Ionic currents in cardiac muscle: a framework for glycoside action.

This paper briefly reviews the current state of understanding of cardiac excitation--contraction coupling and its relation to glycoside action. Evidence that inotropic action of glycosides might result from increased influx of Ca2+ during action potential is reviewed. Recent voltage clamp studies that show little if any direct effect on Ca2+ influx during the action potential are cited. It is suggested that the primary inotropic effects derive from altered ionic exchange mechanisms secondary to inhibition of Na+,K+-ATPase. The role of ionic currents in glycoside toxicity is considered, with discussion of a dynamic, depolarizing current that appears shortly after action potential. This current is apparently an inward movement of positive ions that is strongly mediated by extracellular Ca2+ levels. It is noted that such spontaneous depolarizations of the membrane have been observed in several other circumstances where strong positive inotropism has been induced. The conclusion is reached that membrane ionic currents probably play only a secondary role in glycoside inotropism and in many of the toxic effects.     

Bethanidine increased Na+ and Ca2+ currents and caused a positive inotropic effect in heart cells

The effects of bethanidine sulphate, a pharmacological analog of the cardiac antibrillatory drug, bretylium tosylate, were studied on action potentials (APs) and K+, Na+, and Ca2+ currents of single cultured embryonic chick heart cells using the whole-cell current clamp and voltage clamp technique. Extracellular application of bethanidine (3 X 10(-4) M) increased the overshoot and the duration of the APs and greatly decreased the outward K+ current (IK) and potentiated the inward fast Na+ currents (INa) and the inward slow calcium current (ICa). However, intracellular introduction of bethanidine (10(-4) M) blocked INa. In isolated atria of rat, bethanidine increased the force of contraction in a dose-dependent manner. These findings suggest that when applied extracellularly, bethanidine exerts a potentiating effect on the myocardial fast Na+ current and slow Ca2+ current and an inhibitory effect of IK. The positive inotropic effect of bethanidine could be due, at least in part, to an increase of Ca2+ influx via the slow Ca2+ channel and the Na-Ca exchange. It is suggested that the decrease of IK by bethanidine may account for its antifibrillatory action.     

Mechanistic distinction between activation and inhibition of (Na(+)+K(+))-ATPase-mediated Ca2+ influx in cardiomyocytes

(Na(+)+K(+))-ATPase (NKA) mediates positive inotropy in the heart. Extensive studies have demonstrated that the reverse-mode Na(+)/Ca(2+)-exchanger (NCX) plays a critical role in increasing intracellular Ca(2+) concentration through the inhibition of NKA-induced positive inotropy by cardiac glycosides. Little is known about the nature of the NCX functional mode in the activation of NKA-induced positive inotropy. Here, we examined the effect of an NKA activator SSA412 antibody on (45)Ca influx in isolated rat myocytes and found that KB-R7943, a NCX reverse-mode inhibitor, fails to inhibit the activation of NKA-induced (45)Ca influx, suggesting that the Ca(2+) influx via the reverse-mode NCX does not mediate this process. Nifedipine, an L-type Ca(2+) channel (LTCC) inhibitor, completely blocks the activation of NKA-induced (45)Ca influx, suggesting that the LTCC is responsible for the moderate increase in intracellular Ca(2+). In contrast, the inhibition of NKA by ouabain induces 4.7-fold (45)Ca influx compared with the condition of activation of NKA. Moreover, approximately 70% of ouabain-induced (45)Ca influx was obstructed by KB-R7943 and only 30% was impeded by nifedipine, indicating that both the LTCC and the NCX contribute to the rise in intracellular Ca(2+) and that the NCX reverse-mode is the major source for the (45)Ca influx induced by the inhibition of NKA. This study provides direct evidence to demonstrate that the activation of NKA-induced Ca(2+) increase is independent of the reverse-mode NCX and pinpoints a mechanistic distinction between the activation and inhibition of the NKA-mediated Ca(2+) influx path ways in cardiomyocytes.     

The influence of external cations and membrane potential on Ca- activated Na efflux in Myxicola giant axons

In microinjected Myxicola giant axons with elevated [Na]i, Na efflux was sensitive to Cao under some conditions. In Li seawater, sensitivity to Cao was high whereas in Na seawater, sensitivity to Cao was observed only upon elevation of [Ca]o above the normal value. In choline seawater, the sensitivity of Na efflux to Cao was less than that observed in Li seawater whereas Mg seawater failed to support any detectable Cao-sensitive Na efflux. Addition of Na to Li seawater was inhibitory to Cao-sensitive Na efflux, the extent of inhibition increasing with rising values of [Na]o. The presence of 20 mM K in Li seawater resulted in about a threefold increase in the Cao-activated Na efflux. Experiments in which the membrane potential, Vm, was varied or held constant when [K]o was changed showed that the augmentation of Ca- activated Na efflux by Ko was not due to changes in Vm but resulted from a direct action of K on activation by Ca. The same experimental conditions that favored a large component of Cao-activated Na efflux also caused a large increase in Ca influx. Measurements of Ca influx in the presence of 20 mM K and comparison with values of Ca-activated Na efflux suggest that the Na:Ca coupling ratio may be altered by increasing external [K]o. Overall, the results suggest that the Cao- activated Na efflux in Myxicola giant axons requires the presence of an external monovalent cation and that the order of effectiveness at a total monovalent cation concentration of 430 mM is K + Li greater than Li greater than Choline greater than Na.     

Cyclic GMP regulates free cytosolic calcium in the pancreatic acinar cell

The present studies were performed in order to measure the effects of cyclic GMP (cGMP) on the regulation of free cytosolic calcium [( Ca2+]i) in the pancreatic acinar cell. In guinea pig dispersed pancreatic acini the findings demonstrated that the Ca2+ ionophore, Br A23187, caused a sustained increase in [Ca2+]i in the presence of 3 mM CaCl2 in the media and a transient 20 fold rise in cellular cGMP followed by a sustained 3-4 fold rise in cellular cGMP. Increasing cellular cGMP with nitroprusside, hydroxylamine or dibutyryl cGMP had no effect on resting [Ca2+]i. However, these agents attenuated the increase in [Ca2+]i resulting from Br A23187-induced Ca2+ influx. Nitroprusside also attenuated the carbachol-induced sustained rise in [Ca2+]i that resulted from Ca2+ influx. The nitroprusside effect on carbachol-stimulated acini occurred without decreasing Ca2+ influx across the plasma membrane or alteration in the mobilization of Ca2+ from the intracellular agonist-sensitive pool. Inhibition of the increase in cellular cGMP caused by Br A23187 by the guanylate cyclase inhibitor, 6-anilino-5,8-quinolinedione (LY83583), resulted in augmentation of the increase in [Ca2+]i. This augmentation was reversed with dibutyryl cGMP. These results indicated that cGMP regulated [Ca2+]i in the pancreatic acinar cell. The mechanism involves the removal of Ca2+ from the cytoplasm.     

Early differentiation of vertebrate spinal neurons in the absence of voltage-dependent Ca2+ and Na+ influx

The development of the action potential and responses to neurotransmitters have been described for a population of embryonic spinal neurons developing in vivo. A comparable pattern is seen for spinal neurons developing in dissociated cell culture. The impulse appears very early in this developmental sequence, and the action potential involves a large inward Ca2+ current. Since Ca2+ is a ubiquitous intracellular regulator, we questioned whether a large influx of Ca2+ is necessary for the subsequent differentiation of membrane properties. Embryonic Xenopus neurons grown in normal culture medium do not make Ca2+- or Na+-dependent action potentials in their cell bodies in a Ca2+-free saline containing tetrodotoxin (TTX). To achieve a chronic blockade of impulse activity, neurons were grown in a medium in which Ca2+ was replaced by Mg2+, and to which 1 mM EGTA was added. In some instances TTX was present. Neurons grown in these experimental culture media extend neurites more rapidly than controls. Action potentials cannot be elicited from neurons when examined in experimental medium. However, examination in saline reveals that the change in the ionic dependence of the impulse is indistinguishable from that observed in neurons grown in normal medium. Furthermore, the time of onset of responses to GABA is unaffected by this experimental treatment. Thus the expression of Ca2+- and Na+-dependent action potentials seems not to play a part in the early differentiation of these membrane properties. However, the later development of GABA sensitivity is reduced.     

The Effect of Ca and Antidiuretic Hormone on Na Transport across Frog Skin : I. Examination of interrelationships between Ca and hormone

Ca added to the solution bathing the outside of isolated frog skin causes a decrease in net Na transport across the skin while antidiuretic hormone (ADH) causes an increase. Possible interrelations between the effects of these agents have been examined. The decrease in Na transport caused by Ca was the same before and after treatment of the skin with ADH and the increase in transport caused by ADH was unaffected by the presence of Ca. The relationship between Ca concentration and degree of inhibition of Na transport was not appreciably altered by ADH. These results indicate that Ca and ADH do not compete but act independently at two different sites and these sites appear to be located on the same barrier to Na movement in the skin. Further, Ca causes a decrease in Cl influx across the short-circuited skin but ADH has no effect on Cl movement, again suggesting that the actions of these agents are independent.     

Generation of twitch tension in frog atrial fibers by Na/Ca exchange

Electrical and mechanical responses of frog atrial trabeculae were studied simultaneously using the double-sucrose gap method. Action potentials and twitch tension could be successively generated in fibers in which the slow inward calcium channel current was not observed. As a rule, this could be obtained in the course of a long experiment (3 to 4 hours). Peak tension was shown to increase monotonically with membrane potential in these preparations. In preparations with the slow inward current the total peak tension could be separated into two components. The first component (tonic) monotonically increased with the membrane potential and was probably related to Na/Ca exchange (Horackova 1984). The potential dependency of the second (phasic) component correlated with that of the slow inward calcium current. Only the tonic but not the phasic component could be observed in preparations without the presence of the slow inward calcium current. The tonic component prevailed when both the slow inward current and phasic tension were greatly reduced by nifedipine. Long experiments, long depolarizing clamp pulses, a metabolic inhibitor 2,4-dinitrophenol, inhibitors of Na/K pump ouabain and AR-L57, toxins promoting intracellular sodium accumulation (aconitine, scorpion toxin) were all shown to increase the tonic tension, but not the slow inward current; they induced a transition from biphasic tension-voltage curve into a monotonically increasing one. We concluded that these procedures and agents greatly stimulate Ca influx via Na/Ca exchange. These results show that Na/Ca exchange can function as a reserve system of Ca2+ used for contraction, thus supporting the heart function, especially under unfavourable metabolic conditions.     

Fast Na+ channels in smooth muscle from pregnant rat uterus.

Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L type) Ca2+ channels and fast (T type) Ca2+ channels. Whole-cell voltage clamp was done on single smooth muscle cells isolated from the longitudinal layer of the 18-day pregnant rat uterus. Depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by tetrodotoxin (TTX) (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o (or Ba2+), and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. We conclude that the ion channels that generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells that possess fast Na+ channels. The Ca2+ channel current density was also higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and we suggest that the fast Na+ current may be involved in spread of excitation. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 = 12 mM) and nifedipine (K0.5 = 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions cannot be explained by a stimulation of ICa(s).     

温度和胞内钠、ATP及酸碱度对心室肌细胞钠钙交换电流的调节

心肌缺血损伤过程中,胞内Na^＋、ATP及pH都出现明显变化。钠／钙交换对心肌细胞的钙平衡起重要的调节作用。本实验采用膜片钳全细胞记录豚鼠心室肌细胞钠／钙交换电流,研究温度和胞内Na^＋、ATP及pH对钠／钙交换双向电流的影响。结果表明,温度从22℃升至34℃,钠／钙交换电流增大约4倍,而pH值的改变对钠／钙交换双向电流没有明显的影响。在22～24℃时,同时耗竭胞内ATP和胞内酸化对钠／钙交换双向转运功能影响程度小;而在34—37℃时,同时耗竭胞内ATP和胞内酸化能抑制钠／钙交换双向电流的外向和内向成分,且内向成分抑制程度高于外向成分抑制程度。表明同时耗竭胞内ATP和胞内酸化对钠／钙交换的作用具有温度依赖性。胞内Na^＋超载能使钠／钙交换电流的外向成分增加,但不增加或减少内向电流（即正向转运）成分。因此,胞内酸化及耗竭胞内ATP损伤细胞排钙机制和胞内钠超载通过钠／钙反向交换引起钙内流是引起心肌细胞钙超载的两个独立的重要因素。     

Extracellular ATP induces ion fluxes and inhibits growth of Friend erythroleukemia cells

Extracellular ATP (1 mM) inhibited the growth of Friend virus-infected murine erythroleukemia cells (MEL cells) but had no effect on dimethyl sulfoxide-induced differentiation. ATP (1 mM) also caused changes in the permeability of MEL cells to ions. There was an increased influx of 45Ca2+ from a basal level of 5 pmol/min to 18 pmol/min/10(6) cells to achieve a 2-fold increase in steady-state Ca2+ as measured at isotopic equilibration. Ca2+ influx was blocked by diisothiocyanostilbene disulfonate (DIDS), an inhibitor of anion transport. ATP also stimulated Cl- uptake, and this flux was inhibited by DIDS. The ratio of ATP stimulated Cl- to Ca2+ uptake was 1.6:1. K+ and Na+ influx were also stimulated by ATP, but phosphate uptake was inhibited; the Na+ influx dissipated the Na+ gradient and thus inhibited nutrient uptake. ATP-stimulated K+ influx was ouabain inhibitable; however, the total cellular K+ decreased due to an ATP-stimulated ouabain-resistant K+ efflux. Na+ influx and Ca2+ influx occurred by separate independent routes, since Na+ influx was not inhibited by DIDS. The effects observed were specific for ATP *K1/2 MgATP = 0.7 mM) since AMP, GTP, adenosine, and the slowly hydrolyzable ATP analogue adenyl-5'-yl imidodiphosphate were without effect. The major ionic changes in the cell were a decrease in K+ and increase in Na+; cytoplasmic pH and free Ca2+ did not change appreciably. These ATP-induced changes in ion flux are considered to be responsible for growth inhibition.     

Cholera toxin increases the rate of antigen-stimulated calcium influx in rat basophilic leukemia cells

Cholera toxin pretreatment has been found to cause a 3-fold increase in the initial rate of antigen-stimulated secretion of serotonin from rat basophilic leukemia (RBL) cells. Under similar conditions, cholera toxin enhances the antigen-stimulated rise in cytoplasmic free ionized calcium levels and causes a 2-3-fold increase in the rate of antigen-stimulated influx of 45Ca. In intact RBL cells cholera toxin pretreatment potentiates the antigen-stimulated production of inositol phosphates, but in permeabilized cells, with strongly buffered free calcium levels, no effect of cholera toxin pretreatment on the antigen-stimulated activation of cellular phospholipase activities is observed. In addition, pretreatment of cells with tetradecanoylphorbol acetate inhibits antigen-stimulated production of inositol phosphates by greater than 95%, while the stimulated influx of 45Ca remains unaffected. These data indicate that the antigen-stimulated influx of calcium into RBL cells can be dissociated from the production of inositol phosphates in these cells. The observed effects of cholera toxin on exocytosis and Ca2+ influx in RBL cells are not due to the elevation of cellular cyclic AMP levels since a variety of agents capable of elevating cellular cyclic AMP levels do not mimic these effects. Together, these data suggest that a cholera toxin-sensitive guanine nucleotide-binding protein is involved in the pathway responsible for the antigen-stimulated influx of calcium into RBL cells.     

Effects of pressure overload-induced hypertrophy on TTX-sensitive inward currents in guinea pig left ventricle

We investigated the effects of pressure overload hypertrophy on inward sodium (I Na) and calcium currents (I Ca) in single left ventricular myocytes to determine whether changes in these current systems could account for the observed prolongation of the action potential. Hypertrophy was induced by pressure overload caused by banding of the abdominal aorta. Whole-cell patch clamp experiments were used to measure tetrodotoxin (TTX)-sensitive inward currents. The main findings were that I Ca density was unchanged whereas I Na density after stepping from -80 to -30 mV was decreased by 30% (-9.0 +/- 1.16 pA pF(-1) in control and -6.31 +/- 0.67 pA pF(-1) in hypertrophy, p < 0.05, n = 6). Steady-state activation/inactivation variables of I Na, determined by using double-pulse protocols, were similar in control and hypertrophied myocytes, whereas the time course of fast inactivation of I Na was slowed (p < 0.05) in hypertrophied myocytes. In addition, action potential clamp experiments were carried out in the absence and presence of TTX under conditions where only Ca2+ was likely to enter the cell via TTX-sensitive channels. We show for the first time that a TTX-sensitive inward current was present during the plateau phase of the action potential in hypertrophied but not control myocytes. The observed decrease in I Na density is likely to abbreviate rather than prolong the action potential. Delayed fast inactivation of Na+ channels was not sustained throughout the voltage pulse and may therefore merely counteract the effect of decreased I Na density so that net Na+ influx remains unaltered. Changes in the fast I Na do not therefore appear to contribute to lengthening of the action potential in this model of hypertrophy. However, the presence of a TTX-sensitive current during the plateau could potentially contribute to the prolongation of the action potential in hypertrophied cardiac muscle.     

Reductions in external divalent cations evoke novel voltage-gated currents in sensory neurons

It has long been recognized that divalent cations modulate cell excitability. Sensory nerve excitability is of critical importance to peripheral diseases associated with pain, sensory dysfunction and evoked reflexes. Thus we have studied the role these cations play on dissociated sensory nerve activity. Withdrawal of both Mg(2+) and Ca(2+) from external solutions activates over 90% of dissociated mouse sensory neurons. Imaging studies demonstrate a Na(+) influx that then causes depolarization-mediated activation of voltage-gated Ca(2+) channels (Ca(V)), which allows Ca(2+) influx upon divalent re-introduction. Inhibition of Ca(V) (ω-conotoxin, nifedipine) or Na(V) (tetrodotoxin, lidocaine) fails to reduce the Na(+) influx. The Ca(2+) influx is inhibited by Ca(V) inhibitors but not by TRPM7 inhibition (spermine) or store-operated channel inhibition (SKF96365). Withdrawal of either Mg(2+) or Ca(2+) alone fails to evoke cation influxes in vagal sensory neurons. In electrophysiological studies of dissociated mouse vagal sensory neurons, withdrawal of both Mg(2+) and Ca(2+) from external solutions evokes a large slowly-inactivating voltage-gated current (I(DF)) that cannot be accounted for by an increased negative surface potential. Withdrawal of Ca(2+) alone fails to evoke I(DF). Evidence suggests I(DF) is a non-selective cation current. The I(DF) is not reduced by inhibition of Na(V) (lidocaine, riluzole), Ca(V) (cilnidipine, nifedipine), K(V) (tetraethylammonium, 4-aminopyridine) or TRPM7 channels (spermine). In summary, sensory neurons express a novel voltage-gated cation channel that is inhibited by external Ca(2+) (IC(50)～0.5 μM) or Mg(2+) (IC(50)～3 μM). Activation of this putative channel evokes substantial cation fluxes in sensory neurons.     

Modulation of action potential by [Ca2+]i in modeled rat atrial and guinea pig ventricular myocytes

We simulated mechanisms that increase Ca2+ transients with two models: the Luo-Rudy II model for guinea pig (GP) ventricle (GP model) representing long action potential (AP) myocytes and the rat atrial (RA) model exemplifying myocytes with short APs. The interventions were activation of stretch-gated cationic channels, increase of intracellular Na+ concentration ([Na+]i), simulated bet-adrenoceptor stimulation, and Ca2+ accumulation into the sarcoplasmic reticulum (SR). In the RA model, interventions caused an increase of AP duration. In the GP model, AP duration decreased except in the simulated beta-stimulation where it lengthened APs as in the RA model. We conclude that the changes in the APs are significantly contributed by the increase of the Ca2+ transient itself. The AP duration is controlled differently in cardiac myocytes with short and long AP durations. With short APs, an increase of the Ca2+ transient promotes an inward current via Na+/Ca2+-exchanger lengthening the AP. This effect is similar regardless of the mechanism causing the increase of the Ca2+ transient. With long APs the Ca2+ transient increase decreases the AP duration via inactivation of the L-type Ca2+ current. However, L-type current increase (as with beta-stimulation) increases the AP duration despite the simultaneous Ca2+ transient augmentation. The results explain the dispersion of AP changes in myocytes with short and long APs during interventions increasing the Ca2+ transients.     

Calcium entry through receptor-operated channels in bovine pulmonary artery endothelial cells

The activation of endothelial cells by endothelium-dependent vasodilators has been investigated using bioassay, patch clamp and 45Ca flux methods. Cultured pulmonary artery endothelial cells have been demonstrated to release EDRF in response to thrombin, bradykinin, ATP and the calcium ionophore A23187. The resting membrane potential of the endothelial cells was -56 mV and the cells were depolarized by increasing extracellular K+ or by the addition of (0.1-1.0 mM)Ba2+ to the bathing solution. The electrophysiological properties of the cultured endothelial cells suggest that the membrane potential is maintained by an inward rectifying K+ channel with a mean single channel conductance of 35.6 pS. The absence of a depolarization-activated inward current and the reduction of 45Ca influx with high K+ solution suggests that there are no functional voltage-dependent calcium or sodium channels. Thrombin and bradykinin were shown to evoke not only an inward current (carried by Na+ and Ca2+) but also an increase in 45Ca influx suggesting that the increase in intracellular calcium necessary for EDRF release is mediated by an opening of a receptor operated channel. High doses of thrombin and bradykinin induced intracellular calcium release, however, at low doses of thrombin no intracellular calcium release was observed. We propose that the increased cytosolic calcium concentration in endothelial cells induced by endothelium dependent vasodilators is due to the influx of Ca2+ through a receptor operated ion channel and to a lesser degree to intracellular release of calcium from a yet undefined intracellular store.     

Distinct effects of various amino acids on 45Ca2+ fluxes in rat pancreatic islets.

The effects of three types of amino acids on 45Ca2+ fluxes in rat pancreatic islets have been compared. Alanine, a non-insulinotropic neutral amino acid, transported with Na+, increased 45Ca2+ efflux in the presence or in the absence of extracellular Ca2+, but not in the absence of Na+. Its effects in Na+-solutions were practically abolished by 7 mM-glucose. Alanine slightly stimulated 45Ca2+ influx (5 min uptake) only when Na+ was present. Two insulinotropic cationic amino acids (arginine and lysine) triggered similar changes in 45Ca2+ efflux. They accelerated the efflux in the presence of Ca2+ and inhibited the efflux in a Ca2+-free medium, whether glucose was present or not. In an Na+-free Ca2+-medium, arginine and lysine markedly accelerated 45Ca2+ efflux, but this effect was suppressed by 7 mM-glucose. Arginine stimulated 45Ca2+ influx irrespective of the presence or absence of glucose and Na+. Leucine, a neutral insulinotropic amino acid well metabolized by islet cells, inhibited 45Ca2+ efflux from the islets in a Ca2+-free medium; this effect was potentiated by glutamine. In the presence of Ca2+ and Na+, leucine was ineffective alone, but triggered a marked increase in 45Ca2+ efflux when combined with glutamine. In an Na+-free Ca2+-medium, leucine accelerated 45Ca2+ efflux to the same extent with or without glutamine. Leucine also stimulated 45Ca2+ influx in the presence or in the absence of Na+, but its effects were potentiated by glutamine only in the presence of Na+. The results show that amino acids of various types cause distinct changes in 45Ca2+ fluxes in pancreatic islets. Certain of these changes involve an Na+-mediated mobilization of cellular Ca2+ from sequestering sites where glucose appears to exert an opposite effect.     

A sodium zinc exchange mechanism is mediating extrusion of zinc in mammalian cells

Zinc influx, driven by a steep inward electrochemical gradient, plays a fundamental role in zinc signaling and in pathophysiologies linked to intracellular accumulation of toxic zinc. Yet, the cellular transport mechanisms that actively generate or maintain the transmembrane gradients are not well understood. We monitored Na+-dependent Zn2+ transport in HEK293 cells and cortical neurons, using fluorescent imaging. Treatment of the HEK293 cells with CaPO4 precipitates induced Na+-dependent Zn2+ extrusion, against a 500-fold transmembrane zinc gradient, or zinc influx upon reversal of Na+ gradient, thus indicating that Na+/Zn2+ exchange is catalyzing active Zn2+ transport. Depletion of intracellular ATP did not inhibit the Na+-dependent Zn2+ extrusion, consistent with a mechanism involving a secondary active transporter. Inhibitors of the Na+/Ca2+ exchanger failed to inhibit Na+-dependent Zn2+ efflux. In addition, zinc transport was unchanged in HEK293 cells heterologously expressing functional cardiac or neuronal Na+/Ca2+ exchangers, thus indicating that the Na+/Zn2+ exchange activity is not mediated by the Na+/Ca2+ exchanger. Sodium-dependent zinc exchange, facilitating the removal of intracellular zinc, was also monitored in neurons. To our knowledge, the Na+/Zn2+ exchanger described here is the first example of a mammalian transport mechanism capable of Na+-dependent active extrusion of zinc. Such mechanism is likely to play an important role, not only in generating the transmembrane zinc gradients, but also in protecting cells from the potentially toxic effects of permeation of this ion.     

Prokineticin 2 influences subfornical organ neurons through regulation of MAP kinase and the modulation of sodium channels

Prokineticin 2 (PK2) is a neuropeptide that acts as a signaling molecule regulating circadian rhythms in mammals. We have previously reported PK2 actions on subfornical organ (SFO) neurons, identifying this circumventricular organ as a target at which PK2 acts to influence autonomic control (Cottrell GT, and Ferguson AV. J. Neurosci. 24: 2375-2379, 2004). In this study, we have examined the cellular mechanisms by which PK2 increases the excitability of SFO neurons. Whole cell patch recordings from dissociated rat SFO neurons demonstrated that the mitogen-activated protein (MAP) kinase inhibitor PD-98059 prevented PK2-induced depolarization and decreases in delayed rectifier K(+) current. PK2 also increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in 39% of dissociated SFO neurons (mean increase = 20.8 +/- 5.5%), effects that were maintained in the presence of thapsigargin but abolished by both nifedipine, or the absence of extracellular Ca(2+), suggesting that PK2-induced [Ca(2+)](i) transients resulted from Ca(2+) entry through voltage-gated Ca(2+) channels. Voltage-clamp recordings showed that PK2 was without effects on Ca(2+) currents evoked by voltage ramps, suggesting that PK2-induced Ca(2+) influx was secondary to PK2-induced increases in action potential frequency, an hypothesis supported by data showing that tetrodotoxin abolished effects of PK2 on [Ca(2+)](i). These observations suggested PK2 modulation of voltage-gated Na(+) currents, a possibility confirmed by voltage-clamp experiments showing that PK2 increased the amplitude of both transient and persistent Na(+) currents in 29% of SFO neurons (by 34 and 38%, respectively). These data indicate that PK2 influences SFO neurons through the activation of a MAP kinase cascade, which, in turn, modulates Na(+) and K(+) conductances.     

Autocrine regulation of calcium influx and gonadotropin-releasing hormone secretion in hypothalamic neurons.

Gonadotropin-releasing hormone (GnRH) receptors are expressed in hypothalamic tissues from adult rats, cultured fetal hypothalamic cells, and immortalized GnRH-secreting neurons (GT1 cells). Their activation by GnRH agonists leads to an overall increase in the extracellular Ca2+-dependent pulsatile release of GnRH. Electrophysiological studies showed that GT1 cells exhibit spontaneous, extracellular Ca2+-dependent action potentials, and that their inward currents include Na+, T-type and L-type Ca2+ components. Several types of potassium channels, including apamin-sensitive Ca2+-controlled potassium (SK) channels, are also expressed in GT1 cells. Activation of GnRH receptors leads to biphasic changes in intracellular Ca2+ concentration ([Ca2+]i), with an early and extracellular Ca2+-independent peak and a sustained and extracellular Ca2+-dependent plateau phase. During the peak [Ca2+]i response, electrical activity is abolished due to transient hyperpolarization that is mediated by SK channels. This is followed by sustained depolarization and resumption of firing with increased spike frequency and duration. The agonist-induced depolarization and increased firing are independent of [Ca2+]i and are not mediated by inhibition of K+ currents, but by facilitation of a voltage-insensitive and store depletion-activated Ca2+-conducting inward current. The dual control of pacemaker activity by SK and store depletion-activated Ca2+ channels facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process accounts for the autoregulatory action of GnRH on its release from hypothalamic neurons.