Nimodipine Binding Research Articles

AHR-16303B, a Novel Antagonist of 5-HT2 Receptors and Voltage-Sensitive Calcium Channels

In vivo and in vitro methods were used to characterize AHR-16303B, a novel compound with antagonistic action at 5-HT2 receptors and voltage-sensitive calcium channels. The 5-HT2 receptor-antagonistic properties of AHR-16303B were demonstrated by inhibition of (a) [3H]ketanserin binding to rat cerebral cortical membranes (IC50 = 165 nM); (b) 5-hydroxytryptamine (5-HT)-induced foot edema in rats (minimum effective dose, (MED) = 0.32 mg/kg orally, p.o.); (c) 5-HT-induced vasopressor responses in spontaneously hypertensive rats (SHR) (ID50 = 0.18 mg/kg intravenously (i.v.), 1.8 mg/kg p.o.), (d) 5-HT-induced antidiuresis in rats (MED = 1 mg/kg p.o.), and (e) platelet aggregation induced by 5-HT + ADP (IC50 = 1.5 mM). The calcium antagonist properties of AHR-16303B were demonstrated by inhibition of (a) [3H]nimodipine binding to voltage-sensitive calcium channels on rabbit skeletal muscle membranes (IC50 = 15 nM), (b) KCl-stimulated calcium flux into cultured PC12 cells (IC50 = 81 nM), and (c) CaCl2-induced contractions of rabbit thoracic aortic strips (pA2 = 8.84). AHR-16303B had little or no effect on binding of radioligands to dopamine2 (DA2) alpha 1, alpha 2, H1, 5-HT1 alpha, beta 2, muscarinic M1, or sigma opioid receptors; had no effect on 5-HT3 receptor-mediated vagal bradycardia; and had only minor negative inotropic, chronotropic, and dromotropic effects on isolated guinea pig atria. In conscious SHR, 30 mg/kg p.o. AHR-16303B completely prevented the vasopressor responses to i.v. 5-HT, and decreased blood pressure (BP) by 24% 3 h after dosing.

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.

Nitrendipine decreases benzodiazepine withdrawal seizures but not the development of benzodiazepine tolerance or withdrawal signs

1. The effects of the calcium channel blocking agent, nitrendipine, were studied on seizures in mice produced during withdrawal from chronic benzodiazepine treatment and on the development of tolerance to benzodiazepines. 2. Nitrendipine produced a dose-dependent decrease in seizure incidence, when seizures were produced by the partial inverse agonist FG7142 during withdrawal from seven days treatment with flurazepam. 3. Nitrendipine did not raise the seizure thresholds in naïve mice to the full inverse agonist methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), or to the gamma-aminobutyric acid (GABA) antagonist, bicuculline. 4. When given concurrently with flurazepam for seven days, nitrendipine did not affect the incidence of seizures during flurazepam withdrawal. 5. When given concurrently with the benzodiazepines, nitrendipine did not prevent the development of tolerance to midazolam general anaesthesia or tolerance to the ataxic actions of flurazepam or midazolam. 6. Chronic treatment with flurazepam for seven days did not affect the Kd or Bmax of [3H]-nimodipine binding in mouse whole brain or cerebral cortex. 7. These results with benzodiazepines are partially in contrast with those for ethanol, where nitrendipine not only decreased ethanol withdrawal seizures when given acutely, but also prevented the development of tolerance and withdrawal signs when given concurrently with ethanol. However, they do confirm the selectivity of nitrendipine for withdrawal-induced seizures.

Interaction of 1,4 dihydropyridine calcium channel antagonists with biological membranes: Lipid bilayer partitioning could occur before drug binding to receptors

The binding of dihydropyridine calcium channel agonists and antagonists to receptors in cardiac sarcolemmal membranes is a complex reaction that may involve an interaction with the lipid bilayer matrix of the sarcolemma. Membrane/buffer partition coefficients (λ) for three dihydropyridine calcium channel antagonists were measured directly in the sarcolemma and sarcoplasmic reticulum membranes and found to be in the range of 5000 to 150 000. These drugs interact primarily with the membrane bilayer component of these membranes but may also bind to nonreceptor proteins. The intrinsic forward rate constants for dihydropyridine binding to sarcolemmal calcium channel receptors were apparently not strongly dependent on their membrane partition coefficients. For example, nimodipine (λ = 6300) had a forward rate constant of 6.8 ± 0.6 × 10 6/ m/s, whereas the forward rate constant for Bay P 8857 (λ = 149 000) was 1.4 ± 0.8 × 10 7/ m/s. Model calculations for this binding reaction demonstrated that since these drugs are highly lipid soluble, the dependence of these rates on lipid solubility would probably not be reflected in the experimental forward rate constants. In addition, the intrinsic forward rate constant for nimodipine binding to sarcolemmal calcium channel receptors was found not to be linearly dependent on the viscosity of the buffer medium over a five-fold range. The rate of nonspecific (non-receptor protein) drug binding to highly purified sarcoplasmic reticulum membranes essentially devoid of specific receptors for these drugs appears to be extremely fast, at least 10 3 times faster than specific drug binding to the receptor in the sarcolemma. Thus, it appears that partitioning into the lipid bilayer matrix of the sarcolemma could be a general property of dihydropyridine calcium channel antagonists and may be a prerequisite for their binding to sarcolemmal membrane receptors.

In vivo morphine decreases [3H]nimodipine receptor binding in rat brain regions.

The in vivo effect of the mu agonist morphine and antagonist naloxone on [3H]nimodipine receptor binding in rat brain regions has been investigated. Morphine administration (15 mg/s.c.) for thirty minutes produced a 19% decrease in [3H]nimodipine receptor binding (Bmax 158.2 fmol to 128.9 fmol) in cortex and 29% decrease in cerebellum (65.3 fmol to 46.0 fmol). Lesser changes were observed in hippocampal and striatal regions with no changes in hypothalamus and brain stem. All effects were completely antagonized by naloxone pretreatment (1 mg/kg). The studies suggest that opiates in vivo can alter [3H]nimodipine binding to the Ca2+ channel receptor protein. These findings agree with the previously observed decreases in Ca2+ influx in nerve ending preparations and inhibition of ICa2+ following opiate treatment and suggest opiates reduce Ca2+-dependent neurotransmitter release by altering the Ca2+ channel receptor protein in an allosteric fashion.

Action of pyrethroids on K +-stimulated calcium uptake by, and [ 3H]nimodipine binding to, rat brain synaptosomes

The effects of pyrethroids were studied on K +-stimulated 45Ca 2+ uptake by rat brain synaptosomes. This uptake had low affinity for the inhibitors of voltage-dependent Ca 2+ channels (verapamil, diltiazem, nimodipine, and nifedipine) but was potently inhibited by 2′-4′-dichlorobenzamil (DCB). The characteristics of 45Ca 2+ uptake, measured in the absence of any added ATP, suggested that most of it was a result of the activity of the Na +-Ca 2+ exchanger in these membranes. The pyrethroids were more potent inhibitors of this K +-stimulated 45Ca 2+ uptake than even the “specific” inhibitor DCB. The seven type II pyrethroids (containing α-cyano-3-phenoxybenzyl alcohol) tested (with average IC 50 of 11 μ M) were more potent inhibitors of this 45Ca 2+ uptake than the seven type I pyrethroids (which do not contain an α-cyano substituent). Both toxic and nontoxic cypermethrin isomers inhibited the 45Ca 2+ uptake with similar potencies. Both types of pyrethroids also inhibited voltage-dependent Ca 2+ channels in the membrane, which were detected by their specific binding of [ 3H]nimodipine with the following order of decreasing potencies: pyrethrins>cypermethrin>cyfluthrin>deltamethrin = resmethrin>tetramethrin> S-bioallethrin> allethrin = permethrin>flucythrinate>bioallethrin>fenvalerate = fluvalinate ⪢ tralomethrin. The relatively low potencies of pyrethroids on the K +-stimulated 45Ca 2+ uptake and [ 3H]nimodipine binding, the poor stereospecificity of pyrethroid action, and the poor correlation with their toxicities suggest that neither the Na +-Ca 2+ exchanger nor the voltage-dependent Ca 2+ channel are primary targets for pyrethroid toxicity.

The effect of κ agonist U50-488H on [ 3H]nimodipine receptor binding in rat brain regions

The effects of a κ opiate agonist have been evaluated on [ 3H]nimodipine binding to dihydropyridine receptors for ‘L’-type Ca 2+ channels in rat brain regions. Administration of U50-488H (trans-(±)-3,4-dichloro-N-methyl-N-[2-(1,- pyrolidinyl)-cyclohexyl]benzeneacetamide) produced a 28% decrease in B max in coxtex and a 23% decrease in cerebellum. No changes were seen in the affinity (K d) for [ 3H]nimodipine binding sites. Slight changes in hippocampal and striatal binding capacities were observed with no changes seen in hypothalamus and brainstem. The κ antagonist MR2266 effectively reversed in vivo all changes in [ 3H]nimodipine binding without producing any effect alone. These studies suggest that κ opiate receptors may be directly coupled to L-type calcium channels as evidenced by [ 3H]nimodipine binding studies and may account for the findings that κ opiate agonists inhibit neurotransmitter release by allosterically interferring with the Ca 2+ channel protein in brain membranes.

Pharmacology of nimodipine. A review.

The major pharmacological findings with nimodipine reviewed in this chapter are summarized in TABLE 3. On the basis of these findings, the following conclusions appear to be justified: 1. Nimodipine is a 1,4-dihydropyridine with Ca2+ channel antagonist properties. It is more lipophilic than nifedipine and its distribution volume in the brain of rats is higher than that of nifedipine. 2. Nimodipine dilates cerebral vessels at considerably lower concentrations than required for dilatation of peripheral blood vessels. It can, therefore, improve cerebral blood flow at doses that do not reduce systemic arterial pressure. 3. Nimodipine inhibits 45Ca uptake into vascular smooth muscle and neuronal cells. 4. Nimodipine antagonized postischemic cerebral hypoperfusion in cats and prolonged life of stroke-prone spontaneously hypertensive (SH) rats at doses that have little if any effect on arterial blood pressure. 5. Nimodipine reduced neurological deficits in dogs and monkeys with global cerebral ischemia. In focal ischemia (MCA occlusion) nimodipine reduced infarct size and neurological deficits and normalized intracellular brain pH. 6. In addition to its cerebral vasodilator effect, nimodipine appears to have a direct neuronal action. The suggested evidence for the neuronal site of action of nimodipine includes: a. Presence of nimodipine binding sites in brain. b. Blockade by nimodipine of Ca2+ channels in single nerve cells and in endocrine cells under conditions of sustained depolarization. c. Interactions with centrally acting drugs. d. Effects on release of various neurotransmitters from neuronal tissue or endocrine cells. e. Demonstration of anticonvulsant action of nimodipine. f. Blockade of behavioral effects of Ca2+ channel agonists by calcium channel antagonists.

Morphine Treatment Increases [3H]Nimodipine Binding Sites in Rat Brain

Morphine Treatment Increases [³H]Nimodipine Binding Sites in Rat Brain

Characteristics of [3H]Nimodipine Binding to Sarcolemmal Membranes from Rat Vas Deferens and Its Regulation by Guanine Nucleotide

The binding properties of a 1,4-dihydropyridine (DHP) calcium entry blocker, [3H]nimodipine, to a microsomal fraction from rat vas deferens was characterized. The specific binding was saturable, rapid and reversible. Scatchard analysis of the binding revealed a single binding site, and the dissociation constant and the maximum number of binding sites were 0.31±0.02 nM and 97.0±7.19 fmol/mg protein, respectively. Both the Kd value obtained from the kinetic study and the IC50 value from relaxation of the K+-depolarized organ were approximately 0.4 nM, indicating that the binding site is closely related to the functional Ca2+ channel. The specific [3H]nimodipine binding was displaced by DHP derivatives at low concentration and by verapamil at high concentration, but diltiazem had no effect on the binding. Calcium chelating agents decreased the [3H]nimodipine binding which was restored by adding Ca2+. 5’-Guanylylimidodiphosphate caused a rightward shift of the displacement curve for Bay K 8644 but not for nimodipine, suggesting the involvement of guanine nucleotide binding protein in the signal transduction between the DHP binding site and the Ca2+ channel.

Prolonged morphine treatment increases rat brain dihydropyridine binding sites: possible involvement in development of morphine dependence

Regulation of L-type Ca 2+ channels by morphine in rat brain was determined by the binding of [ 3H]nimodipine. Morphine, administered by subcutaneous pellet implantation, increased the density of [ 3H]nimodipine binding sites in a time- and dose-dependent manner and this effect was reversible upon removal of the pellets. Increases in these dihydropyridine siteswere localized to the cortex, hippocampus, hypothalamus and brainstem but not to the cerebellum and striatum. Additional experiments were performed to test the ability of different Ca 2+ channel antagonists to affect naloxone-precipitated withdrawal in morphine-dependent mice and rats. These drugs effectively reduced the incidence of naloxone-induced jumping in mice and several of the withdrawal signs in rats. Taken together, our study underscores the plasticity of brain L-type Ca 2+ channels and suggests that their upregulation might contribute to morphine dependence.

Regional distribution of calcium channel ligand (1,4-dihydropyridine) binding sites and 45Ca 2+ uptake processes in rat brain

The binding of nimodipine, a 1,4-dihydropyridine Ca 2+ channel antagonist, and of Bay K 8644, a Ca 2+ channel activator, was measured in several regions of rat brain and compared to the distribution of K + depolarization-induced 45Ca 2+ uptake into synaptosomes. The maximum binding densities ( B max) of [ 3H]nimodipine and [ 3H]Bay K 8644 were not significantly different one from the other, but differed according to brain region with binding being highest in the olfactory bulb and hippocampus, intermediate in the caudate nucleus and cerebral cortex (various regions), and lowest in the cerebellum [563 to 107 fmol/mg protein (mean)]. The K D values, [ 3 H] nimodipine = 1.8 × 10 −10 M (mean) and [ 3 H] Bay K 8644 = 1.4 × 10 −9 M ( mean) , did not differ according to region. Depolarizationinduced 45Ca 2+ uptake in synaptosomes occurred as fast (1 sec) and slow (10 sec) components distinguished by their selective occurrence in choline-containing and pre-depolarized preparations respectively. Distribution of the fast component of uptake paralleled that of [ 3H]nimodipine binding, being least in the cerebellum and greatest in the hippocampus and cortex, but the magnitude of the slow phase of 45Ca 2+ uptake did not vary in the three brain regions studied.

A novel kappa agonist inhibits [ 3H]nimodipine binding to a Ca ++ channel receptor protein in rat brain

The novel kappa agonist U50-488H in vitro produced a concentration-dependent decrease (0.25 – 25 μM) in [ 3H]nimodipine binding in neuronal P 2 fraction from rat brain cortex. Kinetic analysis indicates the decrease in binding results from a reduced B max with no change in affinity (K d). The kappa antagonist, MR2266, blocked the decrease in [ 3H]nimodipine binding to membrane fractions. At equimolar concentrations (25 μM), morphine in vitro had no effect on [ 3H]nimodipinebinding, while U50-488H demonstrated potent inhibition. Further kinetic analysis indicates that the IC 50 for U50-488H is (0.5 – 0.7 μM) and a Dixon plot for K I was found to be 1.5 – 1.7 μM. These results suggest that kappa opiate receptors may be coupled to dihydropyridine receptors and as a result modulate Ca ++ entry and neurotransmitter release in brain neurons.

Effects of Ca2+ Channel Blockers on Ca2+ Translocation Across Synaptosomal Membranes

The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.

Solubilization of the bovine cardiac sarcolemmal binding sites for calcium channel blockers.

Nonionic and ionic detergents were used to solubilize the bovine cardiac sarcolemmal binding sites for nimodipine and (-)desmethoxyverapamil in the absence of added ligand. Only Chaps, digitonin and sucrose monolauryl ester were able to solubilize the binding sites in a form that bound radioligands. About 45% of each of the membrane-bound high-affinity site was solubilized by 0.4% Chaps (w/v) in the presence of 48% (w/v) glycerol. The solubilized binding sites were destroyed by trypsin or by a 10-min incubation at 50 degrees C. Calcium stimulated nimodipine binding slightly at 0.3 mM and inhibited (-)desmethoxyverapamil binding completely with an IC50 of 1.2 mM. Nimodipine binding was reduced by 20% in the presence of EGTA. The solubilized receptors sedimented in sucrose density gradients with an apparent s20,w of 21 S. An identical sedimentation value was obtained for the cardiac sarcolemmal and skeletal transverse tubulus receptor which were prelabeled with nitrendipine and solubilized by digitonin. Solubilization reduced the affinity of nimodipine for its high-affinity site slightly from 0.35 nM to 1.2 nM and that for its low-affinity site from 33 nM to 130 nM. Solubilization did not affect significantly the specific density of these sites. Binding of nimodipine to the low-affinity site was completely abolished by 0.1 microM nitrobenzylthioinosine. After solubilization only the high-affinity site for (-)desmethoxyverapamil could be measured with tenfold reduced affinity (Kd = 15.3 nM) but unchanged specific density. Binding to the solubilized high-affinity site for nimodipine and (-)desmethoxyverapamil was stereospecific and showed a similar rank order as the particulate binding sites. Binding of nimodipine was inhibited allosterically by phenylalkylamines. Similarly, (+)PN200-110 inhibited allosterically (-)desmethoxyverapamil binding. d-cis-Diltiazem stimulated nimodipine binding at 20 degrees C 1.2-fold, reduced the dissociation rate from 0.018 min-1 to 0.0083 min-1 and had no effect on the association rate (0.173 min-1. nM-1). The Kd calculated from the rate constants was 0.1 nM and in close agreement with the value of 0.49 nM measured under equilibrium conditions in the presence of nitrobenzylthioinosine. In contrast, desmethoxyverapamil increased the dissociation rate of nimodipine to 0.03 min-1. The association and dissociation rate constants for (-)desmethoxyverapamil were 0.024 min-1. nM-1 and 0.025 min-1, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Nimodipine levels in gerbil brain following parenteral drug administration

Nimodipine binding to the particulate fraction of gerbil brain homogenate was characterized using tritiated (3H)-nimodipine as the radioactive ligand. Binding was monophasic and saturable, with the apparent affinity constant (KD) = 0.4 nM and the maximum number of binding sites (Bmax) = 12 nmol/kg wet wt. A competitive binding assay was validated for the measurement of nimodipine using gerbil brain as the source of receptors for the drug. Binding characteristics were sufficiently similar in specimens from different animals to allow the use of homogenates from individual animals as the source of both membrane-binding sites and competing ligand. Nimodipine could be detected in the brains of animals sacrificed soon after drug injection, and reached a peak level within 15 minutes. Brain drug level at a given time was a linear function of dose administered. One hour after a 1-mg/kg dose, the level of drug measured in brain was approximately 100 nmol/kg wet wt, more that 200 times the KD. Sufficient drug to mediate a maximal pharmacological effect accumulated in brain even after a dose of only 0.25 mg/kg. Thus, in this species, effective tissue nimodipine levels may be achieved at doses which minimize the risk of systemic hypotension.

Human red-blood-cell Ca2+-antagonist binding sites. Evidence for an unusual receptor coupled to the nucleoside transporter.

The human red blood cell ghost Ca2+-antagonist binding sites were characterized with (+/-)-[3H]nimodipine. The labelled 1,4-dihydropyridine bound in a non-cooperative, reversible manner with a Kd of 52 nM at 25 degrees C to 9.65 pmol sites/mg ghost protein. The stereochemistry of the binding domain was evaluated with the optically pure enantiomers of chiral 1,4-dihydropyridines. In contrast to the 1,4-dihydropyridine-selective receptors on Ca2+ channels in electrically excitable tissues, the (+) enantiomer of nimodipine and the (-) enantiomer of the benzoxadiazol 1,4-dihydropyridine (PN 200-110) were bound with higher affinity than the respective optical antipodes. The human red blood cell ghost [3H]nimodipine-labelled sites also interacted with the inorganic Ca2+-antagonist La3+ (increase in the number of binding sites), and were allosterically regulated by the optical enantiomers of the phenylalkylamine-type Ca2+-antagonists (e.g. verapamil, desmethoxyverapamil, methoxyverapamil). The benzothiazepines d- or l-cis-diltiazem were without effect. Nucleosides (adenosine approximately equal to inosine greater than cytidine) were inhibitory at the nimodipine-labelled site, as were the nucleoside uptake inhibitors dipyridamole, hexobendine, dilazep, nitrobenzylthioinosine and nitrobenzylthioguanosine. The binding sites have essential sulfhydryl groups, show trypsin sensitivity, but are relatively heat stable. When nitrobenzylthioinosine was employed as a covalent probe to inactivate the red blood cell ghost nucleoside carrier, [3H]nimodipine binding was irreversibly lost. (+)-Nimodipine greater than (-)-nimodipine inhibited [14C]adenosine transport into human red blood cells. A good correlation between IC50 values for inhibition of [3H]nimodipine binding and IC50 values for inhibition of [14C]adenosine uptake was found for 18 compounds. Sheep red blood cells (which lack the nucleoside transporter) had no detectable [3H]nimodipine binding sites. It is concluded that the Ca2+-antagonist receptor sites of the human erythrocyte are coupled to the nucleoside transporter.

Characterization of the binding sites for nimodipine and (-)-desmethoxyverapamil in bovine cardiac sarcolemma.

The bovine cardiac sarcolemmal binding sites for the dihydropyridine nimodipine and the phenylalkylamine (-)-desmethoxyverapamil were studied. The density of the nimodipine and (-)-desmethoxyverapamil binding sites increased 8.3-fold and 3.4-fold with the sarcolemma. The binding sites for both compounds were destroyed by trypsin. Nimodipine bound in the presence of 1 mM free calcium to a high-affinity and a low-affinity site with apparent Kd values of 0.35 +/- 0.09 nM (n = 9) and 33 +/- 6.0 nM (n = 9) and with apparent densities of 0.3 +/- 0.05 pmol/mg (n = 9) and 8.2 +/- 1.0 pmol/mg (n = 9). The binding to the high-affinity site was abolished by 1 mM EGTA. The binding sites were specific for dihydropyridines. The (-)-isomers of several phenylalkylamines inhibited nimodipine binding by an apparent allosteric mechanism. (-)-Desmethoxyverapamil bound in the presence of 5 mM EGTA to a high-affinity and a low-affinity site with apparent Kd values of 1.4 +/- 0.3 nM (n = 6) and 171 +/- 26 nM (n = 6) and with apparent densities of 0.16 +/- 0.02 pmol/mg (n = 6) and 13.6 +/- 2.7 pmol/mg (n = 6). The binding to both sites was inhibited by calcium with a half-maximal concentration of 4.3 mM. The binding sites were specific for the other phenylalkylamines and had a higher affinity for the (-)-isomers than for the (+)-isomers. Nimodipine inhibited the binding of (-)-desmethoxyverapamil by an apparent allosteric mechanism. d-cis-Diltiazem inhibited non-competitively the binding of (-)-[3H]desmethoxyverapamil with a Ki of 3.7 microM. Diltiazem up to concentrations of 10 microM did not affect the amount of nimodipine bound at equilibrium at 20 degrees C. However, but in agreement with this result, diltiazem decreased threefold at 20 degrees C the dissociation and association rates for the high-affinity nimodipine receptor. These rates were only marginally affected at 4 degrees C and 37 degrees C. d-cis-Diltiazem reversed in a competitive manner the inhibition of nimodipine binding elicited by the addition of (-)-desmethoxyverapamil with a Ka value of 1.6 microM. The amount of nimodipine bound was inhibited by 50% by the adenosine uptake inhibitors nitrobenzylthioinosine and hexobendine with apparent median inhibitory concentrations of 1 nM and 3 nM, respectively. Nitrobenzylthioinosine completely abolished binding of nimodipine to the low-affinity site, but did not affect binding to the high-affinity site.(ABSTRACT TRUNCATED AT 400 WORDS)

Relationship between the stereoselective negative inotropic effects of verapamil enantiomers and their binding to putative calcium channels in human heart

Ventricular preparations from patients with mitral disease and hypertrophic obstructive cardiomyopathy (HOCM) were set up to contract isometrically. Ventricular membrane particles were also prepared and putative calcium channels were labelled with [3H]-nimodipine. Positive staircase was induced by varying the rate of stimulation of isolated strips from 6 min-1 to 120 min-1 in the presence of 6-60 microM (-)-adrenaline or (-)-noradrenaline. (-)-Verapamil 3-5 microM or (+)-verapamil 20-30 microM reversed the force-frequency relationship (i.e. caused negative staircase) in preparations from patients with mitral disease or HOCM. In subendocardial strips of ventricular septum from 5 patients with HOCM paced at 60 min-1, both (-)-verapamil and (+)-verapamil caused cardiodepression. Half-maximal cardiodepression was observed with 0.4 microM (-)-verapamil and with 3 microM (+)-verapamil. [3H]-nimodipine bound to ventricular membrane particles in a saturable, reversible fashion to a high affinity site with an equilibrium dissociation constant of 0.23 nM. The density of these sites was 95 fmol mg-1 of membrane protein. Binding of the tritiated 1,4-dihydropyridine was stereoselectively inhibited by 1,4-dihydropyridine enantiomers and nifedipine. (-)-Verapamil and (+)-verapamil inhibited high affinity [3H]-nimodipine binding in a negative heterotropic allosteric manner with (-)-verapamil being 5 times more potent than (+)-verapamil on an IC50 basis. At a given [3H]-nimodipine concentration, (+)-verapamil inhibited a greater fraction of specific [3H]-nimodipine binding. The allosteric mode of (+)-verapamil inhibition of [3H]-nimodipine binding was confirmed by kinetic studies. (-)-Verapamil shifted (+)-verapamil-binding inhibition curves to the right in an apparently competitive fashion. The inversion of staircase caused by both verapamil enantiomers suggests that they cause a use-dependent channel blockade. The similar potency ratios for binding and for cardiodepression are indicative of a common locus of action for both verapamil enantiomers within the calcium channel.

Molecular approach to the calcium channel.

Tritiated 1,4-dihydropyridines (nimodipine, nitrendipine, nifedipine, PN 200-110) and [3H]D-cis-diltiazem as well as [3H]verapamil were employed to directly identify calcium channels in membranes for excitable tissues. The channels, when probed with 1,4-dihydropyridines, exhibit the following properties: 1,4-Dihydropyridine calcium channel blockers bind in a temperature-dependent, reversible manner and with high affinity (dissociation constants 0.2-2 nM at 37 degrees C) to a finite number of sites. For chiral 1,4-dihydropyridines, the binding is stereoselective. Hill slopes of approximately 1.0 are observed. In brain, heart, and solubilized skeletal-muscle membranes, an absolute requirement for certain divalent cations exists in order to bind the ligands with high affinity. Cooperativity (negative and positive) between Me2+ and 1,4-dihydropyridine binding sites is observed. 1,4-Dihydropyridine binding sites are down-regulated in a complex manner by the optically pure enantiomers of D-600 and verapamil. These channel blockers induce, to a different extent, a low-affinity state of the 1,4-dihydropyridine binding site. It is postulated that this allosteric site, at which these blockers act, is closely coupled to the 1,4-dihydropyridine binding site and that a spectrum of compounds exists that differ in their affinity as well as their intrinsic activity to induce the down-regulation. The 1,4-dihydropyridine binding sites are up-regulated by D-cis-diltiazem and KB-944. The up-regulation is temperature-dependent and induces a high-affinity state for 1,4-dihydropyridine channel blockers, accompanied by distinct alterations of the kinetics as well as the pharmacological profile of the 1,4-dihydropyridine binding sites. Complex interactions exist between the channel blockers that induce up-regulation and those that induce down-regulation of the binding. For a given radiolabeled 1,4-dihydropyridine, a tissue-specific (but not species-specific) equilibrium binding dissociation constant is observed. Thus, all heart (human, rat, guinea pig, frog, bovine) have the same KD (0.25 nM at 37 degrees C) for, [3H]nimodipine. The same is observed for brain (KD = 0.5 nM) and for skeletal muscle (KD = 1-2 nM). Three subtypes of channels can be distinguished on the basis of the KD and the tissue-specific up-regulation by D-cis-diltiazem. Subtype-selective drugs exist; e.g., AQA 39 is an inhibitor of [3H]nimodipine binding at skeletal-muscle calcium channels, but not at brain channels. Despite their different pharmacological and kinetic profiles, calcium channels in skeletal muscle and brain have the same molecular size (Mr) when probed by radiation inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)