Molecular Mechanism Of Iptakalim Hydrochloride, A Novel K_ATP Channel Opener, In The Treatment Of Pulmonary Hypertension

Objective To investigate the therapeutic mechanism of iptakalim(IPT) hydrochloride, a novel K_Atp channel opener(K_ATPCO_s), in the treatment of pulmonary hypertension via a research on the expressions of the proliferating factors which involved proliferating cell nuclear antigen (PCNA) and transforming growth factor-beta1 (TGF-betal) and the expressions of the apoptosis-regulating factors which included Bc1-2 and caspase-3 of hypoxic-cultured pulmonary artery smooth muscle cells (PASMC) in vitra and hypoxia lung tissue in vivo under the treatment of iptakalim hydrochloride. Methods Under a study in vitra, cultured PASMC were divided into 7 groups which consisted of the hypoxia-cultured group, which PASMC exposed to a constant O₂ concentration of (2 ± 0.5)%, the IPT treatment groups which PASMC were hypoxia-cultured with IPT at serial doses of 10^-8mol/L , 10^-7mol/L, 10^-6mol/L, 10^-5mol/L, 10^-4mol/L within 48 hours respectively, and the normoxic-cultured group which PASMC exposed to air. TGF-betal was determined by ELISA in PASMC culture fluid. Caspase-3 activity of PASMC was detected by fluoemetry immunoassay. PCNA, TGF-betal and its receptor TGF- β R I , Bcl-2 and caspsae-3 mRNA expression of PASMC wereevaluated by reverse transcription polymerase chain reaction(RT-PCR). Under another study in vivo, 60 Sprague Dawley(SD) rats were randomly divided into 5 groups , i. e. hypoxic pulmonary hypertension(HPH), hypoxic+0.75mg/kgIPT(IPT interference group 1), hypoxic+1.50mg/kgIPT(IPT interference group2), normoxic+1.50mg/kgIPT(IPT interference group3) and normoxic control. HPH group, IPT interference group 1 and group 2 were placed into a normobaric hypoxic chamber for 811-day"1, 6-day week"1 and lasting for 4 wks. Hypoxic exposure was accomplished by ventilation with room air and N2 resulting in a constant O2 concentration of (10 ± 0.5)%. The O2 concentration was checked by an O2 analyzer (CYES- II ,Shanghai, China). IPT were orally administered in IPT interference group 1 and 2 at the dosage of 0.75mg/kg, 1.50mg/kg respectively, once a day, before the rats being exposed to the hypoxic condition. IPT interference group 3 and normoxic control group were kept under normoxic conditions with 21% O2 in room air . IPT at a dosage of 1.50mg/kg and 0.9% NaCl at a dosage of 5ml/kg were orally administered in IPT interference group 3 and normoxic control group respectively. Mean pulmonary artery pressure (mPAP) was measured by using right heart cathetenzation. TGF-betal was determined by ELISA in serum and bronchoalveolar lavage fluid samples. PCNA, TGF-betal and its receptor TGF- P R I , Bcl-2 and caspsae-3 expressions of PASMC in small to mediate pulmonary arteries were detected by S-P immunohistochemical analysis, and their mRNA expression in lung tissue were assessed by using RT-PCR. Valueswere described as mean+SD and mean values were then analyzed by two-way analysis of variance (ANOVA).Results Studies In vitro: â‘ Effects of IPT on protein level expression of TGF-betal of PASMC: TGF-betal was overexpressed in the hypoxia-cultured group comparing with which in the normoxic-cultured group (q'=10.43,P <0.01). IPT could inhibit TGF-betal overexpression in a dose-dependent manner. 10^-4mol/L IPT completely reversed TGF-betal expression to normal level(q'=2.65,P>0.05 vs the normoxic-cultured group). â‘¡Effects of IPT on caspase-3 activity of PASMC: Caspase-3 activity increased in the hypoxia-cultured group (q'=3.28,P<0.05 vs normoxic-cultured group) .Caspase-3 activity was dose-dependently elevated by IPT and increased slowly while IPT concentration locating from 10^-6 to 10^-4mol/L. (3) Effects of IPT on PCNA mRNA expression of PASMC: PCNA mRNA expressed increasingly in the hypoxia-cultured group(q'=7.86, P <0.01 vs the normoxic-cultured group). PCNA mRNA overexpression could be reduced by IPT and completely recovered to normoxic mRNA level by 10^-5mol/L IPT(q'=2.82, P >0.05 vs the normoxic-cultured group). â‘£Effects of IPT on TGF-betal and its receptor TGF-beta R I mRNA expression of PASMC: TGF-betal mRNA expr...