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Pinacidil, a nonselective ATP-sensitive K + (KATP) channel opener, has cardioprotective effects for hypertension, ischemia/reperfusion injury, and arrhythmia. This agent abolishes early afterdepolarizations, delayed afterdepolarizations (DADs), and abnormal automaticity in canine cardiac ventricular myocytes. DADs are well known to be caused by the Na + /Ca 2+ exchange current (I NCX ). In this study, we used the whole-cell patch-clamp technique and Fura-2/AM (Ca 2+ -indicator) method to investigate the effect of pinacidil on I NCX in isolated guinea pig cardiac ventricular myocytes. In the patch-clamp study, pinacidil enhanced I NCX in a concentration-dependent manner. The half-maximal effective concentration values were 23.5 and 23.0 μM for the Ca 2+ entry (outward) and Ca 2+ exit (inward) components of I NCX , respectively. The pinacidil-induced I NCX increase was blocked by L-NAME, a nitric oxide (NO) synthase inhibitor, by ODQ, a soluble guanylate cyclase inhibitor, and by KT5823, a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor, but not by N-2-mercaptopropyonyl glycine (MPG), a reactive oxygen species (ROS) scavenger. Glibenclamide, a nonselective KATP channel inhibitor, blocked the pinacidil-induced I NCX increase, while 5-HD, a selective mitochondria KATP channel inhibitor, did not. In the Fura-2/AM study pinacidil also enhanced intracellular Ca 2+ concentration, which was inhibited by L-NAME, ODQ, KT5823, and glibenclamide, but not by MPG and 5-HD. Sildenafil, a phosphodiesterase 5 inhibitor, increased further the pinacidil-induced I NCX increase. Sodium nitroprusside, a NO donor, also increased I NCX . In conclusion, pinacidil may stimulate cardiac Na + /Ca 2+ exchanger (NCX1) by opening plasma membrane KATP channels and activating the NO/cGMP/PKG signaling pathway.

Flocalin (FLO) is a new ATP-sensitive K(+) (KATP) channel opener (KCO) derived from pinacidil (PIN) by adding fluorine group to the drug's structure. FLO acts as a potent cardioprotector against ischemia-reperfusion damage in isolated heart and whole animal models primarily via activating cardiac-specific Kir6.2/SUR2A KATP channels. Given that FLO also confers relaxation on several types of smooth muscles and can partially inhibit L-type Ca(2+) channels, in this study, we asked what is the mechanism of FLO action in bladder detrusor smooth muscle (DSM). The actions of FLO and PIN on contractility of rat and guinea pig DSM strips and membrane currents of isolated DSM cells were compared by tensiometry and patch clamp. Kir6 and SUR subunit expression in rat DSM was assayed by reverse transcription PCR (RT-PCR). In contrast to PIN (10 μM), FLO (10 μM) did not produce glibenclamide-sensitive DSM strips' relaxation and inhibition of spontaneous and electrically evoked contractions. However, FLO, but not PIN, inhibited contractions evoked by high K(+) depolarization. FLO (40 μM) did not change the level of isolated DSM cell's background K(+) current, but suppressed by 20 % L-type Ca(2+) current. Determining various Kir6 and SUR messenger RNA (mRNA) expressions in rat DSM by RT-PCR indicated that dominant KATP channel in rat DSM is of vascular type involving association of Kir6.1 and SUR2B subunits. Myorelaxant effects of FLO in bladder DSM are explained by partial blockade of L-type Ca(2+) channel-mediated Ca(2+) influx rather than by hyperpolarization associated with increased K(+) permeability. Thus, insertion of fluorine group in PIN's structure made the drug more discriminative between Kir6.2/SUR2A cardiac- and Kir6.1/SUR2B vascular-type KATP channels and rendered it partial L-type Ca(2+) channel-blocking potency.

Activators of both adenosine 5'-triphosphate (ATP)-sensitive K(+) (KATP) channel and cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel have significant in vivo and in vitro neuroprotection against glutamate-induced death of some neuronal cells. Here, the effect of the KATP channel activator, pinacidil, and the CFTR Cl(-) channel opener, levamisole, against glutamate-induced oxidative stress were investigated in mouse hippocampal cells, HT22. The results from cell viability assay (WST-1) showed that pinacidil and levamisole weakly protected cells against glutamate-induced toxicity at 10 μM and their effect increased in a dose-dependent manner till reach maximum protection at 300 μM. Pretreatment with pinacidil or levamisole significantly suppressed the elevation of reactive oxygen species (ROS) triggered by glutamate through stabilising mitochondrial membrane potential and subsequently protected HT22 cells against glutamate-induced death. HT22 cells viability was maintained by pinacidil and levamisole in presence of glutathione inhibitor, BSO. Also, pinacidil and levamisole pretreatment did not induce recovery of glutathione levels decreased by glutamate Expectedly, this protection was abolished by the KATP and CFTR Cl(-) channels blocker, glibenclamide. Thus, both pinacidil and levamisole protect HT22 cells against glutamate-induced cell death through stabilising mitochondrial membrane potential and subsequently decreasing ROS production.

In the experiments on non-linear white rats with the model of acute hypoxia we have studied the changes of the functional state of kidneys after a single intraventricular administration of the original fluorine-containing KATP-sensitive potassium channels activator flocalin at the dose of 5 mg/kg on the background of induced water load. It has been shown that under the influence of prohypoxic factors: sodium nitrite (50 mg/kg, subcutaneously) and dinitrophenol (3 mg/kg, intraperitoneal) flocalin activates volume-regulating, ion-regulating and excretory functions of kidneys. Renal effects after activation of KATP-sensitive potassium channels in the rats with histohemic hypoxia were induced by the changes of tubular and predominantly glomerular processes. The increase of glomerular filtration rate, restoration of volatile distal reabsorption of potassium channels, decrease of sodium loss with urine, decrease ofproteinuria after a single administration of flocalin under conditions of acute hypoxia complement the range of protective effects of KATP-sensitive potassium channels activator flocalin.

In experiments on the anaesthetized dogs the influence of a new fluorine-containing opener of ATP-sensitive potassium (K(ATP)) channels flocalin on the cardiohemodynamic of great animals in vivo was studied. Flocalin introduced intravenously in doses 0.01 - 1.5 mgs/kg. It is shown that it reduces in dose-dependent manner a system arterial pressure, perfusion pressure in coronary artery and general peripheral resistance of vessels with maximal effects on 56.8 +/- 2.7, 22.4 +/- 4.7 and 47.2% +/- 6.5% accordingly at most dose 1.5 mgs/kg. Flocalin causes development of cardiodepressive reactions in heart, that is exhibited in dose-dependent the decrease of pressure in the left ventricle, speed of growth (dP/dt(max)) and reduction (dP/dt(min)) in it's of pressure with maximal effects on 37.1 +/- 5.1, 51.2 +/- 9.4 and 55.6% +/- 6.9% accordingly at introduction of most dose of flocalin. Diminish of the cardiac out put and heart rate with a maximal effects on 23.1% +/-12.7% and 19.2% +/- 1.7% accordingly at a dose 1.0 mgs/kg was shown. It should be noted that considerable reduction of heart rate and general peripheral resistance of vessels takes place only at the large doses of flocalin - 1 and 1.5 mgs/kg. Thus, it is shown that activation of K(ATP) channels by flocalin causes the dose-dependent decrease of pressure in the system of circulation of blood and contraction activity of myocardium.

The influence of the new cardioprotector flocalin was investigated on the culture of rat's neonatal cardiomyocytes during anoxia-reoxygenation modelling. The mechanisms of apoptosis and necrosis were investigated under influence of ATP-sensitive potassium (K(ATP)) channels activation and in conditions of blocking of the L-type calcium (VGCCs) channels. Flocalin was added in the culture medium in the dose 5 and 20 microM at 2 minutes before anoxia (30 minutes) and following reoxygenation (60 minutes). These doses are near to: the first dose means the opening of K(ATP) channels and the second one means the IC50 block of VGCCs. It is discovered that in dose 5 microM of flocalin drew the change of correlation of living, necrotizing and apoptizing cells drew side-shifting living. The number of live cells was almost the same like in control (experiments without anoxia-reoxygenation modelling). The opening of K(ATP) channels decreases necrosis in two times and fully prevented development of apoptosis which was induced anoxia-reoxygenation modelling. Flocalin depressed the apoptosis of neonatal cardiomyocytes so that he was on to 36% less than in control group (without anoxia-reoxygenation). But in the high dose (20 microM) that provokes not only K(ATP) channels opening but also IC50 block of VGCCs cardioprotection was not detected after modelling of anoxia-reoxygenation. The last can be investigation both enough strong activating of the potassium channels and by investigation of both factors are opening of potassium and inhibition of VGCCs channels and, accordingly, substantial diminishing of level of introcellular Ca2+ and violation of metabolic processes yet to anoxia-reoxygenation. Thus, small doses of flocalin, that induce moderate opening of K(ATP) channels significantly decrease the number of necrotic and apoptotic cells in culture of rat neonatal cardiomyocytes induced by anoxia-reoxygenation.

The drugs that are currently used to treat pulmonary hypertension (PH) lack the ability to inhibit or reverse the pulmonary vascular remodeling that occurs during the course of the disease. We propose a novel method that combines the therapeutic powers of the potassium channel opener pinacidil and the statin drug simvastatin. These two drugs do not share similar mechanisms of treating PH. We used rats with monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) as a model and examined the combined effects of pinacidil and simvastatin on pulmonary vascular remodeling. A series of indicators, including those for pulmonary vascular obstruction, proliferation, and cell phenotype, pulmonary vascular matrix and pulmonary vascular smooth muscle cell phenotype were used to monitor changes in pulmonary structure over the course of disease and treatment in normal controls, untreated PAH rats, pinacidil-treated subjects, simvastatin-treated subjects, and combination-treated subjects. We found that levels of mPAP, right ventricle Fulton index, pulmonary arteriolar wall thickness and muscularization, cell growth rate, transforming growth factor beta (TGF-beta), lung tissue matrix metalloproteinase-2 (MMP-2), MMP-9 and lung tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), vascular smooth muscle cell (VSMC) contractile protein SM-alpha-actin, and SM-alpha-actin mRNA of these different groups were all significantly lower in the combination-treated group than in the untreated group. Subjects in the combination-treated group also showed lower levels than those in either the pinacidil-treated or simvastatin-treated group. These results support our hypothesis and provide basis for a new, more effective therapeutic methods of treating PAH in human patients.

In experiments on the anaesthetized dogs with modeling of experimental ischemia (90 min) and reperfusion (180 min) it was investigated the changes of biochemical processes in the different areas of heart (intact, risk and necrotic zone) during intragastric introduction of medicinal form (tablets) of flocalin (the fluorine-containing opener of ATP-sensitive potassium channels) in a dose 2,2 mg/kg. The data analysis allowed to define a few possible cardioprotective mechanisms of flocalin action at ischemia-reperfusion conditions: the preservation of sufficient levels of de novo (by cNOS) NO synthesis, an inhibition of de novo (by iNOS) and salvage (by NADH-dependent nitratreductase) NO synthesis, an inhibition of L-arginine degradation by arginase, an inhibition of oxidizing metabolism due to limitation of ROS and RNS generation, inhibition of free arachidonic acid and eicosanoids synthesis, inhibition of ATP and GTP degradations and, possibly, stimulation of protective haem degradation. These changes may prevent formation of toxic peroxynitrite and suggest the possibility of participating in flocalin-mediated cardioprotective effects of warning a mitochondrial permeability transition pore (MPTP) opening and inhibition of apoptosis and/or necrosis of cardiomyocytes induced by it.

In experiments on anaesthetized dogs with modeling of experimental ischemia (90 min) and reperfusion (180 min), the cardioprotective influence of the pharmacological preconditioning caused by intragastric (with a help of catheter) introduction of medicinal form (tablets) of new fluorine-containing opener of ATP-sensitive potassium channels flocalin was shown. Flocalin was introduced in a dose 2.2 mg/kg, which in the conditions of physiological norm has a minimum influence on the parameters of cardiohemodynamic. The conducted research allowed to define the changes of these parameters during development of antiischemic protective effect of pharmacological preconditioning, caused by the medicinal form of flocalin, and describes basic cardioprotective mechanisms, related to the changes of cardiohemodynamic in the dynamics of ischemia-reperfusion of myocardium. In our opinion, to positive influences of flocalin, which are possibly related to cardioprotective action, it is possible to add the prevention of an increase of general peripheral resistance, resistance of coronal vessels of heart, and relative preservation of myocardium contractility in the period of reperfusion. Also these positive effects can be explained by moderate decrease of blood pressure that decreases the loading on the damaged heart and allows to preserve cardiac emission in the first period of ischemia. One of the major indexes of development of protective mechanism of pharmacological preconditioning caused by preischemic introduction of medicinal form of flocalin is the diminishing of infarct size of myocardium in experiments with ischemia-reperfusion of myocardium on 42.53% +/- 2.91% versus control experiments.

Objectives: This work evaluated the potential usefulness of pharmacological activation of cardiac ATP-sensitive potassium channels (K(ATP)) in the prevention of drug-induced QT prolongation in anaesthetised guinea-pigs. Prolongation of cardiac repolarisation and QT interval is an adverse effect of many drugs blocking HERG potassium channels. This alteration can be dangerously arrhythmogenic and has been associated with the development of a particular form of ventricular tachyarrhythmia known as torsade de pointes., Methods: The well-known K(ATP) openers aprikalim, cromakalim and pinacidil were used. Moreover, three benzothiazine derivatives, which have been reported as potent activators of K(ATP) channels, were also used., Key Findings: Pharmacological activation of K(ATP) channels caused a reduction of the QT prolongation, induced by astemizole, cisapride, quinidine and thioridazine. In contrast, the QT prolongation induced by haloperidol, sotalol and terfenadine, which are known to block HERG channels but also K(ATP) channels, was not influenced by K(ATP) activation. Glibenclamide and tolbutamide (non-selective blockers of K(ATP) channels expressed both in sarcolemmal and in mitochondrial membranes) antagonised the effects of K(ATP) openers, whereas 5-hydroxydecanoic acid (selective blocker of the mitochondrial K(ATP) channels) failed to antagonise the effects of K(ATP) openers, indicating that only the sarcolemmal K(ATP) is involved in the cardioprotective activity., Conclusions: The data suggest that pharmacological K(ATP) activation might represent an option for treatment of patients exposed to QT-prolonging drugs.

In experiments on the anaesthetized dogs we investigated the influence of a new fluorine-containing opener of ATP-sensitive potassium channels of sarcolemal and mitochondrial membranes flocalin on the level of glucose in arterial blood at physiological conditions and under ischemia (90 min) and reperfusion (180 min) of myocardium. It was shown that intravenous introduction of flocalin in doses 0,1 - 1,0 mg/kg did not change the level of glucose in blood. In experiments with ischemia-reperfusion of myocardium, flocalin also did not increase the level of glucose during all experiment (5,5 hours) after intragastric (with a help of catheter) introduction of drug form (tablets) at cardiorotective dose of 2,2 mg/kg. However, intravenous introduction of flocalin in the dose of 1,5 mg/kg, which 15 times exceeded a cardioprotective dose of 0,1 mg/kg increased the glucose level 1,33 fold. It should be noted that this increase was not sustained and the level of glucose restored to the initial level within 1 hour. Identical changes of indexes of cardiohemodynamic and the level of glucose in arterial blood under introduction of identical doses of flocalin at the beginning and at the end of experiment (total dose of flocalin reached 4 - 4,5 mg/kg) can testify the absence of cumulative effect of flocalin at these experimental conditions. Thus, strong cardioprotective properties, hypotoxicity and the absence of meaningful changes in a carbohydrate exchange allow to consider a new fluorine-containing opener of K(ATP) channels of flocalin as perspective drug for clinical use.

In experiments on anesthetized dogs it was shown that the amplitude and duration of hypotensive effect of flocalin, a fluorine-containing pinacidil analogue, were dose-dependent and similar to those evoked by the known ATP-sensitive potassium channel opener pinacidil. However, flocalin appeared to be 3.5 times less toxic than pinacidil. Registration of systemic arterial pressure (SAP) has shown that following intravenous introduction of the threshold dose of flocalin (0.05 mg/ kg) the dilatation lasts around three minutes with the amplitude 9.52% +/- 2.01% (n=7, P < 0.05). Introduction of flocalin in a dose 0.5 mg/kg and above reduced SAP on more than 37%. Flocalin at 0.5, 0.75, 1.0 and 1.5 mg/kg reduced SAP by 42.07 +/- 6.18 (n=5, P < 0.05); 44.22 +/- 4.87 (n=3, P < 0.05); 44.3 +/- 4.59 (n=5, P < 0.05) and 66.28 +/- 3.15 mm Hg (n=3, P < 0.05), accordingly. Intravenous introduction of high doses of flocalin (0.75-1.5 mg/kgs) quite often reduced SAP to 40-50 mm Hg. However, such dangerous reduction in arterial pressure was comparatively short and lasted not more than 15 minutes, and then (usually within an hour) SAP gradually restored. Introduction of flocalin in hip artery, while measuring the perfusion pressure, produced practically similar results. In our opinion, the optimal cardioprotective doses of flocalin were 0.1 and 0.2 mg/kg. In experiments with acute ischemia and reperfusion of myocardium, preischemic introduction of flocalin at 0.1 and 0.2 mg/kg reduced an infarct size of myocardium by 37-40% and reduced SAP within first 5 and 25 minutes, accordingly.

Objectives: The purpose of this study was to determine whether ATP-sensitive potassium (K(ATP)) channel agonists exert a beneficial effect on the structural, functional, and molecular features of the remodeling heart in infarcted rats., Background: Myocardial K(ATP) channels have been implicated in the ventricular remodeling after myocardial infarction by inhibition of 70-kDa S6 (p70S6) kinase., Methods: Male Wistar rats after induction of myocardial infarction were randomized to either vehicle, agonists of K(ATP) channels nicorandil and pinacidil, an antagonist of K(ATP) channels glibenclamide, or a combination of nicorandil and glibenclamide or pinacidil and glibenclamide for 4 weeks. To verify the role of p70S6 kinase in ventricular remodeling, rapamycin was also assessed., Results: Significant ventricular hypertrophy was detected by increased myocyte size at the border zone isolated by enzymatic dissociation after infarction. Increased synthesis of p70S6 kinase messenger ribonucleic acid after infarction in vehicle-treated rats was confirmed by reverse transcription-polymerase chain reaction, consistent with the results of immunohistochemistry and Western blot for phosphorylated p70S6 kinase. Rats in the nicorandil- and pinacidil-treated groups significantly attenuated cardiomyocyte hypertrophy and phosphorylated p70S6 kinase expression with similar potency, as compared with vehicle. The beneficial effects of nicorandil and pinacidil were abolished by administering either glibenclamide or 5-hydroxydecanoate. Addition of rapamycin attenuated ventricular remodeling and did not have additional beneficial effects compared with those seen in rats treated with either nicorandil or pinacidil alone., Conclusions: Activation of K(ATP) channels by either nicorandil or pinacidil can attenuate ventricular remodeling, probably through a p70S6 kinase-dependent pathway after infarction.

Objective: To investigate effects of K(ATP) opener on the expressions of caspase-12 mRNA and protein, and to explore the role of endoplasmic reticulum (ER) stress pathway in the mechanism of K(ATP) opener protecting against neuronal apoptosis after cerebral ischemia-reperfusion., Methods: Two hundred rats were randomly divided into four groups: sham operation group, ischemia-reperfusion group, K(ATP) opener group, and K(ATP) blocker group. The middle cerebral artery occlusion (MCAO) model was established by intraluminal suture occlusion method; neuronal apoptosis was detected by TUNEL staining. The mRNA and protein expressions of caspase-12 were detected by semi-quantitative RT-PCR and immunohistochemical staining, respectively., Results: In ischemia-reperfusion group, K(ATP) opener group and K(ATP) blocker group, the number of apoptotic cells and the mRNA and protein expressions of caspase-12 gradually increased following cerebral reperfusion, and reached the peak at 24 h. In K(ATP) opener group, the number of apoptotic cells was significantly less than that in ischemia-reperfusion group and K(ATP) blocker group at 12 h, 24 h, 48 h and 72 h (P< 0.05 or P< 0.01); while the mRNA and protein levels of caspase-12 were significantly less than those in ischemia-reperfusion group and K(ATP) blocker group at all times (P< 0.05 or P< 0.01). There were no differences between the ischemia-reperfusion group and K(ATP) blocker group at each time (P> 0.05)., Conclusion: K(ATP) opener may protect neurons from apoptosis following the cerebral ischemia-reperfusion by inhibiting ER stress pathway.