Extragenomic actions of progesterone in human sperm and progesterone metabolites in human platelets.

Progesterone rapidly increased intracellular free calcium ([Ca2+]i) in human sperm, removal of extracellular Ca2+ prevented the increase in [Ca2+]i. The Ca2+ influx was not blocked by the T-type Ca2+ channel blocker mibefradil. However T-type calcium channels do appear to be present in human sperm because the neoglycoprotein mannose-albumin, an inducer of the acrosome reaction, was able to promote Ca2+ influx, which was blocked by mibefradil and more potently inhibited by Ni2+ than Cd2+. The receptor for progesterone that promotes the Ca2+ influx was located on the plasma membrane using FITC-progesterone-albumin. It is concluded that progesterone stimulates Ca2+ influx in human sperm via a unique Ca2+ channel possibly similar to a store-operated channel (SOC) or a receptor-operated channel (ROC). We have found that progesterone metabolites, such as pregnanolone and pregnanediol, promote a rapid rise in [Ca2+]i and aggregation in human platelets, similar to that observed with thrombin. The increase in [Ca2+]i was prevented when extracellular Ca2+ was removed or by the SOC inhibitor SKF-96365. The phospholipase C inhibitor U-73122 also prevented the increase in [Ca2+]i, suggesting that these metabolites interact with a cell surface receptor on the platelet to activate phospholipase C to produce inositol-P3, which mobilizes intracellular Ca2+, thereby activating the SOC in the plasma membrane. Progesterone and estradiol conjugated to albumin, also produced a rapid increase in [Ca2+]i, which was prevented by Ca2+ removal from the medium or when SKF-96365 or U-73122 were added. It is proposed that human platelets possess cell surface receptors for steroids.     

Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels

An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that most cells exhibited spontaneous, extracellular Ca(2+)-dependent action potentials (APs). Spiking was initiated by a slow pacemaker depolarization from a baseline potential between -75 and -50 mV, and AP frequency increased with membrane depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Characterization of the inward currents in GT1 cells revealed the presence of tetrodotoxin-sensitive Na+, Ni(2+)-sensitive T-type Ca2+, and dihydropyridine-sensitive L-type Ca2+ components. The availability of Na+ and T-type Ca2+ channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in cytosolic Ca2+ concentration ([Ca2+]i), with an early, extracellular Ca(2+)-independent peak and a sustained, extracellular Ca(2+)-dependent phase. During the peak [Ca2+]i response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The GnRH-induced change in firing pattern accounted for about 50% of the elevated Ca2+ influx, the remainder being independent of spiking. Basal [Ca2+]i was also dependent on Ca2+ influx through AP-driven and voltage-insensitive pathways. Thus, in both resting and agonist-stimulated GT1 cells, membrane depolarization limits the participation of Na+ and T-type channels in firing, but facilitates AP-driven Ca2+ influx.     

Calcium fluxes in T lymphocytes.

Mechanisms controlling Ca2+ fluxes through the plasma membrane of lymphocytes have been characterized in a human T-cell clone and in the Jurkat T-cell line. Due to endogenous buffers, about 1/125 of the Ca2+ ions that enter the cell are free. Ca2+ fluxes were estimated from the variations in intracellular Ca2+ concentration ([Ca2+]i) elicited by concentration jumps in extracellular Ca2+ ([Ca2+]o). Thapsigargin was used to inhibit Ca2+ uptake into intracellular stores and to stimulate Ca2+ entry. Ca2+ extrusion was strictly due to the activity of plasma membrane Ca(2+)-ATPases since there was no detectable Na+/Ca2+ exchange activity in these cells. The rate of Ca2+ extrusion was mainly influenced by [Ca2+]i and less by [Ca2+]o but was insensitive to cell depolarization. In depolarized cells, thapsigargin-induced Ca2+ influx was reduced to 10% of the value measured in normally polarized cells, suggesting that depolarization not only reduces the electrochemical gradient for Ca2+ ions, but also inhibits Ca2+ permeation. When Ca2+ ions enter the cell, they bind to a site inside the channel, with a Kd of 3.3 mM. Stimulation of clonal T-cells with low concentrations of either anti-CD3 antibodies or thapsigargin elicited Ca2+ oscillations. Both the amplitude and the frequency of CD3-induced Ca2+ oscillations were sensitive to [Ca2+]o. These oscillations were immediately interrupted when extracellular Ca2+ was removed. The properties of Ca2+ oscillations in T lymphocytes suggest that they are mainly due to variations of Ca2+ influx, modulated by variations in [Ca2+]i.     

Relationship of the decreases in protein synthesis and intracellular Na+ during friend murine erythroleukemic cell differentiation

The earliest known ionic event during Friend murine erythroleukemic (MEL) cell differentiation along the erythroid pathway is a 45% drop in intracellular sodium concentration ([Na+]i) due to a decrease in Na+ influx (Lannigan, D. A., and Knauf, P. A. (1985) J. Biol. Chem. 260, 7322-7324). We have analyzed the mechanism of the decreased Na+ influx. The Na+ influx in uninduced cells was insensitive to dimethylamiloride, bumetanide, and diisothiocyanostilbene disulfonate. The intracellular pH (pHi) did not change up to 15 h after dimethyl sulfoxide induction, at which time Na+ influx has decreased by approximately 40%; thus, the decrease in Na+ influx is not coupled to a change in pHi. A substantial amount of the decrease in Na+ influx seems to result from a drop in amino acid-dependent Na+ transport. This reduction in amino acid-dependent Na+ influx reflects a decrease in net Na+ influx rather than solely in Na+/Na+ exchange and can account for an appreciable portion of the reduction in [Na+]i seen during differentiation. The drop in amino acid-dependent Na+ influx could not be explained by membrane depolarization but was correlated with a decrease in protein synthesis. Inhibition of protein synthesis in uninduced cells by cycloheximide also caused a decrease in Na+ influx. We conclude that during differentiation the reduction in protein synthesis decreases amino acid-dependent Na+ influx which in turn causes a drop in [Na+]i leading to a reduction in the Na+/K+ pump rate.     

Thrombin-induced membrane depolarization of platelets and its inhibition by cetiedil

When 50 microM cetiedil alone was added to a platelet suspension, increase in Na+ content, decrease in K+ content, and depolarization of platelet membrane were observed without change in the intracellular concentration of free Ca2+ ([Ca2+]1) or in the morphology of platelets. The cetiedil-induced depolarization was attenuated by the reduction of extracellular sodium concentration, while sodium transport inhibitors such as procaine and tetrodotoxin failed to modify the depolarization. On the other hand, thrombin caused such changes in platelets as increases in Na+ content, 22Na space and [Ca2+]1, decrease in K+ content, and membrane depolarization. All these changes caused by thrombin were inhibited by cetiedil. It is suggested that cetiedil brought the increased ion transport and subsequent partial depolarization, which might lead to modification of the reaction of platelet membrane induced by thrombin.     

Cross-linking of IgG receptors inhibits membrane immunoglobulin- stimulated calcium influx in B lymphocytes

By cross-linking membrane immunoglobulins (mIg), the antigenic stimulation of B lymphocytes induces an increase in intracellular free calcium levels ([Ca2+]i) because of a combination of release from intracellular stores and transmembrane influx. It has been suggested that both events are linked, as in a number of other cases of receptor- induced increase in [Ca2+]i. Conversely, in B lymphocytes, type II receptors for the Fc fragment of IgG (Fc gamma RII) inhibit mIg- mediated signaling. Thus, we have investigated at the level of single cells if these receptors could act on specific phases of mIg Ca2+ signaling. Lipopolysaccharide-activated murine B splenocytes and B lymphoma cells transfected with intact or truncated Fc gamma RII-cDNA were used to determine the domains of Fc gamma RII implicated in the inhibition of the Ca2+ signal. [Ca2+]i was measured in single fura-2- loaded cells by microfluorometry. The phases of release from intracellular stores and of transmembrane influx were discriminated by using manganese, which quenches fura-2, in the external medium as a tracer for bivalent cation entry. The role of membrane potential was studied by recording [Ca2+]i in cells voltage-clamped using the perforated patch-clamp method. Cross-linking of mIgM or mIgG with F(ab')2 fragments of anti-Ig antibodies induced a sustained rise in [Ca2+]i due to an extremely fast and transitory release of Ca2+ from intracellular stores and a long lasting transmembrane Ca2+ influx. The phase of influx, but not that of release, was inhibited by membrane depolarization. The increase in [Ca2+]i occurred after a delay inversely related to the dose of ligand. Co-cross-linking mIgs and Fc gamma RII with intact anti-Ig antibodies only triggered transitory release of Ca2+ from intracellular stores but no Ca2+ influx, even when the cell was voltage-clamped at negative membrane potentials. These transitory Ca2+ rises had similar amplitudes and delays to those induced by cross-linking mIgs alone. Thus, our data show that Fc gamma RII does not mediate an overall inhibition of mIg signaling but specifically affects transmembrane Ca2+ influx without affecting the release of Ca2+ from intracellular stores. Furthermore, this inhibition is not mediated by cell depolarization. Thus, Fc gamma RII represents a tool to dissociate physiologically the phases of release and transmembrane influx of Ca2+ triggered through antigen receptors.     

Kinetic parameters and mechanism of active cation transport in HeLa cells as studied by Rb+ influx

On incubation of HeLa cells in chilled isotonic medium, intracellular Na+ (Nac+) increased and K+ (Kc+) decreased with time, reaching steady levels after 3 h. The steady levels varied in parallel with the extracellular cation concentrations ([Na+]e, [K+]e). The cell volumes and the protein and water contents, respectively, of cells kept for 3 h in chilled media of various [Na+]e and [K+]e were not significantly different. Ouabain-sensitive Rb+ influx took place at the initial rate for a certain period which depended on [Na+]c at the beginning of the assays. The existence of two external K+ loading sites per Na+/K+-pump was demonstrated. The affinities of the sites for Rb+ as a congener of K+ were almost the same. Na+e inhibited ouabain-sensitive Rb+ influx competitively, whereas K+ was not inhibitory. Kinetic parameters were determined: the K 1/2 for Rbe+ in the absence of Na+e was 0.16 mM and th Ki for Na+e was 36.8 mM; the K 1/2 for Na+c was 19.5 mM and the Ki for K+c seemed to be extremely large. The rate equation of the ouabain-sensitive Rb+ influx suggests that Na+ and K+ are exchanged alternately through the pump by a binary mechanism.     

Dual-fluorescence flow cytometric analysis of membrane potential and cytoplasmic free Ca2+ concentration in embryonic rat hippocampal cells

We have demonstrated simultaneous measurement of the membrane potential and cytoplasmic free Ca2+ concentration ([Ca2+]i) by utilizing a dual-laser flow cytometer in embryonic rat hippocampal cell suspensions. Veratrine, a Na+ channel activator, induced both membrane depolarization and elevation of [Ca2+]i. These actions of veratrine were all reversed by the presence of tetrodotoxin (TTX). These findings suggest that Na+ channels are functionally expressed in the cells and the activation of Na+ channels increases [Ca2+]i. The usefulness of the flow cytometric analysis in elucidating the expression of membrane functions in the embryonic central nervous systems (CNS) is discussed.     

Activation of AMPA/kainate receptors but not acetylcholine receptors causes Mg2+ influx into Retzius neurones of the leech Hirudo medicinalis

In Retzius neurones of the medicinal leech, Hirudo medicinalis, kainate activates ionotropic glutamate receptors classified as AMPA/kainate receptors. Activation of the AMPA/kainate receptor-coupled cation channels evokes a marked depolarization, intracellular acidification, and increases in the intracellular concentrations of Na+ ([Na+]i) and Ca2+. Qualitatively similar changes are observed upon the application of carbachol, an activator of acetylcholine receptor-coupled cation channels. Using multibarrelled ion-selective microelectrodes it was demonstrated that kainate, but not carbachol, caused additional increases in the intracellular free Mg2+ concentration ([Mg2+]i). Experiments were designed to investigate whether this kainate-induced [Mg2+]i increase was due to a direct Mg2+ influx through the AMPA/kainate receptor-coupled cation channels or a secondary effect due to the depolarization or the ionic changes. It was found that: (a) Similar [Mg2+]i increases were evoked by the application of glutamate or aspartate. (b) All kainate-induced effects were inhibited by the glutamatergic antagonist DNQX. (c) The magnitude of the [Mg2+]i increases depended on the extracellular Mg2+ concentration. (d) A reduction of the extracellular Ca2+ concentration increased kainate-induced [Mg2+]i increases, excluding possible Ca2+ interference at the Mg2+-selective microelectrode or at intracellular buffer sites. (e) Neither depolarizations evoked by the application of 30 mM K+, nor [Na+]i increases induced by the inhibition of the Na+/K+ ATPase caused comparable [Mg2+]i increases. (f) Inhibitors of voltage-dependent Ca2+ channels did not affect the kainate-induced [Mg2+]i increases. Moreover, previous experiments had already shown that intracellular acidification evoked by the application of 20 mM propionate did not cause changes in [Mg2+]i. The results indicate that kainate-induced [Mg2+]i increases in leech Retzius neurones are due to an influx of extracellular Mg2+ through the AMPA/kainate receptor-coupled cation channel. Mg2+ may thus act as an intracellular signal to distinguish between glutamatergic and cholinergic activation of leech Retzius neurones.     

Effect of the sulfhydryl reagent thimerosal on cytosolic free Ca2+ and membrane potential of thymocytes.

The sulfhydryl reagent thimerosal at concentrations 5-100 microM has been found to induce a variety of changes in ion transport in rat thymocytes. In particular, [Ca2+]i increases about 10-fold from the basal level. The [Ca2+]i response to thimerosal displays a two-stage time course, with the main [Ca2+]i rise during the second stage. Evidence has been obtained for the depletion of intracellular Ca2+ pools in thimerosal-treated cells, however, Ca2+ mobilization from intracellular stores does not contribute significantly into [Ca2+]i rise. Thimerosal elicits permeability not only for Ca2+, but also for Mn2+ and Ni2+, which is Ca(2+)-dependent. We failed to get any evidence on thimerosal-induced inhibition of the plasma membrane Ca(2+)-ATPase. The induction of Ca2+ influx, rather than inhibition of Ca(2+)-ATPase, accounts for the disturbance of [Ca2+]i homeostasis in thimerosal-treated cells. Thimerosal also elicits changes in monovalent ion fluxes resulting in marked depolarization. The latter seems unrelated to the changes in [Ca2+]i and is suggested to be mediated both by increased permeability for Na+ and a decreased one for K+. Thimerosal significantly stimulates AA release from thymocytes. Evidence has been presented that AA metabolite(s), probably, LO product(s), may mediate the changes in the transport of mono- and divalent cations elicited by the sulfhydryl reagent. Prolonged treatment of thymocytes with thimerosal resulted in cell death.     

Ca2+ influx-independent synaptic potentiation mediated by mitochondrial Na(+)-Ca2+ exchanger and protein kinase C

Activity-dependent modulation of synaptic transmission is an essential mechanism underlying many brain functions. Here we report an unusual form of synaptic modulation that depends on Na+ influx and mitochondrial Na(+)-Ca2+ exchanger, but not on Ca2+ influx. In Ca(2+)-free medium, tetanic stimulation of Xenopus motoneurons induced a striking potentiation of transmitter release at neuromuscular synapses. Inhibition of either Na+ influx or the rise of Ca2+ concentrations ([Ca2+]i) at nerve terminals prevented the tetanus-induced synaptic potentiation (TISP). Blockade of Ca2+ release from mitochondrial Na(+)-Ca2+ exchanger, but not from ER Ca2+ stores, also inhibited TISP. Tetanic stimulation in Ca(2+)-free medium elicited an increase in [Ca2+]i, which was prevented by inhibition of Na+ influx or mitochondrial Ca2+ release. Inhibition of PKC blocked the TISP as well as mitochondrial Ca2+ release. These results reveal a novel form of synaptic plasticity and suggest a role of PKC in mitochondrial Ca2+ release during synaptic transmission.     

Mode of Action of Palytoxin on the Release of Acetylcholine from Rat Cerebrocortical Synaptosomes

Palytoxin (PTX; 10(-14)-10(-6) M) caused a dose-dependent increase in the release of [3H]acetylcholine ([3H]ACh), cytosolic free Ca2+ concentration ([Ca2+]i), and uptake of 22Na+ and decrease in membrane potential in rat cerebrocortical synaptosomes. The dose-response curves for the PTX-induced increases in [3H]ACh release and in [Ca2+]i were depressed by removing extracellular Ca2+ or by decreasing extracellular Na+ concentrations. The release of [3H]ACh induced by concentrations of PTX less than 10(-10) M was more dependent on the simultaneous presence of both Ca2+ and Na+ than the release induced by higher concentrations of PTX. The PTX-induced increase both in [3H]ACh release and in [Ca2+]i was almost completely abolished by the combination of Ca2+ deprivation and Na+ concentration reduction. All responses to PTX were highly resistant to 10(-6) M tetrodotoxin. These results suggest that low concentrations of PTX cause depolarization as a result of an increase in Na+ permeability through tetrodotoxin-insensitive channels. This, in turn, increases Ca2+ influx and leads to an increase in the release of ACh. It appears that at high concentrations PTX increases the release of [3H]ACh by directly increasing the influx of Ca2+ into synaptosomes and by releasing Ca2+ from intracellular storage sites via an Na(+)-Ca2+ exchange mechanism.     

Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium

In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.     

Correlation between plasma membrane potential and second messenger generation in the promyelocytic cell line HL-60.

The effects of plasma membrane depolarization on cytosolic free calcium ([Ca2+]i) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) generation were investigated in the human promyelocytic cell line HL-60 differentiated with either dimethyl sulfoxide or retinoic acid into neutrophil-like cells. Increases in [Ca2+]i and accumulation of Ins(1,4,5)P3 were triggered by two chemoattractants fMet-Leu-Phe and leukotriene B4. Plasma membrane potential was depolarized by isoosmotic substitution of NaCl with KCl, by the pore-forming ionophore gramicidin D, or by long term treatment with ouabain. Both Ca2+ mobilization from intracellular stores and Ca2+ influx across the plasma membrane were reduced by prior depolarization of plasma membrane potential regardless of the procedure employed to collapse it. Agonist-induced generation of Ins(1,4,5)P3 was also reduced in parallel in pre-depolarized HL-60 cells. The present findings provide further evidence suggesting that plasma membrane potential can be an important modulator of agonist-activated second messenger generation in myelocytic cells.     

Calcium-activated membrane depolarization via modulation of chloride efflux from parietal cells during gastrin stimulation

In the isolated parietal cell the following observations were made: gastrin led to an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) even in the absence of medium Ca2+ and to transient membrane depolarization in both the absence of the medium Ca2+ and the fura-2-loaded cell. The incorporated Ca2+ chelator BAPTA inhibited the gastrin-induced membrane depolarization. The magnitude of depolarization caused by gastrin was unchanged on removal of medium Na+. Furosemide but not tetraethylammonium inhibited the gastrin-induced depolarization. The results suggest that the Ca2+ released from the store(s) induces membrane depolarization, possibly via modulation of a Cl- efflux across the luminal surface during gastrin stimulation.     

Phospholipase C-beta and ovarian sex steroids in pig granulosa cells.

We compared the membrane effects of estradiol, progesterone, and androstenedione in a single experimental model, the ovarian granulosa cells collected from immature Large White sows. We measured changes in cytosolic free calcium concentration ([Ca2+]i) in confluent Fura-2 loaded cells. We used pharmacological tools and polyclonal phospholipase C-beta (PLC-beta) antibodies. Each steroid (0.1 pM to 1 nM) transiently increased intracellular calcium concentration ([Ca2+]i) within 5 sec. They mobilized Ca2+ from the endoplasmic reticulum as shown by using two phospholipase C inhibitors, neomycin and U-73122. Ca2+ mobilization involved PLC-beta1 for progesterone, PLC-beta2 for estradiol and PLC-beta4 for androstenedione. A pertussis toxin-insensitive G protein was involved in the effects of progesterone on Ca2+ mobilization whereas estradiol and androstenedione effects were mediated via a pertussis toxin-sensitive G-protein. Ca2+ influx from the extracellular milieu was involved in the increase in [Ca2+]i induced by progesterone and estradiol, but not by androstenedione. Influx of Ca2+ was independent of Ca2+ mobilization from calcium stores, and it was suggested that L-type Ca2+ channels for estradiol and T-type Ca2+ channels for progesterone were involved. The three steroids had no effect on cAMP. Rapid effects of progesterone, estradiol, and androstenedione involved a direct action on cell membrane elements such as PLC-beta, G-proteins, and calcium channels, and these mechanisms were hormone-specific.     

Effects of a time-varying strong magnetic field on transient increase in Ca2+ release induced by cytosolic Ca2+ in cultured pheochromocytoma cells

Exposure of pheochromocytoma (PC 12) cells to a time-varying 1.51 T magnetic field inhibited an increase in the intracellular Ca2+ concentration ([Ca2+]i) induced by addition of caffeine to Ca(2+)-free medium. This inhibition occurred after a 15-min exposure and was maintained for at least 2 h. [Ca2+]i sharply increased in cells loaded with cyclic ADP-ribose, and 2-h exposure significantly suppressed the increase. Addition of ATP induced a transient increase in intracellular Ca2+ release mediated by IP3 receptor, and this increase was strongly inhibited by the exposure. Results indicated that the magnetic field exposure strongly inhibited Ca2+ release mediated by both IP3 and ryanodine receptors in PC 12 cells. However, thapsigargin-induced Ca2+ influx (capacitative Ca2+ entry) across the cell membrane was unaffected. The ATP content was maintained at the normal level during the 2-h exposure, suggesting that ATP hydrolysis was unchanged. Therefore, Mg2+ which is known to be released by ATP hydrolysis and inhibit intracellular Ca2+ release may not relate the exposure-caused inhibition. Eddy currents induced in culture medium appear to change cell membrane properties and indirectly inhibit Ca2+ release from endoplasmic reticulum and other Ca2+ stores in PC 12 cells.     

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.     

Increased plasma membrane permeability to Ca2+ in anti-Ig-stimulated B lymphocytes is dependent on activation of phosphoinositide hydrolysis

The biochemical basis of Ca2+ mobilization after anti-Ig binding to B cell Ag-R has been further characterized by flow cytometric analysis of indo-1-loaded B cells. The ability to distinguish intracellular Ca2+ release from extracellular Ca2+ influx by using an extracellular calcium depletion-repletion approach has allowed us to study the relationship between the mobilization of Ca2+ from these sources. Studies involving manipulation of the Ca2+ gradient across the plasma membrane indicate that a significant portion of the Ca2+ mobilization response is preserved even when the normal inwardly directed Ca2+ gradient is reversed. In the presence of an extracellular calcium concentration ([Ca2+]o) of 10 microM, the response to anti-Ig is not blocked by the organic Ca2+ channel blockers. This response is not reduced by further depletion of [Ca2+]o by EGTA Ca2+-binding buffers. Thus, the Ca2+ response that occurs when [Ca2+]o less than or equal to 10 microM represents intracellular calcium release. Analysis of B cells stimulated with anti-Ig in low Ca2+ medium ([Ca2+]o = less than 10 microM) followed by repletion of [Ca2+]o to 1 to 5 mM reveals that a significant increase in permeability of the plasma membrane to Ca2+ develops in the stimulated cells. The resultant Ca2+ influx is nimodipine (20 microM) sensitive. Both intracellular Ca2+ release and Ca2+ influx are reduced in parallel as the concentration of anti-Ig stimulus is decreased, suggesting that Ca2+ influx may be coupled to the release of intracellular stores. Neomycin blocks anti-Ig-stimulated formation of inositol trisphosphate, which mediates release of Ca2+ from the endoplasmic reticulum. It also blocks the anti-Ig-induced release of intracellular Ca2+ stores as well as Ca2+ influx, indicating that both responses may be dependent upon phosphatidylinositol 4,5-bisphosphate hydrolysis.     

Secretagogue-induced mobilization of an intracellular Mg2+ pool in rat sublingual mucous acini.

The regulation of the intracellular free Mg2+ concentration ([Mg2+]i) was monitored in rat sublingual mucous acini using dual wavelength microfluorometry of the Mg(2+)-sensitive dye mag-fura-2. Acini attached to coverslips and superfused continuously with a Mg(2+)-containing medium (0.8 mM) have a steady-state [Mg2+]i of 0.35 +/- 0.01 mM. Adjusting the extracellular Mg2+ concentration to 0 and 10 mM or removing extracellular Na+ did not alter the resting [Mg2+]i. Stimulation with the Ca(2+)-mobilizing, muscarinic agonist, carbachol, induced a sustained increase in [Mg2+]i (approximately 50%; t1/2 < 20 s; Kd approximately 1.5 microM), the magnitude and the duration of which were unchanged in Mg(2+)-depleted medium indicating that the rise in [Mg2+]i was generated by Mg2+ release from an intracellular Mg2+ pool. Forskolin, which increases the intracellular cAMP content, produced a small, transient increase in the [Mg2+]i (< 10%). Muscarinic stimulation in a Ca(2+)-free medium blunted the initial increase in [Mg2+]i by approximately 50%, whereas the sustained increase in [Mg2+]i was lost. When the muscarinic-induced increase in [Ca2+]i was blocked by 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate, an inhibitor of the agonist-sensitive intracellular Ca2+ release pathway, both the initial and the sustained phases of the increase in [Mg2+]i were virtually eliminated. Thapsigargin and 2,5-di-(terbutyl)-1,4-benzohydroquinone, which increase [Ca2+]i by inhibiting microsomal Ca(2+)-ATPase, caused a dramatic increase in [Mg2+]i. Stimulation in a Na(+)-free medium or in the presence of bumetanide, an inhibitor of Na+/K+/2Cl- cotransport, blunted the agonist-induced rise in [Mg2+]i (approximately 50%), whereas ouabain, a Na+,K(+)-ATPase inhibitor, had no significant effect. FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), a mitochondrial uncoupler, mobilized an intracellular Mg2+ pool as well. The carbachol-induced increase in [Mg2+]i was markedly inhibited by FCCP (approximately 80%), suggesting that the same pool(s) of Mg2+ were primarily involved. The above results provide strong evidence that Ca(2+)-mobilizing agonists increase cytoplasmic free [Mg2+] by releasing an intracellular pool of Mg2+ that is associated with a rise in the [Na+]i.