The Effects Of Iptakalim, A Novel K_ATP Channel Opener, On MRNA Level Of SUR2B/Kir6.1 In Primary Cultured Human Pulmonary Artery Smooth Muscle Cells

Pulmonary artery hypertension(PAH)is charactered by contraction and proliferation of small pulmonary artery smooth muscle cells,and remodeling of small pulmonary arteries.However,the role of K⁺ channels in the development of PAH by mediating pulmonary vascular constriction and remodeling has been extensively demonstrated.The dysfunction of K⁺ channels activites L-Ca²⁺channels which induces influx of Ca²⁺and contraction of smooth muscle cells.Ca²⁺is also the important second messenger for some transform factors leading to cell proliferation in cells. On the other hand,a loss of K⁺ in cytoplast inhibits apoptosis factors and promotes cell proliferation.There are at least four types of K⁺ channels in pulmonary artery smooth muscle cells:(1)voltage-gated K⁺(Kv)channels,(2) Ca²⁺-activated K⁺(K_Ca)channels,(3)ATP-sensitive K⁺(K_ATP)channels, (4)two-pore-domain K⁺(K_2P)channels.N_ATPchannels were activated when hypoxia occurred,but they would be mostly inhibited by persistent hypoxia because of the unbalance between endothelium-derived vasoconstrictive substance and vasodilating substance.The increase of vasoconstrictive substance like angiotensinâ…¡(ANGâ…¡),endothelin-1(ET-1),ect.and decrease of vasodilating substance like prostaglandin I₂(PGI₂),nitric oxide(NO),etc.inhibit the expressions and fuctions of K_ATPchannels.Therefore,K_ATPchannels in pulmonary artery smooth muscle cells represent potential therapeutic targets for the control of PAH.Iptakalim,a novel K_ATPopener,which specially target to SUR2B/Kir6.1,can increase the value of outward K⁺ current density,attenuate pulmonary resistance vascular remodeling and prevent chronic PAH and right ventricle hypertrophy in rats in our precious studies.In order to investigate the effect of Iptakalim on human pulmonary smooth muscle cells,serial experiments include this one were performed.Our aim was to investigate the effects of Iptakalim on mRNA expression of SUR2B/Kir6.1 by real-time quantitative reverse transcriptive polymerase chain reaction (RQ-RT-PCR)in primary cultured human pulmonary artery smooth muscle cells induced by ET-1 or hypoxia,and to probe into the molecular biological mechanism of development in chronic PAH,and to accumlate experiment foundation of this new drug.Partâ… The effects of Iptakalim on mRNA expression of SUR2B/Kir6.1 in primary cultured human pulmonary artery smooth muscle cells induced by ET-1 Objective To study the effects of Iptakalim on mRNA expression of SUR2B/Kir6.1 in primary cultured human pulmonary artery smooth muscle cells induced by ET-1.Methods The specimens was obtained from patients who undergoing partly pneumonectomy.Intrapulmonary arteries(3rd～4th division)were opened longitudinally.After removing the adventitia and epithelia by a blade,vessels were cut into pieces of 1～3 mm².The tissues and smooth muscle cells were cultured with DMEM. Cells were incubated with test substances for 24h in normoxia.ET-1(10 nM),Iptakalim or Glyburide in different concentration(0.1μM,1.0μM, 10μM)were added respectively.Total RNA was extracted by Trizol and reverse transcribed into cDNA.RQ-RT-PCR was explored to measure the level of the expression of SUR2B/Kir6.1 mRNA.Results ET-1 suppressed the expression of SUR2B mRNA compared with control group.The expressions of SUR2B mRNA in the groups of ET-1+Iptakalim 0.1μM,ET-1+Iptakalim 1μM,ET-1+Iptakalim 10μM were increased in concentration-dependent manner compared with that in ET-1 group.The expressions of SUR2B mRNA in the groups of ET-1+Iptakalim 10μM+Glyburide 0.1μM,ET-1+Iptakalim 10μM+Glyburide 1μM,ET-1+Iptakalim 10μM+Glyburide 10μM were decreased in concentration-dependent manner compared with that in ET-1+Iptakalim 10μM group.There were no significant differences about the expressions of Kir6.1 mRNA among all groups.Conclusion Iptakalim can increase the expression of SUR2B mRNA of K_ATPchannels in primary cultured human pulmonary artery smooth muscle cells induced by ET-1,which shows a prospect for therapy of PAH.Partâ…¡The effects of Iptakalim on mRNA expression of SUR2B/Kir6.1 in primary cultured human pulmonary artery smooth muscle cells induced by hypoxiaObjective To study the effects of Iptakalim on mRNA expression of SUR2B/Kir6.1 in primary cultured human pulmonary artery smooth muscle cells induced by hypoxia.Methods The specimens was obtained from patients who undergoing partly pneumonectomy.Intrapulmonary arteries(3rd～4th division)were opened longitudinally.After removing the adventitia and epithelia by a blade,vessels were cut into pieces of 1～3 mm².The tissues and smooth muscle cells were cultured with DMEM. Cells were incubated with test substances for 24h in hypoxia(O₂ 5%). Iptakalim or Glyburide in different concentration(0.1μM,1.0μM,10μM)were added respectively.Total RNA was extracted by Trizol and reverse transcribed into cDNA.RQ-RT-PCR was explored to measure the level of the expression of SUR2B/Kir6.1 mRNA.Results Hypoxia suppressed the expression of SUR2B mRNA compared with control group.The expressions of SUR2B mRNA in the groups of hypoxia+Iptakalim 0.1μM,hypoxia+Iptakalim 1μM,hypoxia+Iptakalim 10μM were increased in concentration-dependent manner compared with that in hypoxia group.The expressions of SUR2B mRNA in the groups of hypoxia+Iptakalim 10μM+Glyburide 0.1μM,hypoxia+Iptakalim 10μM+Glyburide 1μM,hypoxia+Iptakalim 10μM+Glyburide 10μM were decreased in concentration-depended manner compared with that in hypoxia+Iptakalim 10μM group.There were no significant differences about the expressions of Kir6.1 mRNA among all groups.Conclusion Iptakalim can increase the expression of SUR2B mRNA of K_ATPchannels in primary cultured human pulmonary artery smooth muscle cells induced by hypoxia,which shows a prospect for therapy of PAH.