Nicorandil opens mitochondrial K<Subscript>ATP</Subscript> channels not only directly but also through a NO-PKG-dependent pathway

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 μM) completely blocked ROS production by nicorandil at all doses. The PKG inhibitor 8-bromoguanosine-3’,5’-cyclic monophosphorothioate, Rpisomer (Rp-8-Br-cGMPs, 50 μM) shifted the dose-ROS production curve to the right with an increase of the EC₅₀ 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 μM). 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.     

Nicorandil inhibits oxidative stress-induced apoptosis in cardiac myocytes through activation of mitochondrial ATP-sensitive potassium channels and a nitrate-like effect

The anti-anginal drug nicorandil has been shown to inhibit apoptosis by activating mitochondrial ATP-sensitive potassium (K(ATP)) channels. The possible contribution of the nitrate moiety of this drug to its anti-apoptotic effect has now been investigated in neonatal rat ventricular myocytes subjected to oxidative stress. Exposure of cultured myocytes to 100 micromol/l hydrogen peroxide (H(2)O(2)) increased the number of nuclei stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique as well as induced internucleosomal DNA fragmentation, loss of mitochondrial membrane potential, cytochrome c release into the cytosol, and activation of caspases-3 and -9, all of which are characteristics of apoptosis. Pretreatment of cells with nicorandil (100 micromol/l) inhibited these effects of H(2)O(2). Both the mitochondrial K(ATP) channel antagonist 5-hydroxydecanoate (5-HD) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, attenuated the anti-apoptotic effect of nicorandil in concentration-dependent manners. Coapplication of ODQ (10 micromol/l) and 5-HD (500 micromol/l) completely abolished nicorandil-induced cytoprotection. The effect of nicorandil was also reduced by an inhibitor of cGMP-dependent protein kinase (KT5823, 1 micromol/l). The nitric oxide donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 50 micromol/l) mimicked the protective effect of nicorandil in a manner sensitive to ODQ but not to 5-HD. A cell-permeable cGMP analog, 8-bromo-cGMP, also reduced H(2)O(2)-induced apoptosis. The inhibition of the H(2)O(2)-induced activation of caspase-3, but not that of caspase-9, by nicorandil in the presence of 5-HD or by SNAP was reversed by the addition of dithiothreitol to the enzyme assay. Nicorandil inhibits oxidative stress-induced apoptosis in cardiac myocytes through a nitric oxide/cGMP-dependent mechanism as well as by activating mitochondrial K(ATP) channels.     

Structure and mechanism of proton transport through the transmembrane tetrameric M2 protein bundle of the influenza A virus

The M2 proton channel from influenza A virus is an essential protein that mediates transport of protons across the viral envelope. This protein has a single transmembrane helix, which tetramerizes into the active channel. At the heart of the conduction mechanism is the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior. Protons are conducted as the total charge of the four His37 side chains passes through 2⁺ and 3⁺ with a pK_a near 6. A 1.65 Å resolution X-ray structure of the transmembrane protein (residues 25–46), crystallized at pH 6.5, reveals a pore that is lined by alternating layers of sidechains and well-ordered water clusters, which offer a pathway for proton conduction. The His37 residues form a box-like structure, bounded on either side by water clusters with well-ordered oxygen atoms at close distance. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters. Thus, the new crystal structure provides a possible unification of the discrete site versus continuum conduction models.     

gamma-tocopherol, but not alpha-tocopherol, potently inhibits neointimal formation induced by vascular injury in insulin resistant rats

Insulin resistance may enhance the neointima formation via increased oxidative stress. However, clinical trials investigating the benefit of antioxidant therapy with alpha-tocopherol showed negative results. Recent studies showed that chemical characteristics of gamma-tocopherol are distinct from those of alpha-tocopherol. We hypothesized that gamma-tocopherol is superior to alpha-tocopherol in preventing the neointima growth after arterial injury in insulin resistance. Male rats were fed with standard chow or a high fructose diet for induction of insulin resistance. Thereafter, the left carotid artery was injured with a balloon catheter. After 2 weeks, the carotid arteries were harvested and histomorphometrically analyzed. The neointima-media ratio of the injured artery was significantly greater in insulin resistance group (n=8, 1.33+/-0.12) than in normal group (n=10, 0.76+/-0.11, p<0.01). gamma-Tocopherol (100 mg/kg/day) reduced the ratio (n=5, 0.55+/-0.21, p<0.01 vs. insulin resistance group), while alpha-tocopherol was without effect (n=7, 1.08+/-0.14). The quantification of plasma phosphatidylcholine hydroperoxide, an indicator of systemic oxidative stress, and dihydroethidium fluorescence staining of the carotid artery, an indicator of the local superoxide production, showed that oxidative stress in the systemic circulation and local arterial tissue was increased in insulin resistance. Both tocopherols decreased plasma phosphatidylcholine hydroperoxide, but failed to suppress the superoxide production in the carotid arteries. Increased 3-nitrotyrosine in neointima by insulin resistance was greatly reduced only by gamma-tocopherol. In conclusion, gamma-tocopherol, but not alpha-tocopherol, reduces the neointima proliferation in insulin resistance, independently of its effects on superoxide production. The beneficial effect may be related with its inhibitory effects on nitrosative stress.     

Myocardial protection by insulin is dependent on phospatidylinositol 3-kinase but not protein kinase C or KATP channels in the isolated rabbit heart

Because tyrosine kinase blockade prevents protection by ischemic preconditioning (PC) in several species, activation of tyrosine kinase appears to be critical for cardioprotection. The tyrosine kinase's identity, however, is unknown. The present study tested whether activation of a receptor tyrosine kinase, the insulin receptor, could mimic PC and if the mechanism of protection was similar to that of PC. Isolated rabbit hearts were subjected to 30 min of regional ischemia and 2 h of reperfusion. Infarct size was determined by triphenyltetrazolium staining and expressed as a percentage of the area at risk. Infarct size in control hearts was 32.6 ± 2.3 %. A 5-min infusion of insulin (5 mU/ml) followed by a 10-min washout period prior to ischemia significantly reduced infarction to 14.7 ± 2.1 % (P < 0.05). The tyrosine kinase inhibitor genistein (50 μM) given around the insulin infusion blocked protection (28.9 ± 2.8 %). However, when present during the onset of ischemia, genistein had no effect on protection triggered by insulin (14.0 ± 2.4 %; P < 0.05). Inhibition of either PKC by polymyxin B (50 μM) or K_ATP channels by 5-hydroxydecanoate (100 μM) also failed to prevent protection by insulin (17.5 ± 3.2 % and 17.6 ± 3.0 %, respectively). However, the reduction in infarct size by insulin was significantly attenuated by wortmannin (100 nM), a selective inhibitor of phosphatidylinositol 3-kinase (P13K, 28.3 ± 2.2 %). Insulin was still able to protect the heart when given only during the reperfusion period (13.2 ± 3.4 %). PC reduced infarction to 12.8 ± 2.0 % (P < 0.05). In conclusion, activation of the insulin receptor reduces infarct size in the rabbit heart even when instituted upon reperfusion. However, the mechanism of protection is quite different from that of PC and involves activation of P13K but not PKC or K_ATP channels. Received: 12 November 1998, Returned for revision: 25 November 1998, Revision received: 8 December 1998, Accepted: 10 December 1998     

SNAP-25, a SNARE protein, inhibits two types of K channels in esophageal smooth muscle

BACKGROUND & AIMS: The plasma membrane-associated soluble N-ethylmaleimide-sensitive factors attachment protein receptors (SNAREs), synaptosome-associated protein of 25 kilodaltons (SNAP-25), and syntaxin 1A, have been found to physically interact with and functionally modify membrane-spanning ion channels. Studies were performed in cat esophageal body and lower esophageal sphincter (LES) smooth muscle to (1) show the presence of SNAP-25, and (2) determine whether SNAP-25 affects K+ channel activity. METHODS: Single circular muscle cells from the esophageal body and sphincter were studied. Cellular localization of SNAP-25 and K+ channel activity were assessed. RESULTS: SNAP-25 was found in the plasma membrane of all regions examined. Outward K+ currents in body circular muscle were mainly composed of large conductance Ca2+-activated channel currents (K(Ca), 40.1%) and delayed rectifier K+ channel currents (K(V), 54.2%). Microinjection of SNAP-25 into muscle cells caused a dose-dependent inhibition of both outward K+ currents, maximal 44% at 10(-8) mol/L. Cleavage of endogenous SNAP-25 by dialyzing botulinum neurotoxin A into the cell interior resulted in a 35% increase in outward currents. CONCLUSIONS: SNAP-25 protein is present in esophageal smooth muscle cells, and inhibits both K(V) and K(Ca) currents in circular muscle cells. The findings suggest a role for SNAP-25 in regulation of esophageal muscle cell excitability and contractility, and point to potential new targets for treatment of esophageal motor disorders.     

SNARE protein regulation of cardiac potassium channels and atrial natriuretic factor secretion

Coordinated cardiac ion channel gating is fundamental for generation of action potential and excitability throughout the myocardium. The interaction of pore-forming ion channels with auxiliary subunits can regulate surface expression, localization and anchoring of these channels to plasma membrane. SNARE (soluble N-ethylmaleimide sensitive factors attachment protein or SNAP receptor) proteins mediate the targeting, docking, and fusion of intracellular vesicles for exocytotic release of neurotransmitters and hormones. In secretory neurons and neuroendocrine cells, some voltage-gated channels are physically coupled with SNARE proteins, resulting in alterations in channel gating and trafficking. Coupling of SNARE proteins to membrane ion channels is however not unique to secretory cells. We have demonstrated the expression of SNARE proteins in rodent myocardial tissue, and more importantly, functional interaction of SNARE proteins with cardiac K_ATP and K_v (K_v1.2, K_v2.1, K_v4.2, K_v4.3, and K_v11.1) channels. SNARE proteins, therefore, have similar fundamental functions in ion channel trafficking and regulation per se, independent of secretion. We now review the body of work of SNARE protein regulation on membrane ion channels in the heart.