Voltage-gated Calcium Channel Blocker Research Articles

Calcium channel antibodies and Lambert-Eaton myasthenic syndrome

The present study investigated the effects of pharmacological spinal inhibition of voltage-gated calcium channels (VGCC) in mouse pruritus. The epidural administration of P/Q-type MVIIC or PhTx3.3, L-type verapamil, T-type NNC 55-0396 or R-type SNX-482 VGCC blockers failed to alter the scratching behavior caused by the proteinase-activated receptor 2 (PAR-2) activator trypsin, injected into the mouse nape skin. Otherwise, trypsin-elicited pruritus was markedly reduced by the spinal administration of preferential N-type VGCC inhibitors MVIIA and Phα1β. Time-course experiments revealed that Conus magus-derived toxin MVIIA displayed significant effects when dosed from 1 h to 4 h before trypsin, while the anti-pruritic effects of Phα1β from Phoneutria nigriventer remained significant for up to 12 h. In addition to reducing trypsin-evoked itching, MVIIA or Phα1β also prevented the itching elicited by intradermal (i.d.) injection of SLIGRL-NH2, compound 48/80 or chloroquine, although they did not affect H2O2-induced scratching behavior. Furthermore, the co-administration of MVIIA or Phα1β markedly inhibited the pruritus caused by the spinal injection of gastrin-releasing peptide (GRP), but not morphine. Notably, the epidural administration of MVIIA or Phα1β greatly prevented the chronic pruritus allied to dry skin model. However, either tested toxin failed to alter the edema formation or neutrophil influx caused by trypsin, whereas they significantly reduced the c-Fos activation in laminas I, II and III of the spinal cord. Our data bring novel evidence on itching transmission mechanisms, pointing out the therapeutic relevance of N-type VGCC inhibitors to control refractory pruritus.

Modulation of Voltage-Gated Calcium Channels by Orphanin FQ in Freshly Dissociated Hippocampal Neurons

Orphanin FQ (OFQ) has recently been reported to be an endogenous ligand for the opioid-like LC132 receptor. The effect of OFQ on high voltage-gated calcium channels (VGCCs) was examined in freshly dissociated rat pyramidal neurons using the whole-cell configuration of the patch-clamp technique. High-threshold Ba2+ currents were reversibly inhibited by OFQ. The depression of the currents was associated with a slowed rate of activation and a change in the activation I-V relationship at step potentials higher than +30 mV. In concentration-response experiments, a mean (+/-SEM) pEC50 value of 7.0 +/- 0.07 and a Hill coefficient of 1.5 +/- 0.08 (n = 5) were obtained. The near-maximum inhibition of the Ba2+ currents by OFQ (1 microM) amounted to 31 +/- 2.2% of control (n = 15). Opioid receptors could not account for the effects of OFQ on VGCCs, because naloxone, a broad spectrum mu-, delta-, and kappa-receptor antagonist, did not reduce the effectiveness of OFQ. When GTP-gamma-S was included in the pipette, the depression of the currents by OFQ was irreversible, whereas currents from neurons preincubated with pertussis toxin were not inhibited by OFQ, consistent with the involvement of a PTX-sensitive G-protein. When selective blockers of VGCCs were used, it was demonstrated that all subtypes of VGCCs were affected by OFQ. In conclusion, the effect of OFQ on VGCCs expressed in hippocampal CA3 and CA1 neurons may play an important role in the regulation of hippocampal cell excitability and neurotransmitter release.

Weaver granule neurons are rescued by calcium channel antagonists and antibodies against a neurite outgrowth domain of the B2 chain of laminin.

The weaver mutation impairs migration of the cerebellar granular neurons and induces neuronal death during the first two weeks of postnatal life. To elucidate the molecular mechanisms for the impaired neuronal migration, we investigated the rescue mechanisms of the weaver (wv/wv) granule neurons in vitro. We found that Fab2 fragments of antibodies against a neurite outgrowth domain of the B2 chain of laminin enhanced neurite outgrowth and neuronal migration of the weaver granule neurons on a laminin substratum and in the established cable culture system. The rescue of the weaver granule neurons by antibodies against the B2 chain of laminin may result from the neutralizing effect of these antibodies against the elevated B2 chain levels of the weaver brain. The L-type calcium channel blocker, verapamil (1-5 microM), also rescued the weaver granule neurons. High concentrations of MK-801 (10-20 microM), a glutamate receptor antagonist and voltage-gated calcium channel blocker, rescued the weaver granule neurons similar to verapamil, but low concentrations of MK-801 (1 microM) had no rescue effect. Simultaneous patch-clamp studies indicated that the weaver granule neurons did not express functional N-methyl-D-aspartate receptors further indicating that the rescue of the weaver granule neurons by MK-801 resulted from its known inhibition of voltage-gated calcium channels. The present results indicate that antibodies against the B2 chain of laminin, verapamil, and high concentrations of MK-801 protect the weaver granule neurons from the otherwise destructive action of the weaver gene. Thus, both the laminin system and calcium channel function contribute to the migration deficiency of the weaver granule neurons.

Long-term potentiation in the hippocampal CA1 region in the presence ofN-methyl-d-aspartate receptor antagonists

The present study deals with the question of whether L-type voltage-gated calcium channels can support the induction of input specific long-term potentiation. Tetanus-induced potentiation of synaptic transmission was examined in the CA1 region of normal and disinhibited guinea-pig hippocampal slices. It was found that afferent tetanization in the presence of 50 microM of the N-methyl-D-aspartate receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid led to a prolonged input specific potentiation. This potentiation was found only in disinhibited slices, its induction required cooperativity, and it was associated with an increase in the early part of the field excitatory postsynaptic potential initial slope. It was not affected by the L-type voltage-gated calcium channel blocker nifedipine, but it was reduced when D(-)-2-amino-5-phosphonopentanoic acid was supplemented with other N-methyl-D-aspartate receptor antagonists. The present study also examined a potentiation that was not restricted to the activated synapses and that was not associated with an increase in the early part of the field excitatory postsynaptic potential initial slope. This potentiation was blocked by the L-type voltage-gated calcium channel antagonist nifedipine. It is concluded that calcium influx through L-type voltage-gated calcium channels participates in the generation of a prolonged potentiation, but not of the input specific N-methyl-D-aspartate receptor-dependent long-term potentiation. The results also suggest that high concentrations of D(-)-2-amino-5-phosphonopentanoic acid can be insufficient to fully prevent N-methyl-D-aspartate receptor activation by strong afferent tetanization in the disinhibited slice.

Hypoxic contraction of isolated rabbit mesenteric veins. Contribution of endothelium and attenuation by volatile anesthetics.

Acute systemic hypoxia induces mesenteric venoconstriction in intact rabbits in part because of an increase in chemoreflex-mediated sympathetic efferent nerve activity. Inhaled anesthetics attenuate this reflex response. The direct effects of hypoxia on mesenteric veins are unknown. The purpose of the current study was to examine the effects of hypoxia on isolated rabbit mesenteric capacitance veins and to determine the effects of halothane, isoflurane, and enflurane on the responses to hypoxia. Isometric tension was measured before, during, and after 10 min of hypoxia in the rings of either quiescent or norepinephrine contracted veins, with or without endothelium. Effects of various pharmacologic agents and volatile anesthetics on the responses to hypoxia were examined. Hypoxia augmented contractions to norepinephrine and phenylephrine only in endothelium-intact veins. The hypoxic response was inhibited by phentolamine (alpha-adrenoceptor antagonist) and abolished in the absence of extracellular Ca2+. There were no effects of propranolol (beta-adrenoceptor antagonist), ryanodine (a sarcoplasmic reticulum Ca2+ depleter), indomethacin (cyclooxygenase inhibitor), or nordihydroguaiaretic acid (lipoxygenase inhibitor). L-NAME (an inhibitor of nitric oxide synthase) enhanced basal sensitivity of veins to norepinephrine but had no effect on the response to hypoxia. Nicardipine (a blocker of voltage-gated calcium channels) depressed the hypoxic contraction by 86 +/- 5%, phosphoramidon (an inhibitor of endothelin-converting enzyme) by 82 +/- 8%, and BQ-123 (a specific endothelin-1 receptor antagonist) by 47 +/- 10%. Volatile anesthetics (1.0 MAC) inhibited responses to hypoxia in the absence as well as presence of L-NAME. These results suggest that in mesenteric capacitance veins of rabbits an intrinsic vascular mechanism contributes to endothelium-dependent hypoxic augmentation of contraction to alpha-adrenergic agonists that involve activation of endothelin-1, an endothelium-derived constricting factor. Inhibition of hypoxic contraction by volatile anesthetics is not mediated by endothelium relaxing factor.

K+ modulation of microglial superoxide production: involvement of voltage-gated Ca2+ channels.

A variety of cytoactive factors produced during injury and inflammation are known to activate the central nervous system (CNS) macrophage, the microglia. Since extracellular potassium levels are known to rise rapidly at sites of injury in the CNS, we examined the possibility that changes in extracellular potassium could mediate changes in microglial function. The effect of an increase in potassium concentration on microglial superoxide anion production was studied in cultured neonatal rat microglia. Rather than directly inducing superoxide anion production, exposure to media containing 25 and 55 mM potassium enhanced the production of superoxide induced by phorbol 12-myristate 13-acetate. This potentiation was blocked by nifedipine, a voltage-gated calcium channel blocker. Treatment of the microglia with BAY K 8644, an agonist for voltage-gated calcium channels, produced an enhancement of superoxide levels similar to that of potassium. Because these data indicated the presence of a voltage-gated calcium channel, we also examined whole cell current in cultured microglia. A small, voltage-dependent inward calcium current was seen that was increased by exposure of the microglia to BAY K 8644. The presence of a small but finite calcium influx via these channels may be an important factor in the regulation of intracellular microglial events such as activation of the NADPH oxidase and the consequent production of superoxide anion.

Brief calcium transients evoked by glutamate receptor agonists in rat dorsal horn neurons: fast kinetics and mechanisms.

1. The calcium indicator dye, indo-1, was used to analyse the receptor-specific mechanisms of intracellular calcium ion ([Ca2+]i) responses evoked by excitatory amino acid (EAA) stimulation of dorsal horn neurons. Measurements of somal changes in [Ca2+]i were made on a subsecond time scale under conditions designed to allow membrane potential to mediate interactions between agonist-gated channels and voltage-gated calcium channels (VGCCs). 2. Voltage-gated calcium channels were activated in a receptor-independent manner using elevated extracellular [K+]. The concentration-dependence of K(+)-evoked [Ca2+]i transients was steep and variable among cells, with a mean maximal [Ca2+]i response of 1400 nM and a rapid maximal rate of rise. These data indicate that VGCCs provide a high-capacity route for Ca2+ entry that is very sensitive to small changes in membrane potential. 3. Stimulation of non-NMDA receptors using the non-desensitizing agonist kainate also evoked large [Ca2+]i responses (mean, 840 nM) that were predominantly due to indirect activation of VGCCs. However, in 60% of neurons tested, a component of the [Ca2+]i transient evoked by kainate at concentrations above 10 microM was not blocked by the potent VGCC blocker, lanthanum (La3+). The La(3+)-resistant [Ca2+]i responses to kainate rose exponentially, required extracellular Ca2+, and were caused neither by evoked release of EAA transmitters nor by reversal of Na(+)-Ca2+ exchange. These responses may be mediated by a Ca(2+)-permeable conformation of non-NMDA receptors and can also be evoked by quisqualate, (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and glutamate. 4. Non-NMDA receptors were activated in a desensitizing manner using quisqualate or AMPA. Quisqualate evoked small [Ca2+]i transients (210 nM) with a slow rate of rise. Typically, above 3 microM quisqualate, the size of the responses decreased, reflecting desensitization of the receptor. Responses to quisqualate were blocked by removal of extracellular Ca2+ indicating that mobilization of intracellular Ca2+ stores does not occur in the majority of dorsal horn neurons. However, trans-(+-)-1-amino-1,3-cyclopentane dicarboxylic acid (trans-ACPD) was occasionally able to evoke modest Ca2+ release. 5. Activation of the Ca(2+)-permeable NMDA receptors evoked [Ca2+]i transients that were large (780 nM), with a moderate rate of rise, and that generally achieved a maximum amplitude at NMDA concentrations around 300 microM. 6. Glutamate was used to examine [Ca2+]i responses to the activation of mixed EAA receptor subtypes by an endogenous ligand.(ABSTRACT TRUNCATED AT 400 WORDS)

Stage-specific effects of cadmium on preimplantation embryo development and implantation in the mouse

The effects of cadmium (Cd) exposure during the preimplantation period of pregnancy on the subsequent development and implantation of mouse embryos were examined. Injection of a high dose of Cd (38 μmol Cd/kg body wt.) on day 2 (D1 = vaginal plug), when the embryo is at the two-cell stage, had little effect on the initiation and maintenance of pregnancy when examined on D8. The initiation of implantation (localized sites of increased uterine vascular permeability) in a similarly treated group of mice was assessed in the morning of D5, and these sites were absent in 62% of the animals examined. Thus, Cd treatment on D2 delayed temporarily, but did not prevent implantation and was not embryolethal. In marked contrast, the same dose Cd administered on D4 caused pregnancy failure in all mice examined on D8. No implantation sites were detected on D5 and the few blastocysts recovered were degenerating. To explore the mechanisms underlying these in vivo stage-specific effects of Cd, preimplantation embryos (two-cell, four-cell, eight-cell and morulae) were exposed in vitro to a high concentration of Cd (50 μM) for 8 h followed by reculture to monitor their potential to develop to the blastocysts stage. Two-cell embryos were remarkably resistant to Cd, but toxicity increased with development, and morulae readily degenerated after Cd exposure. Analysis of the accumulation of 109Cd (50 μM) by preimplantation embryos showed little or none in two-cell embryos, but rapid accumulation and efflux of this metal by blastocysts. Removal of the zona pellucida had no influence on Cd accumulation. Nifedine (500 nM), a potent voltage-gated calcium channel blocker, and zinc (Zn; 100-fold molar excess) each significantly reduced (≈ 50% in 2 h) Cd accumulation by blastocysts, whereas N-ethylymaleimide (NEM; 20 μM) increased it. These results provide evidence that pregnancy failure after Cd exposure during the preimplantation period reflects a direct embryotoxic effect of Cd, although maternal injury by Cd may also contribute. Resistance to Cd at the two-cell stage (D2) reflects a lack of uptake of this metal, whereas sensitivity to Cd at the blastocyst stage (D4) reflects the ability to accumulate Cd.

Age-related changes in the mechanisms of LHRH-stimulated LH release from pituitary cells in vitro

In vitro release of LH in response to LHRH, phorbol myristate acetate (PMA), the ionophore A23187, and nifedipine was evaluated in primary cultures of anterior pituitary cells from intact mature (6 to 7 month) and old (23 to 24 month) male Wistar rats. LH release from pituitary cells is reduced approximately 30% and 60% after 4 and 48 h of 10 −7M LHRH stimulation in cells of old rats, respectively. This impairment may be secondary to a loss of LHRH receptors. LHRH-stimulated LH release from cells of mature rats was inhibited 70% by the voltage-gated calcium channel blocker, nifedipine (10 −6M), whereas LHRH-stimulated LH release from cells of old rat was too low to detect the effects of this drug. Age changes can be partially reversed by A23187 and PMA during 4 h, but not 48 hrs of stimulation. It therefore appears that short- and long-term (4 h and 48 h, respectively) stimulation of LH release may proceed through separate mechanisms that are differentially affected by aging.

Effects of calcium and calcium antagonists against deprivation of glucose and oxygen in guinea pig hippocampal slices

To provide evidence to support the calcium hypothesis of cerebral ischemia, we examined the effects of extracellular calcium and calcium antagonists (verapamil, flunarizine, nicardipine) on in vitro ‘ischemia’ using guinea pig hippocampal slices. As a model of in vivo ischemia we used a state of both glucose and oxygen deprivation. Recovery of dentate antidromic field response and histological changes were used as indices of cell damage. After 10 min of deprivation in standard Krebs-Ringer solution, the field potentials exhibited minimum recovery and dentate neurons were severely damaged. Damaged neurons had pyknotic nuclei and swollen cytoplasms. Drugs were added and the calcium concentration was changed during 30 min of pre-deprivation and during deprivation. In the first experiment we demonstrated that pre-treated calcium antagonists protect the dentate granule cells against glucose and oxygen deprivation. The order of the protective potency was flunarizine>verapamil>nicardipine. In the second experiment we also showed that neuronal damage caused by deprivation is dependent on the extracellular concentration of calcium. Our data show that extracellular calcium is partially responsible for ‘ischemia’ neuronal injury in the hippocampal slice. Both low calcium and voltage-gated calcium channel blockers can preserve an antidromic population spike. Conversely, high calcium in the bath can worsen the damage caused by in vitro ‘ischemia’ to hippocampal slices.

Galanin-induced hyperpolarization of mudpuppy neurons is calcium dependent

The calcium dependence of the hyperpolarization produced by galanin has been studied in parasympathetic postganglionic neurons of the mudpuppy. Necturus maculosus. The galanin-induced hyperpolarization, but not the bethanechol-induced hyperpolarization, was reduced when manganese or magnesium were substituted for external calcium. Also, the galanin-induced, but not the bethanechol-induced hyperpolarization, was decreased in the presence of cadmium or nitrendipine. Because the inhibition of the galanin-induced hyperpolarization developed gradually and reversed slowly in the absence of extracellular calcium or presence of the voltage-gated calcium channel blockers, it is suggested that an intracellular pool rather than an extracellular pool of calcium is involved in the generation of the galanin-induced hyperpolarization.

Hormone secretagogues increase cytosolic calcium by increasing cAMP in corticotropin-secreting cells.

Corticotropin (ACTH)-releasing factor, vasoactive intestinal peptide, and catecholamines--hormones that stimulate ACTH secretion and cAMP generation--increased cytosolic calcium in AtT-20 cells. The increase in intracellular calcium is presumably a consequence of the stimulated cAMP synthesis, since forskolin, an activator of the catalytic unit of adenylate cyclase, and the cAMP analog 8-bromoadenosine 3',5'-cyclic monophosphate (8Br-cAMP) also increased the cytosolic levels of this ion. Pretreatment with somatostatin, a neuropeptide that inhibits stimulation of the adenylate cyclase system and the secretion of ACTH blocked the increase of cytosolic calcium. The effect of 8Br-cAMP, which bypasses the cyclase, was not inhibited by somatostatin pretreatment. The source of the increased calcium appears to be mainly extracellular. This is indicated by the inability of the secretagogues to increase cytosolic calcium in a medium deprived of this ion or in the presence of blockers of voltage-gated calcium channels. The involvement of calcium channels in the calcium rise evoked by the secretagogues was supported by experiments using the whole-cell patch-clamp technique. In these experiments 8Br-cAMP increased voltage-dependent calcium currents. These results suggest the following chain of events in the receptor-mediated elevation of cytosolic calcium and the concomitant release of ACTH from AtT-20 cells: hormone-receptor binding----cAMP synthesis----protein kinase activation----calcium channel activation----increase in cytosolic calcium----many steps----ACTH release. Phorbol myristate acetate, a compound which does not stimulate cAMP generation but enhances the release of ACTH in AtT-20 cells, decreased the cytosolic calcium level.