This paper is to report the exploration of the activation of Rho/ROCK signal pathway in 5-HT-induced proliferation of rat pulmonary artery smooth muscle cells (PASMCs) and the inhibitory effect of m-Nis on this pathway. PASMCs were cultured with the explant technique. MTT assay was used to explore the proliferation of PASMCs after 5-HT treated for different time and the intervening effect of m-Nis. RT-PCR and Western blot were used respectively to explore the mRNA expression of RhoA, ROCK1 and the protein expression of p-MYPT1 in 5-HT-treated PASMCs and intervening effect of m-Nis. The results of MTT assay suggested that 5-HT (1 µmol · L(-1)) treatment for 12-72 h significantly induced the proliferation of rat PASMCs (P<0.05 or P < 0.01), which were inhibited by m-Nis (1 x 10(-5), 1 x 10(-6), l x 10(-7), 1 x10(-8) mol · L(-1)) in dose-dependent manners (P < 0.05 or P < 0.01). Similarly, the mRNA expression of RhoA, ROCK1 and the protein expression of p-MYPT1 were also inhibited by m-Nis in different degrees (P < 0.05 or P < 0.01). Thus, the results of this study suggested that Rho/ROCK pathway played an important role in 5-HT-induced proliferation of rat PASMCs, m-Nis inhibited 5-HT-induced proliferation obviously, which may be related to the blockage of Rho/ROCK signal pathway.
Animals ; Cell Proliferation ; drug effects ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Nisoldipine ; pharmacology ; Protein Phosphatase 1 ; metabolism ; Pulmonary Artery ; cytology ; Rats ; Serotonin ; pharmacology ; Signal Transduction ; rho-Associated Kinases ; metabolism ; rhoA GTP-Binding Protein ; metabolism

The aim of the present study was to explore the effect of cholecystokinin octapeptide (CCK-8) on [Ca(2+)](i) and its signal transduction mechanism in isolated guinea pig cardiomyocytes. [Ca(2+)](i) was measured by laser scanning confocal microscopy in single ventricular myocytes which were dissociated by enzymatic dissociation method and loaded with Fluo 3-AM. The changes in [Ca(2+)](i) were represented by fluorescent intensity (F(i)) or relative fluorescent intensity (F(i)/F(O)%). The results obtained are as follows. (1) In the normal Tyrode's solution containing 1.0 mmol/ L Ca(2+), CCK-8 (1-10(4) pmol/L) elicited a rapid and marked increase in [Ca(2+)](i). (2) When cardiomyocytes were pretreated with the Ca(2+) chelator EGTA (3 mmol/L) and Ca(2+) channel antagonist nisoldipine (0.5 micromol/L) for 5 min, CCK-8 (10(2)pmol/L) caused a slow and small increase in [Ca(2+)](i) (p< 0.01). (3) Pretreatment with the nonselected CCK- receptor (CCK-R) antagonist proglumide (6 micromol/L) or the tyrosine kinase inhibitor genistein (1 micromol/L) for 5 min could inhibit the increase of [Ca(2+)](i) induced by CCK-8 (10(2) pmol/L) (p<0.01). The results suggest that CCK-8 increases the [Ca(2+)](i) via activating the receptor-operated Ca(2+) channel and eliciting the influx of Ca(2+) in isolated guinea pig cardiomyocytes, in which tyrosine kinase may be involved.
Animals ; Calcium ; metabolism ; Calcium Channel Blockers ; pharmacology ; Calcium Channels ; drug effects ; Cell Separation ; Guinea Pigs ; Myocytes, Cardiac ; metabolism ; ultrastructure ; Nisoldipine ; pharmacology ; Protein-Tyrosine Kinases ; metabolism ; Signal Transduction ; Sincalide ; pharmacology

Effect of new calcium antagonist m-nisoldipine (m-Nis) on MCT-induced PH in rats and its mechanisms were investigated. Rats were injected with a single dose (60 mg x kg(-1)) of MCT subcutaneously to induce PH. Pulmonary haemodynamic measurement and lung tissue morphological investigations were undertaken. The MDA production and SOD activity in the serum were tested. PCNA, ERK1 and p-ERK expressions were analyzed by Western blotting. The expressions of 5-HT and PCNA were observed with immunohistochemistry. Results suggested that the PAP, right ventricular index and the degree of muscularization of small pulmonary artery were elevated markedly in MCT group, which was attenuated by m-Nis treatment. A significant reduction in MDA production and an increase in the SOD activity in the serum were also observed in all three m-Nis groups. The number of PCNA and 5-HT positive smooth muscle cells increased significantly in MCT group, and m-Nis treatment attenuated the expression obviously. Western blotting results suggested that the protein expression of PCNA and the ratio of p-ERK/ ERK1 increased markedly in MCT group and decreased by m-Nis. In conclusion, m-Nis protected against MCT-induced PH by decreasing PAP, right ventricular index, PAMSCs proliferation and pulmonary artery remodelling, which may be related to the reduction of 5-HT and the suppression of the ERK/MAPK signal pathway.
Animals ; Antihypertensive Agents ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Hypertension, Pulmonary ; chemically induced ; metabolism ; pathology ; Male ; Monocrotaline ; blood ; Nisoldipine ; pharmacology ; Proliferating Cell Nuclear Antigen ; metabolism ; Pulmonary Artery ; metabolism ; pathology ; Random Allocation ; Rats ; Rats, Wistar ; Serotonin ; metabolism ; Signal Transduction ; Superoxide Dismutase ; blood

This study is to explore the activation of the Ca2+/CaM/CaN signal pathway in 5-HT-induced proliferation of rat pulmonary artery smooth muscle cells (PASMCs) and the inhibitory effect of m-nisoldipine (m-Nis) on this pathway. PASMCs were cultured with the explant technique. The proliferation of PASMCs was evaluated by MTT assay. Confocal microscopy was used to measure the change of [Ca2+]i. The mRNA expression of CaM and CaN was evaluated by RT-PCR and the activity of CaN was measured according to the instruction of kits. The results of MTT assay suggested that 5-HT (1 micromol x L(-1)) significantly induced the proliferation of rat PASMCs (P < 0.01), which was inhibited obviously by m-Nis (P < 0.05 or P < 0.01). Similarly, m-Nis inhibited 5-HT-induced elevation of [Ca2+]i (P < 0.01). The mRNA expression of CaM, CaN and the activation of CaN were also inhibited by m-Nis at different degrees (P < 0.05 or P < 0.01). Thus, the results of this study suggested that Ca2+/CaM/CaN signal pathway played an important role in 5-HT-induced proliferation of rat PASMCs, the inhibition of m-Nis on proliferation of rat PASMCs may be related to the blockage of Ca2+/CaM/CaN signal pathway by inhibiting the elevation of [Ca2+]i.
Animals ; Antihypertensive Agents ; pharmacology ; Calcineurin ; genetics ; metabolism ; Calcium ; metabolism ; Calcium Channel Blockers ; pharmacology ; Calmodulin ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Male ; Myocytes, Smooth Muscle ; cytology ; metabolism ; Nisoldipine ; pharmacology ; Pulmonary Artery ; cytology ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Serotonin ; pharmacology ; Signal Transduction