Gordon T. Bolger

The behavioral effects of the calcium agonist BAY K 8644 in the mouse: antagonism by the calcium antagonist nifedipine

SummaryMice injected with the calcium agonist BAY K 8644 (2–4 mg/kg, i. p.) displayed profound behavioral changes including ataxia, decreased motor activity, Straub tail, arched back, limb clonus and tonus, and an increased sensitivity to auditory stimulation. BAY K 8644 significantly impaired rotorod performance in mice with an ED50 of 0.8 mg/kg. The behavioral effects of BAY K 8644 were antagonized by nifedipine, but not by the non-dihydropyridine calcium channel antagonist verapamil or the α-adrenoceptor antagonist prazosin. Further, the actions of BAY K 8644 were not mimicked by the α-adrenoceptor agonist methoxamine at doses up to 4.5 mg/kg. These observations, coupled with the findings that BAY K 8644 is a potent, competitive inhibitor of [3H]nitrendipine binding to the dihydropyridine binding site in mouse brain (Ki=7.0×10−9M), suggests that BAY K 8644 may produce its behavioral actions via an interaction with the DHP binding site, which has been linked to the control of calcium flux across membranes in peripheral tissues.

Characterization of the binding of [^3H] Ro 5-4864, a convulsant benzodiazepine, to guinea pig brain

Abstract: The density of high affinity binding sites for [3H]4′‐chlorodiazepam ([3H]Ro 5‐4864) in guinea pig cerebral cortex is significantly higher (3.8‐fold) than the density reported in the rat, and is nearly equal to the density of binding sites for other [3H]benzodiazepines (e.g., diazepam, flunitrazepam). The density of these [3H]Ro 5‐4864 binding sites was generally higher in guinea pig brain than in rat brain, with the exception of olfactory bulb. Both the subcellular distribution and pharmacologic profile of these sites in guinea pig brain appears qualitatively similar to observations previously reported in the rat. The high density of binding sites for [3H]Ro 5‐4864, coupled with the potency of this compound as a convulsant in the guinea pig, suggest this species will be a valuable model for elucidating putative pharmacologic and physiologic functions of these sites in brain.

Peripheral-type binding sites for benzodiazepines in brain: relationship to the convulsant actions of Ro 5-4864.

Abstract: Previous studies have shown that Ro 5–4864 is a potent convulsant and increases the firing rate of substantia nigra zona reticulata neurons. The pharmacologic profile of compounds that antagonize these actions suggested that the effects of Ro 5–4864 were not mediated by “brain‐type”benzodiazepine receptors. We examined a number of compounds that are structurally related to Ro 5–4864 for their capacities to displace [3H]Ro 5–4864 from “peripheral‐type”binding sites and their potencies as convulsants (or as antagonists of Ro 5‐4864‐induced convulsions). It was observed that compounds such as KW 3600 (the N‐desmethyl analog of Ro 5–4864), which have very low affinities for “peripheral‐type”sites, are convulsants with a potency nearly equal to that of Ro 5–4864. In contrast, compounds such as Ro 5–6900 and PK 11195, which bind with very high affinities to “peripheral‐type”binding sites, are neither convulsants nor do they antagonize the convulsant actions of Ro 5–4864. Within a series of compounds that are structurally related to Ro 5–4864 there is a good correlation (r = 0.93; p < 0.01) between their potencies as convulsants and their capacities to displace [35S]t‐butylbicyclophosphorothionate from sites that may be associated with the chloride ionophore. Thus, it appears that occupation of “peripheral‐type”binding sites by high‐affinity ligands may not be directly involved in the convulsant actions of Ro 5–4864 and related compounds.

Modulation of neurotransmitter metabolism by dihydropyridine calcium channel ligands in mouse brain

The regional concentrations of dopamine, serotonin, dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindole acetic acid were measured in mouse brain following administration of the dihydropyridine calcium channel activator BAY K 8644, and antagonist, nifedipine. BAY K 8644 (1-8 mg/kg) produced dose- and time-dependent increases in dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid concentrations in the caudate, without altering dopamine and serotonin levels. No changes in 5-hydroxyindoleacetic acid concentration were observed in the raphe nuclei, hypothalamus, hippocampus and frontal cortex. Nifedipine (4 mg/kg) blocked BAY K 8644- (2 mg/kg) elicited increases in dihydroxyphenylacetic acid in the caudate. Furthermore, a higher dose of nifedipine (8 mg/kg) decreased dihydroxyphenylacetic acid and homovanillic acid, but did not affect dopamine, serotonin or 5-hydroxyindoleacetic acid concentrations, while a lower dose of nifedipine (2 mg/kg) significantly increased serotonin, 5-hydroxyindoleacetic acid and homovanillic acid, but did not affect dopamine and dihydroxyphenylacetic acid concentrations. The findings that both BAY K 8644 and nifedipine affect neurotransmitter metabolism in vivo in a dose-, time- and brain region-dependent manner, suggest that high-affinity dihydropyridine calcium channel binding sites play an important role in regulating neurotransmitter turnover in the central nervous system.

Enhancement of brain calcium antagonist binding by phencyclidine and related compounds

The abilities of compounds structurally or pharmacologically related to phencyclidine to increase the apparent affinity of the [3H]dihydropyridine calcium channel antagonist [3H]nitrendipine were examined in lysed synaptosomal membrane preparations of rat brain. The p-bromo analog of phencyclidine (1-(1-(4-bromophenyl)cyclohexyl)piperidine) was the most efficacious compound tested in enhancing the apparent affinity of [3H]nitrendipine. The efficacy of this compound was approximately two-fold greater than PCP. The stereoisomers of PCMP (1-(1-phenylcyclohexyl-3-methylpiperidine) were also more efficacious than phencyclidine, although only a small degree of stereoselectivity was observed. Levoxadrol, dexoxadrol and the enantiomers of ketamine did not potentiate [3H]nitrendipine binding. The enantiomers of SKF 10047 (n-allylormetazocine), dextrorphan, levorphanol and the ion channel toxins histrionicotoxin and pumiliotoxin-B also increased the apparent affinity of [3H]nitrendipine, while several local anesthetics and mu-opiate receptor ligands were without effect. These studies suggest that the ability of phencyclidine and structurally related compounds to increase the apparent affinity of [3H]nitrendipine is not mediated through an interaction with phencyclidine receptors, but may represent a unique site for allosteric modulation of neuronal dihydropyridine calcium channel antagonist binding sites.

Effects of calcium antagonists on phencyclidine behaviors

The calcium antagonists nifedipine and verapamil were evaluated for their potential behavioral interactions with phencyclidine induced changes in mouse rotarod performance and motor activity. Nifedipine (2 and 10 mg/kg) and verapamil (2 mg/kg) significantly potentiated impairment of rotarod performance produced by phencyclidine. These doses of nifedipine and verapamil did not by themselves affect rotarod performance. This action does not appear to be dependent on the hypotensive properties of these drugs, since hypotensive doses of prazosin did not alter the effect of phencyclidine on rotarod performance. Nifedipine, 4.0 mg/kg, antagonized increases in ambulatory motor activity, and potentiated decreases in vertical motor activity (rearing) induced by phencyclidine. The effects of calcium antagonists to alter the behavioral actions of phencyclidine in mice may occur through an interaction with the dihydropyridine calcium antagonist binding site present in the central nervous system.

Phencyclidine increases the affinity of dihydropyridine calcium channel antagonist binding in rat brain

SummaryPhencyclidine (PCP) significantly reduces the apparent dissociation constant (KD) of the dihydropyridine (DHP) calcium channel antagonist, [3H]nitrendipine, in synaptosomal membranes of rat and mouse brain without significantly effecting the maximum binding capacity (Bmax). At an optimum concentration of PCP (10 μM) the apparentKD of [3H]nitrendipine was reduced from 178±9 pM to 112±9 pM in rat forebrain, a 58% increase in affinity. The structural derivatives of PCP, P-Br-PCP {1-[1-(4-bromophenyl-cyclohexyl)piperidine]}, m-NH2-PCP {1-[1-(3-anilo)-cyclohexyl]piperidine}, (±)-PCMP [1-(1-phenyl)-cyclohexyl-3-methylpiperidine] also increased the apparent affinity of [3H]nitrendipine in the following order, p-Br-PCP ≫ PCMp>PCP>m-NH2-PCP. Local anesthetics either reduced the apparent affinity of [3H]nitrendipine or had no effect. Kinetic analysis revealed that PCP both increased the microassociation rate constant and decreased the microdissociation rate contant of [3H]nitrendipine. The magnitude of this enhanced binding varied with the brain region studied; the greatest increase in apparent affinity of [3H]nitrendipine was observed in striatum, while no significant increase in affinity was observed in brainstem. In some brain areas, PCP was more effective in reducing theKD in crude homogenates than in washed tissue. PCP (10 μM) did not alter theKD of [3H]nitrendipine to rat cardiac tissue. Both Ca2+ and Mg2+ inhibited the effect of PCP, while monovalent ions were ineffective in this regard. These data are consistent with an allosteric modulation of DHP calcium channel antagonist binding sites by PCP and structural derivatives that is not mediated through the brain PCP binding site. This modulation of DHP binding sites may account for some of the psychopharmacologic actions PCP and related compounds in vivo.

Differential regulation of μ-opiate receptors in heroin- and morphine-dependent rats

Rats made dependent on heroin and morphine exhibit both qualitative and quantitative differences in the characteristics of radioligand binding to μ‐opioid receptors in the central nervous system. In brain membranes prepared from control animals, [3H]dihydromorphine (DHM) binding was best described by a two‐site model, while in morphine‐dependent rats, [3H]DHM binding was best described by a single‐site model. In contrast, [3H]DHM binding to membranes from heroin‐dependent animals was best described by a two‐site model, with an increased density of the high‐affinity, and no change in the low‐affinity population compared to controls. Furthermore, both the number of binding sites for [3H]DAGO (a ligand that selectively labels a population of high‐affinity μ‐opiate receptors) and the sensitivity of [3H]DHM to sodium ions was increased in heroin; but not in morphine‐dependent rats. These studies demonstrate that opiate receptors are differentially regulated in heroin‐ and morphine‐dependent animals. Such neurochemical changes in μ‐opiate receptors may underlie differences in the behavioral and pharmacological profiles of heroin and morphine reported in man.

Calcium antagonist binding in cat brain tolerant to electroconvulsive shock

Cats subjected to daily (25-30 days) electroconvulsive shock (ECS) demonstrated an elevation of their electroconvulsive threshold or tolerance to ECS. [3H] Nitrendipine binding was measured to brain regions from non-tolerant (sham shocked) and ECS tolerant cats 24 hr following the last shock. ECS produced a significant increase (45%) in the density of [3H] nitrendipine binding sites in the cerebral cortex and a significant decrease (33%) in the apparent affinity of [3H] nitrendipine in the cerebellum. No changes in binding were observed in the hippocampus. The effects of ECS were also investigated in the rat, an animal not displaying tolerance to repeated ECS. [3H] Nitrendipine binding to rat brain was measured 10 min and 24 hr following one shock (acute) or ten shocks delivered transauricularly once daily (chronic). Twenty-four hours following chronic ECS, there was a significant increase (19%) and decrease (11%) in the density, but no change in the apparent affinity of [3H] nitrendipine binding sites in the cerebral cortex and hippocampus respectively. No significant change in [3H] nitrendipine binding was observed in rat cerebellum 24 hr following chronic ECS. There were no changes in [3H] nitrendipine binding in the cerebral cortex and hippocampus 10 min and 24 hr following acute ECS. These results indicate that ECS can alter [3H] nitrendipine binding to calcium channel linked dihydropyridine binding sites in the central nervous system. It is suggested that changes in [3H] nitrendipine binding in the cat cerebellum may be involved in the development of tolerance to ECS.

Acylating phencyclidines irreversibly enhance brain calcium antagonist binding

Phencyclidine was previously shown to allosterically increase the apparent affinity of the dihydropyridine ( [3H]nitrendipine) calcium antagonist binding site in a lysed synaptosomal membrane preparation of rat forebrain. Treatment of a similar preparation of mouse forebrain with 4-isothiocyanato-1-(1-phenylcyclohexyl) piperidine (FOURPHIT), an acylating phencyclidine derivative, resulted in a concentration dependent (0.1-10 microM), irreversible, increase in the apparent affinity of [3H]nitrendipine in contrast to the effects of phencyclidine which were reversible. The FOURPHIT isomer, 1-[1-(3-isothiocyanatophenyl) cyclohexyl] piperidine (METAPHIT), (10 microM) also irreversibly increased the apparent affinity of [3H]nitrendipine, but was much less efficacious than FOURPHIT. Phencyclidine blocked the irreversible increase in the apparent affinity of [3H]nitrendipine produced by FOURPHIT. The interactions of multivalent cations and the calcium antagonist diltiazem with the [3H]nitrendipine binding site were altered following treatment of membranes with FOURPHIT. These studies suggest that FOURPHIT irreversibly interacts with the same sites as PCP, and thus may be a useful tool with which to further probe both the behavioral and biochemical interactions between phencyclidine and the dihydropyridine calcium antagonist binding site.