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Results for “Calcium Channels, Q-Type”

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PubMed1997

Involvement of N- and P/Q- but not L- or T-type voltage-gated calcium channels in ischaemia-induced striatal dopamine release in vitro.

Calcium influx and transmitter efflux are central events in the neuropathological cascade that occurs during and following cerebral ischaemia. This study explored the role of voltage-gated calcium channels (VGCCs) in ischaemia-induced striatal dopamine (DA) release in vitro. Slices (350 microm thickness) of rat neostriatum were superfused (400 ml/h) with an artificial cerebrospinal fluid (aCSF) at 34 degrees C and subjected to episodes of 'ischaemia' by reduction of the glucose concentration from 4 to 2 mM and gassing with 95% N2/5% CO2. DA release was monitored with fast cyclic voltammetry at implanted carbon fibre microelectrodes. The time to onset, time to peak, rate and magnitude of DA release were measured. Non-selective blockade of VGCCs with a high concentration of Ni2+ (2.5 mM), markedly delayed (P < 0.01) and slowed (P < 0.05) DA release but preferential blockade of T-type VGCCs with a lower concentration (200 microM) had no effect. DA release was also unaffected by selective antagonism of L-type VGCCs with nimodipine and nicardipine (10 microM each). Selective blockade of N-type VGCCs with omega-conotoxin GVIA (100 nM) delayed DA release (P < 0.05) but did not affect its rate or magnitude. Blockade of P- and possibly Q-type VGCCs with omega-agatoxin IVA (up to 200 nM) both delayed (P < 0.05) and slowed (P < 0.05) DA release. Preferential blockade of P- type VGCCs with neomycin (500 microM) also delayed (P < 0.05) and slowed (P < 0.05) DA release. These findings suggest that N-, P- and possibly Q- but not L- or T-type VGCCs mediate ischaemia-induced DA release. Although it is not possible to say, on the basis of these results, that the effects are directly upon the dopamine terminals, these calcium channels nevertheless constitute promising targets for therapeutic intervention.

Animals↗

PubMed1997

Identification of calcium channels involved in neuronal injury in rat hippocampal slices subjected to oxygen and glucose deprivation.

The presynaptic Ca2+-influx affecting glutamate release during neuropathological processes is mediated via voltage-sensitive calcium channels (VSCCs). There is controversy, however, over the fractional contribution of the specific channel types involved. We have addressed this by investigating the protective effects of various VSCC blockers on oxygen and glucose-deprived rat hippocampal slices. The viability of treated and non-treated slices was assayed electrophysiologically by measuring the evoked population spike (PS) amplitude in the stratum pyramidale of the CA1 region and by imaging slices loaded with fluorochrome dyes specific for dead (ethidium homodimer) and live (calcein) cells using confocal microscopy. PS amplitudes were significantly (P < 0.01) depressed from 4.4 +/- 0.2 mV (n = 38) to 0.2 +/- 0.1 mV (n = 40) after the deprivation insult. Responses from deprived slices treated with omega-conotoxin MVIIC (100 nM; 4.2 +/- 0.5 mV; n = 20) were not significantly different from control, non-deprived slice responses. In contrast, deprived slices treated with either L-type (0.1 or 1 microM nimodipine) or N-type (0.1 or 3 microM omega-conotoxin MVIIA) blockers showed no significant protection. The viability of CA1 neurons as revealed by the fluorescence live/dead confocal viability assay was consistent with the electrophysiological measurements. By comparison with previous studies using P- and Q-type blockers to attempt neuroprotection against the same deprivation insult, the rank order in which specific Ca2+-channel types contribute to neuronal death due to oxygen and glucose deprivation was determined to be Q > N >> P > L.

Animals↗

PubMed1997

A novel type of calcium channel sensitive to omega-agatoxin-TK in cultured rat cerebral cortical neurons.

We characterized the electrophysiological properties of calcium channels in cultured rat cerebral cortical neurons using omega-agatoxin-TK (omega-Aga-TK) by a patch-clamp technique. Two types of slowly inactivating calcium channels sensitive to omega-Aga-TK were detected. The first type showed high sensitivity to omega-Aga-TK and low recovery from the omega-Aga-TK-induced blockade during washout, corresponding to the P-type channel. The second type showed low sensitivity to omega-Aga-TK and high recovery, resembling the Q-type channel, although it was distinct from the Q-type in terms of slower inactivation kinetics. We designate this channel as Q(L)-type (long-lasting Q channel). The omega-Aga-TK-sensitive calcium channels involved in the glutamatergic synaptic transmission were also divided into two types based on the sensitivity to omega-Aga-TK and reversibility of omega-Aga-TK-induced blockade. We conclude that the Q(L)-type is a novel type of channel, and that both P-type and Q(L)-type channels play a significant role in the cerebral cortical synaptic transmission.

Agatoxins↗

PubMed1997

R- and L-type Ca2+ channels are insensitive to eliprodil in rat cultured cerebellar granule neurons.

We have investigated, by using the whole-cell patch-clamp technique, the Ca2+ channel antagonist properties of eliprodil in cultured cerebellar granule cells which are known to express L-, N-, P- as well as Q- and R-type Ca2+ channels. Eliprodil maximally antagonized 50% of the voltage-dependent Ba2+ current with an IC50 of 4 microM. omega-Conotoxin-GVIA (3.2 microM) and omega-agatoxin-IVA (0.5 microM) blocked 28 and 43% of the current, respectively. When eliprodil (30 microM) was added to omega-conotoxin-GVIA or omega-agatoxin-IVA the magnitude of the maximal inhibition was identical to that obtained with eliprodil alone confirming a full blockade by eliprodil of N-, P- and Q-type Ca2+ channels. The L-type channel antagonist nimodipine (10 microM) blocked 24% of the current; this blockade was fully additive to that of eliprodil, indicating that the nimodipine-sensitive component of the current is eliprodil-insensitive. In the presence of eliprodil and nimodipine a residual Cd2+ sensitive current (25%), identified as the R-type current, remained unblocked. We conclude that in cerebellar granule neurons R- and L-type Ca2+ channels are insensitive to eliprodil. The nimodipine-sensitive channels present in cerebellar granule neurons may represent a neuronal subtype of L channels distinct from that (eliprodil-sensitive/nimodipine-sensitive) present in cortical or hippocampal neurons.

Animals↗

PubMed1997

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, omega-AgaIVA and the P/Q/N-type channel blocker, omega-conotoxin MVIIC. Transmission at inhibitory interneuronal synapses was not suppressed by these toxins, or by the N-type (omega-conotoxins GVIA and MVIIA) or L-type (nimodipine) channel blockers. Inhibitory transmission could be inhibited by Ni2+ and amiloride, but only at concentrations (IC50 approximately 300 microM) 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.

Animals↗

PubMed1997

Inhibition of Ca2+ channel current by mu- and kappa-opioid receptors coexpressed in Xenopus oocytes: desensitization dependence on Ca2+ channel alpha 1 subunits.

1. Desensitization of mu- and kappa-opioid receptor-mediated inhibition of voltage-dependent Ca2+ channels was studied in a Xenopus oocyte translation system. 2. In the oocytes coexpressing kappa-opioid receptors with N- or Q-type Ca2+ channel alpha 1 and beta subunits, the kappa-agonist, U50488H, inhibited both neuronal Ca2+ channel current responses in a pertussis toxin-sensitive manner and the inhibition was reduced by prolonged agonist exposure. 3. More than 10 min was required to halve the inhibition of Q-type channels by the kappa-agonist. However, the half-life for the inhibition of N-type channels was only 6 +/- 1 min. In addition, in the oocytes coexpressing mu-opioid receptors with N-type or Q-type channels, the uncoupling rate of the mu-receptor-mediated inhibition of N-channels was also faster than that of Q-type channels. 4. In the oocytes coexpressing both mu- and kappa-receptors with N-type channels, stimulation of either receptor resulted in a cross-desensitization of the subsequent response to the other agonist. Treatment of oocytes with either H-8 (100 microM), staurosporine (400 nM), okadaic acid (200 nM), phorbol myristate acetate (5 nM) or forskolin (50 microM) plus phosphodiesterase inhibitor did not affect either the desensitization or the agonist-evoked inhibition of Ca2+ channels. 5. These results suggest that the rate of rapid desensitization is dependent on the alpha 1 subtype of the neuronal Ca2+ channel, and that a common phosphorylation-independent mechanism underlies the heterologous desensitization between opioid receptor subtypes.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1996

Voltage-sensitive Ca2+ channels involved in nicotinic receptor-mediated [3H]dopamine release from rat striatal synaptosomes.

The potent nicotinic agonist anatoxin-a elicits mecamylamine-sensitive [3H]dopamine release from striatal synaptosomes, and this action is both Na+ and Ca2+ dependent and is blocked by Cd2+. This suggests that stimulation of presynaptic nicotinic receptors results in Na+ influx and local depolarisation that activates voltage-sensitive Ca2+ channels, which in turn provide the Ca2+ for exocytosis. Here we have investigated the subtypes of Ca2+ channels implicated in this mechanism. [3H]-Dopamine release evoked by anatoxin-a (1 microM) was partially blocked by 20 microM nifedipine, whereas KCl-evoked release was insensitive to the dihydropyridine. However, a 86Rb+ efflux assay of nicotinic receptor function suggested that nifedipine has a direct effect on the receptor, discrediting the involvement of L-type channels. The N-type Ca2+ channel blocker omega-conotoxin GVIA (1 microM) blocked anatoxin-a-evoked [3H]dopamine release by 60% but had no significant effect on 86Rb+ efflux; release evoked by both 15 and 25 mM KCl was inhibited by only 30%. The P-type channel blocker omega-agatoxin IVA (90 nM) also inhibited KCl-evoked release by approximately 30%, whereas anatoxin-a-evoked release was insensitive. The Q-type channel blocker omega-conotoxin MVIIC (1 microM) had no effect on either stimulus. These results suggest that presynaptic nicotinic receptors on striatal nerve terminals promote [3H]dopamine release by activation of N-type Ca2+ channels. In contrast, KCl-evoked [3H]dopamine release appears to involve both N-type and P-type channels.

Animals↗

PubMed1997

The amyloid beta-protein of Alzheimer's disease increases acetylcholinesterase expression by increasing intracellular calcium in embryonal carcinoma P19 cells.

One of the characteristic changes that occurs in Alzheimer's disease is the loss of acetylcholinesterase (AChE) from both cholinergic and noncholinergic neurons of the brain. However, AChE activity is increased around amyloid plaques. This increase in AChE may be of significance for therapeutic strategies using AChE inhibitors. The aim of this study was to examine the effect of amyloid beta-protein (A beta), the major component of amyloid plaques, on AChE expression. A beta peptides spanning residues 1-40 or 25-35 increased AChE activity in P19 embryonal carcinoma cells. A peptide containing a scrambled A beta(25-35) sequence did not stimulate AChE expression. To examine the possibility that the increase in AChE expression was mediated by an influx of calcium through voltage-dependent calcium channels (VDCCs), drugs acting on VDCCs were tested for their effects. Inhibitors of L-type VDCCs (diltiazem, nifedipine, and verapamil), but not N- or P- or Q-type VDCCs, resulted in a decrease in AChE expression. Agonists of L-type VDCCs (maitotoxin and S(-)-Bay K 8644) increased AChE expression. As L-type VDCCs are known to be modulated by cyclic AMP-dependent protein kinase, the effect of the adenylate cyclase activator forskolin was also examined. Forskolin stimulated AChE expression, an action that was blocked by the L-type VDCC antagonist nifedipine. The A beta(25-35)induced increase in AChE expression was mediated by an L-type VDCC, as the effect was also blocked by nifedipine. The results suggest that the increase in AChE expression around amyloid plaques could be due to a disturbance in calcium homeostasis involving the opening of L-type VDCCs.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1995

Regional difference of high voltage-activated Ca2+ channels in rat CNS neurones.

The diversity of high voltage-activated (HVA) Ca2+ channels in rat CNS neurones was investigated with the nystatin perforated patch recording configuration. The neurones were freshly dissociated from rat substantia nigra, ventromedial hypothalamus, tuberomammillary nucleus, nucleus tractus solitarius, hippocampal CA1 region and cerebellum. Five different types of HVA Ca2+ channels were distinguished pharmacologically; dihydropyridine sensitive L-type, omega-conotoxin-GVIA sensitive N-type, omega-agatoxin-IVA sensitive P-type, omega-conotoxin-MVIIC sensitive Q-type, and R-type which is insensitive to these organic Ca2+ antagonists. The results showed clearly that the five subtypes of HVA Ca2+ channels differ considerably in their distribution among various CNS regions.

Animals↗

PubMed1995

Two types of calcium channels sensitive to omega-agatoxin- TK in cultured rat hippocampal neurones.

We characterized the electrophysiological properties of calcium channels in cultured rat hippocampal neurones using omega-agatoxin-TK (omega-Aga-TK) and compared them with those of the P-type channel and the BI (alpha 1A) channel which resembles the Q-type channel. Two types of omega-Aga-TK-sensitive calcium channels were detected in hippocampal neurones. The first type showed slow inactivation, high sensitivity to omega-Aga-TK and low reversibility from omega-Aga-TK-induced block, resembling the P-type channel. The second type showed fast inactivation, low sensitivity to omega-Aga-TK and high reversibility from omega-Aga-TK-induced block. These results suggest that the second type of calcium channel (Q-type-like) plays a prominent role in the hippocampal synaptic transmission.

Agatoxins↗

PubMed1996

Opioid inhibition of Ca2+ channel subtypes in bovine chromaffin cells: selectivity of action and voltage-dependence.

Bovine chromaffin cells possess a mixture of high-voltage-activated Ca2+ channel subtypes: L-type, dihydropyridine-sensitive channels, and N-, P- and Q-types, omega-conotoxin MVIIC-sensitive channels. In these cells, we studied the reversible, naloxone-antagonized inhibition of Ba2+ currents by the opioid agonist met-enkephalin (IC50 = 272 nM). This inhibition could be resolved into a voltage-dependent and a voltage-independent component. The first was revealed by its slow Ba2+ current activation kinetics at 0 mV and by the current facilitation induced by short prepulses to +90 mV. The second was estimated as the residual inhibition persisting after the facilitation protocol. The two inhibitory components varied markedly from cell to cell and each contributed to about half of the total inhibition. Replacement of internal GTP by GDP-beta-S or cell pretreatment with pertussis toxin completely abolished the voltage-dependent inhibition by opioids, partially preserving the voltage-independent component. The opioid-induced inhibition was not selective for any Ca2+ channel subtype, being not prevented after the addition of specific Ca2+ channel antagonists. However, when separately analysing the contribution of each channel type to the voltage-dependent and voltage-independent modulation, a clear-cut distinction could be achieved. The voltage-independent inhibition was effective on all Ca2+ channel subtypes but predominantly on L-type Ca2+ channels. The voltage-dependent process was abolished by omega-conotoxin-MVIIC, but unaffected by nifedipine, and was thus sharply restricted to non-L-type channels (N-, P- and Q-types). Our data suggest a functionally distinct opioid receptor-mediated modulation of L- and non-L-type channels, i.e. of the two channel classes sharing major control of catecholamine secretion from bovine chromaffin cells.

Animals↗

PubMed1997

Multiple types of Ca2+ channels in mouse motor nerve terminals.

We measured neurotransmitter release and motor nerve terminal currents in mouse phrenic nerve-diaphragm and triangularis sterni preparations, to evaluate the role of Ca2+-channel subtypes in regulating transmitter release. Saturated concentrations of either omega-agatoxin IVA [omega-Aga-IVA (0.3 microM), a blocker of P-type Ca2+ channels] or omega-conotoxin MVIIC [omega-CTx-MVIIC (2 microM), a P- and Q-type Ca2+-channel blocker], inhibited nerve-evoked muscle contractions and the amplitude of endplate potentials respectively. In contrast, combined treatment with nifedipine (50 microM, a blocker of L-type Ca2+ channels) plus omega-conotoxin GVIA [omega-CTx-GVIA (2 microM), a blocker of N-type Ca2+ channels] did not elicit inhibitory effects on nerve-evoked muscle contractions, endplate potentials or nerve terminal waveforms. Because of the non-linear relationship between endplate potentials and Ca2+ signals, a small decrease in presynaptic Ca2+ entry can significantly reduce the amplitude of the endplate potential. Thus, we applied 3,4-diaminopyridine (3,4-DAP, a K+-channel blocker) or high Ca2+ (10 mM) to accelerate and amplify the endplate potentials and Ca2+ currents. The endplate potentials amplified by 3,4-DAP or by high Ca2+ correspondingly proved to be quite resistant to both omega-Aga-IVA and omgea-CTx-MVIIC; omega-Aga-IVA exerted only a partial inhibitory effect on endplate potentials, and the omega-Aga-IVA-resistant component was further inhibited by omega-CTx-MVIIC. The component that was resistant to the two toxins could be completely blocked by the non-selective Ca2+ channel blocker Cd2+ (300 microM). A combination of the two toxins had no significant effects on either spontaneous transmitter release or postsynaptic resting membrane potentials of the diaphragm preparation and the Na+ and K+ waveforms of the triangularis sterni preparations. This finding suggests a preferential inhibitory effect at a presynaptic site. Measuring the Ca2+ currents in the triangularis sterni also revealed partial inhibition by omega-CTx-MVIIC with further incomplete inhibition by omega-Aga-IVA. Cd2+ (300 microM) abolished the toxin-resistant component of the Ca2+ current. In contrast, a combination of nifedipine (50 microM) with omega-CTx-GVIA (2 microM) was without inhibitory effect. We conclude that multiple types of Ca2+ channels, i.e. omega-Aga-IVA-sensitive, omega-CTx-MVIIC-sensitive and toxin-resistant Ca2+ channels, coexist in mouse motor nerve terminals.

Animals↗

PubMed1997

Role of Q-type Ca2+ channels in vasopressin secretion from neurohypophysial terminals of the rat.

1. The nerve endings of rat neurohypophyses were acutely dissociated and a combination of pharmacological, biophysical and biochemical techniques was used to determine which classes of Ca2+ channels on these central nervous system (CNS) terminals contribute functionally to arginine vasopressin (AVP) and oxytocin (OT) secretion. 2. Purified neurohypophysial plasma membranes not only had a single high-affinity binding site for the N-channel-specific omega-conopeptide MVIIA, but also a distinct high-affinity site for another omega-conopeptide (MVIIC), which affects both N- and P/Q-channels. 3. Neurohypophysial terminals exhibited, besides L- and N-type currents, another component of the Ca2+ current that was only blocked by low concentrations of MVIIC or by high concentrations of omega-AgaIVA, a P/Q-channel-selective spider toxin. 4. This Ca2+ current component had pharmacological and biophysical properties similar to those described for the fast-inactivating form of the P/Q-channel class, suggesting that in the neurohypophysial terminals this current is mediated by a 'Q'-type channel. 5. Pharmacological additivity studies showed that this Q-component contributed to rises in intraterminal Ca2+ concentration ([Ca2+]i) in only half of the terminals tested. 6. Furthermore, the non-L- and non-N-component of Ca(2+)-dependent AVP release, but not OT release, was effectively abolished by the same blockers of Q-type current. 7. Thus Q-channels are present on a subset of the neurohypophysial terminals where, in combination with N- and L-channels, they control AVP but not OT peptide neurosecretion.

Animals↗

PubMed1997

Different contributions of L- and Q-type Ca2+ channels to Ca2+ signals and secretion in chromaffin cell subtypes.

In this study, we investigated the contribution of different subtypes of voltage-dependent Ca2+ channels to changes in cytosolic free Ca2+ ([Ca2+]i) and secretion in noradrenergic and adrenergic bovine chromaffin cells. In single immunocytochemically identified chromaffin cells, [Ca2+]i increased transiently during high K+ depolarization. Furnidipine and BAY K 8644, L-type Ca2+ channel blocker and activator, respectively, affected the [Ca2+]i rise more in noradrenergic than in adrenergic cells. In contrast, the Q-type Ca2+ channel blocker omega-conotoxin MVIIC inhibited the [Ca2+]i rise more in adrenergic cells. omega-Agatoxin IVA (30 nM), which blocks P-type Ca2+ channels, had little effect on the [Ca2+]i signal. The N-type Ca2+ channel blocker omega-conotoxin GVIA similarly inhibited the [Ca2+]i rise in both cell types. The effects of furnidipine, BAY K 8644, and omega-conotoxin MVIIC on K+-evoked norepinephrine and epinephrine release paralleled those effects on [Ca2+]i signals. However, omega-conotoxin GVIA and 30 nM omega-agatoxin IVA did not affect the secretion of either amine. The data suggest that, in the bovine adrenal medulla, the release of epinephrine and norepinephrine are preferentially controlled by Q- and L-type Ca2+ channels, respectively. P- and N-type Ca2+ channels do not seem to control the secretion of either catecholamine.

Animals↗

PubMed1989

Anoxia on slow inward currents of immature hippocampal neurons.

1. The effects of brief anoxia (2-4 min) on membrane currents--especially the tetrodotoxin (TTX)-insensitive, Cd2+-sensitive slow inward currents, presumed to be Ca2+ currents--were studied by single-electrode voltage clamp in CA1 and CA3 neurons in submerged hippocampal slices from adult and newborn Wistar rats (PN1-13). 2. In mature neurons, anoxia had no effect on Q-type inward relaxations, but slowly activating C-type outward currents were depressed. The most striking change was the suppression of Ca inward currents (especially the slowly inactivating L-type, by greater than 95%). This effect of anoxia was not sensitive to the N-methyl-D-aspartate (NMDA) receptor blocker, D-aminophosphonovalerate. Anoxia also reversibly abolished the NMDA-evoked inward current. 3. In neurons from newborn animals (PN1-6), Q-type inward relaxations and postanoxic outward currents were very small or undetectable. The slow inward (Ca) currents were smaller than in mature cells, but they showed a clearer separation between low-threshold, fast-inactivating and high-threshold, slowly inactivating currents. Both types of current were more resistant to anoxia (mean depression of L-type was by only 53.3 +/- 5.6%, mean +/- SE). 4. In such immature neurons, the NMDA-evoked inward currents were also more resistant to anoxia. 5. By PN7-13, increasing maturation was reflected in 1) larger voltage-dependent inward currents, 2) increasingly evident Q-type relaxations and postanoxic outward currents, and 3) near-complete blockade of inward currents by anoxia (at PN11-13, mean depression of L-type currents was by 98.5 +/- 1.5%).

Aging↗

PubMed1996

Pharmacological dissection of high-voltage-activated Ca2+ current types in acutely dissociated rat supraoptic magnocellular neurons.

1. We used pharmacological blockers and whole cell voltage-clamp recordings from acutely dissociated magnocellular neurons from the rat supraoptic nucleus to test for the presence of L-, N-, P-, Q-, and R-type calcium currents. 2. We found clear evidence for L-type (dihydropyridine sensitive), N-type (blocked by omega-conotoxin (GVIA), and P-type (blocked by omega-agatoxin IVA) currents. Nifedipine blocked 28 +/- 2% (mean +/- SE) of the current, omega-conotoxin GVIA blocked 26 +/- 4%, and omega-agatoxin IVA (25-30 nM) blocked 17 +/- 4%. In addition, omega-conotoxin MVIIC blocked about half (54 +/- 2%) of the current remaining after L-, N-, and P-type currents were blocked, suggesting the presence of Q-type currents. 3. About 20% of the whole cell current was unblocked by the combination of all four blockers, suggesting the presence of a fifth current type. This residual current inactivated slowly at -10 mV.

Animals↗

PubMed1997

Dihydropyridine- and neurotoxin-sensitive and -insensitive calcium currents in acutely dissociated neurons of the rat central amygdala.

The central amygdala (CeA) is an area involved in emotional learning and stress, and identification of Ca2+ currents is essential to understanding interneuronal communication through this nucleus. The purpose of this study was to separate and characterize dihydropyridine (DHP)- and neurotoxin-sensitive and -resistant components of the whole cell Ca2+ current (ICa) in acutely dissociated rat CeA neurons with the use of whole cell patch-clamp recording. Saturating concentrations of nimodipine (NIM, 5 microM), a DHP antagonist, blocked 22% of ICa: this NIM-sensitive (L-type) current was recorded in 68% of CeA neurons. The DHP agonist Bay K 8644 (5 microM) produced a 36% increase in ICa in a similar proportion of CeA neurons (70%). omega-Conotoxin GVIA (CgTx GVIA, 1 microM) in saturating concentrations inhibited 30% of ICa, whereas omega-agatoxin IVA (Aga IVA, 100 nM), in concentrations known to block P-type currents, did not affect ICa. Higher concentrations of Aga IVA (1 microM) alone reduced ICa by 34%, but in the presence of NIM (5 microM) and CgTx GVIA (1 microM) blocked only 18% of ICa. omega-Conotoxin MVIIC (CgTx MVIIC, 250 nM) reduced ICa by 13% in the presence of CgTx GVIA (1 microM). Application of NIM (5 mM), CgTx GVIA (1 microM); and Aga IVA (1 microM) blocked approximately 67% of ICa. A similar portion (63%) of Ca2+ current was blocked with CgTx MVIIC (250 nM) in the presence of NIM (5 microM) and CgTx GVIA (1 microM). The current resistant to NIM and the neurotoxins represented 37% of ICa, whereas in neurons not having L-type currents the resistant current made up approximately 53% of ICa (49 +/- 2%, mean +/- SE). The resistant current activated at around -40 mV and peaked at approximately 0 mV with half-activation and -inactivation potentials of -17 and -58 mV and slopes for activation and inactivation of -5 and 13 mV, respectively. The resistant current was sensitive to Cd2+ (IC50 = 2.5 microM) and Ni2+ (IC50 = 86 microM), was larger in Ca2+ than in Ba2+ (ratio = 1.31:1), and showed a moderate rate of decay. In summary, our results show that the high-voltage-activated calcium current in rat CeA neurons is composed of at least four pharmacologically distinct components: L-type current (NIM sensitive, 22%), N-type current (CgTx GVIA sensitive, 30%), Q-type current [Aga IVA (1 microM) and CgTx MVIIC sensitive, approximately 13-18%], and a resistant current (Non-L, -N, and -Q current, 33 approximately 37%), amounting to 37-53% of the total current. The resistant current has some electrophysiological and pharmacological characteristics in common with doe-1, alpha 1E, and R-type calcium currents, but remains unclassified.

Amygdala↗

PubMed1996

Pituitary adenylate cyclase-activating polypeptide causes rapid Ca2+ release from intracellular stores and long lasting Ca2+ influx mediated by Na+ influx-dependent membrane depolarization in bovine adrenal chromaffin cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to increase intracellular Ca2+ concentrations ([Ca2+]i) and catecholamine release in adrenal chromaffin cells. We measured [Ca2+]i with fura-2 and recorded ion currents and membrane potentials with the whole cell configuration of the patch-clamp technique to elucidate the mechanism of PACAP-induced [Ca2+]i increase in bovine adrenal chromaffin cells. PACAP caused [Ca2+]i to increase due to Ca2+ release and Ca2+ influx, and this was accompanied by membrane depolarization and inward currents. The Ca2+ release was suppressed by ryanodine, an inhibitor of caffeine-sensitive Ca2+ stores, but was unaffected by cinnarizine, an inhibitor of inositol trisphosphate-induced Ca2+ release. Ca2+ influx and inward currents were both inhibited by replacement of extracellular Na+, and Ca2+ influx was inhibited by nicardipine, an L-type Ca2+ channel blocker, or by staurosporine, a protein kinase C (PKC) inhibitor, but was unaffected by a combination of omega- conotoxin-GVIA, omega-agatoxin-IVA, and omega-conotoxin- MVIIC, blockers of N-, P-, and Q-type Ca2+ channels. Moreover, 1-oleoyl-2-acetyl-sn-glycerol, a PKC activator, induced inward currents and Ca2+ influx. These results indicate that PACAP causes both Ca2+ release, mainly from caffeine-sensitive Ca2+ stores, and Ca2+ influx via L-type Ca2+ channels activated by membrane depolarization that depends on PKC-mediated Na+ influx.

Adrenal Glands↗