Voltage-sensitive Calcium Channel Blocker Research Articles

Amyotrophic lateral sclerosis IgG-treated neuromuscular junctions develop sensitivity to L-type calcium channel blocker.

In order to search for early changes induced by the application of human immunoglobulin G (IgG) on motor nerve terminals, IgG from patients with amyotrophic lateral sclerosis (ALS) and control subjects was injected subcutaneously into the levator auris muscle of mice. A week or a month after the last injection, endplate potentials were recorded. No changes in quantal content of transmitter release were observed. In control and ALS IgG-treated muscles, neurotransmitter release remained sensitive to P/Q-type and insensitive to N-type voltage-sensitive calcium channel (VSCC) blockers as in untreated muscles. In contrast, IgG from 5 of 8 different ALS patients induced a significant reduction in quantal content of the evoked response after incubation with nitrendipine, indicating that a novel sensitivity to this calcium channel blocker appears in these motor nerve terminals. These results indicate that ALS IgG induces plastic changes at nerve terminals. The expression of transmitter release coupled to L-type VSCC indicate that ALS IgGs are capable of inducing plastic changes at the nerve terminals that may participate in the process leading to neuronal death.

Interactions of intrathecally administered ziconotide, a selective blocker of neuronal N-type voltage-sensitive calcium channels, with morphine on nociception in rats

Ziconotide is a selective, potent and reversible blocker of neuronal N-type voltage-sensitive calcium channels (VSCCs). Morphine is an agonist of μ-opioid receptors and inhibits N-type VSCC channels via a G-protein coupling mechanism. Both agents are antinociceptive when they are administered intrathecally (spinally). The present study investigated the acute and chronic (7-day) interactions of intrathecally administered ziconotide and morphine on nociception in several animal models of pain. In the acute study, intrathecal bolus injections of morphine and ziconotide alone produced dose-dependent inhibition of formalin-induced tonic flinch responses and withdrawal responses to paw pressure. The combination of ziconotide and morphine produced an additive inhibition of formalin-induced tonic flinch responses and a significant leftward shift of the morphine dose-response curve in the paw pressure test. After chronic (7-day) intrathecal infusion, ziconotide enhanced morphine analgesia in the formalin test. In contrast, chronic intrathecal morphine infusion produced tolerance to analgesia, but did not affect ziconotide antinociception. Antinociception produced by ziconotide alone was the same as that observed when the compound was co-administered with morphine to morphine-tolerant rats. In the hot-plate and tail immersion tests, chronic intrathecal infusion of morphine lead to rapid tolerance whereas ziconotide produced sustained analgesia with no loss of potency throughout the infusion period. Although ziconotide in combination with morphine produced an apparent synergistic analgesic effects during the initial phase of continuous infusion, it did not prevent morphine tolerance to analgesia. These results demonstrate that (1) acute intrathecal administrations of ziconotide and morphine produce additive or synergistic analgesic effects; (2) chronic intrathecal morphine infusion results in tolerance to analgesia but does not produce cross-tolerance to ziconotide; (3) chronic intrathecal ziconotide administration produces neither tolerance nor cross-tolerance to morphine analgesia; (4) intrathecal ziconotide does not prevent or reverse morphine tolerance.

Effects of intrathecal administration of ziconotide, a selective neuronal N-type calcium channel blocker, on mechanical allodynia and heat hyperalgesia in a rat model of postoperative pain

Ziconotide (SNX-111), a selective blocker of neuronal N-type voltage-sensitive calcium channels, is antinociceptive when it is administered intrathecally. It is currently under clinical investigation for the treatment of malignant and non-malignant pain syndromes. The present study was undertaken to compare and contrast antinociceptive properties of ziconotide, morphine and clonidine in a rat model of post-operative pain. Post-operative pain was produced by making a longitudinal incision through the skin, fascia, and muscle of the plantar aspect of the left hindpaw. This procedure produced immediate (0.5 h after surgery) and long-lasting (4–7 days post-surgery) heat hyperalgesia and mechanical allodynia in the injured hindpaw. Pain thresholds in the contralateral hindpaw were unaffected. Administered one day after incisional surgery, intrathecal ziconotide blocked established heat hyperalgesia in the injured hindpaw in a dose-dependent manner yielding an ED50 of 0.1 μg (49 pmol) but had no effect on heat-induced nociceptive responses in the normal hindpaw. Intrathecal ziconotide (0.3 μg (112 pmol)) also produced a long-lasting (>4 h) but reversible (<24 h) blockade of established mechanical allodynia. Administered one day after surgery, intrathecal bolus injection of morphine dose-dependently blocked heat hyperalgesia in the injured hindpaw with an ED50 of 1.6 μg (2.1 nmol) and heat nociceptive responses in the normal hindpaw with an ED50 of 2.7 μg (3.6 nmol). The effects were immediate and short-lasting (≤1 h). Intravenous bolus injection of 3 mg/kg (1.1 μmol/kg) ziconotide, administered either before or after incisional surgery, had no effect on thermal pain thresholds measured in either the injured or normal hindpaw. In contrast, intraperitoneal injections of 2 mg/kg (2.6 μmol/kg) morphine and 2.5 mg/kg (9.4 μmol/kg) clonidine blocked heat hyperalgesia in the injured hindpaw; morphine, but not clonidine, also elevated thermal (heat) nociceptive response thresholds in the normal hindpaw. The results of this study show that intrathecal ziconotide is antinociceptive in a rat incisional model of post-operative pain and is more potent, longer acting, and more specific in its actions than intrathecal morphine.

Synthesis of a series of 4-benzyloxyaniline analogues as neuronal N-type calcium channel blockers with improved anticonvulsant and analgesic properties.

In this article, the rationale for the design, synthesis, and biological evaluation of a series of N-type voltage-sensitive calcium channel (VSCC) blockers is described. N-Type VSCC blockers, such as ziconotide, have shown utility in several models of stroke and pain. Modification of the previously reported lead, 1a, led to several 4-(4-benzyloxylphenyl)piperidine structures with potent in vitro and in vivo activities. In this series, the most interesting compound, (S)-2-amino-1-{4-[(4-benzyloxy-phenyl)-(3-methyl-but-2-enyl)-amino]-p iperidin-1-yl}-4-methyl-pentan-1-one (11), blocked N-type calcium channels (IC(50) = 0.67 microM in the IMR32 assay) and was efficacious in the audiogenic DBA/2 seizure mouse model (ED(50) = 6 mg/kg, iv) as well as the antiwrithing model (ED(50) = 6 mg/kg, iv). Whole-cell voltage-clamp electrophysiology experiments demonstrated that compound 11 blocked N-type Ca(2+) channels and Na(+) channels in superior cervical ganglion neurons at similar concentrations. Compound 11, which showed superior in vivo efficacy, stands out as an interesting lead for further development of neurotherapeutic agents in this series.

Effects of Calcium Channel Blockers on Behaviors Induced by the N-Methyl- d-Aspartate Receptor Antagonist, Dizocilpine, in Rats

The present study assessed the ability of voltage-sensitive calcium channel (VSCC) blockers to affect the behavioral effects of the noncompetitive N-methyl- d-aspartate (NMDA) receptor antagonist, dizocilpine, in male Wistar rats. Dizocilpine produced dose-dependent increases in locomotor activity. Nimodipine, verapamil, and flunarizine suppressed dizocilpine-facilitated vertical activity, while horizontal activity was attenuated by verapamil and nimodipine but not flunarizine. Repeated dizocilpine injections resulted in the development of sensitization to its locomotor stimulating properties. Development of sensitization was not context specific, and was observed following repeated exposures to 0.1 but not 0.056 or 0.3 mg/kg of dizocilpine. Nimodipine retarded the development of sensitization to dizocilpine's stimulating effects on horizontal activity, while verapamil suppressed sensitization to the vertical stimulating effects of dizocilpine. Flunarizine had no significant effects on sensitization to dizocilpine's locomotor stimulating properties. In rats trained to discriminate between injections of 0.056 mg/kg of dizocilpine and vehicle, none of the tested VSCC blockers was able to completely antagonize the discriminative stimulus properties of dizocilpine. Nimodipine, when administered in combination with the training dose of dizocilpine, modestly decreased the dizocilpine-lever selection. Dizocilpine dose dependently decreased the self-determined stimulation threshold implanted in rats with electrodes into the ventral tegmental area. Nimodipine exhibited some tendency to block the facilitating effects of dizocilpine, while verapamil and flunarizine had no effects. In summary, in the present experiments VSCC blockers exerted only modest interactions with the behavioral effects of dizocilpine, and it is unlikely that VSCC blockers have remarkable potential as adjunct treatment aimed at correcting the negative side effects of NMDA receptor antagonists (e.g., dizocilpine).

Cyclosporine A-induced prostacyclin release is maintained by extracellular calcium in rat aortic endothelial cells: role of protein kinase C

Chronic treatment with the immunosuppressive drug Cyclosporine A (CsA) is associated with increased intracellular calcium in vascular smooth muscle cells, which may activate phospholipase A2. We used rat aortic endothelial cells to investigate the role of protein kinase C (PKC) in CsA-induced prostacyclin (PGI2) release. CsA (10−9M) produced a significant increase in PGI2release. CsA-induced PGI2release were inhibited 80–85 % by 10−9M, and 99–100 % by 10−6M pretreatment doses of any of three different PKC inhibitors, i.e. 1-(5-isoquinolinesulfonylmethyl)piperazine(H7), staurosporine or 1-(5-isoquinolinesulfonyl)piperazine. Pretreatment with (10−9M) of diltiazem (a voltage-sensitive L-type calcium channel blocker) completely inhibited both CsA-induced PGI2release. Conversely, pretreatment with (10−9M) of thapsigargin (an intracellular calcium channel blocker) did not alter the action of CsA. These results strongly suggest that PKC, in association with an influx of extracellular calcium, mediates CsA-induced PGI2release in rat aortic endothelial cells.Cyclosporine A; 1-(5-isoquinolinemethyl)piperazine(H7); staurosporine; 1-(5-isoquinolinesulfonyl)piperazine; diltiazem; thapsigargin; rat aortic endothelial cells.

Structure-activity relationship at the proximal phenyl group in a series of non-peptidyl N-type calcium channel antagonists

Selective N-Type Voltage Sensitive Calcium Channel (VSCC) antagonists have shown utility in several models of pain and ischemia. We report the structure-activity relationship at the proximal phenyl group in a series of non-peptidyl VSCC blockers.

Endothelin-induced prostacyclin production in rat aortic endothelial cells: role of calcium

Endothelin (ET) is a potent vasoconstrictor peptide, released from endothelial cells, which is associated with prostaglandin (PG) release. The mechanism by which ET causes the release of PG is not clearly understood. We used rat aortic endothelial cells to investigate the role of calcium (Ca 2+) in ET-1-induced prostacyclin (PGI 2) release. ET-1 (10 −9 M) produced a significant increase in PGI 2release. Pretreatment of rat aortic endothelial cells with different doses (10 −9 M and 10 −6M) of diltiazem (voltage-sensitive L-type calcium channel blocker) produced significant inhibition of ET-1- and PDBu-induced PGI 2release. Inhibition was first noted at 10 −9M and was complete at 10 −6M. Conversely, pretreatment of rat aortic endothelial cells with different doses (10 −9M and 10 −6M) of calcium channel blockers (thapsigargin, an intracellular calcium channel blocker or conotoxin, a voltage-sensitive N-type calcium channel blocker) produced no changes on ET-1- or PDBu-induced PGI 2release. These results provide further support for the concept that PKC mediates ET-induced PGI 2release in rat aortic endothelial cells via an increase in intracellular calcium and this increase is due to the influx of extracellular calcium and not to the release of calcium from the sarcoplasmic reticulum.

Effect of lysophosphatidic acid on the ovum transport in mouse oviducts

The effects of lysophosphatidic acid (LPA) on ovum transport in mouse oviducts were studied. When excised oviducts were incubated at 37°C under 5% CO 2 in humidified air for 24 hours, addition of LPA at 10 μm to the medium significantly accelerated the rate of ovum transport, and 1μM LPA slightly increased the ovum transport rate. These increases were not inhibited by 10 μM indomethacin, a cyclooxygense inhibitor, but were suppressed by 200 ng ml of pertussis toxin or 10 μM verapamil, a voltage-sensitive calcium channel blocker. These data suggested that LPA stimulates mouse ovum transport by contracting oviductual smooth muscle via a voltage-sensitive calcium channel mediated by a pertussis toxin-sensitive G-protein-linked receptor.

Chemosensory response to high pCO is blocked by cadmium, a voltage-sensitive calcium channel blocker

In the dark, during normocapnic ( pCO 2=35 Torr, pH o=7.4) normoxia ( pO 2=100 Torr), high pCO (>300 Torr) causes Ca 2+-dependent photolabile excitation of chemosensors in the carotid body (CB). We previously proposed that the source of this Ca 2+ was the [Ca 2+] i stores because CO would react only intracellularly. However, influx of extracellular Ca 2+ was not excluded. Now, using perfused rat CB ( n=6) in the presence of normal extracellular [Ca 2+] we show that chemosensory response to CO ( pCO∼550 Torr) in normoxic ( pO 2∼100 Torr) normocapnia ( pCO 2∼30 Torr, pH∼7.4) is completely but reversibly inhibited by Cd 2+ (200 μM), a voltage-gated Ca 2+ channel blocker. Thus, extracellular Ca 2+ is necessary for excitatory chemosensory response to high pCO. Cd 2+ block occurs in spite of an enhanced [Ca 2+] i rise. This shows that Ca 2+ rise alone is unable to release neurotransmitter and to elicit a chemosensory response. Therefore, as a corollary, we conclude that Cd 2+ blocks the Ca 2+ flux that is needed for vesicle–membrane fusion for neurotransmitter release and neural discharge.

Prion protein fragment 106-126 induces apoptotic cell death and impairment of L-type voltage-sensitive calcium channel activity in the GH3 cell line.

The prion diseases are transmissible neurodegenerative pathologies characterized by the accumulation of altered forms of the prion protein (PrP), termed PrP(Sc), in the brain. Previous studies have shown that a synthetic peptide homologous to residues 106-126 of PrP (PrP 106-126) maintains many characteristics of PrP(Sc), i.e., the ability to form amyloid fibrils and to induce apoptosis in neurons. We have investigated the intracellular mechanisms involved in the cellular degeneration induced by PrP 106-126, using the GH3 cells as a model of excitable cells. When assayed in serum-deprived conditions (48 hr), PrP 106-126 (50 microM) induced cell death time-dependently, and this process showed the characteristics of the apoptosis. This effect was specific because a peptide with a scrambled sequence of PrP 106-126 was not effective. Then we performed microfluorimetric analysis of single cells to monitor intracellular calcium concentrations and showed that PrP 106-126 caused a complete blockade of the increase in the cytosolic calcium levels induced by K+ (40 mM) depolarization. Conversely, the scrambled peptide was ineffective. The L-type voltage-sensitive calcium channel blocker nicardipine (1 microM) also induced apoptosis in GH3 cells, suggesting that the blockade of Ca2+ entry through this class of calcium channels may cause GH3 apoptotic cell death. We thus analyzed, by means of electrophysiological studies, whether Prp 106-126 modulate L-type calcium channels activity and demonstrated that the apoptotic effect of PrP 106-126 was due to a dose-dependent inactivation of the L-type calcium channels. These data demonstrate that the prion protein fragment 106-126 induces a GH3 apoptotic cell death inducing a selective inhibition of the activity of the L-type voltage-sensitive calcium channels.

Differential effects of intrathecally administered N- and P-type voltage-sensitive calcium channel blockers upon two models of experimental mononeuropathy in the rat

We investigated the effects of intrathecally administered N-type and P-type voltage-sensitive calcium channel (VSCC) blockers on the level of thermal hyperalgesia in two neuropathic pain models: the chronic constriction injury (CCI) model and the partial sciatic nerve injury (PSNI) model. N-type, but not P-type, VSCC blockers attenuated the level of thermal hyperalgesia induced by CCI in a dose-dependent manner. In the PSNI model, both N-type and P-type VSCC blockers had no effect on thermal hyperalgesia. This suggests that some types of neuropathic pain may be treatable with N-type VSCC blockers.

TxA 2-induced pulmonary artery contraction requires extracellular calcium

In order to examine the role of extracellular calcium in the pulmonary arterial vasoconstriction that is elicited by thromboxane A 2 (TxA 2), rabbits were sacrificed and the main trunk of the pulmonary artery removed. Contractile responses of the isolated vessel to the TxA 2 mimetic, U46 619, were measured in a temperature controlled (37°C) organ bath. Compared with control responses, U46 619 (0.5 μM) contractions were nearly eliminated when 1 mM EGTA was added to the buffer. In the presence of normal extracellular calcium concentrations, antagonists of voltage sensitive calcium channels (e.g. verapamil and nifedipine) attenuated the U46 619-induced contractions. These voltage sensitive calcium channel blockers were more effective in eliminating contractile responses to high KCl concentrations (60 or 120 mM KCl). The inability of these calcium channel antagonists to completely eliminate U46 619 responses was confirmed in the anesthetized rabbit where both nifedipine and verapamil failed to block the increase in pulmonary arterial blood pressure resulting from intravenous U46 619 infusion. These results indicate that extracellular calcium is essential for U46 619-induced pulmonary vascular contraction, and that mechanisms in addition to voltage operated calcium channels participate in the movement of extracellular calcium through the plasma membrane.

Effects of the protein tyrosine kinase inhibitor, herbimycin A, on prolactin gene expression in GH 3 and 235-1 pituitary tumor cells

The high basal level of prolactin (PRL) gene expression in rat pituitary GH 3 cells is maintained through the spontaneous activity of voltage-sensitive calcium channels (VSCCs). This can be observed experimentally by addition of 0.5 mM CaCl 2 to GH 3 cells cultured in a low calcium, serum-free medium. CaCl 2 specifically induces PRL gene expression and this induction is inhibited by VSCC blockers. PRL gene expression is also stimulated by several hormones and growth factors. In the present study, we examined the effects of tyrosine kinase inhibitors on the ability of CaCl 2, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and thryrotropin-releasing hormone (TRH) to increase PRL mRNA levels. Of several PTK inhibitors used, one PTK inhibitor, herbimycin A, specifically inhibited the CaCl 2-induced increase in cytoplasmic and nuclear prolactin (PRL) mRNA without affecting cell viability, cell-cell and cell-matrix adhesion, or the expression of several other genes. The effects of herbimycin A were reversible. In cells pretreated with herbimycin A, PRL mRNA levels were reduced by 69±12% ( P<0.001; n=4).Western blot analysis using anti-phosphotyrosine antibody revealed a decrease of 91±1% ( P<0.001; n=4) in the phosphotyrosine content of proteins in the molecular weight range of 130–160 kDa. After changing the medium back to SFM plus 0.5 mM CaCl 2, levels of PRL mRNA increased over a period of several hours, and this increase was accompanied by the tyrosine phosphorylation of two or more proteins in the approximate size range of 130–160 kDa. Herbimycin A also inhibited PRL gene expression in the independently-derived 235-1 lactotrope cell line and lowered the tyrosine specific phosphorylation of protein(s) in a similar size range. Herbimycin A inhibited the ability of bFGF, EGF and TRH to stimulate PRL gene expression in GH 3 cells. Again, in cells pretreated with herbimycin A, bFGF induced a reappearance of tyrosine-specific phosphorylation, followed by a reappearance of PRL mRNA. These findings provide evidence for a role for at least one PTK which is necessary for basal and stimulated PRL gene expression.

Metabotropic Glutamate Receptor Mediated Long-term Depression in Developing Hippocampus

The effects of bath application of the metabotropic glutamate receptor (mGluR) agonist 1 S,3 R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, 10 μM) were studied at the Schaffer collateral-CA1 synapse in hippocampal slices from rats of 8–33 days postnatal age. In immature animals (8–12 days) ACPD induced a biphasic response characterized by an acute decrease in field EPSP slope (∼50–60% of baseline) in the presence of the agonist, followed by long-term depression (LTD, ∼75–80% of baseline) after washout. In animals older than 20 days, ACPD induced a slow onset potentiation or minimal change. Both the acute depression and LTD were blocked by the mGluR antagonist α-methyl-4-carboxyphenyl glycine (MCPG). ACPD-induced LTD was blocked by the N-methyl- d-aspartate receptor (NMDAR) antagonists d(−)-2-amino-5 phosphopentanoic acid (AP5) and dizocilpine maleate (MK-801), and by ethanol. Glutamic pyruvic transaminase, an enzyme that selectively metabolizes endogenous extracellular glutamate, also blocked LTD suggesting that the requisite NMDA currents were tonically activated by extracellular rather than synaptically released glutamate. ACPD-induced LTD was blocked by staurosporine, indicating a requirement for serine-threonine kinase activation, and was unaffected by the L-type voltage sensitive calcium channel blocker nitrendipine and the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT). Because mGluR-mediated LTD was observed only in immature CA1, mGluRs may play a role in hippocampal development, perhaps by contributing to synapse pruning in a temporally restricted fashion. © 1997 Elsevier Science Ltd.

Angiotensin II induces a tachyphylactic calcium response in the rabbit afferent arteriole.

The influence of repeated administration of angiotensin II (AII) on smooth muscle calcium concentration ([Ca2+]i) was studied in isolated rabbit renal afferent arterioles loaded with the calcium-sensitive fluorescent probe Fura-2. [Ca2+]i was evaluated in the proximal and distal parts of the afferent arterioles. AII (10(-8) M) increased the [Ca2+]i in both these regions. A second administration of AII, however, did not elicit any response in [Ca2+]i. The response to noradrenaline administration at the end of the experiment was not affected, i.e. there was no fading or cross-desensitization. Since this desensitization was specific for AII, it was of the tachyphylaxis type. Increasing doses of AII (10(-11)-10(-8) M) did not reverse the tachyphylaxis. However, in the proximal part, pretreatment with the voltage-sensitive calcium channel blocker nifedipine (10(-6) M) blunted the tachyphylactic effect of a second administration of AII. When L-arginine (L-Arg) was administered to the bath solution, thus activating the NO system, the development of tachyphylaxis was suppressed in the proximal region. Pretreatment with the protein kinase C (PKC) inhibitor chelerythrine (10(-6) M) did not affect the tachyphylaxis. We conclude that the calcium response to AII in the isolated rabbit afferent arteriole shows tachyphylaxis. This tachyphylaxis cannot be reversed by applying increasing doses of AII (10(-11)-10(-8) M). PKC does not seem to be involved in the tachyphylactic phenomenon in this preparation. It was also found that nifedipine and NO reduced the tachyphylaxis.

Novel Modulatory Effect of L-Type Calcium Channels at Newly Formed Neuromuscular Junctions

This study aimed to examine changes of presynaptic voltage-sensitive calcium channel (VSCC) subtypes during synapse formation and regeneration in relation to transmitter release at the neuromuscular junction (NMJ). Synaptic potentials were recorded from developing rat NMJs and from regenerating mouse and frog NMJs. As in normal adult NMJs, evoked transmitter release was reduced by an N-type VSCC blocker in the frog and by a P/Q-type VSCC blocker in the mammal at immature NMJs; however, various L-type VSCC blockers, both dihydropyridine and nondihydropyridine antagonists, increased evoked but not spontaneous release in a dose-dependent manner at newly formed NMJs. This presynaptic potentiation disappeared as NMJs matured. A rapid intracellular Ca2+ buffer, bis(O-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM, prevented the potentiation effect of nifedipine, but a slow Ca2+ buffer, EGTA-AM, did not. Thus, the potentiation effect of L-type blockers requires Ca2+ transients. Pretreatment with Ca2(+)-activated K+ channel blockers, iberiotoxin or charybdotoxin, did not prevent potentiation by nifedipine at regenerating frog NMJs. Thus, Ca(2+)-activated K+ channels were not likely involved in this potentiation. In contrast, no additional potentiation by nifedipine was seen in muscles pretreated with pertussis toxin (PTX), a G-protein blocker, which by itself enhances evoked transmitter release at regenerating frog NMJs. These results suggest the existence of multiple subtypes of VSCCs at newly formed motor nerve terminals. In addition to the normal N- or P/Q-type VSCCs that mediate transmitter release, L-type VSCCs may play a novel modulatory role in evoked transmitter release by activating a mechanism linked to PTX-sensitive G-proteins during synapse maturation.

Cyclosporine A-induced contractions and prostacyclin release are maintained by extracellular calcium in rat aortic rings: role of protein kinase C

Chronic treatment with the immunosuppressive drug, Cyclosporine A (CsA), is associated with increased intracellular calcium in vascular smooth muscle cells, which may cause vasoconstriction and/or activate phospholipase A 2. We used rat aortic rings to investigate the role of protein kinase C (PKC) in CsA-induced contractions and secondary prostacyclin (PGI 2) release. CsA (10 −9 M) produced a sustained contraction in rat aortic rings. Both CsA-induced contractions and PGI 2 release were inhibited 84 to 89% by 10 −9 M, and 99 to 100% by 10 −6 M pretreatment doses of any of three different PKC inhibitors, i.e. 1-(5-isoquinolinesulfonylmethyl) piperazine (H7), staurosporine or 1-(5-isoquinolinesulfonyl) piperazine. Pretreatment with (10 −9 M) of diltiazem (a voltage-sensitive L-type calcium channel blocker) completely inhibited both CsA-induced contractions and PGI 2 release. Conversely, pretreatment with (10 −9 M) of thapsigargin (an intracellular calcium channel blocker) did not alter the action of CsA. These results strongly suggest that PKC, in association with an influx of extracellular calcium mediates CsA-induced contractions and secondary PGI 2 release in rat aortic rings.

Selective blockade of N-type voltage-sensitive calcium channels protects against brain injury after transient focal cerebral ischemia in rats

The neuroprotective efficacy of the selective N-type voltage-sensitive calcium channel blocker, SNX-111, was evaluated in spontaneously hypertensive rats subjected to 60 min of focal cerebral ischemia by permanent ligation of the right common carotid artery and temporary occlusion of the right middle cerebral artery. Intravenous infusion of 167 μg/kg per min SNX-111 for 30 min (5 mg/kg), initiated immediately after reperfusion, significantly reduced cortical infarct volumes measured 24 h after the ischemic insult.

Blockade of spinal N- and P-type, but not L-type, calcium channels inhibits the excitability of rat dorsal horn neurones produced by subcutaneous formalin inflammation

The effects of the intrathecal (i.t.) administration of different voltage-sensitive calcium channel (VSCC) blockers were studied in the formalin model of inflammation. The responses of convergent dorsal horn neurones after the subcutaneous injection of formalin (5% formaldehyde, 50 μl volume) were recorded extracellularly in rats under halothane anaesthesia. Administration of the L-type calcium channel blocker verapamil, 5 and 50 μg, before formalin injection had no effect on either the first or second phase of the formalin response. Pre-treatment with the N-type calcium channel blocker ω-Conotoxin-GVIA, 0.1 and 0.4 μg, reduced both phases of the formalin response. The low dose of ω-Conotoxin-GVIA significantly inhibited the first phase response whereas the high dose significantly reduced the second phase. Pre-treatment with the P-type calcium channel blocker ω-Agatoxin-IVA, 0.125 and 0.5 μg, did not cause a significant inhibition of the first phase whereas a marked dose-related reduction in the second phase of formalin response was found with the high dose producing 95% inhibition. These results demonstrate that spinal N- and P-type, but not L-type, VSCCs are involved in the inflammation-evoked hyperexcitability of dorsal horn neurones after peripheral formalin injection. Since selective antagonists for each type of VSCC had differential effects on the formalin response, it is suggested that each type of VSCC could be preferentially regulating or coupled to the release of certain neurotransmitters in the enhanced nociceptive transmission at the spinal level following formalin inflammation.