N-type Channel Blocker Research Articles

Effect of Voltage‐Dependent Calcium Channel Blockers on Ethanol‐Induced β‐Endorphin Release From Hypothalamic Neurons in Primary Cultures

The voltage-dependent calcium channel (VDCC) has been shown to mediate calcium entry into neurons that regulates neurotransmission in many neuronal cells. Four major types of VDCCs (three high-voltage-activated L-, N-, and P-types and one low-voltage-activated T-type) have been identified in neurons. Involvement of the VDCC in ethanol-stimulated beta-endorphin (beta-EP) release from hypothalamic neurons has not been studied. In the present study, the role of VDCC on basal and ethanol-induced beta-EP release was determined by using rat fetal hypothalamic cells in primary cultures. Treatments with a 50 mM dose of ethanol for 3 hr increased immunoreactive beta-EP (IR-beta-EP) release from hypothalamic cells maintained in cultures for 9 days. Ethanol-induced IR-beta-EP release was inhibited by a P/Q-type channel blocker omega-agatoxin TK (0.1-1 microM), an N-type channel blocker omega-conotoxin (0.1-1 microM), an L-type blocker nifedipine (1-10 microM), and a T-type blocker flunarizine (1-10 microM). The minimal effective doses of these blockers that blocked the ethanol response produced no significant effects on basal release of IR-beta-EP; neither did these doses of the blockers produce any significant effects on cell viability. These results suggest that ethanol-stimulated IR-beta-EP release is regulated by extracellular calcium involving P-, N-, L- and T-type channels.

Regulation of Ca 2+ influx by a protein kinase C activator in chromaffin cells: differential role of P/Q- and L-type Ca 2+ channels

Phorbol esters reduce depolarization-evoked Ca 2+ influx in adrenal chromaffin cells, suggesting that voltage-sensitive Ca 2+ channels (VSCCs) are inhibited by protein kinase C-mediated phosphorylation. We now address the possibility that L- and P/Q-type Ca 2+ channel subtypes might be differentially involved in phorbol ester action. In bovine chromaffin cells, short-term (10 min) incubations with phorbol 12-myristate 13-acetate (PMA) inhibited early high K +-evoked rises in cytosolic free Ca 2+ concentration ([Ca 2+] i) and the early component of the depolarization-evoked Mn 2+ quenching of fura-2 fluorescence in a dose-dependent manner (IC 50: 18 and 7 nM; maximal inhibitions: 45 and 48%, respectively). The protein kinase C inhibitor staurosporine (100 nM) reverted the inhibitory action of PMA. PMA (0.1–1 μM) inhibited the early and late phases of the ionomycin (2 μM)-evoked [Ca 2+] i transients by 14–23%. ω-Agatoxin IVA, a blocker of P/Q-type Ca 2+ channels, inhibited high K +-evoked [Ca 2+] i rises in a dose-dependent fashion (IC 50=50 nM). In contrast, 0.1 μM ω-conotoxin GVIA, a blocker of N-type channels, was without effect. A sizeable (<45%) component of early Ca 2+ influx persisted in the combined presence of ω-agatoxin IVA (100 nM) and nitrendipine (1 μM). Simultaneous exposure to ω-agatoxin IVA and PMA inhibited both the early [Ca 2+] i transients and Mn 2+ quenching to a much greater extent than each drug separately. Inhibition of the [Ca 2+] i transients by nitrendipine and PMA did not significantly exceed that produced by PMA alone. It is concluded that phorbol ester-mediated activation of protein kinase C inhibits preferentially L-type VSCCs over P/Q type channels in adrenal chromaffin cells. However, the possibility cannot be ruled out that dihydropyridine-resistant, non-P/Q type channels might also be negatively regulated by protein kinase C. This may represent an important pathway for the specific control of VSCCs by protein kinase C-linked receptors, not only in paraneurones but presumably also in neurones and other excitable cells.

虚血性脳血管障害急性期の病態生理とその臨床応用

The purpose of this lecture is to introduce the results of researches concerning the pathophysiology of ischemic cerebrovascular disease (CVD) in my department and to consider its clinical application.We found that gradual activation of voltage-sensitive Ca channel activity, a sensitive indicator of brain ischemia, occurs when CBF is reduced to 50%. This indicates the brain is more vulnerable to ischemia than had previously been believed.Fig. 1 is a schema showing the release of glutamate, Ca influx, activation of NOS, etc. on ischemia. Administration of an N-type Ca channel blocker or K channel opener could decrease the concentration of glutamate and reduce the infarct volume produced by MCA occlusion in rats. We estimated NOS activity during brain ischemia by measuring nitrotyrosine, which is a footprint of peroxynitrite. nNOS was active in the early phase of ischemia and iNOS became predominant in the later phase of the acute stage of ischemia. Aminoguanidine, a relatively selective iNOS inhibitor, could reduce infarct volume if administered 12 hours after MCA ligation in rats. Cytoxic edema is an other important issue in ischemia. An NOS inhibitor, as well as L-type Ca channel blockers, could delay the shift of water from extra- to intracellular space. Increase in blood viscosity produced by increase in Ht, decrease in RBC deformability, accelerated RBC aggregation time and increase in plasma viscosity, as well as accelerated platelet aggregation, are important factors causing secondary circulatory disturbance after ischemia or inducing reattack.The results are summarized in Fig. 11, and Fig. 10 shows various types of neuronal death after ischemia.Because of the diversity in the pathophysiology, effective treatment may require a cocktail of drugs, but so far we have no idea what would be the best cocktail.

Effect of subcutaneous administration of calcium channel blockers on nerve injury-induced hyperalgesia

Recent studies suggest that calcium contributes to peripheral neural mechanisms of hyperalgesia associated with nerve damage. In this animal behavioural study, we examined further the contribution of calcium in neuropathic pain by testing whether subcutaneous administration of either a calcium chelating agent or voltage-dependent calcium channel blockers attenuate nerve injury-induced hyperalgesia to mechanical stimulation. Studies were carried out in animals with partially ligated sciatic nerves, an established animal model of neuropathic pain. The nociceptive flexion reflex was quantified using an Ugo Basile Analgesymeter. Partial nerve injury induced a significant decrease in mechanical threshold compared to the sham operated controls. Daily subcutaneous injections of the calcium chelating agent, Quin 2 (20 μg/2.5 μl), significantly attenuated the nerve injury-induced hyperalgesia. Similarly, SNX-111, a N-type channel blocker, also significantly attenuated the nerve injury-induced hyperalgesia. SNX-230, a P and/or Q-type channel blocker, and nifedipine, a L-type channel blocker, had no effect on the hyperalgesia to mechanical stimulation. In control experiments, SNX-111 had no effect on mechanical thresholds when administered subcutaneously in either the hindpaw of normal animals or the back of the neck in nerve injury animals. This study shows that neuropathic pain involves a local calcium-dependent mechanism in the receptive field of intact neurons of an injured nerve, since it can be alleviated by subcutaneous injections of either a calcium chelating agent or SNX-111, a N-type calcium channel blocker. These agents may be effective, peripherally acting therapeutic agents for neuropathic pain.

Voltage-dependent calcium channels in cultivated neurons of the rat hippocampus

Calcium currents through the somatic membrane of cultivated (a low-density culture) hippocampal neurons of rats were studied with the use of a patch-clamp technique in the whole-cell configuration. Low- and high-threshold components of calcium currents were found in the somata of all studied cells. Low-threshold currents were activated at a membrane potential of about−75 mV and reached the maximum amplitude at −45±4 mV, while the maximum amplitude of high-threshold currents was observed at 17±6 mV. Low-threshold calcium currents differed from high-threshold current in weak suppression by low Cd2+ concentration (10–20 μM), while Ni2+ inhibited both types of calcium currents to an equal extent. Experiments with organic channel blockers showed that in most neurons at least four channel types were expressed: these were L, N, P, and channels insensitive to the used blockers (presumably, R-type). A blocker of L-type calcium channels, nifedipine (10 μM), blocked, on the average, 22.7±5.2%; a blocker of N-type channels, ω-CTx-GVIA (1.0 μM), blocked 30.0±5.0% and a blocker of P/Q channels, ω-Aga-IVA (200 nM), blocked 37.2±13.3% of the integral high-threshold current. A resistive component equalled 15.7±5.1% of the latter current. It is concluded that hippocampal neurons cultivated with a low density express a pharmacologically heterogeneous population of calcium channels, and the relative proportions of different type channels are close to the earlier described channel type composition in rat hippocampal slices. Our study shows that the low-density culture can be used as an adequate model for studying calcium channels in the somatic membrane of hippocampal neurons.

Functional characterization of ion permeation pathway in the N-type Ca2+ channel.

Multiple types of high-voltage-activated Ca2+ channels, including L-, N-, P-, Q- and R-types have been distinguished from each other mainly employing pharmacological agents that selectively block particular types of Ca2+ channels. Except for the dihydropyridine-sensitive L-type Ca2+ channels, electrophysiological characterization has yet to be conducted thoroughly enough to biophysically distinguish the remaining Ca2+ channel types. In particular, the ion permeation properties of N-type Ca2+ channels have not been clarified, although the kinetic properties of both the L- and N-type Ca2+ channels are relatively well described. To establish ion conducting properties of the N-type Ca2+ channel, we examined a homogeneous population of recombinant N-type Ca2+ channels expressed in baby hamster kidney cells, using a conventional whole cell patch-clamp technique. The recombinant N-type Ca2+ channel, composed of the alpha1B, alpha2a, and beta1a subunits, displayed high-voltage-activated Ba2+ currents elicited by a test pulse more positive than -30 mV, and were strongly blocked by the N-type channel blocker omega-conotoxin-GVIA. In the presence of 110 mM Ba2+, the unitary current showed a slope conductance of 18.2 pS, characteristic of N-type channels. Ca2+ and Sr2+ resulted in smaller ion fluxes than Ba2+, with the ratio 1.0:0. 72:0.75 of maximum conductance in current-voltage relationships of Ba2+, Ca2+, and Sr2+ currents, respectively. In mixtures of Ba2+ and Ca2+, where the Ca2+ concentration was steadily increased in place of Ba2+, with the total concentration of Ba2+ and Ca2+ held constant at 3 mM, the current amplitude went through a clear minimum when 20% of the external Ba2+ was replaced by Ca+2. This anomalous mole fraction effect suggests an ion-binding site where two or more permeant ions can sit simultaneously. By using an external solution containing 110 mM Na+ without polyvalent cations, inward Na+ currents were evoked by test potentials more positive than -50 mV. These currents were activated and inactivated in a kinetic manner similar to that of Ba2+ currents. Application of inorganic Ca2+ antagonists blocked Ba2+ currents through N-type channels in a concentration-dependent manner. The rank order of inhibition was La3+ >/= Cd2+ >> Zn2+ > Ni2+ >/= Co2+. When a short strong depolarization was applied before test pulses of moderate depolarizing potentials, relief from channel blockade by La3+ and Cd2+ and subsequent channel reblocking was observed. The measured rate (2 x 10(8) M-1 s-1) of reblocking approached the diffusion-controlled limit. These results suggest that N-type Ca2+ channels share general features of a high affinity ion-binding site with the L-type Ca2+ channel, and that this site is easily accessible from the outside of the channel pore.

Contribution of T-type calcium channels to afterdischarge generation in rat hippocampal slices

To clarify the possible involvement of Ca 2+ channel subtypes in epileptogenesis, the CA1 region of rat hippocampal slices was examined for the pharmacological effects of Ca 2+ channel blockers on ictal-like afterdischarges (ADs) observed following the repetition of high-frequency electrical stimulation, as well as on interictal-like bursts of population spikes following single stimuli in the presence of bicuculline. Ni 2+ and amiloride, which predominantly block T-type Ca 2+ channels, suppressed the number of spikes in ADs at concentrations of these two channel blockers sufficient to eliminate this type of Ca 2+ channel in acutely dissociated and cultured hippocampal neurons. On the other hand, neither the L-type Ca 2+ channel blocker nifedipine nor the N-type Ca 2+ channel blocker, ω-conotoxin GVIA had any effect on the generation of ADs. In addition, none of these T-, L- and N-type channel blockers had any effect on the bicuculline-induced, interictal-like epileptiform discharges. We therefore conclude that the activation of T-type Ca 2+ channels, which is distinct from the mechanisms involved in interictal activity, may play a causative role in the generation of ictal-like ADs.

Serotonergic Inhibition of the T-Type and High Voltage-Activated Ca2+Currents in the Primary Sensory Neurons ofXenopusLarvae

The primary sensory Rohon-Beard (R-B) neurons of Xenopus larvae are highly analogous to the C fibers of the mammalian pain pathway. We explored the actions of 5-HT by studying the modulation of Ca2+ currents. In approximately 80% of the acutely isolated R-B neurons, 5-HT inhibited the high voltage-activated (HVA) currents by 16% (n = 29) and the T-type currents by 24% (n = 41). The modulation of the T-type and the HVA currents was mimicked by selective 5-HT1A and 5-HT1D agonists: 8-OH-DPAT and L-694,247. The effects of the agonists were blocked by their respective 5-HT1A or 5-HT1D antagonists: p-MPPI and GR127935, suggesting that both 5-HT1A and 5-HT1D receptors were involved. Approximately 70% of the actions of 5-HT on HVA currents was occluded by omega-conotoxin-GVIA (N-type channel blocker), whereas the rest of the modulation ( approximately 30%) was occluded by <100 nM omega-agatoxin-TK (P/Q-type channel blocker). This suggests that 5-HT acts on N- and P/Q-type Ca2+ channels. Neither the modulation of the T-type nor that of the HVA currents was accompanied by changes in their voltage-dependent kinetics. Cell-attached patch-clamp recordings suggest that the modulation of the T-type channel occurs through a membrane-delimited second messenger. We have studied the functional consequences of the modulation of T-type Ca2+ channels and have found that these channels play a role in spike initiation in R-B neurons. Modulation of T-type channels by 5-HT therefore could modulate the sensitivity of this sensory pathway by increasing the thresholds of R-B neurons. This is a new and potentially important locus for modulation of sensory pathways in vertebrates.

Effects of Ca2+ channel blocker neurotoxins on transmitter release and presynaptic currents at the mouse neuromuscular junction.

1. The effects of the voltage-dependent calcium channel (VDCC) blockers omega-agatoxin IVA (omega-AgaIVA), omega-conotoxin GVIA (omega-CgTx), omega-conotoxin MVIIC (omega-MVIIC) and omega-conotoxin MVIID (omega-MVIID) were evaluated on transmitter release in the mouse diaphragm preparation. The effects of omega-AgaIVA and omega-MVIIC were also evaluated on the perineurial calcium and calcium-dependent potassium currents, ICa and IK(Ca), respectively, in the mouse levator auris preparation. 2. The P- and Q-type VDCC blocker omega-AgaIVA (100 nM) and P- Q- and N-type channel blockers omega-MVIIC (1 microM) and omega-MVIID (3 microM) strongly reduced transmitter release (> 80-90% blockade) whereas the selective N-type channel blocker omega-CgTx (5 microM) was ineffective. 3. The process of release was much more sensitive to omega-MVIIC (IC50 = 39 nM) than to omega-MVIID (IC50 = 1.4 microM). After almost completely blocking transmitter release (quantal content approximately 0.3% of its control value) with 3 microM omega-MVIIC, elevating the external [Ca2+] from 2 to 10 mM induced an increase of approximately 20 fold on the quantal content of the endplate potential (e.p.p.) (from 0.2 +/- 0.04 to 4.8 +/- 1.4). 4. Nerve-evoked transmitter release in a low Ca(2+)-high Mg2+ medium (low release probability, quantal content = 2 +/- 0.1) had the same sensitivity to omega-AgaIVA (IC50 = 16.8 nM) as that in normal saline solutions. In addition, K(+)-evoked transmitter release was also highly sensitive to the action of this toxin (IC50 = 11.5 nM; 100 nM > 95% blockade). The action of omega-AgaIVA on transmitter release could be reversed by toxin washout if the experiments were carried out at 31-33 degrees C. Conversely, the effect of omega-AgaIVA persisted even after two hours of toxin washout at room temperature. 5. Both the calcium and calcium-dependent potassium presynaptic currents, ICa and IK(Ca), respectively, were highly sensitive to low concentrations (10-30 nM) of omega-AgaIVA. The ICa and the IK(Ca) were also strongly reduced by 1 microM omega-MVIIC. The most marked difference between the action of these two toxins was the long incubation times required to achieve maximal effects with omega-MVIIC. 6. In summary these results provide more evidence that synaptic transmission at the mammalian neuromuscular junction is mediated by Ca2+ entry through P- and/or Q-type calcium channels.

FK506-binding proteins regulate smooth muscle contractility by altering neurotransmitter release

To determine the roles of FK506-binding proteins, receptors for the immunosuppressant FK506, in tachykinin release, we examined the effects of the FK506 derivative ascomycin, [3S-[3R[E(1S,3S,4S)],4S,5R,8S,9E,12R,14R,15S,16R,18S,19S,26aR]]-8-ethyl-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylethenyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-15,19-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone, on the contractility of the rabbit iris sphincter muscle. Ascomycin (10 −7 to 10 −5 M) caused concentration-dependent contractions, which were greatly attenuated by preexposure to rapamycin (10 −5 M), a FK506 receptor antagonist. Similarly, this contractile effect was abolished by preexposure to FK888 (10 −6 M), a tachykinin receptor antagonist, and to capsaicin (10 −5 M), a tachykinin-depleting agent. L-type voltage-dependent Ca 2+ channel blockers, nicardipine (10 −5 M) and verapamil (5×10 −5 M), inhibited the ascomycin-induced contraction, but the N-type channel blocker ω-conotoxin (10 −6 M) did not. These results suggest that ascomycin stimulates tachykinin release by its binding to FK506-binding proteins and the subsequent activation of L-type Ca 2+ channels. Thus, FK506-binding proteins may regulate muscle contractility by altering transmitter release from peripheral tachykininergic nerves.

Pharmacologically Distinct Presynaptic Calcium Channels in Cerebellar Excitatory and Inhibitory Synapses

We have used whole-cell patch clamp recordings and pharmacological blockers of Ca channels to compare the pharmacology of Ca channels that mediate synaptic transmission at the three types of synapses innervating Purkinje cells in rat cerebellar slices. Both parallel fiber and climbing fiber excitatory synapses were sensitive to the P-type Ca channel blocker, ω-AgaIVA and the P/Q/N-type channel blocker, ω-conotoxin MVIIC. Transmission at inhibitory interneuronal synapses was not suppressed by these toxins, or by the N-type (ω-conotoxins GVIA and MVIIA) or L-type (nimodipine) channel blockers. Inhibitory transmission could be inhibited by Ni 2+ and amiloride, but only at concentrations (IC 50∼300 μM) that affect other types of Ca channels. These results indicate that excitatory and inhibitory presynaptic terminals of the cerebellar cortex possess different types of voltage-gated Ca channels. The excitatory terminals contain P-type, Q-type and N-type Ca channels, with P-type channels playing the most prominent role. The inhibitory terminals possess quite different type(s) of Ca channel. The heterogeneous distribution of Ca channel types should impart unique properties to transmitter release from the excitatory and inhibitory terminals. © 1997 Elsevier Science Ltd.

Cytoplasmic Ca2+ mobilization and Ca2+-dependent membrane currents in dispersed bovine ciliary muscle cells

PURPOSE. The dependence of plasmalemma Ca 2+ influx and Ca 2+ release from intracellular stores on Ca 2+ activated K + channels of bovine ciliary muscle (CM) cells were examined. METHODS. The nystatin-perforated patch clamp technique for the measurement of membrane currents and a microscope based fura-2 fluorescence imaging of [Ca 2+ ] i were applied to CM cells freshly dissociated with collagenase and identified with smooth muscle-specific a-isoactin. RESULTS. At holding voltages (V H) of >260 mV, CM cells showed spontaneous transient outward currents (STOCs) and caffeine (>10 -4 M) induced large transient outward currents (I CAF) . Both STOCs and I CAF were abolished by tetraethylammonium chloride (10 -3 M) and charybdotoxin (10 -7 M), but not by apamin (10 -6 M), suggesting that both currents are mediated by Ca 2+ -activated K + channels similar to those with medium (MK) or large (BK-type) conductance. Both STOCs and I CAF were gradually abolished in the nominally Ca 2+ -free and Co 2+ -containing solutions but were resistant to L-type Ca 2+ channel blockers, including nicardipine, verapamil and diltiazem and a N-type channel blocker, ?-conotoxin. The [Ca 2+ ] i -elevation during high K + (100 mM)-depolarization was prevented by Ca 2+ -free and Co 2+ -containing solutions but not by nicardipine. CONCLUSIONS. These results suggest that CM cells possess MK or BK type-like Ca 2+ -activated K + channels and that L-type Ca 2+ channels play minor roles for the maintenance of Ca 2+ -dependent responses in contrast to other types of smooth muscle cells.

Ca2+ channel currents in type I carotid body cells of normoxic and chronically hypoxic neonatal rats

Whole-cell patch-clamp recordings were used to study voltage-gated Ca2+ channel currents in type I carotid body cells of young rats born and reared in normoxia or in a chronically hypoxic (CH) environment (10% O2). Currents activated at potentials of −40 mV and more positive, and typically peaked at 0 mV in both groups of cells. Steady-state inactivation curves were similar in the two populations. Ca2+ currents were significantly larger in CH type I cells, but this was accounted for by the increased size of CH cells: current density was similar in both cell types. Nifedipine (5 μM) always partially inhibited currents and Bay K 8644 (2–5 μM) always enhanced currents, indicating the presence of L-type channels. In a small number of cells from each group, the N-type channel blocker ω-conotoxin GVIA caused partial, irreversible inhibition, but in most cells was without discernible effect. These results indicate that type I cells possess L-type Ca2+ channels, that N-type are expressed in some cells and that non-L, non-N-type channels are also present. Furthermore, chronic hypoxia does not appear to cause specific adaptive changes in the properties of Ca2+ channels in type I cells.

The influx of Ca 2+ and the release of noradrenaline evoked by the stimulation of presynaptic nicotinic receptors of chick sympathetic neurons in culture are not mediated via L-, N-, or P-type calcium channels

We have shown earlier that nicotinic agonists induce the release of noradrenaline from chick sympathetic neurons in culture in two ways: (a) by activating the postsynaptic nicotinic receptors on nerve cell bodies, giving rise to spreading electrical activity and opening of voltage operated calcium channels in neuronal processes; (b) by activating the presynaptic nicotinic receptors on neuronal processes. In the present work, we investigated the contribution of various pathways to the observed Ca 2+ influx and subsequent noradrenaline release. Sympathetic neurons in culture were stimulated either by the nicotinic agonist dimethylphenylpiperazinium or electrically, in the presence or absence of tetrodotoxin and of specific blockers of calcium or nicotinic channels, and the effects on [Ca 2+] i in the area of neuronal processes and on noradrenaline release were measured. Under control conditions, the N-type channel blocker ω-conotoxin (0.1 μmol/1) diminished the release of noradrenaline and the increase of intraterminal Ca 2+ by 48% and 55%, respectively, whereas the L-type channel blocker (+)Bay k 8644 (1 μmol/1) diminished the release of noradrenaline by 25% and the increase of [Ca 2+] i by 39%. The P-type channel blocker ω-agatoxin (0.3 μmol/1) had no effect. The effects of the L-type channel ligands were complex and could only be explained on the assumption that, at high concentrations, these drugs also act as nicotinic antagonists. Tetrodotoxin blocked the Ca 2+ response evoked by electrical stimulation whereas DMPP applied in the presence of tetrodotoxin still evoked an increase of [Ca 2+] i and the release of noradrenaline (27% and 30% of control without tetrodotoxin, respectively). These residual responses were not blocked by any of the calcium channel blockers used or by their combination. Apparently, a substantial part of the influx of Ca 2+ induced by the activation of presynaptic nicotinic receptors is not carried by the N-, L- or P-type channels and probably occurs directly via the open channels of nicotinic receptors.

Microsphere embolism-induced changes in presynaptic function of the cerebral cortex in rats

The present study was undertaken to elucidate pathophysiological changes in the cortical presynaptic function, K +-stimulated calcium influx, noradrenaline release and noradrenaline uptake, on the 1st and 3rd days after microsphere embolism in rats. Voltage-dependent calcium channels were characterized pharmacologically using three types of calcium channel blockers, L-type (nifedipine and diltiazem), N-type (ω-conotoxin GVIA), and P-type channel (ω-agatoxin IVA) blockers. K +-stimulated calcium influx of the normal rat synaptosome was inhibited by 100 nM ω-agatoxin IVA, but not by 10 μM nifedipine, 10 μM diltiazem and 100 nM ωconotoxin GVIA. Calcium influx of the cortical nerve terminals of the right hemisphere was decreased on the 1st and 3rd days after the embolism. Noradrenaline release and uptake were also decreased on the 1st and 3rd days after the embolism. However, the percent release of noradrenaline was not altered. The results suggest that P-type channels are predominant in the cerebrocortical nerve terminals in rats and that calcium influx, noradrenaline release and uptake in the cerebrocortical nerve terminals are decreased by microsphere embolism. The decrease in noradrenaline release may be mainly due to a reduction in the activity of noradrenaline uptake in cerebrocortical nerve terminals of the microsphere-embolized rat.

Calcium channel blockers and transmitter release at the normal human neurornuscular junction

Transmitter release evoked by nerve stimulation is highly dependent on Ca2+ entry through voltage-activated plasma membrane channels. Calcium influx may be modified in some neuromuscular diseases like Lambert-Eaton syndrome and amyotrophic lateral sclerosis. We studied the pharmacologic sensitivity of the transmitter release process to different calcium channel blockers in normal human muscles and found that funnel web toxin and omega-Agatoxin-IVA, both P-type calcium channel blockers, blocked nerve-elicited muscle action potentials and inhibited evoked synaptic transmission. The transmitter release was not affected either by nitrendipine, an L-type channel blocker, or omega-Conotoxin-GVIA, an N-type channel blocker. The pharmacologic profile of neuromuscular transmission observed in normal human muscles indicates that P-like channels mediate transmitter release at the motor nerve terminals.

Inhibition of calcium channels in rat central and peripheral neurons by omega-conotoxin MVIIC

Inhibition of voltage-dependent calcium channels by omega-conotoxin MVIIC (omega-CTx-MVIIC) was studied in various types of rat neurons. When studied with 5 mM Ba2+ as charge carrier, omega-CTx-MVIIC block of N-type calcium channels in sympathetic neurons was potent, with half-block at 18 nM. Block of N-type channels had a rapid onset (tau approximately 1 sec at 1 microM omega-CTx-MVIIC) and quick reversibility (tau approximately 30 sec). The rate of block was proportional to toxin concentration, consistent with 1:1 binding of toxin to channels, with a rate constant (k on) of approximately 1 X 10(6) M-1. sec-1. Both potency and rate of block were reduced dramatically with increasing concentrations of extracellular Ba2+ omega-CTx-MVIIC also blocked P-type calcium channels in cerebellar Purkinje neurons, but both development and reversal of block were far slower than for N-type channels. The rate of block was proportional to toxin concentration, with k on -1.5 x 10(3) M-1. sec-1 at 5 mM Ba2+. From this value and an unblocking time constant of approximately 200 min, a dissociation constant of approximately 50 nM was estimated. Thus, block of P-type channels is potent but very slow. In hippocampal CA3 pyramidal neurons, omega-CTx-MVIIC blocked approximately 50% of the high-threshold calcium channel current; one component (approximately 20%) was blocked with the rapid kinetics expected for N-type channels, whereas the other component was blocked slowly. The component blocked slowly was reduced but not eliminated by preexposure to 200 nM or 1 microM omega-Aga-IVA.

Spatial and temporal aspects of Ca 2+ oscillations in Xenopus laevis melanotrope cells

Spatio-temporal aspects of Ca 2+ signaling in melanotrope cells of Xenopus laevis have been studied with confocal laser-scanning microscopy. In the whole-frame scanning mode, two major intracellular Ca 2+ compartments, the cytoplasm and the nucleus, were visualized. The basal [Ca 2+] in the nucleus appeared to be lower than that in the cytoplasm and Ca 2+ oscillations seemed to arise synchronously in both compartments. The N-type channel blocker ω-conotoxin eliminated oscillations in both regions, indicating a strong coupling between the two compartments with respect to Ca 2+ dynamics. Line-scanning mode, which gives higher time resolution, revealed that the rise phase of a Ca 2+ oscillation is not a continuous process but consists of 3 or 4 discrete steps. Each step can be seen as a Ca 2+-wave starting at the cell membrane and going through the cytoplasm at a speed of 33.3 ± 4.3 μm/s. Before the Ca 2+-wave enters the nucleus, a delay of 120.0 ± 24.1 ms occurred. In the nucleus, the speed of a wave was 80.0 ± 3.0 μm/s. Treatment with the Ca 2+-ATPase inhibitor thapsigargin (1 μM) almost completely eliminated the apparent difference in the basal [Ca 2+] in the cytoplasm and the nucleus, reduced the delay of a Ca 2+-wave before entering the nucleus to 79.8 ± 8.7 ms, and diminished the nuclear wave speed to 35.0 ± 4.9 μ/s. These results indicate that a cytoplasmic thapsigargin-sensitive ATPase near the nuclear envelope is involved in buffering Ca 2+ before the Ca 2+ wave enters the nucleus. After sensitizing IP 3 receptors by thimerosal (10 μM) the speed of the cytoplasmic Ca 2+-wave was increased to 70.3 ± 3.6 μ/s, suggesting that IP 3 receptors may be involved in the propagation of the cytoplasmic Ca 2+ wave. Our results indicate that in melanotropes the generation and propagation of Ca 2+ oscillations is a complex event involving influx of Ca 2+ through N-type Ca 2+ channels, propagation of the cytoplasmic Ca 2+ wave through mobilization of intracellular stores and a regulated Ca 2+ entry into the nucleus. We propose that Ca 2+-binding proteins may act as a Ca 2+ store for propagation of the wave in the nucleus.

Involvement of P-type calcium channels in high potassium-elicited release of neurotransmitters from rat brain slices

Several types of voltage-dependent calcium channels appear to occur in neurons, although coupling of the particular subtype of calcium channels to the release of neurotransmitter has not been clearly understood. We have examined the effects of subtype-specific inhibitors of the calcium channels on depolarization-induced release of endogenous neurotransmitters from brain slices. High potassium-induced release of glutamate and aspartate from hippocampal and striatal slices was almost completely inhibited by a P-type channel blocker, omega-agatoxin IVA. omega-Agatoxin IVA also completely inhibited the release of serotonin from the hippocampal slices with almost the same potency as in the case of glutamate, whereas the potency in blocking the release of serotonin and dopamine from striatal slices was lower than that from the hippocampal slices. Another calcium channel blocker, omega-agatoxin TK, that was recently found to block P-type channels with very similar selectivity and potency to omega-agatoxin IVA, also inhibited the release of amino acid transmitters and monoamines, though its potency was lower than that of omega-agatoxin IVA. An N-type channel blocker, omega-conotoxin GVIA, partially inhibited the neurotransmitter release, but an L-type channel blocker, nifedipine was ineffective. We propose that the activation of P-type calcium channels makes a major contribution to depolarization-elicited neurotransmitter release in the CNS and that multiple P-type channels sensitive to omega-agatoxin IVA and omega-agatoxin TK modulate the neurotransmitter release.

Antagonists of neuronal calcium channels: structure, function, and therapeutic implications.

This article reviews the structural and functional diversity of neuronal calcium channels and the therapeutic potential of antagonizing such channels. Through spatial and temporal control of intracellular calcium concentration, voltage-sensitive calcium channels regulate a host of neuronal processes, including neurotransmitter secretion, electrical activity, cytoskeletal function, cell metabolism and proliferation, and gene expression. Several genes elaborate a number of calcium channel isoforms or subtypes--each tailored to specific roles in neuronal function and possessing distinct biophysical properties, distribution, modulation, and pharmacological sensitivity. This diversity has raised the possibility that subtype-specific antagonists could provide novel treatments for some neuropathologies. In fact, neuroprotective and analgesic actions of N-type channel blockers in animals appear to confirm this supposition. These properties prompted human clinical studies evaluating these agents for prevention of neuronal degeneration following ischemic brain trauma and for relief of pain. Future medical applications for these blockers and antagonists of other channels subtypes are discussed.