Cardioprotective effect of cromakalim (potassium channel opener) in isoproterenol induced myocardial infarction in rats.

Animals pretreated with cromakalim (1 mg/kg,po) along with isoproterenol (85 mg/kg,sc) showed less myocardial degenerative changes on histopathological examinations when compared with those treated with isoproterenol alone. Cromakalim's beneficial effects on myocardium were in dose-dependent manner. Administration of cromakalim (po) lowered significantly the serum LDH and SGOT and depleted intracytoplasmic glycogen as demonstrated by periodic schiff staining procedure. Increase in blood clotting time was highly significant (P less than 0.001). The results suggest cardioprotective effect of cromakalim in isoproterenol induced myocardial infarction.     

ATP敏感性钾通道开放剂埃他卡林对大鼠低氧性肺动脉高压的影响

目的 :探讨新型ATP敏感性钾通道开放剂 (KATPCO)埃他卡林 (iptkalim ,Ipt)对低氧性肺动脉高压 (HPH)大鼠肺血管重构的影响。方法 :将大鼠置于常压低氧舱内 (O2 1 0 %± 0 .5 % ) ,8h/d ,每周 6d ,4周后测定平均肺动脉压(mPAP)、RV/ (LV +S) ;用图象分析仪测量与呼吸性细支气管伴行的肺小动脉外径 (ED)、动脉中层壁厚 (MT)、动脉管壁中层面积 (MA)、动脉管腔面积 (VA)和血管总面积 (TAA)。结果 :慢性低氧组大鼠的mPAP和RV/ (LV +S)显著高于正常对照组 (P <0 .0 1 ) ;图象分析显示低氧组大鼠肺小动脉中层壁厚与动脉外径百分比 (MT % )、动脉壁中层面积与血管总面积百分比 (MA % )均显著高于对照组 (P <0 .0 1 ) ;慢性低氧组大鼠肺小动脉管腔面积 (VA)与血管总面积 (TAA)百分比显著低于正常组 (P <0 .0 1 )。Ipt 0 .75mg·kg- 1 ·d- 1 和 1 .50mg·kg- 1 ·d- 1 均可显著抑制低氧性肺血管壁重构 ,降低肺动脉压 ,减少右心室肥厚 ,1 .50mg·kg- 1 ·d- 1 则可逆转持续低氧所致的所有病理性变化。结论 :新型KATPCO埃他卡林是一个富有潜力的治疗HPH药物     

Cardioprotective effects of stretch are mediated by activation of sarcolemmal, not mitochondrial, ATP-sensitive potassium channels

To determine whether sarcolemmal and/or mitochondrial ATP-sensitive potassium (K(ATP)) channels (sarcK(ATP), mitoK(ATP)) are involved in stretch-induced protection, isolated isovolumic rat hearts were assigned to the following protocols: nonstretched hearts were subjected to 20 min of global ischemia (Is) and 30 min of reperfusion, and before Is stretched hearts received 5 min of stretch + 10 min of no intervention. Stretch was induced by a transient increase in left ventricular end-diastolic pressure (LVEDP) from 10 to 40 mmHg. Other hearts received 5-hydroxydecanoate (5-HD; 100 microM), a selective inhibitor of mitoK(ATP), or HMR-1098 (20 microM), a selective inhibitor of sarcK(ATP), before the stretch protocol. Systolic function was assessed through left ventricular developed pressure (LVDP) and maximal rise in velocity of left ventricular pressure (+dP/dt(max)) and diastolic function through maximal decrease in velocity of left ventricular pressure (-dP/dt(max)) and LVEDP. Lactate dehydrogenase (LDH) release and ATP content were also measured. Stretch resulted in a significant increase of postischemic recovery and attenuation of diastolic stiffness. At 30 min of reperfusion LVDP and +dP/dt(max) were 87 +/- 4% and 92 +/- 6% and -dP/dt(max) and LVEDP were 95 +/- 9% and 10 +/- 4 mmHg vs. 57 +/- 6%, 53 +/- 6%, 57 +/- 10%, and 28 +/- 5 mmHg, respectively, in nonstretched hearts. Stretch increased ATP content and did not produce LDH release. 5-HD did not modify and HMR-1098 prevented the protection achieved by stretch. Our results show that the beneficial effects of stretch on postischemic myocardial dysfunction, cellular damage, and energetic state involve the participation of sarcK(ATP) but not mitoK(ATP).     

Regulation of the mitochondrial ATP-sensitive potassium channel in rat uterus cells by ROS

In previous study we demonstrated the presence of ATP-sensitive potassium current in the inner mitochondrial membrane, which was sensitive to diazoxide and glybenclamide, in mitochondria isolated from the rat uterus. This current was supposed to be operated by mitochondrial ATP-sensitive potassium channel (mitoK(ATP)). Regulation of the mitoK(ATP) in uterus cells is not studied well enough yet. It is well known that the reactive oxygen species (ROS) can play a dual role. They can damage cells in high concentrations, but they can also act as messengers in cellular signaling, mediating survival of cells under stress conditions. ROS are known to activate mitoK(ATP) during the oxidative stress in the brain and heart, conferring the protection of cells. The present study examined whether ROS mediate the mitoK(ATP) activation in myometrium cells. Oxidative stress was induced by rotenone. ROS generation was measured by 2',7'-dichlorofluorescin diacetate. The massive induction of ROS production was demonstrated in the presence of rotenone. Hyperpolarization of the mitochondrial membrane was also detected with the use of the potential-sensitive dye DiOC6 (3,3'-dihexyloxacarbocyanine iodide). Diazoxide, a selective activator of mitoK(ATP), depolarized mitochondrial membrane either under oxidative stress or under normal conditions, while mitoK(ATP) blocker glybenclamide effectively restored mitochondrial potential in rat myocytes. Estimated value for diazoxide to mitoK(ATP) under normoxia was four times higher than under oxidative stress conditions: 5.01 +/- 1.47-10(-6) M and 1.24 +/- 0.21 x 10(-6) M respectively. The ROS scavenger N-acetylcysteine (NAC) successfully eliminates depolarization of mitochondrial membrane by diazoxide under oxidative stress. These results suggest that elimination of ROS by NAC prevents the activation of mitoK(ATP) under oxidative stress. Taking into account the higher affinity of diazoxide to mitoK(ATP) under stress conditions than under normoxia, we conclude that the oxidative stress conditions are more favourable than normoxia for the activation of mitoK(ATP). Thus we hypothesize that the ROS regulate the activity of the mitoK(ATP) in myocytes.     

Role of sarcolemmal ATP-sensitive potassium channel in oxidative stress-induced apoptosis: mitochondrial connection

From time of their discovery, sarcolemmal ATP-sensitive K+ (sarcK ATP) channels were thought to have an important protective role in the heart during stress whereby channel opening protects the heart from stress-induced Ca2+ overload and resulting damage. In contrast, some recent studies indicate that sarcK ATP channel closing can lead to cardiac protection. Also, the role of the sarcK ATP channel in apoptotic cell death is unclear. In the present study, the effects of channel inhibition on apoptosis and the specific interaction between the sarcK ATP channel and mitochondria were investigated. Apoptotic cell death of cultured HL-1 and neonatal cardiomyocytes following exposure to oxidative stress was significantly increased in the presence of sarcK ATP channel inhibitor HMR-1098 as evidenced by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and caspase-3,7 assays. This was paralleled by an increased release of cytochrome c from mitochondria to cytosol, suggesting activation of the mitochondrial death pathway. sarcK ATP channel inhibition during stress had no effect on Bcl-2, Bad, and phospho-Bad, indicating that the increase in apoptosis cannot be attributed to these modulators of the apoptotic pathway. However, monitoring of mitochondrial Ca2+ with rhod-2 fluorescent indicator revealed that mitochondrial Ca2+ accumulation during stress is potentiated in the presence of HMR-1098. In conclusion, this study provides novel evidence that opening of sarcK ATP channels, through a specific Ca2+-related interaction with mitochondria, plays an important role in preventing cardiomyocyte apoptosis and mitochondrial damage during stress.     

Identification and properties of a novel intracellular (mitochondrial) ATP-sensitive potassium channel in brain.

Protection of heart against ischemia-reperfusion injury by ischemic preconditioning and K(ATP) channel openers is known to involve the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)). Brain is also protected by ischemic preconditioning and K(ATP) channel openers, and it has been suggested that mitoK(ATP) may also play a key role in brain protection. However, it is not known whether mitoK(ATP) exists in brain mitochondria, and, if so, whether its properties are similar to or different from those of heart mitoK(ATP). We report partial purification and reconstitution of a new mitoK(ATP) from rat brain mitochondria. We measured K(+) flux in proteoliposomes and found that brain mitoK(ATP) is regulated by the same ligands as those that regulate mitoK(ATP) from heart and liver. We also examined the effects of opening and closing mitoK(ATP) on brain mitochondrial respiration, and we estimated the amount of mitoK(ATP) by means of green fluorescence probe BODIPY-FL-glyburide labeling of the sulfonylurea receptor of mitoK(ATP) from brain and liver. Three independent methods indicate that brain mitochondria contain six to seven times more mitoK(ATP) per milligram of mitochondrial protein than liver or heart.     

ATP-sensitive potassium channel opener iptakalim protected against the cytotoxicity of MPP+ on SH-SY5Y cells by decreasing extracellular glutamate level

Mounting evidence reveals that ATP-sensitive potassium (K(ATP)) channel openers (KCOs) exert significant neuroprotection in vivo and in vitro in several models of Parkinson's disease (PD). However, the mechanisms are not well understood. In this study, we demonstrated that SH-SY5Y cells expressed mRNA and proteins for Kir6.1, Kir6.2, SUR1 and SUR2 subunits of K(ATP) channels. Moreover, our results showed that 1-methyl-4-phenyl-pyridinium ion (MPP+) induced up-regulation of mRNA for the Kir6.2 subunit and down-regulation of SUR1. It was further found that pretreatment with iptakalim, a novel K(ATP) channel opener, could attenuate increased extracellular glutamate level and decreased cell survival in SH-SY5Y cell culture after exposure to MPP+. Trans-pyrrolidine-2, 4-dicarboxylic acid (t-PDC), a glutamate transporter inhibitor, partially blocked the effect of iptakalim decreasing extracellular glutamate level. Additionally, iptakalim prevented MPP+-induced inhibition of glutamate uptake in primary cultured astrocytes. The beneficial effects of iptakalim on glutamate uptake of astrocytes were abolished by selective mitochondrial K(ATP) (mitoK(ATP)) channel blocker 5-HD. These results suggest (i) K(ATP) channel dysfunction may be involved in the mechanisms of MPP+-induced cytotoxicity and (ii) iptakalim may modulate glutamate transporters and subsequently alleviate the increase of extracellular glutamate levels induced by MPP+ through opening mitoK(ATP) channels, thereby protecting SH-SY5Y cells against MPP+-induced cytotoxicity.     

The mitochondrial complex II and ATP-sensitive potassium channel interaction: quantitation of the channel in heart mitochondria

The mitochondrial ATP-sensitive potassium channel (mK(ATP)) is important in cardioprotection, although the channel remains molecularly undefined. Several studies have demonstrated that mitochondrial complex II inhibitors activate the mK(ATP), suggesting a potential role for complex II in channel composition or regulation. However, these inhibitors activate mK(ATP) at concentrations which do not affect bulk complex II activity. Using the potent complex II inhibitor Atpenin A5, this relationship was investigated using tight-binding inhibitor theory, to demonstrate that only 0.4 % of total complex II molecules are necessary to activate the mK(ATP). These results estimate the mK(ATP) content at 15 channels per mitochondrion.     

Reconstitution of the ATP-sensitive potassium channel of skeletal muscle. Activation by a G protein-dependent process

Potassium channels inhibited by adenosine-5'-trisphosphate, K(ATP), found in the transverse tubular membrane of rabbit skeletal muscle were studied using the planar bilayer recording technique. In addition to the single-channel properties of K(ATP) we report its regulation of Mg2+ and by the guanosine-5'-trisphosphate analogue, GTP-y(gamma)-S. The K(ATP) channel (a) has a conductance of 67 pS in 250 mM internal, 50 mM external KCl, and rectifies weakly at holding potentials more positive than 50 mV, (b) is not activated by internal Ca2+ or membrane depolarization, (c) has a permeability ratio PK/PNa greater than 50, and (d) is inhibited by millimolar internal ATP. Activity of K(ATP), measured as open channel probability as a function of time, was unstable at all holding potentials and decreases continuously within a few minutes after a recording is initiated. After a decrease in activity, GTP-y-S (100 microM) added to the internal side reactivated K(ATP) channels but only transiently. In the presence of internal 1 mM Mg2+, GTP-y-S produced a sustained reactivation lasting 20-45 min. Incubation of purified t-tubule vesicles with AlF4 increased the activity of K(ATP) channels, mimicking the effect of GTP-y-S. The effect of AlF4 and the requirement of GTP-y-S plus Mg2+ for sustained channel activation suggests that a nucleotide-binding G protein regulates ATP-sensitive K channels in the t-tuble membrane of rabbit skeletal muscle.     

Characterization of human cardiac mitochondrial ATP-sensitive potassium channel and its regulation by phorbol ester in vitro

Activation of the mitochondrial ATP-sensitive K+ channel (mitoKATP) and its regulation by PKC are critical events in preconditioning induced by ischemia or pharmaceutical agents in animals and humans. The properties of the human cardiac mitoKATP channel are unknown. Furthermore, there is no evidence that cytosolic PKC can directly regulate the mitoKATP channel located in the inner mitochondrial membrane (IMM) due to the physical barrier of the outer mitochondrial membrane. In the present study, we characterized the human cardiac mitoKATP channel and its potential regulation by PKC associated with the IMM. IMM fractions isolated from human left ventricles were fused into lipid bilayers in symmetrical potassium glutamate (150 mM). The conductance of native mitoKATP channels was usually below 80 pS ( approximately 70%), which was reduced by ATP and 5-hydroxydecanoic acid (5-HD) in a dose- and time-dependent manner. The native mitoKATP channel is activated by diazoxide and inhibited by ATP and 5-HD. The PKC activator phorbol 12-myristate 13-acetate (2 microM) increased the cumulative open probability of the mitoKATP channel previously inhibited by ATP (P < 0.05), but its inactive analog 4alpha-phorbol 12,13-didecanoate had no effect. Western blot analysis detected an inward rectifying K+ channel (Kir6.2) immunoreactive protein at 56 kDa and PKC-delta in the IMM. These data provide the first characterization of the human cardiac mitoKATP channel and its regulation by PKC(s) in IMM. This local PKC control mechanism may represent an alternative pathway to that proposed previously for cytosolic PKC during ischemic/pharmacological preconditioning.     

Role of ATP-sensitive potassium channel activators in liver mitochondrial function in rats with different resistance to hypoxia

Effects of ATP-sensitive potassium (KATP) channels opener pinacidil (0.06 mg/kg) and inhibitor glibenclamide (1 mg/kg) in rats with different resistance to hypoxia on indices of ADP-stimulation of mitochondrial respiration by Chance, calcium capacity and processes of lipid peroxidation in liver has been investigated. We used next substrates of oxidation: 0.35 mM succinate, 1 mM alpha-ketoglutarate. Additional analyses contain the next inhibitors: mitochondrial fermentative complex I-10 mkM rotenone, succinate dehydrogenase 2 mM malonic acid. It was shown that effects of pinacidil induced the increasing of oxidative phosporylation efficacy and ATP synthesis together with lowering of calcium capacity in rats with low resistance to hypoxia. Effects of pinacidil were leveled by glibenclamide. These changes are connected with the increasing of respiratory rate, calcium overload and intensification of lipid peroxidation processes. A conclusion was made about protective effect of pinacidil on mitochondrial functioning by economization of oxygen-dependent processes, adaptive potentialities of organisms with low resistance to hypoxia being increased.     

Comparison of effects of a potassium channel opener BRL34915, a specific potassium ionophore valinomycin and calcium channel blockers on endothelin-induced vascular contraction

The effects of a potassium (K+) channel opener BRL34915 and a specific K+ ionophore valinomycin on vasoconstriction induced by endothelin (ET) were compared with those of calcium (Ca2+) channel blockers, nicardipine and verapamil, using helical strips from rat thoracic aorta. ET induced potent and persistent contraction in control solution and similar but smaller contraction in Ca2+-free solution. BRL34915 and valinomycin inhibited the ET-induced contraction dose-dependently in control solution, but not in Ca2+-free solution. The ET-induced contraction was also inhibited by nicardipine and verapamil, though less strongly. On the other hand, high K+ (35 mM)-induced vasoconstriction was strongly inhibited by nicardipine and verapamil, but not by BRL34915 or valinomycin. These results support the idea that the extracellular Ca2+-dependent component of the ET-induced contraction may be mediated by Ca2+ influx by a route other than voltage-dependent Ca2+-channels.     

MCC-134, a blocker of mitochondrial and opener of sarcolemmal ATP-sensitive K+ channels, abrogates cardioprotective effects of chronic hypoxia

We examined the effect of MCC-134, a novel inhibitor of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels and activator of sarcolemmal ATP-sensitive K(+) (sarcK(ATP)) channels, on cardioprotection conferred by adaptation to chronic hypoxia. Adult male Wistar rats were exposed to intermittent hypobaric hypoxia (7000 m, 8 h/day, 5-6 weeks) and susceptibility of their hearts to ventricular arrhythmias and myocardial infarction was evaluated in anesthetized open-chest animals subjected to 20-min coronary artery occlusion and 3-h reperfusion on the day after the last hypoxic exposure. MCC-134 was administered intravenously 10 min before ischemia and 5 min before reperfusion in a total dose of 0.3 mg/kg or 3 mg/kg divided into two equal boluses. The infarct size (tetrazolium staining) was reduced from 59.2+/-4.4 % of the area at risk in normoxic controls to 43.2+/-3.3 % in the chronically hypoxic group. Chronic hypoxia decreased the reperfusion arrhythmia score from 2.4+/-0.5 in normoxic animals to 0.7+/-0.5. Both doses of MCC-134 completely abolished the antiarrhythmic protection (score 2.4+/-0.7 and 2.5+/-0.5, respectively) but only the high dose blocked the infarct size-limiting effect of chronic hypoxia (54.2+/-3.7 %). MCC-134 had no effect in the normoxic group. These results support the view that the opening of mitoKATP channels but not sarcKATP channels plays a crucial role in the mechanism by which chronic hypoxia improves cardiac tolerance to ischemia/reperfusion injury.     

Cardioselective sulfonylthiourea HMR 1098 blocks mitochondrial uncoupling induced by a KATP channel opener,P-1075, in beating rat hearts

We investigated effects of blockade of cardiac ATP-sensitive potassium channels (KATP) with a novel cardioselective sulfonylthiourea, HMR 1098, on metabolic uncoupling caused by a potent KATP opener, P-1075, in Langendorff-perfused rat hearts. We used (1) 87Rb-NMR to detect activation-deactivation of sarcolemmal KATP, (2) 31P-NMR to monitor high-energy phosphates, (3) oxygen uptake measurements to monitor cellular respiration, and (4) myocardial optical absorbance measurements at 603 nm to follow changes in cytochrome c oxidase redox state. Activation of sarcolemmal KATP by P-1075 (5 microM) and a mitochondrial uncoupler 2,4-dinitrophenol (DNP) (50 microM) stimulated Rb+ efflux from the hearts by 130% and 60%, respectively. HMR 1098 (5 and 30 microM) blocked activation of sarcolemmal KATP in situ. HMR 1098 also prevented cardiac arrest and mitochondrial uncoupling induced by P-1075, such as (a) depletion of phosphocreatine and ATP by 40%, (b) two-fold decrease in venous oxygen, and (c) reduction of cytochrome c oxidase (demonstrated by an increase in 603 nm optical absorbance). The metabolic effects of P-1075 can be readily explained by activation of putative mitochondrial KATP. We concluded that blockade of mitochondrial uncoupling by HMR 1098 included an inhibiting effect of HMR 1098 on sarcolemmal and mitochondrial KATP in beating rat hearts.     

Regulation of the ATP-sensitive potassium channel subunit, Kir6.2, by a Ca2+-dependent protein kinase C

The activity of ATP-sensitive potassium (K(ATP)) channels is governed by the concentration of intracellular ATP and ADP and is thus responsive to the metabolic status of the cell. Phosphorylation of K(ATP) channels by protein kinase A (PKA) or protein kinase C (PKC) results in the modulation of channel activity and is particularly important in regulating smooth muscle tone. At the molecular level the smooth muscle channel is composed of a sulfonylurea subunit (SUR2B) and a pore-forming subunit Kir6.1 and/or Kir6.2. Previously, Kir6.1/SUR2B channels have been shown to be inhibited by PKC, and Kir6.2/SUR2B channels have been shown to be activated or have no response to PKC. In this study we have examined the modulation of channel complexes formed of the inward rectifier subunit, Kir6.2, and the sulfonylurea subunit, SUR2B. Using a combination of biochemical and electrophysiological techniques we show that this complex can be inhibited by protein kinase C in a Ca(2+)-dependent manner and that this inhibition is likely to be as a result of internalization. We identify a residue in the distal C terminus of Kir6.2 (Ser-372) whose phosphorylation leads to down-regulation of the channel complex. This inhibitory effect is distinct from activation which is seen with low levels of channel activity.     

Diversity in cardiac sodium channel disease phenotype in transgenic mice carrying a single SCN5A mutation

Lethal ventricular arrhythmias are increasingly considered an important cause of sudden death in relatively young individuals. A genetic predisposition has been recognised in many cases, and research in the last decade has focused on underlying inherited mutations in cardiac ion channels.     

G protein control of potassium channel activity in a mast cell line

Using the patch-clamp technique, we studied regulation of potassium channels by G protein activators in the histamine-secreting rat basophilic leukemia (RBL-2H3) cell line. These cells normally express inward rectifier K+ channels, with a macroscopic whole-cell conductance in normal Ringer ranging from 1 to 16 nS/cell. This conductance is stabilized by including ATP or GTP in the pipette solution. Intracellular dialysis with any of three different activators of G proteins (GTP gamma S, GppNHp, or AlF-4) completely inhibited the inward rectifier K+ conductance with a half-time for decline averaging approximately 300 s after "break-in" to achieve whole-cell recording. In addition, with a half-time averaging approximately 200 s, G protein activators induced the appearance of a novel time-independent outwardly rectifying K+ conductance, which reached a maximum of 1-14 nS. The induced K+ channels are distinct from inward rectifier channels, having a smaller single-channel conductance of approximately 8 pS in symmetrical 160 mM K+, and being more sensitive to block by quinidine, but less sensitive to block by Ba2+. The induced K+ channels were also highly permeable to Rb+ but not to Na+ or Cs+. The current was not activated by the second messengers Ca2+, inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, or by cyclic AMP-dependent phosphorylation. Pretreatment of cells with pertussis toxin (0.1 microgram/ml for 12-13 h) prevented this current's induction both by guanine nucleotides and aluminum fluoride, but had no effect on the decrease in inward rectifier conductance. Since GTP gamma S is known to stimulate secretion from patch-clamped rat peritoneal mast cells, it is conceivable that K+ channels become inserted into the plasma membrane from secretory granules. However, total membrane capacitance remained nearly constant during appearance of the K+ channels, suggesting that secretion induced by GTP gamma S was minimal. Furthermore, pertussis toxin had no effect on secretion triggered by antigen, and triggering of secretion before electrical recording failed to induce the outward K+ current. Finally, GTP gamma S activated the K+ channel in excised inside-out patches of membrane. We conclude that two different GTP-binding proteins differentially regulate two subsets of K+ channels, causing the inward rectifier to close and a novel K+ channel to open when activated.