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Objective Centrally acting antihypertensive action of moxonidine is a result of activation of Imidazoline-1 receptor (I1R) in the rostral ventrolateral medulla (RVLM). Hypertension shows an increase in reactive oxygen species (ROS) in the RVLM. The present objective was to determine the phosphoinositide-3 kinase (PI3K) signaling pathway involved in the effect of moxonidine on ROS generation in the RVLM of spontaneously hypertensive rat (SHR). Methods Wistar-Kyoto rats and SHR received intracisternal infusion (2 weeks) of tested agents which were subjected to subsequent experiments. In-situ ROS in the RVLM was evaluated by the oxidative fluorescence dye. Western blot and PCR analysis were performed to detect the expression levels of PI3K signaling pathway. Lentivirus was injected bilaterally into the RVLM for silencing PI3K signaling. Results ROS production in the RVLM was dose-dependently reduced in SHRs treated with infusion of moxonidine (20 nmol/day), which was prevented by the I1R antagonist efaroxan but not by the α2-adrenoceptor antagonist yohimbine. Moxonidine pretreatment significantly blunted cardiovascular sensitivity to injection of tempol (5 nmol) or angiotensin II (10 pmol) into the RVLM in SHR. Expression levels of PI3K/Akt, nuclear factor kappa-B (NFκB), NADPHase (NOX4), and angiotensin type I receptor (AT1R) in the RVLM were markedly decreased in SHR treated with moxonidine. Infection of lentivirus containing PI3K shRNA in the RVLM effectively prevented effects of moxonidine on cardiovascular activity and expression levels of Akt, NFκB, NOX4, and AT1R. Conclusion The centrally antihypertensive drug moxonidine decreases ROS production in the RVLM through inactivation of the PI3K/Akt signaling pathway in hypertension.

Angiotensin-1-7 [Ang-(1-7)], acting via the Mas receptor in the central nervous system, is involved in the regulation of cardiovascular activity. Nitric oxide (NO) is implicated as an important modulator in the nucleus tractus solitarii (NTS), a key region involved in control of cardiovascular activity. The aim of the present study was to determine the role of phosphatidylinositol 3-kinase (PI3K) signaling in mediating the effect of Ang-(1-7) on NO generation in the NTS. In Sprague-Dawley rats, acute injection of Ang-(1-7) into the NTS significantly increased NO generation and neuronal/endothelial NO synthase (n/eNOS) activity, which were abolished by the selective Mas receptor antagonist d-Alanine-[Ang-(1-7)] (A-779), the PI3K inhibitor LY294002, or the Akt inhibitor triciribine (TCN). Western blotting analysis further demonstrated that Ang-(1-7) significantly increased levels of Akt/NOS phosphorylation in the NTS, and Ang-(1-7)-induced e/nNOS phosphorylation was antagonized by LY294002 or TCN. Furthermore, gene knockdown of PI3K by lentivirus containing small hairpin RNA in the NTS prevented the Ang-(1-7)-induced increases in NOS/Akt phosphorylation and NO production. The physiological (in vivo) experiments showed that pretreatment with the NOS inhibitor l-NAME, LY294002, or TCN abolished the decreases in blood pressure, heart rate, and renal sympathetic nerve activity induced by Ang-(1-7) injected into the NTS. Our findings suggest that nitric oxide release meditated by the Mas-PI3K-NOS signaling pathway is involved in the cardiovascular effects of Ang-(1-7) in the NTS.

The imbalance between angiotensin II (Ang II) and angiotensin 1–7 (Ang 1–7) in the brain has been reported to contribute to cardiovascular dysfunction in hypertension. Exercise training (ExT) is beneficial to hypertension and the mechanism is unclear. This study was aimed to determine if ExT improves hypertension via adjusting renin angiotensin system in cardiovascular centers including the rostral ventrolateral medulla (RVLM). Spontaneously hypertensive rats (SHR, 8 weeks old) were subjected to low-intensity ExT or kept sedentary (Sed) for 12 weeks. Blood pressure elevation coupled with increase in age was significantly decreased in SHR received ExT compared with Sed. The results in vivo showed that ExT significantly reduced or increased the cardiovascular responses to central application of sarthran (antagonist of Ang II) or A779 (antagonist of Ang 1–7), respectively. The protein expression of the Ang II acting receptor AT1R and the Ang 1–7 acting receptor Mas in the RVLM was significantly reduced and elevated in SHR following ExT, respectively. Moreover, production of reactive oxygen species in the RVLM was significantly decreased in SHR following ExT. The current data suggest that ExT improves hypertension via improving the balance of Ang II and Ang 1–7 and antioxidative stress at the level of RVLM.

The rostral ventrolateral medulla (RVLM) is a key region in cardiovascular regulation. It has been demonstrated that cholinergic synaptic transmission in the RVLM is enhanced in hypertensive rats. Angiotensin-converting enzyme 2 (ACE2) in the brain plays beneficial roles in cardiovascular function in hypertension. The purpose of this study was to determine the effect of ACE2 overexpression in the RVLM on cholinergic synaptic transmission in spontaneously hypertensive rats (SHRs). Four weeks after injecting lentiviral particles containing enhanced green fluorescent protein and ACE2 bilaterally into the RVLM, the blood pressure and heart rate were notably decreased. ACE2 overexpression significantly reduced the concentration of acetylcholine in microdialysis fluid from the RVLM and blunted the decrease in blood pressure evoked by bilateral injection of atropine into the RVLM in SHRs. In conclusion, we suggest that ACE2 overexpression in the RVLM attenuates the enhanced cholinergic synaptic transmission in SHRs.

Background: Hypertension is characterized by sympathetic overactivity, which is associated with an enhancement in angiotensin receptor type I (AT1R) in the rostral ventrolateral medulla (RVLM). β-arrestin1, a canonical scaffold protein, has been suggested to show a negative effect on G protein-coupled receptors via its internalization and desensitization and/or the biased signaling pathway. The major objectives of the present study were to observe the effect of β-arrestin1 overexpression in the RVLM on cardiovascular regulation in spontaneously hypertensive rats (SHR), and further determine the effect of β-arrestin1 on AT1R expression in the RVLM. Methods: The animal model of β-arrestin1 overexpression was induced by bilateral injection of adeno-associated virus containing Arrb1 gene (AAV-Arrb1) into the RVLM of WKY and SHR. Results: β-arrestin1 was expressed on the pre-sympathetic neurons in the RVLM, and its expression in the RVLM was significantly (P < 0.05) downregulated by an average of 64% in SHR than WKY. Overexpression of β-arrestin1 in SHR significantly decreased baseline levels of blood pressure and renal sympathetic nerve activity, and attenuated cardiovascular effects induced by RVLM injection of angiotensin II (100 pmol). Furthermore, β-arrestin1 overexpression in the RVLM significantly reduced the expression of AT1R by 65% and NF-κB p65 phosphorylation by 66% in SHR. It was confirmed that β-arrestin1 overexpression in the RVLM led to an enhancement of interaction between β-arrestin1 and IκB-α. Conclusion: Overexpression of β-arrestin1 in the RVLM reduces BP and sympathetic outflow in hypertension, which may be associated with NFκB-mediated AT1R downregulation.

SummaryAims It has been demonstrated that neuroinflammation is associated with cardiovascular dysfunction. The phosphoinositide-3 kinase (PI3K) signaling in the rostral ventrolateral medulla (RVLM), a key region for sympathetic outflow, is upregulated and contributes to increased blood pressure (BP) and sympathetic outflow in hypertension. This study was designed to determine the role of the PI3K signaling in neuroinflammation in the RVLM of hypertension. Methods The normotensive WKY rats were performed by intracisternal infusion of lipopolysaccharide (LPS) or angiotensin II (Ang II) for inducing neuroinflammation. Elisa was used to determine the level of proinflammatory cytokines. Western blot was employed to detect the protein expression of PI3K signaling pathway. Gene silencing of PI3K p110δ subunit and overexpression of angiotensin-converting enzyme 2 (ACE2) were realized by injecting related lentivirus into the RVLM. Results In the spontaneously hypertensive rats (SHR), the PI3K signaling in the RVLM was upregulated compared with WKY, gene silencing of PI3K in the RVLM significantly reduced BP and renal sympathetic nerve activity (RSNA), but also decreased the levels of proinflammatory cytokines. In the WKY rats, central infusion of LPS and Ang II significantly elevated BP and RSNA, but also increased the levels of proinflammatory cytokines and PI3K signaling activation in the RVLM. These changes in the Ang II-induced hypertension were effectively prevented by gene silencing of PI3K in the RVLM. Furthermore, overexpression of ACE2 in the RVLM significantly attenuated high BP and neuroinflammation, as well as decreased the activation of PI3K signaling in hypertensive rats. Conclusion This study suggests that the PI3K signaling in the RVLM is involved in neuroinflammation in hypertension and plays an important role in the renin–angiotensin system-mediated changes in neuroinflammation in the RVLM.

Nitric oxide (NO) contributes to the central control of cardiovascular activity. The rostral ventrolateral medulla (RVLM) has been recognized as a pivotal region for maintaining basal blood pressure (BP) and sympathetic tone. It is reported that asymmetric dimethylarginine (ADMA), characterized as a cardiovascular risk marker, is an endogenous inhibitor of nitric oxide synthesis. The present was designed to determine the role of ADMA in the RVLM in the central control of BP in hypertensive rats. In Sprague Dawley (SD) rats, microinjection of ADMA into the RVLM dose-dependently increased BP, heart rate (HR), and renal sympathetic never activity (RSNA), but also reduced total NO production in the RVLM. In central angiotensin II (Ang II)-induced hypertensive rats and spontaneously hypertensive rat (SHR), the level of ADMA in the RVLM was increased and total NO production was decreased significantly, compared with SD rats treated vehicle infusion and WKY rats, respectively. These hypertensive rats also showed an increased protein level of protein arginine methyltransferases1 (PRMT1, which generates ADMA) and a decreased expression level of dimethylarginine dimethylaminohydrolases 1 (DDAH1, which degrades ADMA) in the RVLM. Furthermore, increased AMDA content and PRMT1 expression, and decreased levels of total NO production and DDAH1 expression in the RVLM in SHR were blunted by intracisternal infusion of the angiotensin II type 1 receptor (AT1R) blocker losartan. The current data indicate that the ADMA-mediated NO inhibition in the RVLM plays a critical role in involving in the central regulation of BP in hypertension, which may be associated with increased Ang II.

It has been demonstrated that homocysteine (HCY) is a significant risk factor of hypertension, which is characterized by overactivity of sympathetic tone. Excessive oxidative stress in the rostral ventrolateral medulla (RVLM), a key region for control of sympathetic outflow, contributes to sympathetic hyperactivity in hypertension. Therefore, the goal of the present study is to determine the effect of systemic HCY on production of reactive oxygen species (ROS) in the RVLM. In the rat model of the diet-induced hyperhomocysteinemia (L-methionine, 1 g/kg/day, 8 weeks), we found that the HCY resulted in a significant increase (≈3.7-fold, P<0.05) in ROS production in the RVLM, which was paralleled with enhanced sympathetic tone and blood pressure (BP). Compared to the vehicle group, levels of BP and basal renal sympathetic nerve activity in the HCY group were significantly (P<0.05, n=5) increased by an average of 27 mmHg and 31%, respectively. Furthermore, the rats treated with L-methionine (1 g/kg/day, 8 weeks) showed an upregulation of NADPHase (NOX4) protein expression and a downregulation of superoxide dismutase protein expression in the RVLM. The current data suggest that central oxidative stress induced by systemic HCY plays an important role in hypertension-associated sympathetic overactivity.

The rostral ventrolateral medulla (RVLM) plays a key role in cardiovascular regulation. It has been reported that tonically active glutamatergic input to the RVLM is increased in hypertensive rats, whereas angiotensin-converting enzyme 2 (ACE2) in the brain has been suggested to be beneficial to hypertension. This study was designed to determine the effect of ACE2 gene transfer into the RVLM on tonically active glutamatergic input in spontaneously hypertensive rats (SHRs). Lentiviral particles containing enhanced green fluorescent protein (lenti-GFP) or ACE2 (lenti-ACE2) were injected bilaterally into the RVLM. Both protein expression and activity of ACE2 in the RVLM were increased in SHRs after overexpression of ACE2. A significant reduction in blood pressure and heart rate in SHRs was observed 6 wk after lenti-ACE2 injected into the RVLM. The concentration of glutamate in microdialysis fluid from the RVLM was significantly reduced by an average of 61% in SHRs with lenti-ACE2 compared with lenti-GFP. ACE2 overexpression significantly attenuated the decrease in blood pressure and renal sympathetic nerve activity evoked by bilateral injection of the glutamate receptor antagonist kynurenic acid (2.7 nmol in 100 nl) into the RVLM in SHRs. Therefore, we suggest that ACE2 overexpression in the RVLM attenuates the enhanced tonically active glutamatergic input in SHRs, which may be an important mechanism underlying the beneficial effect of central ACE2 to hypertension.

Aims The depressor action of the centrally antihypertensive drug moxonidine has been attributed to activation of I1-imidazoline receptor in the rostral ventrolateral medulla (RVLM). The objective of this study was to determine the role of the γ-aminobutyric acid (GABA) mechanisms in the RVLM in mediating the effect of moxonidine in anaesthetized normotensive rats. Methods and results The relationship between the effects of microinjection or picoinjection of moxonidine and the functional state of GABA receptors at the level of the RVLM or pre-sympathetic neuron was determined. Microdialysis was performed to detect the effect of moxonidine on the release of GABA in the RVLM. Western blot analysis was carried out to test the effect of chronic intracerebroventricular injection of moxonidine on the protein expression of GABA receptors in the RVLM. Pre-treatment with the GABAA or GABAB receptor antagonist bicuculline (5 pmol) or CGP35348 (200 pmol), respectively, microinjected into the RVLM significantly attenuated the decrease in blood pressure and renal sympathetic nerve activity induced by moxonidine. In 22 moxonidine-sensitive pre-sympathetic neurons in the RVLM, picoinjection of bicuculline (100 fmol/5 nL) significantly attenuated the neuronal inhibition evoked by moxonidine (100 pmol/5 nL). The release of GABA in the RVLM was increased after intravenous moxonidine (50 μg/kg). Central infusion of moxonidine upregulated the protein expression of both GABAA and GABAB receptors in the RVLM. Conclusion The current data demonstrate that GABAergic mechanisms in the RVLM are responsible for the hypotension and sympathoinhibition of moxonidine.

Obestatin is a recently discovered 23-amino acid peptide encoded by the same gene that encodes ghrelin. It has been reported that there is a significant negative correlation between the plasma ghrelin concentration and systemic blood pressure in patients with pregnancy-induced hypertension. We investigated the plasma concentration of obestatin in 18 non-pregnant women, 18 normal pregnant women, and 15 patients with pregnancy-induced hypertension. The plasma concentrations of obestatin in these 3 groups of women were 63.4 ± 9.5 pg/ml, 38.1 ± 6.3 pg/ml, and 46.0 ± 9.3 pg/ml, respectively. In non-pregnant women, there was no correlation between the plasma obestatin concentration and the mean arterial pressure. However, there was a positive correlation between the plasma obestatin concentration and the mean arterial pressure in normal pregnant women and pregnant women with pregnancy-induced hypertension. These results suggest that obestatin may have some potential role in the regulation of blood pressure in normal pregnant women and women with pregnancy-induced hypertension.

Aims The central antihypertensive drug moxonidine lowers blood pressure (BP) through stimulating an imidazoline receptor within the rostral ventrolateral medulla (RVLM). Nitric oxide (NO) generated by the inducible NO synthase (iNOS) in the RVLM has been suggested to be involved in tonic sympathetic inhibition. The aim of this study was to determine the role of NO generated by iNOS in mediating moxonidine-induced cardiovascular inhibition in rats. Methods and results In anaesthetized rats, the cardiovascular response to local or systemic injection of moxonidine was observed after treatment with the selective iNOS inhibitor S -methylisothiourea (SMT) in the brain. Using immunohistochemical staining and western blot techniques, the protein expression of iNOS in the RVLM was measured in the moxonidine-infused rats. Intracerebroventricular (ICV) injection of SMT (1–100 nmol) dose-dependently attenuated the moxonidine (20 nmol, ICV)-induced decrease in BP and heart rate. Prior injection of SMT (20 and 200 pmol) into the RVLM also dose-dependently prevented the decrease in BP and renal sympathetic nerve activity evoked by RVLM microinjection of moxonidine (5 nmol) or intravenous injection of moxonidine (50 µg/kg). We further found that expression of iNOS protein following chronic ICV infusion of moxonidine (20 nmol, 2 weeks) is selectively upregulated in the RVLM but not in the nucleus tractus solitarius. Conclusion The present data suggest that an NO mechanism generated by iNOS in the RVLM plays an important role in mediating the sympathetic inhibition of the centrally acting drug moxonidine.

Centrally antihypertensive action of moxonidine has been demonstrated to be resulted from activation of Imidazoline-1 receptor (I1R) in the rostral ventrolateral medulla (RVLM). Angiotensin II (Ang...

Summary Aims It is well known that low-intensity exercise training (ExT) is beneficial to cardiovascular dysfunction in hypertension. The tonically active glutamatergic input to the rostral ventrolateral medulla (RVLM), a key region for control of blood pressure and sympathetic tone, has been demonstrated to be increased in hypertensive rats. The aim of this study was to determine the effect of ExT on the increased glutamatergic input to the RVLM in spontaneously hypertensive rat (SHR). Methods Normotensive rats Wistar-Kyoto (WKY) and SHR were treadmill trained or remained sedentary (Sed) for 12 weeks and classed into four groups (WKY-Sed, WKY-ExT, SHR-Sed, and SHR-ExT). The release of glutamate in the RVLM and its contribution to cardiovascular activity were determined in WKY and SHR after treatment of ExT. Results Blood pressure and sympathetic tone were significantly reduced in SHR after treatment with ExT. Bilateral microinjection of the glutamate receptor antagonist kynurenic acid (2.7 nmol in 100 nL) into the RVLM significantly decreased resting blood pressure, heart rate, and renal sympathetic nerve activity in SHR-Sed but not in WKY groups (WKY-Sed and WKY-ExT). However, the degree of reduction in these cardiovascular parameters evoked by KYN was significantly blunted in SHR-ExT compared with SHR-Sed group. The concentration of glutamate and the protein expression of vesicular glutamate transporter 2 in the RVLM were significantly increased in SHR-Sed compared with WKY-Sed, whereas they were reduced after treatment with ExT. Conclusion Our findings suggest that ExT attenuates the enhancement in the tonically acting glutamatergic input to the RVLM of hypertensive rats, thereby reducing the sympathetic hyperactivity and blood pressure.

Urocortin (Ucn), a member of CRH family, has been implicated to be one of the endogenous regulators in the cardiovascular system and exerts its effects locally via an autocrine/paracrine fashion. Previous studies have shown the gender difference in CRH-induced vasodilation in human skin, which is related to the concentration of estrogens during the menstrual cycle. The aim of this study was to investigate whether estrogens modulate Ucn/CRH receptor type 2 (CRHR2) expression in vascular smooth muscle, thereby leading to vasodilation. We performed sham operation or bilateral ovariectomy (OVX) on female Sprague Dawley rats. OVX rats were sc administered 17β-estradiol (E2) at a dose of 30 μg/kg·d or with placebo for 12 wk. Primary smooth muscle cells of aorta were used for the in vitro study. It was found that the Ucn-induced vasodilation and CRHR2 expression were decreased in OVX rats and restored by E2 replacement treatment for 12 wk. E2 increased the expression of CRHR2 in cultured smooth muscle cells, which was blocked by estrogen receptor-β antagonist. Ucn significantly suppressed the phenylephrine-induced phospholipase Cβ3 activation, inositol 1,4,5-trisphosphate (IP3) production, and intracellular Ca2+ elevation. Ucn stimulated the expression of active GTP-bound Gαs protein and cAMP production. The suppressive effects of Ucn on phenylephrine-induced IP3 production and intracellular Ca2+ elevation were blocked by the inhibitors of adenylate cyclase and protein kinase A. Our results demonstrate that estrogen maintains the expression of CRHR2 in aorta smooth muscle, thereby enhancing vasodilator actions of Ucn. Ucn exerts its vasorelaxant effects via Gαs-cAMP-protein kinase A signaling, leading to down-regulation of the phospholipase Cβ-IP3-Ca2+ signaling pathway.

The high prevalence of obesity and diabetes will lead to higher rates of morbidity and mortality. It is well known that ghrelin plays a potential role in obesity and diabetes. Obestatin, a novel 23 amino acid amidated peptide encoded by the same gene that encodes ghrelin, was initially reported to have opposite actions to ghrelin in the regulation of food intake, emptying of the stomach and body weight. Recent work suggests that obestatin also regulate beta-cell survival and insulin secretion. The ghrelin-obestatin system is, therefore, a promising target for the developing of new drugs for the treatment of obesity and diabetes. This review summarizes the interrelationship between obestatin, obesity and diabetes.

This study aimed to investigate the vasoactivity of sulfur dioxide (SO 2 ), a novel gas identified from vascular tissue, in rat thoracic aorta. The thoracic aorta was isolated, cut into rings, and mounted in organ-bath chambers. After equilibrium, the rings were gradually stretched to a resting tension. Isometric tension was recorded under the treatments with vasoconstrictors, SO 2 derivatives, and various drugs as pharmacological interventions. In endothelium-intact aortic rings constricted by 1 μM phenylephrine (PE), SO 2 derivatives (0.5 - 8 mM) caused a dose-dependent relaxation. Endothelium removal and a NOS inhibitor L-NAME reduced the relaxation to low doses of SO 2 derivatives, but not that to relatively high doses (≥* 2 mM). In endothelium-denuded rings, SO 2 derivatives attenuated vasoconstriction induced by high K + (60 mM) or CaCl 2 (0.01-10 mM). The relaxation to SO 2 derivatives in PE-constricted rings without endothelium was significantly inhibited by blockers of ATP-sensitive K + (K ATP ) and Ca 2 + -activated K + (K ca ) channels, but not by those of voltage-dependent K + channels, Na + -K + -ATPase or Na + -Ca 2+ exchanger. SO 2 relaxed vessel tone via endothelium-dependent mechanisms associated with NOS activation, and via endothelium-independent mechanisms dependent on the inhibition of voltage-gated Ca 2+ channels, and the opening of K ATP and K ca channels.

Obestatin is a bioactive peptide encoded by the same gene that encodes ghrelin. Our aim was to investigate the effect of obestatin on insulin secretion. We evaluated the effects of obestatin on insulin secretion from rat islet cells which had been incubated overnight in the presence of 8.3, 11.1, and 22.2 mmol/l of glucose. In vivo, the serum levels of glucose and insulin were measured 0, 1, 5, 10, 20, 40, and 60 min after the intravenous administration of saline or glucose (1g/kg), with or without obestatin, and the area under the 60 min curve of insulin concentration (AUC(insulin)) was calculated. Obestatin (0.01-100 nmol/l) inhibited insulin secretion from rat islets in a dose-dependent fashion. In vivo, when administered intravenously to rats together with glucose, obestatin (10, 50, and 250 nmol/kg) inhibited both the rapid 1-min insulin response and the AUC(insulin) in a dose-dependent fashion. Our data demonstrate that under glucose-stimulated conditions, exogenous obestatin acts as a potent inhibitor of insulin secretion in anaesthetized rats in vivo as well as in cultured islets in vitro.