No abstract

Effects of cannabinoids on endogenous potassium and calcium currents in HEK293 cells were studied using the whole-cell variant of the patch-clamp technique. The cannabinoid agonists WIN 55,212-2, methanandamide, and anandamide (1 microM) decreased the calcium current by 53.1 +/- 2.6, 47.5 +/- 1.2, and 38.8 +/- 3.1%, respectively, after transfection of human CB1 cannabinoid receptor (hCB1) cDNA into HEK293 cells. The delayed rectifier-like current was not changed after application of these agonists, but the inward rectifier was increased by 94.0 +/- 3.6, 83.7 +/- 5.1, and 63.0 +/- 2.5% after application of WIN 55,212-2, methanandamide, and anandamide, respectively. The effects of the cannabinoid antagonists (AM251, AM281, and AM630) on the inward rectifier and calcium currents were the opposite of those seen with cannabinoid agonists; thus, these compounds act as inverse agonists in this preparation. These results suggest that endogenous inward rectifier and calcium currents are modulated by cannabinoids in HEK293 cells, and that some expressed receptors may be constitutively active.

The aim of this work was to determine whether Capsaicin may exert a vascular regulation through the activation of CB1 and/or CB2 receptors causing vasorelaxation in the rat aorta. Our results show the location of TRPV1 mainly in the endothelial and smooth muscle cells membrane. Nevertheless, Capsaicin caused vasorelaxation of this artery through a mechanism independent of TRPV1, since the specific antagonists Capsazepine and SB-366791 did not block the effect of Capsaicin. Because the significant expression of CB1 and CB2 receptors has been previously reported in the rat aorta, we used antagonists for these two receptors prior to the addition of Capsaicin. In these experiments, we found that the inhibition of CB1 using AM281, decreases the vasorelaxant effect caused by Capsaicin. On the other hand, the vasorelaxant effect is not altered in the presence of the CB2 receptor antagonist AM630. Furthermore, a partial decrease of the effect of Capsaicin was also seen when L-type calcium channels are blocked. A complete block of Capsaicin-induced vasorelaxation was achieved using a combination of Verapamil and AM281. In accordance to our results, Capsaicin-induced vasorelaxation of the rat aorta is neither dependent of TRPV1 or CB2 receptors, but rather it is strongly suggested that a tandem mechanism between inactivation of L-type calcium channels and the direct activation of CB1 receptors is involved. These findings are supported by CB1 docking simulation which predicted a binding site on CB1 receptors for Capsaicin.

This study aimed to investigate the function of the cannabinoid receptor in the neuromuscular junction of the frog (Rana pipiens). Miniature end-plate potentials were recorded using the intracellular electrode recording technique in the cutaneous pectoris muscle in the presence of the cannabinoid agonists WIN55212-2 (WIN; R-(ϩ)- [2,3-dihydro-5-methyl-3-[(morpholinyl) . Adding WIN to the external medium decreased the frequency and amplitude of the miniature end-plate potentials (MEPPs); the WIN EC 50 value was 5.8 Ϯ 1.0 M. Application of ACPA, a selective agonist of cannabinoid receptor CB 1 , also decreased the frequency of the MEPPs; the ACPA EC 50 value was 115.5 Ϯ 6.5 nM. The CB 2 antagonist AM630 did not inhibit the effects of WIN, indicating that its action is not mediated through the CB 2 receptor. However, the CB 1 antagonist AM281 inhibited the effects of WIN and ACPA, suggesting that their actions are mediated through the CB 1 receptor. Pretreatment with the pertussis toxin inhibited the effects of WIN and ACPA, suggesting that their effects are mediated through G i/o protein activation. The N-type Ca 2ϩ channel blocker -conotoxin GVIA (-CgTX) diminished the frequency of the MEPPs, with an -CgTX EC 50 value of 2.5 Ϯ 0.40 M. Blocking the N-type Ca 2ϩ channels with 5 M -CgTX before addition of ACPA to the bath had no additional inhibitory effect on the MEPPs, whereas in the presence of 1 M -CgTX, ACPA had an additional inhibition effect. These results suggest that cannabinoids modulate transmitter release in the end-plate of the frog neuromuscular junction by activating CB 1 cannabinoid receptors in the nerve ending.

The pathologic cardiac remodeling has been widely documented; however, the physiological cardiac remodeling induced by pregnancy and its reversion in postpartum are poorly understood. In the present study we investigated the changes in collagen I (Col I) and collagen III (Col III) mRNA and protein levels in left ventricle from rat heart during pregnancy and postpartum. Col I and Col III mRNA expression in left ventricle samples during pregnancy and postpartum were analyzed by using quantitative PCR. Data obtained from gene expression show that Col I and Col III in left ventricle are upregulated during pregnancy with reversion in postpartum. In contrast to gene expression, the protein expression evaluated by western blot showed that Col I is downregulated and Col III is upregulated in left ventricle during pregnancy. In conclusion, the pregnancy differentially regulates collagens types I and III in heart; this finding could be an important molecular mechanism that regulates the ventricular stiffness in response to blood volume overload present during pregnancy which is reversed in postpartum.