Effects of organic antagonists of Ca(2+), Na(+), and K(+) on chemotaxis and motility of escherichia coli

Various Ca(2+) antagonists used in animal research, many of them known to be Ca(2+) channel blockers, inhibited Escherichia coli chemotaxis (measured as entry of cells into a capillary containing attractant). The most effective of these, acting in the nanomolar range, was omega-conotoxin GVIA. The next most effective were gallopamil and verapamil. At concentrations around 100-fold higher than that needed for inhibition of chemotaxis, each of these antagonists inhibited motility (measured as entry of cells into a capillary lacking attractant). Various other Ca(2+) antagonists were less effective, though chemotaxis was almost always more sensitive to inhibition than was motility. Cells treated with each of these Ca(2+) antagonists swam with a running bias, i.e., tumbling was inhibited. Similarly, some Na(+) antagonists used in animal research inhibited bacterial chemotaxis. E. coli chemotaxis was inhibited by saxitoxin at concentrations above 10(-7) M, while more than 10(-4) M was needed to inhibit motility. Cells treated with saxitoxin swam with a tumbling bias. In the case of other Na(+) antagonists in animals, aconitine inhibited bacterial chemotaxis 10 times more effectively than it inhibited motility, and two others inhibited chemotaxis and motility at about the same concentration. In the case of K(+) antagonists used in animal research, 4-aminopyridine blocked E. coli chemotaxis between 10(-3) M and, totally, 10(-2) M, while motility was not affected at 10(-2) M; on the other hand, tetraethylammonium chloride failed to inhibit either chemotaxis or motility at 10(-2) M.     

The dihydropyridine-sensitive calcium channel subtype in cone photoreceptors

High-voltage activated Ca channels in tiger salamander cone photoreceptors were studied with nystatin-permeabilized patch recordings in 3 mM Ca2+ and 10 mM Ba2+. The majority of Ca channel current was dihydropyridine sensitive, suggesting a preponderance of L- type Ca channels. However, voltage-dependent, incomplete block (maximum 60%) by nifedipine (0.1-100 microM) was evident in recordings of cones in tissue slice. In isolated cones, where the block was more potent, nifedipine (0.1-10 microM) or nisoldipine (0.5-5 microM) still failed to eliminate completely the Ca channel current. Nisoldipine was equally effective in blocking Ca channel current elicited in the presence of 10 mM Ba2+ (76% block) or 3 mM Ca2+ (88% block). 15% of the Ba2+ current was reversibly blocked by omega-conotoxin GVIA (1 microM). After enhancement with 1 microM Bay K 8644, omega-conotoxin GVIA blocked a greater proportion (22%) of Ba2+ current than in control. After achieving partial block of the Ba2+ current with nifedipine, concomitant application of omega-conotoxin GVIA produced no further block. The P-type Ca channel blocker, omega-agatoxin IVA (200 nM), had variable and insignificant effects. The current persisting in the presence of these blockers could be eliminated with Cd2+ (100 microM). These results indicate that photoreceptors express an L-type Ca channel having a distinguishing pharmacological profile similar to the alpha 1D Ca channel subtype. The presence of additional Ca channel subtypes, resistant to the widely used L-, N-, and P-type Ca channel blockers, cannot, however, be ruled out.     

Type IV collagen stimulates an increase in intracellular calcium. Potential role in tumor cell motility.

Type IV collagen (Coll IV), a component of the extracellular matrix, stimulates motility in the A2058 human melanoma cell line, a response that is inhibited by pertussis toxin (PT). Fibronectin (FN)-induced chemotaxis in this cell line is not affected by PT. To understand the mechanism of cellular signaling, single cell intracellular Ca2+ responses to Coll IV and FN were studied using Fura-2 and digital imaging fluorescence microscopy. Coll IV, at a dose that stimulates motility (100 micrograms/ml, 185 nM), induces a significant rise in cytosolic free Ca2+ concentration ([Ca2+]i) within 100 s. This response is not inhibited by PT. Treatment of the cells with FN 30 micrograms/ml (70 nM), a dose that stimulates near-maximal chemotaxis, does not increase [Ca2+]i appreciably. Removal of extracellular Ca2+ fails to inhibit the Coll IV-stimulated rise in Ca2+ in all cells. Depletion of extracellular Ca2+ and pretreatment of cells with Ca2+ channel blockers only partially inhibits Coll IV-induced motility. Depletion of intracellular Ca2+ inhibits both chemotaxis and the Coll IV-induced increase in intracellular Ca2+. Coll IV does not stimulate membrane phosphoinositide hydrolysis. We conclude that Coll IV treatment induces an inositol 1,4,5-trisphosphate-independent release of intracellular Ca2+ stores which appears to play a necessary role in the chemotactic response of A2058 cells but is not mediated by a PT-sensitive G-protein. This response is not seen in cells exposed to FN, suggesting different intracellular signaling mechanisms for stimulated motility between these two extracellular matrix molecules.     

Clostridium perfringens alpha-toxin-induced hemolysis of horse erythrocytes is dependent on Ca2+ uptake

Clostridium perfringens alpha-toxin is able to lyse various erythrocytes. Exposure of horse erythrocytes to alpha-toxin simultaneously induced hot-cold hemolysis and stimulated production of diacylglycerol and phosphorylcholine. When A23187-treated erythrocytes were treated with the toxin, these events were dependent on the concentration of extracellular Ca2+ . Incubation with the toxin of BAPTA-AM-treated horse erythrocytes caused no hemolysis or production of phosphorylcholine, but that of the BAPTA-treated erythrocytes did. When Quin 2-AM-treated erythrocytes were incubated with the toxin in the presence of 45Ca2+, the cells accumulated 45Ca2+ in a dose- and a time-dependent manner. These results suggest that the toxin-induced hemolysis and hydrolysis of phosphatidylcholine are closely related to the presence of Ca2+ in the cells. Flunarizine, a T-type Ca2+ channel blocker, and tetrandrine, an L- and T-type Ca2+ channel blocker, inhibited the toxin-induced hemolysis and Ca2+ uptake. However, L-type Ca2+ channel blockers, nifedipine, verpamil and diltiazem, an N-type blocker, omega-conotoxin SVIB, P-type blockers, omega-agatoxin TK and omega-agatoxin IVA, and a Q-type blocker, omega-conotoxin MVII C, had no such inhibitory effect. The observation suggests that Ca2+ taken up through T-type Ca2+ channels activated by the toxin plays an important role in hemolysis induced by the toxin.     

Chemotactic properties of Escherichia coli mutants having abnormal Ca2+ content.

The calA, calC, and calD mutants of Escherichia coli are known to be sensitive to Ca2+ (R. N. Brey and B. P. Rosen, J. Bacteriol. 139:824-834, 1979). In the absence of any added stimuli for chemotaxis, both the calC and the calD mutants swam with a tumbly bias. Both the calC and the calD mutants were defective in chemotaxis as measured by computer analysis, use of swarm plates, and capillary assays. The calA mutant was only slightly defective in motility and only slightly impaired in chemotaxis. Chemotactically wild-type cells had an intra-cellular free-Ca2+ level of about 105 nM. The intracellular free-Ca2+ levels of the mutants, as determined by use of the fluorescent Ca2+ indicator dye fura-2 or fluo-3, were about 90, about 1,130, and about 410 nM for calA, calC, and calD, respectively. Lowering the intracellular free-Ca2+ levels in wild-type cells and in the tumbly cal mutants by use of Ca2+ chelators promoted running (smooth swimming). Overexpression of CheZ (which causes dephosphorylation of CheY-phosphate) in the wild type and in the tumbly cal mutants decreased the level of tumbliness (which is caused by CheY-phosphate). The calA mutant was 4- to 10-fold more resistant than the wild type to the inhibitory effect of omega-conotoxin on chemotaxis. omega-Conotoxin had no effect on Ca2+ extrusion by wild-type E. coli; that result suggests that omega-conotoxin affects Ca2+ transport at the point of entry instead of exit.     

Separate Binding and Functional Sites for ω co-Conotoxin and Nitrendipine Suggest Two Types of Calcium Channels in Bovine Chromaffin Cells

Purified adrenomedullary plasma membranes contain two high-affinity binding sites for 125I-omega-conotoxin, with KD values of 7.4 and 364 pM and Bmax values of 237 and 1,222 fmol/mg of protein, respectively. Dissociation kinetics showed a biphasic component and a high stability of the toxin-receptor complex, with a t1/2 of 81.6 h for the slow dissociation component. Unlabeled omega-conotoxin inhibited the binding of the radioiodinated toxin, adjusting to a two-site model with Ki1 of 6.8 and Ki2 of 653 pM. Specific binding was not affected by Ca2+ channel blockers or activators, cholinoceptor antagonists, adrenoceptor blockers, Na+ channel activators, dopaminoceptor blockers, or Na+/H+ antiport blockers, but divalent cations (Ca2+, Sr2+, and Ba2+) inhibited the toxin binding in a concentration-dependent manner. The binding of the dihydropyridine [3H]nitrendipine defined a single specific binding site with a KD of 490 pM and a Bmax of 129 fmol/mg of protein. At 0.25 microM, omega-conotoxin was not able to block depolarization-evoked Ca2+ uptake into cultured bovine adrenal chromaffin cells depolarized with 59 mM K+ for 30 s, whereas under the same conditions, 1 microM nitrendipine inhibited uptake by approximately 60%. When cells were hyperpolarized with 1.2 mM K+ for 5 min and then Ca2+ uptake was subsequently measured during additions of 59 mM K+. Omega-conotoxin partially inhibited Ca2+ uptake in a concentration-dependent manner. These results suggest that two different types of Ca2+ channels might be present in chromaffin cells. However, the molecular identity of omega-conotoxin binding sites remains to be determined.     

Different contributions of voltage-sensitive Ca2+ channels to histamine-induced catecholamine release and tyrosine hydroxylase activation in bovine adrenal chromaffin cells.

Histamine stimulates catecholamine release and tyrosine hydroxylase activity in a Ca(2+)-dependent manner in bovine adrenal chromaffin cells. The role of voltage-sensitive Ca2+ channels in these two responses has been investigated. Using an EC50 concentration of histamine, 1 microM, catecholamine release was enhanced by (+/-)BayK8644, and partially inhibited by nitrendipine and omega-agatoxin IVA, blockers of L- and P/Q-type Ca2+ channels. omega-Conotoxin GVIA gave small and variable inhibitory effects. With a maximal histamine concentration, 10 microM, similar results were obtained except that now omega-conotoxin GVIA reliably reduced release. In contrast, neither (+/-)BayK8644 nor any of the individual Ca2+ channel antagonists had any significant effect on tyrosine hydroxylase (TOH) activation induced by either an EC50 or a maximal concentration of histamine. When high concentrations of nitrendipine, omega-conotoxin GVIA and omega-agatoxin IVA were combined with omega-conotoxin MVIIC (a non-selective blocker of N, P and Q channels) to block voltage-sensitive Ca2+ channels in these cells, release induced by K+ depolarization was completely blocked. Release caused by histamine, however, was substantially reduced but not abolished. The combination of antagonists also only partially inhibited TOH activation by histamine. The results show that the G protein-coupled receptor agonist histamine activates several different types of voltage-sensitive Ca2+ channels in chromaffin cells to mediate its cellular effects. Histamine may also activate additional pathways for Ca2+ entry. The results also suggest that the manner by which Ca2+ controls release and TOH activation once it has entered chromaffin cells through these channels are different.     

Pharmacological characterisation of voltage-dependent Ca2+ channels in isolated ganglia from the myenteric plexus

Voltage-dependent Ca2+ channels of fura-2-loaded ganglionic cells from the myenteric plexus of newborn rats were pharmacologically characterised. In contrast to completely dissociated myenteric cells, intact ganglia showed a stronger loading with the Ca2+-sensitive dye and a reproducible stimulation of the fura-2 signal by the cholinergic agonist, carbachol. A depolarisation-induced increase in the intracellular Ca2+ concentration ([Ca2+]i) was induced by superfusion with 35 mmol l(-1) KCl. This increase in [Ca2+]i was sensitive to Ni2+ and Co2+ as well as omega-conotoxin MVIIA, omega-agatoxin IVA, and SNX-482. The strongest inhibition was achieved by nifedipine (5 x 10(-7) mol l(-1)) and omega-conotoxin GVIA (4.3 x 10(-7) mol l(-1)). These two blockers also inhibited the [Ca2+]i increase evoked by nicotinic receptor stimulation. Consequently, isolated myenteric ganglia in culture express different types of voltage-dependent Ca2+ channels, from which the L- and the N-type seem to be the most important. When exposed to mediators of inflammation such as tumor necrosis factor-alpha (TNF-alpha) or different prostaglandins, no pronounced alterations in the fura-2 ratio were observed suggesting that changes in the Ca2+-signalling are not centrally involved in the response of enteric ganglionic cells to these paracrine substances.     

Ca2+ channels in rat central and peripheral neurons: high-threshold current resistant to dihydropyridine blockers and omega-conotoxin.

Block of Ca2+ channel current by dihydropyridines and by omega-conotoxin (omega-CgTx) was studied in a variety of freshly dissociated rat neurons. In most neurons, including those from dorsal root ganglia, sympathetic ganglia, spinal cord, cerebral cortex, and hippocampus, nitrendipine and omega-CgTx each blocked a fraction of the high-threshold current, but a substantial fraction of current remained even when the two blockers were applied together at saturating concentrations. An extreme case was cerebellar Purkinje neurons, in which very little current was blocked by either nitrendipine or omega-CgTx. These results demonstrate the existence in mammalian neurons of high-threshold channels that are resistant to both omega-CgTx and dihydropyridine blockers. Such channels might underlie instances of synaptic transmission and other processes that depend on Ca2+ entry but are not sensitive to these blockers.     

Revisiting the role of H+ in chemotactic signaling of sperm

Chemotaxis of sperm is an important step toward fertilization. During chemotaxis, sperm change their swimming behavior in a gradient of the chemoattractant that is released by the eggs, and finally sperm accumulate near the eggs. A well established model to study chemotaxis is the sea urchin Arbacia punctulata. Resact, the chemoattractant of Arbacia, is a peptide that binds to a receptor guanylyl cyclase. The signaling pathway underlying chemotaxis is still poorly understood. Stimulation of sperm with resact induces a variety of cellular events, including a rise in intracellular pH (pHi) and an influx of Ca2+; the Ca2+ entry is essential for the chemotactic behavior. Previous studies proposed that the influx of Ca2+ is initiated by the rise in pHi. According to this proposal, a cGMP-induced hyperpolarization activates a voltage-dependent Na+/H+ exchanger that expels H+ from the cell. Because some aspects of the proposed signaling pathway are inconsistent with recent results (Kaupp, U.B., J. Solzin, J.E. Brown, A. Helbig, V. Hagen, M. Beyermann, E. Hildebrand, and I. Weyand. 2003. Nat. Cell Biol. 5:109-117), we reexamined the role of protons in chemotaxis of sperm using kinetic measurements of the changes in pHi and intracellular Ca2+ concentration. We show that for physiological concentrations of resact (<25 pM), the influx of Ca2+ precedes the rise in pHi. Moreover, buffering of pHi completely abolishes the resact-induced pHi signal, but leaves the Ca2+ signal and the chemotactic motor response unaffected. We conclude that an elevation of pHi is required neither to open Ca(2+)-permeable channels nor to control the chemotactic behavior. Intracellular release of cGMP from a caged compound does not cause an increase in pHi, indicating that the rise in pHi is induced by cellular events unrelated to cGMP itself, but probably triggered by the consumption and subsequent replenishment of GTP. These results show that the resact-induced rise in pHi is not an obligatory step in sperm chemotactic signaling. A rise in pHi is also not required for peptide-induced Ca2+ entry into sperm of the sea urchin Strongylocentrotus purpuratus. Speract, a peptide of S. purpuratus may act as a chemoattractant as well or may serve functions other than chemotaxis.     

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.     

Limited regulation of somatodendritic dopamine release by voltage-sensitive Ca channels contrasted with strong regulation of axonal dopamine release

The mechanism underlying somatodendritic release of dopamine (DA) appears to differ from that of axon-terminal release. Specifically, somatodendritic DA release in the substantia nigra pars compacta (SNc) persists in low extracellular Ca2+ concentrations that are insufficient to support axonal release in striatum, suggesting that limited Ca2+ entry is necessary to trigger somatodendritic release. Here, we compared the role of voltage-dependent Ca2+ channels in mediating DA release in striatum versus SNc using specific blockers of N-, P/Q-, T-, R- and L-type Ca2+ channels individually and in combination. Release of DA evoked by a single stimulus pulse in the dorsal striatum and SNc of guinea-pig brain slices was monitored in real time using carbon-fiber microelectrodes with fast-scan cyclic voltammetry. Single-pulse evoked DA release was shown to be independent of regulation by concurrently released glutamate or GABA acting at ionotropic receptors in both regions. Under these conditions, striatal DA release was completely prevented by an N-type channel blocker, omega-conotoxin GVIA (100 nm), and was decreased by 75% by the P/Q-type channel blocker omega-agatoxin IVA (200 nm). Blockade of T-type channels with Ni2+ (100 microm) or R-type channels with SNX-482 (100 nm) decreased axonal release in striatum by 25%, whereas inhibition of L-type channels with nifedipine (20 microm) had no effect. By contrast, none of these Ca2+-channel blockers altered the amplitude of somatodendritic DA release in the SNc. Even a cocktail of all blockers tested did not alter release-signal amplitude in the SNc, although the duration of the release response was curtailed. The limited involvement of voltage-dependent Ca2+ channels in somatodendritic DA release provides further evidence that minimal Ca2+ entry is required to trigger the release process, compared with that required for axon-terminal release.     

Ca2+ spikes in the flagellum control chemotactic behavior of sperm

The events that occur during chemotaxis of sperm are only partly known. As an essential step toward determining the underlying mechanism, we have recorded Ca2+ dynamics in swimming sperm of marine invertebrates. Stimulation of the sea urchin Arbacia punctulata by the chemoattractant or by intracellular cGMP evokes Ca2+ spikes in the flagellum. A Ca2+ spike elicits a turn in the trajectory followed by a period of straight swimming ('turn-and-run'). The train of Ca2+ spikes gives rise to repetitive loop-like movements. When sperm swim in a concentration gradient of the attractant, the Ca2+ spikes and the stimulus function are synchronized, suggesting that precise timing of Ca2+ spikes controls navigation. We identified the peptide asterosap as a chemotactic factor of the starfish Asterias amurensis. The Ca2+ spikes and swimming behavior of sperm from starfish and sea urchin are similar, implying that the signaling pathway of chemotaxis has been conserved for almost 500 million years.     

Involvement of Na+ and Ca2+ channel activation and resultant nitric oxide synthesis in glutamate-mediated hypoxic injury in rat cerebrocortical slices

The role of Na+ and Ca2+ channels in glutamate-mediated hypoxic injury was investigated in slices of the rat cerebral cortex. Hypoxic injury was determined by mitochondrial reduction of 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyltetrazolium bromide after exposure of brain slices to 30-min of hypoxia/glucose deprivation followed by 3-h of reoxygenation. Endogenous glutamate release was markedly elevated during hypoxia/glucose deprivation, but it returned almost to basal level during reoxygenation. Hypoxic injury was prevented by MK-801 or 6-cyano-7-nitroquinoxaline-2,3-dione. Combined treatment with omega-conotoxin GVIA, omega-agatoxin IVA, and tetrodotoxin reversed the hypoxic injury, although none of these agents alone or nifedipine was effective. Moreover, a novel Na+/Ca2+ channel blocker NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride] significantly inhibited the hypoxic injury. Several inhibitors of nitric oxide synthase also blocked the hypoxic injury. Consistently, nitric oxide synthesis, as estimated from cyclic GMP formation in the extracellular fluids, was enhanced during hypoxia/glucose deprivation. NS-7 and other Na+ and Ca2+ channel blockers suppressed the enhancement of nitric oxide synthesis, although these compounds alone, or in combination, did not reduce hypoxic glutamate release. These findings suggest that hypoxic injury in rat cerebrocortical slices is triggered by glutamate and subsequent enhancement of nitric oxide synthesis through activation of both Na+ and Ca2+ channels. Thus, the simultaneous blockade of both Na+ channel as well as N-type and P/Q-type Ca2+ channels is required to sufficiently reverse the hypoxic injury.     

Somatostatin decreases voltage-gated Ca2+ currents in GH3 cells through activation of somatostatin receptor 2

The secretion of growth hormone (GH) is inhibited by hypothalamic somatostatin (SRIF) in somatotropes through five subtypes of the somatostatin receptor (SSTR1-SSTR5). We aimed to characterize the subtype(s) of SSTRs involved in the Ca2+ current reduction in GH3 somatotrope cells using specific SSTR subtype agonists. We used nystatin-perforated patch clamp to record voltage-gated Ca2+ currents, using a holding potential of -80 mV in the presence of K+ and Na+ channel blockers. We first established the presence of T-, L-, N-, and P/Q-type Ca2+ currents in GH3 cells using a variety of channel blockers (Ni+, nifedipine, omega-conotoxin GVIA, and omega-agatoxin IVA). SRIF (200 nM) reduced L- and N-type but not T- or P/Q-type currents in GH3 cells. A range of concentrations of each specific SSTR agonist was tested on Ca2+ currents to find the maximal effective concentration. Activation of SSTR2 with 10(-7) and 10(-8) M L-797,976 decreased the voltage-gated Ca2+ current and abolished any further decrease by SRIF. SSTR1, SSTR3, SSTR4, and SSTR5 agonists at 10(-7) M did not modify the voltage-gated Ca2+ current and did not affect the Ca2+ current response to SRIF. These results indicate that SSTR2 is involved mainly in regulating voltage-gated Ca2+ currents by SRIF, which contributes to the decrease in intracellular Ca2+ concentration and GH secretion by SRIF.     

Participation of transient-type Ca2+ channels in the sustained increase of Ca2+ level in GH3 cells

Participation of two types of Ca2+ channels (T- and L-types) in the sustained increase of cytosolic-free Ca2+ concentration [( Ca2+]i) was studied in thyrotropin-releasing hormone (TRH)-stimulated clonal GH3 pituitary cells. The effects of Ca2+ channel blockers were analyzed by measuring Ca2+ channel current and [Ca2+]i, using whole-cell voltage-clamp and Fura-2 fluorometry, respectively. Phenytoin (100 microM) and Ni2+ (100 microM) selectively blocked T-type Ca2+ channels and suppressed the TRH-induced sustained [Ca2+]i increase in single cells. Synthetic omega-conotoxin (omega-CgTX, 2 microM) preferentially blocked L-type Ca2+ channels, but it did not suppress the TRH-induced sustained [Ca2+]i increase. The present results suggest that the sustained elevations of [Ca2+]i triggered by TRH may be mediated by T-type Ca2+ channels in GH3 cells.     

Mouse semaphorin H induces PC12 cell neurite outgrowth activating Ras-mitogen-activated protein kinase signaling pathway via Ca(2+) influx.

We recently showed that mouse semaphorin H (MSH), a secreted semaphorin molecule, acts as a chemorepulsive factor on sensory neurites. In this study, we found for the first time that MSH induces neurite outgrowth in PC12 cells in a dose-dependent manner. Comparison of Ras-mitogen-activated protein kinase (MAPK) signaling pathways between MSH and nerve growth factor (NGF) revealed that these pathways are crucial for MSH action as well as NGF. K-252a, an inhibitor of tyrosine autophosphorylation of tyrosine kinase receptors (Trks), did not inhibit the action of MSH, suggesting that MSH action occurs via a different receptor than NGF. L- and N-types of voltage-dependent Ca(2+) channel blockers, diltiazem and omega-conotoxin, inhibited MSH-induced neurite outgrowth and MAPK phosphorylation in a Ca(2+)-dependent manner. A transient elevation in intracellular Ca(2+) level was observed upon MSH stimulation. These findings suggest that extracellular Ca(2+) influx, followed by activation of the Ras-MAPK signaling pathway, is required for MSH induced PC12 cell neurite outgrowth.     

Ligand-activated signal transduction in the 2-cell embryo

Platelet-activating factor (PAF) is an autocrine trophic/survival factor for the preimplantation embryo. PAF induced an increase in intracellular calcium concentration ([Ca2+]i) in the 2-cell embryo that had an absolute requirement for external calcium. L-type calcium channel blockers (diltiazem, verapamil, and nimodipine) significantly inhibited PAF-induced Ca2+ transients, but inhibitors of P/Q type (omega-agatoxin; omega-conotoxin MVIIC), N-type (omega-conotoxin GVIA), T-type (pimozide), and store-operated channels (SKF 96365 and econazole) did not block the transient. mRNA and protein for the alpha1-C subunit of L-type channels was expressed in the 2-cell embryo. The L-type calcium channel agonist (+/-) BAY K 8644 induced [Ca2+]i transients and, PAF and BAY K 8644 each caused mutual heterologous desensitization of each other's responses. Depolarization of the embryo (75 mM KCl) induced a [Ca2+]i transient that was inhibited by diltiazem and verapamil. Whole-cell patch-clamp measurements detected a voltage-gated channel (blocked by diltiazem, verapamil, and nifedipine) that was desensitized by prior responses of embryos to exogenous or embryo-derived PAF. Replacement of media Ca2+ with Mn2+ allowed Mn2+ influx to be observed directly; activation of a diltiazem-sensitive influx channel was an early response to PAF. The activation of a voltage-gated L-type calcium channel in the 2-cell embryo is required for normal signal transduction to an embryonic trophic factor.     

L-type calcium channels are preferentially coupled to endocytosis in bovine chromaffin cells

Exocytosis and endocytosis are Ca(2+)-dependent processes. The contribution of high-voltage activated Ca(2+) channels subtypes to exocytosis has been thoroughly studied in chromaffin cells. However, similar reports concerning endocytosis are unavailable. Thus, we studied here the effects of blockers of L (nifedipine), N (omega-conotoxin GVIA) and P/Q (omega-agatoxin IVA) Ca(2+) channel on Ca(2+) currents (I(Ca)), Ca(2+) entry (Q(Ca)), as well as on the changes in membrane capacitance (C(m)) in perforated-patch voltage-clamped bovine adrenal chromaffin cells. Using 500-ms pulses to 0 or +10 mV, given from a holding potential of -80 mV and 2 mM Ca(2+) we found that omega-conotoxin GVIA affected little the exo-endocytotic responses while omega-agatoxin IVA markedly blocked those responses. However, nifedipine blocked little exocytosis but almost completely inhibited endocytosis. We conclude that L-type Ca(2+) channels seem to be selectively coupled to endocytosis.     

Nhe1 is essential for potassium but not calcium facilitation of cell motility and the monovalent cation requirement for chemotactic orientation in Dictyostelium discoideum

In Dictyostelium discoideum, extracellular K+ or Ca2+ at a concentration of 40 or 20 mM, respectively, facilitates motility in the absence or presence of a spatial gradient of chemoattractant. Facilitation results in maximum velocity, cellular elongation, persistent translocation, suppression of lateral pseudopod formation, and myosin II localization in the posterior cortex. A lower threshold concentration of 15 mM K+ or Na or 5 mM Ca2+ is required for chemotactic orientation. Although the common buffer solutions used by D. discoideum researchers to study chemotaxis contain sufficient concentrations of cations for chemotactic orientation, the majority contain insufficient levels to facilitate motility. Here it has been demonstrated that Nhe1, a plasma membrane protein, is required for K+ but not Ca2+ facilitation of cell motility and for the lower K+ but not Ca2+ requirement for chemotactic orientation.