Phosphate and thiophosphate group donating adenine and guanine nucleotides inhibit glibenclamide binding to membranes from pancreatic islets

Summary In microsomes obtained from mouse pancreatic islets, the Mg complex of adenosine 5-triphosphate (MgATP) increased the dissociation constant (K _D) for binding of [³H]glibenclamide by sixfold. In the presence of Mg²⁺, not only ATP but also adenosine 5-0-(3-thiotriphosphate) (ATPS), adenosine 5-diphosphate (ADP), guanosine 5-triphosphate (GTP), guanosine 5-diphosphate (GDP), guanosine 5-0-(3-thiotriphosphate) (GTPTS) and guanosine 5-0-(2-thiodiphosphate) (GDP S) inhibited binding of [³H]glibenclamide. These effects were not observed in the absence of Mg²⁺. Half maximally effective concentrations of the Mg complexes of ATP, ADP, ATPS and GDP were 11.6, 19.0, 62.3 and 90.1 mol/l, respectively. The non-hydrolyzable analogues adenosine 5-(,-imidotriphosphate) (AMP-PNP) and guanosine 5-(,-imidotriphosphate) (GMP-PNP) did not alter [³H]glibenclamide binding in the presence of Mg²⁺. MgADP acted much more slowly than MgATP and both MgADP and MgADP did not inhibit [³H]glibenclamide binding when the concentrations of MgATP and MgATP were kept low by the hexokinase reaction. Development of MgATP-induced inhibition of [³H]glibenclamide binding and dissociation of [³H]-glibenclamide binding occurred at similar rates. However, the reversal of MgATP-induced inhibition of [³H]glibenclamide binding was slower than the association of [³H]glibenclamide with its binding site. Exogenous alkaline phosphatase accelerated the reversal of MgATP-induced inhibition of [³H]glibenclamide binding. MgATP enhanced displacement of [³H]glibenclamide binding by diazoxide. The data suggest that sulfonylureas and diazoxide exert their effects by interaction with the same binding site at the sulfonylurea receptor and that protein phosphorylation modulates the affinity of the receptor.Some of the results described here are part of the medical theses of S. Löser and I. Rietze Send offprint requests to M. Schwanstecher at the above address     

二氮嗪对软骨细胞氧化损伤的影响

目的:探讨二氮嗪对双氧水诱导的软骨细胞氧化损伤的影响。方法:取SD乳鼠膝关节软骨细胞进行原代培养,将对数生长期的软骨细胞分成5组,分别为阴性对照组(A组),双氧水组(B组),H2O2+0.1μmol·L-1二氮嗪组(C组),H2O2+1.0μmol·L-1二氮嗪组(D组),H2O2+10μmol·L-1二氮嗪组(E组);A组细胞不做特殊处理,B组用0.3 mmol·L-1双氧水在37℃恒温箱内孵育4 h,C、D、E、F组预先分别用0.1μmol·L-1DZ,1.0μmol·L-1DZ,10μmol·L-1DZ,100μmol·L-1在37℃的恒温箱孵育30分钟,PBS洗涤细胞,再用0.3 mmol·L-1双氧水在37℃恒温箱内孵育4小时。分别检测ABCDE各组细胞的细胞活性,ROS的量,细胞凋亡情况。结果:①MTT法检测各组细胞活性,细胞活性率从大至小依次为D组C组E组F组B组;②用活性氧探针DCFH-DA检测各组细胞中的ROS的量,ROS产量从多至少依次为B组E组D组C组A组;③流式细胞仪检测各组细胞凋亡情况,各组细胞凋亡率由大至小依次为B组E组D组C组。④Western bolt检测各组软骨细胞中HIF-1α蛋白的表达,各组软骨细胞中HIF-1α蛋白表达量依次为C组D组E组A组B组。结论:二氮嗪可降低双氧水诱导的软骨细胞的凋亡率,同时也降低双氧水诱导的软骨细胞ROS的产生,并诱导软骨细胞产生HIF-1α蛋白,可能存在二氮嗪诱导HIF-1α的表达,引起下游相关基因的转录,总体提高软骨细胞对氧化损伤的耐受力,阻碍自由基诱导的软骨细胞的凋亡。     

二氮嗪预处理对心肌再灌注损伤的保护作用

目的观察二氮嗪预处理对在体大鼠心肌缺血再灌注损伤（IRI）的保护作用。方法健康雄性SD大鼠36只,随机分成4组,每组9只。采用结扎左冠状动脉前降支（LAD）法制备心肌IRI模型。记录缺血再灌注前后不同时间点心功能指标,TTC染色测定心肌梗塞面积,检测血清CK-MB及LDH活性。结果结扎左冠状动脉前降支后：IRI组的CK-MB和LDH显著高于Sham组（P〈0.05）;IPC和DPC组较IRI组显著下降（P〈0.05）。与IRI组比较,IPC组及DPC组梗死面积明显降低（P〈0.05）。缺血30min、再灌注60min、120min时,IRI组、IPC组和DPC组与Sham组比较,±dp／＆max、LVSP、LVEDP有显著的差异（P〈0．05）;IPC组和DPC组与IRI组比较,±dp／dtmax、LVSP差别有统计学意义（ P〈0．05）。结论二氮嗪预处理对在体大鼠缺血再灌注损伤心肌具有保护作用,可降低血清心肌酶含量、减少心肌梗死面积,维护心肌收缩和舒张功能。     

ATP-Dependent K+ Channels in Renal Ischemia Reperfusion Injury

ATP-dependent K⁺ channels (K_ATP) account for most of the recycling of K⁺ which enters the proximal tubules cell via Na, K-ATPase. In the mitochondrial membrane, opening of these channels preserves mitochondrial viability and matrix volume during ischemia. We examined K_ATP channel modulation in renal ischemia-reperfusion injury (IRI), using an isolated perfused rat kidney (IPRK) model, in control, IRI, IRI + 200 µM diazoxide (a K_ATP opener), IRI + 10 µM glibenclamide (a K_ATP blocker) and IRI + 200 µM diazoxide + 10 µM glibenclamide groups. IRI was induced by 2 periods of warm ischemia, followed by 45 min of reperfusion. IRI significantly decreased glomerular filtration rate (GFR) and increased fractional excretion of sodium (FE_Na) (p<0.01). Neither diazoxide nor glibenclamide had an effect on control kidney function other than an increase in renal vascular resistance produced by glibenclamide. Pretreatment with 200 µM diazoxide reduced the postischemic increase in FE_Na (p<0.05). Adding 10 µM glibenclamide inhibited the diazoxide effect on postischemic FE_Na (p<0.01). Histology showed that kidneys pretreated with glibenclamide demonstrated an increase in injury in the thick ascending limb of outer medulla (p<0.05). Glibenclamide significantly decreased post ischemic renal vascular resistance (p<0.05), but had no significant effect on other renal function parameters. Our results suggest that sodium reabsorption is improved by K_ATP activation and blockade of K_ATP channels during IRI has an injury enhancing effect on renal epithelial function and histology. This may be mediated through K_ATP modulation in cell and/or mitochondrial inner membrane.     

二氮嗪对幼龄大鼠深低温脑缺血再灌注后氧自由基及细胞凋亡的影响

目的 探讨二氮嗪对幼龄大鼠深低温脑缺血再灌注后氧自由基和细胞凋亡的影响及相关的脑保护作用机制.方法 将54只3周龄健康sD大鼠随机分为假手术组、模型组和二氮嗪组,每组18只,建立深低温脑缺血再灌注模型.于术后24 h处死大鼠,检测脑组织超氧化物歧化酶(SOD)、丙二醛的含量,Western Blot法检测脑组织细胞质细胞色素C含量,免疫组化法检测脑组织细胞质Caspase-3含量.结果 模型组脑组织中SOD含量为(198±41)U/mg,低于假手术组的(321±36)U/rag(P<0.01);丙二醛含量为(212±21)nmol/mg,高于假手术组的(100±23)nmol/mg(P<0.01);细胞色素C蛋白表达(0.72±0.09)和Caspase-3蛋白表达(83±10)均高于假手术组(0.17±0.02和115±9)(P<0.01).二氮嗪组脑组织SOD含量为(264±34)U/rag,高于模型组(P<0.05);而丙二醛含量(174±19)nmol/mg、细胞色素C蛋白表达(0.41±0.05)和Caspase-3蛋白表达(99±11)均低于模型组(P<0.05).结论 二氮嗪对幼龄大鼠深低温脑缺血再灌注损伤具有脑保护作用,其机制与抑制氧自由基产生和细胞凋亡有关.     

二氮嗪预处理对深低温停循环脑早期保护作用的实验研究

目的观察二氮嗪预处理对深低温停循环（DHCA）所致的脑损伤的早期保护作用，并探讨其可能机制。方法健康大耳白兔24只，随机分为3组，每组8只：对照组（安慰剂组）：DHCA＋安慰剂；实验组（二氮嗪组）：DHCA＋二氮嗪（二氮嗪5mg／kg，CPB前15min静脉注入）；拮抗剂组（5-HD组）：DHCA＋5-HD（20mg／kg，给予二氮嗪前静脉注入）＋二氮嗪（5mg／kg，CPB前15min静脉注入）。每组均经CPB降温至鼻咽温18℃，停循环60min，复温，停机，取脑组织。观察指标为脑组织含水量、脑组织匀浆中兴奋性氨基酸（EAA）及丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性的变化，并制作电镜标本行透射电镜观察脑组织超微结构的改变。结果实验组脑组织含水量及MDA含量明显低于对照组（P〈0．05b）及拮抗剂组（P〈0．05）；实验组EAA含量及SOD活性明显高于对照组（P〈0．01）及拮抗剂组（P〈0．05）；拮抗剂组各项指标与对照组比无统计学意义（P〉0．05）；电镜结果示各组脑细胞超微结构明显损伤，总体印象实验组各种神经元细胞器损伤较上述组有所减轻。结论二氮嗪预处理对DHCA引起的神经元损伤具有早期保护作用。     

吡那地尔及二氮嗪对长期低氧大鼠肺动脉平滑肌ＫＡＴＰ通道蛋白表达的影响

目的:研究慢性低氧对大鼠肺动脉平滑肌ATP敏感钾通道(KATP)蛋白表达的影响及KATP开放剂吡那地尔及二氮嗪的作用.方法:SD雄性大鼠35只随机分成对照组、低氧组、吡那地尔干预组[吡那地尔2.0 mg/(kg·d),ig]、二氮嗪干预组[二氮嗪1.5 ms/(ks·d),ig]、5-羟癸酸(5-HD) 二氮嗪干预组[5-HD 3.0 ms/(kg·d),ig;二氮嗪1.5 ms/(ks·d),ig],每组7只.将低氧组和各干预组大鼠放入常压低氧舱内[O2(10.0%±0.5%)],每周6天,每天6 h 4周后测定平均肺动脉压(mPAP)并采用Western-blot技术,分析各组肺动脉主干平滑肌KATP蛋白表达.结果:①慢性低氧组大鼠的mPAP显著高于正常对照组,吡那地尔干预组肺动脉压较低氧组显著下降.二氮嗪干预组加重慢性低氧所致的肺动脉压力升高,5.HD Z.氮嗪干预组的mPAP显著低于二氮嗪干预组,P均<0.05.②低氧组调节亚基磺酰脲受体2B(SUR2B)蛋白水平显著低于正常组,吡那地尔干预组SUR2B显著高于低氧组,二氮嗪干预组SUR2B蛋白水平显著低于低氧组,5-HD 二氮嗪干预组SUR2B显著高于二氮嗪于预组,与低氧组亦有显著统计学差异,P均<0.05.各组内向整流性孔区6.1(Kir6.1)蛋白没有显著差异(P<0.05).结论:慢性低氧抑制KATP通道蛋白的表达,而吡那地尔能提高表达,对慢性低氧所致的肺动脉高压和肺血管壁重构具有较好的预防和逆转作用;二氮嗪能加重低氧性肺动脉压升高,并抑制KATP通道蛋白的表达.     

Dilutional hyponatremia due to diazoxide-produced polydipsia

Subcutaneous injection of diazoxide every 3 hr for a total of 5 doses in 15 hr produced a state of elevated drinking and antidiuresis in rats resulting in a massive, positive, self-imposed water load. Dilutional hyponatremia was present, but not serum hyposmolality, owing to the increased serum glucose and BUN. The mechanism by which diazoxide produces a polydipsia even in the presence of an accumulating water load may illuminate the genesis of other pathophysiological dilutional states.     

Severe transient hyperinsulinaemic hypoglycaemia: two neonates without predisposing factors and a review of the literature

We report on transient hyperinsulinism (HI), presenting as severe congenital HI, in two neonates born without intrauterine growth restriction, maternal diabetes, perinatal asphyxia or Rhesus/platelet isoimmunisation. The neonates developed early (<6 h of life), symptomatic, non-ketotic hypoglycaemia (0–0.66 mmol/l), associated with elevated insulin levels (40–200 mU/l), and required high glucose infusion rates (22–24 mg/kg per min) to maintain normoglycaemia. However, both babies were diazoxide-sensitive and did not require glucose infusions beyond 2 weeks of life. Neither neonate had elevated serum ammonia levels or evidence of a metabolic disorder. Conclusion:transient hyperinsulinism can occur in newborns delivered uneventfully without significant perinatal complications. The unusual sensitivity to medical treatment in these cases of neonatal-onset hyperinsulinaemic hypoglycaemia underscores the importance of careful medical management of severe congenital hyperinsulinism. Careful consideration of the indication and if necessary, timing and extent of pancreatectomy is required, while maintaining euglycaemia to protect the developing brain.Abbreviations AGA appropriate for gestational age - BGL blood glucose level - BWS Beckwith-Wiedemann syndrome - HI hyperinsulinism - HI/HA hyperinsulinism/hyperammonaemia - IUGR intrauterine growth restriction - PHHI persistent hyperinsulinaemic hypoglycaemia of infancy - SGA small for gestational age - SUR sulphonylurea receptor - TNHI transient neonatal hyperinsulinism - UVC umbilical venous catheter