The role of potassium channels in the proliferation and migration of endometrial adenocarcinoma HEC1-A cells.

BACKGROUND: Endometrial cancer is the most common gynecological cancer in developed countries. Potassium channels, which have many types, are suggested to play a major role in cancer progression. However, their role in endometrial cancer has not been fully investigated. We aimed to demonstrate whether the ATP-sensitive potassium channel blocker glibenclamide, voltage-sensitive potassium channel blocker 4-aminopyridine, non-selective (voltage-sensitive and calcium-activated) potassium channels blocker tetraethylammonium and potassium chloride (KCl) have any effect on the proliferation and migration of HEC1-A cells. METHODS AND RESULTS: Proliferation and migration were evaluated by real-time cell analysis (xCELLigence system) and wound healing assays, respectively. Proliferation was reduced by glibenclamide (0.1 and 0.2 mM, P < 0.05 and P < 0.01, respectively), 4-aminopyridine (10 and 20 mM, P < 0.001) and tetraethylammonium (10 and 20 mM, P < 0.01 and P < 0.001, respectively). However, KCl did not change the proliferation. Migration was reduced by glibenclamide (0.01, 0.1 and 0.2 mM, P < 0.001, P < 0.001 and P < 0.01, respectively) and 4-aminopyridine (10 and 20 mM, P < 0.05 and P < 0.01, respectively). Tetraethylammonium did not change migration. However, KCl reduced it (10, 25 and 50 mM, P < 0.05, P < 0.01 and P < 0.01, respectively). Both proliferation and migration were reduced by glibenclamide and 4-aminopyridine. However, tetraethylammonium only reduced proliferation and KCl only reduced migration. CONCLUSIONS: Potassium channels have an important role in HEC1-A cell proliferation and migration and potassium channel blockers needs to be further investigated for their therapeutic effect in endometrial cancer.

Involvement of Rho-kinase/IκB-α/NF-κB activation in IL-1ß-induced inflammatory response and oxidative stress in human chondrocytes.

It has been clearly indicated that osteoarthritis (OA) is an inflammatory and degenerative disease that could be promoted by Rho-kinase (ROCK); however, little is known about the role of ROCK/inhibitor κB alpha (IκB-α)/nuclear factor-κB (NF-κB) p65 pathway activation in interleukin-1ß (IL-1ß) induced inflammatory response and oxidative stress in primary human chondrocytes. To test this hypothesis, we focused on determining ROCK-II, IκB-α, p-IκB-α, NF-κB p65, p-NF-κB p65, IL-6, tumor necrosis factor alpha (TNF-α), cyclooxygenase-2 (COX-2), p22phox, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subtype 4 (NOX4) protein expression, ROCK-II activity, NADPH oxidase levels, and total antioxidant capacity (TAC) in the presence and absence of ROCK-inhibitor fasudil. IL-1ß (2 ng·mL-1, 24 h) increased the expression of ROCK-II, p-IκB-α, NF-κB p65, p-NF-κB p65, IL-6, TNF-α, COX-2, and p22phox proteins, and decreased the expression of IκB-α, and the NOX4 protein level did not alter. ROCK activity and NADPH oxidase levels were increased, whereas the TAC was decreased by IL-1ß. Fasudil (10-5-10-7 M) reversed all these changes induced by IL-1ß. These results demonstrate that ROCK/IκB-α/NF-κB p65 pathway activation contributes to the IL-1ß-induced inflammatory response and oxidative stress, and thus, ROCK inhibition might be a beneficial treatment option for OA patients mainly based on its anti-inflammatory and antioxidant effects.

Effects of Vitamin D Analogs Alfacalcidol and Calcitriol on Cell Proliferation and Migration of HEC1A Endometrial Adenocarcinoma Cells.

Endometrial carcinoma is the most diagnosed among infiltrating tumor of the female genital tract. Vitamin D has antiproliferative and immunomodulatory properties besides its classical effect on calcium and phosphate. We aimed to demonstrate whether alfacalcidol and calcitriol have any effect on proliferation and migration. Endometrial adenocarcinoma HEC1A was used as a cancer cell line. The effect of alfacalcidol (1α-hydroxyvitamin D3) and calcitriol (1α,25-dihydroxyvitamin D3) on proliferation was demonstrated by real-time cell analysis device and migration was shown by a wound healing model. 10-11-10-9M alfacalcidol and calcitriol reduced both proliferation and migration. In contrast, the high concentration of alfacalcidol and calcitriol (10-8-10-6M) increased proliferation and migration. The proliferative effects of alfacalcidol (0-12 h) immediately started earlier than calcitriol (12-48 h). In contrast, the antiproliferative effects of calcitriol (12-24 h) have begun earlier than alfacalcidol (48-60 h). While the high concentrations of alfacalcidol and calcitriol increased the migration relatively earlier (12-24 h), low concentrations decreased the migration at late times (24-48 h). Lower concentrations of vitamin D prevent proliferation and migration in endometrial adenocarcinoma HEC1A cells. In contrast, high concentrations of vitamin D increase proliferation and migration.

Evaluation of the Rho A/Rho-kinase pathway in the uterus of the rat model of polycystic ovary syndrome.

The aim of this study was to investigate the expression of RhoA/Rho-kinase in the uterus and the effect of Rho-kinase inhibitors on uterine contractions of dehydroepiandrosterone (DHEA) induced polycystic ovary syndrome (PCOS) rats. Forty-four female Sprague-Dawley (21 days old) rats divided into three groups: The control group (n = 14, any procedure was not performed), vehicle group (n = 14, 0.2 ml of sesame oil, subcutaneous injection, 20 days) and PCOS group (n = 16, DHEA 6 mg/100 g in 0.2 ml of sesame oil, subcutaneous injection, 20 days). The myometrium thickness and uterine wet weight were assessed. The mRNA and protein expressions of Rho A, the effect of Rho-kinase inhibitors (fasudil and Y-27632) on KCl, carbachol, and PGF2α induced contractions were evaluated in the uterus. In the PCOS group, the myometrium thickness and uterine wet weight significantly increased compared to the control group and vehicle group. The mRNA expression level and the immunoreactive score of Rho A, ROCK 1, ROCK 2 were similar in all groups. In the PCOS group, KCl, carbachol, and PGF2α induced uterine contractions significantly increased compared to the control group and vehicle group. Fasudil and Y-27632 significantly inhibited KCl, carbachol, and PGF2α induced uterine contractions in all groups. In conclusion, the expression of Rho A, ROCK 1, ROCK 2 not changed although myometrium thickness, uterine wet weight and the contractile responses of uterus increased in the PCOS group. The results suggest that the Rho-kinase inhibitors effectively suppressed increased contractions in the PCOS group they might be potential therapeutic agents.

The effects of fibroblast growth factor-2 and pluripotent astrocytic stem cells on cognitive function in a rat model of neonatal hypoxic-ischemic brain injury.

OBJECTIVE: This study aimed to determine the effect of pluripotent astrocytic stem cells (PASCs) and fibroblast growth factor-2 (FGF-2) on cognitive function in neonatal rats with hypoxic-ischemic brain injury (HIBI). METHODS: The study was performed on 7-d-old rats that were randomly divided into four groups. All rats, except those in the sham group, were kept in a hypoxic chamber containing 8% oxygen for 2 h after the ligation of the right carotid artery. Next, 5 d after HIBI was induced, PASCs were administered to the motor cortex, and FGF-2 was administered intraperitoneally to group AF; PASCs were administered to the motor cortex, and salt solution buffered with phosphate was administered intraperitoneally to group A; and fresh cell culture solution (medium) was administered to group M. Immunofluorescence was used to localize the administered PASCs in the brains of rats from groups A and AF. The Morris water maze tank (MWM) test was performed to assess the rats' cognitive functions at week 12. The rats that were administered PASCs were observed for the development of neoplasms and autopsies were performed after 30 months. RESULTS: PASCs migrated to damaged brain regions surrounding the hippocampus in groups A and AF. The mean platform finding time (PFT) significantly decreased over time in each group on day 1-4 of MWM testing (p < 0.001). On day 2-4, the mean PFT was shortest in group S followed by group AF. In group A, the PFT was significantly longer than in group S on day 3-4 (p = 0.01 and 0.007, respectively). On day 5 of the MWM test, the time spent in the eastern quadrant (which previously contained the platform) was longest in group S followed by groups AF, A, and M; however, the differences between groups were not significant (p = 0.51). After 30 months, none of the rats in groups A or AF had benign or malignant neoplasms. CONCLUSIONS: Following the administration of PASCs in rats with experimentally induced HIBI, PASCs migrated to the injured brain regions; however, treatment with PASCs did not have a positive effect on cognitive function. The administration of FGF-2 together with PASCs resulted in positive cognitive results, although not at the level of significance.

Effects of 17ß-estradiol and progesterone on the production of adipokines in differentiating 3T3-L1 adipocytes: Role of Rho-kinase.

Effect of female sex hormones on the production/release of adipocyte-derived cytokines has been debatable. Furthermore, whether the cellular signaling triggered by these hormones involve Rho-kinase has not been investigated yet. Therefore, in this study, effects of 17ß-estradiol and progesterone as well as the Rho-kinase inhibitor, Y-27632 on the level of adipokines such as resistin, adiponectin, leptin, TNF-α and IL-6 were investigated in 3T3-L1-derived adipocytes. Differentiation was induced in the post-confluent preadipocytes by the standard differentiation medium (Dulbecco's modified Eagle's medium with 10% fetal bovine serum together with the mixture of isobutylmethylxanthine, dexamethasone and insulin) in the presence of 17ß-estradiol (10(-8)-10(-7)M), progesterone (10(-6)-10(-5)M), the Rho-kinase inhibitor, Y-27632 (10(-5)M) and their combination for 8days. Measurements of the adipokines were performed in the culturing medium by ELISA kits using specific monoclonal antibodies. 17ß-estradiol elevated resistin but decreased adiponectin and IL-6 levels; however, it did not alter the concentration of leptin and TNF-α. Y-27632 pretreatment inhibited the rise of resistin and the fall of adiponectin by 17ß-estradiol without any effects by its own. Progesterone did not change resistin, leptin and TNF-α level; however, it elevated adiponectin and decreased IL-6 production. Neither 17ß-estradiol nor Y-27632 was able to antagonize the increase of adiponectin and the reduction of IL-6 levels by progesterone. While Y-27632 alone lowered IL-6 level, it increased leptin and TNF-α concentration without altering resistin and adiponectin. In conclusion, 17ß-estradiol could modify adipokine production in 3T3-L1 adipocytes with the actions some of which involve Rho-kinase mediation.

Hyperosmolar glucose induces vasoconstriction through Rho/Rho-kinase pathway in the rat aorta.

Rho/Rho-kinase signalling pathway plays a substantial role in vascular contractions. In this study, we investigated any roles of Rho/Rho-kinase pathway in the vasoconstriction of the rat conductance and capacitance vessels by hyperosmolar glucose solution. Isolated aortic, mesenteric and renal rings were suspended and exposed to hyperosmolar glucose, sucrose and NaCl in the organ chambers filled with Krebs solution gassed with 95% O2 and 5% CO2 and maintained at 37 °C. The effect of a Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10(-5) M), was tested on the contraction induced by hypertonic solutions. Endothelial integrity was also assessed after hyperosmolar glucose exposure. Moreover, the activity and expression of Rho-kinase (ROCK-2) as well as RhoA translocation were detected by Western blotting and enzyme-linked immunosorbent assay-based RhoA activity detection method detection kit. The vessels produced substantial contractions in response to hyperosmolar solutions. Y-27632 significantly reduced hyperosmolarity-induced vasoconstrictions (P < 0.05). Phosphorylation of myosin-phosphatase target 1 increased after hyperosmolar glucose exposure, and this phosphorylation was significantly decreased by Y-27632 (P < 0.05) in the aorta. Furthermore, RhoA translocation but not ROCK-2 expression markedly increased by hyperosmolar glucose solution. These results may indicate that hyperosmolarity could induce vasoconstriction through Rho/Rho-kinase signalling.

Proton pump inhibitors omeprazole, lansoprazole and pantoprazole induce relaxation in the rat lower oesophageal sphincter.

OBJECTIVES: We aimed to investigate effects of the proton pump inhibitors (PPIs) omeprazole, lansoprazole and pantoprazole, which are currently used for the treatment of hyperacidity and gastro-oesophageal reflux, on the reactivity of the isolated rat lower oesophageal sphincter. METHODS: Omeprazole, lansoprazole and pantoprazole (all 10(-9) -10(-3) m, cumulatively) were tested on carbachol-induced (10(-6) m) contraction. In addition, the effects of PPI preincubation (all 10(-3) m) on the contractions induced by cumulative carbachol (10(-9) -10(-5) m), angiotensin-2 (10(-9) -10(-5) m) or electrical field stimulation (EFS; 40 V, 32 Hz, 1 ms, 10 s) were assessed. Finally, the effects of PPI on the spontaneous contractile activity of the tissue were also evaluated. KEY FINDINGS: PPI relaxed precontracted lower oesophageal sphincter in a concentration-dependent manner and suppressed carbachol-, angiotensin- and EFS-induced contractions. Furthermore, PPI attenuated spontaneous contractile activity of the tissue. CONCLUSIONS: Omeprazole, lansoprazole and pantoprazole had a suppressor effect on lower oesophageal sphincter contractions.

Myocardial ischemia during everyday life in patients with arterial hypertension: prevalence, risk factors, triggering mechanism and circadian variability.

INTRODUCTION: The objective of the present study was to investigate the prevalence, the risk factors, the hemodynamic triggering mechanisms, the circadian variability of ST segment depression (ST depression) and the effect of day and night fall in blood pressure on the prevalence of ST depression in hypertensive patients. MATERIALS AND METHODS: In a multicentric study in Germany, 1,244 CardioTens registrations (combined 24-h ambulatory blood pressure measurement/electrocardiography with ST segment triggering; Meditech, Budapest, Hungary) from patients with arterial hypertension were consecutively monitored and evaluated centrally at the University of Bonn. Inclusion criterion was treated or untreated arterial hypertension. The ST segment was measured in accordance with the "1 : 1 : 1 rule" (horizontal or descending ST depression by 1 mm, 1 min duration, 1 min interval from the previous episode). RESULTS: ST segment depression was observed in 250 (20.1%) patients; 90.3% of the transient ST-segment depression was silent (without angina pectoris). Ambulatory 24-h blood pressure measurement, but not office-based blood pressure measurement, was predictive for the occurrence of ST-segment depression. Risk factors for ST-segment depression were the Sokolow index > or =3.5 mV, smoking status, severity of coronary heart disease, use of diuretics, reduced left ventricular function, pulse pressure > or =60 mmHg and increase of double product (1,000 mmHg/min). A significant rise of the systolic/diastolic blood pressure (+8+ or -18/+7+ or -10 mmHg), of the heart rate (+12+ or -13/min) and of the double product (+2,471+ or -2,517 mmHg/min) was found during the transient ST depression as compared with the corresponding 24-h ambulatory blood pressure measurement mean values (P<0.0001 for all parameters specified). In most intermittent ST depressions, a rise of the double product was seen (n=789 episodes), and in the remaining 239 ST depressions, a fall of the double product was observed. ST depressions with fall of the double product showed a circadian distribution with a peak in the late evening. ST depression accompanied by a rise in double product showed two peaks (one in the early morning and one in the late evening). The prevalence of ST depression was significantly higher (28.6%) in extreme dippers than in dippers (18.2%), risers (21.8%) and non-dippers (19.6%). CONCLUSIONS: ST depressions have a high prevalence of 20.1% in hypertensive patients. Clinical predictors for the occurrence of ST-segment depression were classical risk factors and cardiac target organ damage. Office-based blood pressure measurement was not a useful measuring tool for forecasting the likelihood of ST-segment depression. ST depressions were triggered inter alia by variations of blood pressure and the heart rate. The circadian variability of the ST depressions is crucially affected by the pressure double product characteristics on which the ST depression is based.

Testosterone relaxes human internal mammary artery in vitro.

Preliminary clinical studies of testosterone therapy in male patients with coronary artery disease raised promising results. However, there is no study on in vitro effects of testosterone in human isolated arteries. We investigated the effect of testosterone on contractile tone of human isolated internal mammary artery. The responses in human internal mammary artery (IMA) were recorded isometrically by a force-displacement transducer in isolated organ baths. Testosterone (10 nM to 100 microM) was added cumulatively to organ baths either at rest or after precontraction with KCl (68 mM) and PGF2alpha (10 microM). Testosterone-induced relaxations were tested in the presence of cyclooxygenase inhibitor indomethacin (10 microM), nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 1 microM), nonselective large-conductance Ca2+-activated and voltage-sensitive K+ channel inhibitor tetraethylammonium (TEA, 1 mM), ATP-sensitive K+ channel inhibitor glibenclamide (GLI, 100 microM), and voltage-sensitive K+ channel inhibitor 4-aminopyridine (4-AP, 1 mM). Testosterone produced relaxation in human IMA (Emax 33% and 41% of KCl- and PGF2alpha-induced contraction, respectively). Vehicle had no significant relaxant effect. Except for TEA, the relaxation at low concentrations is not affected by either K+ channel inhibitors (GLI and 4-AP) or L-NAME and indomethacin. We report for the first time that supraphysiological concentrations of testosterone induce relaxation in IMA. This response may occur in part via large-conductance Ca2+-activated K+ channel-opening action.

Influence of estradiol pretreatment on antimuscarinic action of oxybutynin in rat detrusor muscle.

OBJECTIVES: To investigate the antimuscarinic effect of oxybutynin in the rat detrusor muscle after estrogen pretreatment because, to our knowledge, no study has been done on the interaction of estrogen with antimuscarinic drugs. Estrogen has been shown to affect muscarinic receptors in the detrusor muscle of animals. In addition, oxybutynin has been shown to block muscarinic receptors in the bladder. METHODS: Estradiol benzoate (150 microg/kg) or saline was given subcutaneously to virgin female Wistar albino rats (n = 6, each group) for 10 consecutive days. On the 11th day, isolated detrusor muscle strips were taken, and acetylcholine (ACh)-induced contractions were evaluated in the absence or presence of oxybutynin (10 and 100 nM). RESULTS: ACh induced concentration-dependent contractions in the detrusor muscle. In the estradiol-pretreated group, the maximum of the ACh-induced contractions was diminished compared with that in the control group (P < 0.05). Oxybutynin (10 and 100 nM) inhibited ACh-induced contractions competitively (pK(B) 8.85). In the estradiol-pretreated group, the concentration-response curve to ACh was shifted further to the right in the presence of oxybutynin (100 nM). CONCLUSIONS: We have demonstrated for the first time that oxybutynin further inhibits ACh-induced and muscarinic receptor-mediated contractions in rat detrusor muscle after pretreatment with estrogen.

The relationship between risk factors and testosterone-induced relaxations in human internal mammary artery.

In human internal mammary artery (IMA), testosterone induces vasodilation that shows marked variability among patients. We aimed to investigate the relationship of this variability with cardiovascular risk factors. Cumulative relaxations to testosterone after precontraction with KCl were examined in IMA segments from patients with identified cardiovascular risk factors such as hypercholesterolemia, diabetes, hypertension, smoking, age, gender, body mass index (BMI), and number of occluded vessels. Testosterone responses were significantly diminished in subjects with 3 compared with 1 risk factor. Hypercholesterolemia independently influenced testosterone responses by significantly decreasing its maximum, and smoking significantly decreased the sensitivity to testosterone. Thus, the variability observed in testosterone-induced vascular relaxations may in part be related to differences in risk factors present among the individuals studied.

Rho-kinase expression and its contribution to the control of perfusion pressure in the isolated rat mesenteric vascular bed.

Rho-kinase expression was investigated in the rat mesenteric artery and the effects of its inhibitors, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632) and fasudil (HA-1077), were examined on the increase in perfusion pressure induced by two different receptor agonists, namely the alpha-adrenoceptor agonist, phenylephrine and, the endothelin ET(A) and ET(B) receptor agonist, endothelin-1. Y-27632 and fasudil produced a concentration-dependent decrease in perfusion pressure. There was no difference between the concentration-response lines of these two inhibitors. The maximum decrease in the perfusion pressure induced by 10(-5) M Y-27632 was 85.8+/-3.7% when the tone was increased by phenylephrine. However, it was 48.1+/-5.4% (P<0.001) when the perfusion pressure was elevated by endothelin-1. Saponin perfusion (100 mg l(-1), for 10 min), which abolished acetylcholine-induced relaxation, did not significantly modify the Y-27632-elicited relaxation. Western blot analysis revealed that rat mesenteric artery expresses Rho-kinase protein with a molecular weight of approximately 160 kDa. These results show that Rho-kinase enzyme is expressed in rat mesenteric artery and that it contributes to the control of vascular resistance. Moreover, endothelium removal had no marked effect on the vasodilatation induced by Y-27632. In addition, the endothelin-1-induced vasoconstriction was more resistant to the Rho-kinase inhibitors than was that induced by phenylephrine, probably because excitatory endothelin receptors are associated with this signal transduction pathway at a different level from that of alpha-adrenoceptors.

Effects of the Rho-kinase inhibitors, Y-27632 and fasudil, on the corpus cavernosum from diabetic mice.

Relaxant responses to two Rho-kinase inhibitors, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632) and fasudil, were compared in the corpus cavernosum obtained from diabetic and non-diabetic mice. Streptozotocin (100 mg kg(-1) day(-1), for 2 days) induced diabetes with a blood glucose level of 318+/-55.4 mg dl(-1); whereas it was 85.4+/-4.1 mg dl(-1) in control mice (P<0.05). Electrical field stimulation (40 V, 0.5 ms, 1, 2, 4, 8, 16 Hz for 15 s) and acetylcholine-induced relaxations were markedly attenuated in the corpus cavernosum from streptozotocin-diabetic mice whereas responses to Y-27632 (10(-9)-3 x 10(-5) M) and fasudil (10(-9)-3 x 10(-5) M) were not altered. EC(50) values for Y-27632 were 2.98+/-0.89 and 4.19+/-2.71 microM in the corpus cavernosum from control and diabetic mice, respectively (P>0.05). The values for fasudil were 7.42+/-4.91 and 3.53+/-1.41 microM in the corpus cavernosum from control and diabetic mice, respectively (P>0.05). These results may suggest that, in diabetes, the relaxant effects of the Rho-kinase inhibitors may not be changed and thus, they may have a beneficial therapeutic effect in diabetic erectile dysfunction.

Mediation of nitric oxide from photosensitive stores in the photorelaxation of the rabbit corpus cavernosum.

Isolated rabbit corpus cavernosum relaxed in response to ultraviolet (UV) light (365 nm). The UV light-induced relaxation (photorelaxation) was diminished on repeated UV irradiation from 30.5+/-4.0% (the first photorelaxation) to 15.5+/-2.7% (the last photorelaxation). Hydroxocobolamine of 100 microM and hemoglobin (Hb) of 10 microM, which are nitric oxide (NO) scavengers, and 10 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor, markedly reduced photorelaxation. However, 300 microM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO) failed to inhibit photorelaxation. NaNO(2) and N(G)-nitro-L-arginine (L-NA) but not 3-nitro-L-tyrosine (3-NT) were found to be photosensitive in that these compounds are photolysed to release NO, as demonstrated by use of an amperometric NO probe; NO signals produced by 500 microM NaNO(2), and 500 microM L-NA were 133.3+/-28.9 and 54.4+/-10.4 pA, respectively. Not 3-NT but the other compounds (all 200 microM) also enhanced photorelaxation of the cavernosal tissue. Based on these findings, the substance, which mediates photorelaxation, could be NO released from putative stores in the rabbit corpus cavernosum, and L-NA as well as NaNO(2) but not 3-NT produce NO under the influence of UV light.

Effects of glibenclamide, metformin and insulin on the incidence and latency of death by oubain-induced arrhythmias in mice.

This study was performed to investigate possible effects of glibenclamide, insulin and metformin on the death latency and incidence caused by a cardiac glycoside, oubain. Mice of both sexes were injected with oubain (i.p. 20 mg x kg (-1), glibenclamide (s.c. 0.1-10 mg x kg (-1), insulin (s.c. 0.3-3 U kg (-)) and metformin (i.p. 200 mg x kg (-1)) and combinations of the last three drugs with oubain. Death latency was measured and lethality incidence was calculated. Death was assessed by visual observation. Plasma glucose level was evaluated from the tail blood. Glibenclamide (0.1 mg x kg (-)) prolonged the latency from 11.3 plus minus 1.2 to 15.8 plus minus 1.8 min but failed to decrease the incidence of death. At higher doses (1--10 mg x kg (-1)) it had no effects on the latency or the incidence. 0.3 U kg (-1)insulin decreased the incidence from 73.7 to 33.3% ( P< 0.05) without affecting the latency. However the higher dose (3 U kg (-1)) did not have any effects on the incidence or the latency. Oubain increased blood glucose level from 114.1 plus minus 3.8 (control) to 152.1 plus minus 5.3 mg x dl (-1). Metformin (200 mg x kg (-1)) did not affect either the latency or the incidence of death. While metformin did not decrease plasma glucose, insulin and higher doses of glibenclamide (1--10 mg x kg (-1)) markedly lowered glucose in blood. However, at the dose of 0.1 mg x kg (-1)glibenclamide did not alter the glucose level in the blood but prevented oubain from increasing it. Insulin (0.3 U kg (-1)) and, to some extent, glibenclamide (0.1 mg x kg (-)) but not metformin could be effective antiarrhythmic agents against oubain-induced arrhythmias.