Induction of a sodium ion influx by progesterone in human spermatozoa

In human spermatozoa, progesterone (P(4)) induces a depolarization of the plasma membrane, a rapid calcium (Ca(2+)) influx, and a chloride efflux. The sodium ion (Na(+)) was partly responsible for the P(4)-induced depolarizing effect but was not required for calcium influx. We used fluorescent probes for spectrofluorometry to investigate whether P(4) induced a Na(+) influx and whether voltage-operated channels were involved in Na(+) and/or Ca(2+) entries. We found that 10 microM P(4) significantly increased intracellular Na(+) concentration from 17.8 +/- 2.0 mM to 27.2 +/- 1. 6 mM (P < 0.001). Prior incubation of spermatozoa with 10 microM flunarizine, a Na(+) and Ca(2+) voltage-dependent channel blocker, inhibited the sodium influx induced by 10 microM P(4) by 84.6 +/- 15.4%. The Ca(2+) influx induced by 10 microM P(4) was also significantly inhibited in a Na(+)-containing medium by 10 microM flunarizine or 10 microM pimozide (P < 0.01). In contrast, flunarizine had no inhibitory effect on the Ca(2+) influx induced by 10 microM P(4) in spermatozoa incubated in Na(+)-depleted medium. The P(4)-promoted acrosome reaction (AR) was significantly higher when spermatozoa were incubated in Na(+)-containing medium as compared to Na(+)-depleted medium. These data demonstrate that P(4) stimulates a Na(+) influx that could be involved in the AR completion. They also suggest that voltage-dependent Na(+) and Ca(2+) channels are implicated in P(4)-mediated signaling pathway in human spermatozoa.     

Calcium permeability and modulation of nicotinic acetylcholine receptor-channels in rat parasympathetic neurons.

Neuronal nicotinic acetylcholine (ACh)-activated currents in rat parasympathetic ganglion cells were examined using whole-cell and single-channel patch clamp recording techniques. The whole-cell current-voltage (I-V) relationship exhibited strong inward rectification and a reversal (zero current) potential of -3.9 mV in nearly symmetrical Na+ solutions (external 140 mM Na+/internal 160 mM Na+). Isosmotic replacement of extracellular Na+ with either Ca2+ or Mg2+ yielded the permeability (Px/PNa) sequence Mg2+ (1.1) > Na+ (1.0) > Ca2+ (0.65). Whole-cell ACh-induced current amplitude decreased as [Ca2+]0 was raised from 2.5 mM to 20 mM, and remained constant at higher [Ca2+]0. Unitary ACh-activated currents recorded in excised outside-out patches had conductances ranging from 15-35 pS with at least three distinct conductance levels (33 pS, 26 pS, 19 pS) observed in most patches. The neuronal nicotinic ACh receptor-channel had a slope conductance of 30 pS in Na+ external solution, which decreased to 20 pS in isotonic Ca2+ and was unchanged by isosmotic replacement of Na+ with Mg2+. ACh-activated single channel currents had an apparent mean open time (tau 0) of 1.15 +/- 0.16 ms and a mean burst length (tau b) of 6.83 +/- 1.76 ms at -60 mV in Na+ external solution. Ca(2+)-free external solutions, or raising [Ca2+]0 to 50-100 mM decreased both the tau 0 and tau b of the nAChR channel. Varying [Ca2+]0 produced a marked decrease in NP0, while substitution of Mg2+ for Na+ increased NP0. These data suggest that activation of the neuronal nAChR channel permits a substantial Ca2+ influx which may modulate Ca(2+)-dependent ion channels and second messenger pathways to affect neuronal excitability in parasympathetic ganglia.     

Modification of cardiac sodium channels by carboxyl reagents. Trimethyloxonium and water-soluble carbodiimide

In TTX-sensitive nerve and skeletal muscle Na+ channels, selective modification of external carboxyl groups with trimethyloxonium (TMO) or water-soluble carbodiimide (WSC) prevents voltage-dependent Ca2+ block, reduces unitary conductance, and decreases guanidinium toxin affinity. In the case of TMO, it has been suggested that all three effects result from modification of a single carboxyl group, which causes a positive shift in the channel's surface potential. We studied the effect of these reagents on Ca2+ block of adult rabbit ventricular Na+ channels in cell-attached patches. In unmodified channels, unitary conductance (gamma Na) was 18.6 +/- 0.9 pS with 280 mM Na+ and 2 mM Ca2+ in the pipette and was reduced to 5.2 +/- 0.8 pS by 10 mM Ca2+. In contrast to TTX-sensitive Na+ channels, Ca2+ block of cardiac Na+ channels was not prevented by TMO; after TMO pretreatment, gamma Na was 6.1 +/- 1.0 pS in 10 mM Ca2+. Nevertheless, TMO altered cardiac Na+ channel properties. In 2 mM Ca2+, TMO-treated patches exhibited up to three discrete gamma Na levels: 15.3 +/- 1.7, 11.3 +/- 1.5, and 9.8 +/- 1.8 pS. Patch-to-patch variation in which levels were present and the absence of transitions between levels suggests that at least two sites were modified by TMO. An abbreviation of mean open time (MOT) accompanied each decrease in gamma Na. The effects on channel gating of elevating external Ca2+ differed from those of TMO pretreatment. Increasing pipette Ca2+ from 2 to 10 mM prolonged the MOT at potentials positive to approximately -35 mV by decreasing the open to inactivated (O-->I) transition rate constant. On the other hand, even in 10 mM Ca2+ TMO accelerated the O-->I transition rate constant without a change in its voltage dependence. Ensemble averages after TMO showed a shortening of the time to peak current and an acceleration of the rate of current decay. Channel modification with WSC resulted in analogous effects to those of TMO in failing to show relief from block by 10 mM Ca2+. Further, WSC caused a decrease in gamma Na and an abbreviation of MOT at all potentials tested. We conclude that a change in surface potential caused by a single carboxyl modification is inadequate to explain the effects of TMO and WSC in heart. Failure of TMO and WSC to prevent Ca2+ block of the cardiac Na+ channel is a new distinction among isoforms in the Na+ channel multigene family.     

A new type of Ca(2+) channel blocker that targets Ca(2+) sensors and prevents Ca(2+)-mediated apoptosis.

Calmodulin (CaM), as well as other Ca(2+) binding motifs (i.e., EF hands), have been demonstrated to be Ca(2+) sensors for several ion channel types, usually resulting in an inactivation in a negative feedback manner. This provides a novel target for the regulation of such channels. We have designed peptides that interact with EF hands of CaM in a specific and productive manner. Here we have examined whether these peptides block certain Ca(2+)-permeant channels and inhibit biological activity that is dependent on the influx of Ca(2+). We found that these peptides are able to enter the cell and directly, as well as indirectly (through CaM), block the activity of glutamate receptor channels in cultured neocortical neurons and a nonselective cation channel in Jurkat T cells that is activated by HIV-1 gp120. As a consequence, apoptosis mediated by an influx of Ca(2+) through these channels was also dose-dependently inhibited by these novel peptides. Thus, this new type of Ca(2+) channel blocker may have utility in controlling apoptosis due to HIV infection or neuronal loss due to ischemia.     

Influence of sodium-calcium exchange on calcium current rundown and the duration of calcium-dependent chloride currents in pituitary cells, studied with whole cell and perforated patch recording

The whole cell patch-clamp technique, in both standard and perforated patch configurations, was used to study the influence of Na+-Ca++ exchange on rundown of voltage-gated Ca++ currents and on the duration of tail currents mediated by Ca++-dependent Cl- channels. Ca++ currents were studied in GH3 pituitary cells; Ca++-dependent Cl- currents were studied in AtT-20 pituitary cells. Na+-Ca++ exchange was inhibited by substitution of tetraethylammonium (TEA+) or tetramethylammonium (TMA+) for extracellular Na+. Control experiments demonstrated that substitution of TEA+ for Na+ did not produce its effects via a direct interaction with Ca++-dependent Cl- channels or via blockade of Na+-H+ exchange. When studied with standard whole cell methods, Ca++ and Ca++-dependent Cl- currents ran down within 5-20 min. Rundown was accelerated by inhibition of Na+-Ca++ exchange. In contrast, the amplitude of both Ca++ and Ca++-dependent Cl- currents remained stable for 30-150 min when the perforated patch method was used. Inhibition of Na+-Ca++ exchange within the first 30 min of perforated patch recording did not cause rundown. The rate of Ca++-dependent Cl- current deactivation also remained stable for up to 70 min in perforated patch experiments, which suggests that endogenous Ca++ buffering mechanisms remained stable. The duration of Ca++-dependent Cl- currents was positively correlated with the amount of Ca++ influx through voltage-gated Ca++ channels, and was prolonged by inhibition of Na+-Ca++ exchange. The influence of Na+-Ca++ exchange on Cl- currents was greater for larger currents, which were produced by greater influx of Ca++. Regardless of Ca++ influx, however, the prolongation of Cl- tail currents that resulted from inhibition of Na+-Ca++ exchange was modest. Tail currents were prolonged within tens to hundreds of milliseconds of switching from Na+- to TEA+-containing bath solutions. After inhibition of Na+-Ca++ exchange, tail current decay kinetics remained complex. These data strongly suggest that in the intact cell, Na+-Ca++ exchange plays a direct but nonexclusive role in limiting the duration of Ca++-dependent membrane currents. In addition, these studies suggest that the perforated patch technique is a useful method for studying the regulation of functionally relevant Ca++ transients near the cytoplasmic surface of the plasma membrane.     

Ionic mechanism of the fertilization potential of the marine worm, Urechis caupo (Echiura)

Microelectrode and tracer flux studies of the Urechis egg during fertilization have shown: (a) insemination causes a fertilization potential; the membrane potential rises from an initial level of -33 +/- 6 mV to a peak at +51 +/- 6 mV (n = 16), falls to a plateau of about +30 mV, then returns to the original resting potential 9 +/- 1 min (n - 10) later; (b) the fertilization potential results from an increase in Na+ permeability, which is amplified during the first 15 s by a Ca++ action potential; (c) the maximum amplitude of the fertilization potential, excluding the first 15 s, changes by 51 mV for a 10-fold change in external [Na+]; (d) in the 10 min period after insemination, both Na+ and Ca++ influxes increase relative to unfertilized egg values by factors of 17 +/- 7 (n = 6) and 34 +/- 14 (n = 4), respectively; the absolute magnitude of the Na+ influx is 16 +/- 6 times larger than that of Ca++; (e) in the absence of sperm these same electrical and ionic events are elicited by trypsin; thus, the ion channels responsible must preexist in the unfertilized egg membrane; (f) increased Na+ influx under conditions of experimentally induced polyspermy indicates that during normal monospermic fertilization, only a fraction of available Na+ channels are opened; we conclude that these channels are sperm-gated; (g) Ca++ influx at fertilization is primarily via the membrane potential-gated channel, because kinetics are appropriate, and influx depends on potential in solutions of varying [Na+], but is independent of number of sperm incorporations in normal sea water.     

Ionic blockage of the light-regulated sodium channels in isolated rod outer segments.

We have investigated, with osmotic techniques, the light-regulated Na+ channels in rod outer segments (ROS) and ROS fragments freshly isolated from the frog retina. Values of Na+ permeability (PNa) similar to those observed electrophysiologically in the retina were observed using the osmotic technique (continuous flow) described by Korenbrot and Cone. In the other osmotic techniques that we explored, PNa was greatly diminished, if not completely suppressed; however, we found with these techniques that antioxidant conditions (N2 atmosphere or EDTA) significantly increased PNa, suggesting that the Na+ channels are highly sensitivive to membrane oxidation. Using the continuous flow technique, we investigated the H+ and Ca++ dependence of the Na+ channels and found that both of these ions, at micromolar activities, can block the channels. Raising the external H+ activity decreases PNa (reversibly) in a single "sigmoidal" response with an apparent pKa of 5.8. Similarly, in the presence of the ionophores X537A or A23187 which allow equilibration of Ca++ across membranes, the Na+ channels are blocked when the external Ca++ activity is increased from 10(-7) to 10(-5) M. This high sensitivity to both H+ and Ca++ ions suggests that high field strength anionic sites may exist in or near the Na+ channels and that the channels are blocked when these sites bind H+ or Ca++ ions.     

Extracellular Ca²+ alleviates NaCl-induced stomatal opening through a pathway involving H₂O₂-blocked Na+ influx in Vicia guard cells

To gain further insights into the function of extracellular Ca2+ in alleviating salt stress, Vicia faba guard cell protoplasts (GCPs) were patch-clamped in a whole-cell configuration. The results showed that 100 mM NaCl clearly induced Na+ influx across the plasma membrane in GCPs and promoted stomatal opening. Extracellular Ca2+ at 10 mM efficiently blocked Na+ influx and inhibited stomatal opening, which was partially abolished by La3+ (an inhibitor of plasma membrane Ca2+ channel) or catalase (CAT, a H?O? scavenger), respectively. These results suggest that the plasma membrane Ca2+ channels and H?O? possibly mediate extracellular Ca2+-blocked Na+ influx in GCPs. Furthermore, extracellular Ca2+ activated the plasma membrane Ca2+ channels under NaCl stress, which was partially abolished by CAT. These results, taken together, indicate that hydrogen peroxide (H?O?) likely regulates Na+ uptake by activating plasma membrane Ca2+ channels in GCPs. In accordance with this hypothesis, H?O? could mimic extracellular Ca2+ to activate Ca2+ channels and block Na+ influx in guard cells. A single-cell analysis of cytosolic free Ca2+ ([Ca2+](cyt)) using Fluo 3-AM revealed that extracellular Ca2+ induced the accumulation of cytosolic Ca2+ under NaCl stress, but had few effects on the accumulation of cytosolic Ca2+ under non-NaCl conditions. All of these results, together with our previous studies showing that extracellular Ca2+ induced the generation of H?O? in GCPs during NaCl stress, indicate that extracellular Ca2+ alleviates salt stress, likely by activating the H?O?-dependent plasma membrane Ca2+ channels, and the increase in cytosolic Ca2+ appears to block Na+ influx across the plasma membrane in Vicia guard cells, leading to stomatal closure and reduction of water loss.     

A peptide derived from the Shaker B K+ channel produces short and long blocks of reconstituted Ca(2+)-dependent K+ channels.

A 20 amino acid synthetic peptide, corresponding to the amino-terminal region of the Shaker B (ShB) K+ channel and responsible for its fast inactivation, can block large conductance Ca(2+)-dependent K+ channels from rat brain and muscle. The ShB inactivation peptide produces two kinetically distinct blocking events in these channels. At lower concentrations, it produces short blocks, and at higher concentrations long-lived blocks also appear. The L7E mutant peptide produces only infrequent short blocks (no long-lived blocks) at a much higher concentration. Internal tetraethylammonium competes with the peptide for the short block, which is also relieved by K+ influx. These results suggest that the peptide induces the short block by binding within the pore of Ca(2+)-dependent K+ channels. The long block is not affected by increased K+ influx, indicating that the binding site mediating this block may be different from that involved in the short block. The short block of Ca(2+)-dependent K+ channels and the inactivation of Shaker exhibit similar characteristics with respect to blocking affinity and open pore blockade. This suggests a conserved binding region for the peptide in the pore regions of these very different classes of K+ channel.     

Hormonal and neurotransmitter regulation of Ca++ influx through voltage-dependent slow channels in cardiac muscle membrane

The voltage- and time-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca++ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. These slow channels behave kinetically as if their gates open, close, and recover more slowly than those of the fast Na+ channels; in addition, the slow channel gates operate over a less negative (more depolarized) voltage range. Tetrodotoxin does not block the slow channels, whereas the calcium antagonistic drugs, Mn++, Co++, and La ions do. The slow channels have some special properties, including their functional dependence on metabolic energy, their selective blockade by acidosis, and their regulation by cyclic AMP level. Because of their regulation by cyclic AMP, it is proposed that either the slow channel protein or an associated regulatory protein must be phosphorylated in order for the channel to be made available for voltage activation during excitation. That is, the dephosphorylated channel would be electrically silent. The requirement for phosphorylation allows the extrinsic control of the slow channels and Ca++ influx by neurotransmitters, hormones, and autacoids that affect the cyclic nucleotide levels.     

Molecular evolution of voltage-sensitive ion channel genes: on the origins of electrical excitability

We have analyzed nucleic acid and amino acid sequence alignments of a variety of voltage-sensitive ion channels, using several methods for phylogenetic tree reconstruction. Ancient duplications within this family gave rise to three distantly related groups, one consisting of the Na+ and Ca++ channels, another the K+ channels, and a third including the cyclic nucleotide-binding channels. A series of gene duplications produced at least seven mammalian homologues of the Drosophila Shaker K+ channel; clones of only three of these genes are available from all three mammalian species examined (mouse, rat, and human), pointing to specific genes that have yet to be recovered in one or another of these species. The Shaw-related K+ channels and the Na+ channel family have also undergone considerable expansion in mammals, relative to flies. These expansions presumably reflect the needs of the high degree of physiological and neuronal complexity of mammals. Analysis of the separate domains of the four-domain channels (Ca++ and Na+) supports their having evolved by two sequential gene duplications and implies the historical existence of a functional two-domain channel.      

Block by calcium of ATP-activated channels in pheochromocytoma cells

We have investigated the effects of Ca2+ on Na+ influx through ATP- activated channels in pheochromocytoma PC12 cells using single channel current recordings. Under cell-attached patch-clamp conditions with 150 mM Na+ and 2 mM Ca2+ in the pipette, the unitary current activity showed an open level of about -4.3 pA at -150 mV. The channel opening was interrupted by flickery noise as well as occasional transition to a subconducting state of about -1.7 pA at -150 mV. The open level was decreased with increased external Ca2+, suggesting that external Ca2+ blocks Na+ permeation. We assessed the block by Ca2+ as the mean amplitude obtained with heavy filtration according to Pietrobon et al. (Pietrobon, D., B. Prod'hom, and P. Hess, 1989. J. Gen. Physiol. 94:1- 21). The block was concentration dependent with a Hill coefficient of 1 and a half-maximal concentration of approximately 6 mM. A similar block was observed with other divalent cations, and the order of potency was Cd2+ > Mn2+ > Mg2+ not equal to Ca2+ > Ba2+. High Ca2+, Mg2+ and Ba2+ did not block completely, probably because they can carry current in the channel. The block by external Ca2+ did not exhibit voltage dependence between -100 and -210 mV. In the inside-out patch-clamp configuration, the amplitude of inward channel current obtained with 150 mM external Na+ was reduced by increased internal Ca2+. The reduction was observed at lower concentrations than that by external Ca2+. Internal Ba2+ and Cd2+ induced similar reduction in current amplitude. This inhibitory effect of internal Ca2+ was voltage dependent; the inhibition was relieved with hyperpolarization. The results suggest that both external and internal Ca2+ can block Na+ influx through the ATP-activated channel. A simple one-binding site model with symmetric energy barriers is not sufficient to explain the Ca2+ block from both sides.     

Divalent cation selectivity for external block of voltage-dependent Na+ channels prolonged by batrachotoxin. Zn2+ induces discrete substates in cardiac Na+ channels

The mechanism of block of voltage-dependent Na+ channels by extracellular divalent cations was investigated in a quantitative comparison of two distinct Na+ channel subtypes incorporated into planar bilayers in the presence of batrachotoxin. External Ca2+ and other divalent cations induced a fast voltage-dependent block observed as a reduction in unitary current for tetrodotoxin-sensitive Na+ channels of rat skeletal muscle and tetrodotoxin-insensitive Na+ channels of canine heart ventricular muscle. Using a simple model of voltage-dependent binding to a single site, these two distinct Na+ channel subtypes exhibited virtually the same affinity and voltage dependence for fast block by Ca2+ and a number of other divalent cations. This group of divalent cations exhibited an affinity sequence of Co congruent to Ni greater than Mn greater than Ca greater than Mg greater than Sr greater than Ba, following an inverse correlation between binding affinity and ionic radius. The voltage dependence of fast Ca2+ block was essentially independent of CaCl2 concentration; however, at constant voltage the Ca2+ concentration dependence of fast block deviated from a Langmuir isotherm in the manner expected for an effect of negative surface charge. Titration curves for fast Ca2+ block were fit to a simplified model based on a single Ca2+ binding site and the Gouy-Chapman theory of surface charge. This model gave similar estimates of negative surface charge density in the vicinity of the Ca2+ blocking site for muscle and heart Na+ channels. In contrast to other divalent cations listed above, Cd2+ and Zn2+ are more potent blockers of heart Na+ channels than muscle Na+ channels. Cd2+ induced a fast, voltage-dependent block in both Na+ channel subtypes with a 46-fold higher affinity at 0 mV for heart (KB = 0.37 mM) vs. muscle (KB = 17 mM). Zn2+ induced a fast, voltage-dependent block of muscle Na+ channels with low affinity (KB = 7.5 mM at 0 mV). In contrast, micromolar Zn2+ induced brief closures of heart Na+ channels that were resolved as discrete substate events at the single-channel level with an apparent blocking affinity of KB = 0.067 mM at 0 mV, or 110-fold higher affinity for Zn2+ compared with the muscle channel. High-affinity block of the heart channel by Cd2+ and Zn2+ exhibited approximately the same voltage dependence (e-fold per 60 mV) as low affinity block of the muscle subtype (e-fold per 54 mV), suggesting that the block occurs at structurally analogous sites in the two Na+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of excessive neuronal apoptosis by the calcium antagonist amlodipine and antioxidants in cerebellar granule cells

Neuronal cell death as a result of apoptosis is associated with cerebrovascular stroke and various neurodegenerative disorders. Pharmacological agents that maintain normal intracellular Ca2+ levels and inhibit cellular oxidative stress may be effective in blocking abnormal neuronal apoptosis. In this study, a spontaneous (also referred to as age-induced) model of apoptosis consisting of rat cerebellar granule cells was used to evaluate the antiapoptotic activities of voltage-sensitive Ca2+ channel blockers and various antioxidants. The results of these experiments demonstrated that the charged, dihydropyridine Ca2+ channel blocker amlodipine had very potent neuroprotective activity in this system, compared with antioxidants and neutral Ca2+ channel blockers (nifedipine and nimodipine). Within its effective pharmacological range (10-100 nM), amlodipine attenuated intracellular neuronal Ca2+ increases elicited by KCl depolarization but did not affect Ca2+ changes triggered by N-methyl-D-aspartate receptor activation. Amlodipine also inhibited free radical-induced damage to lipid constituents of the membrane in a dose-dependent manner, independent of Ca2+ channel modulation. In parallel experiments, spontaneous neuronal apoptosis was inhibited in dose- and time-dependent manners by antioxidants (U-78439G, alpha-tocopherol, and melatonin), nitric oxide synthase inhibitors (N-nitro-L-arginine and N-nitro-D-arginine), and a nitric oxide chelator (hemoglobin) in the micromolar range. These results suggest that spontaneous neuronal apoptosis is associated with excessive Ca2+ influx, leading to further intracellular Ca2+ increases and the generation of reactive oxygen species. Agents such as amlodipine that block voltage-sensitive Ca2+ channels and inhibit cellular oxidative stress may be effective in the treatment of cerebrovascular stroke and neurodegenerative diseases associated with excessive apoptosis.     

Evidence for the activation of two different Ca2+ channels during the egg jelly-induced acrosome reaction of sea urchin sperm

The influx of Ca2+ and its subsequent intracellular increase are required for the acrosome reaction of sea urchin sperm to occur. Spermatozoa must undergo this reaction, which is triggered by the egg jelly, in order to fertilize the egg. Here, the egg jelly-induced Ca2+ influx mechanisms have been studied in sperm loaded with FURA-2 using Mn2+ under the assumption that this divalent ion is an indicator of Ca2+ influx through Ca2+ channels. Egg jelly induced the immediate entry of Ca2+ (mixing time 1 s), however; we found that the influx of Mn2+ increased after a lag time of 5 s. Nisol-dipine (a Ca2+ channel blocker) did not block the Mn2+ influx which was inhibited by 40 mM of external [K+], low Na+, and 5 mM of tetraethylammonium (a K+ channel blocker). These conditions also inhibited the alkalinization and the acrosome reaction. The inhibition of the Mn2+ influx could be overcome by increasing internal pH (pHi) with ammonium (10 mM). On the contrary the influx of Ca2+ during the first 5 s was not inhibited by any of the conditions indicated before, except by nisoldipine. These data could be explained by the activation of two different Ca2+ channels by egg jelly. The first one being a receptor-operator Ca2+ channel that opens when the receptor for egg jelly is occupied independently of the ionic conditions. The other one could be considered as a second messenger-operated Ca2+ channel that requires at least an increase in pHi to open.     

Cleavage of the plasma membrane Na+/Ca2+ exchanger in excitotoxicity

In brain ischemia, gating of postsynaptic glutamate receptors and other membrane channels triggers intracellular Ca2+ overload and cell death. In excitotoxic settings, the initial Ca2+ influx through glutamate receptors is followed by a second uncontrolled Ca2+ increase that leads to neuronal demise. Here we report that the major plasma membrane Ca2+ extruding system, the Na+/Ca2+ exchanger (NCX), is cleaved during brain ischemia and in neurons undergoing excitotoxicity. Inhibition of Ca2+-activated proteases (calpains) by overexpressing their endogenous inhibitor protein, calpastatin or the expression of an NCX isoform not cleaved by calpains, prevented Ca2+ overload and rescued neurons from excitotoxic death. Conversely, down-regulation of NCX by siRNA compromised neuronal Ca2+ handling, transforming the Ca2+ transient elicited by non-excitotoxic glutamate concentrations into a lethal Ca2+overload. Thus, proteolytic inactivation of NCX-driven neuronal Ca2+ extrusion is responsible for the delayed excitotoxic Ca2+ deregulation and neuronal death.     

Effect of inhibition of Na+-K+ ATPase on the prostacyclin generation of cultured human vascular endothelial cells

Prostacyclin (PGI2) generation of cultured human vascular endothelial cells (VEC) was observed coincidentally with the increase of 45Ca net influx. Ca ionophore A23187 enhanced not only PGI2 generation and 45Ca net influx but also 45Ca efflux. PGI2 generation was completely abolished by the pretreatment with Ca++ immobilizer, TMB-8. A Na+-K+ ATPase inhibitor, ouabain increased 45Ca net influx, but decreased 45Ca efflux, and enhanced PGI2 generation. These observation indicate that PGI2 generation of VEC may be regulated by not only Ca++ but also Na+, and it was suggested that enhanced PGI2 generation by ouabain might be derived from the increased cytosolic Ca++concentration by the decreased Ca++ efflux, and it was considered to be originated from the suppression of Na+-Ca++ exchange systems by the increased intracellular Na+ concentration via inhibition of Na+-K+ ATPase activity by ouabain. Enhancement of PGI2 generation of VEC by the increased ouabain like substances (OLS) in hypertension is suspected to be beneficial on the maintenance of vascular homeostasis.     

Open sodium channel properties of single canine cardiac Purkinje cells.

Open channel properties of canine cardiac Purkinje cell Na+ channels were studied with single channel cell-attached recording and with whole cell macroscopic current recording in internally perfused cells. Single channel currents and membrane currents increased with an increase in Na+ concentration, but showed evidence of saturation. Assuming first-order binding, the Km for Na+ was 370 mM. PCs/PNa was 0.020 and PK/PNa was 0.094. The current-voltage relationship for single channels showed prominent flattening in the hyperpolarizing direction. This flattening was accentuated by 10 mM Ca2+ and was greatly reduced in O mM Ca2+, indicating that the rectification was a consequence of Ca2+ block of the Na+ channels. A similar instantaneous current-voltage relationship was seen for the whole cell membrane currents. These results demonstrate that the cardiac channel shows substantial Ca2+ block, although it is relatively insensitive to tetrodotoxin. The Na+ and Ca2+ binding properties could be modeled by the four-barrier Eyring rate theory model, with similar values to those reported for the neuroblastoma Na+ channel (Yamamoto, D.,J.Z. Yeh, and T. Narahashi, 1984, Biophys J., 45:337-344).     

Na(+) current through KATP channels: consequences for Na(+) and K(+) fluxes during early myocardial ischemia

During early myocardial ischemia, the myocytes are loaded with Na(+), which in turn leads to Ca(2+) overload and cell death. The pathway of the Na(+) influx has not been fully elucidated. The aim of the study was to quantify the Na(+) inward current through sarcolemmal KATP channels (IKATP,Na) in anoxic isolated cardiomyocytes at the actual reversal potential (Vrev) and to estimate the contribution of this current to the Na(+) influx in the ischemic myocardium. IKATP,Na was determined in excised single channel patches of mouse ventricular myocytes and macropatches of Xenopus laevis oocytes expressing SUR2A/Kir6.2 channels. In the presence of K+ ions, the respective permeability ratios for Na(+) to K(+) ions, PNa/PK, were close to 0.01. Only in the presence of Na(+) ions on both sides of the membrane was IKATP,Na similarly large to that calculated from the permeability ratio PNa/PK, indicative of a Na(+) influx that is largely independent of the K+ efflux at Vrev. With the use of a peak KATP channel conductance in anoxic cardiomyocytes of 410 nS, model simulations for a myocyte within the ischemic myocardium showed that the amplitude of the Na(+) influx and K(+) efflux is even larger than the respective fluxes by the Na(+) - K(+) pump and all other background fluxes. These results suggest that during early ischemia the Na(+) influx through KATP channels essentially contributes to the total Na+ influx and that it also balances the K(+) efflux through KATP channels.     

Receptor-mediated regulation of calcium channels and neurotransmitter release

Ca2+ influx into the nerve terminal is normally the trigger for the release of neurotransmitters. Many neurons possess presynaptic receptors whose activation results in changes in the quantity of neurotransmitter released by an action potential. This paper reviews studies that show that presynaptic receptors can regulate the activity of Ca2+ channels in the nerve terminal, resulting in changes in the influx of Ca2+ and in neurotransmitter release. Neurons possess several different types of voltage-sensitive Ca2+ channels. Ca2+ influx through N-type channels appears to trigger transmitter release in many instances. In other cases Ca2+ influx through L channels can influence transmitter release. Neurotransmitters can inhibit N channels through a G protein-mediated transduction mechanism. The G proteins are frequently pertussis toxin substrates. Inhibition of N channels appears to involve changes in their voltage dependence. Neurotransmitters can also regulate neuronal K+ channels. Activation of these K+ channels can lead to a reduction in Ca2+ influx and neurotransmitter release; these effects are also mediated by G proteins. Thus neurotransmitters may often regulate both presynaptic Ca2+ and K+ channels. These two effects may be synergistic mechanisms for the regulation of Ca2+ influx and neurotransmitter release.