Decreased PKG transcription mediated by PI3K/Akt/FoxO1 pathway is involved in the development of nitroglycerin tolerance.

Phosphoinositide 3-kinase (PI3K)/Akt plays a pivotal role in the vascular response. The present study is to determine whether PI3K/Akt pathway in vascular smooth muscle cells is involved in nitroglycerin (NTG) tolerance and the underlying mechanism. Nitrate tolerance of porcine coronary arteries in vitro was induced by incubation of NTG (10-5 M) for 24 h. Nitrate tolerance in vivo was obtained by subcutaneous injection of mice with NTG (20 mg kg-1, tid, 3 days) and the aortas were used. Protein levels of total and phosphorylated Akt, forkhead box protein O1 (FoxO1), and cGMP-dependent protein kinase (PKG) were determined by western blot analysis. Isometric vessel tension was recorded by organ chamber technique. PKG mRNA was determined by real-time PCR. The cellular translocation of FoxO1 was observed by immunofluorescence. Reactive oxygen species (ROS) level was measured by DHE staining. The vascular relaxation to NTG was significantly inhibited in in vivo and in vitro NTG tolerant arteries. Meanwhile, the protein level of phosphorylated Akt at Ser473 was increased in the tolerant arteries. The attenuated relaxation and the augmented Akt-p were ameliorated by LY294002, a specific inhibitor of PI3K. The protein and mRNA expression of PKG were significantly down-regulated in NTG tolerant arteries, which were reversed by LY294002. The level of phosphorylated FoxO1 at Ser256 and its translocation from the nucleus to the cytosol were both increased in NTG tolerance and were also inhibited by LY294002. ROS production was significantly increased in NTG tolerant arteries, which was not be affected by LY294002 but inhibited by N-acetyl-L-cysteine. In conclusion, the present study suggests that PI3K/Akt in vascular smooth muscle is involved in the development of NTG tolerance via inhibiting PKG transcription and the effect is mediated by FoxO1.

Rhynchophylline Ameliorates Endothelial Dysfunction via Src-PI3K/Akt-eNOS Cascade in the Cultured Intrarenal Arteries of Spontaneous Hypertensive Rats.

Objectives: To examine the protective effect of Rhynchophylline (Rhy) on vascular endothelial function in spontaneous hypertensive rats (SHRs) and the underlying mechanism. Methods: Intrarenal arteries of SHRs and Wistar rats were suspended in myograph for force measurement. Expression and phosphorylation of endothelial nitric oxide (NO) synthase (eNOS), Akt, and Src kinase (Src) were examined by Western blotting. NO production was assayed by ELISA. Results: Rhy time- and concentration-dependently improved endothelium-dependent relaxation in the renal arteries from SHRs, but had no effect on endothelium-independent relaxation in SHR renal arteries. Wortmannin (an inhibitor of phosphatidylinositol 3-kinase) or PP2 (an inhibitor of Src) inhibited the improvement of relaxation in response to acetylcholine by 12 h-incubation with 300 µM Rhy. Western blot analysis revealed that Rhy elevated phosphorylations of eNOS, Akt, and Src in SHR renal arteries. Moreover, wortmannin reversed the increased phosphorylations of Akt and eNOS induced by Rhy, but did not affect the phosphorylation of Src. Furthermore, the enhanced phosphorylations of eNOS, Akt, and Src were blunted by PP2. Importantly, Rhy increased NO production and this effect was blocked by inhibition of Src or PI3K/Akt. Conclusion: The present study provides evidences for the first time that Rhy ameliorates endothelial dysfunction in SHRs through the activation of Src-PI3K/Akt-eNOS signaling pathway.

[Nitric Oxide Signaling Pathway and Vascular Activity].

Nitric oxide-cyclic guanosine 3',5'-monophosphate (cGMP)-cGMP dependent protein kinase signaling pathway is a key mechanism for the modulation of vascular function. The progress in this area of recent years was briefly summarized in this article, in particular, regarding the redox-dependent dimerization of the relevant signaling molecules and their physiological significance as well as the role of soluble guanylyl cyclase-derived cyclic inosine 3',5'-monophosphate as a new messenger for vasoconstriction.

[Recent progress in the development of atherosclerosis: role of heat shock protein].

Recent studies have shown a close relationship between heat shock proteins (HSPs) and atherosclerosis. HSP60 may promote the development of atherosclerosis by inducing autoimmune response, enhancing inflammatory response, promoting proliferation and migration of vascular smooth muscle cells. While HSP27 may protect the blood vessels from oxidative stress, reduce inflammatory response, inhibit proliferation, migration and apoptosis of vascular smooth muscle cells, and stabilize the atherosclerotic plaque. These new understanding of the role of HSPs provides useful clues for the diagnosis and treatment of atherosclerosis.

Inhibitory effect of rhynchophylline on contraction of cerebral arterioles to endothelin 1: role of rho kinase.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhynchophylline (Rhy) is a major ingredient of Uncaria rhynchophylla (UR) used to reduce blood pressure and ameliorate brain ailments. This study was to examine the role of Rho kinase (ROCK) in the inhibition of Rhy on contraction of cerebral arterioles caused by endothelin 1 (ET-1). MATERIALS AND METHODS: Cerebral arterioles of male Wistar rats were constricted with ET-1 for 10 min followed by perfusion of Rhy for 20 min. Changes in the diameters of the arterioles were recorded. The effects of Rhy on contraction of middle cerebral arteries (MCAs) were determined by a Multi-Myograph. Western blotting and immunofluorescent staining were used to examine the effects of Rhy on RhoA translocation and myosin phosphatase target subunit 1 (MYPT1) phosphorylation. RESULTS: In vivo, Rhy (30-300 µM) relaxed cerebral arterioles constricted with ET-1 dose-dependently. In vitro, Rhy at lower concentrations (1-100 µM) caused relaxation of rat MCAs constricted with KCl and Bay-K8644 (an agonist of L-type voltage-dependent calcium channels (L-VDCCs)). Rhy at higher concentrations (>100 µM) caused relaxation of rat MCAs constricted with ET-1, which was inhibited by Y27632, a ROCK׳s inhibitor. Western blotting of rat aortas showed that Rhy inhibited RhoA translocation and MYPT1 phosphorylation. Immunofluorescent staining of MCAs confirmed that phosphorylation of MYPT1 caused by ET-1 was inhibited by Rhy. CONCLUSIONS: These results demonstrate that Rhy is a potent inhibitor of contraction of cerebral arteries caused by ET-1 in vivo and in vitro. The effect of Rhy was in part mediated by inhibiting RhoA-ROCK signaling.

Enhanced pressor response to acute Ang II infusion in mice lacking membrane-associated prostaglandin E2 synthase-1.

AIM: To examine the contribution of vascular membrane-associated prostaglandin E2 synthase-1 (mPGES-1) to acute blood pressure homeostasis. METHODS: Angiotensin II (AngII, 75 pmol·kg⁻¹·min⁻¹) was continuously infused via the jugular vein into wild-type and mPGES-1(-/-) mice for 30 min, and blood pressure was measured by carotid arterial catheterization. RT-PCR and immunohistochemistry were performed to detect the expression and localization of mPGES-1 in the mouse arterial vessels. Mesenteric arteries were dissected from mice of both genotypes to study vessel tension and measure vascular PGE2 levels. RESULTS: Wild-type and mPGES-1(-/-) mice showed similar blood pressure levels at baseline, and the acute intravenous infusion of AngII caused a greater increase in mean arterial pressure in the mPGES-1(-/-) group, with a similar diuretic and natriuretic response in both groups. mPGES-1 was constitutively expressed in the aortic and mesenteric arteries and vascular smooth muscle cells of wild-type mice. Strong staining was detected in the smooth muscle layer of arterial vessels. Ex vivo treatment of mesenteric arteries with AngII produced more vasodilatory PGE2 in wild-type than in mPGES-1(-/-) mice. In vitro tension assays further revealed that the mesenteric arteries of mPGES-1(-/-) mice exhibited a greater vasopressor response to AngII than those arteries of wild-type mice. CONCLUSION: Vascular mPGES-1 acts as an important tonic vasodilator, contributing to acute blood pressure regulation.

[Phosphodiesterase regulation of cardiovascular functions].

Phosphodiesterases (PDEs) are the enzymes to degrade the intracellular second messengers cAMP and cGMP. They are the targets for drug research and development of great therapeutic interests. Recent studies show that PDEs in cardiomyocytes form a complex with beta-adrenergic receptor and related proteins to ensure precise and localized cAMP signaling. The mechanism may provide new insight for the treatment of chronic heart failure with PDE inhibitors. The functions of blood vessels are regulated by the reactivity and phenotypes of vascular smooth muscle. PDE5 inhibitors, by enhancing the vasodilator effect of cGMP, have been successfully applied to the treatment of erectile dysfunction. The expression of PDE4 and PDE1C is upregulated during proliferation of vascular smooth muscle cells. Selectively inhibiting these PDE subtypes may provide a new way in treating disorders associated with vascular proliferation such as pulmonary hypertension and post-angioplasty restenosis. This review will focus on recent advance in PDE regulation of cardiovascular functions and new applications of PDE inhibitors in cardiovascular diseases.

[Role of cGMP-dependent protein kinase in the cardiovascular system].

cGMP-dependent protein kinase (PKG) is the major intracellular receptor for cyclic guanosine monophosphate (cGMP). Two forms of PKG, PKG-I and PKG-II, occur in mammalian tissues. PKG may mediate nitric oxide-cGMP-induced vasodilation through decreasing intracellular calcium concentration by the activation of calcium-activated potassium channel on the cell membrane and phosphorylation of phospholamban (PLB) and IP3 receptor-associated PKG-I substrate (IRAG) on the sarcoplasmic reticulum. PKG may also decrease the sensitivity of myosin to calcium by stimulating the activity of myosin light chain phosphatase and by inhibiting Rho kinase activity. PKG plays an important role in regulating the gene expression, phenotype, and proliferation of vascular smooth muscle cells. PKG activation inhibits platelet aggregation and myocardial hypertrophy. Recent studies indicate that the alternations of PKG expression and activity are closely related with the pathogenesis of atherosclerosis, restenosis, hypertension, hyperlipemia as well as nitrate tolerance.