Activation Of SUR 2B/Kir 6.1-type K_ATP Channels By Iptakalim In Microcirculation

Hypertension, a condition in which arteries have persistently elevated blood pressure, has become the most important risk factor of cardiovascular diseases. All over the world, more than one billion patients suffer from it and have to accept lifelong medication. Thus an efficient method for prevention and therapy is in urgent need. The resistance vessels mostly consisting of arterioles in the microcirculation are the sections where the overwhelming majority of the pressure drop between arteries and veins occurs. The excessive constriction of resistance vessels, a vital induction factor of pressure increase, illustrates the pathological development and mechanism of hypertension. Hence, a high selective drug which specifically regulates the vascular tone of resistance vessels might present a fundamental strategy for hypertension treatment.ATP sensitive potassium channel(KATP) consists of inward rectifier potassium channel subunits(Kirs) and regulative sulfonylurea receptors(SURs) whose subunit constitution varies in different tissues. SUR 2B/Kir 6.1 and SUR 2B/Kir 6.2 are the specific ones existing in the vessels. Since activation of KATP is crucial to modulate the tone of the resistance vasculature, KATP openers have been employed as an important antihypertensive approach and demonstrated therapeutic efficacy. Unfortunately, the KATP openers currently available in clinic produce many undesirable adverse effects.Iptakalim, a recently developed KATP opener, had been proved to elicit a strong antihypertensive effect in spontaneously hypertensive rats, renal hypertensive dogs and human. Extensive works had been done to illustrate that iptakalim could protect the target organs from damage due to hypertension by ameliorating the endothelial dysfunction. Iptakalim can inhibit the endothelin-1 system and increase the release of NO to restore the balance between the NO and signaling systems in endothelial dysfunction, resulting in endothelial protection. Previously, our research suggested that the diameter of the vessels is related to their sensitivity to iptakalim, with smaller size showing a stronger dilatation. This phenomenon could be blocked by a selective KATP blocker glibenclamide. In researches via heterologous expression of different subtypes of KATP, we found that iptakalim preferential activated the SUR 2B/ Kir 6.1 subtype of KATP.Based on the above, it was reasonable to hypothesize that iptakalim can regulate the mircrovascular tension by activating KATP. In the first part of the present study, we proposed a method to investigate this problem by using patch clamp techniques and cell internal/external perfusion system. We tested the efficiency of iptakalim in mesenteric arterioles smooth muscle cells isolated from rat and Kir 6.1-knockout mice and characterized the function of iptakalim in comparison with pinacidil, a clinical antihypertensive KATP opener, in cultured rat mesenteric arteriolar endothelial cells internally perfused by different nucleotides. We found that iptakalim could enhance the whole cell current recorded from smooth muscle cells of rat, Kir 6.1+/- and WT mice in a concentration-dependent manner but was ineffective in Kir 6.1-/- mice. The results demonstrated that the efficacy of iptakalim in resistance vasculature was mediated by the activation of SUR 2B/ Kir 6.1 subtype of KATP.The activation of endothelial KATP by iptakalim and pinacidil was dependent on the intracellular nucleotides. Iptakalim showed higher selectivity on metabolic supplements than that of pinacidil. In the presence of ATP, ADP or UDP at the concentrations of 100~1000μM, iptakalim could evoke inward currents. Furthermore, Mg-nucleotide hydrolysis was necessary for iptakalim to activate KATP in mesenteric arteriolar endothelial cells.Brain and lung are important organs with active metabolism. Cerebral and pulmonary ischemia/hypoxia injuries are high mortality deseases which threaten human health. In brain ischemia and hypoxia conditions, endothelial cells and neurons, astrocytes are damaged, and then necrosis and apoptosis happen, thus became the cellular pathological basic for brain impairment. The endothelium of pulmonary arterioles can also be damaged under hypoxic state and the endothelial dysfunction plays an important role in the pathological development of pulmonary hypertension. It has been demonstrated that iptakalim selectively increased dilatation effects on hypoxic pulmonary arterioles and the protection of iptakalim on neurovascular unit against ischemic injury was related to the activation of KATP. Therefore, the purpose of the second part is to identify the opening effect of iptakalim on cerebral and pulmonary arteriolar endothelial cells and characterized its function to provide new clues to further study the drug effect in metabolism related vascular diseases.Using internal perfusion system, we recorded the KATP current of cerebral and pulmonary arteriolar endothelial cells. The results indicated that nucleotides and Mg-nucleotide hydrolysis were required for iptakalim activation. Iptakalim could evoke KATP currents in the presence of ATP, ADP or UDP at the concentration of 1000μM in cerebral arteriolar endothelial cells while it activated the pulmonary endothelial KATP in the presence of ATP 100~1000μM, ADP 30~1000μM or UDP 100~1000μM.Conclusion:1 Iptakalim activated the SUR 2B/Kir 6.1-type KATP of mesenteric arterioles in a concentration-dependent manner.2 Iptakalim enhanced KATP currents in mesenteric, cerebral and pulmonary arteriolar endothelial cells, which is dependent on the presence of ATP, ADP or UDP and is corelated with Mg-ATP hydrolysis.3 Extremely stringent requirements on nucleotides were found in the activation of cerebral endothelial KATP by iptakalim. The drug could only stimulate KATP at the ATP, ADP or UDP concentration of 1000μM, indicated that the protection of Iptakalim on brain is highly relevant to energy metabolism...