Single ventricular myocytes were prepared from control db/+ and insulin-resistant diabetic db/db male mice at 6 and 12 weeks of age. Peak and sustained outward potassium currents were measured using whole-cell voltage clamp methods. At 6 weeks currents were fully developed in control and diabetic mice, with no differences in the density of either current. By 12 weeks both currents were significantly attenuated in the diabetic mice, but could be augmented by in vitro incubation with the angiotensin-converting enzyme (ACE) inhibitor quinapril (1 µm, 5-9 h). In cells from female db/db mice (12 weeks of age), K + currents were not attenuated and no effects of quinapril were observed. To investigate whether lack of insulin action accounts for these gender differences, cells were also isolated from cardiomyocte-specific insulin receptor knockout (CIRKO) mice. Both K + currents were significantly attenuated in cells from male and female CIRKO mice, and action potentials were significantly prolonged. Incubation with quinapril did not augment K + currents. Our results demonstrate that type 2 diabetes is associated with gender-selective attenuation of K + currents in cardiomyocytes, which may underlie gender differences in the development of some cardiac arrhythmias. The mechanism for attenuation of K + currents in cells from male mice is due, at least in part, to an autocrine effect resulting from activation of a cardiac renin-angiotensin system. Insulin is not involved in these gender differences, since the absence of insulin action in CIRKO mice diminishes K + currents in cells from both males and females.

Diabetes induces oxidative stress and leads to attenuation of cardiac K + currents. We investigated the role of superoxide ions and angiotensin II (ANG II) in generating and linking oxidative stress to the modulation of K + currents under diabetic conditions. K + currents were measured using patch-clamp methods in ventricular myocytes from streptozotocin (STZ)-induced diabetic rats. Superoxide ion levels, indicating oxidative stress, were measured by fluorescent labelling with dihydroethidium (DHE). ANG II content was measured using enzyme-linked immunosorbent asssay (ELISA). The results showed DHE fluorescence to be significantly higher in cells from diabetic males, compared to controls. Relief of stress by the NADPH oxidase inhibitor apocynin or by superoxide dismutase (SOD) but not by catalase reversed the attenuation of K + currents and reduced DHE fluorescence. In cells from diabetic females, neither apocynin nor SOD augmented K + currents, ANG II was not elevated and DHE fluorescence was significantly weaker than in cells from males. Reduced glutathione (GSH) also augmented K + currents in cells from diabetic males but not females. In ovariectomized diabetic females K + currents were augmented by GSH and apocynin. Current augmentation and the attenuation of DHE fluorescence by apocynin were significantly blunted by excess ANG II (300 nM). Diabetic male rats pretreated with the angiotensin-converting enzyme (ACE) inhibitor quinapril were hyperglycaemic, but their cellular ANG II levels and DHE fluorescence were significantly decreased. In cells from these rats, K + currents were insensitive to apocynin. In conclusion, diabetes-related oxidative stress attenuates K + currents through ANG II-generated increased superoxide ion levels. When ANG II levels are lower, as in diabetic females or following ACE inhibition in males, oxidative stress is reduced, with blunted alterations in K + currents.

The effect of a reduction in protein kinase C activity on the metabolism of exogenous [3H]diC8 by freshly isolated smooth muscle cells from rabbit aorta and cultured A10 smooth muscle cells was determined. The metabolism of [3H]diC8 by both smooth muscle cell preparations was predominantly by hydrolysis to yield monoC8 and glycerol (lipase pathway); very little radioactivity was incorporated into phospholipids. Diacylglycerol lipase activity measured in vitro with A10 cell homogenates was much greater than diacylglycerol kinase activity. The addition of the protein kinase C inhibitor H-7 to incubations of isolated aortic smooth muscle cells and cultured A10 cells had no significant effect on the metabolism of [3H]diC8. Protein kinase C activity in cultured A10 cells preincubated for 20 h with a phorbol ester was reduced to 14% of control as a consequence of down-regulation, but diC8 metabolism was not changed. Therefore, protein kinase C does not regulate the metabolism of diacylglycerols in aortic smooth muscle cells.

The regulatory effects of diacylglycerol (DAG) second messengers will be terminated by metabolism. A long-chain DAG, 1-palmitoyl-2-[1-14C]oleoyl-sn-glycerol (2-[14C]POG), was metabolized by cultured A10 smooth muscle cells after permeabilization by preincubation with 340 U/ml alpha-toxin from Staphylococcus aureus. In contrast to results with the cell-permeable DAG analogue, dioctanoyl-glycerol ([3H]diC8), no appreciable 2-[14C]POG degradation could be detected in control A10 cells not treated with alpha-toxin. With permeabilized A10 cells, 2-[14C]POG was mainly converted into lipolytic products of a lipase pathway, monoacylglycerol (MG) and fatty acid (FA); very little radioactivity was incorporated into triacylglycerol (TG) or phospholipid (PL) via reactions catalyzed by either DAG acyltransferase, cholinephosphotransferase, or DAG kinase. Similar results were obtained in experiments with 1-stearoyl-2-[1-14C]arachidonoyl-sn-glycerol. The conversion of 2-[14C]POG into PL and TG was not enhanced by the addition of 1 mM ATP-MgCl2, 1 mM CDP-choline, or 1 mM oleoyl-CoA to the alpha-toxin-treated A10 cells. The formation of FA and MG by permeabilized A10 cells was inhibited by DAG lipase inhibitors, U-57,908 (50 microM) and tetrahydrolipstatin (1-25 nM). The predominant contribution of the lipase pathway to the metabolism of a long-chain DAG, 2-[14C]POG, by alpha-toxin-treated A10 cells is similar to results for the degradation of [3H]diC8 by intact A10 cells.