Therapeutic effects of a taurine-magnesium coordination compound on experimental models of type 2 short QT syndrome.

Short QT syndrome (SQTS) is a genetic arrhythmogenic disease that can cause malignant arrhythmia and sudden cardiac death. The current therapies for SQTS have application restrictions. We previously found that Mg· (NH2CH2CH2SO3)2· H2O, a taurine-magnesium coordination compound (TMCC) exerted anti-arrhythmic effects with low toxicity. In this study we established 3 different models to assess the potential anti-arrhythmic effects of TMCC on type 2 short QT syndrome (SQT2). In Langendorff guinea pig-perfused hearts, perfusion of pinacidil (20 µmol/L) significantly shortened the QT interval and QTpeak and increased rTp-Te (P<0.05 vs control). Subsequently, perfusion of TMCC (1-4 mmol/L) dose-dependently increased the QT interval and QTpeak (P<0.01 vs pinacidil). TMCC perfusion also reversed the rTp-Te value to the normal range. In guinea pig ventricular myocytes, perfusion of trapidil (1 mmol/L) significantly shortened the action potential duration at 50% (APD50) and 90% repolarization (APD90), which was significantly reversed by TMCC (0.01-1 mmol/L, P<0.05 vs trapidil). In HEK293 cells that stably expressed the outward delayed rectifier potassium channels (IKs), perfusion of TMCC (0.01-1 mmol/L) dose-dependently inhibited the IKs current with an IC50 value of 201.1 µmol/L. The present study provides evidence that TMCC can extend the repolarization period and inhibit the repolarizing current, IKs, thereby representing a therapeutic candidate for ventricular arrhythmia in SQT2.

Initiation of remote microvascular preconditioning requires K(ATP) channel activity.

The purpose of this study was to investigate vascular preconditioning of individual microvascular networks. Prior work shows that exposure of downstream arterioles to specific agonists preconditions upstream arterioles so that they exhibit an altered local vasoactive response [remote microvascular preconditioning (RMP)]. We hypothesized that mitochondrial ATP-sensitive K+ (K(ATP)) channels were involved in stimulation of RMP. Arteriolar diameter (approximately 15 microm) was observed approximately 1,000 microm upstream of the remote exposure site in the cheek pouch of pentobarbital sodium-anesthetized (70 mg/kg) male hamsters (n = 104); all agonists were applied via micropipette. RMP was initiated by application of pinacidil (Pin), diazoxide (DZ), sodium nitroprusside (SNP), or bradykinin (BK) to the downstream vessel. After 15 min, RMP was apparent at the upstream observation site from testing of local vasoactive responses to L-arginine. Pin, DZ, SNP, and BK each stimulated RMP. To evaluate a specific role for mitochondrial K(ATP) channels in this response, 5-hydroxydecanoate was applied (via a 2nd pipette) during downstream stimulation with agonist. 5-Hydroxydecanoate blocked RMP initiated by Pin, DZ, or SNP, suggesting that mitochondrial K(ATP) channels are involved before SNP signal transduction. To verify this, we applied N(omega)-nitro-L-arginine during DZ or SNP stimulation. RMP was blocked during SNP, but not during DZ, stimulation. Thus stimulation of the RMP response requires mitochondrial K(ATP) channel activity after stimulation by nitric oxide donors.

Selective inhibition of pinacidil effects by estrogen in guinea pig heart.

BACKGROUND: Recently, gender related differences in heart function have been extensively studied. Some of them, as differences in repolarization between males and females have been explained by direct effect of estrogen on delayed rectifier K+ channels and Ca2+ channels. It seems that estrogen induces overexpression of SUR2A subunits of ATP-sensitive K+ channels. The aim of this paper was to compare heart rate changes in male and female guinea pigs in the presence of different potassium channel openers (PCOs). METHODS: We used spontaneously beating right atria from control and estrogen receptor modulator-treated male and female guinea pigs (17-beta-estradiol as a stimulator and tamoxifen as a blocker of estrogen receptor located in heart muscle). RESULTS: In control females, rilmakalim and diazoxide, but not pinacidil elicited concentration-dependent decrease of heart rate. On the other hand, all three PCOs induced similar negative chronotropic action in hearts obtained from male control group (Emax was between -40 and -70 bpm, respectively). After two weeks of treatment with 17-beta-estradiol, pinacidil failed to significantly decrease heart rate in males however, tamoxifen-pretreated female group responded by decrease in automatism in the presence of rising concentration of pinacidil (Emax=-45+/-6 bpm, not significantly different from Emax in male control=-40+/-5 bpm, n=7). Interestingly, we observed lower blood concentration of the heart form of lactate dehydrogenase (H-LDH) in female than in male control group. Moreover, H-LDH concentration increased in tamoxifen-pretreated female group and decreased in 17-beta-estradiol-treated male group. CONCLUSION: Our results indicate that estrogen downregulates H-LDH production and specifically modulate pinacidil action in guinea pig right atria, probably by changes of binding site for this drug in SUR2A receptor, but not for rilmakalim and diazoxide.

NADH supplementation decreases pinacidil-primed I K ATP in ventricular cardiomyocytes by increasing intracellular ATP.

1 The aim of this study was to investigate the effect of nicotinamide-adenine dinucleotide (NADH) supplementation on the metabolic condition of isolated guinea-pig ventricular cardiomyocytes. The pinacidil-primed ATP-dependent potassium current I(K(ATP)) was used as an indicator of subsarcolemmal ATP concentration and intracellular adenine nucleotide contents were measured. 2 Membrane currents were studied using the patch-clamp technique in the whole-cell recording mode at 36-37 degrees C. Adenine nucleotides were determined by HPLC. 3 Under physiological conditions (4.3 mM ATP in the pipette solution, ATP(i)) I(K(ATP)) did not contribute to basal electrical activity. 4 The ATP-dependent potassium (K((ATP))) channel opener pinacidil activated I(K(ATP)) dependent on [ATP](i) showing a significantly more pronounced activation at lower (1 mM) [ATP](i). 5 Supplementation of cardiomyocytes with 300 micro g ml(-1) NADH (4-6 h) resulted in a significantly reduced I(K(ATP)) activation by pinacidil compared to control cells. The current density was 13.8+/-3.78 (n=6) versus 28.9+/-3.38 pA pF(-1) (n=19; P<0.05). 6 Equimolar amounts of the related compounds nicotinamide and NAD(+) did not achieve a similar effect like NADH. 7 Measurement of adenine nucleotides by HPLC revealed a significant increase in intracellular ATP (NADH supplementation: 45.6+/-1.88 nmol mg(-1) protein versus control: 35.4+/-2.57 nmol mg(-1) protein, P<0.000005). 8 These data show that supplementation of guinea-pig ventricular cardiomyocytes with NADH results in a decreased activation of I(K(ATP)) by pinacidil compared to control myocytes, indicating a higher subsarcolemmal ATP concentration. 9 Analysis of intracellular adenine nucleotides by HPLC confirmed the significant increase in ATP.

Isoflurane sensitizes the cardiac sarcolemmal adenosine triphosphate-sensitive potassium channel to pinacidil.

BACKGROUND: Cardioprotective effects of isoflurane are partially mediated by the sarcolemmal adenosine triphosphate-sensitive potassium (sarcK ATP ) channel. The authors tested the hypothesis that isoflurane sensitizes sarcK ATP channels to a potassium channel opener, pinacidil, adenosine- and phospholipid-mediated pathways. METHODS: Activation by pinacidil of the K ATP current (I KATP ) was monitored in guinea pig ventricular myocytes at 0.5 and 5 mm intracellular ATP in the whole cell configuration of the patch clamp technique. The sensitization effect was evaluated by pretreating each myocyte with isoflurane (0.57 +/- 0.04 mm) before application of pinacidil (5 micro m) in the continued presence of the anesthetic. To investigate whether intracellular signaling pathways may be involved in isoflurane sensitization, the authors used the adenosine receptor antagonist theophylline (100 micro m) and the phosphatidylinositol kinase inhibitor wortmannin (100 micro m). RESULTS: The density of pinacidil-activated I KATP was higher at 0.5 mm ATP (20.7 +/- 3.2 pA/pF) than at 5 mm ATP (2.0 +/- 0.3 pA/pF). At 0.5 mm ATP, pretreatment with isoflurane caused an increase in density of pinacidil-activated I KATP (42.4 +/- 6.2 pA/pF) and accelerated the rate of current activation (from 5.4 +/- 1.2 to 39.0 +/- 7.9 pA. pF(-1). min(-1) ). Theophylline attenuated current activation by pinacidil (9.4 +/- 3.9 pA/pF) and abolished the sensitization effect of isoflurane on I KATP (10.0 +/- 2.5 pA/pF). Wortmannin did not alter pinacidil activation of I KATP (13.2 +/- 1.7 pA/pF) but prevented sensitization by isoflurane (15.8 +/- 4.5 pA/pF). CONCLUSIONS: These results suggest that isoflurane increases sensitivity of cardiac sarcK ATP channels to the potassium channel opener pinacidil. Blockade of adenosine receptors or phosphatidylinositol kinases abolishes the sensitization effect, suggesting that the adenosine and phospholipid signaling pathways may be involved in the actions by isoflurane.

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.

Dimethylsulfoxide and ethanol, commonly used diluents, prevent dilation of pial arterioles by openers of K(ATP) ion channels.

Ethanol and dimethylsulfoxide are commonly used as diluents for water-insoluble drugs. Both are antioxidants. An earlier study of cats presented pharmacological evidence indicating that oxidants could open the K(ATP) ion channel in cerebral surface arterioles [pial arterioles] and that antioxidants including dimethylsulfoxide and L-cysteine prevented opening of these channels. Ethanol was not tested. The present study extends the older observations to a second species, the rat, and examines ethanol as well as dimethylsulfoxide and L-cysteine. A microscope and image splitter were used to measure arteriolar diameters under a closed cranial window in pentobarbital-anesthetized, paralyzed rats. Drugs were topically applied. Dose-dependent dilations produced by two well-established openers of the K(ATP) ion channel were inhibited in dose-dependent manner by ethanol at doses from 0.01% to 0.075%. Above this dose, the effect disappeared. Dilation by sodium nitroprusside was not affected. Dimethylsulfoxide and L-cysteine inhibited dilation produced by pinacidil. Dimethylsulfoxide inhibited pinacidil in a dose-dependent manner at doses from 0.01% to 0.2%. L-Cysteine inhibited pinacidil. Since all the inhibitory drugs have antioxidant properties, their effect may be a reflection of that property as suggested in an earlier paper. Ethanol and dimethylsulfoxide inhibited in doses frequently present when these agents are used as solvents. When investigators use these solvents to dissolve water-insoluble, topically applied drugs, we suggest that they first test the possibility that their observations are being made under conditions in which opening of the K(ATP) ion channel is inhibited.

ATP-sensitive K+ channel openers prevent Ca2+ overload in rat cardiac mitochondria.

1. Mitochondrial dysfunction, secondary to excessive accumulation of Ca2+, has been implicated in cardiac injury. We here examined the action of potassium channel openers on mitochondrial Ca2+ homeostasis, as these cardioprotective ion channel modulators have recently been shown to target a mitochondrial ATP-sensitive K+ channel. 2. In isolated cardiac mitochondria, diazoxide and pinacidil decreased the rate and magnitude of Ca2+ uptake into the mitochondrial matrix with an IC50 of 65 and 128 microM, respectively. At all stages of Ca2+ uptake, the potassium channel openers depolarized the mitochondrial membrane thereby reducing Ca2+ influx through the potential-dependent mitochondrial uniporter. 3. Diazoxide and pinacidil, in a concentration-dependent manner, also activated release of Ca2+ from mitochondria. This was prevented by cyclosporin A, an inhibitor of Ca2+ release through the mitochondrial permeability transition pore. 4. Replacement of extramitochondrial K+ with mannitol abolished the effects of diazoxide and pinacidil on mitochondrial Ca2+, while the K+ ionophore valinomycin mimicked the effects of the potassium channel openers. 5. ATP and ADP, which block K+ flux through mitochondrial ATP-sensitive K+ channels, inhibited the effects of potassium channel openers, without preventing the action of valinomycin. 6. In intact cardiomyocytes, diazoxide also induced mitochondrial depolarization and decreased mitochondrial Ca2+ content. These effects were inhibited by the mitochondrial ATP-sensitive K+ channel blocker 5-hydroxydecanoic acid. 7. Thus, potassium channel openers prevent mitochondrial Ca2+ overload by reducing the driving force for Ca2+ uptake and by activating cyclosporin-sensitive Ca2+ release. In this regard, modulators of an ATP-sensitive mitochondrial K+ conductance may contribute to the maintenance of mitochondrial Ca2+ homeostasis.

Thiopental and propofol impair relaxation produced by ATP-sensitive potassium channel openers in the rat aorta.

ATP-sensitive potassium channel openers are used as vasodilators in the treatment of cardiovascular disorders. The effects of i.v. anaesthetics on arterial relaxation induced by ATP-sensitive potassium channel openers have not been studied. Therefore, in this study, we have examined if thiopental (thiopentone) and propofol affect the vascular response to the ATP-sensitive potassium channel openers, cromakalim and pinacidil, in the isolated rat aorta. Rings of rat thoracic aortas without endothelium were suspended for isometric force recording. Concentration-response curves were obtained in a cumulative manner. During submaximal contractions with phenylephrine 0.3 mumol litre-1, relaxation after cromakalim 0.1-30 mumol litre-1, pinacidil 0.1-30 mumol litre-1 and papaverine 0.1-300 mumol litre-1 was demonstrated. Thiopental 30-300 mumol litre-1, propofol 10-100 mumol litre-1, 10% Intralipid 45 microliters or glibenclamide 5 mumol litre-1 were applied 15 min before addition of phenylephrine. During contractions with phenylephrine, cromakalim and pinacidil induced concentration-dependent relaxation. A selective ATP-sensitive potassium channel antagonist, glibenclamide 5 mumol litre-1, abolished this relaxation, whereas it did not affect relaxation produced by papaverine. Thiopental concentrations > 30 mumol litre-1 significantly impaired relaxation produced by cromakalim or pinacidil. Propofol concentrations > 10 mumol litre-1 also significantly reduced relaxation produced by cromakalim or pinacidil, whereas Intralipid was ineffective. Thiopental 300 mumol litre-1 and propofol 100 mumol litre-1 did not alter relaxation produced by papaverine. These results suggest that the i.v. anaesthetics, thiopental and propofol, impaired vasodilatation mediated by ATP-sensitive potassium channels in vascular smooth muscle cells.

Potassium channel openers inhibit ATP-induced cytosolic free calcium increase in cultured rabbit aortic smooth muscle cells.

AIM: To study the effects of potassium channel openers (PCO) on cytosolic free calcium ([Ca2+]i) changes and their possible mechanisms in vascular smooth muscle cells (VSMC). METHODS: Cultured rabbit aortic VSMC were treated with Fura-2 AM 2.5 mumol.L-1 at 37 degrees C for 50 min. The PCO were pinacidil (Pin), nicorandil (Nic), lemakalim (Lem), and RP 49356 (RP). [Ca2+]i level was measured by fluorospectrometer. RESULTS: [Ca2+]i increase induced by K+ 30 mmol.L-1 was weakly inhibited by Pin, Nic, Lem, and RP (441 +/- 23, 455 +/- 48, 451 +/- 22, 370 +/- 31 vs 544 +/- 40 nmol.L-1, P < 0.01). ATP (0.1 mmol.L-1)-induced peak and sustained [Ca2+]i increase were inhibited by these agents in a concentration-dependent manner. The effects of Pin, Lem, and RP were completely canceled (peak phase: 549 +/- 39, 540 +/- 30, 564 +/- 13 vs 541 +/- 39 nmol.L-1; sustained phase: 413 +/- 25, 364 +/- 16, 377 +/- 11 vs 380 +/- 8 nmol.L-1), but that of Nic was only partially blocked (peak phase: 453 +/- 31 vs 541 nmol.L-1; sustained phase: 348 +/- 19 vs 380 +/- 8 nmol.L-1, P < 0.01) by glibenclamide (Gli, 10 mumol.L-1). Pretreated with the Pin, Nic, Lem, and RP (10 mumol.L-1), the peak [Ca2+]i elevation induced by ATP was reduced in the Ca(2+)-free solution (129 +/- 17, 142 +/- 21, 136 +/- 14, 114 +/- 9 vs 258 +/- 32 nmol.L-1, P < 0.01). CONCLUSION: Pin, Nic, Lem, and RP inhibited ATP-induced [Ca2+]i increase, associated with decreases of both Ca2+ release from intracellular store and Ca2+ influx from extracellular store.