Estrogen dampens central cannabinoid receptor 1-mediated neuroexcitation and pressor response in conscious female rats

Abstract

Activation of the rostral ventrolateral medulla (RVLM) cannabinoid receptor-1 (CB₁R) causes neuronal nitric oxide synthase (nNOS)-dependent increases in sympathetic activity, blood pressure (BP) and heart rate (HR) in male rats. However, it remains unknown if the CB₁R-mediated neurochemical and cardiovascular responses are influenced by the ovarian sex hormones, particularly estrogen (E₂). Therefore, we studied the effects of intra-RVLM CB₁R activation (WIN 55,212-2) on BP and HR in conscious female rats under the following hormonal states: (1) highest E₂ level (proestrus sham-operated, SO); (2) E₂-deprivation (ovariectomized, OVX); (3) OVX with E₂ replacement (OVXE2). Intra-RVLM WIN55,212-2 elicited dose (100-400 pmol) dependent pressor and tachycardic responses, in OVX rats, which replicated the reported responses in male rats. However, in SO and OVXE2 rats, the CB₁R-mediated pressor response was attenuated and the tachycardic response reverted to bradycardic response. The neurochemical findings suggested a key role for the upregulated RVLM sympathoexcitatory molecules phosphorated protein kinase B, phosphorated nNOS and reactive oxygen species in the exaggerated CB₁R-mediated BP and HR responses in OVX rats, and an E₂-dependent dampening of these responses. The intra-RVLM WIN55212-2-evoked cardiovascular and neurochemical responses were CB₁R-mediated because they were attenuated by prior CB₁R blockade (AM251). Our findings suggest that attenuation of RVLM neuroexcitation and oxidative stress underlies the protection conferred by E₂, in female rats, against the CB₁R-mediated adverse cardiovascular effects.

Keywords: Blood pressure; Cannabinoid receptor-1; Estrogen; Oxidative stress; Rostral ventrolateral medulla.

Fig. 1

A schematic presentation of cardiovascular and biochemical/molecular studies to investigate the effects of intra-RVLM cannabinoid receptor 1 (CB₁R) activation in estrogen (E₂) replete and devoid female rats.

Fig. 2

The plasma E₂ level (A) and the gain in body weight (B) in sham operated (SO), ovariectomized (OVX) and OVX with E₂ supplementation (OVXE2) rats before conducting the hemodynamic experiments. The plasma β-estradiol was measured in the proestrus phase of SO rat. Values are mean ± SEM. *p < 0.05 versus SO, #p < 0.05 versus OVX.

Fig. 3

Time-course and dose dependent changes in mean arterial pressure (ΔMAP) following intra-RVLM microinjection of the following doses of the CB₁R agonist WIN 55212-2: 100 pmol (A), 200 pmol (B) and 400 pmol (C), or vehicle in SO, OVX and OVXE2 rats. The bar graph (D) summarizes the area under the curve of ΔMAP. Values are mean ± SEM. *p < 0.05 versus SO vehicle, #p < 0.05 versus OVX vehicle, $p < 0.05 versus OVXE2 vehicle, ^p < 0.05 versus SO treated with same dose of WIN 55212-2, &p < 0.05 versus OVX treated with same dose of WIN 55212-2.

Fig. 4

Time-course and dose dependent changes in heart rate (ΔHR) following intra-RVLM microinjection of the following doses of the CB₁R agonist, WIN 55212-2: 100 pmol (A), 200 pmol (B) and 400 pmol (C), or vehicle in SO, OVX and OVXE2 rats. The bar graph (D) summarized the area under the curve of ΔHR. Values are mean ± SEM. #p < 0.05 versus OVX vehicle, $p < 0.05 versus OVXE2 vehicle, ^p < 0.05 versus SO treated with same dose of WIN 55212-2, &p < 0.05 versus OVX treated with same dose of WIN 55212-2.

Fig. 5

CB₁R expression in OVX and SO, and OVXE2 (A) and the effect of the CB₁R agonist, (WIN 55212-2) on the phosphorylation of Akt (B) and nNOS (C) in RVLM of OVX, SO and OVXE2 rats. Representative bands are shown under the bar graphs. Data are expressed as mean ± SEM following normalization to β-actin, total AKT or total nNOS, and comparison with SO vehicle values (100%). *p < 0.05 versus SO vehicle, #p < 0.05 versus OVX vehicle, $p < 0.05 versus OVXE2 vehicle, ^p < 0.05 versus SO WIN 55212-2, &p < 0.05 versus OVX WIN 55212-2.

Fig. 6

The effect of the CB₁R agonist WIN 55212-2 on NO and ROS production in the RVLM of in OVX and SO, and OVXE2 rats. Representative images are shown for NO (fluorogenic probes DAF-AM, A) and ROS (fluorogenic probes DCF, C). The bar graphs summarize the fluorescence intensity of NO (B) and ROS (D). *p < 0.05 versus SO vehicle, #p < 0.05 versus OVX vehicle, ^p < 0.05 versus SO WIN 55212-2, &p < 0.05 versus OVX WIN 55212-2.

Fig. 7

The effect of prior CB₁R blockade with AM251 (800 pmol) on intra-RVLM CB₁R (WIN 55212-2; 400 pmol)-evoked mean arterial pressure (ΔMAP, A) and heart rate (ΔHR, B) along with the following associated molecular responses: (1) phosphorylation of Akt (C) and nNOS (D) and (2) levels of NO (E) and ROS (F) in the RVLM of SO rats. Representative bands of p-Akt/AKT and p-nNOS/nNOS are shown under bar graph (C and D). Data were normalized to corresponding total AKT or total nNOS and compared with AM251 values (100%). Representative fluorescence staining images are shown for NO (DAF-AM, E) and ROS (DCF, G). The bar graphs summarize the fluorescence intensity of NO (F) and ROS (H). Data are mean ± SEM. ^p < 0.05, versus baseline. *p < 0.05 versus AM251, #p < 0.05 versus WIN 55212-2.

Fig. 8

Schematic summary of current data. Activation of RVLM CB₁R (WIN55212-2) induces neuronal sympathoexcitatory responses, elevations in sympathoexcitatory mediators (nNOS and Akt phosphorylation, NO and ROS levels), leads to pressor response (BP and HR), which was dampened by E₂ and reminiscent of pharmacological CB1R blockade (AM251).