B.R. de Costa

Cytotoxic effects of sigma ligands: sigma receptor-mediated alterations in cellular morphology and viability

The morphological effects of several neuroleptics as well as other novel and prototypic sigma ligands were examined by addition to cultures of C6 glioma cells. Sigma ligands caused loss of processes, assumption of spherical shape, and cessation of cell division. The time course and magnitude of this effect were dependent on the concentration of sigma ligand. Continued exposure to sigma compounds ultimately resulted in cell death. However, the morphological effect was reversible when sigma ligand was removed shortly after rounding. The potency of compounds to produce these effects generally correlated with binding affinity at sigma receptors of C6 glioma cell membranes labeled with [3H](+)-pentazocine. At a concentration of 100 microM, haloperidol, reduced haloperidol, fluphenazine, perphenazine, trifluoperazine, BD737, LR172, BD1008, and SH344 produced significant effects in 3–6 hr of exposure. Other compounds, such as trifluperidol, thioridazine, and (-)-butaclamol, produced significant effects by 24 hr of exposure. Despite the requirement of micromolar concentrations of ligand (some compounds were effective at 30 microM), the effect showed a remarkable specificity for compounds exhibiting sigma receptor binding affinity. Neuroleptics lacking potent sigma affinity [e.g., (-)- sulpiride, (+)-butaclamol, and clozapine] and other compounds that lack significant sigma affinity but that are agonists or antagonists at dopamine, serotonin, adrenergic, glutamate, phencyclidine, GABA, opiate, or muscarinic cholinergic receptors were without effect on cellular morphology at concentrations up to 300 microM over a period of 72 hr. Likewise, blockers and activators of Na+, K+, and Ca2+ channels and a monoamine oxidase inhibitor devoid of sigma affinity were without effect. Interestingly, 1,3-di-o-tolylguanidine (DTG), (+)-3-(3- hydroxyphenyl)-N-(1-propyl)piperidine [(+)-3-PPP], (+)-pentazocine, (+)- cyclazocine, and other sigma-active benzomorphans and morphinans appeared inactive in up to 72 hr of culture. However, these compounds interacted synergistically with a subeffective dose of BD737 (30 microM) to produce effects usually in 6 hr or less. Also, the pH of the culture medium had a profound effect on the activity of sigma compounds. Increasing the pH from the normal range of 7.2–7.4 to pH 8.3– 8.5 shifted the dose curves (30, 100, 300 microM) for all sigma compounds to the left. Under these conditions, DTG, (+)-3-PPP, and benzomorphans produced effects in 24 hr or less.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuronal localization of cannabinoid receptors and second messengers in mutant mouse cerebellum

Four lines of mutant mice were used to investigate (1) the neuronal localization of cannabinoid receptors in the cerebellar molecular layer and (2) the anatomical association of these receptors with elements of the two second messenger systems in the brain. Two of the mutant lines--Purkinje cell degeneration and nervous--are selectively deficient in Purkinje cells; the other two--weaver and reeler--are deficient in granule cells. In the heterozygous mice, [3H]CP 55,940 binding to cannabinoid receptors was discretely and densely localized to the molecular layer, as was [3H]forskolin binding to adenylate cyclase and [3H]phorbol 12,13-dibutyrate binding to protein kinase C, a component of the phosphoinositide cycle. [3H]CP 55,940 and [3H]forskolin binding was selectively reduced in weaver and reeler homozygous mice but unchanged in Purkinje cell deficient and nervous homozygotes. No decreases in [3H]phorbol 12,13-dibutyrate binding were found in any of the homozygous mutants relative to the heterozygous littermates. The results suggest that cannabinoid receptors and adenylate cyclase are localized to granule cell axons in the molecular layer, whereas protein kinase C is equally distributed in parallel fibers and Purkinje cell dendrites.

Evaluation of U-50,488H analogs for neuroprotective activity in the gerbil

U-50,488H, a kappa (kappa) opioid ligand with moderate potency at sigma (sigma) receptors, protects against mechanical and ischemia-induced injury. The purpose of this study was to evaluate the possibility that sigma-receptors may be involved in mediating the neuroprotective actions of U-50,488H. This possibility was examined by testing the potential of a series of U-50,488H analogs, which are potent sigma-ligands with minimal activity at kappa-opioid receptors, to protect against ischemia-induced neuronal damage in the gerbil. Like U-50,488H, BD-449 (20 mg/kg), the cis-diastereomer of U-50,4888H, protected against ischemia-induced neuronal damage as did BD-737 (50 and 30 mg/kg) and BD-738 (50 mg/kg). All 3 compounds interacted selectively with sigma-receptors. In contrast, BD-601 (50 mg/kg), did not protect against ischemia-induced neuronal damage, although it also interacted potently with sigma-receptors. One difference between the compounds that were neuroprotective and BD-601 is that only BD-601 produced sigma-like behavioral effects in the rat. Thus, it is possible that BD-601 may interact differently or at a different sigma-subtype than BD-449, BD-737 and BD-738 with sigma-receptors. However, these results clearly indicate that an interaction with kappa-opioid receptors is not required for anti-ischemic activity, and that sigma-receptors may play a role in neuroprotection.

Sigma receptors modulate nicotinic receptor function in adrenal chromaffin cells.

Neither the physiological function of sigma (σ) receptors nor the cellular mechanism responsible for the pharmacological effects of σ receptor ligands is known. We now report that σ receptor ligands noncompetitively inhibit nicotine‐stimulated catecholamine release from bovine adrenal chromaffin cells in a concentration‐dependent and reversible manner. The rank order of potency of ligands to inhibit nicotine‐stimulated catecholamine release is significantly correlated (P < 0.005) with that observed in radioligand binding assays selective for the σ1 receptor subtype. This naltrexone‐insensitive effect is paralleled by an inhibition of nicotine‐stimulated increases in [Ca2+]i. Sigma ligands were without effect on catecholamine release or [Ca2+]i in the absence of nicotine. In addition, nicotine accelerated the association of the σ receptor selective radioligand, [3H](+)pentazocine, to adrenal medullary homogenates while having no effect on the rate of ligand dissociation, consistent with a σ ligand binding site closely associated with and allosterically modulated by the nicotinic acetylcholine receptor. Thus, the actions of agonists at the nicotinic acetylcholine receptor in bovine chromaffin cells are modulated by σ1 receptor selective ligands.—Paul, I. A., Basile, A. S., Rojas, E., Youdim, M. B. H., De Costa, B., Skolnick, P., Pollard, H. B., Kuijpers, G. A. J. Sigma receptors modulate nicotinic receptor function in adrenal chromaffin cells. FASEB J. 7: 1171‐1178; 1993.

In vivo study of nmda-sensitive glutamate receptor by fluorothienylcycloexylpiperidine, a possible ligand for positron emission tomography

As a preliminary to positron emission tomography (PET) studies of excitatory amino acid neurotransmission, N-methyl-d-aspartate (NMDA)-sensitive glutamate receptors of mice and rats were labelled in vivo with [3H]fluorothienylcycloexylpiperidine (FTCP), which binds to the phencyclidine site of the NMDA receptor. After intravenous injection, the half-life of clearance of authentic FTCP from blood was 4.2 min in mice, 12 min in rats and 45 min in a rhesus monkey. In rodent brain, the specific binding of [3H]FTCP, 10 min after intravenous injection, was 10–20% of the total binding and no regional differences were observed. However, if animals were treated with NMDA intraperitoneally (0.68 mmol/kg), 10 min before injection of [3H]FTCP, a three- to five-fold increase in specific binding was observed in hippocampus, cerebral cortex and striatum but not in cerebellum. Thus, specific binding of [3H]FTCP in vivo revealed the physiological status of the NMDA receptor: in fact, preliminary PET studies with [18F]FTCP in monkeys indicated increased binding after activation of NMDA receptors. These data suggest that PET with [18F]FTCP can be a tool to evaluate physiological or pathological modifications of the function of NMDA receptors.

Characterization of benzodiazepine receptors with fluorescent ligands.

Fluorescein conjugates of the high‐affinity benzodiazepine receptor ligands Ro 15‐1788 and Ro 7‐1986 were synthesized. The binding of these fluorescent ligands (BD 621 and BD 607) to benzodiazepine receptors was characterized by direct fluorescence measurement. Both the equilibrium dissociation constants (KD) of BD 621 and BD 607 and the maximum number of binding sites (Bmax) estimated by fluorescence monitoring were consistent with values obtained by using radioligand binding techniques. The binding of BD 621 and BD 607 assessed by fluorescence measurement was reversible, abolished by photoaffinity labeling with Ro 15‐4513, and unaffected by a variety of substances that do not bind to benzodiazepine receptors. The potencies of chemically diverse benzodiazepine receptor compounds to inhibit fluorescent ligand binding were highly correlated (r = 0.94, P < 0.001), with potencies obtained from radioligand binding techniques. These findings demonstrate the feasibility of using direct fluorescence measurement techniques to quantitate ligand‐receptor interactions.— McCabe, R. T.; de Costa, B. R.; Miller, R. L.; Havunjian, R. H.; Rice, K. C.; Skolnick, P. Characterization of benzodiazepine receptors with fluorescent ligands. FASEB J. 4: 2934‐2940; 1990.

Wash-resistant inhibition of phencyclidine- and haloperidol-sensitive sigma receptor sites in guinea pig brain by putative affinity ligands: Determination of selectivity

Several putative affinity ligands, based on the structures of phencyclidine etoxadrol, 5-methyl-10,11-dihydro-5H-dibenzo[a,d] cycloheptene-5,10-imine (MK801) and 1,3-di-(2-methylphenyl)guanidine (DTG) were evaluated in vitro for their ability to produce a wash-resistant inhibition of phencyclidine and sigma receptor sites in homogenates of the brain of the guinea pig. All the phencyclidine-based ligands, including 1-[1-(3-isothiocyanatophenyl)cyclohexyl]piperidine (Metaphit) and (+/-)-N-(2-isothiocyanatoethyl) MK801 [(+/-)-MK801-NCS], produced a wash-resistant inhibition of binding sites for phencyclidine, labelled by [3H]-1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) and sigma binding sites, labelled by [3H]DTG. The DTG-based ligands, 1-(4-isothiocyanato-2-methylphenyl)-3-(2-methylphenyl)guanidine (DIGIT) and 1-(4-[2-(2-isothiocyanatoethoxy)ethoxy]-2-methyl-phenyl)-3-(2- methylphenyl)guanidine (DIGIE), produced a wash-resistant inhibition of sigma sites, at concentrations as small as 1 microM and also inhibited binding sites for phencyclidine at larger concentrations (100 microM). Both 1-(3-isothiocyanatophenyl)-1-ethyl-4-(2-piperidyl)-1,3-dioxolane (ETOX-NCS) and 1-[1-(3-bromoacetyloxyphenyl)cyclohexyl]-1,2,3,6-tetrahydropyri din e (Bromoacetyl-PCP) were the most potent and selective inhibitors of the binding of [3H]TCP, while DIGIT was the most selective inhibitor of the binding of [3H]DTG. Future studies will examine the selectivity of these agents in vivo after intracerebroventricular administration.

Effects of σ ligands on rat cerebellar purkinje neuron firing: An iontophoretic study

Abstract The electrophysiological responses of rat cerebellar Purkinje neurons to selective σ ligands applied iontophoretically was examined in urethane anesthetized male Sprague-Dawley rats. 1,3-Di-o-tolylguanidine (DTG), dextrallorphan (DEX), (+)-pentazocine((+)-PENT), (+)-3-(3-Hydroxyphenyl)- N -propylpiperidine ((+)-3-PPP), and the novel diamine BD1008, were ejected from multibarrel pipettes onto individual Purkinje cells. In some neurons, cell firing was inhibited following ejections of all compounds. These inhibitory effects were dose dependent and occurred without changes in spike amplitude or duration, thus ruling out local anesthetic effects as a mechanism. (+)-3-PPP and DEX increased firing rate in 27% and 14% ( n = 15, n = 14, respectively) of cells studied. The results of this study indicate that σ ligands significantly alter the spontaneous firing of Purkinje neurons, consistent with previous work suggesting motor effects of σ ligands via the rubro-cerebellar circuitry.

Fourphit, an Acylating Phencyclidine Derivative, Attenuates Cocaine-Induced Hyperactivity in Rats

Fourphit (4-isothiocyanato-1-[1-phenylcyclohexyl]piperidine), an acylating phencyclidine derivative that irreversibly inhibits stimulant binding to the dopamine transporter in vitro (Schweri et al., J. Pharmacol. Exp. Ther. 261:936-942, 1992), was tested in rats for its ability to block the increased locomotor activity caused by cocaine. Administration of Fourphit (20 mg/kg, i.v.) significantly reduced the hyperactivity caused by challenge with either 15 or 40 mg/kg (-)cocaine x HCl (i.p.) 24 h later. It also increased the amount of thigmotaxis and decreased the rearing frequency of rats given the higher dose of cocaine. Only negligible effects on behavior were found upon acute administration of the compound by itself, or in response to a saline challenge 24 h later. Activity during the dark cycle immediately following Fourphit administration, however, was moderately depressed. Contrary to the results predicted from its activity in vitro, Fourphit did not inhibit the ex vivo binding of [3H]methylphenidate to stimulant receptors in the striatal tissue of treated rats. These results show that Fourphit can antagonize some behavioral actions of cocaine, but these effects are not likely due to covalent modification of the site on the dopamine transporter recognized by cocaine.