ATP-dependent Potassium Research Articles

Activation of ATP-dependent potassium channels is trigger but not mediator of ischemic preconditioning in pigs

Activation of ATP-dependent potassium channels is trigger but not mediator of ischemic preconditioning in pigs

Activation of mitochondrial ATP-dependent potassium channels protects neurons against ischemia-induced death by a mechanism involving suppression of Bax translocation and cytochrome c release.

Neurons express a variety of plasma-membrane potassium channels that play important roles in regulating neuronal excitability and synaptic transmission, but also contain mitochondrial ATP-sensitive potassium channels, the functions of which are unknown. Studies of cardiac cells suggest that similar mitochondrial ATP-sensitive potassium channels are involved in the process of ischemic preconditioning, suggesting a role in regulating cell survival. The authors report that mice given diazoxide, an activator of mitochondrial ATP-sensitive potassium channels, exhibited a large (60% to 70%) decrease in cortical infarct size after permanent occlusion of the middle cerebral artery. Diazoxide decreases neuronal apoptosis and increases astrocyte survival and activation in the penumbral region of the ischemic cortex. The neuroprotective effect of diazoxide is abolished by 5-hydroxydecanoate, a selective antagonist of mitochondrial ATP-sensitive potassium channels. Studies of cultured hippocampal neurons reveal that diazoxide depolarizes mitochondria, prevents cytochrome c release, and protects cells against death induced by staurosporine and chemical hypoxia. Diazoxide increased the levels of Bcl2 and inhibited the association of Bax with mitochondria in neurons exposed to an apoptotic insult, suggesting that activation of mitochondrial ATP-sensitive potassium channels may stabilize mitochondrial function by differentially modulating proapoptotic and antiapoptotic proteins. Collectively, the data suggest that mitochondrial ATP-sensitive potassium channels play a key role in modulating neuronal survival under ischemic conditions, and identify agents that activate mitochondrial ATP-sensitive potassium channels as potential therapeutics for stroke and related neurodegenerative conditions.

No role of calcium- and ATP-dependent potassium channels in insulin-induced vasodilation in humans in vivo.

The mechanism of insulin-induced vasodilation has not been completely clarified, but could be important in future treatment strategies of insulin resistance. Recently, a role for calcium-dependent and ATP-dependent potassium (K(Ca) and K(ATP)) channels in insulin-induced vasodilation has been demonstrated in in vitro studies. A role for these channels has never been confirmed in humans in vivo. Therefore, we investigated the role of these channels in insulin-induced vasodilation in humans in vivo. A hyperinsulinemic euglycemic clamp was combined with intra-arterial infusion of placebo, tetraethylammonium (blocker of K(Ca) channels) or glibenclamide (blocker of K(ATP) channels) in three groups of 12 healthy volunteers. Bilateral forearm blood flow was measured with venous occlusion plethysmography. Systemic hyperinsulinemia induced a 20+/-9% vasodilation (p=0.001). Neither tetraethylammonium nor glibenclamide reduced this vasodilation as compared to placebo. According to the results of the present study, insulin-induced vasodilation seems not to be mediated by the opening of K(Ca) and K(ATP) channels in humans in vivo.

Mechanisms modulating estrogen-induced uterine vasodilation

Estrogen, a potent vasodilator, has its greatest effects in reproductive tissues, e.g., increasing uterine blood flow (UBF) 5- to 10-fold within 90 min after a bolus dose. High-conductance potassium channels and nitric oxide (NO) contribute to the uterine responses, but other factors may be involved. We examined the role of ATP-dependent (ATP-sensitive) and voltage-gated (K v) potassium channels and new protein synthesis in ovariectomized ewes with uterine artery flow probes, infusing intraarterial inhibitors glibenclamide (GLB; K ATP), 4-aminopyridine (4-AP; K v) or cycloheximide, respectively, into one uterine horn before and/or after systemic estradiol-17β (E 2β, 1 μg/kg iv). E 2β alone increased UBF >5-fold and heart rate by 10–25% ( P<.01) within 90 min; mean arterial pressure (MAP) was unaffected. GLB did not alter basal hemodynamic parameters or responses to E 2β. Basal UBF and heart rate were unaffected by 4-AP, but MAP increased by 10% and 25% at 30 and 120 min of infusion ( P<.01), respectively. Although E 2β-induced rises in UBF were unaffected in the control uterine horn, 4-AP dose-dependently inhibited UBF responses in the infused horn ( R=.83, P=.003, n=10). Cycloheximide not only dose-dependently inhibited UBF responses ( R=.57, P=.01, n=18) and increases in uterine cGMP secretion, 23.4±10.7 versus 340±60 pmol/min ( P<.001), but also decreased UBF by 50% and cGMP by ∼90% at the time of maximum UBF. Mechanisms modulating estrogen-induced uterine vasodilation involve signaling pathways that include NO, smooth muscle cGMP, smooth muscle potassium channels and new protein synthesis.

Alterations of purine and pyrimidine nucleotide contents in rat corticoencephalic cell cultures following metabolic damage and treatment with openers and blockers of ATP-sensitive potassium channels

Rat corticoencephalic cell cultures were investigated by high performance liquid chromatography for changes in the levels of adenosine 5′-triphosphate (ATP), guanosine 5′-triphosphate (GTP), uridine 5′-triphosphate (UTP), cytidine 5′-triphosphate (CTP), and the respective nucleoside diphosphates. Hypoxia was induced by gassing the incubation medium for 30 min with 100% argon. Removal of glucose was caused by washing the cultures in glucose-free medium at the beginning of the 30 min incubation period. Whereas hypoxia or glucose-deficiency alone failed to alter the nucleotide levels, the combination of these two manipulations was clearly inhibitory. Diazoxide (300 μM) an opener of ATP-dependent potassium channels (K ATP) did not alter the nucleotide contents either in a normoxic and glucose-containing medium, or a hypoxic and glucose-free medium. By contrast, the K ATP channel antagonist tolbutamide (300 μM) aggravated the hypoxic decrease of nucleotide levels in a glucose-free medium, although it was ineffective in a normoxic and glucose-containing medium. Hypoxia and glucose-deficiency decreased the ATP/ADP and UTP/UDP ratios, but failed to change the GTP/GDP ratio. Diazoxide and tolbutamide (300 μM each) had no effect on the nucleoside triphosphate/diphosphate ratios either during normoxic or during hypoxic conditions. In conclusion, corticoencephalic cultures are rather resistant to in vitro ischemia. Although they clearly respond to the blockade of plasmalemmal K ATP channels (plasmaK ATP) by tolbutamide, these channels appear to be maximally open as a consequence of the fall in intracellular nucleotides and, therefore, diazoxide has no further effect.

KCO912: a potent and selective opener of ATP-dependent potassium (K(ATP)) channels which suppresses airways hyperreactivity at doses devoid of cardiovascular effects.

ATP-sensitive potassium (K(ATP)) channel openers can obviate experimental airways hyperreactivity (AHR) and have shown therapeutic benefit in asthma. However, the clinical potential of such compounds has been compromised by cardiovascular side-effects. We report here the pharmacological properties of (3 S,4 R)-3,4-dihydro-3-hydroxy-2,2-dimethyl-4-(2-oxo-1-piperidinyl)- N-phenyl-2 H-1-benzopyran-6-sulphonamide (KCO912), a K(ATP) channel opener which suppresses AHR at doses devoid of cardiovascular effects.Specific interaction of KCO912 with the native vascular channel and the sulphonylurea receptor subunit (SUR2B) of the vascular K(ATP) channel was shown in radioligand binding assays. In rat aortic strips, KCO912 inhibited specific binding of [3H]P1075 and [3H]glibenclamide with up to 100% efficacy and with p Ki values of 8.28 and 7.96, respectively. In HEK cells transfected with the recombinant vascular K(ATP) channel (Kir6.1 + SUR2B), the compound elicited a concentration-dependent outward current (pEC50 6.8) and in preloaded rat aortic rings it induced a concentration-dependent glibenclamide-sensitive 86Rb+ efflux (pEC50 7.51). Following intratracheal (i.t.) administration of KCO912 to guinea pigs, AHR induced by immune complexes or ozone was rapidly (<5 min) reversed (ED50 values 1 microg/kg and 0.03 microg/kg, respectively). Changes in blood pressure were seen only at doses =100 microg/kg yielding 'therapeutic ratios' of 100 and 3333, respectively. In addition, KCO912 reversed AHR induced by lipopolysaccharide (LPS; ED50 0.5 microg/kg i.t.) and a dose of 1 microg/kg i.t. fully reversed AHR induced by subchronic treatment with salbutamol. At doses which suppressed AHR, KCO912 had no anti-bronchoconstrictor effects in normoreactive guinea pigs. In spontaneously hyperreactive rhesus monkeys, KCO912, given by inhalation, inhibited methacholine-induced bronchoconstriction (ED50 1.2 microg/kg) but had no significant effects on blood pressure or heart rate at all doses tested (therapeutic ratio >100). In rats given 3 mg/kg of KCO912 by inhalation, the ratio of the area under the concentration-time curve (AUC) for lung to the AUC in blood was 190 and the compound was rapidly cleared (initial t1/2 approximately 30 min). Thus, the wide therapeutic window following administration of KCO912 to the lung seems likely to reflect slow or incomplete passage of KCO912 from the lung into the systemic circulation coupled with rapid removal from the systemic circulation.Thus, when given locally to the airways in both guinea pigs and monkeys, KCO912 suppresses AHR at doses devoid of cardiovascular effects and has a significantly better therapeutic window than representative earlier generation K(ATP) channel openers defined in the same models. Given the pivotal role of AHR in the pathophysiology of asthma and the preclinical profile of KCO912, this compound was selected for clinical evaluation.

ATP-dependent potassium channel mediates neuroprotection by chemical preconditioning with 3-nitropropionic acid in gerbil hippocampus

Chemical preconditioning with low dose of 3-nitropropionic acid (3-NPA) prolongs the latency to hypoxic depolarization (HD), which triggers cell death, and also restores the synaptic transmission which disappears during hypoxia in gerbil hippocampal slices. Here we show that these neuroprotective effects are mediated by the activation of K ATP channels. Diazoxide, a K ATP channel opener, prolonged the latency to HD dose-dependently to the same extent as that of the chemical preconditioning with 3-NPA. Glibenclamide, a K ATP channel blocker, abrogated the prolongation of HD with 3-NPA. The hypoxic tolerance of synaptic transmission with 3-NPA was also abolished by glibenclamide. Diazoxide also induced the hypoxic tolerance of synaptic transmission. Theses results suggest that K ATP channel is involved in the neuroprotection afforded by the chemical preconditioning.

Ischemic shortening of action potential duration as a result of KATP channel opening attenuates myocardial stunning by reducing calcium influx.

Action potential duration (APD) shortening due to opening of sarcolemmal ATP-dependent potassium (KATP) channels has been postulated to protect the myocardium against postischemic damage by reducing Ca2+ influx. This hypothesis was assessed, assuming that increased postischemic stunning due to KATP channel inhibition with glibenclamide could be reverted by the addition of the Ca2+ channel blocker diltiazem. Percent wall thickening fraction (%WTh, conscious sheep) and APD (open-chest sheep) were obtained from the following groups: control: 12 min ischemia by anterior descending coronary artery occlusion followed by 2 h reperfusion; glibenclamide: same as control, with glibenclamide (0.4 mg/kg) infused 30 min before ischemia; diltiazem: same as control, with diltiazem (100 microg/kg) administered prior to ischemia; glibenclamide+diltiazem: both drugs infused as in glibenclamide and diltiazem groups. APD was reduced in control ischemia. Conversely, KATP-channel blockade by glibenclamide lengthened APD and increased postischemic stunning (p < 0.01 vs. control); glibenclamide+diltiazem did not shorten APD but enhanced functional recovery (p < 0.01 vs. glibenclamide). Ca2+ channel blockade improvement of increased stunning provoked by KATP channel inhibition supports the hypothesis that APD shortening due to opening of KATP channels protects against postischemic stunning by limiting Ca2+ influx.

Differential Involvement of Guanylate Cyclase and Potassium Channels in Nitric Oxide-Induced Hyporesponsiveness to Phenylephrine in Endotoxemic Rats

This study evaluated the involvement of nitric oxide (NO), guanylate cyclase, and potassium channels in the long-lasting vascular hyporesponsiveness to phenylephrine induced by Escherichia coli lipopolysaccharide (LPS) in vitro and in vivo. Experiments in rat aorta rings with endothelium incubated with LPS (10 microg/mL) for 12 h showed that the hyporesponsiveness depends on guanylate cyclase activity and tetraethylammonium-sensitive, but not voltage- or ATP-dependent, potassium channels. Pressor responses to phenylephrine were reduced by 50% in rats injected 8 and 24 h before with LPS (10 mg/kg, intraperitoneally). Pretreatment with NO synthase inhibitors (iNOS; Nomega-nitro-L-arginine methyl ester [L-NAME], 55 micromol/kg or aminoguanidine, 244 micromol/kg, intraperitoneally) fully prevented LPS-induced hyporesponsiveness. When administered just before phenylephrine, L-NAME (11 micromol/kg, intravenously) reversed the hyporesponsiveness in rats injected 8 h, but not in those injected 24 h before with LPS, whereas 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1 (ODQ, 11 micromol/kg, intravenously) reversed the hyporesponsiveness in animals injected 24 h, but not in those injected 8 h before with LPS. Tetraethylammonium (360 micromol/kg, intravenously) reestablished normal responses to phenylephrine in rats injected 8 and 24 h before with LPS. Again, neither voltage- nor ATP-dependent potassium channels appears to be involved. Western blot showed that iNOS expression peaked at 8 h, decreasing to low levels 24 h after LPS injection. Therefore, NO is important in initiating LPS-induced hyporesponsiveness to vasoconstrictors, but not in maintaining it for long periods. Once NO has exerted its effects and even when iNOS expression is minimal, the long-lasting hyporesponsiveness appears to depend on a complex interplay between guanylate cyclase and potassium channel activation.

Consequences of brief ischemia: stunning, preconditioning, and their clinical implications: part 2.

In experimental studies in the dog, total proximal coronary artery occlusions of up to 15 minutes result in reversible injury, meaning that the myocytes survive this insult. The 15 minutes of ischemia, however, induce numerous changes in the myocardium, including certain monuments to the brief episode of ischemia that may persist for days. One of these monuments is stunned myocardium, which represents "prolonged postischemic contractile dysfunction of myocardium salvaged by reperfusion." The mechanism of stunning involves generation of oxygen radicals as well as alteration in calcium homeostasis and possibly alteration in contractile protein structure. Stunning has been observed in several clinical scenarios, including after percutaneous transluminal coronary angioplasty, unstable angina, stress-induced ischemia, after thrombolysis, and after cardiopulmonary bypass. Oxygen radical scavengers and calcium channel blockers have been shown to enhance function of stunned myocardium in experimental studies, and in a few clinical studies, calcium channel blockers have been shown to ameliorate stunning. Although brief periods of ischemia can contribute to prolonged left ventricular dysfunction and even heart failure, they paradoxically play a cardioprotective role. Episodes of ischemia as short as 5 minutes, followed by reperfusion, protect the heart from a subsequent longer coronary artery occlusion by markedly reducing the amount of necrosis that results from the test episode of ischemia. This phenomenon, called ischemic preconditioning, has been observed in virtually every species in which it has been studied and is a powerful cardioprotective effect. The mechanism of ischemic preconditioning involves both triggers and mediators and involves complex second messenger pathways that appear to involve such components as adenosine, adenosine receptors, the epsilon isoform of protein kinase C, the ATP-dependent potassium channels, as well as others, including a paradoxical protective role of oxygen radicals. Both an early and a late phase of preconditioning have been described, and the mechanisms underlying their induction are under investigation. That preconditioning may occur in humans is suggested by the observations that repetitive balloon inflations in the coronary artery are associated with progressively less chest pain, ST-segment elevation, lactate production, the protective effects of preinfarction angina, the anginal "warm-up phenomenon," and studies performed on human cardiac biopsies that show metabolic properties suggesting preconditioning. Development of pharmacological agents that stimulate second messenger pathways thought to be involved in preconditioning, but without causing ischemia, could result in novel approaches to treating ischemia. Hence, on one hand, brief episodes of ischemia can have a negative effect on the heart: stunning; and on the other hand, they have a protective effect: preconditioning. The future challenge is how to minimize the stunning phenomenon and maximize the preconditioning phenomenon in clinical practice.

Consequences of brief ischemia: stunning, preconditioning, and their clinical implications: part 1.

In experimental studies in the dog, total proximal coronary artery occlusions of up to 15 minutes result in reversible injury, meaning that the myocytes survive this insult. The 15 minutes of ischemia, however, induce numerous changes in the myocardium, including certain monuments to the brief episode of ischemia that may persist for days. One of these monuments is stunned myocardium, which represents "prolonged postischemic contractile dysfunction of myocardium salvaged by reperfusion." The mechanism of stunning involves generation of oxygen radicals as well as alteration in calcium homeostasis and possibly alteration in contractile protein structure. Stunning has been observed in several clinical scenarios, including after percutaneous transluminal coronary angioplasty, unstable angina, stress-induced ischemia, after thrombolysis, and after cardiopulmonary bypass. Oxygen radical scavengers and calcium channel blockers have been shown to enhance function of stunned myocardium in experimental studies, and in a few clinical studies, calcium channel blockers have been shown to ameliorate stunning. Although brief periods of ischemia can contribute to prolonged left ventricular dysfunction and even heart failure, they paradoxically play a cardioprotective role. Episodes of ischemia as short as 5 minutes, followed by reperfusion, protect the heart from a subsequent longer coronary artery occlusion by markedly reducing the amount of necrosis that results from the test episode of ischemia. This phenomenon, called ischemic preconditioning, has been observed in virtually every species in which it has been studied and is a powerful cardioprotective effect. The mechanism of ischemic preconditioning involves both triggers and mediators and involves complex second messenger pathways that appear to involve such components as adenosine, adenosine receptors, the epsilon isoform of protein kinase C, the ATP-dependent potassium channels, as well as others, including a paradoxical protective role of oxygen radicals. Both an early and a late phase of preconditioning have been described, and the mechanisms underlying their induction are under investigation. That preconditioning may occur in humans is suggested by the observations that repetitive balloon inflations in the coronary artery are associated with progressively less chest pain, ST-segment elevation, lactate production, the protective effects of preinfarction angina, the anginal "warm-up phenomenon," and studies performed on human cardiac biopsies that show metabolic properties suggesting preconditioning. Development of pharmacological agents that stimulate second messenger pathways thought to be involved in preconditioning, but without causing ischemia, could result in novel approaches to treating ischemia. Hence, on one hand, brief episodes of ischemia can have a negative effect on the heart: stunning; and on the other hand, they have a protective effect: preconditioning. The future challenge is how to minimize the stunning phenomenon and maximize the preconditioning phenomenon in clinical practice.

Acetylcholine Attenuates Cardiomyocyte Oxidant Stress during Simulated Ischemia and Reoxygenation

We wanted to determine whether oxygen radicals open the mitochondrial ATP-dependent potassium channels (K_ATP) during an ischemic period to reduce cell death and oxidant stress. Chick embryonic cardiomyocytes were used. Cell viability was quantified with propidium iodide (5 µM), and free radicals was measured using 2′,7′-dichlorofluorescin diacetate. Preconditioning was produced by 10 min of simulated ischemia followed by 10 min of reoxygenation. Acetylcholine (1 mM), infused for 10 min instead of preconditioning, reduced cell death similarly (24 ± 5%, n = 7 and 18 ± 2%, n = 7, respectively, vs. controls, 49 ± 6%, n = 8). In control series, 60 min of simulated ischemia and 3 h of reoxygenation generated free radicals more than 300% above the baseline (ischemia: 3.63 ± 0.58, reoxygenation: 3.66 ± 0.47, n = 8). Preconditioning and acetylcholine markedly attenuated the oxidant stress during simulated ischemia (1.18 ± 0.41, n = 6 and 1.34 ± 0.60, n = 7 vs. controls 3.63 ± 0.58, n = 8) and re-oxygenation (1.23 ± 0.36, n = 6 and 1.50 ± 0.59, n = 7 vs. controls 3.66 ± 0.47, n = 8). The protection of acetylcholine was abolished with pretreatment with the antioxidant thiol reductant 2-mercaptopropionyl glycine and posttreatment with 5-hydroxydecanoate, a selective mitochondrial K_ATP channel antagonist (37 ± 7%, n = 7). These results demonstrate that oxygen radicals open mitochondrial K_ATP channels, which mediates the acetylcholine-induced preconditioning effect, and that stimulation of this signaling pathway attenuates oxidant stress.

Brain glucose-sensing mechanisms: ubiquitous silencing by aglycemia vs. hypothalamic neuroendocrine responses.

Interest in brain glucose-sensing mechanisms is motivated by two distinct neuronal responses to changes in glucose concentrations. One mechanism is global and ubiquitous in response to profound hypoglycemia, whereas the other mechanism is largely confined to specific hypothalamic neurons that respond to changes in glucose concentrations in the physiological range. Although both mechanisms use intracellular metabolism as an indicator of extracellular glucose concentration, the two mechanisms differ in key respects. Global hyperpolarization (inhibition) in response to 0 mM glucose can be reversed by pyruvate, implying that the reduction in ATP levels acting through ATP-dependent potassium (K-ATP) channels is the key metabolic signal for the global silencing in response to 0 mM glucose. In contrast, neuroendocrine hypothalamic responses in glucoresponsive and glucose-sensitive neurons (either excitation or inhibition, respectively) to physiological changes in glucose concentration appear to depend on glucokinase; neuroendocrine responses also depend on K-ATP channels, although the role of ATP itself is less clear. Lactate can substitute for glucose to produce these neuroendocrine effects, but pyruvate cannot, implying that NADH (possibly leading to anaplerotic production of malonyl-CoA) is a key metabolic signal for effects of glucose on glucoresponsive and glucose-sensitive hypothalamic neurons.

Sulfonylurea Drugs Do Not Influence Initial Stroke Severity and In-Hospital Outcome in Stroke Patients With Diabetes

Sulfonylurea drugs inhibit ATP-dependent potassium channels and may increase mortality after myocardial infarction. Sulfonylurea drugs also inhibit ischemic preconditioning in experimental models of brain ischemia and in clinical studies in the human heart. In the present study we examined the impact of sulfonylurea drugs on in-hospital mortality and the immediate neurological deficit of diabetic stroke patients. From a larger stroke data bank, we studied 146 diabetic patients with acute hemispheric ischemic stroke. Sixty patients were using sulfonylurea drugs. Major baseline characteristics such as age, blood pressure, admission glucose level, HbA(1c), distribution of cardiovascular risk factors, and presumed stroke etiology (Trial of Org 10172 in Acute Stroke Treatment [TOAST] criteria) were not different. Mortality (15% versus 14%; P=0.86) and initial stroke severity (Canadian Neurological Scale score, 7.4 versus 7.5; P=0.79) were not significantly different between patients with and without sulfonylurea drugs. Further end points such as Rankin Scale score, deteriorating stroke, duration of hospital stay, type of infarcts on CT/MRI, requirement of intensive care, and complications were not different. In a stepwise logistic regression model, sulfonylurea drugs were not independent predictors for increased mortality, deteriorating stroke, or stroke severity. In the present hospital-based study, sulfonylurea drugs in patients with diabetes and stroke are not associated with increased stroke severity, mortality, or a worse in-hospital outcome.

Control of subcellular localization and function of inwardly rectifying K channels by PDZ-anchoring proteins

The control of subcellular localization is an essential mechanism for ion channels to play specific functional roles in various polarized cells, such as neurons and epithelial cells. We found that anchoring proteins that contain PDZ domains such as PSD-95/SAP90 and SAP97 play critical roles in the control of subcellular localization and function of some of inwardly rectifying K (Kir) channels. Kir4.1/KAB-2, cloned by our group, was shown to be predominantly expressed in glial cells in the brain and retina1.Takumi T. Ishii T. Horio Y. et al.A novel ATP-dependent inward rectifier potassium channel expressed predominantly in glial cells.J Biol Chem. 1995; 270: 16339-16346Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 2.Horio Y. Hibino H. Inanobe A. et al.Clustering and enhanced activity of an inwardly rectifying potassium channel, Kir4.1, by an anchoring protein, PSD-95/SAP 90 family.J Biol Chem. 1997; 272: 12885-12888Crossref PubMed Scopus (121) Google Scholar, 3.Ishii M. Horio Y. Tada Y. et al.Expression and clustered distribution of an inwardly rectifying potassium channel, KAB-2/Kir4.1, on mammalian retinal Müller cell membrane: their regulation by insulin and laminin signals.J Neurosci. 1997; 17: 7725-7735Crossref PubMed Google Scholar, 4.Nagelhus E.A. Horio Y. Inanobe A. et al.Subcellular coexpression of the inwardly rectifying K+ channel Kir4.1 and water aquaporin-4 in retinal Müller cells suggests coupling between K+ siphoning and water transport.Glia. 1999; 26: 47-54Crossref PubMed Scopus (388) Google Scholar. We found that (1) Kir4.1 is distributed in a clustered manner on the retinal Mueller cell membrane; (2) the clustering distribution is probably controlled by the interaction between Kir4.1 and SAP97, a PSD anchoring protein; (3) the C-terminal amino acids, SNV, in Kir4.1 may be critical for the protein-protein interaction; (4) laminin and insulin signals are essential for maintaining the clustered distribution of Kir4.1, which involves tyrosine kinase activity; (5) aquaporin 4 (AQP4), whose amino acids at the C-terminal end are LSSV, is co-localized with Kir4.1 on retinal Mueller cells, which seems to be controlled by associated proteins other than SAP97; and (6) SAP97 enhances the channel activity of Kir4.1 in a heterologous expression system. G protein-gated K (KG) channels generate slow inhibitory postsynaptic potentials in the brain5.Yamada M. Inanobe A. Kurachi Y. G protein regulation of potassium ion channels.Pharmacol Rev. 1998; 50: 723-757PubMed Google Scholar. Current opinion suggests that neuronal KG channels are heterotetramers of Kir3.1 and Kir3.2. Kir3.2 has at least four splicing variants named Kir3.2a-Kir3.2d. We found that the KG channels in substantia nigra (SN) are an assembly of Kir3.2a and Kir3.2c6.Inanobe A. Yoshimoto Y. Horio Y. et al.Characterization of G protein-gated K+ channels composed of Kir3.2 subunits in substantia nigra.J Neuroscience. 1999; 19: 1006-1017PubMed Google Scholar. Kir3.2c is 11 amino acids longer than Kir3.2a, which is the only divergent part between the two clones. The C-terminal amino acids of Kir3.2c are ESKV. The channels were localized specifically at the postsynaptic membrane on the dendrites of dopaminergic neurons. Kir3.2c, but not Kir3.2a, could bind a PDZ domain-containing protein, PSD-95. The heterologously expressed KG channels composed of Kir3.2a and Kir3.2c or Kir3.2a alone were activated by G-protein stimulation, but expression of Kir3.2c alone was not. However, the anchoring proteins confer the G-protein sensitivity to the homomeric Kir3.2c channel through their GK domain7.Hibino H. Inanobe A. Tanemoto M. et al.Anchoring proteins confer G protein sensitivity to an inwardly rectifying K+ channel through the GK domain.EMBO J. 2000; 19: 78-83Crossref PubMed Scopus (47) Google Scholar. Therefore, not only subcellular localization, but also the pore function of some Kir channels are critically controlled by PDZ domain-containing anchoring proteins in vivo.

Calcineurin, Mitochondrial Membrane Potential, and Cardiomyocyte Apoptosis

Mitochondria comprise ≈30% of the total intracellular volume within a mammalian cardiomyocyte.1 2 Not surprisingly, subtle alterations in mitochondrial function or membrane potential can have a dramatic influence on cardiomyocyte energy production and, ultimately, the health of an individual cell. Indeed, cellular injury or stress stimulation directly elicits alterations in mitochondrial architecture, membrane potential, and oxidative capacity, which are associated with an irreversible loss of mitochondrial matrix contents and integral membrane protein constituents such as cytochrome c oxidase.3 The release of cytochrome c and/or mitochondrial permeability transition directly mediates cellular apoptosis through calcium-sensitive proteases or through coupling proteins that coordinate the activation of caspases and DNA fragmentation enzymes.3 Given the high intracellular content of mitochondria in cardiomyocytes and the unabated requirement for high-energy phosphate carriers to maintain ionic gradients and active force generation, coordinated disturbances in mitochondrial function can dramatically affect cell survival. Recent investigation has suggested an emerging paradigm whereby stress-responsive intracellular signaling pathways directly and indirectly influence mitochondrial membrane potential, oxidative capacity, and the coupling of apoptosis initiating factors. For example, stress-responsive signaling through the c-Jun N-terminal kinases (JNKs) has been shown to initiate apoptosis in certain cell types by directly influencing proteins within the mitochondrial membranes.4 In addition, increased expression of the mitochondrial stabilizing Bcl proteins can be transcriptionally regulated through stress-responsive signaling pathways, which subsequently antagonize mitochondrial dysfunction and cytochrome c release.5 Activation of the intracellular kinase Akt also directly antagonizes mitochondrial-directed apoptosis by phosphorylating the mitochondrial destabilizing protein Bad, reducing cytochrome c release and caspase activation.6 Lastly, activation of nuclear factor-κB (NF-κB) signaling induces expression of stress-adaptive proteins such as Bcl-2, antioxidants, and calcium-regulating proteins.7 In general, signaling pathways such as the stress- and mitogen-activated protein kinases (SAPKs and MAPKs), protein kinase C (PKC), Akt, NF-κB, and the …

Action potential shortening due to opening of ATP-dependent potassium channels attenuates post-ischemic stunning by reducing calcium influx in conscious sheep

Action potential shortening due to opening of ATP-dependent potassium channels attenuates post-ischemic stunning by reducing calcium influx in conscious sheep

Ischemia tolerance; model for research, hope for clinical practice?

A brief episode of ischemia renders the brain resistant against subsequent, longer ischemic events. This ischemic tolerance has been shown in numerous experimental models of cerebral ischemia. After global cerebral ischemia, ischemic tolerance may protect up to 90% of hippocampal CA1 neurons. In focal ischemia, this phenomenon reduces infarct volume by 20-60%. However, the basic molecular mechanisms of ischemic tolerance are largely unknown. During the induction phase, N-methyl-d-aspartate (NMDA) and adenosine receptors and, possibly, oxygen free radicals and conservation of energy metabolism are required. Protein kinases, transcription factors, and immediate early genes appear to transduce the signal into a tolerant response. Ischemic tolerance can be observed in different phases. The early phase lasts for several hours after the preconditioning stimulus and adenosine receptors and ATP-dependent potassium channels play a role similar to that in cardiac ischemic tolerance. The delayed protection, retained for a maximum of 2-4 days, currently is best explained by genetic remodeling with expression or repression of multiple genes. Several candidates have been identified to date, among them heat-shock proteins, cytokines, and antioxidant enzymes. Several studies have shown that angina pectoris before myocardial infarction represents a clinical correlate of experimental preconditioning protocols. Accordingly, evidence for a possible protective effect of transient ischemic attacks (TIAs) occurring before stroke are accumulating.

Ascorbic acid-induced modulation of venous tone in humans.

Ascorbic acid appears to have vasodilatory properties, but the underlying mechanisms are not well understood. The aims of this study were to define the acute effects of locally infused ascorbic acid in human veins and to explore underlying mechanisms by using pharmacological tools in vivo. Ascorbic acid was infused in dorsal hand veins submaximally preconstricted with the alpha(1)-adrenoceptor agonist phenylephrine or with prostaglandin F(2alpha) in 23 healthy male nonsmokers, and the venodilator response was measured. Ascorbic acid produced dose-dependent dilation with maximum reversal of constriction of 38+/-4% in phenylephrine-preconstricted veins and of 51+/-13% in prostaglandin F(2alpha)-preconstricted veins. Oral pretreatment with the cyclooxygenase inhibitor acetylsalicylic acid or local coinfusion of ascorbic acid and the nitric oxide synthase inhibitor N:(G)-monomethyl-L-arginine had no effect, but coinfusion of ascorbic acid and methylene blue (to inhibit cGMP generation) abolished venodilation. Coinfusion of ascorbic acid and the nonselective potassium channel blocker quinidine abolished venodilation, whereas the inhibitor of ATP-dependent potassium channels glibenclamide had no effect. In cultured bovine endothelial cells, ascorbic acid did not affect intracellular calcium concentration but blunted the response to ATP or digitonin exposure. Ascorbic acid, in millimolar concentrations, dilates human hand veins, presumably by activation of vascular smooth muscle potassium channels through cGMP. This activation is independent of eNOS-mediated nitric oxide synthesis and cyclooxygenase products and does not involve ATP-dependent potassium channels.

Alterations in potassium channel gene expression in atria of patients with persistent and paroxysmal atrial fibrillation: differential regulation of protein and mRNA levels for K+channels

OBJECTIVESOur purpose was to determine whether patients with persistent atrial fibrillation (AF) and patients with paroxysmal AF show alterations in potassium channel expression.BACKGROUNDPersistent AF is associated with a sustained shortening of the atrial action potential duration and atrial refractory period. Underlying molecular changes have not been studied in humans. We investigated whether a changed gene expression of specific potassium channels is associated with these changes in patients with persistent AF and in patients with paroxysmal AF.METHODSRight atrial appendages were obtained from 8 patients with paroxysmal AF, 10 with persistent AF and 18 matched controls in sinus rhythm. All controls underwent coronary artery bypass surgery, whereas most AF patients underwent Cox’s MAZE surgery (atrial arrhythmia surgery to cure AF) (n = 12). All patients had normal left ventricular function. mRNA (ribonucleic acid) levels were measured by semiquantitative polymerase chain reaction and protein content by Western blotting.RESULTSmRNA levels of transient outward channel (Kv4.3), acetylcholine-dependent potassium channel (Kir3.4) and ATP-dependent potassium channel (Kir6.2) were reduced in patients with persistent AF (−35%, −47% and −36%, respectively, p < 0.05), whereas only Kv4.3 mRNA level was reduced in patients with paroxysmal AF (−29%, p = 0.03). No changes were found for Kv1.5 and HERG mRNA levels in either group. Protein levels of Kv4.3, Kv1.5 and Kir3.1 were reduced both in patients with persistent AF (−39%, −84% and −47%, respectively, p < 0.05) and in those with paroxysmal AF (−57%, −64%, and −40%, respectively, p < 0.05).CONCLUSIONSPersistent AF is accompanied by reductions in mRNA and protein levels of several potassium channels. In patients with paroxysmal AF these reductions were observed predominantly at the protein level and not at the mRNA level, suggesting a post-transcriptional regulation.