Role of the NO/cGMP/K(ATP) pathway in the protective effects of sildenafil against ethanol-induced gastric damage in rats

BACKGROUND AND PURPOSE: Sildenafil is a selective inhibitor of cGMP-specific phosphodiesterase. Sildenafil, acting via NO-dependent mechanisms, prevents indomethacin-induced gastropathy. Activation of ATP-sensitive potassium channels (K(ATP)) is involved in gastric defence. Our objective was to evaluate the role of the NO/cGMP/K(ATP) pathway in the protective effects of sildenafil against ethanol-induced gastric damage. EXPERIMENTAL APPROACH: Rats were treated with L-NAME (1 or 3 mg kg(-1), i.p.) or with L-arginine (200 mg kg(-1), i.p.) + L-NAME (3 mg kg(-1), i.p.), the guanylate cyclase inhibitor, ODQ (10 mg kg(-1), i.p.), glibenclamide (0.1, 0.3, 1 or 3 mg kg(-1), i.p.) or with glibenclamide (1 mg kg(-1), i.p.) + diazoxide (3 mg kg(-1), i.p.). After thirty minutes, the rats received sildenafil (1 mg kg(-1), by gavage), followed by intragastric instillation of absolute ethanol (4 ml kg(-1)) to induce gastric damage. One hour later, gastric damage (haemorrhagic or ulcerative lesions) was measured with a planimetry programme. Samples of stomach were also taken for histopathological assessment and for assays of tissue glutathione and haemoglobin.KEY RESULTS: Sildenafil significantly reduced ethanol-induced gastric damage in rats. L-NAME alone, without L-arginine, significantly reversed the protection afforded by sildenafil. Inhibition of guanylate cyclase by ODQ completely abolished the gastric protective effect of sildenafil against ethanol-induced gastric damage. Glibenclamide alone reversed sildenafil's gastric protective effect. However, glibenclamide plus diazoxide did not alter the effects of sildenafil.CONCLUSIONS: Sildenafil had a protective effect against ethanol-induced gastric damage through the activation of the NO/cGMP/K(ATP) pathway.     

Vasodilator effect of nicorandil on retinal blood vessels in rats

We examined the effect of nicorandil on retinal blood vessels in rats in vivo. Male Wistar rats (8 to 10 weeks old) were anaesthetised with thiobutabarbital (120 mg/kg, intraperitoneal). Fundus images were captured with a digital camera that was equipped with a special objective lens. Diameters of retinal blood vessels were measured with a personal computer. Nicorandil (1–300 μg kg⁻¹ min⁻¹, intravenous [i.v.]) increased diameters of retinal blood vessels and decreased systemic blood pressure in a dose-dependent manner. Both responses to nicorandil were attenuated by glibenclamide (20 mg/kg, i.v.), an adenosine triphosphate (ATP)-dependent K⁺ (K_ATP) channel blocker. On the other hand, indomethacin (5 mg/kg, i.v.), a cyclooxygenase inhibitor, attenuated the vasodilation of retinal blood vessels, but not depressor response, to nicorandil and sodium nitroprusside. Pinacidil (1–300 μg kg⁻¹ min⁻¹, i.v.), a K_ATP channel opener, also dilated retinal blood vessels and decreased systemic blood pressure. The responses to pinacidil were prevented by glibenclamide, but not by indomethacin. The vasodilation of retinal arteriole, but not depressor response, to sodium nitroprusside (1–30 μg kg⁻¹ min⁻¹, i.v.), a nitric oxide donor, was attenuated by indomethacin. These results suggest that nicorandil dilates retinal blood vessels through opening of K_ATP channels and production of prostaglandins that are probably generated by nitric oxide.     

Nitric oxide activates glibenclamide-sensitive K+ channels in urinary bladder myocytes through a c-GMP-dependent mechanism

In the present investigation, we used standard patch clamp techniques to test whether nitric oxide (NO) generation has any role to play with either activation or inhibition of ATP-sensitive (KATP) channels in guinea-pig urinary bladder. We found that NO generation leads to activation of KATP channels through a cyclic guanosine monophosphate (c-GMP)-dependent protein kinase. 3-Morpholinosydnonimine (SIN, 100 microM) potentiated activation of an inward current in whole cell patch clamp experiments. Glibenclamide (10 microM) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM) inhibited the SIN-activated current. Both in cell-attached and in inside out patches, SIN (200 microM) potentiated KATP channel activity, and the increased channel activity in inside out patches was suppressed by glibenclamide (50 microM), ATP (1 mM) and (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, 10 nM). 8-Br-cGMP (100 microM) increased the KATP channel activity in cell-attached patches, and this was suppressed by glibenclamide (50 microM). These results suggest that the NO-c-GMP-PKG pathway contributes to activation of K(ATP) channels in guinea-pig urinary bladder myocytes.     

羽扇豆醇联合槲皮素对膀胱癌5637细胞的抑制作用

目的 研究羽扇豆醇(Lupeol)联合槲皮素(Quercetin)对膀胱癌5637细胞增殖抑制的作用,为膀胱癌的治疗提供实验依据.方法 利用噻唑蓝(MTT)法分别检测不同浓度的羽扇豆醇(10、20、40、60、80、100、120μmol/L)及槲皮素(5、10、20、40、60、80μmol/L)对膀胱癌5637细胞...     

The insulin sensitizing effect of topiramate involves KATP channel activation in the central nervous system

BACKGROUND AND PURPOSE

Topiramate improves insulin sensitivity, in addition to its antiepileptic action. However, the underlying mechanism is unknown. Therefore, the present study was aimed at investigating the mechanism of the insulin-sensitizing effect of topiramate both in vivo and in vitro.

EXPERIMENTAL APPROACH

Male C57Bl/6J mice were fed a run-in high-fat diet for 6 weeks, before receiving topiramate or vehicle mixed in high-fat diet for an additional 6 weeks. Insulin sensitivity was assessed by hyperinsulinaemic-euglycaemic clamp. The extent to which the insulin sensitizing effects of topiramate were mediated through the CNS were determined by concomitant i.c.v. infusion of vehicle or tolbutamide, an inhibitor of ATP-sensitive potassium channels in neurons. The direct effects of topiramate on insulin signalling and glucose uptake were assessed in vivo and in cultured muscle cells.

KEY RESULTS

In hyperinsulinaemic-euglycaemic clamp conditions, therapeutic plasma concentrations of topiramate (∼4 μg·mL⁻¹) improved insulin sensitivity (glucose infusion rate + 58%). Using 2-deoxy-D-[³H]glucose, we established that topiramate improved the insulin-mediated glucose uptake by heart (+92%), muscle (+116%) and adipose tissue (+586%). Upon i.c.v. tolbutamide, the insulin-sensitizing effect of topiramate was completely abrogated. Topiramate did not directly affect glucose uptake or insulin signalling neither in vivo nor in cultured muscle cells.

CONCLUSION AND IMPLICATIONS

In conclusion, topiramate stimulates insulin-mediated glucose uptake in vivo through the CNS. These observations illustrate the possibility of pharmacological modulation of peripheral insulin resistance through a target in the CNS.     

In vivo anti-ulcerogenic effect of okra (Abelmoschus esculentus) on ethanol-induced acute gastric mucosal lesions

Context: Okra, Abelmoschus esculentus (L.) (Malvaceae), is a medicinal plant widely used in Turkish traditional medicine for the treatment of various diseases such as ulcers and gastritis.

Objective: In the present study, we evaluated the gastroprotective effect of okra against ethanol-induced acute gastric mucosal injury in animal models.

Materials and methods: Wistar rats were treated with 500, 250 or 100?mg/kg okra; 20?mg/kg famotidine (Fam); and 75?mg/kg quercetin (Que). Following a 60?min period, all the rats were given 1?mL of ethanol (80%). One hour after the administration of ethanol, all groups were sacrificed.

Results: At 5000?mg/kg, the extract produced (okra) no signs of toxicity in animals. Okra 500, 250, 100, Fam 20 and Que 75 inhibited ulcer formation by 81.0, 67.5, 67.0, 76.3 and 72.4%, respectively. Okra 500 significantly decreased edema, hemorrhage and inflammation scores compared with the ethanol group (p?p?p?p?Discussion and conclusions: Our in vivo data indicate that okra has a gastroprotective effect against ethanol and could reduce the gastric ulcer as seen from biochemical and histopathological results. We suggest that okra could be a possible therapeutic antiulcer agent.     

Vasodilator effects of adenosine on retinal arterioles in streptozotocin-induced diabetic rats

Adenosine is a potent vasodilator of retinal blood vessels and is implicated to be a major regulator of retinal blood flow during metabolic stress, but little is known about the impact of diabetes on the role of adenosine in regulation of retinal hemodynamics. Therefore, we examined how diabetes affects adenosine-induced vasodilation of retinal arterioles. Male Wistar rats were treated with streptozotocin (80 mg/kg, intraperitoneally), and experiments were performed 6–8 weeks later. Rats were treated with tetrodotoxin (50 μg/kg, intravenously [i.v.]) to eliminate any nerve activity and prevent movement of the eye and infused with methoxamine continuously to maintain adequate systemic circulation. Fundus images were captured with a digital camera that was equipped with a special objective lens, and diameters of retinal arterioles were measured. Adenosine increased diameters of retinal arterioles and decreased systemic blood pressure. These responses were significantly attenuated by the nitric oxide synthase inhibitor N^G-nitro-l-arginine methyl ester (30 mg/kg, i.v.) and the adenosine triphosphate-dependent K⁺ (K_ATP) channel blocker glibenclamide (20 mg/kg, i.v.). The depressor responses to adenosine were reduced in diabetic rats, whereas diabetes did not alter vasodilation of retinal arterioles to adenosine. In contrast, both depressor response and vasodilation of retinal arteriole to acetylcholine were reduced in diabetic rats. The retinal vasodilator responses to adenosine and acetylcholine observed in diabetic rats were diminished by N^G-nitro-l-arginine methyl ester. There were no differences in the responses to pinacidil, a K_ATP channel opener, between the diabetic and nondiabetic rats. These results suggest that both the activation of nitric oxide synthase and opening of K_ATP channels contribute to the vasodilator effects of adenosine in rats in vivo. However, diabetes has no significant impact on the vasodilation mediated by these mechanisms in the retinal circulation.     

兰索拉唑对乙醇诱导大鼠胃黏膜损伤的保护作用及其机制

目的　研究兰索拉唑 (LP)对乙醇诱导大鼠胃黏膜损伤的保护作用 ,探讨胃泌素受体和环氧化酶 2 (COX 2 )表达在此过程中的作用。方法　大鼠ig给予LP 0 5、5、5 0mg·kg-1·d-1,或ig联合给予LP 5 0mg·kg-1·d-1和胃泌素受体拮抗剂AG 0 4 1R 3、10、30mg·kg-1·d-1,对照组ig给予羧甲基纤维素 (CMC) 2 5mg·kg-1·d-1,连续 14d。末次给药后 8h各组大鼠ig给予无水乙醇 1ml,观察胃损伤指数 (LI)及光镜下的胃黏膜病理学改变。酶免疫方法测定胃黏膜前列腺素E2 (PGE2 )水平 ,WesternBlot和免疫组化检测胃黏膜COX 2表达。评价特异性COX 2抑制剂NS 398对LP诱导的PGE2 合成及胃黏膜保护作用的影响。结果　在 0 5、5、5 0mg·kg-1LP组 ,LI分别为 (2 5 3± 0 33) %、(1 84±0 2 9) %和 (0 83± 0 12 ) % ,小于对照组 (3 6 5± 0 19) % (P<0 0 5 ) ;胃黏膜PGE2 含量分别为 (42 7± 32 ) ,(483± 12 1)和 (6 14± 82 ) pg·g-1wwt ,高于对照组 (2 6 6± 81) pg·g-1wwt(P <0 0 5 )。LP剂量依赖性地增加大鼠胃黏膜COX 2表达。然而 ,同时给予AG 0 4 1R阻断了LP诱导的胃黏膜保护作用、COX 2表达和PGE2 合成。NS 398抑制LP诱导的PGE2 合成及胃黏膜保护作用。结论　LP的胃黏膜保护作用与内源性胃泌素激活胃泌素受     

竹叶黄酮对乙醇诱导小鼠急性胃黏膜损伤的保护作用

目的观察竹叶黄酮对乙醇诱导小鼠急性胃黏膜损伤的保护作用。方法取40只小鼠随机均分为对照组、模型组、维生素E(50 mg/kg)组以及竹叶黄酮25、50 mg/kg组。各组连续ig给药7 d,1次/d。末次给药1 h后,各组按10 mg/kg ig给予无水乙醇,制作胃黏膜损伤模型。建模4 h后,观察胃黏膜损伤面积,计算胃黏膜损伤指数和损伤抑制率。测定小鼠胃黏膜组织中一氧化氮(NO)、前列腺素(PEG2)含量以及血清中超氧化物歧化酶(SOD)、丙二醛(MDA)水平。结果与模型组比较,竹叶黄酮25 mg/kg组小鼠胃黏膜损伤面积减少,50 mg/kg组小鼠损伤程度最小。与模型组比较,竹叶黄酮25、50 mg/kg组小鼠胃黏膜损伤指数均明显降低(P0.01);胃黏膜组织NO、PEG_2含量明显升高(P0.01);血清SOD、MDA水平明显降低(P0.01),SOD/MDA比值明显升高(P0.01)。结论竹叶黄酮能拮抗乙醇诱导的胃黏膜损伤,其机制可能与其抗氧化、抑制氧自由基产生并升高胃黏膜NO、PEG_2含量有关。     

肉苁蓉总苷对原代培养小鼠肝细胞酒精损伤保护作用研究

目的:研究肉苁蓉总苷对原代培养肝细胞的保护作用。方法:采用原位灌流法收集原代肝细胞,MTT法评价肉苁蓉总苷对酒精损伤原代培养肝细胞存活率的影响;荧光显微镜技术评价肉苁蓉总苷对酒精损伤原代培养肝细胞及细胞核形态的影响;流式细胞仪检测肉苁蓉总苷对酒精损伤原代培养肝细胞凋亡的影响;免疫细胞化学染色法检测肉苁蓉总苷对bcl-2和c-fos表达的影响。结果:原代肝细胞经酒精损伤后,存活率降低,出现明显凋亡和坏死改变,凋亡抑制基因bcl-2减弱、促凋亡基因c-fos表达增强。肉苁蓉总苷可明显提高细胞存活率,改善凋亡和坏死情况,增强bcl-2表达,抑制c-fos表达。且作用呈剂量依赖性。结论:肉苁蓉总苷可通过增加凋亡抑制基因bcl-2表达,减少促凋亡基因c-fos表达,减少凋亡和坏死,增加细胞存活率来实现对原代培养肝细胞的保护作用。     

The mechanism of the late preconditioning effect of 3-nitropropionic acid

The aim of the present study was to investigate the mechanism of effect of 3-nitropropionic acid-(3-NP) induced late preconditioning in rat heart. For this purpose 20–30 min before 3-NP (20 mg/kg, i.p.) injection, the rats were treated intraperitoneally with 5-hydroxydecanoate (40 mg/kg, 5-HD, mitochondrial K_ATP-channel blocker), L-NAME (100 mg/kg, NOS inhibitor), N-2-mercaptopropionylglycine (100 mg/kg, MPG, free radical scavenger), or superoxide dismutase+catalase (10000+10000 IU/kg, SOD+CAT). Control rats received saline only without 3-NP pretreatment. After two days, hearts were isolated and perfused at a constant pressure in a Langendorff apparatus. 15-min global ischemia followed by 30-min reperfusion was applied to all hearts. Pretreatment of 3-NP significantly reduced infarct size, creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) levels, and incidence of ventricular tachycardia (VT) compared with the control group receiving saline only. 5-HD, L-NAME, MPG, or SOD+CAT treatment statistically reversed 3-NP-induced reduction in infarct size. Although CK-MB, LDH levels, and incidence of VT were also reduced by L-NAME, MPG, or SOD+CAT treatment, only 5-HD significantly inhibited beneficial effects of 3-NP on all of the parameters above. These results showed that mito-K_ATP channels play a pivotal role in late preconditioning effect of 3-NP in the isolated rat heart. However, other mediators such as reactive oxygen species and NO may be, at least in part, involved in mechanisms of this effect. Preliminary findings of the present study were presented at the 17th Annual Meeting of the Mediterranean Association of Cardiology and Cardiovascular Surgery (September 22–24, 2005, Portorož, Slovenia).     

Dual modulation of the tension of isolated gastric artery and gastric mucosal circulation by hydrogen sulfide in rats

To clarify roles of H₂S in regulation of gastric circulation, we investigated effects of NaHS, a H₂S donor, on tension of isolated rat gastric artery and gastric mucosal blood flow in rats. In the precontracted ring preparations, NaHS caused contraction and relaxation at low and high concentrations, respectively. The NaHS-induced vasorelaxation was only partially blocked by glibenclamide, a K⁺ _ATP channel inhibitor. The contractile activity of NaHS disappeared by removal of the endothelium or by inhibition of nitric oxide synthase and the endothelium-derived hyperpolarizing factor (EDHF) pathways. Intravenous injection of NaHS caused transient increase followed by decrease in gastric mucosal blood flow in rats. Collectively, in the gastric artery, NaHS appears to cause relaxation through both K⁺ _ATP channel-dependent and -independent pathways and contraction through inhibition of NO and EDHF pathways. Together with the in vivo results, our study implies that H₂S plays multiple complex roles in regulation of gastric circulation.     

壳聚糖对乙醇急性损伤时大鼠胃壁细胞的影响

胃酸是胃黏膜最常见的内源性损伤因子,在胃黏膜损伤过程中起着重要作用.壁细胞的主要功能是分泌胃酸,其超微结构与泌酸关系密切.壳聚糖是少见的带正电荷的天然高分子聚合物,有研究发现壳聚糖有促进溃疡愈合和黏膜保护作用.     

Gastroprotective effect of sumatriptan against indomethacin-, stress- and ethanol-induced gastric damage in male rats: Possible modulatory role of 5-hydroxytryptamine 1B/1D receptors and pro-inflammatory cytokines

The current study was aimed to investigate the beneficial effect of sumatriptan, a 5-hydroxytryptamine 1B/1D (5HT_1B/1D) receptor agonist, on gastric ulcer in rats via stimulating 5HT_1B/1D receptors and suppressing pro-inflammatory cytokines. Rats were allocated into three models of gastric ulcer: indomethacin (30 mg/kg, PO), water immersion restraint stress (WRS) and ethanol (5 ml/kg PO). Animals were administered with sumatriptan (0.01, 0.1, 0.3 and 1 mg/kg, i.p) 30 min before gastric ulcer induction. GR-127935 (0.01 mg/kg, i.p, a selective 5HT_1B/1D antagonist) was administered 30 min before sumatriptan (0.1 mg/kg) injection. Macroscopic assessments (J-score), ELISA analysis of tumour necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) and histopathological changes were performed on the rat's stomach tissues. Gastric ulcer induction in three models caused an increase in J-score, TNF-α, IL-1β and microscopic features. Sumatriptan (0.1 mg/kg) significantly improved gastric injury induced by indomethacin, WRS and ethanol through the reduction in the J-score, TNF-α, IL-1β and microscopic lesions. Concurrent administration of GR-127935 (0.01 mg/kg) with sumatriptan (0.1 mg/kg) reversed the gastroprotective effect of sumatriptan in three models. Sumatriptan possessed gastroprotective effects on indomethacin-, WRS- and ethanol-induced gastric damage in rats via the possible involvement of the 5HT_1B/1D receptors.     

Mechanisms involved in the regional haemodynamic effects of intermedin (adrenomedullin 2) compared with adrenomedullin in conscious rats

Background and purpose:

Intermedin (IMD) is a newly identified member of the calcitonin family of peptides that shares structural and functional homology with adrenomedullin (AM). In vivo cardiovascular effects of AM have been described, but relatively little is known of the in vivo actions of IMD. The purpose of this study was to compare the regional haemodynamic effects of IMD with those of AM in conscious rats, and investigate possible underlying mechanisms.

Experimental approach:

Measurements of blood pressure, heart rate and renal, mesenteric and hindquarters haemodynamics were made in conscious, chronically-instrumented rats.

Key results:

IMD caused tachycardia and vasodilatation in all three vascular beds, associated with modest hypotension. At an equimolar dose (1 nmol·kg⁻¹), most of the cardiovascular effects of IMD were greater than those of AM. The AM receptor antagonist, AM_22–52, was equally effective in attenuating the renal and mesenteric vasodilator effects of IMD (1 nmol·kg⁻¹) and AM (3 nmol·kg⁻¹), but inhibition of NO synthase was more effective at reducing the vasodilator effects of IMD than AM. Vascular K_ATP channel blockade with U-37883A did not inhibit the vasodilator effects of either peptide.

Conclusions and implications:

In vivo, the regional haemodynamic profile of IMD resembles that of AM, and some of the vasodilator effects of IMD are mediated by AM receptors and NO, but not by K_ATP channels. The cardiovascular effects of AM have been implicated in various pathological conditions, but whether or not endogenous IMD fulfils a similar role remains to be determined.     

KATP-channel openers for the treatment of breast and endometrial cancer

Frequently, hyperinsulinaemia is the result of obesity, lack of physical activity and a diet rich in carbohydrates, such as glucose or fructose. Chronic exposure to hyperinsulinaemia enhances the bioavailability of insulin-like growth factor-1 (IGF-1). A positive correlation has been demonstrated between high plasma concentration of IGF-1 and insulin and the risk of breast or endometrial cancer. In this patent application, Novo Nordisk claims the use of K_ATP-channel openers (KCOs) to reduce breast and endometrial cancer through a decrease of the insulin plasma level.     

CBS对酒精性胃溃疡大鼠胃粘膜血流的影响

采用激光多普勒法观察了不同剂量胶状铋剂对鼠胃粘膜血流的影响.结果提示:胶状铋剂对酒精造成的胃溃疡具有良好的保护作用.0～90min胃粘膜血流总量在20mg、40mg时明显高于对照组.但用药后15min胃粘膜血流并无增加.认为其保护机理主要是降低胃粘膜血管的通透性。     

Sensitivity of KATP channels to cellular metabolic disorders and the underlying structural basis

Aim:

ATP-sensitive potassium (K_ATP) channels formed by a combination of SUR/Kir6.x subunits play a crucial role in protection against hypoxic or ischemic injuries resulting from cell metabolic disorders. In this study we investigated the effects of Na-azide, a metabolic inhibitor, on K_ATP channels expressed in Xenopus oocytes, and explored the structure basis for their sensitivity to cell metabolic disorders.

Methods:

Six subtypes of K_ATP channels (wild SUR1/Kir6.2, SUR2B/Kir6.2, SUR1/Kir6.1, SUR2B/Kir6.1, SUR2A/Kir6.2 and SUR2A/Kir6.1), as well as eleven subtypes of K_ATP channels with mutant subunits were expressed in Xenopus oocytes. K_ATP currents were recorded using a two-electrode voltage clamp recording technique. The drugs were applied through bath.

Results:

Except SUR2A/Kir6.1, five subtypes of K_ATP channels were activated by Na-azide (3 mmol/L) with an order of the responses: SUR1/Kir6.2>SUR2B/Kir6.2>SUR1/Kir6.1>SUR2B/Kir6.1>SUR2A/Kir6.2, and the opening rate (t_1/2) was SUR1/Kir6.x>SUR2B/Kir6.x>SUR2A/Kir6.2. Furthermore, Kir6.2, rather than Kir6.1, had intrinsic sensitivity to Na-azide, and the residues involved in ATP-binding (R50 and K185) or pH-sensing (H175) were associated with the sensitivity of the Kir6.2 subunit to Na-azide. Moreover, the residues (K707 and K1348) within the Walker A (WA) motifs of two nucleotide-binding domains (NBDs) were essential for SUR2B/Kir6.x (especially SUR2B/Kir6.1) channel activation by Na-azide, suggesting a key role for Mg-adenine nucleotide binding and/or hydrolysis in the SUR2B subunit.

Conclusion:

Among the six subtypes of K_ATP channels, SUR1/Kir6.2 is the most sensitive, whereas SUR2A/Kir6.1 is insensitive, to cell metabolic disorders. The Kir6.2 subunit, rather than the Kir6.1 subunit, has intrinsic sensitivity to cell metabolic disorders. The residues (K707 and K1348) within the WA motifs of SUR2B are important for the sensitivity of SUR2B/Kir6.x channels to cell metabolic disorders.     

Involvement of a glibenclamide-sensitive mechanism in the nitrergic neurotransmission of the pig intravesical ureter

1. The present study was designed to investigate whether potassium (K⁺) channels are involved in the relaxations to nitric oxide (NO) of pig intravesical ureteral preparations suspended in organ baths for isometric tension recordings. In ureteral strips treated with guanethidine (10⁻⁵ M) and atropine (10⁻⁷ M) to block adrenergic neurotransmission and muscarinic receptors, respectively, NO was either released from nitrergic nerves by electrical field stimulation (EFS, 0.5–10 Hz, 1 ms duration, 20 s trains), or exogenously-applied as an acidified solution of sodium nitrite (NaNO₂, 10⁻⁶–10⁻³ M).
2. Incubation with an inhibitor of guanylate cyclase activation by NO, methylene blue (10⁻⁵ M) did not change the basal tension of intravesical ureteral strips but inhibited the relaxation induced by EFS or exogenous NO on ureteral preparations contracted with the thromboxane analogue U46619 (10⁻⁷ M).
3. Incubation with charybdotoxin (3×10⁻⁸ M) and apamin (5×10⁻⁷ M), which are inhibitors of large and small conductance calcium (Ca²⁺)-activated K⁺ channels, respectively, did not modify basal tension or the relaxations induced by EFS and exogenous NO. Treatment with charybdotoxin or apamin plus methylene blue (10⁻⁵ M) significantly reduced the relaxations to EFS and exogenous NO. However, in both cases the reductions were similar to the inhibition evoked by methylene blue alone. The combined addition of charybdotoxin plus apamin did not change the relaxations to EFS or exogenously added NO of the porcine intravesical ureter.
4. Cromakalim (10⁻⁸–3×10⁻⁶ M), an opener of ATP-sensitive K⁺ channels, evoked a dose-dependent relaxation with a pD₂ of 7.3±0.2 and maximum relaxant effect of a 71.8±4.2% of the contraction induced by U46619 in the pig intravesical ureter. The blocker of ATP-sensitive K⁺ channels, glibenclamide (10⁻⁶ M), inhibited markedly the relaxations to cromakalim.
5. Glibenclamide (10⁻⁶ M) had no effect on the basal tone of ureteral preparations but significantly reduced the relaxations induced by both EFS and exogenous NO. Combined treatment with methylene blue (10⁻⁵ M) and glibenclamide (10⁻⁶ M) did not exert an effect greater than that of methylene blue alone on either EFS- or NO-evoked relaxations of the pig ureter.
6. The present results suggest that NO acts as an inhibitory neurotransmitter in the pig intravesical ureter and relaxes smooth muscle through a guanylate cyclase-dependent mechanism which seems to favour the opening of glibenclamide-sensitive K⁺ channels.

     

Inhibitory effect of caffeine on pacemaker activity in the oviduct is mediated by cAMP-regulated conductances

BACKGROUND AND PURPOSE

Spontaneous electrical activity, termed slow waves, drives rhythmic, propulsive contractions in the smooth muscle of the oviduct (myosalpinx). Myosalpinx contractions cause egg transport through the oviduct. Agents that disrupt slow wave pacemaker activity will therefore disrupt myosalpinx contractions and egg transport. Caffeine is commonly used as a ryanodine receptor agonist and has been previously associated with delayed conception. Here we assessed the effects of caffeine on pacemaker activity in the murine myosalpinx.

EXPERIMENTAL APPROACH

The effects of caffeine on electrical pacemaker activity were studied using intracellular microelectrode and isometric force measurements on intact oviduct muscle preparations. Responses to caffeine were compared with responses caused by 3-isobutyl-1-methylxanthine (IBMX) and forskolin.

KEY RESULTS

Caffeine caused hyperpolarization of membrane potential and inhibited slow wave generation and myosalpinx contractions. The effects of caffeine could be mimicked by the K_ATP channel agonist pinacidil and antagonized by the K_ATP channel antagonist glibenclamide. Caffeine is known to inhibit cyclic nucleotide phosphodiesterases (PDEs), leading to an increase in cytosolic cAMP and stimulation of downstream cAMP-dependent mechanisms. The effects of caffeine were mimicked by the PDE inhibitor, IBMX, and the adenylyl cyclase activator forskolin. These effects were also reversed by glibenclamide.

CONCLUSIONS AND IMPLICATIONS

These results suggest that caffeine activates K_ATP channels in oviduct myosalpinx. Since caffeine abolishes slow waves and associated contractions of the myosalpinx, it would have a negative effect on egg transport through the oviduct and may contribute to the documented delayed conception in women consuming caffeinated beverages.