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    • Using RT PCR we demonstrated the expression of

    2022-01-12

    Using RT-PCR we demonstrated the expression of GPR55 receptor mRNA in the ileum and colon of mice, which is in good agreement with previous reports (Lin et al., 2011). The quantitative analysis showed the abundance of GPR55 mRNA in the mucosa of the ileum and colon. In contrast, in LMMP preparations the GPR55 expression was significantly higher in the colon compared to the ileum, where expression is relatively low. Using immunohistochemistry we further revealed that GPR55 immunoreactivity can be localized on ganglion PHA-793887 and nerve fibres in the myenteric plexus of the colon, but hardly in the ileum myenteric plexus, strengthening our findings based on the RT PCR technique. We were also interested whether the GPR55 localized on myenteric neurons is involved in the regulation of intestinal motility and whether the differences in receptor expression translates to differences in pharmacological response to GPR55 activation. For the studies on the in vitro and in vivo motility we used O-1602, a GPR55 agonist, and a GPR55-selective antagonist CBD (Ryberg et al., 2007). We found no effect of O-1602 or CBD on basal tone or on pharmacologically stimulated smooth muscle, suggesting that a direct action of O-1602 on the smooth muscle was unlikely. These findings were supported by our immunohistochemical studies showing the lack of GPR55 staining on smooth muscle cells. O-1602 significantly reduced electrically evoked contractions suggesting a neural site of the GPR55 receptor. This effect was far more pronounced in the colon than in the ileum, showing distinct regional differences of action of O-1602 in the GI tract. In the colon the effect of O-1602 was comparable to that of classical cannabinoids, such as the CB1/CB2 agonist WIN55,212-2. Interestingly, the difference between the effects observed in the ileum and the colon was less pronounced for WIN55,212-2 than O-1602, suggesting significant differences between the mechanisms by which these different classes of cannabinoids exert their action. For atypical cannabinoids, such as O-1602, the responding receptors are only poorly characterized. Functional studies indicate that atypical cannabinoids interact with CB1, CB2 and GPR55 receptors, as well as other structures. In our hands, the actions of O-1602 were neither altered in the presence of a CB1 or a CB2 antagonist, nor observed in tissues of CB1,2−/− mice. This indicates that the O-1602-mediated effects are independent of the classical cannabinoid receptors. As some recent studies suggested that commonly used CB1 antagonists may bind and have effects at receptors other than CB1, we were also interested whether these antagonists would influence O-1602 effects in the GI tissue of CB1,2−/− mice. In our hands such effects were not observed. Our pharmacological in-vitro studies confirm a recent publication where comparable experiments resulted in comparable effects and where O-1602 effects were additionally shown to be absent in tissues from GPR55−/− mice (Ross et al., 2012). The logical next question is whether these GPR55 mediated effects are of relevance in in vivo motility and to address this, we performed in-vivo motility studies in wild type and in GPR55−/− mice. CBD was recently reported to have antagonist effect at the GPR55 receptor (Ryberg et al., 2007). In our study the in vitro effects of O-1602 in the ileum and colon were antagonized by CBD, confirming that GPR55 mediates its actions. CBD did not alter the effects of WIN55,212-2 indicating that the WIN55,212-2 effect on GI motility does not involve GPR55 activation, which is in agreement with receptor binding studies suggesting that WIN55,212-2 does not bind to the GPR55 receptor (Ryberg et al., 2007). We further investigated the actions of O-1602 in vivo. O-1602 slowed whole gut transit and delayed colonic expulsion and, in contrast to the effects of WIN55,212-2 on in vivo motility, these effects were not sensitive to CB1 receptor antagonists. We also extended our in vitro observations to the in vivo finding that the effects of O-1602, but not those of WIN55,212, were antagonized by CBD. Again, this supports the idea of GPR55 mediating the inhibitory effects of O-1602 on GI motility.