Hyperpolarizing current of the Na/K ATPase contributes to the membrane polarization of the Purkinje cell in rat cerebellum

 The contribution of the Na/K ATPase (pump) current to the polarization of the Purkinje cell has been studied using slices of the rat cerebellum by blocking the pump with dihydro-ouabain (DHO) while recording the membrane potential with microelectrodes in the somata. From our recordings, it appeared that blocking the pump depolarized the Purkinje cells more rapidly than might be expected from shifts in Na⁺ and K⁺ concentrations, suggesting the removal of a hyperpolarizing current. Application of DHO, in the presence of tetrodotoxin (TTX), led to calcium spike firing and plateau-like discharges suggesting activation of voltage-dependent calcium channels in the dendrites. Adding 2 mM Co²⁺ to the medium did not prevent the depolarizations. Removing calcium from the bathing medium containing 2 mM Co²⁺ blocked the spiking activity but DHO application still produced a depolarization. Experiments to measure the current inhibited by DHO indicated that the Na/K pump supplies a constant current of 240 pA. Substitution of the sodium with choline produced a hyperpolarization, during which DHO had no effect on the membrane potential. Substitution of the sodium with lithium produced only a slowly developing depolarization. It is concluded that in the cerebellar Purkinje cell, a continuous sodium ion influx activates the pumps which produce a current that directly contributes to the membrane polarization. Possible pathways for this sodium influx are discussed. Received: 3 April 1997 / Received after revision and accepted: 12 May 1997     

A rapidly inactivating Ca2+-dependent K+ current in pheochromocytoma cells (PC12) of the rat

The membrane electrical properties of undifferentiated pheochromocytoma cells of the rat (PC12) were studied using both current-and voltage-clamp techniques with the use of low-resistance blunt-tipped micropipettes (patch electrodes). In the presence of tetrodotoxin (TTX, 2–3 M), a spike-like wave form with a prominent after-hyperpolarization (AHP) was recorded following brief (< 10 ms) depolarizing current pulses. The inorganic divalent cations, Cd²⁺ (0.5 mM), Mn²⁺ (4mM), and 0 mM Ca²⁺/4 mM Mg²⁺ solution prolonged the duration, attenuated the AHP, slowed the rate of repolarization, and slightly enhanced the amplitude of this wave form. A rapidly inactivating outward current was recorded in over 70% of the cells under voltage-clamp conditions. This transient current was elicited at about ±30 mV, and was blocked by tetraethylammonium (5 mM), inorganic divalent cations (Cd²⁺, 0.5 mM; Mn²⁺, 4 mM; Ba²⁺, 3 mM), and removal of Ca²⁺ (0 mM Ca²⁺/4 mM Mg²⁺) from the local perfusion medium. In addition, 4-aminopyridine (5 mM), which blocks the transient outward K⁺ current I_A in a variety of excitable cells, did not have any appreciable effect on this rapidly inactivating current. Moreover, it was possible to elicit the current at a holding potential of ±40 mV. The reversal potential of this current was ±90 mV, and shifted positively when extracellular K⁺ concentrations were elevated. It is concluded that PC12 cells have a rapidly inactivating Ca²⁺ -dependent K⁺ current. A possible explanation for the transient nature of this current may be the presence of an effective intracellular Ca²⁺ buffering (uptake or extrusion) system.     

Demonstration of an inwardly rectifying K+ current component modulated by thyrotropin-releasing hormone and caffeine in GH3 rat anterior pituitary cells

 Reduction of an inwardly rectifying K⁺ current by thyrotropin-releasing hormone (TRH) and caffeine has been considered to be an important determinant of electrical activity increases in GH₃ rat anterior pituitary cells. However, the existence of an inwardly rectifying K⁺ current component was recently regarded as a misidentification of an M-like outward current, proposed to be the TRH target in pituitary cells, including GH₃ cells. In this report, an inwardly rectifying component of K⁺ current is indeed demonstrated in perforated-patch voltage-clamped GH₃ cells. The degree of rectification varied from cell to cell, but both TRH and caffeine specifically blocked a fraction of current with strong rectification in the hyperpolarizing direction. Use of ramp pulses to continuously modify the membrane potential demonstrated a prominent blockade even in cells with no current reduction at voltages at which M-currents are active. Depolarization steps to positive voltages at the maximum of the inward current induced a caffeine-sensitive instantaneous outward current followed by a single exponential decay. The magnitude of this current was modified in a biphasic way according to the duration of the previous hyperpolarization step. The kinetic characteristics of the current are compatible with the possibility that removal from inactivation of a fast-inactivating delayed rectifier causes the hyperpolarization-induced current. Furthermore, the inwardly rectifying current was blocked by astemizole, a potent and selective inhibitor of human ether-á-go-go -related gene (HERG) K⁺ channels. Along with other pharmacological and kinetic evidence, this indicates that the secretagogue-regulated current is probably mediated by a HERG-like K⁺ channel. Addition of astemizole to current-clamped cells induced clear increases in the frequency of action potential production. Thus, an inwardly-rectifying K⁺ current and not an M-like outward current seems to be involved in TRH and caffeine modulation of electrical activity in GH₃ cells. Received: 15 May 1997 / Received after revision and accepted: 24 July 1997     

Caffeine and histamine-induced oscillations of K(Ca) current in single smooth muscle cells of rabbit cerebral artery

In the present experiment, we characterized the intracellular Ca²⁺ oscillations induced by caffeine (1 mM) or histamine (1–3 M) in voltage-clamped single smooth muscle cells of rabbit cerebral (basilar) artery. Superfusion of caffeine or histamine induced periodic oscillations of large whole-cell K⁺ current with fairly uniform amplitudes and intervals. The oscillatory K⁺ current was abolished by inclusion of ethylenebis(oxonitrilo)tetraacetate (EGTA, 5 mM) in the pipette solution. Caffeine- and histamine-induced periodic activation of the large-conductance Ca²⁺-activated K⁺ [K_(Ca)] channel was recorded in the cell-attached patch mode. These results suggest that the oscillations of K⁺ current are carried by the K_(Ca) channel and reflect the oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Ryanodine (1–10 M) abolished both caffeine- and histamine-induced oscillations. Caffeine- induced oscillations were abolished by the sarcoplasmic reticulum Ca²⁺-adenosine 5-triphosphatase (Ca²⁺-ATPase) inhibitor, cyclopiazonic acid (10 M), and a high concentration of caffeine (10 mM). Inclusion of heparin (3 mg/ml) in the pipette solution blocked histamine-induced oscillations, but did not block caffeine-induced oscillations. By the removal of extracellular Ca²⁺, but not by the addition of verapamil and Cd²⁺, the caffeine-induced oscillations were abolished. Increasing Ca²⁺ influx rate increased the frequencies of caffeine-induced oscillations. Spontaneous oscillations were also observed in cells that were not superfused with agonists, and had similar characteristics to the caffeine-induced oscillations. From the above results, it is concluded, that in smooth muscle cells of the rabbit cerebral (basilar) artery, ryanodine-sensitive Ca²⁺-induced Ca²⁺ release pools play key roles in the generation of caffeine- and histamine-induced intracellular Ca²⁺ oscillations.     

A long lasting Ca2+ -activated outward current in guinea-pig atrial myocytes

Among other characteristics, the steady-state current-voltage relationship of patch-clamped single atrial myocytes from guinea-pig hearts is defined by an outward current hump in the potential region –15 to +40mV. This hump was reversibly suppressed by Co²⁺ (3 mM) or nitrendipine (5 M) and enhanced by Bay K 8644 (5 M). The maintained outward current component suppressed by Co²⁺ extended between –15.2±1.9 mV and +39.5 ±1.7 mV (mean±SEM of 14 cells) and has an amplitude of 95.7±9.4 pA at +10 mV. In isochronal I-V curves, the hump was already visible at 400 ms with essentially the same amplitude as at 1500 ms. The Co²⁺ -sensitive outward current underlying the hump was poorly time-dependent during 1.5 s voltage pulses but slowly relaxed upon repolarization. Tail currents reversed near the K⁺ equilibrium potential under our experimental conditions. The current hump of the steady-state I-V curve was also abolished by caffeine (10 mM) or ryanodine (3 M), both drugs that interfere with sarcoplasmic reticulum function. Apamin (1 M) or quinine (100 M) but not TEA (5–50 mM) markedly reduced its amplitude. However, at similar concentrations as required to inhibit the hump, both apamin and quinine appeared to be poorly specific for Ca²⁺ -activated K⁺ currents in heart cells since they also inhibited the L-Type Ca²⁺ current. It is concluded that a long lasting Ca²⁺ -activated outward current, probably mainly carried by K⁺ ions but not sensitive to TEA, exists in atrial myocytes which is responsible for the current hump of the background I-V curve.     

Dual effect of temperature on the human epithelial Na+ channel

The amiloride-sensitive epithelial sodium channel (ENaC) is the rate-limiting step for sodium reabsorption in the distal segments of the nephron, in the colon and in the airways. Its activity is regulated by intracellular and extracellular factors but the mechanisms of this regulation are not yet completely understood. Recently, we have shown that the fast regulation of ENaC by the extracellular [Na⁺], a phenomenon termed self-inhibition, is temperature dependent. In the present study we examined the effects of temperature on the single-channel properties of ENaC. Single-channel recordings from excised patches showed that the channel open probability (P _o, estimated from the number of open channels N·P _o, where N is the total number of channels) increased on average two- to threefold while the single-channel conductance decreased by about half when the temperature of the perfusion solution was lowered from ~30 to ~15 °C. The effects of temperature on the single-channel conductance and P _o explain the changes of the macroscopic current that can be observed upon temperature changes and, in particular, the paradoxical effect of temperature on the current carried by ENaC.     

Classifying cases of fetal wolff-parkinson-white syndrome by estimating the accessory pathway from fetal magnetocardiograms

The paper presents an evaluation of the possibility of using fetal magnetocardiogram (FMCG) signals to estimate and classify the accessory pathway in fetal Wolff-Parkinson-White (WPW) syndrome. The FMCG signals of two fetuses with WPW syndrome (type A) were detected using a 64-channel superconducting quantum-interference device system. An average across the cycles of these signals was taken to obtain clear WPW signals. To determine the direction and position of the accessory pathway in a fetal heart accurately, the accessory pathway and activated pathway at the peak of the QRS complex thus obtained were estimated for each fetus, using a single-dipole model. The phase angle (about 90^o) between the equivalent current dipoles (ECDs) was the same for both fetuses. This angle suggested that the accessory pathway is in the left side of the heart, i.e. that the pathway exists in the position of the accessory pathway in a fetus with WPW syndrome from the angle between the ECD of the accessory pathway and the ECD of the peak in the QRS complex was thus demonstrated.     

The volume-activated chloride current in human endothelial cells depends on intracellular ATP

We have studied the effect of intracellular ATP on volume-activated Cl^–-currents in endothelial cells from human umbilical veins by means of the whole-cell patch clamp technique. The run-down of this current in ruptured patches during repetitive applications of hypotonic solutions (HTS) could be significantly reduced if the cells were internally perfused with a pipette solution that contained 4 mmol/l ATP. This run-down was much less pronounced if currents were recorded using nystatin-perforated patches. The amplitude of the current was drastically reduced and its activation became slower if the cells were superfused with a glucose-free medium with 1 mmol/l KCN. Adding 4 mmol/l ATPS, a poorly hydrolyzable ATP-analogue, to the patch pipette prevented run-down of the current during repetitive activations by HTS, even if the cells were superfused with glucose-free solution with 1 mmol/l KCN. It is concluded that activation of the mechanosensitive Cl^– conductance in human endothelial cells requires the presence of intracellular ATP, but not its hydrolysis.     

人体神经定量电流感觉检测系统的研制

电流感觉阈值测试是人体神经感觉功能的定量感觉测试方法的一种．它采用特定频率的正弦恒电流刺激人体神经末梢感受器，检测人体对电流刺激的最小感受量，用于定量评估神经功能。本文回顾了国际上电流感觉阈值测试技术的发展与现状，介绍了我们开发的人体神经定量电流感觉检测系统。该系统采用生理心理统计算法过滤人体主观感受的影响，具有双盲全自动测量功能，测量结果具有很好的重复性。     

Amiloride-sensitive Na+ transport across cultured renal (A6) epithelium: evidence for large currents and high NaK selectivity

Electrical techniques were used to determine the NaK selectivity of the amiloride-sensitive pathway and to characterize cellular and paracellular properties of A6 epithelium. Under control conditions, the mean transepithelial voltage (V _T) was –57±5 mV, the short-circuit current (I _sc) averaged 23±2 A/cm² and the transepithelial resistance (R _T) was 2.8±0.3 kcm² (n=13). V _T and I _sc were larger than reported in previous studies and were increased by aldosterone. The conductance of the amiloride-sensitive pathway (G _amil) was assessed before and after replacement of Na⁺ in the mucosal bath by K⁺, using two independent measurements: (1) the slope conductance (G _T), determined from current-voltage (I-V) relationships for control and amiloride-treated tissues and (2) the maximum amiloride-sensitive conductance (G _max) calculated from the amiloride dose-response relationship. The ratio of G _amil in mucosal Na⁺ solutions to G _amil for mucosal K⁺ solutions was 22±6 for G _T measurements and 15±2 for G _max data. Serosal ion replacements in tissues treated with mucosal nystatin indicated a potassium conductance in the basolateral membrane. Equivalent circuit analyses of nystatin and amiloride data were used to resolve the cellular (E _c) and paracellular (R _j) resistances (5 kcm² and 8–9 kcm², respectively). Analysis I-V relationships for tissues depolarized with serosal K⁺ solutions revealed that the amiloride-sensitive pathway could be described as a Na⁺ conductance with a permeability coefficient (P _Na)=1.5±0.2× 10^–6 cm/s and the intracellular Na⁺ concentration (Na_i)=5±1 mM (n=5), similar to values from other tight epithelia. We conclude that A6 epithelia are capable of expressing large amiloride-sensitive currents which are highly Na⁺ selective.