The Effects of Iptakalim, a Novel K_(ATP) Opener, on the Expressions of TGF-β_1 and PCNA mRNA in Primary Cultured Human Pulmonary Artery Smooth Muscle Cells

Pulmonary artery hypertension(PAH)has been demonstrated to be the key event in the process of chronic obstructive pulmonary disease (COPD)to chronic cor pulmonale.There is still no ideal treatment for PAH at present.It is of great significance to elucidate the mechanism of PAH and search for one new treatment for this debilitating disease.Pulmonary artery vasoconstriction and vascular remodeling greatly contribute to a sustained elevation of pulmonary vascular resistance (PVR)and pulmonary arterial pressure(PAP)in patients with PAH. Pulmonary artery remodeling is characterized largely by medial hypertrophy due to enhanced vascular smooth muscle cell proliferation and/or attenuated apoptosis.K~+ channels in pulmonary arterial smooth muscle cells may play a unique and important role in the regulation of pulmonary artery remodeling.Decreased K~+ channel activity reduces K~+ efflux through plasmalemmal K~+ channels and causes cellular membrane depolarization.Membrane depolarization,therefore,induce cytoplasmic free Ca~(2+)concentration([Ca~(2+)]_(cyt))rise in pulmonary arterial smooth muscle cells.The increased[Ca~(2+)]_(cyt)stimulates pulmonary artery smooth muscle cell proliferation,ultimately contributing to the development of pulmonary artery remodeling.On the other hand, dysfunction of K~+ channels leads to inhibition of apoptosis by decelerating apoptotic volume decrease(AVD)and inhibiting the activity of cytoplasmic caspases,and promotes pulmonary artery medial hypertrophy.There are mainly four types of K~+ channels in pulmonary arterial smooth muscle cells:(ⅰ)voltage-gated K~+(K_v)channels; (ⅱ)Ca~(2+)-activated K~+(K_(Ca))channels;(ⅲ)two-pore domain K~+(K_(2P)) channels;and(ⅳ)inwardly rectifying K~+(K_(IR))channels,of which ATP-sensitive K~+(K_(ATP))channel is a member.K_(ATP)channels can be inhibited by micromolar concentrations of intracellular ATP.They play important roles in the physiology and pathophysiology of vascular smooth muscle cells by coupling the metabolic state of the cell to its electrical activity.Iptakalim(IPT),a newly selective K_(ATP)opener,is designed and synthesized by native researchers.Our previous studies have shown that IPT treatment not only could decrease rat pulmonary artery pressure induced by hypoxia and reverse rat hypoxic pulmonary artery remodeling and the right ventricular hypertrophy,but also inhibit human or rabbit pulmonary arterial smooth muscle cell proliferation and the [Ca~(2+)]_(cyt)rise induced by endothelin-1(ET-1).Recent studies suggested that transforming growth factor beta-1 (TGF-β_1)and proliferating cell nuclear antigen(PCNA)might be important cytokines which regulate the pulmonary artery remodeling. Moreover,our previous studies also found that IPT treatment could decrease the mRNA expression of TGF-β_1 and PCNA in rat pulmonary artery induced by hypoxia.However,whether IPT has effects on the expressions of TGF-β_1 and PCNA in human pulmonary artery smooth muscle cells requires further investigation.In the present study,the effects of IPT on their expression induced by ET-1 and hypoxia, respectively were explored so as to elucidate the mechanism of K_(ATP) channels regulating pulmonary artery remodeling.PartⅠ:The effects of iptakalim,a novel K_(ATP)opener,on the expressions of TGF-β_1 and PCNA mRNA in primary cultured human pulmonary artery smooth muscle cells induced by ET-1Objective To explore the effects of IPT,a novel K_(ATP)opener,on expressions of TGF-β_1 and PCNA mRNA in primary cultured human pulmonary artery smooth muscle cells induced by ET-1.Methods Under germfree conditions,the smooth muscular layers were separated from human pulmonary arterioles(3rd～4th divisions).Human pulmonary artery smooth muscle cells were cultured by tissue block method,and the cells was induced by ET-1 in vitro.Fluorescence quantitative reverse transcription- polymerase chain reaction(FQ- RT- PCR)was used to detect the levels of TGF-β_1 and PCNA mRNA in human pulmonary artery smooth muscle cells.Results Human pulmonary artery smooth muscle cells in the medium with ET-1(10~(-5)mol·L~(-1))showed higher expressions of TGF-β_1 and PCNA mRNA than those in control group. Compared with the value of the cells treated with ET-1 only,IPT at the concentrations of 10~(-7),10~(-6)and 10~(-5)mol·L~(-1)down-regulated the expressions of TGF-β_1 and PCNA mRNA in a concentration-dependent manner.Glibenclimide(GLI),a selective K_(ATP)channel antagonist,at the concentrations of 10~(-7),10~(-6)and 10~(-5)mol·L~(-1)antagonized the effects of IPT on the expressions of TGF-β_1 and PCNA mRNA in a concentration-dependent manner.Conclusion IPT has an inhibitory effect on the expressions of TGF-β_1 and PCNA mRNA in human pulmonary artery smooth muscle cells induced by ET-1 through activating K_(ATP)channels. PartⅡ:The Effects of iptakalim,a novel K_(ATP)opener,on the expressions of TGF-β_1 and PCNA mRNA in primary cultured human pulmonary artery smooth muscle cells induced by hypoxiaObjective To investigate the effects of IPT,a novel K_(ATP)opener,on the expressions of TGF-β_1 and PCNA mRNA in primary cultured human pulmonary artery smooth muscle cells induced by hypoxia.Methods Under germfree conditions,the smooth muscular layers were separated from human pulmonary arterioles(3rd～4th divisions).Human pulmonary artery smooth muscle cells were cultured by tissue block method.FQ-RT-PCR was used to measure the expression level of TGF-β_1 and PCNA mRNA.Results The expressions of TGF-β_1 and PCNA mRNA in hypoxia group were higher than those in control group.IPT at the concentrations of 10~(-7),10~(-6)and 10~(-5)mol·L~(-1)lowered the expressions of TGF-β_1 and PCNA mRNA in a concentration-dependent manner. Glibenclimide(GLI),a selective K_(ATP)channel antagonist,at the concentration of 10~(-5)mol·L~(-1)antagonized the effect of IPT with the same concentration on the expressions of TGF-β_1 and PCNA mRNA.Conclusion The expressions of TGF-β_1 and PCNA mRNA in primary cultured human pulmonary artery smooth muscle cells under hypoxic conditions increased,while IPT has an inhibitory effect on the expressions of TGF-β_1 and PCNA mRNA in a concentration-dependent manner through activating K_(ATP)channels.