Nimodipine Binding Research Articles

Invitro and invivo evaluation of hypothermia on pharmacokinetics and pharmacodynamics of nimodipine in rabbits.

ObjectiveTo investigate the effect of hypothermia on the pharmacokinetics and pharmacodynamics of nimodipine in rabbits using in vivo and in vitro methods.MethodsFive healthy New Zealand rabbits received a single dose of nimodipine (0.5 mg/kg) intravenously under normothermic and hypothermic conditions. Doppler ultrasound was used to monitor cerebral blood flow, vascular resistance, and heart rate. In vitro evaluations of protein binding, hepatocyte uptake and intrinsic clearance of liver microsomes at different temperatures were also conducted.ResultsPlasma concentrations of nimodipine were significantly higher in hypothermia than in normothermia. Nimodipine improved cerebral blood flow under both conditions, but had a longer effective duration during the hypothermic period. Low temperature decreased the intrinsic clearance of liver microsomes, with no change in protein binding or hepatocyte uptake of nimodipine.ConclusionNimodipine is eliminated at a slower rate during hypothermia than during normothermia, mainly due to the decreased activity of cytochrome P450 enzymes. This results in elevated system exposure with little enhancement in pharmacological effect.

ChemInform Abstract: Synthesis and Pharmacological Effects of Optically Active 2-(4-(4- Benzhydryl-1-piperazinyl)phenyl)ethyl Methyl 1,4-Dihydro-2,6-dimethyl- 4-(3-nitrophenyl)-3,5-pyridinedicarboxylate Hydrochloride.

Optically active 2-[4-(4-benzhydryl-1-piperazinyl)phenyl]ethyl methyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate [(S)-(+)-1 and (R)-(-)-1] hydrochlorides were synthesized with high optical purities from (R)-(-)- and (S)-(+)-1,4-dihydro-5-methoxycarbonyl-2,6-dimethyl-4-(3-nitrophenyl)- 3-pyridinecarboxylic acids [(R)-(-)-6 and (S)-(+)-6], which are available from (+/-)-6 by optical resolution using quinidine and cinchonidine, respectively. From pharmacological investigations of (S)-(+)-1 and (R)-(-)-1 such as the antihypertensive effect on spontaneously hypertensive rats and inhibition of [3H]nimodipine binding to rat cardiac membrane homogenate, the active form of 1 was defined to be the (4S)-(+)-enantiomer of 1.

Increase in receptor binding affinity for nimodipine in the rat brain with permanent occlusion of bilateral carotid arteries

The permanent occlusion of bilateral common carotid arteries (2VO) in rats has been shown to cause progressive and long-lasting cognitive deficits which may be due to impairment of memory retention and/or memory recall process. To clarify the function of voltage dependent calcium channels and the receptor binding of nimodipine by chronic cerebral ischemia, we examined specific (+)-[ 3H]PN 200-110 binding and the effect of oral administration of nimodipine in brain regions and hearts of rats, at 2 weeks to 4 months after permanent 2VO. There was no significant difference in either dissociation constant (Kd) or maximal number of binding sites (Bmax) for (+)-[ 3H]PN 200-110 in the cerebral cortex, hippocampus, corpus striatum and thalamus between 2VO and sham rats. In addition, in vitro inhibitory effect of nimodipine on cerebral cortical (+)-[ 3H]PN 200-110 binding in 2VO rats was similar to that in sham rats. Compared to control rats, oral administration of nimodipine to both 2VO and sham rats at 2 months after permanent 2VO brought about a significant increase in Kd values of specific (+)-[ 3H]PN 200-110 binding in the cerebral cortex, hippocampus, thalamus and myocardium, and the increase in Kd values was much larger in brain regions of 2VO rats than sham rats. However, the increase in Kd values in the myocardium did not differ between 2VO and sham rats. This observation suggests an increased in vivo binding affinity for nimodipine in chronic ischemic brain. In conclusion, the present study has shown that oral administration of nimodipine may cause a greater occupation in vivo of 1,4-dihydropyridine (DHP) calcium channel antagonist receptors in brains of permanent 2VO rats than in sham rats. Thus, nimodipine may be pharmacologically effective in preventing brain dysfunction due to cerebral ischemia in vivo.

Autoradiographic [3H]nimodipine distribution after experimental spinal cord injury in rats.

Because of its potential for augmentation of blood flow and protection of neurons after neurological insult, nimodipine has been investigated as a treatment of spinal cord injury (SCI). The results have been inconsistent, possibly because of poor delivery of nimodipine to the injured spinal cord. The following study was designed to determine the delivery of nimodipine to the injured spinal cord. It was also hoped that information about the temporal and spatial pattern of binding of nimodipine after SCI might further elucidate the relationship between calcium channel activation and injury. Fourteen female Wistar rats were divided into three groups: control (n = 3), 30 min post-SCI (n = 6); and 4 h post-SCI (n = 5). The injury was produced by acute clip compression for 1 min at T1. [3H]Nimodipine was administered 5 min after laminectomy in the control group, and at the above-specified times after injury in the SCI groups. The drug was then allowed to equilibrate for 30 min before the animals were killed. The spatial patterns and concentrations of [3H]nimodipine in various segments of the spinal cord were autoradiographically determined. The highest concentrations of [3H]nimodipine were at the injury site after SCI. Also, the mean [3H]nimodipine concentrations in all sites in each animal were higher in the injury groups than in the control group (p < 0.05). This study indicates that delivery of this agent to the injured cord is possible, and provides evidence of widespread Ca2+ channel activation in the first 4 h after injury.

Ischemic penumbra: the therapeutic window.

The concept of ischemic penumbra has extended our understanding of the focal ischemic process and in many ways has revitalized our research. Simply stated, the penumbra is the part of the brain that is sandwiched between brain regions committed to die and those that receive enough blood to communicate. Therefore, it is ischemic brain tissue that has just enough energy to survive for a short time but not enough to communicate and function. The life expectancy of the penumbra is short. Although the penumbra is an elegant concept, in practice it has been a difficult one to exploit. For one thing, it is unstable in both time and space. Depending on the severity and the duration of the focal ischemia, it may be anywhere in the ischemic brain. We believe that nimodipine binding experiments have taught us a great deal about the ischemic penumbra. Second, cells in the penumbra may die not by necrosis but by apoptosis. If that is true, then our concepts about the benign transient ischemic attack may need revision. Third, the penumbra may regain its ability to survive not only through reperfusion but also by interrupting the process of commitment to apoptosis.

Regional activation of L-type voltage-sensitive calcium channels in experimental thiamine deficiency.

During pyrithiamine-induced thiamine deficiency (PTD), specific regions of the brain develop histological damage. The basis of this selective vulnerability is unknown but the mechanism may involve a glutamate-mediated excitotoxic process in affected structures, leading to alterations in membrane potential and disturbances in calcium homeostasis. In this study, we have examined the volume of distribution of [3H]nimodipine, an L-type voltage-sensitive calcium channel (VSCC) antagonist, in the brain of the PTD rat. An increase in specific binding of [3H]nimodipine was detected only in the posterior thalamus at the symptomatic stage, immediately following the loss of righting reflexes (P < 0.0001). There was also an increase in nonspecific binding in the medial geniculate and inferior colliculi. Replenishment with thiamine at the symptomatic stage returned [3H]nimodipine binding to normal levels. These findings provide evidence that depolarization and activation of L-type VSCCs occur in the posterior thalamus and may contribute to the appearance of histological lesions in this structure during experimental thiamine deficiency.

Postischemic changes of

Sequential alterations of [3H]nimodipine and [3H]ryanodine binding in gerbils were investigated in selectively vulnerable regions, such as the striatum and hippocampus, 1 h to 7 days after 10 min of transient cerebral ischemia. [3H]Nimodipine binding showed no significant changes in the striatum and hippocampus up to 48 h after ischemia. Seven days after ischemia, however, a severe reduction in [3H]nimodipine binding was observed in the dorsolateral striatum, hippocampal CA1 (stratum oriens, stratum pyramidale and stratum radiatum) and hippocampal CA3 sector. On the other hand, [3H]ryanodine binding showed a significant increase in the hippocampus 1 h after ischemia. Five hours after ischemia, a significant reduction in [3H]ryanodine binding was observed only in the hippocampal CA1 sector. Thereafter, the striatum and hippocampus showed no significant alterations in [3H]ryanodine binding up to 48 h after ischemia. After 7 days, a marked reduction in [3H]ryanodine binding was observed in the striatum and hippocampus which were particularly vulnerable to ischemia. These results demonstrate that postischemic alteration in [3H]nimodipine and [3H]ryanodine binding is produced with different processes in the hippocampus. They also suggest that the mechanism for striatal cell damage caused by transient cerebral ischemia may, at least in part, differ from that for hippocampal neuronal damage. Furthermore, our findings suggest that abnormal calcium release from intracellular stores may play a pivotal role in the development of hippocampal neuronal damage. Copyright Rapid Science Ltd

Age-related changes in [3H]nimodipine and [3H]rolipram binding in the rat brain.

Ageing is associated with changes in neurotransmission which might be correlated with abnormal calcium metabolism. Because there is evidence that nimodipine can enhance the learning abilities of ageing animals and rolipram can enhance the excitability of neurons, providing a functional basis for cognition-enhancing activity, age-related alterations in the binding of voltage-dependent L-type calcium channels and calcium/calmodulin-independent cyclic adenosine monophosphate-selective phosphodiesterase (cyclic-AMP PDE) were studied in 3-week- and 6-, 12-, 18- and 24-month-old Fisher 344 rats by use of receptor autoradiography. [3H]Nimodipine and [3H]rolipram were used to label the voltage-dependent L-type calcium channels and calcium/calmodulin-independent cyclic-AMP PDE, respectively. [3H]Nimodipine binding showed no obvious change in all brain areas of 12- and 18-month-old rats, as compared with 6-month-old animals. In 24-month-old rats, however, [3H]nimodipine binding increased significantly in the striatum and hippocampal CA3 sector. In contrast, [3H]rolipram binding showed no significant change in most brain areas during ageing, except for a transient change only in the hippocampal CA1 sector of 12-month-old animals. [3H]Nimodipine and [3H]rolipram binding showed a significant increase in some brain areas of 3-week-old rats compared with 6-month-old animals. The results indicate that in rats voltage-dependent L-type calcium channels are more susceptible to ageing processes than calcium/calmodulin-independent cyclic-AMP PDE. Our data also demonstrate that voltage-dependent L-type calcium channels and calcium/calmodulin-independent cyclic-AMP PDE might play roles in developmental processes. These findings might help further elucidation of the relationship between age-related neurological deficits and behavioural pharmacology including cognitive function.

In Vitro Binding of [ 3H]Nimodipine and [ 3H]CGS-19755 to Rat Brain in Focal Cerebral Ischemia

Focal cerebral ischemia results in increased in vivo binding of calcium channel antagonists to both the L-type voltage sensitive calcium channel (VSCC) and the N-methyl- d-aspartate (NMDA) receptor-linked calcium channel. It was the aim of this study to investigate the effect of focal cerebral ischemia on the in vitro binding of calcium channel antagonists to rat brain. Quantitative autoradiography was used to measure regional in vitro binding of the L-type VSCC antagonist [ 3H]nimodipine and the competitive NMDA receptor antagonist [ 3H]CGS-19755 to rat brain following 4 h of irreversible focal cerebral ischemia. [ 3H]Nimodipine binding to the nonischemic hemisphere was characterized by one binding site with regional binding affinity ( K D ) estimates ranging from 221 to 482 p M and maximal binding site densities ( B max ) ranging from 13.2 (9.6-17.5) pmol/g tissue (estimate and 95% confidence interval) in CA1 to 32.5 (26.5-39.9) pmol/g tissue in dentate. [ 3H]CGS-19755 binding to the nonischemic hemisphere was characterized by K D estimates ranging from 59 to 97 n M and B max values ranging from 143 (108-192) pmol/g tissue in cortex to 569 (515-641) pmol/g tissue in CA1. For [ 3H]CGS-19755 a model of two binding sites was applicable in several brain regions. No difference in binding site densities or binding affinities between ischemic and paired nonischemic structures (cortex and striatum) was observed with either ligand. In vitro binding of [ 3H]nimodipine and [ 3H]CGS-19755 to ischemic brain failed to identify ischemic-induced changes in calcium channel function previously reported by in vivo binding methods.

In vivo binding of [3H]nimodipine in rat brain after transient forebrain ischemia.

We report the regional variation in relative in vivo binding of the L-type voltage sensitive calcium channel (VSCC) antagonist [3H]nimodipine to brain following transient forebrain ischemia in the rat. At 30-min of reperfusion after 20 min of forebrain ischemia, [3H]nimodipine binding was significantly increased in striatum, CA3 and CA4, and dentate relative to binding in sham-operated rats, suggesting that VSCCs were responding to ischemic depolarization. Two h following ischemia, binding in all brain structures returned to normal levels indicating repolarization of cell membranes. At 24 h of recirculation, increased [3H]nimodipine binding was again observed in striatum and dentate. Binding remained elevated in the striatum and dentate, and increased binding became evident in the CA1 region of the hippocampus after 48 h of reperfusion. With the exception of the dentate gyrus, the second rise in [3H]nimodipine binding anticipated or coincided with the observed regional ischemic cell changes. These observations in global cerebral ischemia support previous work indicating that in vivo binding of [3H]nimodipine to the L-type VSCC may be an early and sensitive indicator of impending ischemic injury. Such measurements may be of use in identifying vulnerable brain regions and defining a therapeutic window of opportunity in models of cerebral ischemia.

The interaction of the NK1 receptor antagonist CP‐96,345 with L‐type calcium channels and its functional consequences

1. We investigated the effects of the non-peptide NK1 receptor antagonist, CP-96,345, its inactive enantiomer CP-96,344, and the racemic mixture (+/-)-CP-96,345, on the binding of [3H]-nimodipine and [3H]-diltiazem to L-type calcium channels in rat cerebral cortex membranes. In isolated peripheral tissues containing tachykinin receptors, the effects of (+/-)-CP-96,345 have been compared with those of diltiazem. 2. In guinea-pig trachea, (+/-)-CP-96,345 produced antagonism of responses to the selective NK1 agonists [Sar9, Met(O2)11]SP and substance P-methyl ester that was apparently competitive in nature (pKB 7.0-7.5), while in guinea-pig ileum the antagonism was not surmountable. 3. The reduction of maximum responses by (+/-)-CP-96,345 in the guinea-pig ileum was not selective; it was obtained with muscarinic agonists and other agents, and was also observed in the portal vein of the rat where NK1 receptors are not present. 4. The tissue-specific reduction of maximum responses by (+/-)-CP-96,345 in ileum was reproduced by diltiazem. 5. (+/-)-CP-96,345 produced a concentration-dependent enhancement of [3H]-nimodipine binding to rat cerebral cortex membranes with a maximal stimulation of 186 +/- 29% above control (EC50 83.2 nM). Scatchard analysis revealed that (+/-)-CP-96,345 increased the affinity of [3H]-nimodipine for its binding sites without affecting Bmax (control: KD = 0.32 nM; with 100 nM (+/-)-CP-96,345: KD = 0.074 nM). 6. CP-96,345, CP-96,344, and the racemate all inhibited [3H]-diltiazem binding in rat cerebral cortex membranes with Ki values of 22.5 nM, 34.5 nM and 29.9 nM respectively; a similar value was obtained for diltiazem itself (33.6 nM). In comparison, CP-96,345 and ( +/- )-CP-96,345 inhibited the binding of[125I]-Bolton-Hunter-conjugated substance P in this tissue with Ki values of 59.6 nM and 82.0 nM respectively, while CP-96,344 had no measurable affinity (IC50> 10 microM).7. Substance P and a range of ligands selective for NK1, NK2, or NK3 receptors had no significant effect at 10 microM on either [3H]-diltiazem or [3H]-nimodipine binding.8. The results indicate that in addition to possessing affinity for the NK1 receptor, the non-peptide antagonist, CP-96,345, displays high affinity for [3H]-diltiazem binding sites on L-type calcium channels.The functional effect that may be observed in integrated models will be a consequence of either property, or be a composite effect of NK1 receptor antagonism and L-channel blockade.

Blockade by calmodulin inhibitors of Ca2+ channels in smooth muscle from rat vas deferens

1. Effects of three compounds which are used as calmodulin inhibitors (trifluoperazine, W-7 and calmidazolium) on Ca2+ channels were investigated in smooth muscle from rat vas deferens. 2. All three calmodulin inhibitors relaxed the smooth muscle precontracted by a high concentration of KCl (63.7 mM). The order of potency for the relaxation was trifluoperazine > W-7 > calmidazolium. 3. In binding studies using a microsomal fraction of vas deferens, all these calmodulin inhibitors displaced specific [3H]-nimodipine binding. Trifluoperazine and W-7 inhibited the binding at concentrations that relaxed the smooth muscle whereas calmidazolium inhibited at concentrations much lower than those necessary for muscle relaxation. 4. Ba2+ current flowing through voltage-gated Ca2+ channels was measured under whole-cell voltage-clamp conditions in isolated smooth muscle cells. The Ba2+ current was suppressed by the three calmodulin inhibitors in the concentration-range where inhibition of [3H]-nimodipine binding was observed. Neither voltage-dependence nor the inactivation time course of Ba2+ current were affected by these compounds. 5. The results suggest that the calmodulin inhibitors directly block Ca2+ channels in the smooth muscle cells. The channel inhibition by trifluoperazine and W-7, but perhaps not that by calmidazolium, may be responsible for the muscle relaxation observed with these compounds.

Reversibility of nimodipine binding to brain in transient cerebral ischemia.

Using autoradiography, we have measured the in vivo binding of [3H]nimodipine to brain in a rat model of reversible cerebral ischemia. Ischemia was induced by simultaneous occlusion of the middle cerebral artery (MCA) and ipsilateral common carotid artery by microaneurysm clips. Rats were studied after 15 min of ischemia (ischemic group) or after 45 min of reperfusion following 15 min of ischemia (reperfused group). Regional cerebral blood flow (CBF) was determined autoradiographically using [14C]iodoantipyrine in both ischemic (n = 6) and reperfused (n = 6) groups. During ischemia blood flow in the territory of the MCA was depressed and recovered to normal only in the distal territory of the MCA following reperfusion. [3H]Nimodipine binding in the ischemic group (n = 12) was elevated in ischemic brain regions and declined significantly (p < 0.01) in these regions in the reperfused group (n = 11). The ratio of the volume of cortex showing increased binding to the total volume of the forebrain was 0.113 +/- 0.025 (mean +/- SD) in the ischemic group and declined to 0.080 +/- 0.027 following reperfusion (p < 0.005). In general, infarct was only observed in regions showing persistent elevation of nimodipine binding following reperfusion as determined by histology performed in a separate group of rats (n = 8) after 24 h of reperfusion. We conclude that increased nimodipine binding to ischemic tissue is initially reversible with prompt reestablishment of CBF and is a sensitive indicator of early and reversible ischemia-induced cerebral dysfunction.

High-performance liquid chromatographic determination of the calcium channel blocker nimodipine in monkey plasma

A new high-performance liquid chromatographic (HPLC) assay was developed for the determination of nimodipine in monkey plasma. An ethyl acetate extraction procedure was employed with a reversed-phase HPLC separation for the analysis. Absolute recovery of nimodipine from plasma was over 95% with a lower limit of quantitation of 10 ng/ml. This method was applied to a preliminary pharmacokinetic study in which 0.25 mg/kg nimodipine was administered intravenously to three monkeys. Protein binding and stability of nimodipine in monkey plasma were also examined. The pharmacokinetic parameters of nimodipine in monkeys were similar to those obtained in humans and indicate that monkeys are an appropriate animal model for further pharmacokinetic investigations.

Multiple types of high-threshold calcium channels in rabbit sensory neurons: high-affinity block of neuronal L-type by nimodipine

Whole-cell and cell-attached patch recording have been used to characterize multiple types of voltage-dependent calcium channels in neurons freshly dispersed from rabbit dorsal root ganglia. In whole-cell patch recordings, high-threshold current, strongly resistant to inactivation by depolarized holding potentials (L-type; V1/2 = -27.2 mV), was potently inhibited by nimodipine. Assuming 1:1 binding, the dissociation constant for nimodipine binding to the inactivated state of the L-type calcium channel (KI) was 5.3 nM (n = 8). In contrast, a second type of high-threshold current less resistant to inactivation by depolarized holding potentials (N-type; V1/2 = -56.9 mV) was not blocked by nimodipine. Nimodipine-resistant N-type calcium current was inhibited by omega-conotoxin (5 microM). Cell-attached patch recordings of single calcium channel currents demonstrated the existence of three different unitary conductances; 7.4 pS, 13.1 pS, and 24.1 pS. The 24.1 pS high-threshold channel was enhanced by (-) BAY K 8644 and inhibited by nimodipine in a concentration- and voltage-dependent manner. Hyperpolarization reversed this block. These results demonstrate that, as in cardiac and smooth muscle, there is a component of neuronal high-threshold current corresponding to the L-type calcium channel that can be blocked with high affinity by nimodipine.

Molecular basis for the inhibition of 1,4-dihydropyridine calcium channel drugs binding to their receptors by a nonspecific site interaction mechanism

The "membrane bilayer" pathway (Rhodes, D. G., J. G. Sarmiento, and L. G. Herbette. 1985. Mol. Pharmacol. 27:612-623.) for 1,4-dihydropyridine calcium channel drug (DHP) binding to receptor sites in cardiac sarcolemmal membranes has been extended to include the interaction of amphiphiles within the lipid bilayer. These studies focused on the ability of the Class III antiarrhythmic agents bretylium and clofilium to nonspecifically inhibit DHP-receptor binding in canine cardiac sarcolemma. Clofilium was found to inhibit nimodipine binding with an inhibition constant of approximately 5 microM, whereas bretylium had no effect on nimodipine binding. Small angle x-ray diffraction was then used to examine the differential ability of these two Class III agents to inhibit DHP-receptor binding. The time-averaged locations of bretylium, clofilium, and nimodipine in bovine cardiac phosphatidylcholine (BCPC) bilayers (supplemented with 13 mol% cholesterol) were determined to a resolution of 9 A. The location of bretylium as dominated by its phenyl ring in BCPC bilayers was found to be at the hydrocarbon core/water interface, similar to that of the dihydropyridine ring of nimodipine. The location of clofilium as dominated by its phenyl ring was found to be below the hydrocarbon/core water interface within the hydrocarbon chain region of the bilayer, similar to that of the phenyl ring of nimodipine. The location of the dihydropyridine ring portion of nimodipine has previously been shown by neutron diffraction to be located at the hydrocarbon core/water interface of native sarcoplasmic reticulum, consistent with the small angle x-ray data from model membranes in this paper. Therefore, we speculate that the nonspecific inhibition arises from the interaction of clofilium's phenyl ring with the site on the calcium channel receptor where the phenyl ring portion of nimodipine must interact. The DHP-receptor binding pathway would then involve both nonspecific (membrane) and specific (protein) binding components, both of which are necessary for receptor binding.

In vivo Binding of Nimodipine in the Brain: I. The Effect of Focal Cerebral Ischemia

We report the regional variation in [3H]nimodipine binding in vivo during focal cerebral ischemia. After intravenous injection, 30 min of circulation of [3H]nimodipine was sufficient to establish a secular equilibrium of distribution in the brain. Rats sustained left middle cerebral and common carotid artery occlusions for 5 min, and 4, 24, and 48 h (n greater than or equal to 6 epr group). They were decapitated 30 min after injection of 250 microCi of [3H]nimodipine and their brains were submitted to autoradiography. The concentrations of [3H]nimodipine in plasma and brain structures, corrected for metabolism of nimodipine, were used to calculate the regional volumes of distribution (V) in the ischemic left (L) and control right (R) hemispheres. Log (VL/VR) was then defined as the group mean of the logarithms of the left-to-right ratio of V of [3H]nimodipine. In the lateral caudate, binding of [3H]nimodipine on the ischemic side was highest within 5 min of occlusion. Log (VL/VR) in this region for the combined sham-operated and normal control rats and those after 5 min and 4 and 24 h of ischemia were -0.014 +/- 0.025, 0.137 +/- 0.056*, -0.201 +/- 0.367, and -0.049 +/- 0.370 (mean +/- SD, *represents p less than 0.01 compared with controls). By contrast, in the superior frontal cortex, values for log (VL/VR) in the same sequence were -0.016 +/- 0.025, 0.028 +/- 0.056, 0.284 +/- 0.228*, and 0.224 +/- 0.069*, thus showing a significant rise in [3H]nimodipine binding only at 4 h.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo Binding of Nimodipine in the Brain: II. Binding Kinetics in Focal Cerebral Ischemia

We report the binding characteristics of [3H]nimodipine to normal and ischemic brain in vivo. We used the 1,4-dihydropyridine, nimodipine, to label the L-type voltage-sensitive calcium channel in focal cerebral ischemia after occlusion of both the middle cerebral and ipsilateral common carotid arteries in rats. Varying concentrations of [3H]nimodipine were infused 3.5 h after the onset of ischemia and circulated for 30 min before the brain was obtained for autoradiography and determination of regional nimodipine content. In separate sets of experiments, the metabolites of nimodipine were determined and the conditions for equilibrium of nimodipine distribution were established. Increased nimodipine uptake was observed in ischemic regions. This increased binding was saturable and specific with an affinity constant, KD, of 0.45 nM and a maximal regional binding capacity, Bmax, ranging from 3.1 to 10.9 pmol/g. Only binding to ischemic tissue was specific and saturable whereas that in nonischemic tissue was nonspecific. In vivo binding of nimodipine may be used to identify cell membrane depolarization and calcium channel activation in focal cerebral ischemia.

Reductions in Calcium Uptake Induced in Rat Brain Synaptosomes by Ionizing Radiation

Gamma irradiation (60Co) reduced KCl-stimulated voltage-dependent 45Ca2+ uptake in whole-brain, cortical, and striatal synaptosomes. The time course (3, 10, 30, and 60 s) of calcium uptake by irradiated (3 Gy) and nonirradiated synaptosomes, as well as the effect of KCl (15-65 mM), was measured in whole-brain synaptosomes. The fastest and highest rate of depolarization-dependent calcium uptake occurred at 3 s with 65 mM KCl. Irradiation reduced calcium uptake at all incubation times and KCl concentrations. Bay K 8644 enhancement of KCl-stimulated calcium influx was also reduced by radiation exposure. Nimodipine binding to dihydropyridine (DHP) L-type calcium channel receptors was not altered following radiation exposure. These results demonstrate an inhibitory effect of ionizing radiation on the voltage-sensitive calcium channels in rat brain synaptosomes that are not mediated by DHP receptors.

A pharmacological and biochemical examination of AHR-16462B, a novel calcium antagonist coronary vasodilator/antihypertensive

A series of pharmacological and biochemical studies were conducted to characterize AHR-16462B 4-[bis(4-fluorophenyl)methyl]-1-(2-naphthalenylmethyl)piperidine (HCI), a novel antagonist of voltage-sensitive calcium channels. AHR-16462B displaced [3H]-nimodipine binding from calcium channels on rabbit skeletal muscle membranes (IC50 = 118 nM) and inhibited depolarization-induced changes in intracellular free calcium in cultured PC-12 cells (IC50 = 125 nM), but had virtually no effects on binding of specific radioligands to 5-HT1a, 5-HT2, alpha1, alpha2, dopamine2, beta1, beta2, muscarinic M1, or histamine H1 receptors. AHR-16462B antagonized CaCI2-induced contractions of depolarized rabbit aortic strips (pA2 = 8.83), but had no negative chronotropic, and only slight negative inotropic effects on isolated guinea pig atria. In anesthetized dogs, cumulative doses of 0.1, 0.4, and 1.4 mg/kg, i. v., AHR-16462B decreased coronary vascular resistance and blood pressure; the magnitude and duration of these effects were dose related. Heart rate and Lead 2 ECG waveforms remained unaltered at all doses. In conscious spontaneously hypertensive rats (SHR), 3 to 100 mg/kg, p. o., AHR-16462B produced marked, long-lasting, and dose-related antihypertensive actions. Heart rate remained unaltered following 3, 10, and 30 mg/kg AHR-16462B, and bradycardia was evoked by 100 mg/kg. Effective antihypertensive doses of AHR-16462B had modest natriuretic and/or chloruretic actions.