5-HT_1B/1D-receptor Mechanism Of Pulmonary Artery Smooth Muscle Cell Proliferation And Mitogenesis In The Rat

ObjectiveSerotonin (5 - hydroxytryptamine, 5 - HT) plays an important role in pulmonary hypertension. It was reported that there was an increased expression of 5 - HT1B receptor mRNA in monocrotaline (MCT) induced pulmonary hypertension rats. Also, hypoxia increased pulmonary vascular remodeling in wild - type mice and this was reduced in the 5 - HT1B receptor knockout mice. Furthermore , hypoxia increased 5 - HT1 receptor - mediated constriction of pulmonary arteries from the wild - type mice and this was attenuated in the 5 - HT1B receptor knockout mice. These evidences suggested 5 - HT1B receptor plays an important role in the development of PHT. Serotonin is one of vasoactive substances and has been recognized to cause proliferation of a variety of cells. Several studies have shown that the mitogenic action of serotonin is initiated through its binding to cells surface receptors. Yet, the 5 - HT receptor mechanism of pulmonary artery smooth muscle cell proliferation and mitogenesis has not yet been fully e-licited. So, the objetive of the present study covers the following aspects.1. To investigate the mitogenetic effects of 5 - HT and, a 5 - HT1B/1D receptor agonist sumatriptan (Sum) on pulmonary artery smooth muscle cells (PASMC) , to identify the role of 5 - HT1B/1D receptors in PASMC proliferationand pulmonary remodeling.2. To observe the effects of selective 5 - HT1B receptor antagonist SB224289 and selective 5 - HT1D receptor antagonist BRL15572 on PASMC proliferation induced by 5 - HT and sumatriptan, investigate the role of 5 - HT1B receptor in PASMC proliferation and pulmonary remodeling.3. To observe the effect of 5 - HT and 5 - HT1B/1D receptor agonist sumatriptan on ASMC proliferation and DNA synthesis, observe the difference reaction of PASMC and ASMC to 5 - HT and 5 - HT1B/1D receptor agonist. Study the effect of selective 5 - HT1B receptor antagonist SB224289 and selective 5 - HT1D receptor antagonist BRL15572 on ASMC proliferation induced by 5 - HT, investigate the mitogenic mechanism of ASMC.4. According to the different mitogenic mechanism between ASMC and PASMC, research high selectivity and efficiency new medicine for the treatment of pulmonary remodeling and pulmonary hypertension, especially from the point of 5 - HT1B receptor and 5 - HT1B receptor antagonists.Methods1. PASMC and ASMC were isolated from rat pulmonary artery and aorta. DMEM culture media with 20% FBS were used in primary culture; DMEM culture media with 5% FBS were used as vehicle; 5-HT, sumatriptan, 5 - HT1Breceptor antagonist SB22489 and selective 5 - HT1D receptor antagonist BRL15572 were used as treatments.2. Proliferation of cultured PASMC and ASMC were evaluated by MTT assay , cells DNA synthesis were evaluated by 3 H - TdR incorporation, and cell cycle analysis, proliferation index (PI) , s - phase cell fraction (SPF) were performed by flow cytometry (FCM).3. All data were expressed as mean value standard deviation. Statistic a-nalysis was done by Statistical Program for Social Sciences (SPSS) Software. One Way ANOVA analyze multi - groups comparisons. Comparisons between groups were tested by LSD (least significant difference) test. P < 0. 05 wereconsidered to be statistical significant.Results1. SMC culture and evaluation of cell proliferation methodPI and SPF performed by FCM were closely correlated with cpm that was acquired from 3H -TdR incorporation tests. It is a better way to use MTT assay and FCM method in observing cells proliferation and mitogenesis than to use them alone.2. Mechanism of PASMC mitogenesis induced by 5 - HT(1) Effect of 5 - HT and sumatriptan on PASMCIt was found that 5 - HT 10-6 - 10-8 mole/L stimulated proliferation of PASMC by MTT assay. The proliferation rates ( PR) of 5 - HT 10 -6-10-8mol/ L treatments were 256%±18% ; 228%±29% and 201% ±17% respectively. Sumatriptan 10 -6-10-8mol/L mimicked the mitogenesis effect of 5 - HT. The proliferation rates of Sum were 245%±21% ; 220%±25% ; and 199%±25...