The Study On The Effects Of Novel ATP-sensitive Potassium Channel Opener, Iptakalim, On Human Pulmonary Arterial Constriction And Proliferation Of Pulmonary Arterial Smooth Muscle Cells

Hypoxic pulmonary hypertension(HPH)is the common cause of hospital admissions.HPH has been demonstrated to be important pathophysiological process and the key event in the process of chronic obstructive pulmonary disease(COPD)to chronic cor pulmonale.The treatment of pulmonary hypertension(PH)has improved significantly over the past ten years.Currently available medications are prostanoids, phosphodiesterase-5 inhibitors,and endothelin receptor antagonists. However,since the primary etiology and mechanism of HPH remain unclear,the treatment has remained primarily palliative.It is very important to elucidate the mechanism of HPH and search for new treatment options based on the causative factors of the pulmonary hypertension.Recently,many experts have known that the plasmalemmal K⁺ channels in pulmonary arterial smooth muscle cells(PASMCs)might play an important role in the regulation of pulmonary vasoconstriction and vascular remodeling.The suppression of function and gene expression of K⁺ channels will cause a decrease in trans-membrane K⁺ current,which results in a less negative Em,leading to cellular membrane depolarization,activation of the L-type Ca²⁺channels,and influx of Ca²⁺ in the cell down a 10000-fold gradient.Elevated[Ca²⁺]is implicated in stimulating vascular arterial smooth muscle cell proliferation and triggers vasoconstriction,leading to pulmonary hypertension.Furthermore,the decreased activity of K⁺ channel also helps to maintain a high concentration of K⁺ in the cytoplasm and inhibit apoptosis,thereby promoting vascular remodeling.Therefore,the therapeutic potent of K⁺ channel openers for pulmonary hypertension is worth to be further evaluated.There are at least three types of K⁺ channels in PASMCs:(1) voltage-gated K⁺(Kv)channels,(2)Ca²⁺-activated K⁺(K_Ca)channels,and (3)ATP-sensitive K⁺(K_ATP)channels.Among them,K_ATPchannels are now thought to play an important role as mediators of the response of vascular smooth muscle to a variety of pharmacological and endogenous vasodilators.They play important roles in the physiology and pathophysiology of vascular smooth muscle cells(SMCs)by coupling the metabolic state of the cell to its electrical activity.Therefore,K_ATP channels in PASMCs represent potential therapeutic targets for PH.The cellular signal mechanism of K_ATPchannels on the proliferation and contraction of the human PASMCs is unclear.Iptakalim designed and synthesized by native researchers is a fatty para-amino compound with low molecular weight.It has been confirmed by substantial pharmacological,biochemical,and electrophysiological studies as well as receptor-combining test as a newly selective K_ATPchannel opener (K_ATPCO).In the present study,the effects of iptakalim on pulmonary hypertension were explored so as to evaluate its therapeutic value and elucidate the mechanism of K_ATPchannels on the proliferation and constriction of the human PASMCs.The study included following 3 experiments.Partâ… The effects of iptakalim on pulmonary artery ring constriction induced by ET-1 in humanAIM:To investigate the effects of iptakalim on ET-1-induced constriction of human small pulmonary arteries ring.METHODS:Human pulmonary arteries(PA)were dissected free from connective tissues and cut into cylindrical segments(2～3 mm in width).Then the PA rings were mounted on two stainless steel hooks suspended in Krebs-Henseleit(K-H)solution (composition in mol·L^-1:NaCl 119,KCl 4.7,MgSO₄ 0.6,NaHCO₃ 25, KH₂PO₄ 1.2,CaCl₂ 2.5 and Glucose 11.1,PH 7.4)oxygenated continuously with 95%O₂-5%CO₂ gas mixture at 37℃and connected to a force-displacement transducer for tension recording with a resting tension of 800 mg and were equilibrated for 1hr before the beginning of each experiment.Vascular responses to ET-1 were examined by cumulative application of increasing concentrations of ET-1 in the range 0.05～50 nmol·L^-1,and concentration-response curves were obtained.For the study of dilatory response,PA rings were preconstricted with ET-1, then exposed to cumulative concentrations of iptakalim or pinacidil (10^-13～10^-3mol·L^-1).The vasodilatory responses to K_ATPCO(iptakalim or pinacidil)were expressed as relaxation percentage of the preconstricted values.RESULTS:The concentration-dependent human PA constriction was evoked by different ET-1 concentrations in the range 0.05～50 nmol·L^-1,EC₅₀values(95%confidence limits)were 10.19(8.93-11.45) nmol·L^-1.Slope B and standard error of B were 0.905 and 0.186, respectively.The correlation coefficient of cumulative concentration-response curve was 0.91.Iptakalim antagonized vasoconstriction induced by ET-1 with the concentration of 10^-13～10^-3 nmol·L^-1in a concentration-dependent manner.The IC₅₀value for dilating pulmonary arterial rings-preconstricted with ET-1 was 27.11 nmol·L^-1. Pinacidil also antagonized vasoconstriction induced by ET-1 in a concentration-dependent manner.There is no difference in the dilating effect between iptakalim and pinacidil(P＞0.05).CONCLUSION: Iptakalim antagonized vasoconstriction induced by ET-1 via opening K_ATP channel of human PASMCs.Iptakalim could dilate effectively human pulmonary artery.It is a promising candidate for pulmonary hypertension. Partâ…¡The effects of iptakalim on the proliferation of primary cultured human PASMCs induced by ET-1AIM:To explore the effects of iptakalim on the proliferation of primary cultured human PASMCs induced by ET-1.METHODS:The experimental models of proliferation of primary cultured human PASMCs induced by ET-1 in vitro were established.[³H]Thymidine([³H]-TdR) incorporation was used to evaluate DNA synthesis and cell proliferation. Cell cycle phase distribution was used to detect cell proliferation and analyzed by flow cytometry.RESULTS:ET-1(10 nM)stimulated [³H]-thymidine incorporation,propelled cell cycle from static phase (G₀/G₁)to DNA synthesis(S)and mitotic phase(G₂/ M)in human PASMCs.Iptakalim inhibited[³H]-TdR incorporation of human PASMCs and hold-back cells from static phase(G₀/G₁)to DNA synthesis(S)and mitotic phase(G₂/M)in a dose-dependent manner.Pinacidil also inhibited ET-1-induced[³H]-TdR incorporation and the transition of cell cycle phase.Glibenclimide,a selective K_ATPchannel blocker,abolished the effect of iptakalim and pinacidil on the cell proliferation in a concentration-dependent manner.CONCLUSION:Iptakalim acts as a specific K_ATPchannel opener to antagonize the proliferative effect of ET-1 on the human PASMCs.This study provides further evidence that iptakalim may serve as another candidate drug to treat pulmonary hypertension.Partâ…¢The effects of iptakalim on changes of[Ca²⁺]_cytand phosphorylation of ERK1/2 in primary cultured human PASMCs induced by ET-1AIM:To explore the effects of iptakalim on changes of[Ca²⁺]_cytand phosphorylation of ERK1/2 in primary cultured human PASMCs induced by ET-1.METHODS:The experimental models of primary cultured human PASMCs induced by ET-1 in vitro were established.Changes in [Ca²⁺]_cytwere measured using laser scanning confocal microscope.The phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2)were analyzed by western blot.RESULTS:The treatment with ET-1 resulted in a transient raise for[Ca²⁺]_cytin human PASMCs and this raise could be blocked by iptakalim(10μM).ET-1 induced phosphorylation of ERK1/2 from 2 to 30 min with a peak response observed at 10 min.Iptakalim inhibited ET-1-induced ERK1/2 phosphorylation in a dose-dependent manner.CONCLUSION:Thus, we can presume that iptakalim may be capable of increasing K_ATP expression and function,and that these processes cause membrane hyperpolarization,a decrease in[Ca²⁺]and inhibition of mitogen-activated protein kinases(MAPKs)activation.These actions promote the vasorelaxation of PASMCs and result in the inhibition of proliferation in PASMCs.Altogether,following conclusion are demonstrated by the results in the present studies:1.Iptakalim could antagonize vasoconstriction induced by ET-1 via opening K_ATPchannel of human PASMCs and dilate effectively human pulmonary artery.2.Iptakalim inhibited[³H]-TdR incorporation of human PASMCs and hold-back cells from static phase(G₀/G₁)to DNA synthesis(S) and mitotic phase(G₂/M).Thus,iptakalim could inhibited the proliferation of human PASMCs.3.Iptakalim may be capable of increasing the expression and function of K_ATPchannels,and that these processes cause a decrease in[Ca²⁺]and inhibition of ERK1/2 phosphorylation.4.The novel K_ATPchannel opener,iptakalim,is an promising candidate for the treatment of pulmonary hypertension because of its effects on both constriction of human pulmonary artery ring and proliferation of human PASMCs.