Channel Blocker Glibenclamide Research Articles

Mechanisms of the vasorelaxing effects of CORM-3, a water-soluble carbon monoxide-releasing molecule: interactions with eNOS

The purpose of the present work was to elucidate the mechanisms underlying the endothelium-dependent and endothelium-independent components of the vascular relaxation induced by a water-soluble and ruthenium-based carbon monoxide (CO)-releasing agent, tricarbonylchloro(glycinato)ruthenium(II) (CORM-3). Changes in isometric tension and cyclic guanosine monophosphate (cGMP) production were measured in isolated aortic rings from normotensive Wistar-Kyoto rats. Nitric oxide (NO) generation was assessed in cultured human umbilical vein endothelial cells (HUVEC) by electron spin resonance. In rat aortic rings, CORM-3, but not the inactivated compound, iCORM, induced relaxations. In rings with but not in those without endothelium relaxations were partially inhibited by L-nitro-arginine (L-NA), 1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (ODQ), or hydroxocobalamin, inhibitors of NO-synthase, soluble guanylyl cyclase, and scavenger of NO, respectively. In rings with and without endothelium, deoxyhemoglobin abolished the relaxations. A combination of potassium channel blockers (barium, glibenclamide, and iberiotoxin) blunted the relaxation in rings without endothelium. CORM-3 produced an endothelium-dependent generation of cGMP that was inhibited by L-NA. CORM-3, but not iCORM, inhibited the endothelium-dependent relaxation to acetylcholine without affecting the response to sodium nitroprusside. In HUVEC, CORM-3 produced a concentration-dependent release of NO. Therefore, CORM-3-induced relaxations involve the soluble guanylyl cyclase-independent activation of smooth muscle potassium channels. Additionally, CO can produce concomitantly activation and inhibition of NO synthase, the former being responsible for the endothelium- and cGMP-dependent effect of CORM-3, the latter for the inhibition of acetylcholine-induced endothelium-dependent relaxations.

Effect of potential-dependent potassium uptake on calcium accumulation in rat brain mitochondria

The effect of potential-dependent potassium uptake at 0-120 mM K+ on matrix Ca2+ accumulation in rat brain mitochondria was studied. An increase in oxygen consumption and proton extrusion rates as well as increase in matrix pH with increase in K+ content in the medium was observed due to K+ uptake into the mitochondria. The accumulation of Ca2+ was shown to depend on K+ concentration in the medium. At K+ concentration ≤30 mM, Ca2+ uptake is decreased due to K+-induced membrane depolarization, whereas at higher K+ concentrations, up to 120 mM K+, Ca2+ uptake is increased in spite of membrane depolarization caused by matrix alkalization due to K+ uptake. Mitochondrial K+(ATP)-channel blockers (glibenclamide and 5-hydroxydecanoic acid) diminish K+ uptake as well as K+-induced depolarization and matrix alkalization, which results in attenuation of the potassium-induced effects on matrix Ca2+ uptake, i.e. increase in Ca2+ uptake at low K+ content in the medium due to the smaller membrane depolarization and decrease in Ca2+ uptake at high potassium concentrations because of restricted rise in matrix pH. The results show the importance of potential-dependent potassium uptake, and especially the K+(ATP) channel, in the regulation of calcium accumulation in rat brain mitochondria.

Warifteine, a bisbenzylisoquinoline alkaloid, induces relaxation by activating potassium channels in vascular myocytes

The present study used functional and electrophysiological approaches to investigate the mechanisms by which warifteine, a bisbenzylisoquinoline alkaloid isolated from Cissampelos sympodialis Eichl., causes vasorelaxation of the rat thoracic aorta. Warifteine (1 pmol/L-10 μmol/L) induced concentration-dependent relaxation (pD(2) = 9.40 ± 0.06; n = 5) of endothelium-intact aortic rings precontracted with noradrenaline (10-100 μmol/L). The relaxation effects were not attenuated by removal of the endothelium. Warifteine also induced the relaxation of prostaglandin F(2α) (1-10 mmol/L)-precontracted rings (pD(2) = 9.2 ± 0.2; n = 8). In contrast, the relaxant activity of warifteine was nearly abolished in high K(+) (80 mmol/L)-precontracted aortic rings. In preparations incubated with 20 mmol/L KCl or with the K(+) channel blockers tetraethylammonium (1, 3 and 5 mmol/L), iberiotoxin (20 nmol/L), 4-aminopyridine (1 mmol/L) or glibenclamide (10 μmol/L), the vasorelaxant activity of warifteine was markedly reduced. However, BaCl(2) (1 mmol/L) had no effect on the relaxant effects of warifteine. In vascular myocytes, warifteine (100 nmol/L) significantly increased whole-cell K(+) currents (at 70 mV). Under nominally Ca(2+) -free conditions, warifteine did not reduce extracellular Ca(2+) -induced contractions in rings precontracted with high K(+) or noradrenaline (100 μmol/L). Together, the results of the present study indicate that warifteine induces potent concentration-dependent relaxation in the rat aorta via an endothelium-independent mechanism that involves the activation of K(+) channels.

Ex Vivo Study of the Vasorelaxant Activity Induced by Phenanthrene Derivatives Isolated from Maxillaria densa

The phenanthrenes gymnopusin (1), fimbriol A (2), and erianthridin (3) from Maxillaria densa were found to induce significant relaxant effects in a concentration-dependent and endothelium-independent manner on aortic rings precontracted with norepinephrine (NE, 0.1 μM) and KCl (80 mM). Compound 1 was the most active and also inhibited the cumulative concentration-response contraction of NE or CaCl(2). Contractions induced by FPL 64176, an agonist of L-type voltage-dependent calcium channels, were blocked by 1. The potassium channel blockers glibenclamide and TEA (tetraethylammonium) reduced the relaxations induced by 1. Nevertheless, the effect of 1 was not modified by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a specific soluble guanylate cyclase inhibitor. The functional results obtained suggest that 1 induces relaxation through an endothelium-independent pathway by the control of cationic channels (calcium channel blockade and potassium channel opening) in the myogenic response of rat aortic rings.

Coronary response to diadenosine triphosphate after ischemia–reperfusion in the isolated rat heart

Diadenosine triphosphate (Ap3A) is a vasoactive mediator stored in platelet granules that may be released during coronary ischemia-reperfusion. To study its coronary effects in such circumstances, rat hearts were perfused in a Langendorff preparation and the coronary response to Ap3A (10(-7)-10(-5) mol/L) was recorded. Both at basal coronary resting tone and after precontraction with 11-dideoxy-1a,9a-epoxymethanoprostaglandin F(2)(α) (U46619), Ap3A produced concentration-dependent vasodilation in the heart, which was attenuated following ischemia-reperfusion. Ap3A-induced relaxation was also attenuated in control conditions and after ischemia-reperfusion by the purinergic P2Y antagonist reactive blue 2 (2 × 10(-6) mol/L), the P2Y(1) antagonist MRS 2179 (10(-5) mol/L), the nitric oxide synthesis inhibitor N-omega-nitro-l-arginine methyl ester (l-NAME; 10(-4) mol/L) and the ATP-dependent potassium channel blocker glibenclamide (10(-5) mol/L). These results suggest that Ap3A induces coronary vasodilation, an effect attenuated by ischemia-reperfusion due to the functional impairment of purinergic P2Y receptors and K(ATP) channels, and/or reduced nitric oxide release. This impairment of vasodilation may contribute to the coronary dysregulation that occurs during ischemia-reperfusion.

Arecoline improves vascular endothelial function in high fructose-fed rats via increasing cystathionine-γ-lyase expression and activating KATP channels

To investigate the effect of arecoline, a major component of betel nut, on vascular endothelial function in high fructose-fed rats and the potential mechanisms underlying the effect. Male Wistar rats were fed a high-fructose or control diet for 16 weeks. At the beginning of week 13, the rats were injected ip with low (0.5 mg·kg(-1)·d(-1)), medium (1.0 mg·kg(-1)·d(-1)) or high (5.0 mg·kg(-1)·d(-1)) doses of arecoline for 4 weeks. At the termination of the treatments, blood was collected, fasting blood glucose (FBG) and serum insulin (FSI) levels were measured, and insulin sensitivity index (ISI) was calculated. The thoracic aortas were isolated and aortic rings were prepared for studying ACh-induced endothelium-dependent vasorelaxation (EDVR). The mRNA and protein expression of cystathionine-γ-lyase (CSE) in the thoracic aortas was analyzed using RT-PCR and Western blot analysis, respectively. In high fructose-fed rats, the levels of FBG and FSI were remarkably increased, whereas the ISI and the mRNA and protein expression of CSE were significantly decreased. ACh-induced EDVR in the aortic rings from high fructose-fed rats was remarkably reduced. These changes were reversed by treatment with high dose arecoline. Pretreatment of the aortic rings rings from high fructose-fed rats with the CSE inhibitor propargylglycine (10 mmol/L) or the ATP-sensitive potassium (K(ATP)) channel blocker glibenclamide (10 mmol/L) abolished the restoration of ACh-induced EDVR by high dose arecoline. On the contrary, treatment with high dose arecoline significantly impaired ACh-induced EDVR in the aortic rings from control rats, and pretreatment with propargylglycine or glibenclamide did not cause further changes. Arecoline treatment improves ACh-induced EDVR in high fructose-fed rats, and the potential mechanism of action might be associated with increase of CSE expression and activation of K(ATP) channels by arecoline.

The Vasorelaxant Mechanisms of a Rho Kinase Inhibitor DL0805 in Rat Thoracic Aorta

Rho-kinase has been suggested as a potential therapeutic target in the treatment of cardiovascular diseases. The Rho-kinase signaling pathway is substantially involved in vascular contraction. The aim of the present study was to evaluate the vasorelaxant effects of Rho kinase inhibitor DL0805 in isolated rat aortic rings and to investigate its possible mechanism(s). It was found that DL0805 exerted vasorelaxation in a dose-dependent manner in NE or KCl-induced sustained contraction and partial loss of the vasorelaxation under endothelium-denuded rings. The DL0805-induced vasorelaxation was significantly reduced by the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester, the guanylate cyclase inhibitor methylene blue and the cyclooxygenase inhibitor indomethacin. The voltage-dependent K+ channel blocker 4-aminopyridine remarkably attenuated DL0805-induced relaxations. However, the ATP-sensitive K+ channel blocker glibenclamide and Ca2+-activated K+ channel blocker tetraethylammonium did not affect the DL0805-induced relaxation. In the endothelium-denuded rings, DL0805 also reduced NE-induced transient contraction and inhibited contraction induced by increasing external calcium. These findings suggested that DL0805 is a novel vasorelaxant compound associated with inhibition of Rho/ROCK signaling pathway. The NO-cGMP pathway may be involved in the relaxation of DL0805 in endothelium-intact aorta. The vasorelaxant effect of DL0805 is partially mediated by the opening of the voltage-dependent K+ channels.

The Relaxant Action of Nicorandil in Bovine Tracheal Smooth Muscle

The relaxant mechanisms of nicorandil were examined by comparing its effects with those of sodium nitroprusside and cromakalim in bovine tracheal smooth muscle. In preparations contracted with methacholine (0.3 µmol/l) or high K⁺ (40 mmol/l), nicorandil and sodium nitroprusside caused concentration-dependent relaxations. Their relaxant effects on high K⁺-contracted preparations were smaller than those on methacholine-contracted muscle. Cromakalim relaxed methacholine-contracted preparations, whereas it had no effect on high K⁺-contracted muscle. The inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 µmol/l) completely prevented the relaxation induced by lower concentrations (<30 µmol/l) of nicorandil, whereas it partially attenuated relaxation caused by higher concentrations. The ATP-sensitive K⁺ (K_ATP) channel blocker glibenclamide only partially attenuated the relaxant responses to nicorandil (at 100 and 300 µmol/l). Combination treatment with ODQ and glibenclamide almost completely prevented nicorandil-induced relaxations. The large-conductance Ca²⁺-activated K⁺ channel (Maxi K⁺ channel) inhibitor iberiotoxin significantly prevented the relaxations induced by lower concentrations (3 and 10 µmol/l) of nicorandil. The preventive effect of iberiotoxin was markedly enhanced under the blockade of K_ATP channels with glibenclamide. These results suggest that nicorandil relaxes bovine tracheal smooth muscle through 2 mechanisms: opening of K_ATP channels and activation of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. Nicorandil may also activate Maxi K⁺ channels, possibly through the NO-cGMP pathway, and the interaction of K_ATP channels and Maxi K⁺ channels may affect the relaxant effect of nicorandil in bovine tracheal smooth muscle.

Non-genomic vasorelaxant effects of 17β-estradiol and progesterone in rat aorta are mediated by L-type Ca2+ current inhibition

The sex hormones 17β-estradiol (βES) and progesterone (PRG) induce rapid non-genomic vasodilator effects which could be protective for the cardiovascular system. The purpose of this study was to analyze the mechanisms underlying their vasodilator effect in rat aortic smooth muscle preparations. Endothelium-denuded aorta artery rings were prepared from male Wistar rats and incubated in an organ bath. The contractions of the preparation were recorded through isometric transducers. The effects of the hormones on K(+) current and L-type Ca(2+) current (LTCC) were analyzed by using the whole cell voltage-clamp technique in A7r5 cells. Both βES and PRG (1-100 μmol/L) concentration-dependently relaxed the endothelium-denuded aortic rings contracted by (-)-Bay K8644 (0.1 μmol/L) or by KCl (60 mmol/L). The IC(50) values of the two hormones were not statistically different. The K(V) channel blocker 4-aminopyridine (2 mmol/L), BK(Ca) channel blocker tetraethylammonium (1 mmol/L) and K(ATP) channel blocker glibenclamide (10 μmol/L) did not significantly modify the relaxant effect of the hormones. On the other hand, the blockage of the intracellular βES and PRG receptors with estradiol receptor antagonists ICI 182,780 (1 μmol/L) and PRG receptor antagonist mifepristone (30 μmol/L), respectively, did not significantly modify the relaxant action of the hormones. In A7r5 cells, both the hormones (1-100 μmol/L) rapidly and reversibly inhibited the basal and BAY-stimulated LTCC. However, these hormones had no effect on the basal K(+) current. The vasorelaxant effects of βES and PRG are due to the inhibition of LTCC. The K(+) channels are not involved in the effects.

Endothelium‐dependent and independent effects of isoquercitrin in rat mesenteric bed involve nitric oxide production and direct activation of ATP‐sensitive K+ channels

The vascular effect of isoquercitrin (ISQ), a flavonoid with putative cardioprotective effects, was investigated in perfused mesenteric vascular bed removed from naïve Wistar rats (females, 3–4 months). The perfusion pressure (PP) was measured under continuous flux of physiological saline solution (PSS, 4 ml/min) containing phenylephrine (10 μM). In endothelium‐intact tissue, PP was reduced by ISQ around 13, 33 and 60% at doses of 0.1, 0.3 and 1 μmol, respectively. Chemical removal of endothelium by sodium deoxycholate, as well as L‐NAME, an inhibitor of nitric oxide (NO) synthase, and the K+ channel blockers tetraethylammonium or iberiotoxin, partially avoided (~40%) the effects of 1 μmol of ISQ. The ATP‐sensitive K+ channel blocker glibenclamide (GLB, 10 μM) vanished the effects of ISQ at 0.1 and 0.3 μmol, but was ineffective against 1 μmol. However, infusion of GLB + L‐NAME, or high K+ (40 mM) solution, fully prevented the effects of ISQ. These results suggest that, in addition to previously showed angiotensin‐converting enzyme inhibition, the vascular effects of ISQ includes direct opening of ATP‐sensitive K+ channels and increased production of NO in resistance arteries.Research support: DEGPP/UNIPAR and CNPq/Brazil.

Impaired K+ channel function leads to increased catecholamine secretion by adrenal chromaffin cells in DOCA‐salt hypertension

Sympathetic nerve activity is increased in deoxycorticosterone acetate (DOCA) salt model of hypertension and catecholamine secretion from adrenal chromaffin cells is increased in DOCA‐salt rats. This includes the number of vesicles released by stimulation (acetylcholine, 1 mM). We tested the hypothesis that individual chromaffin granule size was increased and that K+ channel function was impaired in chromaffin cells from DOCA‐salt rats. Using the charge value obtained from individual events a vesicle radius can be determined by taking the cubed root of the charge. These data showed a positive shift in the mean Q1/3 value for DOCA‐salt release events compared to sham events suggesting that the increase in charge observed is due in part to increased vesicle radii. To determine why DOCA‐salt cells have increased release frequency compared to sham cells, K+ channel function was investigated. We used the K+ channel blockers paxilline (BK), 4‐aminopyridine (Kv1), glibenclamide (ATP‐sensitive) and apamin (SK). Paxilline and glibenclamide (but not apamin or glibenclamide) increased the number of secretory events and total number of catecholamines oxidized from sham chromaffin cells, however did not effect release from DOCA‐salt cells. We conclude that increased vesicle radius and impaired potassium channel function contribute to increased secretion and quantal charge in DOCA‐salt chromaffin cells.

Participation of the NO/cGMP/K+ATP pathway in the antinociception induced by Walker tumor bearing in rats

Implantation of Walker 256 tumor decreases acute systemic inflammation in rats. Inflammatory hyperalgesia is one of the most important events of acute inflammation. The L-arginine/NO/cGMP/K+ATP pathway has been proposed as the mechanism of peripheral antinociception mediated by several drugs and physical exercise. The objective of this study was to investigate a possible involvement of the NO/cGMP/K+ATP pathway in antinociception induced in Walker 256 tumor-bearing male Wistar rats (180-220 g). The groups consisted of 5-6 animals. Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. Walker tumor (4th and 7th day post-implantation) reduced prostaglandin E2- (PGE2, 400 ng/paw; 50 µL; intraplantar injection) and carrageenan-induced hypernociception (500 µg/paw; 100 µL; intraplantar injection). Walker tumor-induced analgesia was reversed (99.3% for carrageenan and 77.2% for PGE2) by a selective inhibitor of nitric oxide synthase (L-NAME; 90 mg/kg, ip) and L-arginine (200 mg/kg, ip), which prevented (80% for carrageenan and 65% for PGE2) the effect of L-NAME. Treatment with the soluble guanylyl cyclase inhibitor ODQ (100% for carrageenan and 95% for PGE2; 8 µg/paw) and the ATP-sensitive K+ channel (KATP) blocker glibenclamide (87.5% for carrageenan and 100% for PGE2; 160 µg/paw) reversed the antinociceptive effect of tumor bearing in a statistically significant manner (P < 0.05). The present study confirmed an intrinsic peripheral antinociceptive effect of Walker tumor bearing in rats. This antinociceptive effect seemed to be mediated by activation of the NO/cGMP pathway followed by the opening of KATP channels.

Nicorandil Attenuates Monocrotaline-Induced Vascular Endothelial Damage and Pulmonary Arterial Hypertension

BackgroundAn antianginal KATP channel opener nicorandil has various beneficial effects on cardiovascular systems; however, its effects on pulmonary vasculature under pulmonary arterial hypertension (PAH) have not yet been elucidated. Therefore, we attempted to determine whether nicorandil can attenuate monocrotaline (MCT)-induced PAH in rats.Materials and MethodsSprague-Dawley rats injected intraperitoneally with 60 mg/kg MCT were randomized to receive either vehicle; nicorandil (5.0 mg·kg−1·day−1) alone; or nicorandil as well as either a KATP channel blocker glibenclamide or a nitric oxide synthase (NOS) inhibitor N ω-nitro-l-arginine methyl ester (l-NAME), from immediately or 21 days after MCT injection. Four or five weeks later, right ventricular systolic pressure (RVSP) was measured, and lung tissue was harvested. Also, we evaluated the nicorandil-induced anti-apoptotic effects and activation status of several molecules in cell survival signaling pathway in vitro using human umbilical vein endothelial cells (HUVECs).ResultsFour weeks after MCT injection, RVSP was significantly increased in the vehicle-treated group (51.0±4.7 mm Hg), whereas it was attenuated by nicorandil treatment (33.2±3.9 mm Hg; P<0.01). Nicorandil protected pulmonary endothelium from the MCT-induced thromboemboli formation and induction of apoptosis, accompanied with both upregulation of endothelial NOS (eNOS) expression and downregulation of cleaved caspase-3 expression. Late treatment with nicorandil for the established PAH was also effective in suppressing the additional progression of PAH. These beneficial effects of nicorandil were blocked similarly by glibenclamide and l-NAME. Next, HUVECs were incubated in serum-free medium and then exhibited apoptotic morphology, while these changes were significantly attenuated by nicorandil administration. Nicorandil activated the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways in HUVECs, accompanied with the upregulation of both eNOS and Bcl-2 expression.ConclusionsNicorandil attenuated MCT-induced vascular endothelial damage and PAH through production of eNOS and anti-apoptotic factors, suggesting that nicorandil might have a promising therapeutic potential for PAH.

Site-specific regulation of ion transport by prolactin in rat colon epithelium

The effect of prolactin (PRL) on ion transport across the rat colon epithelium was investigated using Ussing chamber technique. PRL (1 μg/ml) induced a sustained decrease in short-circuit current (I(sc)) in the distal colon with an EC(50) value of 100 ng/ml and increased I(sc) in the proximal colon with an EC(50) value of 49 ng/ml. In the distal colon, the PRL-induced decrease in I(sc) was not affected by Na(+) channel blocker amiloride or Cl(-) channel blockers, NPPB, DPC, or DIDS, added mucosally. However, the response was inhibited by mucosal application of K(+) channel blockers glibenclamide, quinidine, and chromanol 293B, whereas other K(+) channel blockers, Ba(2+), tetraethylammonium, clotrimazole, and apamin, failed to have effects. The PRL-induced decrease in I(sc) was also inhibited by Na(+)-K(+)-2Cl(-) transporter inhibitor bumetanide, Ba(2+), and chromanol 293B applied serosally. In the transverse and proximal colon, the PRL-induced increase in I(sc) was suppressed by DPC, glibenclamide, and bumetanide, but not by NPPB, DIDS, or amiloride. The PRL-induced changes in I(sc) in both distal and proximal colon were abolished by JAK2 inhibitor AG490, but not BAPTA-AM, the Ca(2+) chelating agent, or phosphatidylinositol 3-kinase inhibitor wortmannin. These results suggest a segment-specific effect of PRL in rat colon, by activation of K(+) secretion in the distal colon and activation of Cl(-) secretion in the transverse and proximal colon. Both PRL actions are mediated by JAK-STAT-dependent pathway, but not phosphatidylinositol 3-kinase pathway or Ca(2+) mobilization. These findings suggest a role of PRL in the regulation of electrolyte transport in mammalian colon.

Glutamate oxidative injury to RGC-5 cells in culture is necrostatin sensitive and blunted by a hydrogen sulfide (H2S)-releasing derivative of aspirin (ACS14)

Oxidative stress to RGC-5 cells in culture was delivered by exposure to a combination of glutamate (Glu) and buthionine-S,R-sulfoximine (BSO). The effect of the insult on cell survival was quantified by the resazurin-reduction and a dead/live assays. Moreover, breakdown of DNA, the localisation of phosphatidylserine and reactive radical species (ROS) and its quantification were determined. In addition, various proteins and mRNAs were studied using Western blot, real time PCR and immunocytochemistry. ACS14, its sulfurated moiety ACS1 and aspirin were tested for their ability to blunt the negative effects of Glu/BSO on RGC-5 cells. In addition assays were carried out to see whether any of these substances influenced glutathione (GSH).Glu/BSO dose-dependently kills RGC-5 cells by a mechanism that involves an elevation of ROS accompanied by a breakdown of DNA, expression of phosphatidylserine and the activation of p38 MAPK. The process is unaffected by the pan caspase inhibitor z-VAD-fmk, does not involve the activation of apoptosis inducing factor (AIF) but is sensitive to active necrostatin-1. In cell viability studies (resazurin-reduction assay), ACS1 and ACS14 equally counteracted the negative effects of 5mM Glu/BSO to RGC-5 cells but aspirin was only effective with a milder oxidative stress (1mM Glu/BSO). In all other assays ACS14 was very much more effective than aspirin at counteracting the influence of 5mM Glu/BSO. Moreover, ACS14 and ACS1 directly stimulated GSH while aspirin was ineffective. In addition the neuroprotecive effect of ACS14 was specifically blunted by the non-specific potassium channel blocker glibenclamide. Also the up-regulation of Bcl-2, HO-1 and XIAP induced by 5mM Glu/BSO were all attenuated to a greater extent by ACS14 (20μM) than aspirin (20μM). These data show that ACS14 is a very effective neuroprotectant when compared with aspirin. ACS14 maintains its aspirin characteristics and has the ability to release H2S. The combined multiple actions of aspirin and H2S in the form of ACS14 is worthy to consider for possible use in the treatment of glaucoma.

PPARα-Independent Arterial Smooth Muscle Relaxant Effects of PPARαAgonists

We sought to determine direct vascular effects of peroxisome proliferator-activated receptor alpha (PPARα) agonists using isolated mouse aortas and middle cerebral arteries (MCAs). The PPARα agonists GW7647, WY14643, and gemfibrozil acutely relaxed aortas held under isometric tension and dilated pressurized MCAs with the following order of potency: GW7647≫WY14643>gemfibrozil. Responses were endothelium-independent, and the use of PPARα deficient mice demonstrated that responses were also PPARα-independent. Pretreating arteries with high extracellular K+ attenuated PPARα agonist-mediated relaxations in the aorta, but not in the MCA. In the aorta, the ATP sensitive potassium (KATP) channel blocker glibenclamide also impaired relaxations whereas the other K+ channel inhibitors, 4-aminopyridine and Iberiotoxin, had no effect. In aortas, GW7647 and WY14643 elevated cGMP levels by stimulating soluble guanylyl cyclase (sGC), and inhibition of sGC with ODQ blunted relaxations to PPARα agonists. In the MCA, dilations were inhibited by the protein kinase C (PKC) activator, phorbol 12,13-dibutyrate, and also by ODQ. Our results demonstrated acute, nonreceptor-mediated relaxant effects of PPARα agonists on smooth muscle of mouse arteries. Responses to PPARα agonists in the aorta involved KATP channels and sGC, whereas in the MCA the PKC and sGC pathways also appeared to contribute to the response.

Magnesium increases iberiotoxin-sensitive large conductance calcium activated potassium currents on the basilar artery smooth muscle cells in rabbits

Objectives: Magnesium has been known for treating vasospasm following subarachnoid hemorrhage. However, its action mechanism in cerebral vascular relaxation is not clear. Potassium channels play a pivotal role in the relaxation of smooth muscle cells. To investigate their role in magnesium-induced relaxation of basilar smooth muscle cells, we examined the effect of magnesium on potassium channels using the patch clamp technique on acutely isolated smooth muscle cells from rabbit basilar artery.Method: Fresh smooth muscle cells were isolated from the basilar artery by enzyme treatment. To identify which potassium channels are involved in the magnesium-induced currents, we used the potassium channel blockers tetraethylammonium (TEA), glibenclamide, apamin and iberiotoxin (IBX).Results: Magnesium (5 mM) increased the step pulse-induced outward K+ currents by 46% over control level (P<0·01). The outward K+ current was decreased to 22% (P<0·01) by TEA (10 mM), a non-specific K+ channel blocker, and to 60% of control level (P<0·01) by IBX (0·1 μM,), a large-conductance Ca2+-activated K+ (BK) channel blocker, but was not inhibited by apamin (1 nM), a small-conductance Ca2+-activated potassium (SK) channel blocker, or glibenclamide (3 mM), an adenosine triphosphate (ATP)-sensitive K+ channel blocker. Caffeine (3 mM) enhanced outward K+ currents. Magnesium-induced increase of outward K+ currents persisted in the presence of apamin. However, magnesium failed to increase the outward K+ currents in the presence of IBX.Discussion: These results demonstrate that BK channels are functionally expressed in rabbit basilar smooth muscle cells and suggest that BK channels may play a pivotal role in magnesium-induced relaxation.

Synthesis and Vasorelaxant Effect of 9-aryl-1,8-acridinediones asPotassium Channel Openers in Isolated Rat Aorta.

ATP-sensitive potassium (KATP) channel openers have a relaxation effect due to the lower cellular membrane potential and inhibit calcium influx. There has been considerable interest in exploring KATP channel openers in the treatment of various diseases such as cardiovascular, cerebrovascular, and urinary system disease and premature labor. The purpose of this study was to synthesize 3,3,6,6-tetramethy l-9-aryl-octahydro-1,8-acridindiones and investigate their effects on vascular potassium channels and mechanism of induced relaxations on phenylephrine-induced contractile responses in isolated rings of rat aortic smooth muscle. In this study, four new derivatives of 3,3,6,6-tetramethy l-9-aryl-octahydro-1,8-acridindione [2a-d] were synthesized by the reaction of 5, 5-dimethyl-1,3-cyclohexanedione with an aromatic aldehyde, 2-alkylthio-1-(4-fluorobenzyl)-5-formylimidazole or 3-substituted benzaldehyde, in the presence of ammonia in methanol. Their effects on vascular potassium channels and mechanism of induced relaxations on phenylephrine-induced contractile responses in isolated rat aorta were investigated. Minoxidil was used as a standard potassium channel opener and Glibenclamide was used as a standard potassium channel blocker. The effects of compounds on KCl-induced contractile response which is an indicator of ca-channel blocking activity was also investigated and compared to that of nifedipine as a standard calcium channel blocker. Compounds 3a-d and Minoxidil relaxed the contractions exerted by using phenylephrine with the potency order as follows: Minoxidil > 3c > 3d > 3a > 3b. This effect was sensitive to the potassium channel blocker Glibenclamide. It can be concluded that these compounds act via ATP-sensitive potassium (KATP) channels. Selectivity index (SI) for these compounds and Minoxidil also shows that these compounds are selective to ATP-sensitive potassium (KATP) channels and the selectivity of compounds 3a-d is less than Minoxidil.

Activation of KATP channels suppresses glucose production in humans

Increased endogenous glucose production (EGP) is a hallmark of type 2 diabetes mellitus. While there is evidence for central regulation of EGP by activation of hypothalamic ATP-sensitive potassium (K(ATP)) channels in rodents, whether these central pathways contribute to regulation of EGP in humans remains to be determined. Here we present evidence for central nervous system regulation of EGP in humans that is consistent with complementary rodent studies. Oral administration of the K(ATP) channel activator diazoxide under fixed hormonal conditions substantially decreased EGP in nondiabetic humans and Sprague Dawley rats. In rats, comparable doses of oral diazoxide attained appreciable concentrations in the cerebrospinal fluid, and the effects of oral diazoxide were abolished by i.c.v. administration of the K(ATP) channel blocker glibenclamide. These results suggest that activation of hypothalamic K(ATP) channels may be an important regulator of EGP in humans and that this pathway could be a target for treatment of hyperglycemia in type 2 diabetes mellitus.

Electrophysiological effects of hydrogen sulfide on human atrial fibers.

It has been reported that endogenous or exogenous hydrogen sulfide (H(2)S) exerts physiological effects in the vertebrate cardiovascular system. We have also demonstrated that H(2)S acts as an important regulator of electrophysiological properties in guinea pig papillary muscles and on pacemaker cells in sinoatrial nodes of rabbits. This study was to observe the electrophysiological effects of H(2)S on human atrial fibers. Human atrial samples were collected during cardiac surgery. Parameters of action potential in human atrial specialized fibers were recorded using a standard intracellular microelectrode technique. NaHS (H(2)S donor) (50, 100 and 200 µmol/L) decreased the amplitude of action potential (APA), maximal rate of depolarization (V(max)), velocity of diastolic (phase 4) depolarization (VDD) and rate of pacemaker firing (RPF), and shortened the duration of 90% repolarization (APD(90)) in a concentration-dependent manner. ATP-sensitive K(+) (K(ATP)) channel blocker glibenclamide (Gli, 20 µmol/L) partially blocked the effects of NaHS (100 µmol/L) on human atrial fiber cells. The L-type Ca(2+) channel agonist Bay K8644 (0.5 µmol/L) also partially blocked the effects of NaHS (100 µmol/L). An inhibitor of cystathionine γ-lyase (CSE), DL-propargylglycine (PPG, 200 µmol/L), increased APA, V(max), VDD and RPF, and prolonged APD(90). H(2)S exerts a negative chronotropic action and accelerates the repolarization of human atrial specialized fibers, possibly as a result of increases in potassium efflux through the opening of K(ATP) channels and a concomitant decrease in calcium influx. Endogenous H(2)S may be generated by CSE and act as an important regulator of electrophysiological properties in human atrial fibers.