Molecular Mechanism For Protecting Vascular Endothelial Cells By SUR2B/Kir6.1 Channel Openers

Vascular endothelial cells, although only a simple monolayer of vascular, are able to respond to multiple physical and chemical signals by production of a wide range of factors including nitric oxide(NO), endothelin-1(ET-1), prostacyclin(PGI2) and thromboxane A2(TXA2),which regulating vascular tone, thromboresistance, cellular adhesion, smooth muscle cell proliferation, and vessel wall inflammation. Endothelial dysfunction associated with various cardiovascular diseases. On the one hand, many cardiovascular diseases caused endothelial dysfunction, leading to the imbalance of regulation and secretion of endothelial cells; the other hand, endothelial dysfunction also contribute to pathological processes of such cardiovascular diseases. Extensive clinical investagations have shown that endothelial dysfunction plays a key event in the pathogenesis of various forms of cardiovascular diseases such as hypertension, congestive heart failure, atherosclerosis, primary pulmonary hypertension, and others. Endothelial dysfunction is characterized of reduction of NO bioavailability. Taken hypertension as an example, patients with hypertension showed decreased NO bioavailability secondary to increased oxidative stress products and vascular inflammation. Shear stress or acetylcholine induced endothelium-dependent vasodilation of vasculature from spontaneously hypertensive rats(SHRs) reduced significantly, indicating decreased NO production caused by impaired endothelial cells. Stimulated by some chemical and physical factors, dysfunctional endothelial cells would increase the production of endothelium-derived contracting factors, including ET-1, angiotensinⅡ(AngⅡ), PGE2, PGD2 and superoxide anion which can contribute to moment to moment changes in contractile activity of the underlying vascular smooth muscle cells. In addition, endothelial dysfunction is an early marker of atherosclerosis, and risk factor of cardiovascular events. Patients with congestive heart failure caused endothelial dysfunction shows damaged coronary artery and decreased exercise capacity. Therefore, correction of endothelial dysfunction plays an important role on treatment of cardiovascular events, disease treatment, prognosis and prevention.Iptakalim(Ipt), a novel hypertensive drug belongs to 1.1 class of new drugs was designed and synthesized by Chinese scholar. It belongs to the ATP sensitive potassium channel opener(KCO) and now has completed phase III clinical trials of anti-hypertension. Previous studies of iptakalim have suggested that it has complete new chemical structure and new pharmacological characteristics: on the one hand, the molecular structure of iptakalim belongs to nonyl aliphatic secondary amine derivative, different from the previously reported eight types of KCOs; on the other hand, iptakalim selective activation of SUR2B/Kir6.1subtype of KATP channel. The property of iptakalim selective activation of SUR2B/Kir6.1 is the pharmacological basis of its dilation of the resistance vessels and reduction of adverse effects. In addition, in vitro studies have shown that iptakalim protects against endothelial dysfunction induced by high uric acid, high glucose, high DL-homocysteine, and high ox-Low density lipoprotein(LDL) through increasing NO production associated with increased intracellular calcium concentration([Ca2+]i), reducing ET-1secretion and the expression of monocyte chemoattractant protein-1(MCP-1), intercellular adhesion molecule-1(ICAM-1) and vascular cell adhesion factor-1(VCAM-1). In vivo study showed that activation of KATP channel by iptakalim prevents insulin resistance associated with hypertension, protects hypertension caused kidney injury induced by hyperuricemia, and reverses pressure overload-induced cardiac hypertrophy and heart failure through protecting vascular endothelial cells. It also can protect against ischemia-reperfusion caused brain injury via protecting neurovascular unit, which including middle cerebral artery microvascular endothelial cells, astrocytes and neurons. Natakalim, a derivative of iptakalim shows higher selectivity of SUR2B/Kir6.1 channel and beneficial protective effects on endothelim.Based on previous studies, in this study we aim to to investigate the selectivity of iptakalim and natakalim on subtypes of KATP channels of endothelial cells and to elucidate the molecular mechanism underlying of their protective effects on endothelial cells. The main focus of the study included two channels, SUR2B/Kir6.1 subtype of KATP channel and a calcium ion permeability TRPV1 channel. 1. The selectivity of iptakalim and natakalim on subtypes of KATP channelIn the present we designed and synth specific small interfering RNA(siRNA) targeting to each subunit of KATP channel expressed on EA.hy926 cells to decrease the expression of each subunit protein respectively. After successful knockdown of each subunit with specific siRNA,the endothelial cells were incubated with different concentrations of KCOs, including iptakalim, natakalim, pinacidil and diazoxide, at the same time, the control group were treated with non-specific siRNA and were incubated with the same drugs and the same concentrations, then detect the NO content in the media, the effective molecular of endothelial protection. Compare the difference between RNAi group and the non-specific group with the same drug and same doses. The results showed that iptakalim and natakalim at the concentration of 10μmol/L, pinacidil and diazoxide at the concentration of 1mmol/L can significantly promote the release of NO from endothelial cells. NO production from endothelial cells induced by iptakalim, natakalim, pinacidil and diazoxide were inhibited significantly after knockdown of endothelial SUR2 B subunits. NO production induced by iptakalim, natakalim and diazoxide have no significant difference between SUR2 A knockdown group and non-specific group, but significantly reduced in the group treated with pinadicil after SUR2 A knockdown. NO production from endothelial cells induced by iptakalim, natakalim, pinacidil and diazoxide were decreased significantly after knockdown of endothelial Kir6.1 subunits. NO production induced by iptakalim, natakalim and diazoxide except for pinadicil have no significant difference between Kir6.2 knockdown group and non-specific group. The results indicated that iptakalim, natakalim and diazoxide promote NO release from endothelial cells need the coexistence of SUR2 B and Kir6.1 subunits. They selectively activated SUR2B/Kir6.1 subtype of KATP channels of endothelial cells. However, pinadicil showed nonselective effects on subtype of KATP channels.2. Effects of iptakalim and natakalim on endothelial Ca2+ signaling pathwayIt has been proved that iptakalim and natakalim relax resistance vessels in dose dependently and endothelium dependently manners. Their endothelium dependent vasodilation effect may be contributed by increased intracellular Ca2+ concentration of endothelial cells that promote the release of endothelium derived relaxing factor(EDRF) and endothelium derived hyperpolarizing factor(EDHF). Endothelial cells which belong to non excitable cells and lack of voltage dependent calcium channel(VDCC), modulate calcium permeation through membrane potential. Endothelial hyperpolarization will itself tend to increase [Ca2+]i by enhancing the electrochemical gradient that drives transmembrane Ca2+ entry into endothelial cell. It has been reported that hyperpolarization induced by KCOs caused endothelium [Ca2+] increased. However, which channels mediated Ca2+ influx into endothelial cells induced by KCOs and the underlying mechanism is still unclear. We choose TRPV1 and TRPV4 channel both of which can mediate endothelium dependent vasodilation from the TRP channel family. Incubation of isolated mesenteric arterial rings with TRPV4 channel blocker RN1734, vasodilative effects of iptakalim and natakalim have no obvious changes. However, incubation of isolated mesenteric arterial rings with TRPV1 channel blockers SB366791 or capsazepine, vasodilative effects of iptakalim and natakalim were significantly inhibited. The results indicated endothelium dependent vasodilation of iptakalim and natakalim through activating endothelial cell TRPV1 channel.We dectected the [Ca2+]i in endothelial cells by confocal laser and the effect of iptakalim and natakalim were tested. The results showed that iptakalim and natakalim at the concentrations of 1, 10 and 100 μmol/L could dose dependently induced elevation of [Ca2+]i. However the TRPV1 channel blocker SB366791 or capsazepine inhibited the effects of iptakalim and natakalim in a dose dependent manner, suggesting that iptakalim and natakalim induced extracellular calcium influx into cells by opening endothelium TRPV1 channel. The further explore the role of TRPV1 channel in iptakalim and natakalim induced elevation of [Ca2+]i, siRNA targeted TRPV1 channel were used to knockdown of expression of the TPRV1 protein, and the iptakalim and natakalim effects were significantly inhibited. In addition, the KATP channel blocker glibenclamide(Gli) can effectively reduce the effects of iptakalim and natakalim on elevation of [Ca2+]i. These results suggest that iptakalim and natakalim open endothelial cell TRPV1 channel to promote the calcium ions influx into cells by activating KATP channel. 3. Protective effects of iptakalim and natakalim on endothelial cellsOur previous studies suggest that activation of endothelial KATP channel protect against endothelial dysfunction induced by cardiovascular risk factors such as glucose, uric acid, homocysteine and low density lipoprotein(LDL). Increased of endothelial [Ca2+]i increase NO production, reduce the secretion of ET-1. NO releasing from endothelial cells plays a pivotal role in regulating the vascular tone, platelet aggregation and thrombosis, proliferation of smooth muscle and vascular fibrosis, anti-inflammation and the decreased of NO bioavailability is an important characteristics of endothelial dysfunction.In present study, we found that iptakalim and natakalim promote endothelial cells to release of NO, this effects can be blocked by 10 μmol/L KATP channel blocker glibenclamide. Iptakalim and natakalim induced calcium influx through TRPV1 channel which was inhibited by SB366791, the NO production from endothelial cells also decreased, suggesting that increased NO production is associated with [Ca2+]i in endothelial cells. 1mmol/L Homocysteine can induce dysfunction and death of endothelial cells, preincubation of iptakalim or natakalim can reverse the homocysteine induced decrease of cell viability, however, glibenclamide and SB366791 can significantly antagonize the protective effect of iptakalim and natakalim on endothelium. Based on the previous study, iptakalim and natakalim have high selectivity of endothelial SUR2B/Kir6.1 subtype KATP channel, suggesting that SUR2B/Kir6.1 channel and TRPV1 channel are important molecular targets of iptakalim and natakalim endothelial protective effects. In summary, the conclusions of present study are as follows:1. In the present study, we firstly used RNAi technology to reduce the expression of SUR2 B, SUR2 A, Kir6.1 and Kir6.2 subunits of KATP channel expressed on EA.hy926 cells. In addition, we proved that iptakalim and natakalim have high selectivity of SUR2B/Kir6.1 channel directly on endothelial cells, and they promote endothelial cells to release NO by selective activation of SUR2B/Kir6.1 channel;2. For the first time we identified iptakalim and natakalim induced endothelial [Ca2+]i increased through TRPV1 channel. Iptakalim and natakalim selectively activation of endothelial SUR2B/Kir6.1 channel, K+ outflow and causes hyperpolarization, increases of electrochemical gradient which drives the extracellular calcium ions into cytoplasm through the TRPV1 channel, increased of [Ca2+]i binding with calmodulin changes the phosphatestate eNOS, enhancing the activity of eNOS, finally increased the synthesis and secretion of NO. At the same time, increased [Ca2+]i can activate Ca2+ activated K+ channel(KCa), including small-conductance potassium channel(SKCa) and intermediated-conductance potassium channel(IKCa) as EDHF to participate in the endothelium dependent vasodilation of iptakalim and natakalim with EDRF.3. For the first time we have proved that TRPV1 channel is involved in the protective effects of iptakalim and natakalim on endothelial cells. Increased endothelial [Ca2+]i induced by iptakalim and natakalim was inhibited by TRPV1 channel blockers SB366791, and the NO release from endothelial cells decreased at the same time. In addition, iptakalim and natakalim reverse the homocysteine induced endothelial dysfunction through TRPV1 channel.4. Three-part studis of of this article further to supply the molecular mechanisms of protective effect on endothelial cells the, dilation of resistance vessels, treatment of hypertension and congestive heart failure and other effects by iptakalim and natakalim. Due to the high activation SUR2B/Kir6.1,their adverse reaction is much less than the other non-selective KCOs. While treatments of cardiovascular disease associated with endothelial dysfunction, they can effectively against the disease by reversing endothelial dysfunction and restoring endothelial function, to inhibit the development of the pathophysiology of the diseases, reduce the incidence of complications and improve outcome and prognosis of the disease.