Renin activates PI3K-Akt-eNOS signalling through the angiotensin AT? and Mas receptors to modulate central blood pressure control in the nucleus tractus solitarii.
ABSTRACT: 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 ¹¹? and Akt-Ser??³ 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 AT? and Mas receptor-mediated activation of G(q) and Ras to evoke PI3K-Akt-eNOS signalling.
Project description: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 ?7 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-Ca(2+) -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.
Project description: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 Y(1) and Y(2) 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 Y(1) 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 (4.65pmol/60nL) into the NTS of urethane-anesthetized Wistar-Kyoto rats markedly decreased blood pressure and heart rate. Microinjection of the Y(1) receptor antagonist BIBP3226 or the G(i) /G(o) -protein inhibitor, Pertussis toxin, into the NTS attenuated these NPY-induced hypotensive effects. A selective Y(1) 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 Ser(1177) . 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 Y(1) receptor-PKC-ERK-RSK-eNOS and Ca(2+) -eNOS signalling pathways, which are involved in regulation of blood pressure in the NTS.
Project description: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.
Project description:The carcinoembryonic antigen-related cell adhesion molecule 1 regulates insulin sensitivity by promoting hepatic insulin clearance. Mice bearing a null mutation of Ceacam1 gene (Cc1(-/-)) develop impaired insulin clearance followed by hyperinsulinemia and insulin resistance, in addition to visceral obesity and increased plasma fatty acids. Because insulin resistance is associated with increased blood pressure, we investigated whether they develop higher blood pressure with activated renal renin-angiotensin system and whether this is mediated, in part, by the upregulation of renal (pro)renin receptor (PRR) expression. Compared with age-matched wild-type littermates, Cc1(-/-) mice exhibited increased blood pressure with increased activation of renal renin-angiotensin systems and renal PRR expression. Cytoplasmic and nuclear immunostaining of phospho-PI3K p85? and phospho-Akt was enhanced in the kidney of Cc1(-/-) mice. In murine renal inner medullary collecting duct epithelial cells with lentiviral-mediated small hairpin RNA knockdown of carcinoembryonic antigen-related cell adhesion molecule 1, PRR expression was upregulated and phosphorylation of PI3K (Tyr508), Akt (Ser473), NF-?B p65 (Ser276), cAMP response element-binding protein/activated transcription factor (ATF)-1 (Ser133), and ATF-2 (Thr71) was enhanced. Inhibiting PI3K with LY294002 or Akt with Akt inhibitor VIII attenuated PRR expression. In conclusion, global null deletion of Ceacam1 caused an increase in blood pressure with increased renin-angiotensin system activation together with upregulation of PRR via PI3K-Akt activation of cAMP response element-binding protein 1, ATF-1, ATF-2, and NF-?B p65 transcription factors.
Project description:Tetrahydropalmatine (THP) is an active natural alkaloid isolated from Corydalis yanhusuo W.T. Wang which has been widely used for treating pain and cardiovascular disease in traditional Chinese medicine. Previous studies suggested THP have various pharmacological effects in neural and cardio tissue while the vascular reactivity of THP was not fully established. The present study found that THP relaxed rat aorta which contracted by phenylephrine (Phe), KCl, and U46619. The vascular relaxation effect of THP was partially attenuated by PI3K inhibitor wortmannin, Akt inhibitor IV, endothelial nitric oxide synthetase (eNOS) inhibitor L-NAME, guanylate cyclase inhibitors and the mechanical removal of endothelium. Also, the eNOS substrate L-arginine reversed the inhibition effect of L-NAME on THP-induced vascular relaxation. THP also induced intracellular NO production in human umbilical vein endothelial cells. However, Pre-incubation with ?-adrenergic receptor blocker propranolol, angiotensin II receptor 1 (AT1) inhibitor losartan, angiotensin II receptor 2 (AT1) inhibitor PD123319 or angiotensin converting enzyme inhibitor enalapril enhanced the vascular relaxation effect of THP. THP did not affect the angiotensin II induced vascular contraction. Cyclooxygenase-2 (COX2) inhibitor indomethacin did not affect the vascular relaxation effect of THP. Furthermore, pre-treatment THP attenuated KCl and Phe induced rat aorta contraction in standard Krebs solution. In Ca2+ free Krebs solution, THP inhibited the Ca2+ induced vascular contraction under KCl or Phe stress and reduced KCl stressed Ca2+ influx in rat vascular smooth muscle cells. THP also inhibited intracellular Ca2+ release induced vascular contraction by blocking Ryr or IP3 receptors. In addition, the voltage-dependent K+ channel (Kv) blocker 4-aminopyridine, ATP-sensitive K+ channel (KATP) blocker glibenclamide and inward rectifying K+ channel blocker BaCl2 attenuated THP induced vascular relaxation regardless of the Ca2+-activated K+ channel (KCa) blocker tetraethylammonium. Thus, we could conclude that THP relaxed rat aorta in an endothelium-dependent and independent manner. The underlying mechanism of THP relaxing rat aorta involved PI3K/Akt/eNOS/NO/cGMP signaling path-way, Ca2+ channels and K+ channels rather than COX2, ?-adrenergic receptor and renin-angiotensin system (RAS). These findings indicated that THP might be a potent treatment of diseases with vascular dysfunction like hypertension.
Project description:The renin-angiotensin system (RAS) in the brain is a critical determinant of blood pressure, but the mechanisms regulating RAS activity in the brain remain unclear. Expression of brain renin (renin-b) occurs from an alternative promoter-first exon. The predicted translation product is a nonsecreted enzymatically active renin whose function is unknown. We generated a unique mouse model by selectively ablating the brain-specific isoform of renin (renin-b) while preserving the expression and function of the classical isoform expressed in the kidney (renin-a). Preservation of renal renin was confirmed by measurements of renin gene expression and immunohistochemistry. Surprisingly, renin-b-deficient mice exhibited hypertension, increased sympathetic nerve activity to the kidney and heart, and impaired baroreflex sensitivity. Whereas these mice displayed decreased circulating RAS activity, there was a paradoxical increase in brain RAS activity. Physiologically, renin-b-deficient mice exhibited an exaggerated depressor response to intracerebroventricular administration of losartan, captopril, or aliskiren. At the molecular level, renin-b-deficient mice exhibited increased expression of angiotensin-II type 1 receptor in the paraventricular nucleus, which correlated with an increased renal sympathetic nerve response to leptin, which was dependent on angiotensin-II type 1 receptor activity. Interestingly, despite an ablation of renin-b expression, expression of renin-a was significantly increased in rostral ventrolateral medulla. These data support a new paradigm for the genetic control of RAS activity in the brain by a coordinated regulation of the renin isoforms, with expression of renin-b tonically inhibiting expression of renin-a under baseline conditions. Impairment of this control mechanism causes neurogenic hypertension.
Project description:A high-fat diet (HFD) induces an increase in arterial pressure and a decrease in baroreflex function, which may be associated with increased expression of angiotensin type 1 receptor (AT1R) and pro-inflammatory cytokine genes and reduced expression of the angiotensin type 2 receptor (AT2R) gene within the nucleus of the solitary tract (NTS), a key area of the brainstem involved in cardiovascular control. Thus, in the present study, we evaluated the changes in arterial pressure and gene expression of components of the renin-angiotensin system (RAS) and neuroinflammatory markers in the NTS of rats fed a HFD and treated with either an AT1R blocker or with virus-mediated AT2R overexpression in the NTS. Male Holtzman rats (300-320?g) were fed either a standard rat chow diet (SD) or HFD for 6 weeks before commencing the tests. AT1R blockade in the NTS of HFD-fed rats attenuated the increase in arterial pressure and the impairment of reflex bradycardia, whereas AT2R overexpression in the NTS only improved the baroreflex function. The HFD also increased the hypertensive and decreased the protective axis of the RAS and was associated with neuroinflammation within the NTS. The expression of angiotensin-converting enzyme and neuroinflammatory components, but not AT1R, in the NTS was reduced by AT2R overexpression in this site. Based on these data, AT1R and AT2R in the NTS are differentially involved in the cardiovascular changes induced by a HFD. Chronic inflammation and changes in the RAS in the NTS may also account for the cardiovascular responses observed in HFD-fed rats.
Project description:Tumour cells become addicted to the expression of initiating oncogenes like Ras, such that loss of oncogene expression in established tumours leads to tumour regression. HRas, NRas or KRas are mutated to remain in the active GTP-bound oncogenic state in many cancers. Although Ras activates several proteins to initiate human tumour growth, only PI3K, through activation of protein kinase B (PKB; also known as AKT), must remain activated by oncogenic Ras to maintain this growth. Here we show that blocking phosphorylation of the AKT substrate, endothelial nitric oxide synthase (eNOS or NOS3), inhibits tumour initiation and maintenance. Moreover, eNOS enhances the nitrosylation and activation of endogenous wild-type Ras proteins, which are required throughout tumorigenesis. We suggest that activation of the PI3K-AKT-eNOS-(wild-type) Ras pathway by oncogenic Ras in cancer cells is required to initiate and maintain tumour growth.
Project description:Angiotensin II is the principle effector molecule of the renin angiotensin system (RAS). It exerts its various actions on the cardiovascular and renal system, mainly via interaction with the angiotensin II type-1 receptor (AT1R), which contributes to blood pressure regulation and development of hypertension but may also mediate effects on the immune system. Here we study the role of the RAS in myelin-oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE), a model mimicking many aspects of multiple sclerosis. Quantitative RT-PCR analyses showed an up-regulation of renin, angiotensin-converting enzyme, as well as AT1R in the inflamed spinal cord and the immune system, including antigen presenting cells (APC). Treatment with the renin inhibitor aliskiren, the angiotensin II converting-enzyme inhibitor enalapril, as well as preventive or therapeutic application of the AT1R antagonist losartan, resulted in a significantly ameliorated course of MOG-EAE. Blockade of AT1R did not directly impact on T-cell responses, but significantly reduced numbers of CD11b+ or CD11c+ APC in immune organs and in the inflamed spinal cord. Additionally, AT1R blockade impaired the expression of CCL2, CCL3, and CXCL10, and reduced CCL2-induced APC migration. Our findings suggest a pivotal role of the RAS in autoimmune inflammation of the central nervous system and identify RAS blockade as a potential new target for multiple sclerosis therapy.
Project description:Studies involving the use of prenatally programmed hypertension have been shown to potentially contribute to prevention of essential hypertension (EH). Our previous research has demonstrated that prenatal inflammatory stimulation leads to offspring's aortic dysfunction and hypertension in pregnant Sprague-Dawley rats challenged with lipopolysaccharide (LPS). The present study found that prenatal LPS exposure led to NF-κB dyshomeostasis from fetus to adult, which was characterized by PI3K-Akt activation mediated degradation of IκBα protein and impaired NF-κB self-negative feedback loop mediated less newly synthesis of IκBα mRNA in thoracic aortas (gestational day 20, postnatal week 7 and 16). Prenatal or postnatal exposure of the IκBα degradation inhibitor, pyrollidine dithiocarbamate, effectively blocked NF-κB activation, endothelium dysfunction, and renin-angiotensin system (RAS) over-activity in thoracic aortas, resulting in reduced blood pressure in offspring that received prenatal exposure to LPS. Surprisingly, NF-κB dyshomeostasis and RAS over-activity were only found in thoracic aortas but not in superior mesenteric arteries. Collectively, our data demonstrate that the early life NF-κB dyshomeostasis induced by prenatal inflammatory exposure plays an essential role in the development of EH through triggering RAS over-activity. We conclude that early life NF-κB dyshomeostasis is a key predictor of EH, and thus, NF-κB inhibition represents an effective interventional strategy for EH prevention.