Modulation of potassium currents by angiotensin and oxidative stress in cardiac cells from the diabetic rat

Abstract

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.

Figure 1. Comparison of DHE fluorescence in control and diabetic myocytes

A, Images of myocytes from control (a) and diabetic (b) male rats. Panels c (control) and d (diabetic) show the uppermost nuclei in (a) and (b) after a lower threshold was chosen and pixels below threshold were set to an intensity of 0. All compared images were collected at the same camera settings. Scale bar in b, 10 μm. B, mean results obtained by analysis of DHE fluorescence images of nuclei from control and male diabetic rats. For this analysis, the mean fluorescence intensity of the pixels in seven nuclei in myocytes from non-diabetic animals (normalized as 100%) and 10 nuclei from male diabetic animals were compared using the threshold method of analysis. Mean DHE fluorescence intensity in nuclei from the diabetic myocytes was 155 ± 10%, compared to nuclei from the control rats (P < 0.003). Similar results were obtained when the nuclear images were analysed using a second method (see Methods), in which the fluorescence intensity in nuclei from the male diabetic rats was 151 ± 6% of that in nuclei from control rats.

Figure 2. Effects of the NADPH inhibitor apocynin on outward currents in myocytes from male diabetic rats

A, current traces (obtained in response to pulses from −80 mV to potentials ranging from −10 to +50 mV) from two cells in the absence (left) or following 8 h in the presence of 300 μm apocynin (right). B, mean current densities at +50 mV for I_peak (left) and I_sus (right) in the absence (open bars) or presence (hatched bars) of apocynin, which significantly enhances (P < 0.005) both currents.

Figure 3. Effects of the NADPH inhibitor apocynin on DHE fluorescence (indicating superoxide anion levels)

Several cells and nuclei from diabetic male rats are shown either in the absence (A) or in the presence (B) of apocynin (300 μm, 6 h). Scale bar, 10 μm. C, mean (±s.e.m.) DHE fluorescence intensity in the absence (open bar, normalized as 100%) and presence of the drug (hatched bar), for one set of cells. *P < 0.05. A similar significant reduction of DHE intensity by apocynin was obtained in five other experiments.

Figure 4. Effects of SOD and catalase on K⁺ currents

A, superoxide dismutase (SOD, 300 U ml⁻¹, 5–9 h) significantly augments K⁺ currents in myocytes from male diabetic rats. Mean I_peak (left) and I_sus (right) current densities (at +50 mV) are shown in the absence (open bars) and presence (hatched bars) of PEG-SOD. **P < 0.005; ***P < 0.0005. B, in contrast, catalase (500 U ml⁻¹, 5–9 h exposure) has no effect on I_peak (left) or I_sus (right).

Figure 5. Absence of effects of oxidative stress inhibitors in cells from diabetic females

A, current traces (same protocol as Fig. 2) from two cells, in the absence (left) or following (right) 9 h exposure to apocynin (300 μm). B, mean current densities (at +50 mV) in the absence (open bars) or presence (hatched bars) of apocynin (5–9 h). C, superoxide dismutase (300 U ml⁻¹, 5–9 h) has no significant effect on I_peak (left) or I_sus (right), in marked contrast to the augmentation observed in cells from diabetic males.

Figure 6. Sex-dependent effects of reduced glutathione (GSH) on outward currents in ventricular cells from STZ-induced diabetic rats

A, current traces obtained from two cells obtained from males, in response to 500-ms voltage steps from −80 mV to potentials ranging from −10 to +50 mV, in the absence (left) or following 5.5 h in the presence of 1 mm GSH (right). Below are the current–voltage relationships, with mean (±s.e.m.) current densities plotted against membrane potentials in the absence (•) or following 5–8 h the presence of in GSH (▪). GSH significantly augments both I_peak (left) and I_sus (right). B, Current–voltage relationships obtained in cells from diabetic females (current traces omitted for clarity). No effects of GSH on I_peak or I_sus were observed in these cells. C, in contrast, the effects of GSH are restored in diabetic females that had been ovariectomized. *P < 0.05; **P < 0.005.

Figure 7. Comparison of superoxide ion levels in diabetic males and females

A, DHE fluorescence images of myocytes from male (a) and female (b) diabetic rats. Images were collected at the same camera settings. Scale bar in b, 10 μm. B, results of analysis of images of nuclei from male and female diabetic rats. For this analysis, the mean fluorescence intensity of the pixels in 18 nuclei in myocytes from male diabetic animals and 19 nuclei from female diabetic animals were compared using the threshold method of analysis. The mean DHE fluorescence intensity in the nuclei from the female animals was 64 ± 6% of that in the nuclei from the male animals (normalized as 100%), which was significantly (P < 0.001) different. Similar results were obtained when the nuclear images were analysed using the second method (see Methods). This method of analysis indicated that the fluorescence intensity in the nuclei from the female animals was 63 ± 6% of that in the nuclei from male animals.

Figure 8. The effects of apocynin are blunted by angiotensin in cells from male diabetic rats

A, mean current densities (at +50 mV) are shown in the absence of drugs (−) apo., after 5–8 h incubation with 300 μm apocynin (+) apo., and in the presence of apocynin and 300 nm ANG II, added 1 h before apocynin (apo. + ANGII). ANG II significantly (P < 0.05) reversed current augmentation elicited by apocynin. I_peak is shown on the left and I_sus on the right. B, mean DHE fluorescence intensity mirrored these changes. Apocynin reduces DHE fluorescence in cells from diabetic rats (100%), but this effect is blunted by 300 nm ANG II.

Figure 9. Effects of quinapril treatment on superoxide levels

A, DHE fluorescence images from myocytes obtained from a diabetic rat (a) and from a diabetic rat after in vivo quinapril treatment (b). Scale bar, 10 μm. B, summary data obtained from analysis of the fluorescence of 78 nuclei (42 from untreated rats, normalized as 100%, and 36 from quinapril-treated rats). The intensity of fluorescence following quinapril-treatment was reduced by 43.2% (P < 0.0001).