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Abstract:Recent studies have shown that the pharmacological tolerance observed after prolonged exposure to synthetic or plant-derived cannabinoids in adult rats is accompanied by down-regulation/desensitization of brain cannabinoid receptors. However, no evidence exists on possible changes in the contents of the endogenous ligands of cannabinoid receptors in the brain of cannabinoid-tolerant rats. The present study was designed to elucidate this possibility by measuring, by means of isotope dilution gas chromatography/mass spectrometry, the contents of both anandamide (arachidonoylethanolamide; AEA) and its biosynthetic precursor, N-arachidonoylphosphatidylethanolamine (NArPE), and 2-arachidonoylglycerol (2-AG) in several brain regions of adult male rats treated daily with ⌬ 9 -tetrahydrocannabinol (⌬ 9 -THC) for a period of 8 days. The areas analyzed included cerebellum, striatum, limbic forebrain, hippocampus, cerebral cortex, and brainstem. The same regions were also analyzed for cannabinoid receptor binding and WIN-55,212-2-stimulated guanylyl-5Ј-O-(␥-[ 35 S]thio)-triphosphate ([ 35 S]GTP␥S) binding to test the development of the well known down-regulation/desensitization phenomenon. Results were as follows: As expected, cannabinoid receptor binding and WIN-55,212-2-stimulated [ 35 S]GTP␥S binding decreased in most of the brain areas of ⌬ 9 -THC-tolerant rats. The only region exhibiting no changes in both parameters was the limbic forebrain. This same region exhibited a marked (almost fourfold) increase in the content of AEA after 8 days of ⌬ 9 -THC treatment. By contrast, the striatum exhibited a decrease in AEA contents, whereas no changes were found in the brainstem, hippocampus, cerebellum, or cerebral cortex. The increase in AEA contents observed in the limbic forebrain was accompanied by a tendency of NArPE levels to decrease, whereas in the striatum, no significant change in NArPE contents was found. The contents of 2-AG were unchanged in brain regions from ⌬ 9 -THCtolerant rats, except for the striatum where they dropped significantly. In summary, the present results show that prolonged activation of cannabinoid receptors leads to decreased endocannabinoid contents and signaling in the striatum and to increased AEA formation in the limbic forebrain. The pathophysiological implications of these findings are discussed in view of the proposed roles of endocannabinoids in the control of motor behavior and emotional states. Key Words: Endocannabinoids-Anandamide-2-Arachidonoylglycerol-Cannabinoid receptor binding-Tolerance-⌬ 9 -Tetrahydrocannabinol.

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Most data on effects of natural and synthetic cannabinoids on anterior pituitary hormone secretion point out to a primary impact on the hypothalamus. There is also some evidence, however, of possible direct actions of these compounds on the anterior pituitary, although the presence of cannabinoid receptors in the pituitary has not been documented as yet. In the present study, we evaluated the presence of cannabinoid CB₁ receptor-mRNA transcripts in the pituitary gland by in situ hybridization. We observed CB₁ receptor-mRNA transcripts in the anterior pituitary and to a lesser extent in the intermediate lobe whereas they were absent in the neural lobe. We then examined whether CB₁ receptor-mRNA levels in both pituitary lobes responded to chronic activation by a specific agonist, as did receptors located in adjacent hypothalamic nuclei and in other brain regions. Daily administration of CP-55,940 for 18 days produced a small, but statistically significant paradoxical increase in CB₁ receptor-mRNA levels in the anterior pituitary, with no changes in the intermediate lobe, in contrast to reduced CB₁ receptor-mRNA levels observed in the ventromedial hypothalamic nucleus (VMN), and to decreased CB₁ receptor binding in the VMN and the arcuate nucleus. The time-course of up-regulation of CB₁ receptor-mRNA transcripts in the anterior lobe was biphasic; daily administration of Δ⁹-tetrahydrocannabinol produced an early and marked decrease in CB₁ receptor-mRNA levels after 1 and 3 days, followed by normalization after 7 days and by a small increase after 14 days. We also checked whether endogenous cannabinoid ligands are present in the anterior pituitary and the hypothalamus. Although anandamide itself was detected only in trace amounts, concentrations of its precursor N-arachidonoyl-phosphatidyl-ethanolamine and of 2-arachidonoyl-glycerol were found in both tissues, suggesting that endocannabinoids may be synthetized in the anterior pituitary. In summary, CB₁ receptors and corresponding ligands seem to be expressed in cells of the anterior and intermediate lobes of the pituitary, but the response of CB₁ receptor-mRNA transcripts in the anterior lobe to chronic agonist activation is different than the desensitization observed in hypothalamic nuclei.

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