Enhancement of Rostral Ventrolateral Medulla Neuronal Nitric-Oxide Synthase–Nitric-Oxide Signaling Mediates the Central Cannabinoid Receptor 1-Evoked Pressor Response in Conscious Rats

Abstract

Our recent studies implicated brainstem GABAergic signaling in the central cannabinoid receptor 1 (CB 1 R)-mediated pressor response in conscious rats. Given the well established link between neuronal nitric-oxide synthase (nNOS)/nitric oxide (NO) signaling and GABAergic transmission in brainstem cardiovascular regulating areas, we elucidated the role of nNOSgenerated NO in the central CB 1 R-elicited pressor response. Compared with vehicle, intracisternal (i.c.) microinjection of themethanone mesylate (WIN55212-2) (15 g/rat) significantly enhanced nNOS phosphorylation as well as the total nitrate and nitrite content in the rostral ventrolateral medulla (RVLM) at 5, 10, and 30 min, which paralleled the elicited pressor response. These findings were corroborated by: 1) the parallel dose-related increases in blood pressure and RVLM-NO levels, measured in real time by in vivo electrochemistry, elicited by intra-RVLM WIN55212-2 (100, 200, or 300 pmol /80 nl; n ϭ 5) in conscious rats; and 2) the significantly higher phosphorylated nNOS (pnNOS) levels in the WIN55212-2-injected RVLM compared with the contralateral RVLM. Subsequent neurochemical studies showed that WIN55212-2 (15 g/rat i.c.) significantly increased the number and percentage of neurons immunostained for nNOS (nitroxidergic neurons) and c-Fos (marker of neuronal activity) within the RVLM. The increases in blood pressure and the neurochemical responses elicited by intracisternal WIN55212-2 were attenuated by prior central CB 1 R blockade by N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251; 30 g/rat i.c.) or selective nNOS inhibition by N -propyl-L -arginine (1 g/rat i.c.). These findings implicate RVLM p-nNOS/NO signaling as a molecular mechanism in the central CB 1 R-evoked pressor effect in conscious rats.

A pivotal role for enhanced brainstem Orexin receptor 1 signaling in the central cannabinoid receptor 1-mediated pressor response in conscious rats

“…Coronal brainstem sections from naïve non-treated animals ( n =4) were prepared for multiple co-localization of CB 1 R/ orexin-A or TH/OX 1 R/CB 1 R within the C1 area of RVLM, as described under methods and in our previous studies (Ibrahim and Abdel-Rahman, 2011, 2012b). Dual labeling immunofluorescence studies in the RVLM (Fig.…”

“…( n =7). The AM251 dose and time interval were based on our previous findings (Ibrahim and Abdel-Rahman, 2011, 2012b). Further, we directly investigated the role of RVLM OX 1 R in the pressor response caused by the activation of RVLM CB 1 R. In this latter experiment, two groups of conscious rats ( n = 6) received WIN55,212-2 (0.1 µg/200 pmol) 10 min after vehicle (control) or SB-408124 (1 nmol; based on preliminary experiment); injections were made unilaterally into the right RVLM in 80 nl and vehicle microinjection in such volume had no impact on BP in our previous studies.…”

“…Further, we directly investigated the role of RVLM OX 1 R in the pressor response caused by the activation of RVLM CB 1 R. In this latter experiment, two groups of conscious rats ( n = 6) received WIN55,212-2 (0.1 µg/200 pmol) 10 min after vehicle (control) or SB-408124 (1 nmol; based on preliminary experiment); injections were made unilaterally into the right RVLM in 80 nl and vehicle microinjection in such volume had no impact on BP in our previous studies. The intra-RVLM WIN55,212-2 dose was based on our previous studies (Ibrahim and Abdel-Rahman, 2012b). As shown in Figs.…”

“…A total of 17 rats, divided into 4 groups ( n =3–5 each), were used for the measurement of RVLM orexin-A level in treatment and control groups according to the protocol followed in the first part of Section 2.3 above and in our previous studies (Ibrahim and Abdel-Rahman, 2012a, 2012b). Two groups received WIN55,212-2 (15 µg, i.c.)…”

“…Our recent studies implicated the activation (phosphorylation) of the neuronal nitric oxide synthase (nNOS) and extracellular signal-regulated kinase1/2 (ERK1/2) in the central cannabinoid receptor 1 (CB 1 R)-evoked pressor response in conscious freely moving rats (Ibrahim and Abdel-Rahman, 2012a, 2012b). However, the exact mechanisms of such response remain unclear.…”