Roles of ATP sensitive potassium channel in modulating gastric tone and accommodation in dogs.

OBJECTIVE: The ATP sensitive potassium (KATP) channel plays an important role in the regulation of resting membrane potential and membrane excitability. The role of the KATP channel in modulating gastric motility is unclear. The aim of this study was to investigate the role and mechanism of the KATP channel in modulating gastric tone and accommodation in dogs. MATERIALS AND METHODS: Gastric volume under a constant pressure reflecting gastric tone was measured using a barostat device in dogs equipped with a gastric cannula. Gastric accommodation was evaluated by the difference in gastric volume before and after a liquid meal. The roles of cholinergic and nitrergic pathways in the inhibitory effect of pinacidil (a KATP opener) were assessed. RESULTS: 1) Pinacidil dose-dependently decreased gastric tone at a dosage of 30 (p = 0.628), 100 (p = 0.013) and 300 µg kg-1 (p < 0.001). 2) Glibenclamide, a KATP blocker, completely blocked the inhibitory effect of pinacidil on gastric tone. 3) Atropine did not block the inhibitory effect of pinacidil on gastric tone but Nω-Nitro-L-arginine methyl ester markedly attenuated the inhibitory effect of pinacidil (p = 0.004). 4) Glibenclamide significantly reduced gastric accommodation (p < 0.001) while pinacidil had no effects on gastric accommodation. 5) Glibenclamide significantly reduced nitric oxide donor sodium nitroprusside-induced gastric relaxation. CONCLUSIONS: These findings indicate that the KATP channel plays an important role in modulating gastric tone and accommodation in dogs. The inhibitory effect of pinacidil on gastric tone was through the nitrergic pathway as well as acting directly on smooth muscle cells.

Functional role for mouse cerebellar NO/cGMP/KATP pathway in ethanol-induced ataxia.

BACKGROUND: We have previously shown that brain adenosine A1 receptors and nitric oxide (NO) play an important role in ethanol (EtOH)-induced cerebellar ataxia (EICA) through glutamate/NO/cGMP pathway. I now report possible modulation of EICA by the cerebellar NO/cGMP/K(ATP) pathway. METHODS: EICA was evaluated by Rotorod in CD-1 male mice. All drugs (K(ATP) activators pinacidil, 0.05, 0.1, 0.5 nmol; minoxidil, 0.01, 0.1, 1.0 pmol; antagonists glipizide/glibenclamide, 0.01, 0.05, 0.1 nmol; NO donor l-arginine, 20 nmol; NOS inhibitors [iNOS] inhibitor L-NAME, 50 nmol; glutamate, 1.5 nmol; adenosine A1 receptor agonist N(6) -cyclohexyladenosine [CHA], 6, 12 pmol; antagonist DPCPX, 0.1 or 0.4 nmol) were given by direct intracerebellar microinfusion via stereotaxically implanted guide cannulas, except EtOH (2 g/kg, i.p.). RESULTS: Pinacidil and minoxidil dose-dependently accentuated, whereas glipizide and glibenclamide markedly attenuated EICA, indicating tonic participation of K(ATP) channels. Glipizide abolished the pinacidil potentiation of EICA, which confirmed both drugs acted via K(ATP) channels. A possible link between K(ATP) channels and glutamate/NO pathway was suggested when (i) CHA (12 pmol) totally abolished l-arginine-induced attenuation of EICA; (ii) L-NAME abolished l-arginine-induced attenuation of EICA associated with further increase in EICA; and (iii) the combined l-arginine and glutamate infusion virtually abolished EICA. Also, whereas CHA abolished glibenclamide-induced attenuation and potentiated pinacidil/minoxidil-induced accentuation of EICA, the effects of DPCPX were just the opposite to those of CHA. CONCLUSIONS: The results with CHA therefore suggest a functional link between K(ATP) and A1 receptors and between K(ATP) and glutamate/NO and as an extension may involve participation of NO/cGMP/K(ATP) pathway in EICA.

Effect of pinacidil on rat ventricular myocytes during transient hypoxia and reperfusion.

The aim of this study was to evaluate the cardioprotective effect of pinacidil postconditioning on rat hearts with transient hypoxia and reperfusion. An acute myocardial anoxia-reperfusion rat model was created by ligating coronary arteries for 10 min and subsequent reperfusion for 60 min. Twenty-four rats in 4 groups received different treatments: normal hearts as control (N = 6), anoxia-reperfusion (A/R) only (N = 6), pinacidil postconditioning (N = 6), and pinacidil plus adenosine triphosphate-sensitive potassium channel inhibitors (glibenclamide) (N = 6). The kinetic parameters and electrophysiological properties, including early apoptosis protein expression changes of Bax, Bcl-2, and FN were examined using the isolated perfusion and patch-clamp technique and immunohistochemistry. The left ventricular systolic pressure and maximum -dp/dt in A/R groups were significantly higher than those in the control group (P < 0.05). The left ventricular developing pressure, maximum +dp/dt, and heart rate in the A/R group were slightly decreased. The pinacidil-postconditioned group has better cardiac function recovery after ischemia/reperfusion than the A/R group (P < 0.01). In addition, using the patch-clamp technique, the mean open time and conductance values are significantly higher in the pinacidil postconditioning group, compared with those in the A/R group. The expression of apoptosis proteins (Bax, FN) increased during A/R, while Bcl-2 protein expression decreased. A significant difference was found in the pinacidil treatment group relative to the A/R group. Pinacidil postconditioning can exert cardioprotective effects on A/R-injured rat hearts, which may indicate a potential application of pinacidil postconditioning to protect A/R-injured hearts.

The effect of pinacidil on postshock activation and ventricular defibrillation threshold in canine hearts.

AIM: To determine the postshock activation patterns with both successful and failed shocks in a canine model of ventricular fibrillation, and whether piniacidil, an early after-depolarization (EAD) inhibitor, altered the defibrillation threshold (DFT) and postshock activation patterns. METHODS: In 6 beagles, a basket catheter with 64 unipolar electrodes was placed in the LV for global endocardial mapping, a monophasic action potential catheter was inserted into the LV apex, and a catheter with the negative electrode in the right ventricle and the positive electrode in the superior vena cava was inserted for defibrillation. The DFT, 90% action potential duration (APD(90)) and activation recovery interval (ARI) were evaluated before and after pinacidil administration (loading dosage 0.5 mg/kg and maintenance dosage 0.5 mg·kg(-1)·h(-1), iv). Electrical heterogeneities were defined with the dispersion of ARI. After successful and failed shocks with near-DFT strength, the earliest postshock activation patterns (focal or nonfocal endocardial activation), interval and location were detected. RESULTS: Pinacidil significantly decreased APD(90) (from 178±16 ms to 168±18 ms) and ARI from (152±10 ms to 143±10 ms) at pacing cycle length of 300 ms. The drug significantly increased VF activation rate (from 10.0±1.9 Hz to 10.8±2.0 Hz). The drug did not affect the dispersion of ARI, neither it changed DFT (baseline: 480±110 V; pinacidil: 425±55 V, P>0.05). The earliest postshock activation arose locally on the LV apical endocardium before and after the drug treatment. Pinacidil significantly prolonged the postshock cycle length of cycles 2 to 5 for the successful episodes but not for the failed episodes. CONCLUSION: Pinacidil increases the postshock cycle length suggesting that EAD may play a role in postshock activation, while it fails to alter DFT suggesting that EAD produced by shock does not determine a defibrillation success or failure.

Role of vascular K(ATP) channels in blood pressure variability after sinoaortic denervation in rats.

AIM: To investigate the role of ATP-sensitive potassium (K(ATP)) channels on blood pressure variability (BPV) in sinoaortic denervated (SAD) rats. METHODS: SAD was performed on male Sprague-Dawley rats 4 weeks before the study. mRNA expression of Kir6.1, Kir6.2 and SUR2 in aorta and mesenteric artery was determined using real-time quantitative polymerase chain reaction, and confirmed at the protein level using Western blotting and laser confocal immunofluorescence assays. Concentration-response curves of isolated aortic and mesenteric arterial rings to adenosine and pinacidil were established. Effects of K(ATP) channel openers and blocker on BPV were examined in conscious SAD rats. RESULTS: Aortic SUR2 expression was significantly greater, while Kir6.1 was lower, in SAD rats than in sham-operated controls. In contrast, in the mesenteric artery both SUR2 and Kir6.1 expression were markedly lower in SAD rats than controls. For both arteries, Kir6.2 expression was indistinguishable between sham-operated and SAD rats. These findings were confirmed at the protein level. Responses of the aorta to both adenosine and pinacidil were enhanced after SAD, while the mesenteric response to adenosine was attenuated. Pinacidil, diazoxide, nicorandil, and glibenclamide significantly decreased BPV. CONCLUSION: These findings indicate that expression of vascular K(ATP) channels is altered by chronic SAD. These alterations influence vascular reactivity, and may play a role in the increased BPV in chronic SAD rats.

K(ATP) channel openers protect mesencephalic neurons against MPP+-induced cytotoxicity via inhibition of ROS production.

Opening of ATP-sensitive potassium (K(ATP)) channels has been demonstrated to exert significant neuroprotection in in vivo and in vitro models of Parkinson's disease (PD), but the exact mechanism remains unclear. In the present study, various K(ATP) channel openers (KCOs) sensitive to diverse K(ATP) subunits were used to clarify the protective role of K(ATP) channel opening in 1-methyl-4-phenylpyridinium (MPP(+))-induced oxidative stress injury in mouse primary cultured mesencephalic neurons. The results showed that pretreatment with nonselective KCO pinacidil (Pin) or diazoxide (Dia), a KCO sensitive to Kir6.2/SUR1 K(ATP) channels, protected mesencephalic neurons, especially dopaminergic neurons, against MPP(+)-induced injury in a concentration-dependent manner. However, cromakalim (Cro), an opener of Kir6.1/SUR2 but not Kir6.2/SUR1 K(ATP) channels, failed to protect against MPP(+)-induced cytotoxicity. Furthermore, Pin and Dia but not Cro significantly suppressed the elevation of reactive oxygen species (ROS) triggered by MPP(+) and prevented the loss of mitochondrial member potential (Delta Psi m) and the release of mitochondrial cytochrome c. Consequently, opening of K(ATP) channels expressed in neurons could protect primary mesencephalic neurons against MPP(+)-induced cytotoxicity via inhibiting ROS overproduction and subsequently ameliorating mitochondrial function.

The nitric oxide-cyclic GMP-protein kinase G-K+ channel pathway participates in the antiallodynic effect of spinal gabapentin.

The possible participation of the nitric oxide (NO)-cyclic GMP-protein kinase G (PKG) pathway on gabapentin-induced spinal antiallodynic activity was assessed in spinal nerve injured rats. Intrathecal gabapentin, diazoxide or pinacidil reduced tactile allodynia in a dose-dependent manner. Pretreatment with NG-L-nitro-arginine methyl ester (L-NAME, non-specific inhibitor of NO synthase NOS), 7-nitroindazole (neuronal NO synthase inhibitor), 1H-[1,2,4] -oxadiazolo [4,3-a] quinoxalin-1-one (ODQ, guanylyl cyclase inhibitor) or (9S, 10R, 12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo-[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT-5823, specific PKG inhibitor), but not NG-D-nitro-arginine methyl ester (D-NAME) or okadaic acid (protein phosphatase 1 and 2 inhibitor) prevented gabapentin-induced antiallodynia. Pinacidil activity was not blocked by L-NAME, D-NAME, 7-nitroindazole, ODQ, KT-5823 or okadaic acid. Moreover, KT-5823, glibenclamide (ATP-sensitive K+ channel blocker), apamin and charybdotoxin (small- and large-conductance Ca2+-activated K+ channel blockers, respectively), but not margatoxin (voltage-gated K+ channel blocker), L-NAME, 7-nitroindazole, ODQ or okadaic acid, reduced diazoxide-induced antiallodynia. Data suggest that gabapentin-induced spinal antiallodynia could be due to activation of the NO-cyclic GMP-PKG-K+ channel pathway.

Effects of pinacidil on electrophysiological properties of epicardial border zone of healing canine infarcts: possible effects of K(ATP) channel activation.

BACKGROUND: K(ATP) channels, activated by ischemia, participate in the arrhythmogenic response to acute coronary occlusion. The function of these channels in border zones of healing infarcts, where arrhythmias also arise, has not been investigated. Do these channels remain maximally activated during infarct healing, or do they downregulate after a period of time? Both might preclude further activation. METHODS AND RESULTS: Myocardial infarction was produced in dogs by ligation of the left anterior descending coronary artery. Impulse propagation in the epicardial border zone (EBZ) of 4-day-old healing infarcts was mapped during administration of pinacidil, a K(ATP) channel activator, directly into the EBZ coronary blood supply. Pinacidil restored conduction and excitability when the EBZ was initially inexcitable and had large regions of block (6 of 8 experiments). This allowed reentrant circuits to form in the EBZ, causing tachycardia (4 of 8 experiments). In hearts with an initially excitable EBZ, pinacidil shortened the effective refractory period and abolished conduction block at short cycle lengths (7 experiments). This effect prevented initiation of reentry (1 of 2 experiments). CONCLUSIONS: The response to pinacidil indicates that K(ATP) channels in the EBZ remain functional and can be activated to influence electrophysiological properties and arrhythmogenesis.

Effects of pinacidil on changes to the microenvironment around the incision site, of a skin/muscle incision and retraction, in a rat model of postoperative pain.

The aim of the present study was to evaluate the influence of the microenvironment around an incision site, on peripheral and central sensitization. The effects of pinacidil activation of ATP-sensitive potassium (KATP) channels prior to skin/muscle incision and retraction (SMIR) surgery were assessed. A total of 24 male Sprague Dawley rats were randomly assigned to four groups: Control, sham (incision operation), SMIR (incision plus retraction 1 h after the skin/muscle incision) and pinacidil (SMIR plus pinacidil). The rats in the pinacidil group were intraperitoneally injected with pinacidil prior to the SMIR procedure. The mechanical withdrawal threshold (MWT) was determined at each time point. The microvessel density (MVD) value was determined by immunohistochemistry, and western blotting was performed to analyze the relative protein expression levels of nerve growth factor (NGF), glucose transporter protein-1 (GLUT1) and C-jun N-terminal kinases. There was a significant reduction in the levels of MVD, GLUT1 and MWT following SMIR surgery as compared with the incision alone, and a significant increase in the NGF protein expression levels. In the SMIR group, the MVD value was significantly increased seven days after surgery, as compared with three days after surgery. Additionally, intraperitoneal administration of pinacidil prior to the SMIR surgery inhibited the SMIR­induced reduction in MWT and MVD and attenuated the SMIR­induced GLUT1 reduction. The results of the present study suggest that the microenvironment around an incision site may affect the development of peripheral and central sensitization. In addition, pinacidil had an inhibitory effect on the formation of the inflammatory microenvironment around the incision site through activation of KATP channels, thereby inhibiting peripheral and central sensitization.

ATP-sensitive potassium channels alleviate postoperative pain through JNK-dependent MCP-1 expression in spinal cord.

Although adenosine triphosphate-sensitive potassium (KATP) channels have been proven to be involved in regulating postoperative pain, the underlying mechanism remains to be investigated. In this study, we aimed to determine the role of spinal KATP channels in the control of mechanical hypersensitivity in a rat pain model, in which rats were subjected to skin/muscle incision and retraction (SMIR) surgery, as well as in LPS-stimulated astrocytes. The results showed that KATP channel subunits Kir6.1, SUR1 and SUR2 were normally expressed in the spinal cord and significantly downregulated after SMIR. SMIR caused a marked increase in monocyte chemoattractant protein-1 (MCP-1) mRNA expression and in the protein level of p-JNK in the spinal cord. Intrathecal administration of a KATP channel opener pinacidil (Pina) suppressed mechanical allodynia after SMIR and significantly downregulated the MCP-1 mRNA expression and the protein level of p-JNK induced by SMIR. Inverted fluorescence microscopy showed that Kir6.1 was co-localized with astrocytes only and SUR2 was co-localized primarily with neurons, in a small amount with astrocytes. Furthermore, in vitro studies showed that following incubation with LPS, the astrocytic MCP-1 mRNA expression and p-JNK content were markedly increased, whereas the mRNA levels of Kir6.1 and SUR2 were significantly downregulated in astrocytes. KATP channel opener pinacidil inhibited the LPS-triggered MCP-1 and p-JNK elevation in rat primary astrocytes. The results suggested that KATP channel opener treatment is an effective therapy for postoperative pain in animals, through the activation of the JNK/MCP-1 pathway in astrocytes.

Altered effects of potassium channel modulation in the coronary circulation in experimental hypercholesterolemia.

OBJECTIVE: To evaluate the role of potassium channels in the regulation of coronary hemodynamics in experimental hypercholesterolemia. BACKGROUND: Potassium (K(+)) channels play an important role in coronary vasoregulation. It has previously been demonstrated that experimental hypercholesterolemia is associated with altered coronary vasomotion; however, the role of K(+) channels in modulating coronary blood flow in this pathophysiologic state has not been evaluated. METHODS AND RESULTS: Pinacidil (group 1, n=5) at 2 microg/kg per min, glibenclamide (group 2, n=5), or N-monomethyl-L-arginine (LNMMA) (group 3, n=4) at 50 microg/kg per min were infused into the left anterior descending artery of pigs prior to and following 10 weeks of 2% cholesterol diet. After 10 weeks of cholesterol feeding, intracoronary pinacidil resulted in a significant increase in coronary blood flow (CBF) and coronary artery diameter (CAD) compared to the normolipidemic state (111+/-10 versus 59+/-12%, and 6+/-1.1 versus 2.7+/-1.0%, respectively, P<0.05 for both comparisons), whereas intracoronary glibenclamide resulted in a significant decrease in CBF and CAD compared to the normolipidemic state (-17+/-5 versus 5+/-6%, and -0.8+/-1.4 versus 3.6+/-1.6%, respectively, P<0.05 for both comparisons). The effect of intracoronary LNMMA on CBF and CAD was significantly attenuated after 10 weeks of cholesterol feeding as compared to the normolipidemic state (-47+/-5.4 versus -0.8+/-6.8%, and -19.4+/-5.7 versus -2.3+/-3.3%, respectively, P<0.05 for both comparisons). Furthermore, pretreatment with intracoronary LNMMA did not alter the CBF response to pinacidil in normal pigs (group 4, n=4) (57.4+/-19 versus 59+/-12%, P=NS). CONCLUSIONS: The current study demonstrates an enhanced effect of coronary K(+) channel modulation and confirms the attenuated basal NO activity previously reported in experimental hypercholesterolemia. Acute withdrawal of basal NO activity alone, however, does not explain the enhanced effect of coronary K(+) channel modulation. These findings underscore the importance of the K(+) channel pathway in the regulation of coronary vasomotor tone in pathophysiologic states.

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

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

Contribution of Na+ -Ca2+ exchanger to pinacidil-induced relaxation in the rat mesenteric artery.

1 Pinacidil relaxes blood vessels through opening the K(ATP) channels with a resultant membrane hyperpolarization and inhibition of Ca(2+) influx. The aim of this study was to examine the mechanisms thereby pinacidil induces K(+) channel-independent relaxation in isolated endothelium-denuded rat mesenteric artery. 2 Pinacidil-induced relaxation was inhibited by glibenclamide (1-10 micro M) in phenylephrine-preconstricted rings, but was unaffected by glibenclamide after inhibition of K(+) channels and VGCCs. Pinacidil-induced K(+) channel-independent relaxation remained unchanged after treatment with cyclopiazonic acid (10 micro M), thapsigargin (1 micro M), ouabain (100 micro M), propranolol (10 micro M), Rp-cAMPS triethylamine (30 micro M), L-NNA (100 micro M), or ODQ (10 micro M). 3 Pinacidil induced more relaxant effect in the presence of nifedipine than in the presence of 60 mM K(+) plus nifedipine. Pretreatment with Na(+)-Ca(2+) exchanger inhibitors, nickel (30-300 micro M) or benzamil (20 micro M) attenuated pinacidil-induced relaxation in normal or in nifedipine-containing solution. Pinacidil (1 micro M) produced less relaxant effect with decreasing extracellular Na(+) concentration. Na(+)-free condition abolished the inhibitory effect of benzamil. Both nickel and benzamil inhibited pinacidil-induced relaxation in the presence of glibenclamide (10 micro M). Nickel (300 micro M) did not affect the relaxant response to sodium nitroprusside. 4 Pinacidil relaxed the rings preconstricted by active phorbol and U46619 with similar potency. 5 The present results indicate that stimulation of the forward mode Na(+)-Ca(2+) exchange pathway is in part responsible for pinacidil-induced K(+) channel-independent vasorelaxation. Pinacidil also induces K(+) channel-dependent but VGCCs-independent relaxation. The PKC-mediated cellular pathway may be a target site for pinacidil only in higher concentrations.

Effect of diabetes on pinacidil-induced antinociception in mice.

The antinociceptive effects of pinacidil, an adenosine triphosphate (ATP)-sensitive K(+)i (K(ATP)) channel opener, were examined using the tail-flick test in non-diabetic and diabetic mice. Pinacidil i.c.v. produced dose-dependent antinociception in both non-diabetic and diabetic mice. There was no significant difference between the antinociceptive effect of i.c.v. pinacidil in non-diabetic mice and diabetic mice. The i.t. administration of pinacidil also produced dose-dependent antinociception in both non-diabetic and diabetic mice, however, the antinociceptive effect of i.t. pinacidil in diabetic mice was significantly greater than that in non-diabetic mice. The antinociceptive effect of i.c.v. or i.t. pinacidil was significantly antagonized by i.c.v. or i.t. glibenclamide, a K(ATP) channel blocker in both non-diabetic and diabetic mice. In non-diabetic mice, the antinociceptive effect of i.c.v. or i.t. administration of pinacidil was significantly antagonized by beta-funaltrexamine, a mu-opioid receptor antagonist, 7-benzylidenenaltrexone, a delta1-opioid receptor antagonist, naltriben, a delta2-opioid receptor antagonist, and nor-binaltorphimine, a kappa-opioid receptor antagonist. In diabetic mice, the antinociceptive effect of i.c.v. pinacidil was significantly reduced by 7-benzylidenenaltrexone, naltriben, and nor-binaltorphimine. However, beta-funaltrexamine had no effect on antinociception induced by i.c.v. pinacidil in diabetic mice. On the other hand, the antinociceptive effect of i.t. pinacidil was significantly antagonized by beta-funaltrexamine, 7-benzylidenenaltrexone, naltriben, and nor-binaltorphimine in diabetic mice. These results indicated that pinacidil produced antinociception through the release of opioid peptides acting at mu-, delta- and kappa-opioid receptors in surpraspinal and spinal cord of non-diabetic mice. On the other hand, in diabetic mice, the antinociception-induced by pinacidil was mediated through the release of opioid peptides acting at delta- and kappa-opioid receptors supraspinally, whereas pinacidil produced antinociception through the release of opioid peptides acting at mu-, delta-, and kappa-opioid receptors spinally.

Monolithic matrix type transdermal drug delivery systems of pinacidil monohydrate: in vitro characterisation.

The monolithic matrix type transdermal drug delivery systems of pinacidil monohydrate (PM) were prepared by film casting technique on mercury substrate and characterised in vitro by drug release studies using paddle over disc assembly, skin permeation studies using Keshary and Chein diffusion cell on albino rat skin and drug-excipient interaction analysis. Four formulations were developed which differed in the ratio of matrix forming polymers, Eudragit RL-100 and PVP K-30, i.e. 8:2, 4:6, 2:8 and 6:4 and were coded as B-1, B-2, B-3 and B-4, respectively. All the four formulations carried 20% w/w of PM, 5% w/w of plasticiser, PEG-400 and 5% w/w of DMSO (based on total polymer weight) in isopropyl alcohol: dichloromethane (40:60) solvent system. Cumulative % of drug released in 48 h from the four formulations was 63.96, 55.95, 52.26 and 92.18%. The corresponding values for cumulative amount of drug permeated for the said formulations were 57.28, 50.35, 46.38 and 86.54%, respectively. On the basis of in vitro drug release and skin permeation performance, formulation B-4 was found to be better than the other three formulations and it was selected as the optimised formulation. The interaction studies carried out by comparing the results of assay, ultraviolet, infrared and TLC analyses for the pure drug, medicated and placebo formulations indicated no chemical interaction between the drug and excipients.

Fabrication and evaluation of polymeric films for transdermal delivery of pinacidil.

The objective of the present work was to fabricate Eudragit RL 100-polyvinyl acetate films and evaluate their potential for transdermal drug delivery in a quest to develop a suitable transdermal therapeutic system for pinacidil. The polymeric films (composed of Eudragit RL100 and polyvinyl acetate in 2:8, 4:6, 6:4, 8:2 ratios in films P-1, P-2, P-3, P-4 respectively, together with 5% w/w of pinacidil and 5% w/w of dibutylphthalate in all the films) were cast on a glass substrate and evaluated for physicochemical parameters viz. thickness, weight, folding endurance (a measure of fragility), percent elongation at break (a measure of flexibility), drug content uniformity, water absorption capacity, moisture vapour transmission, drug-polymer interaction, in vitro drug release and skin permeation profiles. The films were also evaluated for appearance, smoothness and transparency. The film finally selected was assessed for its skin irritation potential, and its stability on storage under accelerated temperature and humidity conditions. The values of thickness, weight, folding endurance, percent elongation at break, percentage water absorbed, moisture vapour transmission, cumulative amount of drug released and permeated for different films were in the following order: P-1 < P-2 < P-3 < P-4. The results suggest that Eudragit RL 100, a freely permeable polymer, has a major influence on the physicochemical profile of the films. The higher the quantity of Eudragit RL100 in the film, the better its strength and flexibility as well as its higher drug release and skin permeation potential. The final optimized film (with a composition of Eudragit RL 100: polyvinyl acetate: pinacidil monohydrate: dibutylphthalate in 8.0:2.0:0.5:0.5 ratio) was found to be the best in terms of drug release (cumulative amount of drug released in 48 h was 96.09%) and skin permeation (permeability coefficient, 0.0164 cm/h). There was no apparent drug-polymer interaction in the films. The optimized film was seemingly free of potentially hazardous skin irritation. The film was found to be stable and intact at ambient temperature and humidity conditions. The films hold promise for the development of a matrix type transdermal therapeutic system for pinacidil.

[The functional state of the kidneys after adenosine triphosphate-sensitive potassium channels activation in experimental acute hypoxia].

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.

Effects of fluorine-containing opener of ATP-sensitive potassium channels, pinacidil-derivative flocalin, on cardiac voltage-gated sodium and calcium channels.

Fluorine-containing pinacidil-derivative flocalin is an effective adenosine triphosphate-sensitive potassium (K(ATP))-channel opener with pronounced vasodilatory, cardioprotective effects and low general toxicity. By activating cardiac K(ATP) channels, flocalin hyperpolarizes cardiac myocytes, decreases their excitability, reduces Ca(2+) entry, and inhibits Ca(2+)-dependent signalling processes. Since our previous studies indicated that the drug also influences the rate of rise and amplitude of the cardiomyocyte's action potential, here we have investigated its possible actions on depolarizing inward currents through voltage-gated sodium (VGSC) and L-type calcium (VGCC) channels. Experiments were conducted on cultured cardiac myocytes prepared from the whole hearts of neonatal rats and maintained in culture for 1-3 days using whole-cell patch-clamp technique with no distinction of myocyte's type. Flocalin concentration dependently inhibited the Na(+) inward current through VGSCs with IC(50) = 17.4 µM and a maximal extent of 0.54, slowed down its inactivation kinetics, and hyperpolarized steady-state inactivation by 5.6 mV. The drug also inhibited calcium current through L-type VGCCs with IC(50) = 24.1 µM and a maximal block of 0.38, without affecting its inactivation but producing 5.3-mV hyperpolarization shifting of steady-state activation. Inhibition of both depolarizing currents by flocalin in addition to its ability to open K(ATP) channels enhances the suppressive action of the drug on cardiac excitability and broadens its pharmacological effects. Since, according to our previous data, cardiac K(ATP)-channel opening by flocalin occurs with ЕC(50) = 8 µM, the possibility of partial blockade of VGSC and L-type VGCCs should be considered when determining the therapeutic concentrations of the compound during its use as a cardioprotector.

[The changes of metabolism in myocardium at ischemia-reperfusion and activating of the ATP-sensitive potassium channels].

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.