Influx of Ca2+ via Cav1.3 calcium channels in satellite cells of muscle fibers in rats

Voltage-dependent L-type Ca_v1.3 channels have been detected in satellite cells localized to muscle fibers. It was established that the action of carbachol, which activates nicotinic acetylcholine receptors and causes cell membrane to depolarize, resulted in the activation of these channels. In addition, verapamil and amlodipine, selective L-type calcium channel blockers, suppressed extracellular calcium influx into the cytoplasm. It was noted that in a calcium-free medium, carbachol had no influence on the concentration of calcium in the cytoplasm of satellite cells, whereas adrenaline induced calcium efflux from intracellular stores. In addition, calcium influx into the cytoplasm was not suppressed by verapamil and amlodipine under the action of adrenaline and noradrenalin in a medium with calcium, and an ICI-118551 blocker of β₂-adrenoreceptros significantly decreased the increase in the concentration of calcium in the cytoplasm.     

Structure-activity study of L-amino acid-based N-type calcium channel blockers

Synthesis and structure-activity relationship (SAR) study of L-amino acid-based N-type calcium channel blockers are described. The compounds synthesized were evaluated for inhibitory activity against both N-type and L-type calcium channels focusing on selectivity to reduce cardiovascular side effects due to blocking of L-type calcium channels. In the course of screening of our compound library, N-(t-butoxycarbonyl)-L-aspartic acid derivative 1a was identified as an initial lead compound for a new series of N-type calcium channel blockers, which inhibited calcium influx into IMR-32 human neuroblastoma cells with an IC(50) of 3.4 microM. Compound 1a also exhibited blockade of N-type calcium channel current in electrophysiological experiment using IMR-32 cells (34% inhibition at 10 microM, n=3). As a consequence of conversion of amino acid residue of 1a, compound 12a, that include N-(t-butoxycarbonyl)-L-cysteine, was found to be a potent N-type calcium channel blocker with an IC(50) of 0.61 microM. Thus, L-cysteine was selected as a potential structural motif for further modification. Optimization of C- and N-terminals of L-cysteine using S-cyclohexylmethyl-L-cysteine as a central scaffold led to potent and selective N-type calcium channel blocker 21f, which showed improved inhibitory potency (IC(50) 0.12 microM) and 12-fold selectivity for N-type calcium channels over L-type channels.     

The lipid peroxidation product 4-hydroxynonenal facilitates opening of voltage-dependent Ca2+ channels in neurons by increasing protein tyrosine phosphorylation

Calcium influx through voltage-dependent calcium channels (VDCCs) mediates a variety of functions in neurons and other excitable cells, but excessive calcium influx through these channels can contribute to neuronal death in pathological settings. Oxyradical production and membrane lipid peroxidation occur in neurons in response to normal activity in neuronal circuits, whereas excessive lipid peroxidation is implicated in the pathogenesis of of neurodegenerative disorders. We now report on a specific mechanism whereby lipid peroxidation can modulate the activity of VDCCs. The lipid peroxidation product 4-hydroxy-2,3-nonenal (4HN) enhances dihydropyridine-sensitive whole-cell Ca2+ currents and increases depolarization-induced increases of intracellular Ca2+ levels in hippocampal neurons. Prolonged exposure to 4HN results in neuronal death, which is prevented by treatment with glutathione and attenuated by the L-type Ca2+ channel blocker nimodipine. Tyrosine phosphorylation of alpha1 VDCC subunits is increased in neurons exposed to 4HN, and studies using inhibitors of tyrosine kinases and phosphatases indicate a requirement for tyrosine phosphorylation in the enhancement of VDCC activity in response to 4HN. Phosphorylation-mediated modulation of Ca2+ channel activity in response to lipid peroxidation may play important roles in the responses of neurons to oxidative stress in both physiological and pathological settings.     

Calbindin-D28k decreases L-type calcium channel activity and modulates intracellular calcium homeostasis in response to K+ depolarization in a rat beta cell line RINr1046-38

Calbindin-D(28k), acts as a modulator of depolarization induced calcium transients in the pancreatic beta cell. However, specific mechanisms have not been defined. Here we show for the first time that the calcium binding protein calbindin-D(28k) acts by affecting calcium influx through voltage-dependent calcium channels in RIN pancreatic beta cells. Whole-cell patch-clamp recordings revealed that Ca(2+) current amplitudes of calbindin-D(28k) expressing RINr1046-38 beta cells were smaller than the Ca(2+) current amplitudes in control cells in response to depolarizing pulses. The peak current was observed at +20mV and the average amplitude was approximately 50pA in the calbindin expressing cells compared to approximately 250pA in control cells. In calbindin-D(28k) expressing cells, the channels had enhanced sensitivity to Ca(2+) dependent inactivation and currents decayed much more rapidly than in control cells. The Ca(2+) channels affected by calbindin were found to have biophysical properties consistent with dihydropyridine-sensitive L-type calcium channels. In response to depolarizing concentrations of K(+), calbindin expression caused a five-fold decrease in the rate of rise of [Ca(2+)](i) and decay was slower in the calbindin expressing cells. Application of verapamil resulted in a drop in the [Ca(2+)](i) signal to pre-stimulation levels indicating that the Ca(2+) channel responsible for the depolarization evoked Ca(2+) entry, modulated by calbindin, is the L-type. Co-immunoprecipitation and GST pull-down assays indicate that calbindin-D(28k) can interact with the alpha(1) subunit of Ca(v)1.2. We thus conclude that calbindin-D(28k) can regulate calcium influx via L-type calcium channels. Our findings suggest a role for calbindin-D(28k) in the beta cell in modulating Ca(2+) influx via L-type voltage-dependent calcium channels.     

L-Cysteine based N-type calcium channel blockers: structure-activity relationships of the C-terminal lipophilic moiety,and oral analgesic efficacy in rat pain models

This study was performed to determine the structure-activity relationships (SAR) of L-cysteine based N-type calcium channel blockers. Basic nitrogen was introduced into the C-terminal lipophilic moiety of L-cysteine with a view toward improvement of its physicochemical properties. L-Cysteine derivative 9 was found to be a potent and selective N-type calcium channel blocker with IC(50) of 0.33 microM in calcium influx assay using IMR-32 cells and was 15-fold selective for N-type calcium channels over L-type channels. Compound 9 showed improved oral analgesic efficacy in the rat formalin induced pain model and the rat chronic constriction injury (CCI) model, which is one of the most reliable models of chronic neuropathic pain, without any significant effect on blood pressure or neurological behavior.     

Stable and functional expression of the calcium channel alpha 1 subunit from smooth muscle in somatic cell lines.

Voltage-activated calcium channels are membrane spanning proteins that allow the controlled entry of Ca2+ into the cytoplasm of cells. The principal channel forming subunit of an L-type calcium channel is the alpha 1 subunit. Transfection of Chinese hamster ovary (CHO) cells with complementary DNA encoding the calcium channel alpha 1 subunit from smooth muscle led to the expression of functional calcium channels which bind calcium channel blockers and show the voltage-dependent activation and slow inactivation and unitary current conductance characteristic of calcium channels in smooth muscle. The currents mediated by these channels are sensitive towards dihydropyridine-type blockers and agonists indicating that the calcium channel blocker receptor sites were present in functional form. The smooth muscle alpha 1 subunit cDNA alone is sufficient for stable expression of functional calcium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.     

Atrial natriuretic factor (ANF) effects on L-, N-, and P/Q-type voltage-operated calcium channels

1. We have previously reported that atrial natriuretic factor (ANF) decreases neuronal norepinephrine (NE) release. The mechanism that mediates NE release from presynaptic membrane to synaptic cleft is a strongly calcium-dependent process. The modulator effect of ANF may be related to modifications in calcium influx at the presynaptic nerve ending by interaction with voltage-operated calcium channels (VOCCs).2. On this basis we investigated the effects of ANF on K⁺-induced ⁴⁵Ca²⁺ uptake and evoked neuronal NE release in the presence of specific L-, N-, and P/Q-type calcium channel blockers in the rat hypothalamus.3. Results showed that ANF inhibited K⁺-induced ⁴⁵Ca²⁺ uptake in a concentration-dependent fashion. Concentration–response curves to VOCC blockers nifedipine (NFD, L-type channel blocker), -conotoxin GVIA (CTX, N-type channel blocker), and -agatoxin IVA (AGA, P/Q-type channel blocker) showed that all the blockers decreased NE release. Incubation of ANF plus NFD showed an additive effect as compared to NFD or ANF alone. However, when the hypothalamic tissue was incubated in the presence of ANF plus CTX or AGA there were no differences in neuronal NE release as compared to calcium channel blockers or ANF alone.4. These results suggest that ANF decreases NE release by an L-type calcium channel independent mechanism by inhibiting N- and/or P/Q-type calcium channels at the neuronal presynaptic level. Thus, ANF modulates neuronal NE release through different mechanisms involving presynaptic calcium channel inhibition.     

Effects of voltage-gated calcium channel subunit genes on calcium influx in cultured C. elegans mechanosensory neurons

Voltage-gated calcium channels (VGCCs) serve as a critical link between electrical signaling and diverse cellular processes in neurons. We have exploited recent advances in genetically encoded calcium sensors and in culture techniques to investigate how the VGCC alpha1 subunit EGL-19 and alpha2/delta subunit UNC-36 affect the functional properties of C. elegans mechanosensory neurons. Using the protein-based optical indicator cameleon, we recorded calcium transients from cultured mechanosensory neurons in response to transient depolarization. We observed that in these cultured cells, calcium transients induced by extracellular potassium were significantly reduced by a reduction-of-function mutation in egl-19 and significantly reduced by L-type calcium channel inhibitors; thus, a main source of touch neuron calcium transients appeared to be influx of extracellular calcium through L-type channels. Transients did not depend directly on intracellular calcium stores, although a store-independent 2-APB and gadolinium-sensitive calcium flux was detected. The transients were also significantly reduced by mutations in unc-36, which encodes the main neuronal alpha2/delta subunit in C. elegans. Interestingly, while egl-19 mutations resulted in similar reductions in calcium influx at all stimulus strengths, unc-36 mutations preferentially affected responses to smaller depolarizations. These experiments suggest a central role for EGL-19 and UNC-36 in excitability and functional activity of the mechanosensory neurons.     

Mechanisms of p-methoxycinnamic acid-induced increase in insulin secretion

p-Methoxycinnamic acid (p-MCA) is a cinnamic acid derivative that shows various pharmacologic actions such as hepatoprotective and antihyperglycemic activities. The present study was to elucidate the mechanisms by which p-MCA increases [Ca2?]i and insulin secretion in INS-1 cells. p-MCA (100 μM) increased [Ca2?]i in INS-1 cells. The p-MCA-induced insulin secretion and rise in [Ca2?]i were markedly inhibited in the absence of extracellular Ca2? or in the presence of an L-type Ca2? channel blocker nimodipine. These results suggested that p-MCA increased Ca2? influx via the L-type Ca2? channels. Diazoxide, an ATP-sensitive K? channel opener, did not alter p-MCA-induced insulin secretion, nor [Ca2?]i response. In addition, p-MCA enhanced glucose-, glibenclamide-induced insulin secretion whereas it also potentiated the increase in insulin secretion induced by arginine, and Bay K 8644, an L-type Ca2? channel agonist. Taken together, our results suggest that p-MCA stimulated insulin secretion from pancreatic β-cells by increasing Ca2? influx via the L-type Ca2? channels, but not through the closure of ATP-sensitive K? channels.     

Tryptophan hydroxylase is modulated by L-type calcium channels in the rat pineal gland

Calcium is an important second messenger in the rat pineal gland, as well as cAMP. They both contribute to melatonin synthesis mediated by the three main enzymes of the melatonin synthesis pathway: tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase. The cytosolic calcium is elevated in pinealocytes following alpha(1)-adrenergic stimulation, through IP(3)-and membrane calcium channels activation. Nifedipine, an L-type calcium channel blocker, reduces melatonin synthesis in rat pineal glands in vitro. With the purpose of investigating the mechanisms involved in melatonin synthesis regulation by the L-type calcium channel, we studied the effects of nifedipine on noradrenergic stimulated cultured rat pineal glands. Tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase activities were quantified by radiometric assays and 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin contents were quantified by HPLC with electrochemical detection. The data showed that calcium influx blockaded by nifedipine caused a decrease in tryptophan hydroxylase activity, but did not change either arylalkylamine N-acetyltransferase or hydroxyindole-O-methyltransferase activities. Moreover, there was a reduction of 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin intracellular content, as well as a reduction of serotonin and melatonin secretion. Thus, it seems that the calcium influx through L-type high voltage-activated calcium channels is essential for the full activation of tryptophan hydroxylase leading to melatonin synthesis in the pineal gland.     

Vasorelaxant effect in rat aortic rings through calcium channel blockage: A preliminary in vitro assessment of a 1,3,4-oxadiazole derivative

The study was undertaken on the basis of several reports in the literature that relaxation of vascular smooth muscles is a good treatment strategy in hypertension, angina and other cardiovascular disorders. Oxadiazoles have been reported to have effect on vascular smooth muscles and calcium influx. The goals of our current in vitro study were to investigate the effect of a 1,3,4-oxadiazole derivative on vascular smooth muscles in rat aorta, and to elucidate the associated signaling pathway. NOX-1 induced a relaxation of vascular smooth muscles in both endothelium intact and denuded rat aortic rings precontracted with norepinephrine or phenylephrine or KCl. NOX-1 also significantly antagonized cumulative dose-response effect of norepinephrine, phenylephrine, KCl or calcium with reduction in submaximal contractions. Verapamil, an L-type of calcium channel blocker, effectively attenuated phenylephrine and calcium induced contractions in aortic rings. Incubation with NOX-1 and verapamil did not significantly alter the dose-response curve of phenylephrine or calcium compared to verapamil treatment alone indicating L-type Ca²⁺ channel blockage leads to loss of NOX-1 activity. Hence it can be concluded NOX-1 exhibited vasorelaxant action by inhibiting calcium influx from extracellular space to intracellular space through L-type of calcium channels.     

MicroRNAs are essential for stretch-induced vascular smooth muscle contractile differentiation via microRNA (miR)-145-dependent expression of L-type calcium channels

Stretch of the vascular wall is an important stimulus to maintain smooth muscle contractile differentiation that is known to depend on L-type calcium influx, Rho-activation, and actin polymerization. The role of microRNAs in this response was investigated using tamoxifen-inducible and smooth muscle-specific Dicer KO mice. In the absence of Dicer, which is required for microRNA maturation, smooth muscle microRNAs were completely ablated. Stretch-induced contractile differentiation and Rho-dependent cofilin-2 phosphorylation were dramatically reduced in Dicer KO vessels. On the other hand, acute stretch-sensitive growth signaling, which is independent of influx through L-type calcium channels, was not affected by Dicer KO. Contractile differentiation induced by the actin polymerizing agent jasplakinolide was not altered by deletion of Dicer, suggesting an effect upstream of actin polymerization. Basal and stretch-induced L-type calcium channel expressions were both decreased in Dicer KO portal veins, and inhibition of L-type channels in control vessels mimicked the effects of Dicer deletion. Furthermore, inhibition of miR-145, a highly expressed microRNA in smooth muscle, resulted in a similar reduction of L-type calcium channel expression. This was abolished by the Ca(2+)/calmodulin-dependent protein kinase II inhibitor KN93, suggesting that Ca(2+)/calmodulin-dependent protein kinase IIδ, a target of miR-145 and up-regulated in Dicer KO, plays a role in the regulation of L-type channel expression. These results show that microRNAs play a crucial role in stretch-induced contractile differentiation in the vascular wall in part via miR-145-dependent regulation of L-type calcium channels.     

Regulation of calcium signalling by 1-O-alkylglycerols in human Jurkat T lymphocytes

We studied the role of natural occurring 1-O-alkylglycerols on the calcium signalling in Jurkat T-cells. Alkylglycerols evoked an increase in free intracellular calcium concentration [Ca2+]i, in a dose-dependent manner. When the experiments were performed in calcium-free buffer, the alkylglycerol response on the rise of [Ca2+]i was wholly abolished compared with the one in calcium-containing buffer, suggesting that these etherlipids induce a calcium influx by the opening of Ca2+ channels. We further employed inhibitors of voltage-gated calcium channels. We observed that omega-conotoxin, a blocker of N-type voltage-activated Ca2+ channels, but not verapamil, a blocker of L-type voltage-activated Ca2+ channels, curtailed significantly the calcium rise evoked by the lipid agents. Alkylglycerols also induced plasma membrane depolarisation, known to be involved in the opening of the voltage-gated calcium channels. Our study shows that alkylglycerols increase [Ca2+]i influx in human Jurkat T-cells possibly by modulating the permeability of calcium channels.     

Modulation of Ca2+ channels by atrial natriuretic peptide in the bovine adrenal glomerulosa cell.

In the bovine adrenal glomerulosa cell, calcium influx through voltage-dependent calcium channels is critical to maintaining an aldosterone secretory response. In patch clamp, atrial natriuretic peptide (ANP) inhibits T-type calcium channel current yet stimulates L-type calcium channel current. In the present study the channel effects of ANP observed in the patch-clamp configuration were extended and related to populations of cells. We observed the following. (i) The effect of ANP on T-channel current resulted in the reduction in the open state probability. ANP decreased the mean open state duration from 14.2 to 1.8 ms/sweep. (ii) In the weakly depolarized cell stimulated by 8 mM K+, ANP reduced the level of aequorin luminescence (a measure of cytosolic calcium) and completely inhibited the stimulated rate of aldosterone secretion, returning it to prestimulation values. These effects are consistent with a decrease in net calcium channel influx and the reported inhibition of T-channel current. In contrast, the calcium channel blocker, nitrendipine, which at low dose selectively blocks L-type calcium channel flux, only slightly reduced luminescence, and partially inhibited the sustained secretory response. (iii) In the strongly depolarized cell, stimulated by 60 mM K+, ANP increased the level of aequorin luminescence consistent with an increase in net calcium channel influx and the reported stimulation of L-channel current. These results indicate that under physiological conditions the inhibition of T-type calcium channels may be involved in the inhibition of the aldosterone secretion induced by ANP.     

The relationship between calcium and the metabolism of plasma membrane phospholipids in hemolysis induced by brown spider venom phospholipase-D toxin

Brown spider venom phospholipase-D belongs to a family of toxins characterized as potent bioactive agents. These toxins have been involved in numerous aspects of cell pathophysiology including inflammatory response, platelet aggregation, endothelial cell hyperactivation, renal disorders, and hemolysis. The molecular mechanism by which these toxins cause hemolysis is under investigation; literature data have suggested that enzyme catalysis is necessary for the biological activities triggered by the toxin. However, the way by which phospholipase-D activity is directly related with human hemolysis has not been determined. To evaluate how brown spider venom phospholipase-D activity causes hemolysis, we examined the impact of recombinant phospholipase-D on human red blood cells. Using six different purified recombinant phospholipase-D molecules obtained from a cDNA venom gland library, we demonstrated that there is a correlation of hemolytic effect and phospholipase-D activity. Studying recombinant phospholipase-D, a potent hemolytic and phospholipase-D recombinant toxin (LiRecDT1), we determined that the toxin degrades synthetic sphingomyelin (SM), lysophosphatidylcholine (LPC), and lyso-platelet-activating factor. Additionally, we determined that the toxin degrades phospholipids in a detergent extract of human erythrocytes, as well as phospholipids from ghosts of human red blood cells. The products of the degradation of synthetic SM and LPC following recombinant phospholipase-D treatments caused hemolysis of human erythrocytes. This hemolysis, dependent on products of metabolism of phospholipids, is also dependent on calcium ion concentration because the percentage of hemolysis increased with an increase in the dose of calcium in the medium. Recombinant phospholipase-D treatment of human erythrocytes stimulated an influx of calcium into the cells that was detected by a calcium-sensitive fluorescent probe (Fluo-4). This calcium influx was shown to be channel-mediated rather than leak-promoted because the influx was inhibited by L-type calcium channel inhibitors but not by a T-type calcium channel blocker, sodium channel inhibitor or a specific inhibitor of calcium activated potassium channels. Finally, this inhibition of hemolysis following recombinant phospholipase-D treatment occurred in a concentration-dependent manner in the presence of L-type calcium channel blockers such as nifedipine and verapamil. The data provided herein, suggest that the brown spider venom phospholipase-D-induced hemolysis of human erythrocytes is dependent on the metabolism of membrane phospholipids, such as SM and LPC, generating bioactive products that stimulate a calcium influx into red blood cells mediated by the L-type channel.     

Calcium entry through L-type calcium channels causes mitochondrial disruption and chromaffin cell death

Sustained, mild K+ depolarization caused bovine chromaffin cell death through a Ca(2+)-dependent mechanism. During depolarization, Ca(2+) entered preferentially through L-channels to induce necrotic or apoptotic cell death, depending on the duration of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) signal, as proven by the following. (i) The L-type Ca(2+) channel activators Bay K 8644 and FPL64176, more than doubled the cytotoxic effects of 30 mm K+; (ii) the L-type Ca(2+) channel blocker nimodipine suppressed the cytotoxic effects of K+ alone or K+ plus FPL64176; (iii) the potentiation by FPL64176 of the K+ -evoked [Ca(2+)](c) elevation was totally suppressed by nimodipine. Cell exposure to K+ plus the L-type calcium channel agonist FPL64176 caused an initial peak rise followed by a sustained elevation of the [Ca(2+)](c) that, in turn, increased [Ca(2+)](m) and caused mitochondrial membrane depolarization. Cyclosporin A, a blocker of the mitochondrial transition pore, and superoxide dismutase prevented the apoptotic cell death induced by Ca(2+) overload through L-channels. These results suggest that Ca(2+) entry through L-channels causes both calcium overload and mitochondrial disruption that will lead to the release of mediators responsible for the activation of the apoptotic cascade and cell death. This predominant role of L-type Ca(2+) channels is not shared by other subtypes of high threshold voltage-dependent neuronal Ca(2+) channels (i.e. N, P/Q) expressed by bovine chromaffin cells.     

Voltage-dependent facilitation of a neuronal alpha 1C L-type calcium channel.

Calcium entry into excitable cells through voltage-gated calcium channels can be influenced by both the rate and pattern of action potentials. We report here that a cloned neuronal alpha 1C L-type calcium channel can be facilitated by positive pre-depolarization. Both calcium and barium were effective as charge carriers in eliciting voltage-dependent facilitation. The induction of facilitation was shown to be independent of intracellular calcium levels, G-protein interaction and the level of phosphatase activity. Facilitation was reduced by the injection of inhibitors of protein kinase A and required the coexpression of a calcium channel beta subunit. In contrast, three neuronal non-L-type calcium channels, alpha 1A, alpha 1B and alpha 1E, were not subject to voltage-dependent facilitation when coexpressed with a beta subunit. The results indicate that the mechanism of neuronal L-type calcium channel facilitation involves the interaction of alpha 1 and beta subunits and is dependent on protein kinase A activity. The selective voltage-dependent modulation of L-type calcium channels is likely to play an important role in neuronal physiology and plasticity.     

Stimulation of arterial smooth muscle L-type calcium channels by hydrogen peroxide requires protein kinase C

Changes in intracellular calcium regulate countless biological processes. In arterial smooth muscle, voltage-dependent L-type calcium channels are major conduits for calcium entry with the primary function being determination of arterial diameter. Similarly, changes in intracellular redox status, either discrete controlled changes or global pathological perturbations, are also critical determinants of cell function. We recently reported that in arterial smooth muscle cells, local generation of hydrogen peroxide leads to colocalized calcium entry through L-type calcium channels. Here we extend our investigation into mechanisms linking hydrogen peroxide to calcium influx through L-type calcium channels by focusing on the role of protein kinase C (PKC). Our data indicate that stimulation of L-type calcium channels by hydrogen peroxide requires oxidant-dependent increases in PKC catalytic activity. This effect is independent of classical cofactor-dependent activation of PKC by diacylglycerol. These data provide additional experimental evidence supporting the concept of oxidative stimulation of L-type calcium channels.