ATP Channel Opener Research Articles

Antidiabetic Activity of Momordica charantia L. and Mechanism of Insulin Secretion of 1-Butanol Soluble Part on Type 2 Diabetic Model Rats

The in vivo effects of fruit pulp juice (MC-PJ) of Momordica charantia and its 1-butanol soluble part (MC-BP) and aqueous soluble part (MC-AP) on blood glucose of type 2 diabetic rats were studied. In vitro insulin secretion in response to MC-BP and MC-AP from whole perfused pancreas was measured. For elucidating the mechanism of insulinotropic action, the insulin secretory activity of MC-BP in the presence of 11 mM glucose, 50 μM verapamil (Ca++ channel blocker), 8 mM diazoxide (K+ ATP channel opener) and 10 mM theophylline (cAMP phosphodiesterase inhibitor) were studied. Serum glucose was measured by glucose oxidase-peroxidase method and rat insulin was assayed by specific ELISA. In the in vivo study, MC-BP significantly opposed the rise of serum glucose compared to control at 105 min (p<0.05). Although the MC-AP and MC-PJ lowered the serum glucose both at 60 and 105 min, these were not statistically significant. In the in vitro study, only MC-BP produced 22-fold increase in insulin secretion from the perfused pancreas at nonstimulatory glucose level, which was significant (basal vs. MC-BP, 0.071±0.009 vs. 1.563±0.150 ng/ml, p<0.001). The MC-BP also enhanced the insulin secretion from the glucose-stimulated pancreas (p<0.001). The MC-BP induced insulin secretion was not affected in presence of diazoxide and verapamil. The obtained results also showed that MC-BP enhanced the insulin secretory effect of theophylline (p<0.001). The findings indicate that MC-BP has stimulatory effects on physiological pathways of insulin secretion which may underlie its reported antidiabetic action.
 Dhaka Univ. J. Pharm. Sci. 19(2): 111-117, 2020 (December)

Vasodilator Therapy: Nitrates and Nicorandil.

Nitrates have been used to treat symptoms of chronic stable angina for over 135 years. These drugs are known to activate nitric oxide (NO)-cyclic guanosine-3′,-5′-monophasphate (cGMP) signaling pathways underlying vascular smooth muscle cell relaxation, albeit many questions relating to how nitrates work at the cellular level remain unanswered. Physiologically, the anti-angina effects of nitrates are mostly due to peripheral venous dilatation leading to reduction in preload and therefore left ventricular wall stress, and, to a lesser extent, epicardial coronary artery dilatation and lowering of systemic blood pressure. By counteracting ischemic mechanisms, short-acting nitrates offer rapid relief following an angina attack. Long-acting nitrates, used commonly for angina prophylaxis are recommended second-line, after beta-blockers and calcium channel antagonists. Nicorandil is a balanced vasodilator that acts as both NO donor and arterial K+ ATP channel opener. Nicorandil might also exhibit cardioprotective properties via mitochondrial ischemic preconditioning. While nitrates and nicorandil are effective pharmacological agents for prevention of angina symptoms, when prescribing these drugs it is important to consider that unwanted and poorly tolerated hemodynamic side-effects such as headache and orthostatic hypotension can often occur owing to systemic vasodilatation. It is also necessary to ensure that a dosing regime is followed that avoids nitrate tolerance, which not only results in loss of drug efficacy, but might also cause endothelial dysfunction and increase long-term cardiovascular risk. Here we provide an update on the pharmacological management of chronic stable angina using nitrates and nicorandil.

Studies of Ischemic Preconditioning Mechanisms in Langendorff Rat Heart Model the Impact of Phosphocreatin Kinase and ATP Sensitive Potasium Channels Pharmachological Openers and Blockers on Cardioprotection

Abstract Ischemic preconditioning (IPC) induced by administration of brief episodes of ischemia-reperfusion represents a protective mechanism of the heart against prolonged episodes of ischemia. Although the mechanism of ischemic preconditioning has been extensively studied, however, until now it is insufficient elucidated. Using a Langendorff rat heart model with 45 minutes ischemia followed by 120 minute reperfusion we aimed to evaluate the role of Phosphocreatin Kinase-C (PCK) in ischemic rat myocardium and demonstration of its involvment in the path of pharmachological preconditioning (PP) by using PKC activators 1,2-dioctanoyl-sn-glycerol (DOG) and inhibitors chelerythrine (CHE) and evaluation of the role of K ATP channels in pharmachological preconditioning (PP) mechanism by administration of a K ATP channel opener (Cromakalim) (CRK) or by blocking the opening of K ATP channels with glibenclamide(GLB). The activators of PCK(DOG) and of K ATP channel (CRK) limited the infarct size when perfused before lethal ischemia, mimicking the ischemic preconditioning in rat heart When activator of PCK DOG + GLB, a K ATP channel inhibitor were coperfused before the lethal ischemia, there was an increase in myocardial infarct size, expressed as a percentage of the area at risk, versus control, the cardioprotective effect of DOG being abolished by GLB. The same results were obtained when CHE the inhibitor of PCK was coperfused with CRK the activator of K ATP channel. The effect of CHE and GLB perfused at the beginning of IPC resulted in loss of protection accompanied by a significant increase in infarct size area. The finding that treatment with a DOG PKC activator and CRK activator of K ATP channel gives a similar degree of protection against infarction as that seen after ischemic preconditioning and that this protection can be blocked by CHE a PCK pharmachological inhibitor provides support for the hypothesis that PKC plays a pivotal role in ischemic preconditioning. Our data may have a significance in pharmachological preconditioning (PP) with decrease in infarct size as an end point.

Effects of levosimendan on heart failure in normotensive patients: does loading dose matter?

Levosimendan is a calcium sensitizer and K(+)-ATP channel opener with inotropic and vasodilatatory effects irrespective of myocardial oxygen consumption, used for treatment of heart failure (HF). A loading dose is usually given by infusion for 12 h; however, profound lowering of blood pressure often disrupts or prolongs the infusion. The aim of this study was to assess clinical, biochemical and myocardial differences between different regimes of levosimendan therapy, with or without loading dose, and compared to standard therapy in heart failure. Fifty-seven patients (mean age ± SD: 60.9 ± 9.3 years, 45 males) with HF, New York Heart Association (NYHA) III-IV, reduced left ventricular ejection fraction (LVEF) were included. Twenty patients (NB group) were given levosimendan without loading dose, 14 patients (B group) were given levosimendan with loading dose, and 23 patients (C group) were given standard therapy. Clinical, biochemical and echocardiographic characteristics at baseline and one week after treatment were evaluated. Groups were similar at baseline. After one week NHYA class (P < 0.001), NT pro-BNP (P < 0.001), LVEF (P = 0.045), E/A (P = 0.048) E/e' (P < 0.001), and PAPs (P < 0.001) decreased. DT (P = 0.011) and TAPSE (P = 0.035) increased in all groups. Levosimendan, as well as standard therapy, improves myocardial function and symptoms of HF, irrespective of the loading dose administration. Treatment options for patients with end-stage heart failure refractory to conventional medical therapy are limited. Inotropic drugs play an important role in heart failure (HF).

The effect of ATP-dependent K(+)-channel opener on transmembrane potassium exchange and reactive oxygen species production upon the opening of mitochondrial pore

The effect of mitochondrial ATP-dependent K(+)-channel (K(+)ATP-channel) opener diazoxide (DZ) on transmembrane potassium exchange and reactive oxygen species (ROS) formation under the opening of mitochondrial permeability transition pore (MPTP) was studied in rat liver mitochondria. The activation of K(+)-cycling (K(+)-uptake and K(+)/H(+)-exchange) by DZ was established with peak effect at < or = 500 nM. It was shown that MPTP opening as well resulted in the activation of K(+)-cycling together with simultaneous activation of Ca(2+)-cycle in mitochondria. In the absence of depolarization Ca(2+)-cycle is supported by MPTP and Ca(2+)-uniporter. The stimulation of K(+)/H(+)-exchange by MPTP opening led to the activation of K(+)-cycle, but further activation of K(+)/H(+)-exchange resulted in MPTP inhibition. Under the same conditions the decrease in mitochondrial ROS production was observed. It was proposed that the decrease in ROS formation together with K(+)/H(+)-exchange activation could be the constituents of the complex effect of MPTP inhibition induced by K(+)ATP-channel opener.

Transgenic Expression of Kir6.1 Gain-of-Function Mutations in the Mouse Heart Result in Increased L-Type Calcium Current in Ventricular Myocytes

Background The role of sarcolemmal K ATP current (I KATP ) in myocardial excitability remains unclear. Previously, mice with gain-of-function (GOF) 6.2 mutations (Kir6.2 GOF) were engineered to help elucidate I KATP function in the heart. Surprisingly, L-type calcium channel current (LTCCC) was increased in Kir6.2 GOF ventricular myocytes (VM), suggesting that increased I KATP secondarily leads to increased LTCCC. Methods and Results To further test this hypothesis, Kir6.1 GOF mice expressing a Kir6.1 GOF mutation (G343D) under αMHC promoter control have been generated. Patch-clamp data demonstrate reduced sensitivity of K ATP to inhibition by ATP in VM (EC 50 = 43.5 ± 6.1 µM in GOF, 8.3 ± 3.1 µM in wild type (WT); P = .0007). As with Kir6.2 GOF VM, Kir6.1 GOF VM showed markedly increased LTCCC density (pA/pF), comparable to that of WT VM treated with isoproterenol (ISO): (@ –15 mV (pA/pF): WT: –9.6 ± 1.1, n=12; WT+iso: –18.7 ± 2.7, n=4; Kir 6.1 GOF: –17.7 ± 3.6, n=15; Kir 6.1+ISO: –25.0 ± 8.3, n=9, analysis of variance [ANOVA], P P = NS). LTCCC was also recorded from VM expressing dominant-negative (DN) Kir6.1 [AAA] subunits (Kir6.1 DN). In contrast to Kir6.1 GOF VM, LTCCC was not increased in Kir6.1 DN VM: (@–15 mV (pA/pF): WT: –9.6 ± 1.1, n=12; WT+ISO: –18.7 ± 2.7, n=4; Kir 6.1DN: –14.4 ± 1.5, n=10; Kir 6.1 DN+ISO: –8.4 ± 3.8, n=4, ANOVA, P P = significant), suggesting that increased LTCCC in the former results from increased I KATP and not from increased protein per se. Slow-release minoxidil (MIN, a K ATP channel opener) pellets or placebo (P) pellets were also implanted in WT mice for 3 weeks. LTCCC was subsequently recorded to test whether pharmacologically increased I KATP results in increased LTCCC. As with the genetic models, the MIN model also showed increased LTCCC similar to WT+ISO VM: (@–15 mV (pA/pF): WT+P: –11.9 ± 1.1, n=6; WT+P+ISO: –25.4 ± 1.7, n=3; WT+MIN: –22.0 ± 7.6, n=7; WT+MIN+ISO: –35.4 ± 12.3, n=6, ANOVA, P P = NS). Conclusions The results support the idea that K ATP GOF leads to constitutive upregulation of LTCCC and suggest a potential mechanism underlying ventricular arrhythmias in Kir6.1-associated Brugada and early repolarization syndromes.

Preconditioning with Associated Blocking of Ca2+ Inflow Alleviates Hypoxia-Induced Damage to Pancreatic β-Cells

ObjectiveBeta cells of pancreatic islets are susceptible to functional deficits and damage by hypoxia. Here we aimed to characterize such effects and to test for and pharmacological means to alleviate a negative impact of hypoxia.Methods and DesignRat and human pancreatic islets were subjected to 5.5 h of hypoxia after which functional and viability parameters were measured subsequent to the hypoxic period and/or following a 22 h re-oxygenation period. Preconditioning with diazoxide or other agents was usually done during a 22 h period prior to hypoxia.ResultsInsulin contents decreased by 23% after 5.5 h of hypoxia and by 61% after a re-oxygenation period. Preconditioning with diazoxide time-dependently alleviated these hypoxia effects in rat and human islets. Hypoxia reduced proinsulin biosynthesis (3H-leucine incorporation into proinsulin) by 35%. Preconditioning counteracted this decrease by 91%. Preconditioning reduced hypoxia-induced necrosis by 40%, attenuated lowering of proteins of mitochondrial complexes I–IV and enhanced stimulation of HIF-1-alpha and phosphorylated AMPK proteins. Preconditioning by diazoxide was abolished by co-exposure to tolbutamide or elevated potassium (i.e. conditions which increase Ca2+ inflow). Preconditioning with nifedipine, a calcium channel blocker, partly reproduced effects of diazoxide. Both diazoxide and nifedipine moderately reduced basal glucose oxidation whereas glucose-induced oxygen consumption (tested with diazoxide) was unaffected. Preconditioning with diaxoxide enhanced insulin contents in transplants of rat islets to non-diabetic rats and lowered hyperglycemia vs. non-preconditioned islets in streptozotocin-diabetic rats. Preconditioning of human islet transplants lowered hyperglycemia in streptozotocin-diabetic nude mice.Conclusions1) Prior blocking of Ca2+ inflow associates with lesser hypoxia-induced damage, 2) preconditioning affects basal mitochondrial metabolism and accelerates activation of hypoxia-reactive and potentially protective factors, 3) results indicate that preconditioning by K+-ATP-channel openers has therapeutic potential for islet transplantations.

The Mechanism by which ATP Binding Opens IKs Channels

In cardiac myocytes KCNQ1 and KCNE1 form the IKs channel that is important in terminating action potentials. KCNQ1 has a common Kv channel membrane topology with the central pore domain (PD, S5-S6) and the voltage-sensing domain (VSD, S1-S4). We have demonstrated that ATP directly binds to the cytosolic C-terminus of KCNQ1 to regulate IKs channel function. Here we study if ATP regulates VSD movements or pore opening. using inside-out patch clamp techniques, we found that the steady state G-V relationship and current kinetics of the WT IKs channel are not changed by [ATP]. Consistent with these results, mutations in the putative ATP binding site reduce ATP sensitivity of the current amplitude but do not alter G-V relationship or activation kinetics. Furthermore, using voltage clamp fluorometry, we measured ionic current and VSD movements of the WT and mutant channels in which ATP binding is eliminated; and our results show that florescence signals remain the same, while the ionic current is completely absent in the mutant channels due to the lack of ATP binding. These results suggest that ATP does not regulate VSD movements but is required for pore opening by directly pulling the activation gate to open from the binding site located downstream from S6. Supporting this mechanism, a mutation located between S6 and the putative ATP binding site is found to alter the coupling between ATP binding and channel opening by reducing ATP sensitivity and shifting the G-V relationship to more positive voltages at a given [ATP]; however, increasing [ATP] shifts the G-V back toward negative voltages. The effects of this mutation can be explained by an allosteric model for the coupling between ATP binding and channel opening in which the mutation reduces the allosteric factor for the coupling.

Pharmacological basis for the medicinal use of &lt;i&gt;Morus alba&lt;/i&gt; in gut and airways disorders

Crude extract of Morus alba at 100 mg/kg exhibited protective effect against castor oil-induced diarrhea in mice. In isolated rabbit jejunum, M. alba (0.3-10 mg/mL) inhibited the spontaneous contractions and caused glibenclamide-sensitive inhibition of low K + (20 mM)-induced contractions, with mild effect on high K + (80 mM). Similarly, cromakalim caused inhibition of low K + , but not of high K + , while verapamil did not differentiate in its inhibitory effect on two concentrations of K + . M. alba (3.0-30 mg/kg) caused suppression of carbachol (100 µg/kg)-induced increase in inspiratory pressure of anaesthetized rats. In guinea-pig trachea, M. alba completely inhibited low K + contractions, with partial effect on high K + . M. alba (0.3-1.0 mg/mL) caused leftward shift of isoprenaline-induced inhibitory concentration response curves, like papaverine. These results indicate that M. alba possesses a combination of K ATP channel opening, weak Ca ++ -antagonist and phosphodiesterase inhibitory mechanisms, which explain its medicinal use in hyperactive gut and airways disorders.

Diabetes reduces β-cell mitochondria and induces distinct morphological abnormalities, which are reproducible by high glucose in vitro with attendant dysfunction

We investigated the impact of a diabetic state with hyperglycemia on morphometry of β cell mitochondria and modifying influence of a K+-ATP channel opener and we related in vivo findings with glucose effects in vitro. For in vivo experiments islets from syngeneic rats were transplanted under the kidney capsule to neonatally streptozotocin-diabetic or non-diabetic recipients. Diabetic recipients received vehicle, or tifenazoxide (NN414), intragastrically for 9 weeks. Non-diabetic rats received vehicle. Transplants were excised 7 d after cessation of treatment (wash-out) and prepared for electron microscopy. Morphological parameters were measured from approx. 25,000 mitochondria. Rat islets were cultured in vitro for 2–3 weeks at 27 or 11 (control) mmol/l glucose. Transplants to diabetic rats displayed decreased numbers of mitochondria (-31%, p < 0.05), increased mitochondrial volume and increased mitochondrial outer surface area, p < 0.001. Diabetes increased variability in mitochondrial size with frequent appearance of mega-mitochondria. Tifenazoxide partly normalized diabetes-induced effects, and mega-mitochondria disappeared. Long-term culture of islets at 27 mmol/l glucose reproduced the in vivo morphological abnormalities. High-glucose culture was also associated with reduced ATP and ADP contents, reduced oxygen consumption, reduced signaling by MitoTracker Red and reduction of mitochondrial proteins (complexes I–IV), OPA 1 and glucose-induced insulin release. We conclude that (1) a long-term diabetic state leads to a reduced number of mitochondria and to distinct morphological abnormalities which are replicated by high glucose in vitro; (2) the morphological abnormalities are coupled to dysfunction; (3) K+-ATP channel openers may have potential to partly reverse glucose-induced effects.

Reopening of ATP-sensitive potassium channels reduces neuropathic pain and regulates astroglial gap junctions in the rat spinal cord

Adenosine triphosphate–sensitive potassium (K ATP) channels are suggested to be involved in pathogenesis of neuropathic pain, but remain underinvestigated in primary afferents and in the spinal cord. We examined alterations of K ATP channels in rat spinal cord and tested whether and how they could contribute to neuropathic pain. The results showed that protein expression for K ATP channel subunits SUR1, SUR2, and Kir6.1, but not Kir6.2, were significantly downregulated and associated with thermal hyperalgesia and mechanical allodynia after sciatic nerve injury. Spinal administration of a K ATP channel opener cromakalim (CRO, 5, 10, and 20 μg, respectively) prevented or suppressed, in a dose-dependent manner, the hyperalgesia and allodynia. Nerve injury also significantly increased expression and phosphorylation of connexin 43, an astroglial gap junction protein. Such an increase of phosphorylation of connexin 43 was inhibited by CRO treatment. Furthermore, preadministration of an astroglial gap junction decoupler carbenoxolone (10 μg) completely reversed the inhibitory effects of CRO treatment on the hyperalgesia and allodynia and phosphorylation of NR1 and NR2B receptors and the subsequent activation of Ca 2+-dependent signals Ca 2+/calmodulin-dependent kinase II and cyclic adenosine monophosphate (cAMP) response element binding protein. These findings suggest that nerve injury–induced downregulation of the K ATP channels in the spinal cord may interrupt the astroglial gap junctional function and contribute to neuropathic pain, thus the K ATP channels opener can reduce neuropathic pain probably partly via regulating the astroglial gap junctions. This study may provide a new strategy for treating neuropathic pain using K ATP channel openers in the clinic.

Development and Functional Characterization of Insulin-releasing Human Pancreatic Beta Cell Lines Produced by Electrofusion

Three novel human insulin-releasing cell lines designated 1.1B4, 1.4E7, and 1.1E7 were generated by electrofusion of freshly isolated of human pancreatic beta cells and the immortal human PANC-1 epithelial cell line. Functional studies demonstrated glucose sensitivity and responsiveness to known modulators of insulin secretion. Western blot, RT-PCR, and immunohistochemistry showed expression of the major genes involved in proinsulin processing and the pancreatic beta cell stimulus-secretion pathway including PC1/3, PC2, GLUT-1, glucokinase, and K-ATP channel complex (Sur1 and Kir6.2) and the voltage-dependent L-type Ca(2+) channel. The cells stained positively for insulin, and 1.1B4 cells were used to demonstrate specific staining for insulin, C-peptide, and proinsulin together with insulin secretory granules by electron microscopy. Analysis of metabolic function indicated intact mechanisms for glucose uptake, oxidation/utilization, and phosphorylation by glucokinase. Glucose, alanine, and depolarizing concentrations of K(+) were all able to increase [Ca(2+)](i) in at least two of the cell lines tested. Insulin secretion was also modulated by other nutrients, hormones, and drugs acting as stimulators or inhibitors in normal beta cells. Subscapular implantation of the 1.1B4 cell line improved hyperglycemia and resulted in glucose lowering in streptozotocin-diabetic SCID mice. These novel human electrofusion-derived beta cell lines therefore exhibit stable characteristics reminiscent of normal pancreatic beta cells, thereby providing an unlimited source of human insulin-producing cells for basic biochemical studies and pharmacological drug testing plus proof of concept for cellular insulin replacement therapy.

Eicosapentaenoic acid reduces ischemic ventricular fibrillation via altering monophasic action potential in pigs

Although high intake of n-3 fatty acids is associated with reduced mortality of patients with ischemic heart disease, especially reduction in sudden cardiac death (SCD), the detailed mechanisms remain to be elucidated. Thus, the present study was designed to examine whether long-term treatment with eicosapentaenoic acid (EPA), a major component of n-3 fatty acids, reduces ischemia-induced ventricular fibrillation (VF) in pigs in vivo, and if so, what molecular mechanisms are involved. Male pigs were treated with either a control chow (control group) or a control chow plus EPA (600 mg/kg/day, PO, EPA group) for 3 weeks and were subjected to myocardial ischemia for 90 min (n = 8 each) with measurement of the monophasic action potential (MAP), as a marker of ventricular electrophysiological activities. The EPA treatment significantly attenuated the occurrence of VF (control 5.1 ± 1.7 vs. EPA 1.5 ± 0.8 times/animal, P < 0.05) and markedly reduced the mortality (control 50% vs. EPA 0%, P < 0.05), with the attenuation of MAP duration shortening during ischemia (control − 28.1 ± 3.0% vs. EPA − 18.2 ± 1.4%, P < 0.05). These beneficial effects of EPA were abolished by pre-treatment with cromakalim, a K ATP channel opener (0.3 μg/kg/min, IC). Furthermore, EPA significantly inhibited the mRNA and protein expression of Kir6.2, a major component of sarcolemmal K ATP channels, in both the ischemic region and non-ischemic regions. These results indicate that long-term treatment with EPA reduces ischemia-induced VF and SCD in pigs in vivo, for which attenuation of MAP duration shortening may be involved.

Modulation of the 6-position of benzopyran derivatives and inhibitory effects on the insulin releasing process

The synthesis of different series of 4- and 6-substituted R/ S-3,4-dihydro-2,2-dimethyl-2 H-1-benzopyrans is described. All of these new benzopyran derivatives were bearing, at the 4-position, a phenylthiourea moiety substituted on the phenyl ring by a meta or a para-electron-withdrawing group such as Cl or CN. The study aimed at exploring the influence of the nature of the substituent at the 6-position in order to develop new benzopyran-type K ATP channel activators exhibiting an improved selectivity towards the insulin secreting cells. The original compounds were examined in vitro on rat pancreatic islets (inhibition of insulin release) as well as on rat aorta rings (vasorelaxant effect) and their activity was compared to that of the reference K ATP channel activators (±)-cromakalim, (±)-pinacidil, diazoxide and to previously synthesized cromakalim analogues. Structure–activity relationships indicated that the inhibitory effect on the insulin secreting cells was related to the lipophilicity of the molecules and to the size of the substituent located at the 6-position. A marked inhibitory activity on the insulin secretory process was obtained with molecules bearing a bulky tert-butyloxycarbonylamino group at the 6-position ( 20– 23). The latter compounds were found to have the same efficacy on the pancreatic endocrine tissue than some previously described molecules. Lastly, radioisotopic experiments further identified R/ S- N-4-chlorophenyl- N′-(6- tert-butyloxycarbonylamino-3,4-dihydro-2,2-dimethyl-2 H-1-benzopyran-4-yl)thiourea (23) as a K ATP channel opener.

Pharmacological preconditioning with nicorandil and pioglitazone attenuates myocardial ischemia/reperfusion injury in rats

The present investigation was designed to study the cardioprotective effects of nicorandil and pioglitazone preconditioning in myocardial ischemia/reperfusion-induced hemodynamic, biochemical and histological changes in rats. Oral doses of nicorandil (3 or 6 mg/kg) and pioglitazone (10 or 20 mg/kg) were administered once daily for 5 consecutive days. Rats were then subjected to myocardial ischemia/reperfusion (40 min/10 min). Heart rate and ventricular arrhythmias were recorded during ischemia/reperfusion progress. At the end of reperfusion, plasma creatine kinase-MB activity and total nitrate/nitrite were determined. In addition, lactate, adenine nucleotides, thiobarbituric acid reactive substances, reduced glutathione and myeloperoxidase activity were estimated in the heart left ventricle. Finally, histological examination was performed to visualize the protective cellular effects of different pretreatments. Nicorandil (3 or 6 mg/kg) was effective in attenuating the ischemia/reperfusion-induced ventricular arrhythmias, creatine kinase-MB release, lactate accumulation and oxidative stress. Nicorandil (3 mg/kg) was more effective in improving the energy production and lowering the elevated myeloperoxidase activity. Both doses of pioglitazone (10 or 20 mg/kg) were equally effective in reducing lactate accumulation and completely counteracting the oxidative stress. Pioglitazone (10 mg/kg) was more effective in improving energy production and reducing ventricular arrhythmias, plasma creatine kinase-MB release and total nitrate/nitrite. It seems that selective mitochondrial K ATP channel opening by lower doses of nicorandil and pioglitazone in the present study provided more cardioprotection against ventricular arrhythmias and biochemical changes induced by ischemia/reperfusion. Histological examination revealed also better improvement by the lower dose of nicorandil than that of pioglitazone.

Role of reactive oxygen species in the regulation of cardiac contractility

Increased production of reactive oxygen species (ROS) has been linked to the pathogenesis of contractile dysfunction in heart failure. However, it is unclear whether ROS can regulate physiological cellular processes in the myocardium. Here, we characterized the role of endogenous ROS production in the acute regulation of cardiac contractility in the intact rat heart. In isolated perfused rat hearts, endothelin-1 (ET-1, 1 nmol/L) stimulated ROS formation in the left ventricle, which was prevented by the antioxidant N-acetylcysteine and the NAD(P)H oxidase inhibitor apocynin. N-acetylcysteine, the superoxide dismutase mimetic MnTMPyP, and apocynin significantly attenuated ET-1-mediated inotropic effect, which was accompanied by inhibition of extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation. Moreover, the mitochondrial K ATP channel blocker 5-HD, and the mitochondrial large conductance calcium activated potassium channel blocker paxilline, but not the sarcolemmal K ATP channel blocker HMR 1098 attenuated the inotropic response to ET-1. However, ET-1-induced ROS generation was not abolished by inhibiting mitochondrial K ATP channel opening. In contrast to ET-1 stimulation, the positive inotropic effect of β 1-adrenergic receptor agonist dobutamine (250 nmol/L) was significantly augmented by N-acetylcysteine and apocynin. Moreover, dobutamine-induced phospholamban phosphorylation was markedly enhanced by apocynin. In conclusion, NAD(P)H oxidase-derived ROS play a physiological role in the acute regulation of cardiac contractility in the intact rat heart. Our results reveal that ET-1-induced increase in cardiac contractility is partially dependent on enhanced ROS generation, which in turn, activates the ERK1/2 pathway. On the other hand, β-adrenergic receptor-induced positive inotropic effect and phospholamban phosphorylation is enhanced by NAD(P)H oxidase inhibition.

Gastroprotective potential of risperidone, an atypical antipsychotic, against stress and pyloric ligation induced gastric lesions

Risperidone has been used in some stress disorders and may be potentially protective against stress-induced gastric lesions. Thus, the aim of the present study is to investigate, whether risperidone, a D 2 receptor and 5-HT 2A receptor antagonist, would be able to result in gastroprotective effect in stress-induced lesions and also explore the possible mechanism of action behind its gastroprotective activity. Gastroprotective activity of risperidone was evaluated both by single treatment and 21 days repeated (0.03, 0.1, 0.3 and 1 mg/kg, p.o.) treatment in the cold restraint stress (CRS) model and 21 days repeated treatment in the pyloric ligation (PL) model and compared with that of sulpiride (D 2 receptor antagonist) and ketanserin (5-HT 2 receptor antagonist) as standard. Histopathological assessment was done to evaluate the gastroprotective activity of risperidone in CRS model. The roles of nitric oxide (NO), sulfhydryl (SH) group, ATP-sensitive K + channels (K ATP channels) and prostaglandins (PGs) in the gastroprotective effect of risperidone against CRS were also investigated. PGE 2, hexosamine as a marker of mucus barrier and microvascular permeability were also estimated. Results show that repeated treatment of risperidone, sulpiride and ketanserin exhibited a gastroprotective effect against CRS-induced lesions while single administration of risperidone was found to be ineffective. Moreover, repeated treatment of risperidone and ketanserin was found to be ineffective in case of PL in contrast to sulpiride. Risperidone pretreatment reverses the stress induced alteration in hexosamine, PGE 2 and microvascular permeability. Pretreatment with l-NAME, NEM, glibenclamide and indomethacin reversed the gastroprotective effect of risperidone. The results suggest that risperidone has significant gastroprotective effects in CRS-induced gastric lesions models, which appears to be mediated by endogenous NO, SH, PGs and K ATP channel opening.

Levosimendan preserves the contractile responsiveness of hypoxic human myocardium via mitochondrial K ATP channel and potential pERK 1/2 activation

This study investigated the role of levosimendan, a mitochondrial K ATP channel opener, during hypoxia–reoxygenation injury in human isolated tissue. The activation of preconditioning pathways, and the release of mitochondrial cytochrome c were determined. Human right atrial trabeculae were mounted in an organ bath, electrically paced and contractile force measured. Tissue was subjected to hypoxia–reoxygenation, and isoproterenol concentration–response experiments were performed as an index of contractile viability. The intracellular activities of Akt, ERK 1/2, P38, caspase 3, and cytochrome c were assayed by western blot. Following hypoxia–reoxygenation, the maximal contractile response of trabeculae to isoproterenol was significantly increased with levosimendan pretreatment compared to the hypoxia–reoxygenation control (0.88 ± 0.02 versus 0.60 ± 0.01 g, P < 0.01). This enhanced response was blocked by 5-hydroxydecoanate (0.54 ± 0.09 g, P < 0.01). A significant increase in both phosphorylated and total ERK 1/2 and P38 occurred at 60 min reoxygenation, compared to control tissue. No difference was observed in phosphorylated or total Akt, though there was a trend for increased levels in hypoxic tissue. Cytochrome c was detected at 60 min post reoxygenation, in both levosimendan treated and untreated tissue. No increase in cleaved-caspase 3 activity was observed. Our findings suggest that levosimendan preserves the contractile force to isoproterenol after hypoxia–reoxygenation, a response mediated via mK ATP channel activation. The significant increase in the activity of prosurvival mediators ERK 1/2 and P38 following hypoxia indicates a potential mechanism of action for levosimendan-induced cardioprotection.

The protective roles of mitochondrial ATP-sensitive potassium channels during hypoxia–ischemia–reperfusion in brain

The role of ATP-sensitive potassium (K ATP) channels in cerebral ischemia–reperfusion has been well documented. K ATP channel openers protect neuron by mimicking ischemic preconditioning. However, the different protection between the mitochondrial and sarcolemma K ATP openers has been seldom studied. In the experiment, we investigated the effects of K ATP channel openers diazoxide and pinacidil on the hypoxia–ischemia–reperfusion in cultured hippocampal neurons and gerbil brain. The cultured hippocampal neurons and gerbil brain were pretreated with diazoxide or pinacidil before oxygen-glucose deprivation (OGD) and cerebral ischemia–reperfusion, respectively. Survival rate, apoptosis rate and lactate dehydrogenase (LDH) releasing after the reperfusion were subsequently detected. Then the subunits mRNA was detected by RT-PCR. The survival rate and LDH content in diazoxide group increased more than that in pinacidil group (86.21 ± 2.73% vs. 78.59 ± 1.94%, P < 0.05; 133.29 ± 15.00 U/L vs. 193.47 ± 3.39 U/L, P < 0.01). The apoptosis rate in diazoxide group decreased significantly more than that in pinacidil group (23.82 ± 0.14% vs. 37.05 ± 0.67%, P < 0.01). Diazoxide pretreatment increased the expression of Kir6.1 mRNA obviously. The results suggested that mitoK ATP channels opener diazoxide played a major protective role on cerebral ischemia–reperfusion. Furthermore, diazoxide might become a new treatment for cerebral ischemia diseases through increasing the expression of Kir6.1 mRNA.

High-content screening of feeder-free human embryonic stem cells to identify pro-survival small molecules

The propensity of human embryonic stem cells to die upon enzymatic disaggregation or low-density plating is an obstacle to their isolation and routine use in drug discovery and basic research. Equally, the very low rate of establishment of implanted cells hinders cell therapy. In the present study we have developed a high-content assay for human embryonic stem cell survival and used this to screen a range of libraries of 'lead-like' small molecules and known bioactives. From this we identified 18 confirmed hits with four structural classes being represented by multiple compounds: a series of 5-(acyl/alkyl-amino)indazoles, compounds with a 4-(acylamino)pyridine core, simple N⁶,N⁶-dialkyladenines and compounds with a 5-(acylamino)indolinone core. In vitro kinase profiling indicated that the ROCK (Rho-associated kinase)/PRK2 (protein kinase C-related kinase 2) protein kinases are of pivotal importance for cell survival and identified previously unreported compound classes that inhibited this important biological activity. An evaluation using an extensive panel of protein kinases showed that six of our hit compounds exhibited better selectivity for ROCK inhibition than the routinely used commercially available ROCK inhibitor Y-27632. In this screen we also identified the K(+)-ATP channel opener pinacidil and show that it probably promotes cell survival, by 'off-target' inhibition of ROCK/PRK2. We have therefore identified novel pro-survival compounds of greater specificity, equivalent potency and reduced toxicity relative to the routinely employed ROCK inhibitor Y-27632.