The Role of Diacylglycerol Lipase in Constitutive and Angiotensin AT1 Receptor-stimulated Cannabinoid CB1 Receptor Activity

Abstract

The cannabinoid CB1 receptor (CB1R) is a G protein-coupled receptor, which couples to the G i/o family of heterotrimeric G proteins. The receptor displays both basal and agonist-induced signaling and internalization. Although basal activity of CB1Rs is attributed to constitutive (agonist-independent) receptor activity, studies in neurons suggested a role of postsynaptic endocannabinoid (eCB) release in the persistent activity of presynaptic CB1Rs. To elucidate the role of eCBs in basal CB1R activity, we have investigated the role of diacylglycerol lipase (DAGL) in this process in Chinese hamster ovary (CHO) cells, which are not targeted specifically with eCBs. Agonist-induced G protein activation was determined by detecting dissociation G protein subunits expressed in CHO cells with bioluminescence resonance energy transfer (BRET), after labeling the ␣ and ␤ subunits with Renilla luciferase and enhanced yellow fluorescent protein (EYFP), respectively. Preincubation of the cells with tetrahydrolipstatin (THL), a known inhibitor of DAGLs, caused inhibition of the basal activity of CB1R. Moreover, preincubation of CHO and cultured hippocampal neurons with THL increased the number of CB1Rs on the cell membrane, which reflects its inhibitory action on CB1R internalization in non-simulated cells. In CHO cells co-expressing CB1R and angiotensin AT 1 receptors, angiotensin II-induced G o protein activation that was blocked by both a CB1R antagonist and THL. These data indicate that cell-derived eCB mediators have a general role in the basal activity of CB1Rs in non-neural cells and neurons, and that this mechanism can be stimulated by AT 1 receptor activation.

Angiotensin II-induced activation of central AT1 receptors exerts endocannabinoid-mediated gastroprotective effect in rats

“…Namely, it was observed that inhibition of DAGL in these cells led to inhibition of the constitutive activity of CB 1 receptors, and similar results were obtained in HEK-293 cells (Turu et al, 2007(Turu et al, , 2009). The transactivation of cannabinoid CB1 receptors by Ang II is supported by the finding that showed an inhibitory effect of AM 251, inverse agonist of CB 1 receptors, on the hypertensive effect of Ang II injected directly into the hypothalamic paraventricular nucleus of anaesthetized rats (Gyombolai et al, 2012).…”

“…It was recently shown that activation of the Gq/11-coupled angiotensin AT 1 receptor by angiotensin II resulted in activation of cannabinoid CB 1 receptors via a DAGL-dependent mechanism in CHO cells (Turu et al, 2007). Namely, it was observed that inhibition of DAGL in these cells led to inhibition of the constitutive activity of CB 1 receptors, and similar results were obtained in HEK-293 cells (Turu et al, 2007(Turu et al, , 2009).…”

“…Paracrine transactivation of CB 1 receptors by co-expressed type 1 angiotensin receptors (AT 1 ) in Chinese hamster ovary (CHO) cells was observed recently (Turu et al, 2007). AT 1 receptor is a Gq-protein-coupled receptor.…”

“…From DAG 2-AG is generated by diacylglycerol lipases (DAGLs). Accordingly, inhibition of DAGL in CHO cells interfered with the activation of CB 1 receptors (Turu et al, 2007). The inhibitory effect of DAGL-inhibitor on these processes suggests that these actions are mediated mainly by 2-AG (Bisogno et al, 2005;Makara et al, 2005;Turu et al, 2009).…”

Differential β-arrestin2 requirements for constitutive and agonist-induced internalization of the CB1 cannabinoid receptor

“…spontaneous internalization in the absence of CB 1 R agonists) has also been detected in hippocampal neurons, as well as in CHO and HEK cells (Leterrier et al, 2004(Leterrier et al, , 2006McDonald et al, 2007a;Turu et al, 2007). It has been suggested that constitutive CB 1 R internalization is the consequence of its basal activity, since inverse agonist treatment or inhibition of basal activity with a DAG lipase inhibitor (e.g.…”