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Background and Purpose μ-Opioid receptors, pro-opiomelanocortin and pro-enkephalin are highly expressed in the nucleus tractus solitarii (NTS) and μ receptor agonists given to the NTS dose-dependently increased BP. However, the molecular mechanisms of this process remain unclear. In vitro, μ receptors heterodimerize with α2A-adrenoceptors. We hypothesized that α2A-adrenoceptor agonists would lose their depressor effects when their receptors heterodimerize in the NTS with μ receptors.

Background and Purpose Oxidative stress is an important pathogenic factor in the development of hypertension. Resveratrol, the main antioxidant in red wine, improves NO bioavailability and prevents cardiovascular disease. The aim of this study was to examine whether resveratrol decreases the generation of reactive oxygen species (ROS), thereby reducing BP in rats with fructose-induced hypertension. Experimental Approach Rats were fed 10% fructose with or without resveratrol (10 mg·kg−1·day−1) for 1 week or for 4 weeks with resveratrol treatment beginning at week 2; systolic BP (SBP) was measured by tail-cuff method. Endogenous in vivo O2− production in the nucleus tractus solitarii (NTS) was determined with dihydroethidium. Real-time PCR and immunoblotting analyses were used to quantify RNA and protein expression levels. Key Results In fructose-fed rats, ROS levels in the NTS were higher, whereas the NO level was significantly decreased. Also, RNA and protein levels of NADPH oxidase subunits (p67, p22-phox) were elevated, superoxide dismutase 2 (SOD2) reduced and AMP-activated PK (AMPK) T172 phosphorylation levels in the NTS were lower in fructose-fed rats. Treatment with the AMPK activator resveratrol decreased levels of NADPH oxidase subunits and ROS, and increased NO and SOD2 levels in the NTS of fructose-fed rats. Administration of resveratrol, in combination with fructose at week 0 and later at week 2, significantly reduced the SBP of fructose-fed rats. Conclusions and Implications Collectively, resveratrol decreased BP through the phosphorylation of AMPK, Akt and neuronal NOS in fructose-fed rats. These novel findings suggest that resveratrol may be a potential pharmacological candidate for the treatment of hypertension.

Background and Purpose Clinical studies indicate that statins have a BP-lowering effect in hypercholesterolemic individuals with hypertension. Specifically, statins modulate BP through the up-regulation of endothelial NOS (eNOS) activation in the brain. However, the signalling mechanisms through which statins enhance eNOS activation remain unclear. Therefore, we examined the possible signalling pathways involved in statin-mediated BP regulation in the nucleus tractus solitarii (NTS). Experimental Approach To investigate the involvement of Ras and other signalling pathways in simvastatin-induced effects on BP, BP and renal sympathetic nerve activity (RSNA) were determined in spontaneously hypertensive rats (SHRs) before and after i.c.v. administration of simvastatin in the absence and presence of a Ras-specific inhibitor (farnesyl thiosalicylic acid, FTS), a geranylgeranyltransferase inhibitor (GGTI-2133), a PI3K inhibitor (LY294002) or a MAPK-ERK kinase (MEK) inhibitor (PD98059). Key Results FTS significantly attenuated the decrease in BP and increased NO evoked by simvastatin and reversed the decrease in basal RSNA induced by simvastatin. Immunoblotting and pharmacological studies showed that inhibition of Ras activity by FTS significantly abolished simvastatin-induced phosphorylation of ERK1/2, ribosomal protein S6 kinase (RSK), Akt and decreased eNOS phosphorylation. Likewise, administration of Akt and ERK1/2 signalling inhibitors, LY294002 and PD98059, attenuated the reduction in BP evoked by simvastatin. Furthermore, i.c.v. simvastatin decreased Rac1 activation and the number of ROS-positive cells in the NTS. Conclusions and Implications Simvastatin modulates central BP control in the NTS of SHRs by increasing Ras-mediated activation of the PI3K-Akt and ERK1/2-RSK signalling pathways, which then up-regulates eNOS activation.

BACKGROUND AND PURPOSE Neuropeptide Y (NPY) is a 36-amino acid polypeptide found abundantly in the central and peripheral nervous systems. NPY exerts a potent depressor effect via the activation of both Y1 and Y2 receptors in the nucleus tractus solitarii (NTS) of rats. However, the precise mechanisms involved in this NPY-mediated action remained unclear. EXPERIMENTAL APPROACH Effects of a selective antagonist of Y1 receptors, a PKC inhibitor, a PI3 kinase inhibitor, a NOS inhibitor, an endothelial NOS (eNOS)-selective inhibitor, a neuronal NOS (nNOS)-specific inhibitor or a MAPK inhibitor, on responses to microinjection of NPY into the NTS of Wistar-Kyoto rats were studied to determine the underlying mechanisms. Blood pressure and heart rate were measured and, in NTS, protein phosphorylation assessed by immunohistochemical techniques. KEY RESULTS Unilateral microinjection of exogenous NPY (4.65 pmol/60 nL) into the NTS of urethane-anesthetized Wistar-Kyoto rats markedly decreased blood pressure and heart rate. Microinjection of the Y1 receptor antagonist BIBP3226 or the Gi/Go-protein inhibitor, Pertussis toxin, into the NTS attenuated these NPY-induced hypotensive effects. A selective Y1 receptor agonist increased expression of ERK1/2, ribosomal protein S6 kinase (RSK) and the phosphorylation of eNOS. RSK also bound directly to eNOS and induced its phosphorylation at Ser1177. Pretreatment of the NTS with an eNOS inhibitor, but not a nNOS inhibitor, attenuated the NPY-induced hypotensive effects. CONCLUSIONS AND IMPLICATIONS Together, these results suggested that NPY-induced depressor effects were mediated by activating NPY Y1 receptor-PKC-ERK-RSK-eNOS and Ca2+-eNOS signalling pathways, which are involved in regulation of blood pressure in the NTS.

Background and purpose The renin-angiotensin system (RAS) is critical for the control of blood pressure by the CNS. Recently, direct renin inhibitors were approved as antihypertensive agents. However, the signalling mechanism of renin, which regulates blood pressure in the nucleus tractus solitarii (NTS) remains unclear. Here we have investigated the signalling pathways involved in renin-mediated blood pressure regulation, at the NTS. Experimental approach Depressor responses to renin microinjected into the NTS of Wistar-Kyoto rats were elicited in the absence and presence of the endothelial nitric oxide synthase (eNOS)-specific inhibitor, N(5)-(-iminoethyl)-L-ornithine, Akt inhibitor IV and LY294002, a PI3K inhibitor and GP antagonist-2A [G(q) inhibitor]. Lisinopril (angiotensin converting enzyme inhibitor), losartan, valsartan (angiotensin AT(1) receptor antagonists), D-Ala7-Ang-(1-7) (angiotensin-(1-7) receptor antagonist) were used to study the involvement of RAS on renin-induced depressor effects. Key results Microinjection of renin into the NTS produced a prominent depressor effect and increased NO production. Pretreatment with G(q) -PI3K-Akt-eNOS pathway-specific inhibitors significantly attenuated the depressor response evoked by renin. Immunoblotting and immunohistochemical studies further showed that inhibition of PI3K significantly blocked renin-induced eNOS-Ser ¹¹⁷ and Akt-Ser⁴⁷³ phosphorylation in situ. In addition, pre-treatment of the NTS with RAS inhibitors attenuated the vasodepressor effects evoked by renin. Microinjection of renin also increased Ras activation in the NTS. Conclusions and implications Taken together, these results suggest renin modulated blood pressure at the NTS by AT₁ and Mas receptor-mediated activation of G(q) and Ras to evoke PI3K-Akt-eNOS signalling.

BACKGROUND AND PURPOSE Recent evidence has suggested that nicotine decreases blood pressure (BP) and heart rate (HR) in the nucleus tractus solitarii (NTS), indicating that nicotinic acetylcholine receptors (nAChRs) play an important role in BP control in the NTS. However, the signalling mechanisms involved in nAChR-mediated depressor effects in the NTS are unclear. Hence, the aim of this study was to investigate these signalling mechanisms. EXPERIMENTAL APPROACH Depressor responses to nicotine microinjected into the NTS of Wistar-Kyoto rats were elicited in the absence and presence of an antagonist of α7 nAChR, the calcium chelator ethylene glycol tetraacetic acid, a calmodulin-specific inhibitor, nitric oxide (NO) synthase (NOS) inhibitor, endothelial NOS (eNOS)-selective inhibitor or neuronal NOS (nNOS)-specific inhibitor. KEY RESULTS Microinjection of nicotine into the NTS produced a dose-dependent decrease in BP and HR, and increased nitrate levels. This depressor effect of nicotine was attenuated after pretreatment with a nAChR antagonist or blockers of the calmodulin-eNOS pathway. In contrast, N5-(1-Imino-3-butenyl)-L-ornithine (vinyl-L-NIO), nNOS-specific inhibitor, did not diminish these nicotine-mediated effects. Calmodulin was found to bind eNOS after nicotine injection into NTS. However, nicotine did not affect the eNOS phosphorylation level or eNOS upstream extracellular signal-regulated kinases (ERK)1/2 and Akt phosphorylation levels. Furthermore, pretreatment with an ERK1/2 or Akt inhibitor did not attenuate nicotine-induced depressor effects in the NTS. CONCLUSIONS AND IMPLICATIONS These results suggest that the nAChR-Ca2+-calmodulin-eNOS-NO signalling pathway, but not nNOS, plays a significant role in central BP regulation, and neither the ERK1/2 nor Akt signalling pathway are significantly involved in the activation of eNOS by nAChRs in the NTS.

μ-Opioid receptors, pro-opiomelanocortin and pro-enkephalin are highly expressed in the nucleus tractus solitarii (NTS) and μ receptor agonists given to the NTS dose-dependently increased BP. However, the molecular mechanisms of this process remain unclear. In vitro, μ receptors heterodimerize with α2A -adrenoceptors. We hypothesized that α2A -adrenoceptor agonists would lose their depressor effects when their receptors heterodimerize in the NTS with μ receptors.We microinjected μ-opioid agonists and antagonists into the NTS of rats and measured changes in BP. Formation of μ receptor/α2A -adrenoceptor heterodimers was assessed with immunofluorescence and co-immunoprecipitation methods, along with proximity ligation assays.Immunofluorescence staining revealed colocalization of α2A -adrenoceptors and μ receptors in NTS neurons. Co-immunoprecipitation revealed interactions between α2A -adrenoceptors and μ receptors. In situ proximity ligation assays confirmed the presence of μ receptor/α2A -adrenoceptor heterodimers in the NTS. Higher levels of endogenous endomorphin-1 and μ receptor/α2A -adrenoceptor heterodimers were found in the NTS of hypertensive rats, than in normotensive rats. Microinjection of the μ receptor agonist [D-Ala(2) , MePhe(4) , Gly(5) -ol]-enkephalin (DAMGO), but not that of the α2A -adrenoceptor agonist guanfacine, into the NTS of normotensive rats increased μ receptor/α2A -adrenoceptor heterodimer formation and BP elevation. The NO-dependent BP-lowering effect of α2A -adrenoceptor agonists was blunted following increased formation of μ receptor/α2A -adrenoceptor heterodimers in the NTS of hypertensive rats and DAMGO-treated normotensive rats.Increases in endogenous μ receptor agonists in the NTS induced μ receptor/α2A -adrenoceptor heterodimer formation and reduced the NO-dependent depressor effect of α2A -adrenoceptor agonists. This process could contribute to the pathogenesis of hypertension.

Oxidative stress is an important pathogenic factor in the development of hypertension. Resveratrol, the main antioxidant in red wine, improves NO bioavailability and prevents cardiovascular disease. The aim of this study was to examine whether resveratrol decreases the generation of reactive oxygen species (ROS), thereby reducing BP in rats with fructose-induced hypertension.Rats were fed 10% fructose with or without resveratrol (10 mg·kg(-1) ·day(-1) ) for 1 week or for 4 weeks with resveratrol treatment beginning at week 2; systolic BP (SBP) was measured by tail-cuff method. Endogenous in vivo O2 (-) production in the nucleus tractus solitarii (NTS) was determined with dihydroethidium. Real-time PCR and immunoblotting analyses were used to quantify RNA and protein expression levels.In fructose-fed rats, ROS levels in the NTS were higher, whereas the NO level was significantly decreased. Also, RNA and protein levels of NADPH oxidase subunits (p67, p22-phox) were elevated, superoxide dismutase 2 (SOD2) reduced and AMP-activated PK (AMPK) T172 phosphorylation levels in the NTS were lower in fructose-fed rats. Treatment with the AMPK activator resveratrol decreased levels of NADPH oxidase subunits and ROS, and increased NO and SOD2 levels in the NTS of fructose-fed rats. Administration of resveratrol, in combination with fructose at week 0 and later at week 2, significantly reduced the SBP of fructose-fed rats.Collectively, resveratrol decreased BP through the phosphorylation of AMPK, Akt and neuronal NOS in fructose-fed rats. These novel findings suggest that resveratrol may be a potential pharmacological candidate for the treatment of hypertension.