Five mutations in the GABA A alpha6 gene 5' flanking region are associated with a reduced basal and ethanol-induced alpha6 upregulation in mutated Sardinian alcohol non-preferring rats.

The presence of four nucleotide changes and a three base-pair deletion in the GABA A alpha6-subunit promoter is described in Sardinian alcohol non-preferring rats, selectively bred for their ethanol aversion. These mutations are associated with the R100Q alpha6 intragenic mutation that was previously characterized in the same animals. The possibility that these mutated nucleotides alter the ethanol-induced upregulation of the alpha6 gene was investigated by measuring cerebellar alpha6 mRNA levels after a chronic ethanol liquid diet in sNP rat. Real-time quantitative PCR showed an increased alpha6 gene expression after ethanol ingestion in normal and mutated rats. However, lower amounts of alpha6 mRNA levels were detected both in control and in ethanol-treated sNP rats carrying the five promoter and the intragenic mutations in a homozygous state. Using the electromobility shift assay, specific DNA binding sites were found in cerebellar extracts of the alpha6 regions comprising the five mutations. These results suggest that one or more of the mutated binding sites that were found in the 5' flanking alpha6 region may be a consensus sequence for regulatory factors which are responsible for both basal and ethanol-induced alpha6 gene expression.

The cerebellar GABAA alpha6 subunit is differentially modulated by chronic ethanol exposure in normal (R100R) and mutated (Q100Q) sNP rats.

Sardinian alcohol non-preferring (sNP) rats carry a point mutation (R100Q) in the cerebellar expressed GABAA receptor alpha6 subunit gene, leading to a higher sensitivity to ethanol and diazepam. The role of the alpha6 subunit gene cluster in the ethanol non-preferring phenotype was here investigated by measuring the levels of alpha1, alpha6 and gamma2 peptide in the cerebellum of normal (RR) and mutated (QQ) sNP rats after 2 weeks of chronic ethanol administration. Western blot analysis revealed that the alpha6 subunit is increased in RR sNP rats after chronic ethanol exposure (25.44%+/-8.69 versus control), while it remained unchanged in mutated QQ sNP rats. Interestingly, chronic ethanol administration decreased alpha1 peptide levels in the cerebellum of both rat lines to a similar extent (30.99%+/-6.74 and 27.12%+/-9.83 in RR and QQ rats, respectively), while gamma2 peptide levels remained unchanged. To further correlate the genetic and biochemical difference of the normal and mutated sNP rats with their aversive phenotype, we exposed sNP rats to a protocol of acquisition and maintenance of ethanol drinking. QQ sNP rats drank less ethanol than RR rats during the acquisition phase, but such difference was lost during the maintenance phase. These data may contribute to elucidating the mechanisms of alcohol avoidance in rat lines selected for this behavior when exposed to ethanol solution.

Characterization of wild-type (R100R) and mutated (Q100Q) GABAA alpha 6 subunit in Sardinian alcohol non-preferring rats (sNP).

Sardinian alcohol non-preferring (sNP) rats, selected for their low ethanol preference and consumption, carry a point mutation (R100Q) in the gene coding for GABA(A) receptor alpha(6) subunit, which becomes more sensitive to diazepam-evoked GABA currents. We performed binding studies in the cerebellum of normal (RR) and mutated (QQ) sNP rats using [3H]Ro 15-4513, an inverse agonist for the benzodiazepine site which binds both diazepam insensitive and diazepam sensitive sites. Saturation curves performed on cerebellar membrane from genotyped rats indicated an higher affinity of [3H]Ro 15-4513 for GABA(A) receptors in QQ with respect to RR rats (K(d) values 4.0+/-0.67 and 6.24+/-0.95 nM, respectively), with similar B(max) values (3.5+/-0.25 and 3.9+/-0.39 pmol/mg protein, respectively). Diazepam displacement curves showed a two component model for both genotypes, with similar K(i1) values for QQ and RR (3.6+/-0.62 and 4.9+/-0.33 nM, respectively). In QQ rats diazepam is able to completely displace [3H]Ro 15-4513 (K(i2)=1.48+/-0.27 microM), while in RR rats the diazepam sensitive sites are still present (K(i2)>10 microM). The basal mRNA and protein expression level of the alpha(6) subunit were similar in RR and QQ rats. The electrophysiological profile of oocytes of Xenopus laevis injected with cerebellar synaptosomes showed that ethanol positively modulated GABA-evoked currents significantly more in QQ than in RR rats. These data contribute to the characterization of the function of GABA(A) alpha(6) subunit and its involvement in determining alcohol related behavior.

Molecular characterization of new polymorphisms at the beta2, alpha1, gamma2 GABA(A) receptor subunit genes associated to a rat nonpreferring ethanol phenotype.

Recent preclinical and clinical studies have indicated a possible involvement of the genes encoding for the GABA(A) receptor subunits alpha6, beta2, alpha1 and gamma2 in the genetic susceptibility to alcohol abuse. We have recently found an (R) to (Q) mutation in codon 100 of the alpha6 GABA(A) subunit, that segregated in a rat line selectively bred for its voluntary ethanol aversion, Sardinian alcohol nonpreferring (sNP), but not in their Sardinian alcohol preferring (sP) counterpart, selected for its ethanol preference. In the present study the molecular composition of other GABA(A) subunits (beta2, alpha1 and gamma2) were analyzed in order to further investigate the involvement of the GABA(A) receptors in the genetic predisposition to voluntary alcohol intake. Automated sequencing analysis indicated the presence of six new silent substitutions (289 T-->C in the beta2 gene; 115 G-->A in the alpha1 gene; 157 G-->A, 174 C-->T, 347 A-->G and 385 A-->T in the gamma2 gene), in sNP but not in sP rats. These polymorphisms were linked to the alpha6 R100Q mutation previously described in sNP rats. The strict association between the alpha6 point mutation and the new polymorphisms found in the beta2, alpha1 and gamma2 genes, demonstrate that such genes belong to the same cluster and are inherited together in the rat. These results sustain the synteny for these clusters between the rodent and human genomes, and suggest that mutated GABA(A) beta2, alpha6, alpha1 and gamma2 subunit genes might contribute to the expression of an ethanol nonpreferring phenotype in a rat line that voluntarily avoids alcoholic solutions.

Differential distribution of functional cannabinoid CB1 receptors in the mouse gastroenteric tract.

Recently, the gastrointestinal pharmacology of cannabinoid CB(1) receptors has been extensively explored. We employed western blotting and immunohistochemistry techniques to study the distribution of the cannabinoid CB(1) receptor protein in the mouse gastroenteric tract. The cannabinoid CB(1) receptor peptide was detected by western blotting only in its glycosylated form (63 kDa) with a significant differential distribution. The highest levels of expression were detected in the stomach and in the colon, while the pyloric valve was devoid of any cannabinoid CB(1) receptor protein. The immunohistochemical study showed intense cannabinoid CB(1) receptor immunoreactivity in ganglia subadjacent to the gastric epithelium and in the smooth muscle layers of both the small and large intestine. Only the small intestine showed (-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)-phenyl]-4-(3-hydroxylpropyl) cyclohexan-1-ol) ([3H]CP 55,940) specific binding (27%). These receptors mediated pharmacologically significant effects since the cannabinoid CB(1) receptor agonist R(-)-7-hydroxy-delta-6-tetra-hydrocannabinol-dimethylheptyl (HU 210) dose dependently inhibited gastrointestinal transit up to 70%, while the cannabinoid CB(1) receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide (SR 141716A) increased gastrointestinal transit. Moreover, the dose of 0.3 microg/kg of HU 210, devoid per se of any activity on mouse intestinal propulsion, blocked the increased gastroenteric transit induced by the cannabinoid CB(1) antagonist SR 141716A.

Evidence for functional CB1 cannabinoid receptor expressed in the rat thyroid.

OBJECTIVE: Previous reports have shown that the Delta(9)-tetrahydrocannabinol (Delta(9)TCH), the major psychoactive cannabinoid components of marijuana, is able [corrected] to inhibit thyroid hormonal activity. The aim of this study was to characterize the CB1 functional expression in the rat thyroid by a multi-methods approach. METHODS AND RESULTS: RT-PCR was used to detect the mRNA expression of the CB1 cannabinoid receptor (17.8+/-4.0% of the normalizing reference gene beta(2) microglobulin), as well as the expression of the endocannabinoid hydrolyzing enzyme, fatty acid amide hydrolase (46.9+/-4.3% of beta(2) microglobulin), in the rat thyroid gland. The CB1-encoded protein was detected in its glycosylated form (63 kDa) by Western blot, employing a polyclonal antibody, while CB1 immunohistochemical localization showed an intracellular positive staining in both follicular and parafollicular cells. In addition, a 30% decrease in serum levels of both 3,5,3' tri-iodothyronine (T(3)) and thyroxine (T(4)) was detected 4 h after the administration of the synthetic cannabinoid receptor agonist, WIN 55,212-2 (10 mg/kg i.p.). These effects were antagonized by pretreatment with the CB1 antagonist SR 141716A (3 mg/kg i.p.); thyrotrophin levels were unaffected by both treatments. CONCLUSION: These data indicate that functional CB1 receptors which are able to modulate the release of T(3) and T(4) are expressed in the rat thyroid, and suggest a possible role of cannabinoids in the regulation of rat thyroid hormonal activity.