Nicorandil opens mitochondrial KATP channels not only directly but also through a NO-PKG-dependent pathway
Dept. of Physiology, MSB 3070, College of Medicine University of South Alabama, Mobile, AL 36688, USA. Archiv für Kreislaufforschung
(Impact Factor: 5.41).
02/2007; 102(1):73-9. DOI: 10.1007/s00395-006-0612-5
Nicorandil, a hybrid of nitrate generator and potassium channel opener, protects ischemic myocardium by opening mitochondrial ATP sensitive potassium (mitoK(ATP)) channels. We recently found that nitric oxide (NO) opened K(ATP) channels in rabbit hearts by a protein kinase G (PKG) mechanism. This study examined whether the NO-donor property of nicorandil also contributes to opening of mitoK(ATP) channels through PKG. MitoK(ATP) channel opening was monitored in adult rabbit cardiomyocytes by measuring reactive oxygen species (ROS) production, an established marker of channel opening. Nicorandil increased ROS production in a dose-dependent manner. The selective mitoK(ATP) channel inhibitor 5-hydroxydecanoate (200 microM) completely blocked ROS production by nicorandil at all doses. The PKG inhibitor 8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rpisomer (Rp-8-Br-cGMPs, 50 microM) shifted the dose-ROS production curve to the right with an increase of the EC(50) from 2.4 x 10(-5) M to 6.9 x 10(-5) M. Rp- 8-Br-cGMPs did not affect the increase in ROS production by the selective mitoK(ATP) channel opener diazoxide while it completely blocked increased ROS production from the NO donor S-nitroso-N-acetylpenicillamine (1 microM). Furthermore ODQ, an antagonist of soluble guanylyl cyclase, blocked nicorandil's ability to increase ROS generation. These results indicate that nicorandil, in addition to its direct effect on the channels, opens mitoK(ATP) channels indirectly via a NO-PKG signaling pathway.
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ABSTRACT: Preconditioning is the phenomenon whereby tolerance to lethal insults is induced by exposing the tissue to a prior sublethal stimulus. This exists in several forms, such as ischaemic preconditioning, adenosine preconditioning and excitotoxic preconditioning. Adenosine preconditioning is known to be mediated by activation of A1 receptors and ATP-sensitive potassium channels whilst excitotoxic preconditioning mainly involves stimulation of NMDA receptors, nitric oxide and most likely ATP-sensitive potassium channel activation. ATP-sensitive potassium channel openers such as pinacidil and diazoxide are also known to exert preconditioning against various types of insults. There have been several models of ischaemia used to study preconditioning in vivo and in vitro leading to some confusion over the effects of preconditioning agents. High concentrations of glutamate or NMDA have been used as models of excitotoxicity in many experimental paradigms. Some molecular changes are associated with preconditioning phenomena, the most prominent being an increased expression of heat shock protein 72 (HSP72). The aims of the current study were to: 1) investigate the effects of exogenous glutamate and other depolarizing agents in the slice preparation and their validity for use as toxic agents 2) examine any potential preconditioning neuroprotection induced by adenosine against various depolarizing agents and elucidate the underlying mechanisms where relevant 3) examine the excitotoxic preconditioning phenomenon and possible underlying mechanisms 4) look at the effectiveness of other known preconditioning agents e.g. ATP-sensitive potassium channel openers against depolarizing agents and identify the underlying mechanisms of protection 5) identify any molecular changes that may occur during acute models of chemical ischemia or acute preconditioning. The rat hippocampal slice preparation was used to investigate the effects of depolarizing agents and preconditioning paradigms upon the extracellularly evoked field epsps, orthodromic and antidromic population spikes. Western blotting was used to detect any changes in the levels of HSP72 in the slices that may have occurred as a result of the depolarizing agents or the preconditioning treatments. It was first established that 5mM and 10mM glutamate induced depressions in the amplitudes of orthodromic population spikes which recovered to a stable plateau. The degree of recovery of the spikes depended partially upon the initial size of the response. As adenosine is known to be released in response to glutamate receptor stimulation, the effects of 5mM glutamate upon the orthodromic spikes were studied in the presence of the A1 receptor antagonist, DPCPX. It was observed that DPCPX did not attenuate the depression of the response during glutamate perfusion but there was a significant elevation in the post-glutamate recovery of the response. This effect was not observed when the protocol was applied to antidromic population spikes and field epsps, both of which showed a depression in response during 5mM glutamate perfusion but recovered fully when glutamate was removed. The field epsps showed a trend whereby smaller epsps recovered to a far greater degree than population spikes. Although this effect was not significant, the NMDA receptor blocker, MK-801, was co-perfused with glutamate during epsp recordings to examine this further. The degree to which MK-801 alone affected the response correlated with the post-glutamate recovery. To study this effect, isolated NMDA-receptor mediated epsps were recorded and the effects of 5mM glutamate upon them were studied. There was a similar tendency for small NMDA-receptor mediated epsps to recover to a higher level following glutamate treatment compared with larger potentials. In the presence of DPCPX, the larger potentials showed a significant elevation in recovery following treatment with glutamate. It was also shown that the post-5mM glutamate recovery of the orthodromic population spikes was elevated by the presence of the A2a receptor antagonist, SCH 58261. Further experiments using the ATP-sensitive potassium channel blocker, glibenclamide, indicated that this effect may be due to increasing the opening of these channels. Adenosine preconditioning was attempted using 10mM glutamate as an insult. It was shown that adenosine could not precondition against this effect in antidromic or orthodromic population spikes. The effects of the sodium-potassium ATPase inhibitor, ouabain, upon the evoked responses were studied as an alternative insult. It was shown that ouabain induced depressions in field epsps, orthodromic and antidromic population spikes. The antidromic population spikes showed significantly smaller depressions than the orthodromic responses. Further experiments using the glutamate receptor antagonist, kynurenic acid, showed that glutamate receptors mediated the effects of ouabain upon the orthodromic population spikes but not the antidromic spikes. Adenosine preconditioning was attempted against ouabain. It was shown that adenosine preconditioned against the effects of ouabain upon orthodromic and antidromic population spikes but not field epsps. Further experiments were conducted using antidromic population spikes. It was shown using various antagonists, that adenosine protection against ouabain was mediated by A1 receptors, ATP-sensitive potassium channels, NMDA receptors and nitric oxide. To extend these results further, preconditioning using the ATP-sensitive potassium channel opener, pinacidil, was attempted against 10mM glutamate and ouabain. It was shown that pinacidil was able to precondition the antidromic population spike against either insult. Using the NMDA receptor antagonist, DL-AP5, showed that the preconditioning effect of pinacidil against ouabain was mediated by NMDA receptors. Another preconditioning paradigm was attempted to see if glutamate could precondition against ouabain. It was shown that pre- treatment with glutamate resulted in enhancing the depressant effect of ouabain upon field epsps and antidromic population spikes. To further examine the effects of ouabain upon antidromic population spikes, ouabain was co-perfused in the presence of the intracellular calcium chelator, BAPTA-AM. This resulted in enhancing the depressant effect of ouabain upon the response. A similar result was observed when the calcium concentration in the perfusion medium was lowered to 0.5mM from 2.5mM whereas increasing the concentration to 5mM attenuated the depressant effect. Ouabain was also co-perfused in the presence of charybdotoxin, a blocker of large-conductance calcium activated potassium channels. It was observed that charybdotoxin enhanced the effect of ouabain upon the antidromic spikes. No changes were detected in HSP72 expression in the slices in response to ouabain treatment, 10mM glutamate treatment, pinacidil preconditioning treatment or glutamate preconditioning. The present results show that glutamate and ouabain can induce depressions in the evoked responses from the rat hippocampal slice and that the effects of 5mM glutamate can be attenuated by adenosine receptor antagonists. In addition, adenosine can precondition against ouabain but not glutamate and this effect involves A1 receptors, NMDA receptors, nitric oxide and ATP-sensitive potassium channels. It has also been observed that pinacidil can precondition against ouabain or glutamate and NMDA receptors may be involved in this effect. The inability of glutamate to precondition against ouabain in evoked responses was also demonstrated. The study highlights the effectiveness of preconditioning agents against different depolarizing agents and the interactions between adenosine and glutamate receptors which play a role in preconditioning.
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ABSTRACT: Acute myocardial infarction (AMI) following coronary artery occlusion is a common cause of mortality and morbidity world-wide. Patients currently receive reperfusion therapy as the only anti-infarct intervention. A number of agents have been evaluated to further improve myocardial salvage, but until recently, none has demonstrated clear efficacy in clinical trials. A new target of cardioprotection, the Reperfusion Injury Salvage Kinase (RISK) pathway, has been proposed. These kinases are involved in mediating the cardioprotection of myocardial preconditioning and postconditioning induced by short non-lethal cycles of ischemia/reperfusion performed before (preconditioning) or just after (postconditioning) a lethal ischemic insult. Many pharmacological interventions are now available that protect the heart by activating the RISK pathway at the time of reperfusion. The present review will examine the efficacy of several strategies that have been proposed to protect the acutely ischemic myocardium including (1) those intended to directly alter adverse reperfusion events (e.g., calcium overload and free radical attack), (2) those based on activation of the RISK pathway including postconditioning, and (3) myocardial cooling.
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ABSTRACT: To investigate the effects of nicorandil, an ATP-sensitive potassium (K(ATP)) channel opener with a nitric oxide (NO) donor property, on overactive bladder (OAB) in animal models. Nicorandil is currently used clinically to treat ischaemic heart disease.
Three animal OAB models were used: (i) C-fibre mediated bladder overactivity by infusion of a low concentration of acetic acid (AA) into the bladder in female Wistar rats; (ii) bladder outlet obstruction (BOO) created by partial urethral obstruction in female Wistar rats; and (iii) neuronal NO synthase (nNOS) knockout (KO) mice with urinary frequency. The effects of nicorandil and KRN2391, both of which act as K(ATP) channel openers and NO donors, on the OAB models were examined.
Cystometry showed that intravesical instillation of nicorandil and KRN2391 successfully inhibited OAB induced by intravesical instillation of AA. In the BOO model compared with untreated BOO rats, both nicorandil (1 and 3 mg/kg, orally) and KRN2391 (1 mg/kg, orally) significantly reduced the voiding frequency. Compared with wild-type mice, nNOS KO mice had urinary frequency with no change in the total urine volume. Nicorandil (3 mg/kg, orally) and KRN2391 (1 mg/kg, orally) significantly reduced the voiding frequency in nNOS KO mice.
Our in vivo results show that nicorandil, a K(ATP) channel opener with a NO donor property, can suppress OAB from both neurogenic and myogenic causes. Nicorandil appears to be a promising candidate for clinical use in patients with OAB.
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