Susan Jean Ward

Identification of cannabinoid receptors in cultures of rat cerebellar granule cells

G protein-linked cannabinoid receptors are present in high density in cerebellum, where they inhibit adenylyl cyclase. This study explored whether cannabinoid receptors are co-localized with GABAB receptors on cerebellar granule cells. In rat cerebellar membranes, receptor-coupled G protein function was assayed by agonist stimulation of low Km GTPase as well as agonist-inhibited adenylyl cyclase. Addition of cannabinoid agonists together with the GABAB agonist, baclofen, produced additive responses with stimulation of low Km GTPase but only partially additive responses with inhibition of adenylyl cyclase. In Weaver and Staggerer but not Nervous mutant mice, cannabinoid-inhibited adenylyl cyclase was significantly decreased in cerebellar but not striatal membranes compared to littermate controls. In primary cultures of rat cerebellar granule cells, cannabinoids inhibited forskolin-stimulated cAMP levels, with IC50 values ranging from 0.1 to 2.0 microM. Cannabinoid inhibition of intracellular cAMP levels was blocked by pretreatment of cell cultures with pertussis toxin. Addition of baclofen and cannabinoid agonists together in cultured granule cells produced no additivity in response for inhibition of intracellular cAMP levels. These data confirm that G protein-linked cannabinoid receptors are present in cerebellar granule cells and may share adenylyl cyclase catalytic units with GABAB receptors.

Opioid and Cannabinoid Receptor Inhibition of Adenylyl Cyclase in Braina

Both opioids and cannabinoids bind to G-protein-coupled receptors to inhibit adenylyl cyclase in neurons. These reactions were assayed in brain membranes, where maximal inhibitory activity occurred in the following regions: mu-opioid inhibition in rat thalamus, delta-opioid inhibition in rat striatum, kappa-opioid inhibition in guinea pig cerebellum, and cannabinoid inhibition in cerebellum. The inhibition of adenylyl cyclase by both cannabinoid and opioid agonists was typical of G-protein-linked receptors: they required GTP, they were not supported by non-hydrolyzable GTP analogs, and they were abolished (in primary neuronal cell culture) by pertussis toxin treatment. The immediate targets of this system were determined by assaying protein phosphorylation in the presence of receptor agonists and App(NH)p, a substrate for adenylyl cyclase. In striatal membranes, opioid agonists inhibited the phosphorylation of at least two bands of MW 85 and 63 kDa, which may be synapsins I and II, respectively. Other experiments determined the long-term effects of this second messenger system. In primary neuronal cultures, opioid-inhibited adenylyl cyclase attenuated forskolin-stimulated pro-enkephalin mRNA levels, thus providing a feedback regulation of opioid synthesis. Finally, in cerebellar granule cells, both cannabinoid and opioid receptors may exist on the same cells. In these cells, agonists which bind to different receptor types may produce similar biological responses.

C-ATTACHED AMINOALKYLINDOLES : POTENT CANNABINOID MIMETICS

Abstract Aminoalkylindoles (AAIs) with potent cannabinoid agonist activity have been synthesized where the aminoalkyl chain is attached to the indole ring via a carbon atom of the cyclic amine.

The effects of serotonin antagonists in a behavioral despair procedure in mice

Six serotonin antagonists (pizotifen, mianserin, cyproheptadine, ketanserin, trazodone and methysergide) were tested in mice in a behavioral despair procedure. The behavioral despair procedure detects most antidepressant compounds. Pizotifen, mianserin and cyproheptadine were found to be active and the others were inactive. The serotonin binding potency at serotonin1 or serotonin2 receptors, or the ratio of potency at these receptors did not correlate with activity in the behavioral despair procedure. However, the serotonin antagonists that were active in the behavioral despair procedure were all found to be potent antagonists at histamine1 receptors. It is suggested that the activity of some serotonin antagonists in the behavioral despair procedure is best explained by their antihistaminergic potency.

Peptide azoles: A new class of biologically-active dipeptide mmetics.

Abstract Appropriately substituted thiazoles, imidazoles and oxazoles have been designed to be useful mimetics of dipeptide moieties. These “peptide azoles” have been shown to be potent antagonists of substances P as measured by isolated tissue assays such as the isolated guinea-pig ileum. A novel, chiral synthesis of these peptide azoles from aminoacids has been developed.

Multiple opioid receptor profile in vitro and activity in vivo of the potent opioid antagonist win 44,441-3

Win 44,441-3 is a pure opioid antagonist in rodents in vivo and in isolated tissue preparations in vitro. Win 44,441-3 produced a weak inhibition of electrically-stimulated twitch contractions of the mouse vas deferens (MVD) and guinea-pig ileum (GPI) preparations that was not prevented by naloxone, suggesting that these effects were not mediated through opioid receptors. Similarly, Win 44,441-3 produced a weak, but nonstereoselective antiwrithing effect in the ACh-induced writhing assay. Win 44,441-3 produced a concentration-related antagonism of mu, kappa and delta agonist actions in the MVD and GPI, and was about 10 times more potent than naloxone at each receptor. Also Win 44,441-3 dissociated from mu, kappa and delta receptors more slowly than naloxone. In vivo, Win 44,441-3 produced a dose-related antagonism of morphine and phenazocine-induced antinociception in the tail flick test, and was equipotent with naloxone following s.c. administration. Win 44,441-3 was active orally and demonstrated a significantly longer duration of action than a pharmacologically equivalent oral dose of naloxone vs morphine and phenzaocine in the tail flick test. It can be concluded that Win 44,441-3 is a pure opioid antagonist, 10 times more potent than, with a receptor selectivity profile similar to, that of naloxone in vitro and is of similar potency to, but of longer duration than, naloxone in vivo.

Morpholinoalkylindenes as antinociceptive agents: Novel cannabinoid receptor agonists

Abstract Indence analogs of pravadoline exhibited antinociceptive activity in several animal models. The inhibition of prostaglandin (PG) synthesis in mouse brain microsomes was diminished in these pravadoline analogs, but they were potent in inhibiting electrically stimulated contractions in the mouse vas deferens (MVD) preparations. Binding studies with ligand WIN 55212-2 have aided to demonstrate that the morpholinoalkyl-indene binding site is functionally equivalent with cannabinoid binding site. The antinociceptive activity of the indene derivatives appears to be mediated by increased affinity for the cannabinoid receptor.

Opioid agonist affinity in the guinea-pig ileum and mouse vas deferens

The affinity of morphine, normorphine, methadone, Tyr-D-Ala-Gly-MePhe-NH(CH2)2(N-O)(CH3)2 (RX 783030), [D-Ala2,D-Leu5]enkephalin (DADLE), ketazocine and ethylketocyclazocine (EKC) were determined for their pharmacological receptors in two bioassay tissues, the guinea-pig ileum and the mouse vas deferens (MVD). The method involved the use of the irreversible antagonist, beta-chlornaltrexamine (beta-CNA), and the method of partial receptor blockade. The agonist concentration-effect curves were displaced to the right with decreasing maximum effect, a pattern typical of partial, irreversible blockade of receptors. The concentrations of beta-CNA required to produce a rightward displacement in the concentration-effect curves for different agonists, ranged between 2 and 3000 nM. No similarity was found between the IC50 and the dissociation constant (KA), values predicted to be equivalent only if a linear relationship exists between receptor occupation and observed effect; the dissociation constant for the agonists were between 3 and 218 times larger than the IC50 values. When methadone was used as the agonist in the guinea-pig ileum, beta-CNA produced parallel displacement of the concentration-effect curve, regardless of the blocking concentration chosen, preventing the determination of KA for this agonist, in this tissue; this problem was not encountered in the mouse vas deferens. The KA of morphine, RX 783030 and ketazocine were found not to differ in the guinea-pig ileum and mouse vas deferens. As expected, DADLE had significantly different affinity in the two tissues, showing 117-fold lower affinity in the guinea-pig ileum. Surprisingly, the normorphine affinity was found to be 7-fold higher in the guinea-pig ileum. While the difference in affinity of DADLE may be due to the suggested lack of functional delta receptors in the guinea-pig ileum, the difference in affinity seen with normorphine, but not morphine, in the two tissues is difficult to explain. Taken together with the insensitivity of methadone to beta-CNA blockade in the guinea-pig ileum, but not mouse vas deferens, the difference in the affinity of normorphine in these tissues may suggest the possibility of differences in local milieu of mu receptors or of mu receptor subtypes in the two tissues. The results provide fundamental information regarding opioid agonist affinity in two standard bioassays in vitro, and support the view of (1) a difference in receptors activated by DADLE in the guinea-pig ileum (mu) and mouse vas deferens (delta), as well as (2) possible differences in mu-receptors in these tissues.

Lack of correlation between σ binding potency and inhibition of contractions in the mouse vas deferens preparation

The existence of sigma receptors in the mouse, rat and guinea pig vasa deferentia has previously been proposed, although drug effects are inconsistent and generally occur only at high concentrations. The purpose of the present study was to evaluate lower, physiologically relevant concentrations of ligands for possible sigma effects on electrically stimulated twitch contractions in the mouse vas deferens (MVD). Putative sigma agonists and antagonists all inhibited 0.1 Hz electrically stimulated twitch contractions in nM concentrations. Inhibitory activity plateaued between 20 and 60% for all compounds except 1,3-di(2-tolyl)guanidine (DTG), which had a shallow concentration-effect curve. Subsequent to the plateau, higher concentrations (30 microM) of rimcazole and haloperidol fully inhibited electrically stimulated twitch contractions. There was no correlation between inhibitory potency or maximal effect in the MVD and binding potency at sigma sites in either MVD or guinea pig brain. The inhibitory effects of R(+)-3-(3-hydroxyphenyl)-N-1-propylpiperidine ((+)3-PPP) or DTG on electrically stimulated twitch contractions were not antagonized by the putative sigma antagonists DTG, haloperidol, rimcazole or BMY-14802, nor by alpha 2-adrenoceptor, dopamine D1, dopamine D2 or opiate antagonists. Although the mechanism of sigma ligand effects in the MVD has not been established, the data caution against a presumption that effects of sigma ligands on electrically stimulated twitch contractions in this preparation are mediated by sigma receptors.

Pharmacological profiles of tonazocine (Win 42156) and zenazocine (Win 42964)

The effects of tonazocine and zenazocine, two mixed agonist/antagonist analgesics, have been evaluated in a range of antinociceptive assays and in isolated tissue preparations in vitro. Both tonazocine and zenazocine were antinociceptive in writhing tests and in the i.a. bradykinin test, and were antagonists in the rat tail flick test. Additionally, zenazocine demonstrated some antinociceptive activity in the rat tail flick test. In vitro, both tonazocine and zenazocine demonstrated agonist and antagonist properties at mu receptors in the GPI and agonist properties at delta receptors in the MVD. In general, the agonist properties of zenazocine were more marked than those of tonazocine, and the antagonist properties of tonazocine were more marked than those of zenazocine.