| Background and Objective: The renin-angiotensin system is considered one of the most important regulatory systems for vascular homeostasis. Angiotensin II (AngII) acts at specific receptors, and these interactions can be blocked by a variety of peptide or nonpeptide AngII antagonists. There are several signal transduction mechanisms mediating the physiological and pathophysiological actions of AngII in vascular smooth muscle cells including protein kinase C(PKC), mitogen-activated protein/extracellular signal- regulated kinases (ERK). AngII induces the G-protein-mediated activation of phospholipase C causing the breakdown of phosphatidylinositol-4, 5-bisphosphate to generate inositole trisphosphate and diacylglycerol. The AngII-induced production of diaclyglycerol together with increased cytosolic calcium activates the PKC. Activation of PKC causes phosphorylation of several tyrosine kinases, including Src tyrosine kinases and ERK1/2. Recent studies showed that AngII-induced ERK1/2 activation is mediated by two distinct pathways, one being PKC-dependent, and the other being calcium- and c-Src-dependent. AngII induced activation of ERK1/2 is associated with vasoconstriction, cellular growth and cellular differentiation . Angiotensin I can also be processed into the N-terminal heptapeptide angiotensin-(1-7) (Ang-(1-7))by neutral endopeptidases (EC 3.4.24.11), carboxypeptidases, or metalloendopeptidases. Increased plasma Ang-(1-7) levels have been observed after inhibition of angiotensin-converting enzyme. Ang-(1-7) induces relaxation in porcine coronary arteries and blocks AngII-induced vasoconstrictions of human internal mammary arteries.Short-term intravenous infusion of Ang-(1-7) potentiated the hypotensive effect of bradykinin in normotensive and hypertensive rats. Long-term intravenous infusion of Ang-(1-7) decreased mean arterial blood pressure in spontaneously hypertensive rats. A recent study showed that intra-arterial infusion of Ang-(1-7) blocked the AngII induced vasoconstriction in human forearm resistant vessels, whereas others did not observe any effect of Ang-(1-7) on human forearm blood flow. Additional studies have demonstrated that Ang-(1-7) inhibits growth of vascular smooth muscle cells. It has been suggested that Ang-(1-7) may interact with multiple binding sites. Now, we investigated the effects of Ang-(1-7) on AngII-induced vasoconstriction, growth of vascular smooth muscle cells, and AngII-induced signal transduction pathways.Methods:1. Animals and hemodynamic measurements Male 7-week-old Wistar rats weighting 200-250g were housed under a 12h/12h day/night cycle. Hemodynamic measurements were done according to Symons et al. The thoracic aorta was dissected, carefully freed from connective tissue, and placed in phosphate buffer solution, pH 7.4 at 37°C. Isometric force of the aortic rings was measured according to established methods using a force transducer connceted with a polygraph. 2. Culture of vascular smooth muscle cells (VSMC) Vascular smooth muscle cells (VSMC) were obtained from thoracic aortas and cultured by tissue explant method. Cultures were maintained at 37°C in a humidified atmosphere of 95% air/5% CO2. To verify that cultured cells were VSMC, immunocytochemical localization of smooth muscle-specific alpha actin was done. 3. Cytosolic calcium of VSMC was measured using PTI ratio fluorescence spectrometers. 4. DNA and protein synthesis DNA and protein synthesis were measured according to Touyz et al. Cells were treated with increasing angiotensin-(1-7) concentrations, and the incorporation of [3H] thymidine and[3H] leucine in response to AngII were measured in a liquid scintillation counter.5. Western blotting for proliferation celluar nucleus antigen(PCNA), protein kinase C, extracellular signal-regulated kinase ERK1 and ERK2 Immunoblottings of PCNA,protein kinase C and ERK1/2 were performed in cultured VSMC according to Lia et al. Membranes were incubated with mouse monoclonal anti-PCNA,anti-protein kinase...
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