Simultaneous recording of ATP-sensitive K+ current and intracellular Ca2+ in anoxic rat ventricular myocytes. Effects of glibenclamide

We investigated the temporal relationship between the adenosine triphosphate-sensitive K current (K _ATP current), hypoxic shortening and Ca accumulation in cardiomyocytes exposed to anoxia or metabolic inhibition. Whole-cell, patch-clamp experiments were performed with nonstimulated isolated rat heart ventricular muscle cells loaded with the Ca-sensitive fluorescent dye 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy) ethane-N,N,N′,N′-tetraacetic acid (fura-2) via the patch pipette. After approximately 8 min anoxia, the K _ATP current started to rise and reached a maximum of 21.3 ± 3.7 nA (n = 5, recorded at 0 mV clamp potential) within 1–3 min. At that time hypoxic contracture also occurred. Resting cytoplasmic free calcium (Ca_i) did not change significantly before hypoxic shortening. After hypoxic contracture, the K _ATP current decreased and Ca_i started to rise, reaching about 1 μmol/l. The presence of glibenclamide (10 μmol/l) in the bath reduced the anoxia-induced K _ATP current by more than 50%, but did not significantly influence the time dependence of current, hypoxic shortening and Ca_i, or the magnitude of Ca_i. Metabolic inhibition with 1.5 mmol/l CN resulted in K _ATP current increase and hypoxic shortening, occurring somewhat earlier than under anoxia, but all other parameters were comparable. In non-patch-clamped cells loaded with fura-2 AM ester and field-stimulated with 1 Hz, 1 μmol/l glibenclamide had no significant effect on the magnitude of the Ca_i increase caused by exposure of the cells to 1.5 mmol/l CN⁻. After CN⁻ wash-out in non-patch-clamped cells, Ca_i declined, oscillated and finally returned to control values. It can be concluded that glibenclamide inhibits anoxia-induced K _ATP currents only partially and has no significant effect on anoxia-induced rise in resting Ca_i. Received: 3 November 1995/Received after revision: 9 January 1996/Accepted: 16 January 1996     

Changes in the infectivity, pyruvate kinase activity, acid phosphatase activity and P-glycoprotein expression in glibenclamide-resistant Leishmania mexicana

We previously demonstrated susceptibility of Leishmania sp. to glibenclamide, a K⁺-ATP transport blocker which interacts with members of the superfamily of adenosine 5′ triphosphate-binding cassette transporters. In order to characterize the molecular differences between a sensitive Leishmania strain, NR(Gs), and an experimentally selected glibenclamide-resistant strain, NR(Gr), specific biochemical and functional parameters have been evaluated both in the wild type and in the resistant strain. Most noteworthy, NR(Gr) exhibit an increased expression of P-glycoprotein and a decreased activity of functional key enzymes such as acid phosphatase, a prominent virulent factor of the parasite, and pyruvate kinase, a key control enzyme for both carbohydrate and protein metabolism. The specific biochemical, metabolic and functional changes observed in the resistant strain correlated with a reduced infectivity of stationary phase NR(Gr) in J774 macrophages and suggested a mechanism to overcome the effect of glibenclamide. Received: 21 January 2000 / Accepted: 1 March 2000     

Thymoquinone-induced relaxation of isolated rat pulmonary artery

Aim of the study

The effect of thymoquinone (TQ), the main constituent of the volatile oil of black seed (Nigella sativa L. family Ranunculaceae), on the isolated rat pulmonary arterial rings was investigated.

Materials and methods

Isolated rat pulmonary arterial rings were precontracted with phenylephrine and concentration–response curves to TQ were constructed. The effects of different receptors antagonists or enzyme inhibitors were examined.

Results

TQ caused a concentration-dependent decrease in the tension of the pulmonary arterial rings precontracted by phenylephrine. The effects of TQ were not influenced by pretreatment of the rings with propranolol (a non-selective β-blocker), atropine (a non-selective blocker for muscarinic receptors), theophylline (an adenosine receptor antagonist), indomethacin (a cyclooxygenase inhibitor), L-NAME (a NO synthase inhibitor), methylene blue (an inhibitor of soluble guanylyl cyclase) and nifedipine (a Ca²⁺ channel blocker). The effects of TQ were significantly potentiated by bosentan (an ET_A/ET_B receptor antagonist). The effects of TQ were slightly abolished by pretreatment of the rings with glibenclamide (a non-selective blocker of ATP-sensitive K+ channels). TQ totally abolished the pressor effects of serotonin and phenylephrine on the isolated rat pulmonary arterial rings.

Conclusion

The results of the present study suggest that TQ-induced relaxation of the precontracted pulmonary artery is probably mediated, at least in part, by activation of ATP-sensitive potassium channels and possibly by non-competitive blocking of serotonin, α1 and endothelin receptors.     

Controlled extension of oral antidiabetic therapy on former insulin dependent diabetics by means of the combined i.v. glibenclamide-glucose-response-test

Summary A new sulphonylurea response test is described for predicting the results of long-term treatment with a recently developed sulphonylurea compound, glibenclamide, particularly in insulin-dependent tolbutamide-non-responsive elderly diabetics. The test is based on the observation that the insulin-stimulating capacity of glucose and the determination of the insulin increases are strikingly potentiated following glibenclamide plus glucose i.v. (25 plus 0.33 g/kg body weight) in serum samples where insulin binding antibodies have been removed. 11 out of 40 diabetics demonstrating between 60 and 90 min following injection, a mean increase of insulin of more than 500 per cent above the initial values, correlated satisfactorily with successful long-term oral treatment with glibenclamide. A positive glibenclamide-glucose-response test contrasted with primary failure of glibenclamide therapy in only one patient suffering from haemochromatosis. Oral treatment with glibenclamide may have certain advantages over insulin therapy, especially in elderly diabetics suffering from visual impairment, who are unable to inject themselves with insulin.Support of this Study by Deutsche Forschungsge-meinschaft (Pf 38/28) is gratefully acknowledged.     

Secondary sulfonylurea failure: comparison of period until insulin treatment between diabetic patients treated with gliclazide and glibenclamide

We retrospectively evaluated a possible difference in periods until start of insulin treatment between type 2 diabetic patients treated with gliclazide (GCZ) and glibenclamide (GBC), because GCZ might be protective for beta cells than GBC. Subjects were Japanese patients. GCZ group consisted of patients treated with GCZ alone or with GCZ and GBC in the separate treatment periods in combination with or without other oral hypoglycemic agents (OHAs), while GBC group consisted of patients with GBC alone or in combination with other OHAs except GCZ. The periods until the treatment of insulin commenced were calculated using the Kaplan-Meier method. Proportional hazards models were used to adjust the differing variables between GCZ and GBC groups. The periods until the start of insulin treatment from diabetes onset, diabetes treatment, or GCZ or GBC treatment were significantly longer in the GCZ group than those in GBC group (P < 0.001 in each group). Independent variables affecting the period were average HbA1c levels during GCZ or GBC treatment (hazard ratio = 2.5 per %), other OHAs combined (hazard ratio = 1.9 on combination), and difference between GCZ and GBC groups (hazard ratio = 0.5 on GCZ). These results imply that GCZ may be more protective against secondary beta cell failure than GBC.     

2例新生儿糖尿病患者致病基因型及疗效分析

目的：分析2例新生儿糖尿病患者的致病基因型及探讨格列苯脲的疗效。方法选取2013年4月至2014年10月本院收治并确诊的新生儿糖尿病患儿2例,对其进行常见致病基因KCNJ11、ABCC8、INS和GCK 4个基因测序分析,并复习相关文献,根据基因结果给予硫脲类药物试验性治疗。结果病例1的KCNJ11基因检测到3个杂合突变,其中Arg201His为已报道的永久性新生儿糖尿病致病突变,余2个位点为无致病性的单核苷酸多态性,ABCC8、INS和GCK基因检测未发现突变;病例2的KCNJ11基因检测到2个杂合突变,位点与病例1非致病性突变位点相同,余基因测序未发现异常。给予病例1格列苯脲口服疗效满意,胰岛素减停后随访血糖平稳;给予病例2格列苯脲试验性用药,疗效不佳。结论新生儿糖尿病的遗传发病机制复杂,KCNJ11基因突变可导致新生儿糖尿病的发生,格列苯脲适于用治疗KCNJ11基因突变阳性病例。     

Differential antagonism by glibenclamide of the relaxant effects of cromakalim,pinacidil and nicorandil on canine large coronary arteries

Summary The relaxant mechanisms of action of cromakalim, pinacidil and nicorandil, potassium channel openers, on large epicardial coronary arteries were investigated in isolated canine left circumflex arteries contracted by 10^–7 mol/l U46619, a thromboxane A₂ analogue, or addition of 25 mmol/l KCl in comparison with nitroglycerin.Cromakalim (3 × 10^–8–3 × 10^–5 mol/l), pinacidil (10^–6–10^–4 mol/l), nicorandil (3 × 10^–6–10^–3 mol/l) and nitroglycerin (3 × 10^–8–10^–5 mol/l) all produced a concentration-dependent relaxation in both U46619- or KCl-contracted arteries. At their maximum effects pinacidil, nicorandil and nitroglycerin produced full relaxation in arteries contracted by either means. In contrast, cromakalim produced about a 73% relaxation in KCl-contracted arteries, although it caused full relaxation in U46619-contracted ones. In the presence of glibenclamide the concentration-relaxation curves for cromakalim in U46619- or KCl-contracted arteries underwent rightward parallel shifts. Schild regression had a slope of 1.00 and yielded a pA₂ of 7.47 for glibenclamide in U46619-contracted arteries, and corresponding values obtained in KCl-contracted arteries were 0.86 (not significantly different from unity) and 7.28. The concentration-relaxation curves for pinacidil in U46619-contracted arteries also underwent rightward parallel shifts in the presence of glibenclamide, however, Schild regression had a slope of 0.60. The concentration-relaxation curves for pinacidil in KCl-contracted arteries underwent rightward parallel shifts only to a limited extent in the presence of glibenclamide. The concentration-relaxation curves for nicorandil and nitroglycerin in U46619- or KCl-contracted arteries were not affected by glibenclamide in concentrations which antagonized cromakalim. The concentration-relaxation curves for nicorandil or nitroglycerin in U46619-contracted arteries were shifted by methylene blue (10^–5 mol/l) to the right without suppression of the their maximum effects. Similar curves for cromakalim were not affected at all by this concentration of methylene blue. The concentration-relaxation curves for nicorandil in U46619-contracted arteries determined in the presence of methylene blue (10^–5 mol/l) and glibenclamide (3 × 10^–7, 10^–6 and 3 × 10^–6 mol/l) were not significantly different from those in the presence of methylene blue alone.These results indicate the following: In canine large epicardial coronary arteries (1) cromakalim produced relaxation by the mechanism antagonized by glibenclamide, probably opening ATP-sensitive potassium channels, (2) pinacidil did so by the mechanism shared with cromakalim and by one not antagonized by glibenclamide as well, and (3) nicorandil did so exclusively by the mechanism of action as a nitrate. Send offprint requests to N. Taira at the above address     

Cl− channel inhibition by glibenclamide is not specific for the CFTR-type Cl− channel

As long as the question of which channels are responsible for cAMP-mediated epithelial Cl^– secretion remains unsolved, it is still important to search for specific inhibitors that might help to relate macroscopic to microscopic events. Following the report by Sheppard and Welsh (J Gen Physiol 100: 573, 1992) that glibenclamide inhibits whole-cell Cl^– currents in genetically manipulated fibroblasts expressing the cystic fibrosis transmembrane conductance regulator (CFTR), we have studied the effect of glibenclamide on different types of Cl^– channels of HT₂₉ and T84 cells at the single-channel level. Our results confirm that micromolar concentrations of glibenclamide inhibit the linear, low-conductance Cl-channel, which appears to represent CFTR and show that the inhibition results from a typical flicker block. However, the same concentrations of glibenclamide inhibit also the outwardly rectifying intermediate conductance Cl^– channel which, potentially, may contribute to transepithelial Cl^– secretion.     

Inclusion complexation of glibenclamide with 2-hydroxypropyl-β-cyclodextrin in solution and in solid state

Phase solubility diagrams have been used to investigate complexation between 2-hydroxypropyl-β-cyclodextrin (HPBCD) and glibenclamide (GM) in aqueous medium. More stable GM-HPBCD complexes were formed in alkaline medium (in which the drug is in ionized form) than in acid medium (in which the drug is in non-ionized form). The formation of solid GM-HPBCD inclusion complexes has been evaluated by using kneading, spray-drying and freeze-drying methods. Characterization of the resulting mixtures by X-ray diffraction, infrared spectroscopy and differential scanning calorimetry indicated that inclusion complexes can be obtained by spray drying and freeze drying but not by kneading. According to the phase solubility results, drug solubility in alkaline medium was greatly improved by inclusion with HPBCD, whereas in acid medium inclusion with HPBCD had no appreciable effect. Cyclodextrin complexation of ionized drug molecules in alkaline medium resulted in greater total solubilization, i.e., solubilization of the drug due to both cyclodextrin complexation and ionization.     

The effect of glibenclamide and metformin on serum lipoproteins in type 2 diabetes

In a cross-over study, the effects of 3 months treatment with metformin or glibenclamide on body weight, blood glucose control, and serum lipoproteins were compared in 35 Type 2 diabetic patients, inadequately controlled by dietary therapy alone. Glibenclamide alone increased body weight (mean change +2.75 kg; 95% confidence intervals +1.95 to +3.55 kg; p less than 0.0001). Glibenclamide and metformin achieved equivalent blood glucose control, independent of initial body mass index. Neither drug affected serum triglyceride concentration. Metformin alone significantly reduced low density lipoprotein cholesterol (mean change -0.34 mmol l-1; 95% confidence intervals -0.12 to -0.57 mmol l-1; p less than 0.01). Neither drug altered high density lipoprotein or subfraction cholesterol.