Effect of Type-2 Diabetes Mellitus on the Expression and Function of Smooth Muscle ATP-Sensitive Potassium Channels in Human Internal Mammary Artery Grafts.

Here we have shown for the first time altered expression of the vascular smooth muscle (VSM) KATP channel subunits in segments of the human internal mammary artery (HIMA) in patients with type-2 diabetes mellitus (T2DM). Functional properties of vascular KATP channels in the presence of T2DM, and the interaction between its subunits and endogenous ligands known to relax this vessel, were tested using the potassium (K) channels opener, pinacidil. HIMA is the most commonly used vascular graft in cardiac surgery. Previously it was shown that pinacidil relaxes HIMA segments through interaction with KATP (SUR2B/Kir6.1) vascular channels, but it is unknown whether pinacidil sensitivity is changed in the presence of T2DM, considering diabetes-induced vascular complications commonly seen in patients undergoing coronary artery bypass graft surgery (CABG). KATP subunits were detected in HIMA segments using Western blot and immunohistochemistry analyses. An organ bath system was used to interrogate endothelium-independent vasorelaxation caused by pinacidil. In pharmacological experiments, pinacidil was able to relax HIMA from patients with T2DM, with sensitivity comparable to our previous results. All three KATP subunits (SUR2B, Kir6.1 and Kir6.2) were observed in HIMA from patients with and without T2DM. There were no differences in the expression of the SUR2B subunit. The expression of the Kir6.1 subunit was lower in HIMA from T2DM patients. In the same group, the expression of the Kir6.2 subunit was higher. Therefore, KATP channels might not be the only method of pinacidil-induced dilatation of T2DM HIMA. T2DM may decrease the level of Kir6.1, a dominant subunit in VSM of HIMA, altering the interaction between pinacidil and those channels.

Pinacidil ameliorates cardiac microvascular ischemia-reperfusion injury by inhibiting chaperone-mediated autophagy of calreticulin.

Calcium overload is the key trigger in cardiac microvascular ischemia-reperfusion (I/R) injury, and calreticulin (CRT) is a calcium buffering protein located in the endoplasmic reticulum (ER). Additionally, the role of pinacidil, an antihypertensive drug, in protecting cardiac microcirculation against I/R injury has not been investigated. Hence, this study aimed to explore the benefits of pinacidil on cardiac microvascular I/R injury with a focus on endothelial calcium homeostasis and CRT signaling. Cardiac vascular perfusion and no-reflow area were assessed using FITC-lectin perfusion assay and Thioflavin-S staining. Endothelial calcium homeostasis, CRT-IP3Rs-MCU signaling expression, and apoptosis were assessed by real-time calcium signal reporter GCaMP8, western blotting, and fluorescence staining. Drug affinity-responsive target stability (DARTS) assay was adopted to detect proteins that directly bind to pinacidil. The present study found pinacidil treatment improved capillary density and perfusion, reduced no-reflow and infraction areas, and improved cardiac function and hemodynamics after I/R injury. These benefits were attributed to the ability of pinacidil to alleviate calcium overload and mitochondria-dependent apoptosis in cardiac microvascular endothelial cells (CMECs). Moreover, the DARTS assay showed that pinacidil directly binds to HSP90, through which it inhibits chaperone-mediated autophagy (CMA) degradation of CRT. CRT overexpression inhibited IP3Rs and MCU expression, reduced mitochondrial calcium inflow and mitochondrial injury, and suppressed endothelial apoptosis. Importantly, endothelial-specific overexpression of CRT shared similar benefits with pinacidil on cardiovascular protection against I/R injury. In conclusion, our data indicate that pinacidil attenuated microvascular I/R injury potentially through improving CRT degradation and endothelial calcium overload.

Role of ROS in attenuation of hypoxia-reoxygenation injury in rat cardiomyocytes by pinacidil postconditioning: relationship with Nrf2-ARE signaling pathway / 中华麻醉学杂志

Objective:To evaluate the role of reactive oxygen species (ROS) in attenuation of hypoxia-reoxygenation (H/R) injury in rat cardiomyocytes by pinacidil postconditioning and the relationship with nuclear factor erythrid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway.Methods:Adult rat cardiomyocytes were isolated and cultured and then divided into 4 groups ( n=20 each) by a random number table method: control group (group C), H/R group, pinacidil postconditioning group (group P) and reactive oxygen scavenger N-(2-mercaptopropionyl)-glycine(MPG)+ pinacidil postconditioning group (group MPG+ P). Group C was continuously exposed to 95%O 2+ 5%CO 2 in an incubator at 37 ℃ for 105 min. The cells were exposed to 5%CO 2+ 1%O 2+ 94%N 2 in an incubator at 37 ℃ for 45 min followed by reoxygenation for 60 min to prepare H/R injury model. The cells were exposed to hypoxia for 45 min and then treated with pinacidil 50 μmol/L for 5 min followed by reoxygenation for 60 min in group P. The cells were exposed to hypoxia for 45 min, treated with MPG 2 mmol/L for 10 min, and then treated with pinacidil for 5 min followed by reoxygenation for 60 min in group MPG+ P. The content of Ca 2+ and activity of Nrf2 in cardiomyocytes were measured at the end of reoxygenation. The ultrastructure of cardiomyocytes was observed, and mitochondrial ultrastructure was evaluated using mitochondrial Flameng score. The expression of Nrf2, superoxide dismutase (SOD1), quinone oxidoreductase 1 (NQO1), and heme oxygenase 1 (HO-1) protein and mRNA was detected using Western blot and real-time polymerase chain reaction. Results:Compared with group C, the Ca 2+ content, Nrf2 activity and mitochondrial Flameng score were significantly increased, the expression of Nrf2, SOD1, NQO1 and HO-1 protein and mRNA was down-regulated ( P<0.05), and the damage to the ultrastructure of cardiomyocytes was aggravated in group H/R. Compared with H/R group, the Ca 2+ content and mitochondrial Flameng score were significantly decreased, the Nrf2 activity was increased, the expression of Nrf2, SOD1, NQO1 and HO-1 protein and mRNA was up-regulated ( P<0.05), and the damage to the ultrastructure of cardiomyocytes was attenuated in P group. Compared with P group, the Ca 2+ content and mitochondrial Flameng score were significantly increased, the Nrf2 activity was decreased, the expression of Nrf2, SOD1, NQO1 and HO-1 protein and mRNA was down-regulated ( P<0.05), and the damage to the ultrastructure of cardiomyocytes was aggravated in MPG+ P group. Conclusions:ROS is involved in attenuation of H/R injury by pinacidil postconditioning, which is associated with activation of the Nrf2-ARE signaling pathway in rat cardiomyocytes.

Prenatal exposure to alcohol impairs responses of cerebral arterioles to activation of potassium channels: Role of oxidative stress.

BACKGROUND: Potassium channels play an important role in the basal tone and dilation of cerebral resistance arterioles in response to many stimuli. However, the effect of prenatal alcohol exposure (PAE) on specific potassium channel function remains unknown. The first goal of this study was to determine the influence of PAE on the reactivity of cerebral arterioles to activation of ATP-sensitive potassium (KATP ) and BK channels. Our second goal was to determine whether oxidative stress contributed to potassium channel dysfunction of cerebral arterioles following PAE. METHODS: We fed Sprague-Dawley dams a liquid diet with or without alcohol (3% EtOH) for the duration of their pregnancy (21 to 23 days). We examined in vivo responses of cerebral arterioles in control and PAE male and female offspring (14 to 16 weeks after birth) to activators of potassium channels (Iloprost [BK channels] and pinacidil [KATP channels]), before and following inhibition of oxidative stress with apocynin. RESULTS: We found that PAE impaired dilation of cerebral arterioles in response to activation of potassium channels with iloprost and pinacidil, and this impairment was similar in male and female rats. In addition, treatment with apocynin reversed the impaired vasodilation to iloprost and pinacidil in PAE rats to levels observed in control rats. This effect of apocynin also was similar in male and female rats. CONCLUSIONS: PAE induces dysfunction in the ability of specific potassium channels to dilate cerebral arterioles which appears to be mediated by an increase in oxidative stress. We suggest that these alterations in potassium channel function may contribute to the pathogenesis of cerebral vascular abnormalities and/or behavioral/cognitive deficits observed in fetal alcohol spectrum disorders.

Beneficial In Vitro Effects of a Low Myo-Inositol Dose in the Regulation of Vascular Resistance and Protein Peroxidation under Inflammatory Conditions.

Oxidative stress induces functional changes in arteries. Therefore, the effect of myo-inositol, a possible anti-inflammatory/antioxidant agent was studied on human plasma and rat thoracic arteries. Aortic rings from male Wistar rats (3 months of age) were incubated with myo-inositol (1, 10 and 100 µM, 120 min) and analyzed using the gas chromatography (GC) method. In another experiment, aortic rings were protected first with myo-inositol (1 µM, 60 min) and then subjected to a thromboxane receptor agonist (U-46619, 0.1 nM, 60 min). Therefore, these four groups under the following conditions were studied: (i) the control in the vehicle; (ii) myo-inositol; (iii) the vehicle plus U-46619; (iv) myo-inositol plus U-46619. The hemostatic parameters of human plasma and an H2O2/Fe2+ challenge for lipid and protein peroxidation were also performed. Myo-inositol was not absorbed into the pre-incubated aortic rings as measured by the GC method (0.040 µg/mg, p ≥ 0.8688). The effect of myo-inositol was more significant in the impaired arteries due to U-46619 incubation, which resulted in an improved response to acetylcholine (% Emax: 58.47 vs. 86.69), sodium nitroprusside (logEC50: −7.478 vs. −8.076), CORM-2 (% Emax: 44.08 vs. 83.29), pinacidil (logEC50: −6.489 vs. −6.988) and noradrenaline (logEC50: −7.264 vs. −6.525). This was most likely a possible response to increased nitric oxide release (×2.6-fold, p < 0001), and decreased hydrogen peroxide production (×0.7-fold, p = 0.0012). KCl-induced membrane depolarization was not modified (p ≥ 0.4768). Both the plasma protein carbonylation (×0.7-fold, p = 0.0006), and the level of thiol groups (×3.2-fold, p = 0.0462) were also improved, which was not significant for TBARS (×0.8-fold, p = 0.0872). The hemostatic parameters were also not modified (p ≥ 0.8171). A protective effect of myo-inositol was demonstrated against prooxidant damage to human plasma and rat thoracic arteries, suggesting a strong role of this nutraceutical agent on vasculature which may be of benefit against harmful environmental effects.

The Effect of Hemp (Cannabis sativa L.) Seeds and Hemp Seed Oil on Vascular Dysfunction in Obese Male Zucker Rats.

Seeds of industrial hemp (Cannabis sativa L.) contain a large amount of protein (26.3%), dietary fiber (27.5%), and fatty acids (33.2%), including linoleic, α-linolenic, and some amount of γ-linolenic acid. In our study, obese male Zucker rats (n = 6) at 8 weeks of age were supplemented for a further 4 weeks with either ground hemp seeds (12% diet) or lipid fractions in the form of hemp seed oil (4% diet). Hemp oil decreased blood plasma HDL-cholesterol (x0.76, p ≤ 0.0001), triglycerides (x0.55, p = 0.01), and calculated atherogenic parameters. Meanwhile, hemp seeds decreased HDL-cholesterol (x0.71, p ≤ 0.0001) and total cholesterol (x0.81, p = 0.006) but not the atherogenic index. The plasma antioxidant capacity of water-soluble compounds was decreased by the seeds (x0.30, p = 0.0015), which in turn was associated with a decrease in plasma uric acid (x0.18, p = 0.03). Dietary hemp seeds also decreased plasma urea (x0.80, p = 0.02), while the oil decreased the plasma total protein (x0.90, p = 0.05). Hemp seeds and the oil decreased lipid peroxidation in the blood plasma and in the heart (reflected as malondialdehyde content), improved contraction to noradrenaline, and up-regulated the sensitivity of potassium channels dependent on ATP and Ca2+. Meanwhile, acetylcholine-induced vasodilation was improved by hemp seeds exclusively. Dietary supplementation with ground hemp seeds was much more beneficial than the oil, which suggests that the lipid fractions are only partially responsible for this effect.

ROS­associated mechanism of different concentrations of pinacidil postconditioning in the rat cardiac Nrf2­ARE signaling pathway.

Previous studies have confirmed that 50 µmol/l pinacidil postconditioning (PPC) activates the nuclear factor­E2 related factor 2 (Nrf2)­antioxidant responsive element (ARE) pathway, which protects the myocardium from ischemia­reperfusion (IR) injury; however, whether this is associated with reactive oxygen species (ROS) generation remains unclear. In the present study, a Langendorff rat model of isolated myocardial IR was established to investigate the mechanism of PPC at different concentrations, as well as the association between the rat myocardial Nrf2­ARE signaling pathway and ROS. A total of 48 rats were randomly divided into the following six groups (n=8 per group): i) Normal; ii) IR iii) 10 µmol/l PPC (P10); iv) 30 µmol/l PPC (P30); v) 50 µmol/l PPC (P50); and vi) N­(2­mercaptopropionyl)­glycine (MPG; a ROS scavenger) + 50 µmol/l pinacidil (P50 + MPG). At the end of reperfusion (T3), compared with the IR group, the P10, P30 and P50 groups exhibited improved cardiac function, such as left ventricular development pressure, heart rate, left ventricular end­diastolic pressure, +dp/dtmax, myocardial cell ultrastructure and mitochondrial Flameng score. Furthermore, the P10 and P50 groups demonstrated the weakest and most marked improvements, respectively. Additionally, in the P10, P30 and P50 groups, the residual ROS content at the end of reperfusion was highly negatively correlated with relative expression levels of Nrf2 gene and protein. Higher pinacidil concentration was associated with higher ROS generation at 5 min post­reperfusion (T2), although this was significantly lower compared with the IR group, as well as with increased expression levels of antioxidant proteins and phase II detoxification enzymes downstream of the Nrf2 and Nrf2­ARE pathways. This result was associated with a stronger ability to scavenge ROS during reperfusion, leading to lower levels of ROS at the end of reperfusion (T3) and less myocardial damage. The optimal myocardial protective effect was achieved by 50 mmol/l pinacidil. However, cardiac function of the P50 + MPG group was significantly decreased, ultrastructure of cardiomyocytes was significantly impaired and the relative expression levels of genes and proteins in the Nrf2­ARE pathway were decreased. The aforementioned results confirmed that different PPC concentrations promoted early generation of ROS and activated the Nrf2­ARE signaling pathway following reperfusion, regulated expression levels of downstream antioxidant proteins and alleviated myocardial IR injury in rats. Treatment with 50 mmol/l pinacidil resulted in the best myocardial protection.

Pregnancy-induced hypertension decreases Kv1.3 potassium channel expression and function in human umbilical vein smooth muscle.

Voltage-gated potassium (Kv) channels are the largest superfamily of potassium (K) channels. A variety of Kv channels are expressed in the vascular smooth muscle cells (SMC). Studies have shown that gestational diabetes mellitus (GDM) and pregnancy-induced hypertension (PIH) cause various changes in the human umbilical vein (HUV). Recently, we have shown that 4-AP, a nonspecific Kv1-4 channel inhibitor, significantly decreases vasorelaxation induced by K channel opener pinacidil in vascular SMCs of the HUVs from normal pregnancies, but not in GDM and PIH. The goal of this study was to provide more detailed insight in the Kv channel subtypes involved in pinacidil-induced vasodilation of HUVs, as well as to investigate potential alterations of their function and expression during GDM and PIH. Margatoxin, a specific blocker of Kv1.2 and Kv1.3 channels, significantly antagonized pinacidil-induced vasorelaxation in normal pregnancy, while in HUVs from GDM and PIH that was not the case, indicating damage of Kv1.2 and Kv1.3 channel function. Immunohistochemistry and Western blot revealed similar expression of Kv1.2 channels in all groups. The expression of Kv1.3 subunit was significantly decreased in PIH, while it remained unchanged in GDM compared to normal pregnancy. Phrixotoxin, specific blocker of Kv4.2 and Kv4.3 channels, did not antagonize response to pinacidil in any of the groups. The major novel findings show that margatoxin antagonized pinacidil-induced relaxation in normal pregnancy, but not in GDM and PIH. Decreased expression of Kv1.3 channels in HUV during PIH may be important pathophysiological mechanism contributing to an increased risk of adverse pregnancy outcomes.

Resveratrol modulates the blood plasma levels of Cu and Zn, the antioxidant status and the vascular response of thoracic arteries in copper deficient Wistar rats.

Copper (Cu) deficiency plays an important role in the development of cardiovascular disorders. Resveratrol (RSV) possesses pleiotropic cardiovascular benefits; however, the mechanism(s) by which RSV exerts protective effects are not completely understood. Male Wistar rats at 6 weeks of age were fed for 8 weeks with a Cu deficient diet (no added Cu, Cu = 0). In addition, Cu deficient rats were supplemented with RSV (500 mg/kg of diet, n = 9). Blood and intestinal samples were taken for further analysis together with internal organs and thoracic arteries. RSV supplementation resulted in elevated blood plasma levels of Cu (x2.1) and Zn (x1.1), in an increased activity of superoxide dismutase (SOD, x1.5) and ferric reducing antioxidant power (FRAP, x1.2). Meanwhile, markers of lipid peroxidation expressed as malondialdehyde (MDA, x1.5) and lipid hydroperoxides (LOOH, x1.1) were also increased in a significant way. Food intake, body weight, blood glucose, catalase, ceruloplasmin, lipid profile and intestinal samples were not modified. RSV enhanced the vasoconstriction of isolated thoracic arteries to noradrenaline (x1.4), potentiated the vasodilation to acetylcholine (ACh, x1.4) and increased the sensitivity to sodium nitroprusside (SNP). In addition, preincubation with the cyclooxygenase (COX)-inhibitor, indomethacin, potentiated the ACh-induced vasodilation, which was more pronounced in animals not supplemented with RSV. The KATP channel opener, pinacidil, induced a similar response in both studied groups. In conclusion, this study demonstrates that RSV supplementation influences oxidative stress and the antioxidant status, which may modify the vascular response in Cu deficiency.

Effect of gestational diabetes mellitus and pregnancy-induced hypertension on human umbilical vein smooth muscle KATP channels.

Gestational diabetes mellitus (GDM) and pregnancy-induced hypertension (PIH) can jeopardize mother and/or fetus. Vascular ATP-sensitive potassium (KATP) channels most likely participate in the processes of diabetes and hypertension. The aim of this research was to examine whether GDM and PIH cause changes in the expression and function of KATP channels in vascular smooth muscle of human umbilical vein (HUV). Western blot and immunohistochemistry detected significantly decreased expression of Kir6.1 subunit of KATP channels in GDM and PIH, while the expression of SUR2B was unchanged. In GDM, a K+ channel opener, pinacidil caused reduced relaxation of the endothelium-denuded HUVs compared to normal pregnancy. However, its effects in HUVs from PIH subjects were similar to normal pregnancy. In all groups KATP channel blocker glibenclamide antagonized the relaxation of HUV induced by pinacidil without change in the maximal relaxations indicating additional KATP channel-independent mechanisms of pinacidil action. Iberiotoxin, a selective antagonist of large-conductance calcium-activated potassium channels, inhibited the relaxant effect of pinacidil in PIH, but not in normal pregnancy and GDM. Experiments performed in K+-rich solution confirmed the existence of K+-independent effects of pinacidil, which also appear to be impaired in GDM and PIH. Thus, the expression of KATP channels is decreased in GDM and PIH. In GDM, vasorelaxant response of HUV to pinacidil is reduced, while in PIH it remains unchanged. It is very likely that KATP channels modulation and more detailed insight in KATP channel-independent actions of pinacidil may be precious in the therapy of pathological pregnancies.

Copper nanoparticles modify the blood plasma antioxidant status and modulate the vascular mechanisms with nitric oxide and prostanoids involved in Wistar rats.

BACKGROUND: We aimed to analyze whether a diet supplemented with a standard dose of copper (Cu) in the form of nanoparticles, as an alternative to carbonate, exerts beneficial effects within the vasculature and improves the blood antioxidant status. METHODS: Male Wistar rats were fed for 8 weeks with a diet supplemented with Cu (6.5 mg Cu/kg in the diet) either as nanoparticles (40 nm diameter) or carbonate - the control group. Moreover, a negative control was not supplemented with Cu. At 12 weeks of age, blood samples, internal organs and thoracic aorta were taken for further analysis. Blood antioxidant mechanism was measured together with Cu and Zn. RESULTS: Diet with Cu as nanoparticles resulted in an elevated catalase activity and ferric reducing ability of plasma, however decreased Cu (plasma), and ceruloplasmin (Cp) compared to carbonate. The participation of vasoconstrictor prostanoid was increased, as indomethacin did not modify the acetylcholine (ACh)-induced response. Arteries from Cu nanoparticle and carbonate rats exhibited a reduced maximal contraction to potassium chloride and an increased response to noradrenaline. The endothelium-dependent vasodilation to ACh was enhanced while exogenous NO donor, sodium nitroprusside, did not modify the vascular response. Down-regulation of BKCa channels influenced hyperpolarizing mechanism. The superoxide dismutase and HDL-cholesterol were decreased opposite to an increased lipid hydroperoxides, malondialdehyde, Cu (plasma and liver) and Cp. CONCLUSION: Despite the increased antioxidant capacity in blood of Cu nanoparticle fed rats, vasoconstrictor prostanoids and NO are involved in vascular regulation.

Pinacidil, a KATP channel opener, stimulates cardiac Na+/Ca2+ exchanger function through the NO/cGMP/PKG signaling pathway in guinea pig cardiac ventricular myocytes.

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+/Ca2+ exchange current (INCX). In this study, we used the whole-cell patch-clamp technique and Fura-2/AM (Ca2+-indicator) method to investigate the effect of pinacidil on INCX in isolated guinea pig cardiac ventricular myocytes. In the patch-clamp study, pinacidil enhanced INCX in a concentration-dependent manner. The half-maximal effective concentration values were 23.5 and 23.0 µM for the Ca2+ entry (outward) and Ca2+ exit (inward) components of INCX, respectively. The pinacidil-induced INCX 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 INCX increase, while 5-HD, a selective mitochondria KATP channel inhibitor, did not. In the Fura-2/AM study pinacidil also enhanced intracellular Ca2+ 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 INCX increase. Sodium nitroprusside, a NO donor, also increased INCX. In conclusion, pinacidil may stimulate cardiac Na+/Ca2+ exchanger (NCX1) by opening plasma membrane KATP channels and activating the NO/cGMP/PKG signaling pathway.

Investigations on effect of taurine-magnesium coordination compound against SQT2 in isolated hearts of guinea pig / 中国药理学通报

Aim To investigate the effect of taurine-magnesium coordination compound (TMCC) on elec-trocardiogram of isolated guinea pig hearts, hoping to describe a primary research on its characteristic of anti-short QT syndrome. Methods The isolated guinea pig heart was retrograde perfused using Langendorff tech-nique. In order to determine the effects of TMCC on QT interval, transmural dispersion of repolarization, effective refractory period, instability of RR interval and instability of QT interval in the presence of potassi-um channel opener pinacidil, the electrocardiogram of isolated guinea pig hearts was recorded using Biopac physiological recorder. Results The shortened QT in-terval and the effective refractory period induced by pinacidil could be prolonged by TMCC; the increased transmural dispersion of repolarization induced by pinacidil could be decreased by TMCC; the increased instability of RR and QT interval induced by pinacidil could be decreased by TMCC. Conclusion TMCC has the effects of anti-SQT2 by prolonging the QT inter-val and the effective refractory period, reducing the transmural dispersion of repolarization and instability.

Role of mito-KATP channel in pinacidil postconditioning-induced reduction of myocardial ischemia-reperfusion injury in rats: an in vitro experiment / 中华麻醉学杂志

Objective To evaluate the role of mitochondrial KATP (mito-KATP) channel in pinacidil postconditioning-induced reduction of myocardial ischemia-reperfusion (I/R) injury in rats.Methods SPF healthy male Sprague-Dawley rats,aged 16-20 weeks,weighing 250-300 g,were anesthetized with pentobarbital.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95％ O2-5％ CO2 at 36.5-37.5 ℃.Thirty-two Langendorff-perfused hearts were divided into 4 groups (n =8 each) using a random number table:control group (group C),group I/R,pinacidil postconditioning group (group P) and 5-hydroxy decanoic acid plus pinacidil postconditioning group (group 5-HD+P).Myocardial ischemia was induced by interrupting perfusion for 40 min followed by 60 min reperfusion.Immediately after onset of reperfusion,hearts were perfused with K-H solution containing 50 μmol/L pinacidil for 2 min and then with K-H solution for 58 min in group P,hearts were perfused with K-H solution containing 100 μmol/L 5-HD for 5 min,with K-H solution containing 50 μmol/L pinacidil for 2 min and then with K-H solution for 53 min in group 5-HD+P.The heart rate (HR),left ventricular developed pressure (LVDP),left ventricular end-diastolic pressure (LVEDP) and the maximum rate of increase in left ventricular pressure (+dp/dtmax) were recorded at 20 min of equilibration (T1) and at the end of reperfusion (T2).Myocardial tissues were obtained at T2 for determination of myocardial infarct size and for examination of myocardial ultrastructure and Flameng scoring of the mitochondria was performed.Results Compared with group C,the HR,LVDP and +dp/dtmax were significantly decreased,and the LVEDP,myocardial infarct size and mitochondrial Flameng score were increased at T2 in group I/R (P＜0.05).Compared with group I/R,the HR,LVDP and +dp/dtmax were significantly increased and the LVEDP,myocardial infarct size and mitochondrial Flameng score were decreased at T2 (P＜0.05),and the pathological changes of myocardium were significantly attenuated in group P,and no significant change was found in the parameters mentioned above in group 5-HD+P (P＞0.05).Compared with group P,the HR,LVDP and + dp/dtmax were significantly decreased and the LVEDP,myocardial infarct size and mitochondrial Flameng score were increased at T2 (P＜O.05),and the pathological changes of myocardium were accentuated in group 5-HD+P.Conclusion The whole mechanism by which pinacidil postconditioning reduces myocardial I/R injury is related to promoting opening of mito-KATP channel in rats.

ATP-sensitive inwardly rectifying potassium channel modulators alter cardiac function in honey bees.

ATP-sensitive inwardly rectifying potassium (KATP) channels couple cellular metabolism to the membrane potential of the cell and play an important role in a variety of tissue types, including the insect dorsal vessel, making them a subject of interest not only for understanding invertebrate physiology, but also as a potential target for novel insecticides. Most of what is known about these ion channels is the result of work performed in mammalian systems, with insect studies being limited to only a few species and physiological systems. The goal of this study was to investigate the role that KATP channels play in regulating cardiac function in a model social insect, the honey bee (Apis mellifera), by examining the effects that modulators of these ion channels have on heart rate. Heart rate decreased in a concentration-dependent manner, relative to controls, with the application of the KATP channel antagonist tolbutamide and KATP channel blockers barium and magnesium, whereas heart rate increased with the application of a low concentration of the KATP channel agonist pinacidil, but decreased at higher concentrations. Furthermore, pretreatment with barium magnified the effects of tolbutamide treatment and eliminated the effects of pinacidil treatment at select concentrations. The data presented here confirm a role for KATP channels in the regulation of honey bee dorsal vessel contractions and provide insight into the underlying physiology that governs the regulation of bee cardiac function.

Vascular KATP channels mitigate severe muscle O2 delivery-utilization mismatch during contractions in chronic heart failure rats.

The vascular ATP-sensitive K+ (KATP) channel is a mediator of skeletal muscle microvascular oxygenation (PO2mv) during contractions in health. We tested the hypothesis that KATP channel function is preserved in chronic heart failure (CHF) and therefore its inhibition would reduce PO2mv and exacerbate the time taken to reach the PO2mv steady-state during contractions of the spinotrapezius muscle. Moreover, we hypothesized that subsequent KATP channel activation would oppose the effects of this inhibition. Muscle PO2mv (phosphorescence quenching) was measured during 180s of 1-Hz twitch contractions (∼6V) under control, glibenclamide (GLI, KATP channel antagonist; 5mg/kg) and pinacidil (PIN, KATP channel agonist; 5mg/kg) conditions in 16 male Sprague-Dawley rats with CHF induced via myocardial infarction (coronary artery ligation, left ventricular end-diastolic pressure: 18±1mmHg). GLI reduced baseline PO2mv (control: 28.3±0.9, GLI: 24.8±1.0mmHg, p<0.05), lowered mean PO2mv (average PO2mv during the overall time taken to reach the steady-state; control: 20.6±0.6, GLI: 17.6±0.3mmHg, p<0.05), and slowed the attainment of steady-state PO2mv (overall mean response time; control: 66.1±10.2, GLI: 93.6±7.8s, p<0.05). PIN opposed these effects on the baseline PO2mv, mean PO2mv and time to reach the steady-state PO2mv (p<0.05 for all vs. GLI). Inhibition of KATP channels exacerbates the transient mismatch between muscle O2 delivery and utilization in CHF rats and this effect is opposed by PIN. These data reveal that the KATP channel constitutes one of the select few well-preserved mechanisms of skeletal muscle microvascular oxygenation control in CHF.

In vitro effect of nicorandil on the carbachol-induced contraction of the lower esophageal sphincter of the rat.

The lower esophageal sphincter (LES) is a specialized region of the esophageal smooth muscle that allows the passage of a swallowed bolus into the stomach. Nitric oxide (NO) plays a major role in LES relaxation. Nicorandil possesses dual properties of a NO donor and an ATP-sensitive potassium channel (KATP channel) agonist, and is expected to reduce LES tone. This study investigated the mechanisms underlying the effects of nicorandil on the LES. Rat LES tissues were placed in an organ bath, and activities were recorded using an isometric force transducer. Carbachol-induced LES contraction was significantly inhibited by KATP channel agonists in a concentration-dependent manner; pinacidil >> nicorandil ≈ diazoxide. Nicorandil-induced relaxation of the LES was prevented by pretreatment with glibenclamide, whereas N(G)-nitro-l-arginine methyl ester (l-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and iberiotoxin were ineffective at preventing nicorandil-induced LES relaxation. Furthermore, nicorandil did not affect high K(+)-induced LES contraction. Reverse-transcription polymerase chain reaction analysis and immunohistochemistry revealed expression of KCNJ8 (Kir6.1), KCNJ11 (Kir6.2), ABCC8 (SUR1) and ABCC9 (SUR2) subunits of the KATP channel in the rat lower esophagus. These findings indicate that nicorandil causes LES relaxation chiefly by activating the KATP channel, and that it may provide an additional pharmacological tool for the treatment of spastic esophageal motility disorders.

Synthesis, characterization and biological evaluation of benzothiazoles and tetrahydrobenzothiazoles bearing urea or thiourea moieties as vasorelaxants and inhibitors of the insulin releasing process.

A series of 1,3-benzothiazoles (series I) and 4,5,6,7-tetrahydro-1,3-benzothiazoles (series II) bearing an urea or a thiourea moiety at the 2-position were synthesized and tested as myorelaxants and inhibitors of insulin secretion. Several compounds (i.e. 13u and 13v) from series I showed a marked myorelaxant activity. Benzothiazoles bearing a strong electron withdrawing group (NO2, CN) at the 6-position and an alkyl group linked to the urea or the thiourea function at the 2-position were found to be the most potent compounds. The weak vasorelaxant activity of series II compounds evidenced the necessity of the presence of a complete aromatic heterocyclic system. The myorelaxant activity of some active compounds was reduced when measured on aorta rings precontracted by 80 mM KCl or by 30 mM KCl in the presence of 10 µM glibenclamide, suggesting the involvement of KATP channels in the vasorelaxant effect. Some compounds of series I tested on rat pancreatic islets provoked a marked inhibition of insulin secretion, among which 13a exhibited a clear tissue selectivity for pancreatic ß-cells.

Myorelaxant action of fluorine-containing pinacidil analog, flocalin, in bladder smooth muscle is mediated by inhibition of L-type calcium channels rather than activation of KATP channels.

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.

Pinacidil-postconditioning is equivalent to ischemic postconditioning in defeating cardiac ischemia-reperfusion injury in rat.

Ischemic postconditioning (IPO) had been reported as a promising method against myocardial ischemia-reperfusion (I/R) injury, but IPO was later proved with poor clinical benefit. In this study, we compared the protective effects of pinacidil-postconditioning (PPO) and IPO against myocardial I/R injury. Langendorff rat hearts were randomly assigned to one of the following groups (n=8 each): Control group, I/R group (40min ischemia and 60min reperfusion), IPO group (6 successive cycles of 10s reperfusion per 10s occlusion before fully reperfusion), PPO group (perfused with 50µM pinacidil for 5min before reperfusion). Heart performance, infarct size and mitochondrial respiratory function were evaluated, and target genes/proteins of well-known Nuclear Factor-E2 Related Factor 2 (Nrf2) were assessed. Both IPO and PPO preserved heart function and myocardial ultrastructure at the end of reperfusion (all P<0.05 vs. I/R). The expression of Nrf2, NADH-quinone oxidoreductase-1 (NQO1), heme oxygenase 1 (HO-1) and superoxide dismutase 1 (SOD1) were similarly increased after IPO and PPO treatment (all P<0.05 vs. I/R). PPO exerted solid effect in defeating cardiac ischemia-reperfusion injury in rat.