Molecular aspects of cannabinoid receptors.

Two cannabinoid receptors are reviewed with regard to their primary structure, ligand-binding properties, and signal transduction systems. Both receptors have been cloned; therefore, the expression of their genes and the functional domains within the proteins can be examined. Binding of tritiated agonists has localized these receptors to the central nervous and immune systems. The CBI receptor is predominantly expressed in brain tissues and is found in both glial elements and neurons; subcellular localization to axons and terminals is evident. This receptor is found in motor, limbic, associative, cognitive, sensory, and autonomic brain structures. CBI receptors modulate the activities of calcium and potassium channels. The CB2 receptor is predominantly expressed in the immune system and is found in spleen, tonsils, thymus, mast cells, and blood cells. Although receptors appear to be involved in cannabimimetic-induced modulation of immune cell function, the receptor subtype that is principally involved in specific effects is difficult to determine because both receptors are often coexpressed in the same cells. Cannabimimetic-induced effects on mast cells and B cells appear, however, to be mediated by CB2 receptors.

Cannabinoid receptor binding and messenger RNA expression in human brain: an in vitro receptor autoradiography and in situ hybridization histochemistry study of normal aged and Alzheimer's brains.

The distribution and density of cannabinoid receptor binding and messenger RNA expression in aged human brain were examined in several forebrain and basal ganglia structures. In vitro binding of [3H]CP-55,940, a synthetic cannabinoid, was examined by autoradiography in fresh frozen brain sections from normal aged humans (n = 3), patients who died with Alzheimer's disease (n = 5) and patients who died with other forms of cortical pathology (n = 5). In the structures examined--hippocampal formation, neocortex, basal ganglia and parts of the brainstem--receptor binding showed a characteristic pattern of high densities in the dentate gyrus molecular layer, globus pallidus and substantia nigra pars reticulata, moderate densities in the hippocampus, neocortex, amygdala and striatum, and low densities in the white matter and brainstem. In situ hybridization histochemistry of human cannabinoid receptor, a ribonucleotide probe for the human cannabinoid receptor messenger RNA, showed a pattern of extremely dense transcript levels in subpopulations of cells in the hippocampus and cortex, moderate levels in hippocampal pyramidal neurons and neurons of the striatum, amygdala and hypothalamus, and no signal over dentate gyrus granule cells and most of the cells of the thalamus and upper brainstem, including the substantia nigra. In Alzheimer's brains, compared to normal brains, [3H]CP-55,940 binding was reduced by 37-45% in all of the subfields of the hippocampal formation and by 49% in the caudate. Lesser reductions (20-24%) occurred in the substantia nigra and globus pallidus, internal segment. Other neocortical and basal ganglia structures were not different from control levels. Levels of messenger RNA expression did not differ between Alzheimer's and control brains, but there were regionally discrete statistically significant losses of the intensely expressing cells in the hippocampus. The reductions in binding did not correlate with or localize to areas showing histopathology, estimated either on the basis of overall tissue quality or silver staining of neuritic plaques and neurofibrillary tangles. Reduced [3H]55,940 binding was associated with increasing age and with other forms of cortical pathology, suggesting that receptor losses are related to the generalized aging and/or disease process and are not selectively associated with the pathology characteristic of Alzheimer's disease, nor with overall decrements in levels of cannabinoid receptor gene expression.

Localization of cannabinoid receptor mRNA in rat brain.

Cannabinoid receptor mRNA was localized in adult rat brain by 35S-tailed oligonucleotide probes and in situ hybridization histochemistry. Labelling is described as uniform or non-uniform depending on the relative intensities of individual cells expressing cannabinoid receptor mRNA within a given region or nucleus. Uniform labelling was found in the hypothalamus, thalamus, basal ganglia, cerebellum and brainstem. Non-uniform labelling that resulted from the presence of cells displaying two easily distinguishable intensities of hybridization signals was observed in several regions and nuclei in the forebrain (cerebral cortex, hippocampus, amygdala, certain olfactory structures). Olfactory-associated structures, basal ganglia, hippocampus, and cerebellar cortex displayed the heaviest amounts of labelling. Many regions that displayed cannabinoid receptor mRNA could reasonably be identified as sources for cannabinoid receptors on the basis of well documented hodologic data. Other sites that were also clearly labelled could not be assigned as logical sources of cannabinoid receptors. The localization of cannabinoid receptor mRNA indicates that sensory, motor, cognitive, limbic, and autonomic systems should all be influenced by the activation of this receptor by either exogenous cannabimimetics, including marijuana, or the yet unknown endogenous "cannabinoid" ligand.

Cannabinoid agonists stimulate both receptor- and non-receptor-mediated signal transduction pathways in cells transfected with and expressing cannabinoid receptor clones.

The physiologic activity of (-)-delta 9-tetrahydrocannabinol, the most active component of marijuana, and of many synthetic cannabimimetics may be mediated either through receptor binding and functional coupling to specific signal transduction pathways or through nonspecific interaction with cell membrane components. The cloning of the human and rat cannabinoid receptors has provided the opportunity to investigate the binding properties and signal transduction pathways directly associated with these receptors. Cannabinoid receptor cDNA was transfected into and stably expressed in fibroblast cell lines that do not contain native cannabinoid receptors, thus allowing comparison with untransfected cells. Binding constants measured using [3H]CP55,940 indicated that the rat and human cloned cannabinoid receptors were similar to native cannabinoid receptors measured in brain and neural cell lines. The cloned receptors coupled to the inhibition of cAMP accumulation, as previously demonstrated. CP55,940 binding and inhibition of cAMP accumulation were absent in untransfected cells. Cannabinoid agonist-stimulated release of arachidonic acid and increase in intracellular calcium were observed in both transfected and untransfected cells. Stereoselectivity of cannabinoid agonists was demonstrated for binding and functional inhibition of cAMP accumulation, but not for the release of arachidonic acid and intracellular calcium. Therefore, cannabinoid agonists can stimulate signaling pathways through both receptor- and non-receptor-mediated pathways in the same cell.

Cannabinoid receptors: which cells, where, how, and why?

Localization of the mRNA for this receptor has identified many regions of the rat brain in which the gene for this receptor is active. Several of these regions are consistent with the cannabinoid- or marijuana-induced effects that occur in both laboratory animals and humans. However, other labeled regions are not easily associated with well-known effects of marijuana (Matsuda et al., submitted for publication). Although great progress has been achieved in elucidating the mechanism of action of cannabis in recent years (Howlett et al. 1990), much remains to be discovered about the expression of cannabinoid receptors in the brain and exactly how this receptor influences numerous brain functions.

Structure of a cannabinoid receptor and functional expression of the cloned cDNA.

Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS) in a complex and dose-dependent manner. Although CNS depression and analgesia are well documented effects of the cannabinoids, the mechanisms responsible for these and other cannabinoid-induced effects are not so far known. The hydrophobic nature of these substances has suggested that cannabinoids resemble anaesthetic agents in their action, that is, they nonspecifically disrupt cellular membranes. Recent evidence, however, has supported a mechanism involving a G protein-coupled receptor found in brain and neural cell lines, and which inhibits adenylate cyclase activity in a dose-dependent, stereoselective and pertussis toxin-sensitive manner. Also, the receptor is more responsive to psychoactive cannabinoids than to non-psychoactive cannabinoids. Here we report the cloning and expression of a complementary DNA that encodes a G protein-coupled receptor with all of these properties. Its messenger RNA is found in cell lines and regions of the brain that have cannabinoid receptors. These findings suggest that this protein is involved in cannabinoid-induced CNS effects (including alterations in mood and cognition) experienced by users of marijuana.

Neurochemical effects of amphetamine metabolites on central dopaminergic and serotonergic systems.

Intrastriatal administration of the hydroxylated metabolites of amphetamine, p-hydroxyamphetamine (p-OHA) or p-hydroxy-norephedrine (p-OHNor), decreased local concentrations of dopamine and serotonin in a dose-dependent manner. Although both compounds reduced concentrations of the metabolites of dopamine, 5-hydroxyindoleacetic acid concentrations were elevated. After systemic treatment with p-OHA, striatal dopamine was also reduced. In contrast, only hypothalamic and hippocampal serotonin stores were altered significantly in rats treated with p-OHA systemically. Neither compound decreased the activities of tryptophan hydroxylase or tyrosine hydroxylase. Because p-OHA is metabolized to p-OHNor via dopamine beta-hydroxylase present in noradrenergic neurons, the direct effects of these compounds on dopaminergic and serotonergic variables can be observed in rats which receive intrastriatal drug treatment. p-OHA and p-OHNor were equally potent in decreasing dopamine concentrations. However, p-OHNor was more potent than p-OHA in decreasing serotonin concentrations. Both compounds more readily depleted dopamine compared to serotonin stores. Complete recovery of p-OHA-induced decreases in striatal dopamine occurred within 48 hr of intrastriatal administration and concurrent treatment with the dopamine uptake blocker, amfonelic acid, significantly attenuated the p-OHA-induced effects on dopamine.

Effects of methamphetamine on central monoaminergic systems in normal and ascorbic acid-deficient guinea pigs.

Repeated injections (s.c.) of methamphetamine (METH) were administered to normal and ascorbic acid-deficient (scorbutic) guinea pigs to assess a potential role for ascorbic acid in the METH-induced effects in central monoaminergic systems. The ascorbic acid-deficient condition differentially influenced the METH-induced responses of dopaminergic and serotonergic variables in the striatum: drug-induced changes in dopaminergic variables were identical in normal and scorbutic animals; METH-induced decreases in serotonergic variables [tryptophan hydroxylase activity, serotonin (5-HT) and 5-hydroxyindoleacetic acid concentrations], however, were prevented in scorbutic animals. The scorbutic condition did not alter significantly the distribution of METH in the brain, nor were striatal concentrations of dopamine (DA) or 5-HT affected. In vitro, ascorbic acid increased significantly DA-mediated [3H]5-HT release from striatal slices, thus suggesting a potential role for ascorbate in DA-mediated actions of METH on serotonergic systems. Although supplemental ascorbate failed to restore the METH-induced serotonergic effects in scorbutic guinea pigs, these data suggest that, in a normal animal, the effects of multiple injections of METH, on serotonergic systems, involve ascorbic acid.

Characterization of dopaminergic influence on striatal-nigral neurotensin systems.

Dopamine depletion by treatment with alpha-methyl-p-tyrosine had no effect on methamphetamine-mediated increases in striatal neurotensin (NT) concentrations but significantly attenuated nigral increases; the attenuation in the nigral response was reversed by L-DOPA. Blockade of D2-receptors, with sulpiride, by itself increased striatal NT levels, while having no effect on the nigral NT system or its response to methamphetamine. In contrast, D1 blockade with SCH 23390 had no effect of its own on NT levels but significantly blocked the methamphetamine-induced actions in both the striatal and nigral NT systems.

Effects of cocaine on methamphetamine-induced neurochemical changes: characterization of cocaine as a monoamine uptake blocker.

Many of the rewarding aspects of cocaine are thought to be due to the ability of this stimulant to block reuptake of monoamines. However, because of its ability also to cause transmitter release, it is difficult to examine the properties of cocaine as an uptake blocker using in vitro techniques such as tissue slices or synaptosomes. Thus, we have evaluated cocaine as a blocker of dopamine and 5-hydroxytryptamine uptake processes by determining the in vivo effect of concurrent administrations of cocaine on the neurochemical effects of methamphetamine treatments. These findings demonstrated that cocaine like 5-hydroxytryptamine uptake blockers such as citalopram, greatly attenuated or blocked decreases in striatal and cortical tryptophan hydroxylase activities and concentrations of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid induced by multiple and single methamphetamine administrations. In contrast to other dopamine uptake blockers, such as amfonelic acid, cocaine did not attenuate the methamphetamine effects on striatal tyrosine hydroxylase activity and the concentrations of dopamine, dihydroxyphenylacetic acid and homovanillic acid. The neurochemical findings were correlated with behavioral analyses.

Ascorbic acid-deficient condition alters central effects of methamphetamine.

Effects of multiple doses of methamphetamine (METH) were examined in normal and ascorbic acid-deficient (scorbutic) guinea pigs. METH-induced decreases of striatal serotonin concentrations were completely antagonized in scorbutic animals. Elevations of nigral substance P-like immunoreactivity also differed significantly in METH-treated scorbutic compared to METH-treated normal animals. Various lines of evidence indicate that dopamine is an essential mediator of METH-induced effects in both serotonergic and substance P systems in the brain areas examined; however, results from the present study indicate that, along with dopamine, ascorbic acid also plays a role in mediating the effects of METH in the central nervous system.

Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on striatal tyrosine hydroxylase and tryptophan hydroxylase in rat.

The results reported here indicate that treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) caused significant changes in the dopamine-synthesizing enzyme, tyrosine hydroxylase. The authors examined the effects of two doses of MPTP on the activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) in the striatum, and also the time-course of these effects. Rats received an intraperitoneal loading dose, followed by a 24-hr infusion of MPTP (total doses of 21 or 42 mg) from subcutaneously-implanted osmotic pumps. Seven days after treatment, the activity of tyrosine hydroxylase was decreased by MPTP (42 mg); however, the activity of tryptophan hydroxylase was not affected. In time-course experiments, the activity of tyrosine hydroxylase was maximally reduced at 3 and 7 days after treatment with MPTP (42 mg). The activity of tryptophan hydroxylase did not significantly change at any time-point. Concurrent administration of haloperidol (HALO; 2 mg/kg, 4 doses) with MPTP significantly enhanced the depression of the activity of tyrosine hydroxylase in the striatum caused by MPTP, while treatment with haloperidol alone had no such effect. Concentrations of dopamine in the striatum were maximally decreased to approx. 50% of control in animals treated with haloperidol and MPTP (42 mg), whereas treatment with MPTP alone decreased concentrations of dopamine to approx. 70% of control.

In vitro release of tritiated monoamines from rat CNS tissue by the neurotoxic compound 1-methyl-phenyl-tetrahydropyridine.

The effects of the neurotoxic compound, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (1-MPTP), on monoamine release was determined using slices of rat corpus striatum or cerebral cortex preloaded with [3H]dopamine or [3H]serotonin, respectively. 1-MPTP released both monoamines in a concentration-dependent manner. [3H]Dopamine release was unaffected by tetrodotoxin, the absence of Ca2+, and nicotinic or muscarinic cholinergic antagonists. Dopamine uptake inhibitors had no effect on release from normal striatal slices but appeared to attenuate 1-MPTP-induced release from striatal slices of reserpine-pretreated rats.