Involvement Of Voltage-dependent Calcium Channels Research Articles

Dose-Dependent Differential Effects of In Vivo Exposure of Cadmium on Myometrial Activity in Rats: Involvement of VDCC and Ca2+-Mimicking Pathways.

Present study was undertaken to study the effect of 28-days exposure of female adult rats to cadmium (Cd) in drinking water @ 3, 10 and 30parts per million (ppm) on myometrial responsiveness to different spasmogens and unravel the possible mechanism of alterations in myometrial activity. Cadmium and Ca2+ levels in blood and uterus were measured by atomic absorption spectroscopy while isometric tension in myometrial strips was measured using data acquisition system-based physiograph. Dose-dependent increase in levels of cadmium was observed in both blood and uterus while calcium was increased only in the uterus as compared to those in control. Significant increase in absolute tension and mean integral tension along with non-significant increase in frequency of myometrial contraction was observed in rats of Cd-treated groups. As compared to the control, cadmium decreased and increased the effects of calcium chloride, 80mM KCl, histamine (0.1μM) and oxytocin (10-2IU/ml) in lower-dose (3ppm) and higher-dose groups (10 and 30ppm), respectively. Cadmium potentiated and inhibited the relaxant response to phenylephrine in myometrium of rats at lower-dose (3ppm) and highest-dose (30ppm) Cd-treated groups, respectively. Results of our study revealed that Cd accumulates in the myometrium of rats and alters its responsiveness to oxytocin, histamine, 80mM KCl, calcium chloride and phenylephrine, and these effects are differentially mediated depending on levels of exposure possibly through voltage-dependent calcium channel (VDCC) and Ca2+-mimicking pathways.

Antioxidants and Gabapentin Prevent Heat Hypersensitivity in a Neuropathic Pain Model

ABSTRACTBackground: The involvement of voltage-dependent calcium channels and reactive oxygen species in the pathophysiology of neuropathic pain might justify the preventative administration of antioxidant enzymes, at low doses, in combination with gabapentin (GaP) to maximize its analgesic effect in an experimental model of neuropathic pain in rats. Methods: This work investigated the analgesic effects of these drugs, alone or in combination, by intraperitoneal administration for three consecutive days before (Series I) or after (Series II) a peripheral neuropathy induced by left sciatic nerve ligation. A prospective randomized study was conducted using 96 Wistar rats (50 males and 46 females). Mechanical hypersensitivity was measured with a dynamic plantar anesthesiometer. A hot plate analgesia meter calculated the thermal sensitivity. Side effect profiles for drug combinations were evaluated with a conventional scale to assess levels of sedation interfering with postural control and righting reflexes. Results: Preinjury administration (Series I) of GaP alone prevented the development of mechanical hypersensitivity at the nerve-damage hind paw. When GaP was administered concurrently with superoxide dismutase and catalase, its preventive analgesic effect did not increase. Antioxidants administered alone were completely ineffective at modulating the mechanical or thermal hypersensitivity. When treatments were only delivered following surgery (Series II), only the group receiving combined GaP and antioxidants treatment prior to nerve injury showed higher thermal thresholds from postsurgery days 7–30. Conclusions: These results suggest that preventive antioxidants in combination with GaP provided a synergistic suppression of thermal hypersensitivity that GaP alone cannot produce.

Identification of a CACNA2D4 deletion in late onset bipolar disorder patients and implications for the involvement of voltage‐dependent calcium channels in psychiatric disorders

The GWAS-based association of CACNA1C with bipolar disorder (BPD) is one of the strongest genetic findings to date. CACNA1C belongs to the family of CACN genes encoding voltage-dependent calcium channels (VDCCs). VDCCs are involved in brain circuits and cognitive processes implicated in BPD and schizophrenia (SZ). Recently, it was shown that rare copy number variations (CNVs) are found at an increased frequency in SZ and to a lesser extent also in BPD, suggesting the involvement of CNVs in the causation of these diseases. We hypothesize that CNVs in CACN genes can influence the susceptibility to BPD, SZ, and/or schizoaffective disorder (SZA). A search for CNVs in eight CACN genes in a patient-control sample of European decent was performed. A total of 709 BP patients, 645 SZ patients, 189 SZA patients, and 1,470 control individuals were screened using the Multiplex Amplicon Quantification (MAQ) method. We found a rare, partial deletion of 35.7 kb in CACNA2D4 in two unrelated late onset bipolar I patients and in one control individual. All three deletions shared the same breakpoints removing exons 17-26 of CACNA2D4, comprising part of the CACHE domain. Based on the data we cannot claim causality to BPD of the identified CACNA2D4 deletion but nevertheless this deletion can be important in unraveling the underlying processes leading to psychiatric diseases in general and BPD in particular.

1α,25-Dihydroxyvitamin D3 Signaling Pathways on Calcium Uptake in 30-Day-Old Rat Sertoli Cells

1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is the active metabolite of vitamin D(3) and the major calcium regulatory hormone in tissues. The aim of this work was to investigate the mechanism of action of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells from 30-day-old rats. Results showed that 10(-9) and 10(-12) M 1,25D(3) increased the rate of (45)Ca(2+) uptake 5 and 15 min after hormone exposure and that 1α,25(OH)(2) lumisterol(3) (JN) produced a similar effect suggesting that 1,25D(3) action occurs via a putative membrane receptor. The involvement of voltage-dependent calcium channels (VDCC) in 1,25D(3) action was evidenced by using nifedipine, while the use of Bapta-AM demonstrated that intracellular calcium was not implicated. Moreover, the incubation with ouabain and digoxin increased the rate of (45)Ca(2+) uptake, indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, we demonstrated that the mechanism underlying the hormone action involved extracellular signal-regulated kinase (ERK) and protein kinase C (PKC) activation in a phospholipase C-independent way. Furthermore, a local elevation of the level of cAMP, as demonstrated by incubating cells with dibutyryl cAMP or a phosphodiesterase inhibitor, produced an effect similar to that of 1,25D(3), and the inhibition of protein kinase A (PKA) nullified the hormone action. In conclusion, the stimulatory effect of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells occurs via VDCC, as well as PKA, PKC, and ERK activation. These protein kinases seem to act by inhibiting Na(+)/K(+)-ATPase or directly phosphorylating calcium channels. The Na(+)/K(+)-ATPase inhibition may result in Na(+)/Ca(2+) exchanger activation in reverse mode and consequently induce the uptake of calcium into the cells.

Low-voltage-activated calcium channels are not involved in capacitation and biological response to progesterone in human sperm.

The involvement of voltage-dependent calcium channels in the biological effects exerted by progesterone (P) on human spermatozoa is still a controversial issue. We have investigated the involvement of T-type calcium channels [voltage-operated calcium channels (VOCCT)] in two biological functions of human sperm, responsiveness to P and capacitation, by employing three different pharmacological antagonists of VOCCT, namely mibefradil (Ro 5967), pimozide and amiloride. Intracellular calcium [Ca(2+)]i increase in response to P was essentially unaffected by pre-treatment with mibefradil and pimozide at concentrations previously shown to prevent [Ca(2+)]i increase in response to zona proteins. Amiloride could not be tested in these experiments because it was found to interfere with fura-2 fluorescence. The increase in tyrosine phosphorylation stimulated by P in a protein of about 97 kDa was unaffected by the three antagonists. Acrosome reaction (AR) induced by P was also unaffected by mibefradil or pimozide but was significantly inhibited by amiloride at high concentrations (100 and 500 but not 10 microM). At 100 and 500 microM amiloride also inhibited Na/H exchanger as assessed by a fluorimetric method. We conclude that VOCCT are not involved in calcium increase and AR stimulated by P in human sperm. We next investigated the effect of the three VOCCT inhibitors on sperm capacitation by evaluating tyrosine phosphorylation and AR in basal conditions and in response to P. We found that the presence of pimozide and amiloride during capacitation stimulated a higher increase of tyrosine phosphorylation, whereas mibefradil was less effective. The ability of P to induce the AR, considered an index of occurrence of capacitation, was not affected by pimozide and mibefradil, whereas was inhibited by amiloride at concentrations that inhibit Na/H exchanger. In conclusion, our results do not support a major role of low-voltage-activated calcium channels in capacitation and response to P of human spermatozoa.

Induction by convulsant drugs of brain-derived neurotrophic factor in the rat brain: an involvement of voltage-dependent calcium channels

Induction by convulsant drugs of brain-derived neurotrophic factor in the rat brain: an involvement of voltage-dependent calcium channels

Effects of N- and L-type calcium channel antagonists on the responses of nociceptive spinal cord neurons to mechanical stimulation of the normal and the inflamed knee joint.

1. The present study addresses the involvement of voltage-dependent calcium channels of the N and L type in the spinal processing of innocuous and noxious input from the knee joint, both under normal conditions and under inflammatory conditions in which spinal cord neurons become hyperexcitable. In 30 anesthetized rats, extracellular recordings were performed from single dorsal horn neurons in segments 1-4 of the lumbar spinal cord. All neurons had receptive fields in the ipsilateral knee joint. In 22 rats, an inflammation was induced in the ipsilateral knee joint by kaolin and carrageenan 4-16 h before the recordings. The antagonist at N-type calcium channels, omega-conotoxin GVIA (omega-CTx GVIA), was administered topically in solution to the dorsal surface of the spinal cord at the appropriate spinal segments in 6 rats with normal joints and in 12 rats with inflamed knee joints. The antagonist at L-type channels, nimodipine, was administered topically in 5 rats with normal joints and in 11 rats with inflamed knee joints. In another five rats with inflamed joints, antagonists at L-type calcium channels (diltiazem and nimodipine) and omega-CTx GVIA were administered ionophoretically with multibarrel electrodes close to the neurons recorded. 2. The topical administration of omega-CTx GVIA to the spinal cord reduced the responses to both innocuous and noxious pressure applied to the knee joint in a sample of 11 neurons with input from the normal joint and in a sample of 16 neurons with input from the inflamed joint (hyperexcitable neurons). The responses were decreased to approximately 65% of the predrug values within administration times of 30 min. A similar reduction of the responses to innocuous and noxious pressure was observed when omega-CTx GVIA was administered ionophoretically to nine hyperexcitable neurons. In neurons with input from the normal or the inflamed knee joint, the administration of omega-CTx GVIA led also to a reduction of the responses to innocuous and noxious pressure applied to the noninflamed ankle joint. 3. The topical administration of nimodipine decreased the responses to innocuous and noxious pressure applied to the knee in a sample of 9 neurons with input from the normal joint and in a sample of 16 neurons with input from the inflamed knee joint (hyperexcitable neurons). Within administration times of 30 min, the responses were reduced to approximately 70% of the predrug values. In hyperexcitable neurons, the responses to innocuous and noxious pressure applied to the knee were also decreased during ionophoretic administration of nimodipine (6 neurons) and diltiazem (9 neurons). When the noninflamed ankle was stimulated, the responses to innocuous pressure were reduced neither in neurons with input from the normal knee nor in neurons with input from the inflamed knee, but the responses of hyperexcitable neurons to noxious pressure onto the ankle were reduced. The ionophoretic administration of the agonist at the L-type calcium channel, S(-)-Bay K 8644, enhanced the responses to mechanical stimulation of the knee joint in all 14 hyperexcitable neurons tested. The effect of S(-)-Bay K 8644 was counteracted by both diltiazem (in 6 of 6 neurons) and nimodipine (in 5 of 5 neurons). 4. These data show that antagonists at both the N- and the L-type voltage-dependent calcium channels influence the spinal processing of input from the knee joint. The data suggest, therefore, that voltage-dependent calcium calcium channels of both the N and the L type are important for the sensory functions of the spinal cord. They are involved in the spinal processing of nonnociceptive as well as nociceptive mechanosensory input from the joint, both under normal and inflammatory conditions. The present results show in particular that N- and L-type channels are likely to be involved in the generation of pain evoked by noxious mechanical stimulation in normal tissue as well as in the mechanical hyperalgesia that is usually pres

S.12.01 - Molecular genetic studies in affective disorders

The excitotoxic hypothesis postulates a central role for the excitatory amino acids (EAAs) and their receptors in the neuronal damage that ensues cerebral ischemia-hypoxia and numerous other brain disorders. A major premise of the excitotoxic hypothesis is that neuronal protection can be achieved via blockade of EAA receptors with specific antagonists. This paper describes the use of the rat hippocampal slice preparation in the evaluation of various EAAs and their analogues for their potency as excitotoxins (agonists) and antagonists of the NMDA and the kainate/AMPA glutamate receptor subtypes. The hypersensitivity of hypoxic hippocampal slices to the presence of excitotoxins provided us with an inexpensive, sensitive tool to distinguish between structurally similar compounds. Moreover, these studies indicate that hypoxic neuronal damage cannot solely result from an excitotoxic mechanism; the involvement of voltage-dependent calcium channels in such damage is likely, as is evident from experiments performed in calcium-depleted medium and with the non-competive NMDA antagonist MK-801. At sub-toxic doses, quinolinate, a tryptophan metabolite implicated in Huntington's disease, appears to be a strong potentiator of the toxicity of all excitotoxins tested.

Flow-cytometric estimation on glutamate- and kainate-induced increases in intracellular Ca 2+ of brain neurons: a technical aspect

Effects of glutamate and kainate on the intracellular Ca 2+ concentration ([Ca 2+] i) in a large population (several thousand) of dissociated cerebellar granule cell neurons were evaluated using a flow-cytometer and a combination of two fluorescent dyes, fluo-3-AM for estimating [Ca 2+] i and ethidium bromide for removing neurons that had compromised membranes from the cell population examined. The number of neurons responding to glutamate or kainate in augmenting the fluo-3 fluorescence increased in a dose-dependent manner. The number of neurons responding to kainate was much greater than that to glutamate. CNQX, a blocker of non-NMDA receptors, completely blocked the response elicited by kainate while the complete blockade of this glutamate-induced response was made by a combination of MK-801, a NMDA receptor blocker, and CNQX. Nicardipine, a calcium antagonist, decreased the number of neurons responding to glutamate and kainate, suggesting involvement of voltage-dependent calcium channels. These results indicate that the flow-cytometric measurement of glutamate and kainate responses has the potential to provide answers to such questions as what percentage of the population of neurons respond to these amino acids and what is the resulting distribution of [Ca 2+] i.

Flow-cytometric estimation on glutamate- and kainate-induced increases in intracellular Ca2+ of brain neurons: a technical aspect

Effects of glutamate and kainate on the intracellular Ca2+ concentration ([Ca2+]i) in a large population (several thousand) of dissociated cerebellar granule cell neurons were evaluated using a flow-cytometer and a combination of two fluorescent dyes, fluo-3-AM for estimating [Ca2+]i and ethidium bromide for removing neurons that had compromised membranes from the cell population examined. The number of neurons responding to glutamate or kainate in augmenting the fluo-3 fluorescence increased in a dose-dependent manner. The number of neurons responding to kainate was much greater than that to glutamate. CNQX, a blocker of non-NMDA receptors, completely blocked the response elicited by kainate while the complete blockade of this glutamate-induced response was made by a combination of MK-801, a NMDA receptor blocker, and CNQX. Nicardipine, a calcium antagonist, decreased the number of neurons responding to glutamate and kainate, suggesting involvement of voltage-dependent calcium channels. These results indicate that the flow-cytometric measurement of glutamate and kainate responses has the potential to provide answers to such questions as what percentage of the population of neurons respond to these amino acids and what is the resulting distribution of [Ca2+]i.

Hypoxia, excitotoxicity, and neuroprotection in the hippocampal slice preparation

The excitotoxic hypothesis postulates a central role for the excitatory amino acids (EAAs) and their receptors in the neuronal damage that ensues cerebral ischemia-hypoxia and numerous other brain disorders. A major premise of the excitotoxic hypothesis is that neuronal protection can be achieved via blockade of EAA receptors with specific antagonists. This paper describes the use of the rat hippocampal slice preparation in the evaluation of various EAAs and their analogues for their potency as excitotoxins (agonists) and antagonists of the NMDA and the kainate/AMPA glutamate receptor subtypes. The hypersensitivity of hypoxic hippocampal slices to the presence of excitotoxins provided us with an inexpensive, sensitive tool to distinguish between structurally similar compounds. Moreover, these studies indicate that hypoxic neuronal damage cannot solely result from an excitotoxic mechanism; the involvement of voltage-dependent calcium channels in such damage is likely, as is evident from experiments performed in calcium-depleted medium and with the non-competive NMDA antagonist MK-801. At sub-toxic doses, quinolinate, a tryptophan metabolite implicated in Huntington's disease, appears to be a strong potentiator of the toxicity of all excitotoxins tested.

Endogenous ganglioside GM1 modulates L-type calcium channel activity in N18 neuroblastoma cells

Digital imaging fluorescence microscopy was used to investigate the effect of the B subunit of cholera toxin on calcium homeostasis in neuroblastoma N18 cells. The B subunit, which binds specifically to ganglioside GM1 in the outer leaflet of the cell membrane, was found to induce a sustained increase of intracellular calcium concentration ([Ca2+]i). The increase in [Ca2+]i was not observed in the absence of extracellular calcium, or in the presence of the calcium chelator EGTA, and was blocked by nickel. The B subunit was also found to induce an influx of manganese ions, as indicated by a quench of the intracellular fura-2 fluorescence. These data suggest that the B subunit induces an increase in calcium influx in N18 cells. Potassium-induced depolarization also stimulated manganese influx; however, after the onset of depolarization-induced influx, the B subunit had no further effect. This occlusion suggests involvement of voltage-dependent calcium channels. Treatment with BayK8644, a dihydropyridine agonist selective for L-type calcium channels, induced manganese influx that was not altered by the B subunit and apparently blocked the effect of the B subunit itself. Furthermore, the dihydropyridine L-type channel antagonists niguldipine or nicardipine completely inhibited B subunit-induced manganese influx. Thus, the B subunit-induced manganese influx is likely due to activation of an L-type voltage-dependent calcium channel. Spontaneous influx of manganese ions was also inhibited by nicardipine or niguldipine and by exogenous gangliosides. Ganglioside GM1 was more potent than GM3, but globoside had no significant effect. The modulation of L-type calcium channels by endogenous ganglioside GM1 has important implications for its role in neural development, differentiation, and regeneration and also for its potential function in the electrical excitability of neurons.

Protein kinase C activates capacitative calcium entry in the insulin secreting cell line RINm5F

This study examines the calcium store-regulated (capacitative) calcium influx pathway in the endocrine pancreatic cell line RINm5F, utilizing thapsigargin. After preincubation of the cells with the phorbol ester TPA, thapsigargin induced a sustained elevation of cytosolic calcium as well as a sustained stimulation of manganese entry, the latter being used to assess calcium influx. Thapsigargin given alone provoked a smaller and only transient elevation of cytosolic calcium and stimulation of manganese entry. The protein kinase C inhibitor staurosporine antagonized the effect of the phorbol ester. Verapamil, nifedipine, or measures to hyperpolarize the cells exerted no inhibitory action against this effect, which excludes an involvement of voltage-dependent calcium channels. In conclusion, our data shows for the first time that protein kinase C stimulation activates the capacitative calcium influx pathway of endocrine pancreatic insulin-producing cells.

Differential involvement of voltage-dependent calcium channels in apomorphine-induced hypermotility and stereotypy

The involvement of the voltage-dependent calcium channel in behavioral effects of apomorphine was tested in naive rats and in animals which were morphine-abstinent or were subjected to chronic electroconvulsive treatment (ECS). In naive rats a calcium channel blocker, nifedipine, which by itself does not affect locomotor activity, inhibited the locomotor stimulation induced by apomorphine, while it facilitated stereotyped behavior. Morphine-abstinent and ECS-treated rats displayed elevated responsiveness to apomorphine, reflected by hypermotility and stereotyped behavior after a dose of 1 mg/kg IP that does not produce overt behavioral effects in naive animals. Nifedipine, 5 mg/kg IP, significantly reduced hypermotility produced by apomorphine in morphine abstinent or ECS-treated rats. The calcium channel blocker did not, however, antagonize enhanced stereotyped behavior. The results indicate that apomorphine hypermotility is controlled by dihydropyridine calcium channels and that enhancement of calcium channel density produced by morphine abstinence and by chronic ECS potentiates the hypermotility response. Calcium channels seem to be differently involved in control of apomorphine-induced hypermotility and stereotypy.

The role of P-type calcium channels in the depolarization-induced activation of nitric oxide synthase in frontal cortex.

In this study we demonstrate that 50 mM K+ stimulates the conversion of L-[3H]arginine to L-[3H]citrulline and that this effect is blocked by 10 microM N omega-nitro-L-arginine, a nitric oxide synthase inhibitor, and Ca(2+)-free conditions. Amiloride (1 mM) and low Na+ conditions were used to test the possible involvement of the Na(+)-Ca2+ exchanger. These treatments were without effect. The calcium channel blockers 10 mM Mg2+, 100 microM Cd2+, and 10 mM Co2+ also blocked the K+ response, suggesting the involvement of voltage-dependent calcium channels (VDCCs). The specific VDCC involved seems to be the P type, as funnel-web spider toxin blocked the response whereas 200 microM Ni2+, 10 microM nifedipine, and 100 nM omega-conotoxin did not.

Involvement of voltage-dependent calcium channels (VDCC) in the action of GnRH on GtH release in common carp (Cyprinus carpio L): comparison with K+ action

The involvement of different types of voltage-dependent calcium channels (VDCC) in the stimulatory action of GnRH (in comparison with K+) on maturational gonadotropin (GtH) release was investigated using superfused carp pituitary cells. The action of these 2 stimulants was not modified either by D600 or nifedipine (drugs blocking L-type of VDCC). Cadmium (Cd2+), which blocks all types of VDCC indifferently, provoked a dose-dependent stimulation of GtH secretion. Cd2+ action was not altered by addition of sGnRH in any of the doses. Similar results were obtained using K+ as a secretagogue, but only the highest dose of Cd2+ (200 mumol/l) was able to completely block K+ action. Low doses (0.1 and 1 mumol/l) of the L-type VDCC activator BAY-K8644 did not change basal GtH secretion and had no effect on sGnRH-stimulated GtH secretion. Surprisingly, doses (10 mumol/l and higher) of BAY-K8644 evoked dose-dependent inhibition of GtH secretion. On the other hand, a higher concentration (20 mumol/l) of nifedipine provoked a stimulation of GtH release. Our results indicate that the stimulatory action of GnRH and K+ involves activation of a certain type of cadmium-sensitive VDCC (probably T- or N-type VDCC) whereas dihydropyridine and diphenylalkylamine sensitive VDCC (L-type VDCC) does not participate in this phenomenon. The inhibitory action of BAY-K8644 and, on the other hand, the stimulatory action of nifedipine indicate that L-type VDCC probably play a role in other physiological pathways regulating GtH release in carp.