Effect Of Agmatine On Proliferation Of Rat Pulmonary Artery Smooth Muscle Cells And Its Mechanism

Hypoxic pulmonary hypertension plays a key role in the development of high altitude pulmonary edema, high altitude heart disease and chronic pulmonary heart disease. Many recent data indicate that pulmonary vascular contraction, especially pulmonary arteriole smooth muscle, and pulmonary vascular remodeling are the base of maintenance and development of hypoxic pulmonary hypertension. Pulmonary artery smooth muscle cells can sense hypoxia directly. Hypoxia can stimulate proliferation of PASMCs in vitro. Ang-II and PDGF are involved in the mechanism of hypoxia stimulating proliferation of PASMCs. Agmatine, a product derived from the decarboxylation of L-arginine by arginine decarboxylase, has been recently identified in various mammalian organs and tissues. Concentrations of agmatine in different organs and tissues are different. It has been reported that agmatine can inhibit proliferation of many types of cells. The mechanism of agmatine regulating proliferation of PASMCs is not well understood.To determine whether agmatine can inhibit proliferation of rat PASMCs induced by hypoxia and understand the mechanism of agmatine regulating proliferation of rat PASMCs induced by hypoxia, we observed growth curve, concentration of lactate dehydrogenase, 3H-TdR incorporation, cell cycle, PCNA protein expression of rat PASMCs cultured under hypoxic condition and MAPK protein expression of rat PASMCs stimulated by Ang-II or PDGF.Primary culture of rat PASMCs were prepared from adult male Wistar rat pulmonary artery by the method of tissue block anchorage. 3H-TdR incorporation was measured by scintillation counting. Cell cycle was measured by flow cytometry. PCNA protein expression and MAPK protein expression were measured by image analysis.This experiment was divided into three parts. The first part was: primary culture, immunohistochemical identification and growth curve measure of rat PASMCs. When concentration of lactate dehydrogenase and growth curves were tested, rat PASMCs were divided into two groups: control group and agmatine administration group. The second part was: effect of agmatine on proliferation of rat PASMCs induced by serum or hypoxia. This part was divided into two units: normoxia unit and hypoxia unit. When cell cycle and PCNA content of rat PASMCs were tested, rat PASMCs were divided into two groups: control group and agmatine administration group(100(mol/L). When 3H-TdR incorporation was tested, rat PASMCs were divided two groups: control group and agmatine administration group. Agmatine administration group was subdivided into four groups by the concentration of agmatine: 50(mol/L, 100(mol/L, 200(mol/L and 500(mol/L. The third part was: the mechanism of agmatine regulating the proliferation of rat PASMCs. This part was divided into two units: normoxia unit and hypoxia unit. When 3H-TdR incorporation was tested, rat PASMCs were divided two groups: control group and agmatine administration group(100(mol/L). When MAPK content of rat PASMCs cultured under normoxic condition was tested, rat PASMCs were divided two groups: control group and agmatine administration group(100(mol/L).Main results: 1. Exposure of rat PASMCs to agmatine (1mmol/L) for 24h did not increase the concentration of lactate dehydrogenase in the medium or change the morphology of cells.2. Agmatine dose-dependently decreased 3H-TdR incorporation, PCNA protein expression of rat PASMCs cultured under hypoxic condition or stimulated by 10%NCS. Agmatine increased the percentage of G0/G1 cells and decreased the percentage of G2/M and S cells cultured under hypoxic condition or stimulated by 10%NCS. 3. Agmatine decreased 3H-TdR incorporation of rat PASMCs stimulated by Ang-II or PDGF. 4. Agmatine decreased MAPK protein expression of rat PASMCs stimulated by Ang-II or PDGF.Conclusion: 1.Agmatine up to 1mmol/L was not cytotoxic to cultured rat PASMCs. 2.Agmatine could inhibit proliferation of rat PASMCs induced either by hypoxia or by 10%NCS in a dose dependent manner. 3.Agmatine could inhibit proliferation of rat P...