Opening of mitochondrial K(ATP) channels triggers the preconditioned state by generating free radicals

The critical time for opening mitochondrial (mito) K(ATP) channels, putative end effectors of ischemic preconditioning (PC), was examined. In isolated rabbit hearts 29+/-3% of risk zone infarcted after 30 minutes of regional ischemia. Ischemic PC or 5-minute exposure to 10 micromol/L diazoxide, a mito K(ATP) channel opener, reduced infarction to 3+/-1% and 8+/-1%, respectively. The mito K(ATP) channel closer 5-hydroxydecanoate (200 micromol/L), bracketing either 5-minute PC ischemia or diazoxide infusion, blocked protection (24+/-3 and 28+/-6% infarction, respectively). However, 5-hydroxydecanoate starting 5 minutes before long ischemia did not affect protection. Glibenclamide (5 micromol/L), another K(ATP) channel closer, blocked the protection by PC only when administered early. These data suggest that K(ATP) channel opening triggers protection but is not the final step. Five minutes of diazoxide followed by a 30-minute washout still reduced infarct size (8+/-3%), implying memory as seen with other PC triggers. The protection by diazoxide was not blocked by 5 micromol/L chelerythrine, a protein kinase C antagonist, given either to bracket diazoxide infusion or just before the index ischemia. Bracketing preischemic exposure to diazoxide with 50 micromol/L genistein, a tyrosine kinase antagonist, did not affect infarction, but genistein blocked the protection by diazoxide when administered shortly before the index ischemia. Thus, although it is not protein kinase C-dependent, the protection by diazoxide involves tyrosine kinase. Bracketing diazoxide perfusion with N:-(2-mercaptopropionyl) glycine (300 micromol/L) or Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (7 micromol/L), each of which is a free radical scavenger, blocked protection, indicating that diazoxide triggers protection through free radicals. Therefore, mito K(ATP) channels are not the end effectors of protection, but rather their opening before ischemia generates free radicals that trigger entrance into a preconditioned state and activation of kinases.     

Increased mitochondrial K(ATP) channel activity during chronic myocardial hypoxia: is cardioprotection mediated by improved bioenergetics?

Increased resistance to myocardial ischemia in chronically hypoxic immature rabbit hearts is associated with activation of ATP-sensitive K(+) (K(ATP)) channels. We determined whether chronic hypoxia from birth alters the function of the mitochondrial K(ATP) channel. The K(ATP) channel opener bimakalim (1 micromol/L) increased postischemic recovery of left ventricular developed pressure in isolated normoxic (FIO(2)=0.21) hearts to values (42+/-4% to 67+/-5% ) not different from those of hypoxic controls but did not alter postischemic recovery of developed pressure in isolated chronically hypoxic (FIO(2)=0.12) hearts (69+/-5% to 72+/-5%). Conversely, the K(ATP) channel blockers glibenclamide (1 micromol/L) and 5-hydroxydecanoate (5-HD, 300 micromol/L) attenuated the cardioprotective effect of hypoxia but had no effect on postischemic recovery of function in normoxic hearts. ATP synthesis rates in hypoxic heart mitochondria (3.92+/-0.23 micromol ATP. min(-1). mg mitochondrial protein(-1)) were significantly greater than rates in normoxic hearts (2.95+/-0.08 micromol ATP. min(-1). mg mitochondrial protein(-1)). Bimakalim (1 micromol/L) decreased the rate of ATP synthesis in normoxic heart mitochondria consistent with mitochondrial K(ATP) channel activation and mitochondrial depolarization. The effect of bimakalim on ATP synthesis was antagonized by the K(ATP) channel blockers glibenclamide (1 micromol/L) and 5-HD (300 micromol/L) in normoxic heart mitochondria, whereas glibenclamide and 5-HD alone had no effect. In hypoxic heart mitochondria, the rate of ATP synthesis was not affected by bimakalim but was attenuated by glibenclamide and 5-HD. We conclude that mitochondrial K(ATP) channels are activated in chronically hypoxic rabbit hearts and implicate activation of this channel in the improved mitochondrial bioenergetics and cardioprotection observed.     

Long-chain acyl-coenzyme A esters and fatty acids directly link metabolism to K(ATP) channels in the heart

ATP-sensitive K (K(ATP)) channels are inhibited by cytosolic ATP, a defining property that implicitly links these channels to cellular metabolism. Here we report a direct link between fatty acid metabolism and K(ATP) channels in cardiac muscle cells. Long-chain (LC) acyl-coenzyme A (CoA) esters are synthesized from fatty acids and serve as the principal metabolic substrates of the heart. We have studied the effects of LC acyl-CoA esters and LC fatty acids on K(ATP) channels of isolated guinea pig ventricular myocytes and compared them with the effects of phosphatidylinositol 4,5-bisphosphate (PIP(2)). Application of oleoyl-CoA (0.2 or 1 micromol/L), a naturally occurring acyl-CoA ester, to the cytosolic side of excised patches completely prevented rundown of K(ATP) channels, but not of Kir2 channels. The open probability of K(ATP) channels measured in the presence of oleoyl-CoA or PIP(2) was voltage dependent, increasing with depolarization. Oleoyl-CoA greatly reduced the ATP sensitivity of K(ATP) channels. At a concentration of 2 micromol/L, oleoyl-CoA increased the half-maximal inhibitory concentration of ATP >200-fold. The time course of the decrease in ATP sensitivity was much faster during application of oleoyl-CoA than during application of PIP(2). The effects of PIP(2), but not of oleoyl-CoA, were inhibited by increasing Ca(2+) to 1 mmol/L. Oleate (C18:1; 10 micromol/L), the precursor of oleoyl-CoA, inhibited K(ATP) channels activated by oleoyl-COA: Palmitoleoyl-CoA and palmitoleate (C16:1) exerted similar reciprocal effects. These findings indicate that LC fatty acids and their CoA-linked derivatives may be key physiological modulators of K(ATP) channel activity in the heart.     

Morphine mimics preconditioning via free radical signals and mitochondrial K(ATP) channels in myocytes

BACKGROUND: We tried to determine whether morphine mimics preconditioning (PC) to reduce cell death in cultured cardiomyocytes and whether opioid delta(1) receptors, free radicals, and K(ATP) channels mediate this effect. METHODS AND RESULTS: Chick embryonic ventricular myocytes were studied in a flow-through chamber while flow rate, pH, and O(2) and CO(2) tension were controlled. Cardiomyocyte viability was quantified with propidium iodide (5 micromol/L), and production of free radicals was measured with 2',7'-dichlorofluorescin diacetate. PC with 10 minutes of simulated ischemia before 10 minutes of reoxygenation or morphine (1 micromol/L) or BW373U86 (10 pmol/L) infusion for 10 minutes followed by a 10-minute drug-free period before 1 hour of ischemia and 3 hours of reoxygenation reduced cell death to the same extent (*P:<0.05) (PC, 20+/-1%, n=7*; morphine, 32+/-4%, n=8*; BW373U86, 21+/-6%; controls, 52+/-5%, n=8). Like PC, morphine and BW373U86 increased free radical production 2-fold before ischemia (0.35+/-0.10, n=6*; 0.41+/-0.08, n=4* versus controls, 0.15+/-0.05, n=8, arbitrary units). Protection and increased free radical signals during morphine infusion were abolished with either the thiol reductant 2-mercaptopropionyl glycine (400 micromol/L), an antioxidant; naloxone (10 micromol/L), a nonselective morphine receptor antagonist; BNTX (0.1 micromol/L), a selective opioid delta(1) receptor antagonist; or 5-hydroxydecanoate (100 micromol/L), a selective mitochondrial K(ATP) channel antagonist. CONCLUSIONS: These results suggest that direct stimulation of cardiocyte opioid delta(1) receptors leads to activation of mitochondrial K(ATP) channels. The resultant increase of intracellular free radical signals may be an important component of the signaling pathways by which morphine mimics preconditioning in cardiomyocytes.     

Alpha1-adrenoceptor-mediated breakdown of phosphatidylinositol 4,5-bisphosphate inhibits pinacidil-activated ATP-sensitive K+ currents in rat ventricular myocytes

Phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates ATP-sensitive K+ (K(ATP)) channel activity. Because phospholipase C (PLC) hydrolyzes membrane-bound PIP2, which in turn may potentially decrease K(ATP) channel activity, we investigated the effects of the alpha1-adrenoceptor-G(q)-PLC signal transduction axis on pinacidil-activated K(ATP) channel activity in adult rat and neonatal mouse ventricular myocytes. The alpha1-adrenoceptor agonist methoxamine (MTX) reversibly inhibited the pinacidil-activated K(ATP) current in a concentration-dependent manner (IC50 20.9+/-6.6 micromol/L). This inhibition did not occur when the specific alpha1-adrenoceptor antagonist, prazosin, was present. An involvement of G proteins is suggested by the ability of GDPbetaS to prevent this response. Blockade of PLC by U-73122 (2 micromol/L) or neomycin (2 mmol/L) attenuated the MTX-induced inhibition of K(ATP) channel activity. In contrast, the MTX response was unaffected by protein kinase C inhibition or stimulation by H-7 (100 micro mol/L) or phorbol 12,13-didecanoate. The MTX-induced inhibition became irreversible in the presence of wortmannin (20 micro mol/L), an inhibitor of phosphatidylinositol-4 kinase, which is expected to prevent membrane PIP2 replenishment. In excised inside-out patch membranes, pinacidil induced a significantly rightward shift of ATP sensitivity of the channel. This phenomenon was reversed by pretreatment of myocytes with MTX. Direct visualization of PIP2 subcellular distribution using a PLCdelta pleckstrin homology domain-green fluorescent protein fusion constructs revealed reversible translocation of green fluorescent protein fluorescence from the membrane to the cytosol after alpha1-adrenoceptor stimulation. Our data demonstrate that alpha1-adrenoceptor stimulation reduces the membrane PIP2 level, which in turn inhibits pinacidil-activated K(ATP) channels.     

Role of soluble epoxide hydrolase in postischemic recovery of heart contractile function

Cytochrome P450 epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which are converted to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (Ephx2, sEH). To examine the functional role of sEH in the heart, mice with targeted disruption of the Ephx2 gene were studied. Hearts from sEH null mice have undetectable levels of sEH mRNA and protein and cannot convert EETs to DHETs. sEH null mice have normal heart anatomy and basal contractile function, but have higher fatty acid epoxide:diol ratios in plasma and cardiomyocyte cell culture media compared with wild type (WT). sEH null hearts have improved recovery of left ventricular developed pressure (LVDP) and less infarction compared with WT hearts after 20 minutes ischemia. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 to 100 nmol/L) before ischemia abolishes this cardioprotective phenotype. Inhibitor studies demonstrate that perfusion with phosphatidylinositol-3 kinase (PI3K) inhibitors wortmannin (200 nmol/L) or LY294002 (5 micromol/L), the ATP-sensitive K+ channel (K(ATP)) inhibitor glibenclamide (1 micromol/L), the mitochondrial K(ATP) (mitoK(ATP)) inhibitor 5-hydroxydecanoate (100 to 200 micromol/L), or the Ca2+-sensitive K+ channel (K(Ca)) inhibitor paxilline (10 micromol/L) abolishes the cardioprotection in sEH null hearts. Consistent with increased activation of the PI3K cascade, sEH null mice exhibit increased cardiac expression of glycogen synthase kinase-3beta (GSK-3beta) phospho-protein after ischemia. Together, these data suggest that targeted disruption of sEH increases the availability of cardioprotective EETs that work by activating PI3K signaling pathways and K+ channels.     

Functional Communication Between Cardiac ATPSensitive K+ Channel and Na/K ATPase

K_ATP Channel and Na/K ATPase. Introduction: Functional interaction between K_ATP channel and Na/K ATPase was studied in single guinea pig ventricular myocytes because both membrane molecules are known to he involved in ischemic episodes. Methods and Results: K_ATP channel currents were recorded at 36°C by using whole cell, cell attached, inside-out, and open cell-attached modes of patch clamp techniques on enzymatically isolated ventricular myocytes. In the whole cell mode, ouabain (1 μM) reversibly inhibited the K_ATP currents induced by metabolic stress (ATP-free pipette solution and 1 mM NaCN), but not those activated by cromakalim (100 μM), a K_ATP channel opener. In the cell-attached mode, ouabain concentration dependently inhibited K_ATP, channel opening induced by metabolic suppression (5.5 μM 2-deoxyglucose and 1 mM CN). Half-inhibition concentration for ouabain was 21.0 ± 5.5 nM and the Hill coefficient was 0.8 ± 0.1 (n = 26). However, ouabain did not have an effect on the channel activity induced by cromakalim (100 μM). In the inside-out mode, ouabain applied to the internal side of membrane did not affect the channel. In the open cell-attached mode made by preincubation with streptolysin-0 (0.08 U/mL), the K_ATP channels were not activated by the metabolic inhibitors but were by reducing extracellular ATP concentrations, because subsarcolenimal ATP concentration could he controlled through tiny membrane holes. The channels thus activated were not suppressed by ouabain. Conclusion: The inhibition of Na/K ATPase by ouahain appeared to block the K_ATP channels by accumulating subsarcolemmal ATP caused by a decrease of the transition from ATP to ADP. In the presence of ischemic episodes, the administration of digitalis compounds may affect the opening of K_ATP channels, which is primarily protective against the development of irreversible myocardial damage.     

Differential sensitivity of atrial and ventricular K(ATP) channels to metabolic inhibition

OBJECTIVE: The aim is to compare the activation of ATP-sensitive potassium channels (K(ATP) channels) in intact and metabolically impaired atrial and ventricular myocytes. METHODS: The K(ATP) channel current is measured by whole cell and gramicidin-perforated patch clamp recordings in 164 cultured neonate rat cardiomyocytes. RESULTS: In whole cell recordings with 84 micromol/l ADP in pipette, spontaneous activity is significantly higher in atrium than ventricle, and EC(50) for the K(ATP) channel opener diazoxide is 0.13 micromol/l (atrium) versus 3.1 micromol/l (ventricle). With an ATP-regenerating system in pipette, EC(50) for diazoxide is 19.7 micromol/l (atrium) versus 54.9 micromol/l (ventricle). In gramicidin-perforated patch recordings, atrial myocytes respond significantly to 100 nmol/l of the mitochondrial protonophore CCCP, while ventricular myocytes do not. EC(50) for diazoxide is 129 micromol/l (atrium) versus >2500 micromol/l (ventricle) for myocytes exposed to CCCP, and 676 versus >2500 micromol/l, respectively, without CCCP. CONCLUSIONS: (1) K(ATP) channels are significantly more sensitive to metabolic inhibition in atrial than ventricular myocytes. (2) Sensitivity of atrium versus ventricle to the channel opener diazoxide increases from 3:1 to > or = 24:1 with ADP or metabolic inhibition. If extended to intact hearts, the results would predict a higher atrial sensitivity to ischemia, and a high sensitivity of the ischemic atrium to K(ATP) channel openers.     

Local Ca2+ entry through L-type Ca2+ channels activates Ca2+-dependent K+ channels in rabbit coronary myocytes.

Large-conductance Ca2+-dependent K+ channels (KCa), which are abundant on the sarcolemma of vascular myocytes, provide negative feedback via membrane hyperpolarization that limits Ca2+ entry through L-type Ca2+ channels (ICaL). We hypothesize that local accumulation of subsarcolemmal Ca2+ during ICaL openings amplifies this feedback. Our goal was to demonstrate that Ca2+ entry through voltage-gated ICaL channels can stimulate adjacent KCa channels by a localized interaction in enzymatically isolated rabbit coronary arterial myocytes voltage clamped in whole-cell or in cell-attached patch clamp mode. During slow-voltage-ramp protocols, we identified an outward KCa current that is activated by a subsarcolemmal Ca2+ pool dissociated from bulk cytosolic Ca2+ pool (measured with indo 1) and is dependent on L-type Ca2+ channel activity. Transient activation of unitary KCa channels in cell-attached patches could be detected during long step depolarizations to +40 mV (holding potential, -40 mV; 219 pS in near-symmetrical K+). This local interaction between the channels required the presence of Ca2+ in the pipette solution, was enhanced by the ICaL agonist Bay K 8644, and persisted after impairment of the sarcoplasmic reticulum by incubation with 10 micromol/L ryanodine and 30 micromol/L cyclopiazonic acid for at least 60 minutes. Furthermore, we provide the first direct evidence of simultaneous openings of single KCa (67 pS) and ICaL (3.9 pS) channels in near-physiological conditions, near resting membrane potential. Our data imply a novel sensitive mechanism for regulating resting membrane potential and tone in vascular smooth muscle.     

Effect of ceramide on KCa channel activity and vascular tone in coronary arteries.

A sphingomyelin metabolite, ceramide, serves as a second messenger in a variety of mammalian cells. Little is known regarding the production and actions of this novel intracellular signaling lipid molecule in the vasculature. The present study was designed to test the hypothesis that a ceramide-mediated signaling pathway is present in coronary arterial smooth muscle and that ceramide serves as an inhibitor of the large-conductance Ca2+-activated potassium (KCa) channels and mediates vasoconstriction in coronary circulation. We found that C2-ceramide produced a concentration-dependent decrease in KCa channel activity in vascular smooth muscle cells from small bovine coronary arteries. The average channel activity of the KCa channels in cell-attached patches decreased from 0.046+/-0.01 to 0. 008+/-0.001 at a C2-ceramide concentration of 10 micromol/L. In inside-out patches, C2-ceramide (1 micromol/L) reduced the average channel activity of the KCa channels from 0.06+/-0.007 to 0.016+/-0. 004. Dithiothreitol, an inhibitor of acidic sphingomyelinase (1 mmol/L), increased the average channel activity of the KCa channels in cell-attached patches from 0.05+/-0.02 of control to 0.26+/-0.04, a 5-fold increase that was reversed by addition of 1 micromol/L ceramide. Glutathione, an inhibitor of neutral sphingomyelinase, was without effect. C2-ceramide significantly reduced the diameter of isolated perfused small coronary arteries in a concentration-dependent manner. Addition of 1 micromol/L C2-ceramide decreased average arterial diameter by 28%. When 14C-sphingomyelin was incubated with coronary arterial homogenates at pH 7.4 and pH 5. 0, 14C-choline phosphate and ceramide were produced. The conversion rates of 14C-sphingomyelin into 14C-choline phosphate and ceramide were 65.1+/-1.0 fmol/min per milligram protein at pH 7.4 and 114. 6+/-8.3 fmol/min per milligram protein at pH 5.0. We conclude that both acidic and neutral sphingomyelinases are present in the bovine coronary arteries and that ceramide inactivates the KCa channel in arterial smooth muscle cells and hence exerts a tonic vasoconstrictor action in coronary microcirculation.     

Effects of vasopressin on portal-systemic collaterals in portal hypertensive rats: role of nitric oxide and prostaglandin.

This study investigated the effect of vasopressin on portal-systemic collaterals in portal hypertensive rats and the influence of nitric oxide (NO) and prostaglandin on the responsiveness of collateral vessels to vasopressin. The vascular responsiveness to graded concentrations of vasopressin was tested with or without the incubation of n(omega)-nitro-L-arginine (NNA) (100 micromol/L) and/or indomethacin (10 micromol/L) in perfused collateral vascular beds of rats with portal hypertension induced by partial portal vein ligation. In addition, concentration-response curves to vasopressin with incubation of a vasopressin V(1) receptor antagonist d(CH(2))(5)Tyr(Me) arginine vasopressin and concentration-response curves to a V(2) receptor agonist 1-desamino-8-D-arginine vasopressin were performed. Vasopressin significantly increased the perfusion pressure of collaterals, and this effect was suppressed by the addition of the V(1) receptor antagonist. Perfusion with the V(2) receptor agonist had no effect on the collaterals. Incubation with NNA, indomethacin, or both significantly potentiated the response of collaterals to vasopressin. In addition, the pressor response to vasopressin in the combination group was significantly higher than that in the NNA-alone group. The results show that vasopressin produces a direct vasoconstrictive effect on the portal-systemic collaterals of portal hypertensive rats. This effect is mediated by the vasopressin V(1,) but not V(2), receptors. The attenuation of the response to vasopressin by NO and prostaglandin suggest a function role of both mediators in the regulation of the portal-systemic collateral circulation in portal hypertensive rats.     

14,15-epoxyeicosa-5(Z)-enoic-mSI: a 14,15- and 5,6-EET antagonist in bovine coronary arteries

Endothelium-dependent hyperpolarizations and relaxation of vascular smooth muscle induced by acetylcholine and bradykinin are mediated by endothelium-derived hyperpolarizing factors (EDHFs). In bovine coronary arteries, arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs), function as EDHFs. The 14,15-EET analog 14,15-epoxyeicosa-5(Z)-enoic-methylsulfonylimide (14,15-EEZE-mSI) was synthesized and tested for agonist and antagonist activity. In U46619-preconstricted bovine coronary arterial rings, 14,15-, 11,12-, 8,9-, and 5,6-EET induced maximal concentration-related relaxation averaging 75% to 87% at 10 micromol/L, whereas, 14,15-EEZE-mSI induced maximal relaxation averaging only 7%. 14,15-EEZE-mSI (10 micromol/L) preincubation inhibited relaxation to 14,15- and 5,6- EET but not 11,12- or 8,9- EET. 14,15-EEZE-mSI also inhibited indomethacin-resistant relaxation to arachidonic acid and indomethacin-resistant and l-nitroarginine-resistant relaxation to bradykinin and methacholine. It did not alter the relaxation to sodium nitroprusside, iloprost, or the K+ channel openers bimakalim or NS1619. In cell-attached patches of isolated bovine coronary arterial smooth muscle cells, 14,15-EEZE-mSI (100 nmol/L) blocked the 14,15-EET-induced (100 nmol/L) activation of large-conductance, calcium-activated K+ channels. Mass spectrometric analysis of rat renal cortical microsomes incubated with arachidonic acid showed that 14,15-EEZE-mSI (10 micromol/L) increased EET concentrations while decreasing the concentrations of the corresponding dihydroxyeicosatrienoic acids. Therefore, 14,15-EEZE-mSI inhibits relaxation to 5,6- and 14,15- EET and the K+ channel activation by 14,15-EET. It also inhibits the EDHF component of bradykinin-induced, methacholine-induced, and arachidonic acid-induced relaxation. These results suggest that 14,15- or 5,6 -EET act as an EDHF in bovine coronary arteries.     

Effects of KCNQ1 channel blocker, 293B, on the acetylcholine-induced Cl- secretion of rat pancreatic acini

In rat pancreatic acini (RPAs), acetylcholine (ACh) typically induces a tonic depolarization of membrane potential (Vm) via increasing cytoplasmic Ca2+ concentration and subsequent activation of Cl- channels. In this study, to investigate the role of K+ channels during the ACh-induced Cl- secretion, the intracellular Cl- concentration ([Cl-]i) of RPAs was monitored using SPQ, a fluorescent dye quenchable by Cl-, and the effects of K+ channel blockers were examined. Also, the secretion of fluid and enzyme from the whole pancreas of rat was measured. The fluorescence of RPAs loaded with SPQ (FSPQ) was slightly increased by the application of ACh (ACh-Delta FSPQ), indicating net secretion of Cl-. However, the relative change of FSPQ normalized to the control fluorescence (F/F0) of RPAs was only about 20% of the effect observed in rat submandibular gland acinus. The ACh-Delta FSPQ of RPAs was not influenced by the pretreatment with 293B (20 micromol/L), a blocker of KCNQ-type K+ channels. Even the cocktail of K+ channel blockers (10 mmol/L TEA, 3 mmol/L Ba2+, 20 micromol/L 293B) exerted only minute inhibitory effects on ACh-Delta FSPQ in RPAs. In the vascularly perfused rat pancreas, the fluid and enzyme secretion induced by ACh was directly measured. 293B and HMR-1556, both specific blockers of KCNQ1 channel, did not block but even enhanced the secretion of fluid and amylase. These results suggest that the role of KCNQ1 channels may not be essential in the Ca2+-mediated Cl- secretion in rat pancreatic acini.     

A TREK-1-like potassium channel in atrial cells inhibited by beta-adrenergic stimulation and activated by volatile anesthetics

Many members of the two-pore-domain potassium (K(+)) channel family have been detected in the mammalian heart but the endogenous correlates of these channels still have to be identified. We investigated whether I(KAA), a background K(+) current activated by negative pressure (stretch) and by arachidonic acid (AA) and sensitive to intracellular acidification, could be the native correlate of TREK-1 in adult rat atrial cells. Using the inside-out configuration of the patch-clamp technique, we found that I(KAA), like TREK-1, was outwardly rectifying in physiological K(+) conditions, with a conductance of 41 pS at +50 mV. Like TREK-1, I(KAA) was reversibly activated by clinical concentrations of volatile anesthetics (in mmol/L, chloroform 0.18, halothane 0.11, and isoflurane 0.69). In cell-attached experiments, I(KAA) was inhibited by chlorophenylthio-cAMP (500 micromol/L) and also by stimulation of beta-adrenergic receptors with isoproterenol (1 micromol/L). In addition, TREK-1 mRNAs were detected in all cardiac tissues, and the TREK-1 protein was immunolocalized in isolated atrial myocytes. Such a background potassium channel might contribute to the positive inotropic effects produced by beta-adrenergic stimulation of the heart. It might also be involved in the regulation of the atrial natriuretic peptide secretion.     

Angiotensin-(1-7) reduces norepinephrine release through a nitric oxide mechanism in rat hypothalamus

Angiotensin (Ang)-(1-7) elicits a facilitatory presynaptic effect on peripheral noradrenergic neurotransmission, and because biological responses to the heptapeptide on occasion are tissue specific, the present investigation was undertaken to study its action on noradrenergic neurotransmission at the central level. In rat hypothalamus labeled with [(3)H]-norepinephrine, 100 to 600 nmol/L Ang-(1-7) diminished norepinephrine released by 25 mmol/L KCl. This effect was blocked by the selective angiotensin type 2 receptor antagonist PD 123319 (1 micromol/L) and by the specific Ang-(1-7) receptor antagonist ([D-Ala(7)]Ang-(1-7) (1 micromol/L) but not by losartan (10 nmol/L to 1 micromol/L), a selective angiotensin type 1 receptor antagonist. The inhibitory effect on noradrenergic neurotransmission caused by Ang-(1-7) was prevented by 10 micromol/L N(omega)-nitro-L-arginine methylester, an inhibitor of nitric oxide synthase activity, and was restored by 100 micromol/L L-arginine, precursor of nitric oxide synthesis. Methylene blue (10 micromol/L), an inhibitor of guanylate cyclase considered as the target of nitric oxide action, as well as Hoe 140 (10 micromol/L), a bradykinin B(2)-receptor antagonist, prevented the inhibitory effect of the heptapeptide on neuronal norepinephrine release, whereas no modification was observed in the presence of 0.1 to 10 micromol/L indomethacin, a cyclooxygenase inhibitor. Our results indicate that Ang-(1-7) has a tissue-specific neuromodulatory effect on noradrenergic neurotransmission, being inhibitory at the central nervous system by a nitric oxide-dependent mechanism that involves angiotensin type 2 receptors and local bradykinin production.     

Nicorandil, a potent cardioprotective agent, acts by opening mitochondrial ATP-dependent potassium channels

OBJECTIVES: To determine the mechanism of cardioprotection afforded by nicorandil, an orally efficacious antianginal drug, we examined its effects on ATP-dependent potassium (K(ATP)) channels. BACKGROUND: Nicorandil can mimic ischemic preconditioning, while mitochondrial K(ATP) (mitoK(ATP)) channels rather than sarcolemmal K(ATP) (surfaceK(ATP)) channels have emerged as the likely effectors. METHODS: Flavoprotein fluorescence and membrane current in intact rabbit ventricular myocytes were measured simultaneously to assay mitoK(ATP) channel and surface K(ATP) channel activities, respectively. In a cell-pelleting model of ischemia, cells permeable to trypan blue were counted as killed by 60 and 120 min of ischemia. RESULTS: Nicorandil (100 micromol/liter) increased flavoprotein oxidation but not membrane current; a 10-fold higher concentration recruits both mitoK(ATP) and surfaceK(ATP) channels. Pooled dose-response data confirm that nicorandil concentrations as low as 10 micromol/liter turn on mitoK(ATP) channels, while surfaceK(ATP) current requires exposure to millimolar concentrations. Nicorandil blunted the rate of cell death in a pelleting model of ischemia; this cardioprotective effect was prevented by the mitoK(ATP) channel blocker 5-hydroxydecanoate but was unaffected by the surfaceK(ATP) channel blocker HMR1098. CONCLUSIONS: Nicorandil exerts a direct cardioprotective effect on heart muscle cells, an effect mediated by selective activation of mitoK(ATP) channels.     

Acetylcholine, bradykinin, opioids, and phenylephrine, but not adenosine, trigger preconditioning by generating free radicals and opening mitochondrial K(ATP) channels

It has been assumed that all G(i)-coupled receptors trigger the protective action of preconditioning by means of an identical intracellular signaling pathway. To test this assumption, rabbit hearts were isolated and perfused with Krebs buffer. All hearts were subjected to a 30-minute coronary artery occlusion followed by 120 minutes of reperfusion. Risk area was measured with fluorescent particles and infarct size with triphenyltetrazolium chloride staining. Control hearts showed 29.1+/-2.8% infarction of the risk zone. A 5-minute infusion of acetylcholine (0.55 mmol/L) beginning 15 minutes before the 30-minute occlusion resulted in significant protection (9.2+/-2.7% infarction). This protection could be blocked by administration of 300 micromol/L N-2-mercaptopropionyl glycine (MPG), a free radical scavenger, or by 200 micromol/L 5-hydroxydecanoate (5-HD), a mitochondrial K(ATP) antagonist, for 15 minutes beginning 5 minutes before the acetylcholine infusion (35.2+/-3.9% and 27.8+/-2.4% infarction, respectively). Similar protection was observed with other known triggers, ie, bradykinin (0.4 micromol/L), morphine (0.3 micromol/L), and phenylephrine (0.1 micromol/L), and in each case protection was completely abrogated by either MPG or 5-HD. In contrast, protection by adenosine or its analog N(6)-(2-phenylisopropyl) adenosine could not be blocked by either MPG or 5-HD. Therefore, whereas most of the tested agonists trigger protection by a pathway that requires opening of mitochondrial K(ATP) channels and production of free radicals, the protective action of adenosine is not dependent on either of these steps. Hence, it cannot be assumed that all G(i)-coupled receptors use the same signal transduction pathways to trigger preconditioning.     

Attenuation by 4-aminopyridine of delayed vasorelaxation by troglitazone

Troglitazone and other thiazolidinediones (TZDs) are thought to relax arterial smooth muscle by directly inhibiting calcium channels in smooth muscle cell membranes. However, until recently such inhibition was only examined acutely, ie, within only seconds or minutes after administration of these agents to arterial smooth muscle preparations. Recently, a novel experiment was reported in which troglitazone caused a 2-phase relaxation of perfused resistance arteries, namely, an acute relaxation (within the first 20 minutes of treatment), which was blocked by a nonselective calcium channel blocker and a delayed relaxation (after 2 hours), which was not. We sought to determine if any of the 4 major potassium (K) channels in vascular smooth muscle play a role in the delayed relaxation. We incubated vascular contractile rings prepared from ventral tail arteries of rats with physiological buffer containing either 0 or 4 micromol/L troglitazone for 3 hours (4 micromol/L is typical of plasma levels from diabetic patients). Different K channel inhibitors (1 mmol/L 4-aminopyridine [4AP]; 1 mmol/L tetraethylammonium [TEA]; 5 micromol/L glyburide; 20 micromol/L barium) were coadministered with each level of troglitazone in additional preparations. Then these arterial rings were contracted with either norepinephrine (NE), arginine vasopressin (AVP), or high-K buffer. All contractions were significantly relaxed by troglitazone (P <.05). Only 4AP significantly attenuated troglitazone's relaxation of NE and AVP contractions (P <.05), though not high-K-induced contractions. TEA, glyburide, and barium had no such influence. Thus, for both adrenergic (NE) and nonadrenergic (AVP) contractions, the delayed arterial vasorelaxation by troglitazone may be mediated at least in part by activation of 4AP-sensitive K channels. Furthermore, the specific subtype of the channels involved is most likely those bound in the outer cell membrane where their effectiveness in terms of mediating relaxation would depend on an intact transmembrane K ion gradient.     

Modulation of nucleotide sensitivity of ATP-sensitive potassium channels by phosphatidylinositol-4-phosphate 5-kinase

ATP-sensitive potassium channels (K(ATP) channels) regulate cell excitability in response to metabolic changes. K(ATP) channels are formed as a complex of a sulfonylurea receptor (SURx), a member of the ATP-binding cassette protein family, and an inward rectifier K(+) channel subunit (Kir6.x). Membrane phospholipids, in particular phosphatidylinositol (PI) 4,5-bisphosphate (PIP(2)), activate K(ATP) channels and antagonize ATP inhibition of K(ATP) channels when applied to inside-out membrane patches. To examine the physiological relevance of this regulatory mechanism, we manipulated membrane PIP(2) levels by expressing either the wild-type or an inactive form of PI-4-phosphate 5-kinase (PIP5K) in COSm6 cells and examined the ATP sensitivity of coexpressed K(ATP) channels. Channels from cells expressing the wild-type PIP5K have a 6-fold lower ATP sensitivity (K(1/2), the half maximal inhibitory concentration, approximately 60 microM) than the sensitivities from control cells (K(1/2) approximately 10 microM). An inactive form of the PIP5K had little effect on the K(1/2) of wild-type channels but increased the ATP-sensitivity of a mutant K(ATP) channel that has an intrinsically lower ATP sensitivity (from K(1/2) approximately 450 microM to K(1/2) approximately 100 microM), suggesting a decrease in membrane PIP(2) levels as a consequence of a dominant-negative effect of the inactive PIP5K. These results show that PIP5K activity, which regulates PIP(2) and PI-3,4,5-P(3) levels, is a significant determinant of the physiological nucleotide sensitivity of K(ATP) channels.     

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.