Blockade by nifedipine of repeated restraint stress-induced delayed hyperactivity in rats

Male Wistar rats subjected to repeated restraint stress (1 h per day for 6 days) displayed increasing locomotor horizontal and vertical activity, measured for 1 h in an autotrack actometer 2 days after the last restraint. The administration of L-type Ca2+ channel blocker nifedipine (5 mg/kg i.p.) before each immobilization session completely prevented the stress-related augmentation of the locomotor activity. This shows that functional Ca2+ channels are required for development of stress-related motor activation and suggests that some effects of repeated stress may be prevented by application of Ca2+ channel blockade before stressful stimuli.     

Involvement of both L- and N-type voltage-dependent Ca2+ channels in KCl- and veratridine-evoked transmitter release from non-adrenergic, non-cholinergic nerves in the rabbit iris sphincter muscle

To determine which types of voltage-dependent Ca2+ channels mediate tachykinin release in the isolated rabbit iris sphincter muscle, we examined the effects of several Ca2+ channel modulators on contractions induced by either an elevation of the extracellular KCl concentration or application of the Na+ channel activator veratridine. Contractions caused by either 45.9 mM KCl or veratridine (10 microM) were inhibited by spantide (10 microM), a tachykinin receptor antagonist, and capsaicin (10 microM), a tachykinin-depleting agent, but were not changed by atropine. Nicardipine, an L-type Ca2+ channel blocker, inhibited contractions induced by KCl and veratridine in a concentration-dependent manner. omega-Conotoxin GVIA (1 microM), an N-type Ca2+ channel blocker, inhibited only contractions induced by lower concentrations of KCl, both when applied alone and when combined with nicardipine. Bay K 8644, an L-type Ca2+ channel activator, caused a spantide- and nicardipine-sensitive contraction in muscles partially depolarized with 15.9 mM KCl, and enhanced contractions induced by 15.9 mM KCl and veratridine (2 microM). omega-Agatoxin IVA (0.3 microM), a P-type voltage-dependent Ca2+ channel blocker, did not affect contractions induced by either KCl or veratridine. Contractions induced by exogenous substance P were not modified by any of the Ca2+ channel blockers or by Bay K 8644. These results suggest that, in the rabbit iris sphincter muscle. L- and N-type voltage-dependent Ca2+ channels are involved in neurotransmitter release from tachykininergic nerves elicited by high KCl and by veratridine.     

Effect of fantofarone, a new Ca2+ channel antagonist, on angioplasty-induced vasospasm in an atherosclerotic rabbit model

In order to prevent and treat angioplasty-induced vasospasm, we investigated the effects of a new Ca2+ channel antagonist, fantofarone, a nondihydropyridine compound with a novel site of action on the L-type Ca2+ channel, in an animal model of angioplasty in rabbits with femoral atherosclerotic lesions. Vasospasm which occurred in saline-treated animals following angioplasty was markedly reduced by fantofarone (50 microg/kg, i.v.) at both the distal and proximal sites. Although it totally inhibited distal vasospasm, isosorbide dinitrate (0.3 mg/kg, i.v.) did not significantly affect proximal diameter decrease. Verapamil (0.2 mg/kg, i.v.) was much less potent than fantofarone in reducing angioplasty-induced vasospasm. Our results confirm the preventive effects of Ca2+ blockers on this phenomenon and extend this observation to a potent compound: fantofarone.     

Neuronal synchronization and ionic mechanisms for propagation of excitation in the functional network of immortalized GT1-7 neurons: optical imaging with a voltage-sensitive dye

Immortalized gonadotropin releasing hormone (GnRH) neurons (GT1 cell line) in culture release GnRH in a pulsatile manner, suggesting that GT1 cells form a functional neuronal network. Optical imaging techniques and a voltage-sensitive fluorescent dye (RH795) were used to study the mechanism of neuronal synchronization and intercellular communication in cultured GT1-7 cells (one of the subclones of the GT1 cell line). The majority (79%) of GT1-7 cells in contact with one another revealed synchronized fluctuations in spontaneous neuronal activity. When a cell in contact with other cells was electrically stimulated, the evoked excitation was propagated to neighbouring cells. The ionic mechanisms involved in the propagation of electrical signals between interconnected GT1-7 cells were investigated using various blockers of Na+, Ca2+ and K+ channels. The propagation of stimulus-evoked excitation was prevented by the voltage-dependent Na+ channel blocker tetrodotoxin. It was also prevented by the voltage-dependent Ca2+ channel blockers, Ni+ (nonselective), nimodipine (L-type) and flunarizine (T-type > L-type), but not apparently affected by omega-agatoxin IVA (P- and Q-type) and omega-conotoxin MVIIA (N-type). The propagation was not influenced by the K+ channel blockers, quinine, tetraethylammonium and Ba2+, but in some cases, it was enhanced by 4-aminopyridine (4-AP) and prevented by apamin. These results suggest that voltage-dependent Na+ channels and L- and T-type Ca2+ channels are involved in the propagation of electrical signals in the GT1-7 neuronal network. Ionic mechanisms, through 4-AP- or apamin-sensitive K+ channels, also seem to be involved in the regulation of signal propagation. These mechanisms may underlie the functioning of the neuronal network formed by immortalized GnRH neurons.     

Mibefradil, A T-type calcium channel antagonist in Y1 cells

The effect of mibefradil, a new nondihydropyridine Ca2+ channel antagonist, was investigated on Y1 cells which exhibited T-and L-type Ca2+ currents. In the great majority of these cells, mibefradil rapidly and selectively blocked T-type Ca2+ current in a dose-dependent manner with a half maximum action at 10-7 M. Furthermore, the specific L-type Ca2+ channel inhibitor, nifedipine, blocked the Ca2+ inward current remaining after the action of mibefradil. Mibefradil does not modify the voltage-dependent characteristics of the current/voltage relationship. However, mibefradil is more effective at depolarized membrane potential.     

Skeletal muscle fiber degeneration in mdx mice induced by electrical stimulation

We present an in vitro model in which mouse skeletal muscle fibers undergo degeneration by increasing the current strength of tetanic stimulation. To understand the mechanisms of muscle fiber necrosis in Duchenne muscular dystrophy patients, the process of fiber degeneration was compared between mdx and control mice. The process consisted of four steps, beginning with muscle fiber contraction and extending to onset of myofibril disruption. The four processes were not observed in fibers in Krebs-HEPES (-Ca2+) buffer, nor in the presence of L-type Ca2+ channel blockers. These results suggest that this degenerative phenomenon is regulated by intracellular Ca2+, which moved into fibers mainly through voltage-dependent L-type Ca2+ channels. With the exception of myofibril disruption, mdx mice also exhibited the three other steps, but at a significantly lower current strength than in the fibers in the control mice. We postulate that excess Ca2+ flux occurs in fibers, mainly through abnormal L-type Ca2+ channels, and that the excessively accumulated calcium results in premature degeneration of the fibers by tetanic contraction. This study would provide a clue to investigate and prevent the degeneration processes in Duchenne muscular dystrophy.     

Modulation of neurotransmitter release by dihydropyridine-sensitive calcium channels involves tyrosine phosphorylation

Cultured rat cerebellar granule cells depolarized by high KCl, display a large component of Ca2+ influx through L-type voltage-dependent Ca2+ channels as defined by a sensitivity to 1 microM nifedipine. This Ca2+ influx is not coupled to neurotransmitter exocytosis but has implications for neuronal development. KCl stimulation in the absence of external Ca2+ followed by the readdition of Ca2+ allows the coupling of a class of L-type Ca2+ channels to neurotransmitter exocytosis as assessed by loading of glutamatergic pools with [3H]-D-aspartate. KCl stimulation in the absence of external Ca2+ ('predepolarization') enhances tyrosine phosphorylation of several cellular proteins, and inhibitors of tyrosine kinases block both phosphorylation and the neurotransmitter release coupled to the L-type Ca2+ channel. More specifically, an inhibitor of src family tyrosine kinases, PP1, blocks the effects of predepolarization suggesting a role for a src family kinase in the process. Furthermore, L-type Ca2+ channel recruitment and modulation of release could be activated with the tyrosine phosphatase inhibitor sodium orthovanadate. The phosphoproteins enhanced by predepolarization, which include the cytoskeletal proteins focal adhesion kinase (FAK) and vinculin, are also highly phosphorylated early on in culture when neurite outgrowth occurs. As the neurons develop a network of neurites, both tyrosine phosphorylation and L-type Ca2+ channel activity decrease. These results show a novel mechanism for the recruitment of L-type Ca2+ channels and their coupling to neurotransmitter release which involves tyrosine phosphorylation. This phenomenon has a role in cerebellar granule cell development.     

Effects of the immunosuppressant cyclosporin A on neurotransmitter release from peripheral non-adrenergic, non-cholinergic nerves

This study was undertaken to determine the effect of the immunosuppressant cyclosporin A on neurotransmitter release from non-adrenergic, non-cholinergic nerves (tachykininergic nerves) in the rabbit iris sphincter muscle. Cumulative application of cyclosporin A (0.1 to 10 microM) caused a slow onset of contraction in a concentration-dependent manner. Both FK888 (1 microM) and capsaicin (10 microM), a substance P receptor antagonist and a substance P-depleting agent, respectively, inhibited the contractile effect of cyclosporin A, whereas atropine (1 microM) had no effect. Both cyclosporin A and capsaicin (10 microM) stimulated the release of substance P-like immunoreactivity in the iris. Neither the sodium channel blocker tetrodotoxin (1 microM), the N-type voltage-dependent Ca2+ channel blocker omega-conotoxin GVIA (1 microM) nor the P-type channel blocker omega-agatoxin IVA (0.2 microM) affected cyclosporin A (1 microM)-induced contraction. In contrast, the L-type Ca2+ channel blocker nicardipine (10 microM) inhibited this contractile effect. These results suggest that cyclosporin A stimulates substance P-like tachykinin release by activating L-type voltage-dependent Ca2+ channels, resulting in contraction of the rabbit iris sphincter muscle.     

Anticonvulsant and adverse effects of MK-801, LY 235959, and GYKI 52466 in combination with Ca2+ channel inhibitors in mice

This study was designed to investigate the influence of the calcium (Ca2+) channel inhibitors nicardipine, nifedipine, and flunarizine on the protective action of MK-801, LY 235959 [N-methyl-D-aspartate (NMDA) receptor antagonists], and GYKI 52466 (a non-NMDA receptor antagonist) against electroconvulsions in mice. Unlike nicardipine (15 mg/kg) or flunarizine (10 mg/kg) nifedipine (7.5 and 15 mg/kg) potentiated the protective potency of MK-801 (0.05 mg/kg), as reflected by significant elevation of the convulsive threshold (a CS50 value of the current strength in mA producing tonic hind limb extension in 50% of the animals). The protective activity of LY 235959 and GYKI 52466 was reflected by their ED50 values in mg/kg, at which the drugs were expected to protect 50% of mice against maximal electroshock-induced tonic extension of the hind limbs. Nicardipine (3.75 15 mg/kg), nifedipine (0.94-15 mg/kg), and flunarizine (2.5-10 mg/kg) in a dose-dependent manner markedly potentiated the antiseizure efficacy of LY 235959. Flunarizine (5 and 10 mg/kg) was the only Ca2+ channel inhibitor to enhance the protective action of GYKI 52466 against electroconvulsions. Except with MK-801 + flunarizine (motor performance) or GYKI 52466 + flunarizine (long-term memory), combination of NMDA or non-NMDA receptor antagonists with Ca2+ channel inhibitors produced an impairment of motor performance (evaluated in the chimney test) and long-term memory acquisition (measured in the passive avoidance task) as compared with vehicle treatment.     

Parallel dose-response studies of the voltage-dependent Na+ channel antagonist BW619C89, and the voltage-dependent Ca2+ channel antagonist nimodipine, in rat transient focal cerebral ischaemia

We have compared two classes of putative neuroprotectants, the voltage-dependent Na+ channel antagonist BW619C87 [4-amino-2-(4-methyl-1-piperazinyl)-5-(2,3,5-trichlorophenyl) pyrimidine], and the voltage-dependent Ca2+ channel antagonist nimodipine, in a rat model of transient focal cerebral ischaemia. BW619C87 (10-50 mg/kg) or nimodipine (10-100 microg/kg) were injected intravenously 5 min before induction of 2 h transient focal cerebral ischaemia via intraluminal thread occlusion of the middle cerebral artery. BW619C87 was a potent neuroprotectant over the range tested, maximally reducing the volume of hemispheric ischaemic damage by 51% at the 50 mg/kg dose. Nimodipine maximally reduced ischaemic damage by 33% at the 50 microg/kg dose, although the maximal level of neuroprotection afforded by BW619C89 and nimodipine was not significantly different. This is the first study to compare these two classes of drug directly in a model of middle cerebral artery occlusion with reperfusion, and it supports the effectiveness of both as neuroprotectants.     

Bifurcated endovascular grafting for abdominal aortic aneurysm

Tottering mice inherit a recessive mutation of the calcium channel alpha1A subunit that causes ataxia, polyspike discharges, and intermittent dystonic episodes. The calcium channel alpha1A subunit gene encodes the pore-forming protein of P/Q-type voltage-dependent calcium channels and is predominantly expressed in cerebellar granule and Purkinje neurons with moderate expression in hippocampus and inferior colliculus. Because calcium misregulation likely underlies the tottering mouse phenotype, calcium channel blockers were tested for their ability to block the motor episodes. Pharmacologic agents that specifically block L-type voltage-dependent calcium channels, but not P/Q-type calcium channels, prevented the inducible dystonia of tottering mutant mice. Specifically, the dihydropyridines nimodipine, nifedipine, and nitrendipine, the benzothiazepine diltiazem, and the phenylalkylamine verapamil all prevented restraint-induced tottering mouse motor episodes. Conversely, the L-type calcium channel agonist Bay K8644 induced stereotypic tottering mouse dystonic at concentrations significantly below those required to induce seizures in control mice. In situ hybridization demonstrated that L-type calcium channel alpha1C subunit mRNA expression was up-regulated in the Purkinje cells of tottering mice. Radioligand binding with [3H]nitrendipine also revealed a significant increase in the density of L-type calcium channels in tottering mouse cerebellum. These data suggest that although a P/Q-type calcium channel mutation is the primary defect in tottering mice, L-type calcium channels may contribute to the generation of the intermittent dystonia observed in these mice. The susceptibility of L-type calcium channels to voltage-dependent facilitation may promote this abnormal motor phenotype.     

L-type Ca2+ channel is involved in the regulation of spontaneous transmitter release at developing neuromuscular synapses

Involvement of an L-type Ca2+ channel in the regulation of spontaneous transmitter release was studied in Xenopus nerve-muscle cultures. The frequency of spontaneous synaptic currents, which reflects impulse-independent acetylcholine release from the nerve terminals, showed a marked increase in high-K+ medium or after treatment with a phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate, a drug that activates protein kinase C and depolarizes the presynaptic neuron. The potentiation effect of high K+ and 12-O-tetradecanoyl-phorbol 13-acetate requires Ca2+ influx through the L-type Ca2+ channel in the plasma membrane, since it was significantly reduced by the presence of nifedipine, verapamil or diltiazem and enhanced by Bay K 8644, an L-type Ca2+ channel agonist. It was shown recently that adenosine 5'-triphosphate markedly potentiates the spontaneous acetylcholine release at these synapses through the binding of P2-purinoceptors and the activation of protein kinase C. We found in the present study that potentiation effects of adenosine 5'-triphosphate are inhibited by L-type Ca2+ channel blockers, suggesting that the L-type Ca2+ channel is responsible for the positive regulation of spontaneous acetylcholine secretion by adenosine 5'-triphosphate at the developing neuromuscular synapses. Our data suggest that modulation of the L-type Ca2+ channel in embryonic motor nerve terminals is important for the regulation of spontaneous transmitter release.     

Non-amphetaminic mechanism of stimulant locomotor effect of modafinil in mice

Previously established dose-response curves indicated that modafinil 20-40 mg/kg i.p. elicited in mice an obvious stimulation of locomotor activity roughly similar to that induced by (+)amphetamine 2-4 mg/kg. The effects of various agents modifying dopamine transmission were compared on the locomotor response to both drugs. The preferential D2 dopamine receptor antagonist haloperidol 37.5-150 micrograms/kg i.p. suppressed the stimulant effect of (+)amphetamine in a dose dependent manner, but not that of modafinil. The D1 dopamine receptor antagonist SCH 23390 (7.5-30 micrograms/kg s.c.) reversed the (+)amphetamine but not the modafinil induced hyperactivity. The tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine (200 mg/kg) suppressed the hyperactivity induced by 4 mg/kg dexamphetamine but not that induced by 20 mg/kg modafinil. Associating L-DOPA 150 mg/kg and benserazide 37.5 mg/kg with (+)amphetamine 2 mg/kg resulted in stereotyped climbing behavior, that was not observed with modafinil 10-80 mg/kg. The profound akinesia induced by reserpine (4 mg/kg s.c.; 5 h before testing) was reversed by (+)amphetamine 2 mg/kg but not by modafinil 40 mg/kg. Finally, on synaptosomes prepared from mouse striata preloaded with [3H]dopamine, modafinil 10(-5) M did not increase the spontaneous [3H]dopamine release whereas (+)amphetamine, at the same concentration, doubled it. From all these differences between the two drugs, it is concluded that the mechanism underlying the modafinil induced stimulant locomotor effect differs completely from that of (+)amphetamine.     

Specificity in the interaction of HVA Ca2+ channel types with Ca2+-dependent AHPs and firing behavior in neocortical pyramidal neurons

Intracellular recordings and organic and inorganic Ca2+ channel blockers were used in a neocortical brain slice preparation to test whether high-voltage-activated (HVA) Ca2+ channels are differentially coupled to Ca2+-dependent afterhyperpolarizations (AHPs) in sensorimotor neocortical pyramidal neurons. For the most part, spike repolarization was not Ca2+ dependent in these cells, although the final phase of repolarization (after the fast AHP) was sensitive to block of N-type current. Between 30 and 60% of the medium afterhyperpolarization (mAHP) and between approximately 80 and 90% of the slow AHP (sAHP) were Ca2+ dependent. Based on the effects of specific organic Ca2+ channel blockers (dihydropyridines, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC), the sAHP is coupled to N-, P-, and Q-type currents. P-type currents were coupled to the mAHP. L-type current was not involved in the generation of either AHP but (with other HVA currents) contributes to the inward currents that regulate interspike intervals during repetitive firing. These data suggest different functional consequences for modulation of Ca2+ current subtypes.     

Calcium oxalate crystal attachment to cultured kidney epithelial cell lines

Lamprey spinal neurons exhibit a fast afterhyperpolarization and a late afterhyperpolarization (AHP) which is due to the activation of apamin-sensitive SK Ca2+-dependent K+ channels (KCa) activated by calcium influx through voltage-dependent channels during the action potential (Hill et al. 1992, Neuroreport, 3, 943-945). In this study we have investigated which calcium channel subtypes are responsible for the activation of the KCa channels underlying the AHP. The effects of applying specific calcium channel blockers and agonists were analysed with regard to their effects on the AHP. Blockade of N-type calcium channels by omega-conotoxin GVIA resulted in a significant decrease in the amplitude of the AHP by 76.2+/-14.9% (mean +/- SD). Application of the P/Q-type calcium channel blocker omega-agatoxin IVA reduced the amplitude of the AHP by 20.3+/-10.4%. The amplitude of the AHP was unchanged during application of the L-type calcium channel antagonist nimodipine or the agonist (+/-)-BAY K 8644, as was the compound afterhyperpolarization after a train of 10 spikes at 100 Hz. The effects of calcium channel blockers were also tested on the spike frequency adaptation during a train of action potentials induced by a 100-200 ms depolarizing pulse. The N- and P/Q-type calcium channel antagonists decreased the spike frequency adaptation, whereas blockade of L-type channels had no effect. Thus in lamprey spinal cord motor- and interneurons, apamin-sensitive KCa channels underlying the AHP are activated primarily by calcium entering through N-type channels, and to a lesser extent through P/Q-type channels.     

Flanking proline residues identify the L-type Ca2+ channel binding site of calciseptine and FS2

Calciseptine and FS2 are 60-amino acid polypeptides, isolated from venom of the black mamba (Dendroaspis polylepis polylepis), that block voltage-dependent L-type Ca2+ channels. We predicted that these polypeptides contain an identical functional site between residues 43 and 46 by searching for proline residues that mark the flanks of protein-protein interaction sites [Kini, R. M., and Evans, H. J. (1966) FEBS Lett. 385, 81-86]. The predicted Ca2+ channel binding site also occurs in closely related toxins, C10S2C2 and S4C8. Therefore, it is likely that these toxins also will block L-type Ca2+ channels. To test the proposed binding site on calciseptine and FS2, an eight-residue peptide, named L-calchin (L-type calcium channel inhibitor), was synthesized and examined for biological activity. As expected for an L-type Ca2+ channel blocker, L-calchin reduced peak systolic and developed pressure in isolated rat heart Langendorff preparations without affecting diastolic pressure or heart rate. Furthermore, L-calchin caused a voltage-independent block of L-type Ca2+ channel currents in whole-cell patch-clamped rabbit ventricular myocytes. Thus the synthetic peptide exhibits the L-type Ca2+ channel blocking properties of the parent molecules, calciseptine and FS2, but with a lower potency. These results strongly support the identification of a site in calciseptine and FS2 that is important for binding to L-type Ca2+ channels and reinforce the importance of proline brackets flanking protein-protein interaction sites.     

Does dizocilpine (MK-801) inhibit the development of morphine-induced behavioural sensitization in rats?

Intermittent morphine pretreatment (10 mg/kg/day for 14 days) induced long-lasting (one month post-treatment) sensitization to the locomotor effects of morphine and amphetamine in rats. Co-administration of the non-competitive NMDA-receptor antagonist dizocilpine (MK-801) (0.1 mg/kg) with morphine did not prevent the development of long-term behavioural sensitization. However, this dose of MK-801 did cause long-term sensitization to its own locomotor effects. Co-administration of 0.25 mg/kg MK-801 with morphine caused death in 60% of the animals. In the animals that survived MK-801 plus morphine pretreatment, neither short-term (3 days) nor long-term morphine-induced sensitization was observed. MK-801 alone (0.25 mg/kg/day for 14 days) induced short-term cross-sensitization to morphine. Thus, the development of long-term morphine-induced locomotor sensitization could only be prevented by a dose of MK-801 that yields a lethal combination with morphine. In addition, MK-801 induced sensitization to its own locomotor effects and cross-sensitization to morphine. These findings seriously question whether MK-801 can be used to study the development of morphine-induced behavioural sensitization.     

Comprehensive study on the membrane currents of porcine granulosa cells in culture

The membrane currents of primary cultured porcine granulosa cells have been studied using the whole-cell configuration of the patch-clamp technique. And effects of K+ channel blockers upon progesterone production of the cells have been also studied. The author has identified and characterized two types of K+ currents, transient outward current (Ito) and a delayed rectifier K+ current (Ik), and Ca2+ current (Ica). Ito and Ik were voltage -and calcium-dependent. Both of the currents were blocked by 4-aminopyridine (4-AP), a K+ channel blocker, but only Ik was sensitive to tetraethylammonium (TEA), another K+ channel blocker. Ica was inactivated within 50 ms of the test pulse. Nifedipine and verapamil, L-type Ca2+ channel blockers, did not suppress Ica even at a concentration of 10 microM. Tetramethrin (1 microM), a T-type Ca2+ channel blocker, decreased Ica. These findings suggested that the current was T-type Ca2+ current. LH and dibutyryl cAMP, potent stimulants of steroid production, attenuated Ito by 13.9 +/- 1.8% (n = 7) and 21.0 +/- 1.5% (n = 4), respectively. However, they did not affect Ik and Ica. These results indicated that LH did not modulate Ca2+ current directly, but it suppressed Ito through cAMP. 4-AP (0.2-5 mM) suppressed basal and LH-induced progesterone production of porcine granulosa cells dose-dependently, but TEA (2-10 mM) did not influence progesterone production. These data suggest that Ito may play a role in steroid secretion or other functions in granulosa cells.     

Structure and expression of the human SM22alpha gene, assignment of the gene to chromosome 11, and repression of the promoter activity by cytosine DNA methylation

The pore-forming alpha 1 subunit of L-type calcium (Ca2+) channels is the molecular target of Ca2+ channel blockers such as phenylalkylamines (PAAs). Association and dissociation rates of (-)devapamil were compared for a highly PAA-sensitive L-type Ca2+ channel chimera (Lh) and various class A Ca2+ channel mutants. These mutants carry the high-affinity determinants of the PAA receptor site in a class A sequence environment. Apparent drug association and dissociation rate constants were significantly affected by the sequence environment (class A or L-type) of the PAA receptor site. Single point mutations affecting the high-affinity determinants in segments IVS6 of the PAA receptor site, introduced into a class A environment, reduced the apparent drug association rates. Mutation I1811M in transmembrane segment IVS6 (mutant AL25/-I) had the highest impact and decreased the apparent association rate for (-)devapamil by approximately 30-fold, suggesting that this pore-lining isoleucine in transmembrane segment IVS6 plays a key role in the formation of the PAA receptor site. In contrast, apparent drug dissociation rates of Ca2+ channels in the resting state were almost unaffected by point mutations of the PAA receptor site.     

The effect of collagenase and temperature on mitochondrial respiratory parameters in saponin-skinned cardiac fibers

OBJECTIVE: To determine the contribution of fast and slow inward channels to the electrocardiogram (ECG) of ventricular fibrillation. METHODS: Ventricular fibrillation was induced by endocardial electrical stimulation in pigs anaesthetised with pentobarbitone sodium (30 mg/kg intravenously). ECGs simultaneously recorded from the body surface (lead II) and from the endocardium were studied by power spectrum analysis (0-40 Hz). RESULTS: The mean (SEM) dominant frequency of fibrillation (9.0 (1.1) Hz in lead II at 0-40 s) did not change significantly with time in pigs given intravenous saline. However, the dominant frequency was significantly reduced by intravenous pretreatment with the class I antiarrhythmic drugs, lignocaine (3 mg/kg, 6.5 (0.5) Hz; 10 mg/kg, 4.2 (0.6) Hz), mexiletine (3 mg/kg, 6.2 (0.4) Hz; 10 mg/kg, 5.5 (0.4) Hz), and disopyramide (2.5 mg/kg, 5.4 (0.6) Hz). After flecainide (3 mg/kg, 6.9 (0.5) Hz) the reduction in frequency was not significant. Similar data were obtained with endocardial recordings. In contrast pre-treatment with verapamil (0.2 mg/kg, 11.7 (0.8) Hz; and 1.0 mg/kg, 12.9 (1.6) Hz) produced a significantly higher dominant frequency of fibrillation than saline and widened the bandwidth of frequencies around the dominant frequency. CONCLUSIONS: These results indicate that voltage-dependent sodium channel currents contribute to the rapid frequencies of ventricular fibrillation. Blockade of L-type inward calcium channel activity increases the fibrillation frequency and fractionates the frequencies of the fibrillation wavefronts.