BACKGROUND AND PURPOSE The cannabinoid CB1 receptor is primarily thought to be functionally coupled to the Gi form of G proteins, through which it negatively regulates cAMP accumulation. Here, we investigated the dual coupling properties of CB1 receptors and characterized the structural determinants that mediate selective coupling to Gs and Gi. EXPERIMENTAL APPROACH A cAMP‐response element reporter gene system was employed to quantitatively analyze cAMP change. CB1/CB2 receptor chimeras and site‐directed mutagenesis combined with functional assays and computer modelling were used to determine the structural determinants mediating selective coupling to Gs and Gi. KEY RESULTS CB1 receptors could couple to both Gs‐mediated cAMP accumulation and Gi‐induced activation of ERK1/2 and Ca2+ mobilization, whereas CB2 receptors selectively coupled to Gi and inhibited cAMP production. Using CB1/CB2 chimeric receptors, the second intracellular loop (ICL2) of the CB1 receptor was identified as primarily responsible for mediating Gs and Gi coupling specificity. Furthermore, mutation of Leu‐222 in ICL2 to either Ala or Pro switched G protein coupling from Gs to Gi, while to Ile or Val led to balanced coupling of the mutant receptor with Gs and Gi. CONCLUSIONS AND IMPLICATIONS The ICL2 of CB1 receptors and in particular Leu‐222, which resides within a highly conserved DRY(X)5PL motif, played a critical role in Gs and Gi protein coupling and specificity. Our studies provide new insight into the mechanisms governing the coupling of CB1 receptors to G proteins and cannabinoid‐induced tolerance.

This study suggested that NPR3 rs2270915 polymorphism was associated with decreased SBP level marginally in EH patients in a Chinese Han population, and the polymorphism may function through decreasing NPR3 mRNA expression and ANP level.

Exposure to Clopidogrel Through Modulation of P-Glycoprotein but Does Not Alter Its Antithrombotic Activity" by Oh et al., which elucidated the effect of aspirin coadministration on the pharmacokinetics/ pharmacodynamics of clopidogrel in healthy subjects. 1 The results of their study demonstrated that the clopidogrel area under the concentration-time curve (AUC) significantly decreased, but the AUC of H4 remained unchanged and RPI increased after aspirin administration. In addition, the plasma level of miR-27a, a microRNA involved in regulation of P-glycoprotein (P-gp) expression, was significantly increased after aspirin administration. However, no change in plasma MDR1 mRNA or miR-451 was observed. The authors proposed that coadministration of low-dose aspirin decreased the systemic exposure to clopidogrel, but the antithrombotic effect of clopidogrel was not affected.Previous study has indicated that prolonged use of aspirin may reduce the intestinal absorption of clopidogrel by inducing P-gp expression in human and rat intestinal cells. 2 And a similar trend in the PK profile of clopidogrel was also observed in the study of Oh et al., but the decrements were not statistically significant due to an insufficient dose of aspirin. Although the exposure to clopidogrel was lowered, the level of H4 was not changed after aspirin administration. For these seemingly contradictory results, the authors give two explanations. One is that the decrement in exposure to clopidogrel might not be sufficient to alter the exposure to H4. The other is bioactivation of effluxed clopidogrel to H4 in the intestine by CYP3A4. But we consider that there is another more reasonable explanation for this phenomenon. In a study presented previously, we demonstrated that low-dose aspirin significantly increased the activity of CYP2C19 and CYP3A4 in healthy volunteers. 3 As 15% of the clopidogrel converted to the biologically active thiol metabolite via a two-step CYP-dependent oxidative pathway, even small changes in activities of CYP isoenzymes seem to influence formation of its active metabolite. 4 Given the fact that oxidation of clopidogrel is catalyzed by CYP2C19, CYP3A4, and CYP3A5, 5 increased CYP2C19 and CYP3A4 activity by low-dose aspirin likely increases the activation of clopidogrel, thus enhancing the efficacy of this antiplatelet prodrug. So aspirin coadministration eventually does not alter the exposure of H4 by the combined two effects of aspirin. Further studies are needed to confirm the clinical significance regarding aspirin-increased CYP2C19 and CYP3A4 activity on clopidogrel activation. ACKNOWLEDGMENTS