²⁺ and Cs⁺ is strongly voltage-dependent. The interaction of both blockers with the K⁺ channels was studied by recording relations between the K⁺ currents (I _K) and the transepithelial and intracellular potential. Mucosal Ba²⁺ and Cs⁺ depress I _K, hyperpolarize the cell and induce pronounced nonlinearities in the current/voltage (I/V) relations. The nonlinearities are caused by the voltage-dependent interaction of Ba²⁺ and Cs⁺ with the binding site. Consequently, the apical membrane resistance not only depends on the blocker concentration but also on the apical membrane potential. Also the fractional resistance, fR _a, and the voltage divider ratio, fV _a, will change with blocker concentration and voltage. Owing to this non-ohmic behaviour, measurements of fV _a in the presence of Ba²⁺ deviate markedly from the expected fR _a values. The inhibitory effect of Ba²⁺ and Cs⁺ was analysed at different transepithelial and apical membrane voltages. The relation between the Michaelis-Menten constants and the voltage could be fitted with equations based on Eyring rate theory with the assumption of a single binding site. With this model we calculated the relative electrical position of the binding site for the blocker (), referred to the extracellular side of the channel. We obtained for Ba²⁺, =0.34±0.05 and for Cs⁺, =0.81±0.01. Comparison of the results from apical and transepithelial I/V relations demonstrates that the analysis of the transepithelial data provides overestimated values of the Hill coefficient and results in an underestimation of .     

Transient changes in the size of the extracellular space in the sensorimotor cortex of cats in relation to stimulus-induced changes in potassium concentration

Summary The time course of local changes of the extracellular space (ES) was investigated by measuring concentration changes of repeatedly injected tetramethylammonium (TMA⁺) and choline (Ch⁺) ions for which cell membranes are largely impermeable. After stimulus-induced extracellular [K⁺] elevations the [TMA⁺] and [Ch⁺] signals recorded with nominally K⁺-selective liquid ion-exchanger microelectrodes increased by up to 100%, thus indicating a reduction of the ES down to one half of its initial size. The shrinkage was maximal at sites where the K⁺ release into the ES was also largest. At very superficial and deep layers, however, considerable increases in extracellular K⁺ concentration were not accompanied by significant reductions in the ES. These findings can be explained as a consequence of K⁺ movement through spatially extended cell structures. Calculations based on a model combining the spatial buffer mechanism of Kuffler and Nicholls (1966) to osmolarity changes caused by selective K⁺ transport through primarily K⁺ permeable membranes support this concept.Following stimulation additional iontophoretically induced [K⁺]_o rises were reduced in amplitude by up to 35%, even at sites where maximal decreases of the ES were observed. This emphasizes the importance of active uptake for K⁺ clearance out of the ES.This investigation was supported by DFG grants Lu 158/10 and He 1128/1