Modulation By Iptakalim On Gene Expression Of ATP-sensitive Potassium Channel In Cardiac Muscle And Smooth Muscle And Its Tissue Selectivity

ATP-sensitive potassium channel (K_atp), hetero-octameric complexes consisting of four pore-forming subunits (Kir6.1 or Kir6.2), and four sulfonylurea receptors (SUR1 or SUR2), is the target for potassium channel openers (KCOs). KCO is a novel new class of compounds in the treatment of a range of cardiovascular disorders, particularly hypertension. However, the effective antihypertensive effects of KCOs are usually accompanied by other side effects, such as hyperglycemia, cardiotoxicity, and heart rate raising, due to the wide spread of K_Atp in all kinds of tissues and a lack of selectivity in tissues. Iptakalim (Ipt), a novel structural KCO designed and synthesized in our academy, shows stable, long-lasting antihypertensive effect accompanied by some selective characteristics, such as little influence on rat heart, stronger effect in hypertensive animals than that in normal pressure animals, relaxation selectivity for small arteries but not for large arteries, no influence on K_Atp currents in skeletal smooth muscle cells. Based on these functional results, we aim to investigate the mechanism on K_Atp gene expression changes underlying its functional selectivity and its tissue selectivity.1. Regulation on Katp gene expressions by iptakalim in heart, aortic smooth muscle and tail artery smooth muscle from normotensive ratsNormotensive rats (NTR) were divided into three groups: normal control group,positive drug group (pinacidil-treatment, Pin), and iptakalim group. Pinacidil or iptakalim were given for 1 week. The transcript levels for SUR1, SUR2, Kir6.1 and Kir6.2 were determined by reverse transcription-polymerase chain reaction (RT-PCR).Results showed that KAtp were not affected by iptakalim or pinacidil in heart significantly (P > 0.05 vs. control). In aortic smooth muscles, Katp gene expressions were not significantly changed by iptakalim, while SUR2 was increased by 19% after pinacidil administration (P < 0.05 vs. control). In tail artery smooth muscles, the mRNA levels of Kir6.1 and Kir6.2 were reduced by 37% and 29%, respectively, after Ipt administration (p < 0.05 vs. normal control), meanwhile, Kir6.1 and SUR2 mRNA expressions were decreased by 31% and 20%, respectively, after pinacidil administration. In conclusion, iptakalim shows tissue selectivity for cardiovascular Katp gene expressions, characterized by the selective influence on Kir6.1/Kir6.2 gene expressions in small arteries, but not in heart and large arteries, however, pinacidil shows effects on KAtp genes both in large and small arteries, characterized by SUR2 gene expression changes.2. Regulation on KAtp gene expressions by iptakalin in heart, aortic smooth muscle and tail artery smooth muscle from spontaneously hypertensive ratsSpontaneously hypertensive rats (SHRs) were divided into untreated and iptakalim-treated SHR groups. Iptakalim was given by gastric gavage at a dose of 3 mg/kg body weight per day for 12 weeks. Age-matched Wistar-Kyoto rats (WKY) were included as the normotensive control group.The gene expression levels of SUR2, Kir6.1, and Kir6.2 were elevated in SHR, and reduced in iptakalim-treated SHR. In the three kinds of tissues detected, the levels of SUR2 and Kir6.2 mRNA were increased significantly (P < 0.05 vs. WKY), and reduced in iptakalim-treated SHR (P < 0.05 vs. untreated-SHR). No differences in the normalized expression of SUR2 or Kir6.2 were found between the iptakalim-treated SHR and WKY. The expression of Kir6.1 between groups showed no significant difference in the three kinds of tissues. In conclusion, the characteristic changes of cardiovascular Katp inhypertension are related to the significant increasing of SUR2 and Kir6.2 gene expressions. Iptakalim shows reversing effects on the up-regulated SUR2 and Kir6.2 gene expressions.3. Effects of iptakalim on intracellular calcium, protein kinase A and protein kinase C activities in rat tail artery smooth muscle cellsIntracellular calcium ([Ca2+]0 is thought to be a critical factor for vasorelaxation by modulating activities of protein kinases, including protein kinase A, and protein kinase C. To further identify the mechanisms underlying its vasorelaxation selectivity, we investigate effects of iptakalim and the other two structurally distinct KCOs, pinacidil (cyanoguanidines) and diazoxide (benzothiadiazines), on [Ca2+]j and activities of PKA and PKC in smooth muscle cells primarily cultured from rat tail arteries (RTA-SMCs), the typical type of small arteries.3.1 Effects of iptakalim on intracellular calcium concentration in rat tail artery smooth muscle cellsContinuous application of iptakalim at concentrations of 0.1, 1, and 10 umol-L" decreased [Ca2+]j dose-dependently (P < 0.05 compared with the baseline). However, in the presence of glibenclamide (1 umol-L"1), a selective antagonist of Katp channels, [Ca2+]i did not decrease significantly after iptakalim administration concentrations of 0.1 and 1 umol-L"1. Similar to iptakalim, pinacidil dose-dependently decreased [Ca2+]i significantly in the absence of glibenclamide, and this effect was inhibited by 1 umol-L"1 glibenclamide. Diazoxide at a concentration of 0.1 umol-L"1 did not affect [Ca2+]j, whereas concentrations of 1 and 10 umol-L"1 significantly decreased [Ca2+]j (P < 0.05), and the effect was reversed by 1 umol-L"1 glibenclamide preincubation.3.2 Effects of iptakalim on protein kinase A and protein kinase C activities in rat tail artery smooth muscle cellsPKA and PKC activities were increased by 36% and 51%, respectively, after iptakalim treatment at a concentration of 1 umol-L'1 for 1 hour (P < 0.05 vs.untreated normal control), and 39% and 50% respectively after pinacidil treatment. The sameconcentration of diazoxide significantly increased PKC activity (P < 0.01) but not PKA activity.In conclusion, as far as [Ca2+]j, PKA and PKC activities, the pharmacological characteristics in small artery smooth muscle cells, iptakalim is similar to pinacidil, but different from diazoxide.4. Effects of iptakalim on tissue tensions in stomach, ileum and bladder, the non-vascular smooth muscles from normotensive ratsIn the widespread non-vascular smooth muscle tissues, the effects of iptakalim on KAtp and tensions are largely undetermined. To further understand the relaxation selectivity of iptakalim in non-vascular smooth muscles, we investigated the changes of tension and Katp gene expression induced by iptakalim in stomach, ileum, and bladder, and compared its effects to pinacidil and diazoxide.Iptakalim at concentrations of 0.01-100 umol-L"1 caused no relaxation effects on strips of stomach, ileum and bladder, after contraction with 10 umol-L"1 acetylcholine (ACh). The same results as iptakalim were detected in diazoxide-treated tissue strips. Pinacidil showed no effects on strips of stomach and bladder, however, pinacidil at concentrations of 10 and 100 umol-L'1 induced significant relaxations on ileum strips, and the relaxation rates were 28% and 52%, respectively, (P < 0.01, P < 0.05 compared with the former concentration). In conclusion, iptakalim shows no effects on tensions of stomach, ileum, and bladder smooth muscles, which is similar to diazoxide, but different from pinacidil that induced significant relaxation in ileum smooth muscle.5. Effects of iptakalim on KAtp gene expressions in stomach, ileum and bladder smooth muscles from normotensive ratsSUR2 and Kir6.1 are the major subunits in stomach and ileum smooth muscles, and the gene expressions of SUR2 and Kir6.1 were not significantly changed by iptakalim or pinacidil treatment (P > 0.05 vs. control). In bladder smooth muscles, the major Katp subunits of SUR2 and Kir6.2 were changed significantly by iptakalim treatment for 1 week (P < 0.05 vs. control), and meanwhile, SUR2 gene expression was...