The Effect of Chronic Activation of the Novel Endocannabinoid Receptor GPR18 on Myocardial Function and Blood Pressure in Conscious Rats

Abstract

Although acute activation of the novel endocannabinoid receptor GPR18 causes hypotension, there are no reports on GPR18 expression in the heart or its chronic modulation of cardiovascular function. In this study, after demonstrating GPR18 expression in the heart, we show that chronic (2 weeks) GPR18 activation with its agonist abnormal cannabidiol (abn-cbd; 100 µg·kg·d; i.p) produced hypotension, suppressed the cardiac sympathetic dominance, and improved left ventricular (LV) function (increased the contractility index dp/dtmax and reduced LV end-diastolic pressure, LVEDP) in conscious rats. Ex vivo studies revealed increased: (1) cardiac and plasma adiponectin (ADN) levels; (2) vascular (aortic) endothelial nitric oxide synthase (eNOS) expression, (3) vascular and serum nitric oxide (NO) levels; (4) myocardial and plasma cyclic guanosine monophosphate (cGMP) levels; (5) phosphorylation of myocardial protein kinase B (Akt) and extracellular signal regulated kinase 1/2 (ERK1/2) along with reduced myocardial reactive oxygen species (ROS) in abn-cbd treated rats. These biochemical responses contributed to the hemodynamic responses and were GPR18-mediated because concurrent treatment with the competitive GPR18 antagonist (O-1918) abrogated the abn-cbd-evoked hemodynamic and biochemical responses. The current findings present new evidence for a salutary cardiovascular role for GPR18, mediated, at least partly, via elevation in the levels of adiponectin.

Figure 1

Effect of 2-week treatment with the GPR18 agonist abn-cbd, its antagonist O-1918 (100 µg/kg/day, i.p, each) or their combination on mean arterial pressure (MAP; A) and heart rate (HR; B) in conscious male rats (n = 6–8). Values are mean ± SEM. *P < 0.05 versus control.

Figure 2

Effect of 2-week treatment with the GPR18 agonist abn-cbd, its antagonist O-1918 (100 µg/kg/day, i.p, each) or their combination on left ventricular (LV) maximum contraction velocity (dp/dt_max; A); LV maximum relaxation velocity (dp/dt_min; B); and LV end diastolic pressure (LVEDP; C) in conscious male rats (n = 6–8). Values are mean ± SEM. *P < 0.05 versus control.

Figure 3

(A) Low-frequency component of spectral analysis of RRI (0.25 to 0.75 Hz), index of cardiac sympathetic tone; (B) high-frequency component of the spectral analysis of RRI (0.75 to 3 Hz), index of cardiac vagal tone, and (C) LF_RRI/HF_RRI ratio as a measure of sympathovagal balance in conscious male rats (n = 6–8) treated with GPR18 agonist abn-cbd, its antagonist O-1918 (100 µg/kg/day, i.p, each) or their combination for 2 weeks. Values are mean ± SEM. *P < 0.05 versus control.

Figure 4

(A) Expression of GPR18 (38 kDa) in the rat heart compared with expression in the spleen (positive controls) and liver (negative control) (tissues are obtained from naïve untreated male rats n = 5); (B) Dual-labeled immunofluorescence of naïve rat heart showing GPR18 expression in cardiomyocytes in presence or absence of the specific GPR18 antibody blocking peptide and (C) Western blot showing the effect of 2-weeks treatment with the GPR18 agonist abn-cbd, its antagonist O-1918 or their combination (100 µg/kg/day, i.p, each) on myocardial GPR18 expression in male rats (n = 6). Protein expression was presented as percent of control. Values are mean ± SEM.

Figure 5

Effect of 2-week treatment with the GPR18 agonist abn-cbd, its antagonist O-1918 (100 µg/kg/day, i.p, each) or their combination on the levels of plasma adiponectin (ADN; A); serum nitric oxide (NO; B); vascular (aortic) NO (C); and vascular eNOS expression (Western blot; D) in male rats (n = 6). Values are mean ± SEM. *P < 0.05 versus control, and ^#P < 0.05 versus abn-cbd.

Figure 6

Effect of 2-week treatment with the GPR18 agonist abn-cbd, its antagonist O-1918 (100 µg/kg/day, i.p, each) or their combination on cardiac adiponectin (ADN) levels measured by western blot analysis (A) and by immunohistochemical staining (B) in male rats (n = 6). Values are mean ± SEM. *P < 0.05 versus control.

Figure 7

Western blot analyses showing the effect of 2-week treatment with the GPR18 agonist abn-cbd, its antagonist O-1918 (100 µg/kg/day, i.p, each) or their combination on the myocardial: p-Akt (A), p-ERK1/2 (B), eNOS expression (C) and p-eNOS (D). Data represent mean values of the ratio of p-Akt, p-ERK1/2 or p-eNOS normalized to the corresponding total protein while eNOS values are normalized to GAPDH in male rats (n = 8). Protein expression was presented as percent of control. Values are mean ± SEM. *P < 0.05 versus control.

Figure 8

Effect of 2-week treatment with the GPR18 agonist abn-cbd, its antagonist O-1918 (100 µg/kg/day, i.p, each) or their combination on myocardial NO (A), myocardial ROS (B), myocardial cGMP (C) and plasma cGMP (D) levels in male rats (n = 8). Values are mean ± SEM. * P < 0.05 versus control and ^#P < 0.05 versus abn-cbd.

Figure 9

Suggested mechanisms for chronic GPR18-mediated favorable hemodynamic and cardio-protective effects in conscious rats. The sequence of events is based on the findings that chronic (2 weeks) GPR18 activation (abn-cbd): (i) reduced BP and sympathetic prevalence (lower LF_RRI/HF_RRI ratio) (Figs. 1 and 3), and improved cardiac function (Fig. 2) in conscious rats; (ii) increased plasma and cardiac adiponectin (ADN) (Figs. 5A and 6) and enhanced vascular eNOS expression along with elevated vascular and plasma NO levels (Fig. 5); (iii) reduced myocardial reactive oxygen species (ROS) level (Fig. 8B); (iv) enhanced Akt and ERK1/2 phosphorylation (Figs. 7A and B). These abn-cbd evoked biochemical and cardiovascular responses were abrogated by O-1918, the selective GPR18 antagonist (see text for details).