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Gender differences in ANG II levels and action on multiple K⁺ current modulation pathways in diabetic rats

Cardiovascular Research Group, Department of Physiology and Biophysics, University of Calgary, Alberta, Canada T2N 4N1

Submitted 22 December 2003 ; accepted in final form 18 February 2004

	ABSTRACT

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Gender differences were studied in ventricular myocytes from insulin-deficient (Type 1) diabetic rats. Cells were obtained by enzymatic dispersion of hearts from control male and female rats and from rats made diabetic with streptozotocin (100 mg/kg) 7–14 days before experiments. ANG II content, measured by ELISA, was augmented in diabetic males but unaltered in diabetic females. In diabetic ovariectomized females, ANG II levels were augmented as in males. ANG II affects multiple cellular pathways including activation of protein kinase C (PKC) and several tyrosine kinases as well as inhibition of protein kinase A (PKA). The involvement of these pathways in modulating outward K⁺ currents was studied. Transient and sustained outward K⁺ currents were measured using the whole cell voltage-clamp method. In males, these currents are attenuated under diabetic conditions but are augmented by the ANG II-converting enzyme inhibitor quinapril. Activation of PKA by 8-bromo-cAMP enhanced both K⁺ currents in cells from diabetic males. The augmentation of these currents by quinapril was blocked when PKA inhibition was maintained with the Rp isomer of 3',5'-cyclic monophosphorothioate. Inhibition of tyrosine kinases by genistein also augmented K⁺ currents in cells from diabetic males. Action potentials were abbreviated by 8-bromo-cAMP and genistein. However, both genistein and 8-bromo-cAMP had no effect on K⁺ currents in cells from diabetic females. In cells from ovariectomized diabetic females, 8-bromo-cAMP and genistein enhanced these K⁺ currents as in males. Inhibition of PKC augmented the transient and sustained K⁺ currents in cells from diabetic males and females. A contribution of non-ANG II-dependent activation of PKC is suggested. These results describe some of the mechanisms that may underlie gender-specific differences in the development of cardiac disease and arrhythmias.

angiotensin II; streptozotocin; diabetes mellitus; cardiovascular

Diabetes has been shown to induce activation of a cardiac renin-angiotensin system (9, 29). In earlier work (32), we suggested that an autocrine/paracrine action of ANG II is partially responsible for the attenuation of repolarizing K⁺ currents and for action potential prolongation in cardiomyocytes isolated from insulin-deficient (Type 1) diabetic rats and from Type 2 diabetic db/db mice. In vitro inhibition of the angiotensin-converting enzyme (ACE) or blockade of ANG II receptors produced augmentation of K⁺ currents due to enhanced synthesis of channel proteins (34).

More recently, we determined that there is a striking gender dependence associated with this effect (35). In myocytes from streptozotocin (STZ)-treated diabetic females, K⁺ currents were not affected by ACE inhibition. Estrogen, which is known to interact with the angiotensin pathway (6, 11), was shown to play a role in the gender-selective effects of ACE inhibition on K⁺ currents. Thus in ovariectomized (Ovx) diabetic female rats that have greatly reduced estrogen levels, ACE inhibition augmented these K⁺ currents as in diabetic males. In addition, in vitro incubation with 17-estradiol was found to augment these currents, presumably due to suppression of the renin-angiotensin system. However, this effect was inhibited by addition of ANG II (35).

In our previous studies (34, 35), the effects of ANG II were inferred indirectly through these pharmacological interventions. In the present study, we set out to directly measure ANG II content in myocytes from control and diabetic rats and to determine whether gender differences could be established. In addition, we aimed to determine some of the consequences of gender-selective activation of angiotensin-dependent pathways.

ANG II has a large number of diverse cellular effects (37). These involve several distinct pathways utilizing different second-messenger systems. In cardiac cells, ANG II activates protein kinase C (PKC; Ref. 20) as well as a variety of tyrosine kinases (12, 13). ANG II also inhibits the action of protein kinase A (PKA; Ref. 3). The ANG II-dependent activation of PKC and inhibition of PKA have been linked to activation or inhibition of several ionic channels (1, 14, 24).

The present study addressed several issues that are of great interest for understanding pathological mechanisms that are activated in the setting of diabetes. The questions addressed were the following: 1) Are there gender differences in ANG II content in cardiac myocytes in diabetes? 2) Are (some of the) ANG II-activated transduction pathways linked to the attenuation of K⁺ currents in diabetes? 3) Are there differences between the actions of these pathways in myocytes from control and diabetic rats? and 4) Are there gender differences in the actions of these pathways? The results presented here provide affirmative answers to these questions.

	METHODS

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This study, approved by the University of Calgary Animal Care Committee, conforms to the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health.

Animals. The study was performed on male and female Sprague-Dawley rats (body wt, 220–300 g) that were randomly separated into control and diabetic groups. Insulin-deficient diabetes was induced with a single injection of STZ (100 mg/kg iv) given 7–14 days before isolation of ventricular cells. The diabetic state (elevated glucose and reduced insulin in plasma samples) was confirmed in our earlier studies (e.g., Ref. 33). Another group of Ovx female rats was also used. These were made diabetic with STZ 2–3 wk after ovariectomy, and cells were isolated 7–14 days after STZ injection.

Cell isolation. Single ventricular myocytes were obtained by enzymatic dispersion. Rats were anesthetized by CO₂ inhalation and then killed by cervical dislocation. The hearts were removed and the aortas cannulated on a Langendorff apparatus for retrograde coronary perfusion. The hearts were perfused for 3–5 min (at 37°C, bubbled with a 95% O₂-5% CO₂ mixture) with a control solution that consisted of (in mM) 121 NaCl, 5.4 KCl, 2.8 sodium acetate, 1 MgSO₄, 5 Na₂HPO₄, 24 NaHCO₃, 5 glucose, and 1 CaCl₂ (brought to a pH of 7.4 with NaOH). The solution was switched to a calcium-free solution (other constituents were unchanged). After 10 min, this was changed to the same basic solution that also contained 0.015 mg/ml collagenase (Yakult Honsha), 0.0075 mg/ml protease (Sigma type XIV), 20 mM taurine, and 40 µM CaCl₂. After 8 min, the free wall of the right ventricle was cut into small chunks, which were further incubated in a shaker bath (at 37°C) in a solution that contained 0.3 mg/ml collagenase, 0.15 mg/ml protease, 20 mM taurine, and 10 mg/ml albumin. Aliquots of cells were removed over the next 10–20 min and stored at room temperature in a solution that contained no enzymes, 20 mM taurine, 5 mg/ml albumin, and 0.1 mM CaCl₂. Cells were divided into groups that consisted of untreated cells or cells incubated with various drugs. On each experimental day, treated cells were compared with untreated cells. This allowed for variations in the degree of diabetic conditions in different rats. Results were pooled from different days and are given as means ± SE.

Current recording. Aliquots of cells were placed on the stage of an inverted microscope and perfused (at 21–22°C) with a solution that contained (in mM) 150 NaCl, 5.4 KCl, 1 CaCl₂, 1 MgCl₂, 5 HEPES, and 5 glucose (brought to a pH of 7.4 with NaOH); 0.3 mM CdCl₂ was then added to block L-type calcium currents.

The whole cell voltage-clamp method was used to record currents, which were elicited by 500-ms pulses to membrane potentials ranging from –110 to +50 mV. Recording pipettes (2–3 M resistance) contained filling solutions of (in mM) 120 potassium aspartate, 30 KCl, 4 Na₂-ATP, 10 HEPES, 10 EGTA, 1 CaCl₂, and 1 MgCl₂. Peak outward current (I_peak) and sustained outward current (I_sus, measured at the end of the voltage step), the key determinants of action potential repolarization and duration, were measured and compared.

Currents were digitized at 2 kHz and normalized to cell size by dividing current amplitude by cell capacitance (measured by integrating current traces obtained in response to 5-mV steps from –80 mV). Results are thus presented as current density.

Cell capacitance. Changes in current density may reflect changes in cell size, particularly under diabetic conditions, where ANG II may have hypertrophic effects. Insulin deficiency may also act to reduce cell size. However, measurements of cell capacitance, which is an index of cell size, showed (by ANOVA) no significant differences (P > 0.25) between any of the groups studied. Table 1 shows these values.

ANG II content. ANG II was measured in isolated myocytes by ELISA using a kit from Peninsula Laboratories. Cell extracts (4–6 million rod-shaped striated cells per rat) were first run through C₁₈ Sep columns. Six-point standard curves were obtained for each trial using known concentrations with optical densities plotted against (log) concentrations. Experimental samples, tested in triplicate, fell within the linear ranges of these curves. ANG II levels were normalized for protein content, measured in the same samples using a bicinchoninic acid protein assay kit (Pierce). The values obtained were very similar to those reported by Fiordaliso et al. (9).

Statistics. Mean values were compared using Student's t-test or ANOVA with Student-Newman-Keuls multiple comparisons test. Differences with P values <0.05 were considered significant.

	RESULTS

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The first set of experiments measured ANG II content in isolated ventricular myocytes using ELISA. Fiordaliso et al. (9) had previously shown that ANG II content was elevated in myocytes from male STZ-treated diabetic rats. We confirmed this and showed a significant (P < 0.01 by ANOVA) elevation of ANG II content. We subsequently measured ANG II content in cells from female STZ-treated diabetic rats. Our earlier work showed gender-selective effects in which the ACE inhibitor quinapril augmented K⁺ currents only in cells from diabetic males (35). This suggested that the renin-angiotensin system was not activated in diabetic females. In the present work, we found that ANG II content was indeed not different in control and STZ-treated diabetic females. Our earlier work also showed that ACE inhibition does augment K⁺ currents in cells from Ovx diabetic females (35). This suggested that estrogen plays a major role in suppressing ANG II activation. In assays performed with cells from Ovx diabetic females, we found that ANG II content was significantly (P < 0.001) elevated (compared with control and diabetic females) and comparable to levels found in diabetic males. These results are shown in Fig. 1.

View larger version (23K): [in this window] [in a new window]   Fig. 1. ANG II content in isolated myocytes. ELISA was used to compare ANG II in myocyte extracts from male (A) and female (B) control and diabetic rats. Augmentation of ANG II content occurs only in males. As shown, ANG II content in cells from ovariectomized (Ovx) diabetic female rats is similar to that in diabetic males. ANG II content was measured in triplicate for each sample and normalized for protein content; n = no. of rats, shown in parentheses below each bar. *P < 0.05.

Incubation of cells from male rats with 1 µM quinapril for 5–9 h did not alter I_peak but significantly augmented I_sus. Mean I_peak densities (at +50 mV) were 22.8 ± 1.3 (n = 24) and 20.1 ± 1.5 pA/pF (n = 21) in the absence of and after incubation with quinapril, respectively (P > 0.05). The corresponding values for I_sus were 6.2 ± 0.3 and 7.3 ± 0.3 pA/pF (P < 0.02). In cells from control females, quinapril did not alter either current (P > 0.05). I_peak densities were 20.1 ± 1.2 (n = 24) and 23.1 ± 1.1 pA/pF (n = 20). The values for I_sus in these groups were 7.9 ± 0.3 and 8.0 ± 0.4 pA/pF. This result suggests the presence of a baseline production level of ANG II (leading to some I_sus suppression) in control males but not in females. Diabetes enhances the production of ANG II only in males, which leads to suppression of I_peak and I_sus.

We subsequently proceeded to investigate whether some of the second-messenger systems affected by ANG II are involved in suppression of these currents.

The first set of experiments was based on work showing the involvement of ANG II-linked suppression of PKA (14). The hypothesis was that if PKA inhibition by ANG II were linked to current attenuation, it would be possible to augment these currents by PKA activation. Cells from male STZ-treated diabetic rats were incubated with the cell-permeable PKA activator 8-bromo-cAMP (100 µM) for 5–9 h. This produced significant augmentation of both I_peak and I_sus. At +50 mV, I_peak densities were 13.3 ± 0.9 and 16.7 ± 1.3 pA/pF in the absence (n = 39) or presence (n = 28) of 8-bromo-cAMP (P < 0.03). The corresponding values for I_sus were 4.5 ± 0.2 and 7.1 ± 0.5 pA/pF (P < 0.0001). Sample current traces and the mean current densities are shown in Fig. 2.

View larger version (38K): [in this window] [in a new window]   Fig. 2. A: effects of protein kinase A (PKA) activation by 8-bromo-cAMP (8 Br-cAMP). Current traces (obtained in response to 500-ms pulses to potentials ranging from –10 to +50 mV) in cells from diabetic male rats in the absence of 8-bromo-cAMP and following 6 h of incubation with 8-bromo-cAMP (100 µM). B: mean (± SE) current densities (at +50 mV) for peak outward current (Ipeak) and sustained outward current (Isus) in the absence of or 5–9 h after addition of 8-bromo-cAMP. *P < 0.05; **P < 0.005.

This result suggests that PKA is indeed suppressed in cells from diabetic rats (see DISCUSSION). However, this is not necessarily linked to activation of the renin-angiotensin system. To establish such a link, an additional set of experiments was performed. The ACE inhibitor quinapril was found earlier to augment I_peak and I_sus (32, 35). We hypothesized that if PKA suppression were one of the links between ANG II activation and current attenuation, then maintaining PKA suppression in the presence of quinapril may prevent current augmentation. To test this, cells from diabetic rats were exposed to quinapril in the absence or presence of the cell-permeable PKA inhibitor Rp-CAMPS (100 µM), which is the Rp isomer of 3',5'-cyclic monophosphorothioate. Maintained PKA suppression was found to block the augmentation of both I_peak and I_sus by quinapril. These results are shown in Fig. 3. Figure 3A shows sample current traces, whereas Fig. 3B shows current-voltage relationships in the absence or presence of quinapril, and with quinapril and Rp-CAMPS. ANOVA analysis showed that current augmentation by quinapril (for I_peak and I_sus) was significant at membrane potentials ranging from –30 to +50 mV, whereas reduction of the effect of quinapril by Rp-CAMPS was significant between –10 and +50 mV.

View larger version (26K): [in this window] [in a new window]   Fig. 3. Effects of PKA inhibition on augmentation of currents by the ANG II-converting enzyme inhibitor quinapril. A: current traces (same protocol as Fig. 2) in cells obtained from a male diabetic rat in the absence of drugs, after 7-h incubation in 1 µM quinapril, or after 6-h incubation in quinapril and 100 µM Rp-CAMPS (Rp isomer of 3',5'-cyclic monophosphorothioate), which is a PKA inhibitor that was added 1 h before quinapril. B: current-voltage relationships obtained for the same three conditions. Augmentation of both Ipeak and Isus by quinapril is significantly attenuated by Rp-CAMPS.

In this set of experiments, cells from male STZ-treated diabetic rats were incubated with 100 µM genistein for 5–9 h. This produced significant augmentation of both I_peak and I_sus as shown in Fig. 4. At +50 mV, I_peak density was increased from 11.3 ± 0.7 (n = 21) to 17.0 ± 1.0 pA/pF (n = 18; P < 0.001), whereas I_sus was increased from 4.8 ± 0.2 to 5.7 ± 0.3 pA/pF (P < 0.025). When cells from diabetic rats were exposed to the inactive analog genistin (100 µM), there was no effect on either current. I_peak densities at +50 mV were 18.6 ± 1.3 and 18.8 ± 1.5 pA/pF in the absence (n = 22) and presence (n = 21) of genistin, respectively. The corresponding values for I_sus were 4.5 ± 0.2 and 4.9 ± 0.2 pA/pF (P > 0.05). An additional set of experiments used myocytes from control rats. Incubation of these cells with genistein (100 µM) also had no effects on I_peak or I_sus, presumably because tyrosine kinases are not sufficiently activated in control conditions to affect K⁺ currents (see discussion).

View larger version (39K): [in this window] [in a new window]   Fig. 4. Effects of tyrosine kinase inhibition on K+ currents in cells from diabetic rats. A: current traces (same protocol as above) obtained in the absence of or after 6.5-h incubation in 100 µM genistein. B: mean current densities at +50 mV for Ipeak and Isus in the absence of or after 5–9 h of incubation in 100 µM genistein. Significant augmentation of both currents was attributed to tyrosine kinase inhibition (reversing tyrosine kinase activation by ANG II) because the inactive analog genistin was without effect (not shown). *P < 0.05; **P < 0.005.

In female diabetic rats, however, ANG II levels are unchanged (see Fig. 1), and ACE inhibition has no effect on I_peak and I_sus in females (35). It was therefore hypothesized that changes in currents due to ANG II-dependent effects on PKC, tyrosine kinases, and PKA would be absent in females.

The changes in currents described in Figs. 1–4 were relatively small. However, we have shown previously (32) that changes of this magnitude (for example, after addition of the ACE inhibitor quinapril) are sufficient to alter action potential duration (see Fig. 3). In the present experiments, we also measured action potential durations in the absence of or after 5–7 h of incubation in either 8-bromo-cAMP or genistein. The increase in K⁺ currents by either of these compounds was found to significantly abbreviate action potential duration measured at –60 mV. The mean values were 55.6 ± 5.7 (n = 20), 33.2 ± 1.8 (n = 14; P < 0.005), and 36.3 ± 3.9 (n = 9; P < 0.04) ms in the absence of drug, with 100 µM 8-bromo-cAMP, or with 100 µM genistein, respectively. These results are shown in Fig. 5.

View larger version (18K): [in this window] [in a new window]   Fig. 5. Abbreviating effects of 8-bromo-cAMP and genistein on action potential duration. Superimposed action potentials (left) were obtained in myocytes from diabetic rats (stimulation rate of 1 Hz) in the absence of or after 6-h incubation in 100 µM 8-bromo-cAMP. Mean (± SE) values for action potential duration (measured at –60 mV) in untreated cells and in the presence of 100 µM 8-bromo-cAMP or 100 µM genistein are shown (right). With both drugs, the action potential is significantly abbreviated in comparison to the duration in untreated cells. P < 0.05, 8-bromo-cAMP vs. untreated cells; P < 0.04, genistein vs. untreated cells.

View larger version (35K): [in this window] [in a new window]   Fig. 6. Absence of effects of 8-bromo-cAMP on outward currents in cells from female streptozotocin (STZ)-treated diabetic rats. A: sample current traces in the absence of or after 5.5-h incubation in 100 µM 8-bromo-cAMP. B: mean current densities at +50 mV for Ipeak and Isus. In contrast to the augmentation of both currents in cells from males, no significant changes were observed in females.

View larger version (24K): [in this window] [in a new window]   Fig. 7. Lack of effect of genistein in cells from diabetic females. A: sample current traces obtained in the absence of or after 7-h incubation in 100 µM genistein. B: current-voltage relationships obtained from 16 untreated cells and 13 cells exposed to genistein. Mean values (± SE) for Ipeak (left) and Isus (right) are shown.

View larger version (24K): [in this window] [in a new window]   Fig. 8. Effects of the protein kinase C (PKC) inhibitor bis-indolylmaleimide (bis-indol) in cells from STZ-treated diabetic females. A: sample current traces in the absence of drugs or after 6-h incubation with 100 nM PKC inhibitor. B: current-voltage relationships (Ipeak, left; Isus, right). PKC inhibition augmented both currents with significant differences obtained at potentials ranging from 0 to +50 mV.

The final experiments examined whether the gender differences in the linkage between PKA inhibition, tyrosine kinase activation, and current attenuation could be attributed to estrogen. Our earlier work suggested that estrogen, which is known to suppress the angiotensin pathway (11, 23), accounts for the gender differences in autocrine modulation of K⁺ currents. Thus ACE inhibition augmented I_peak and I_sus in diabetic Ovx rats in contrast to the lack of effect in diabetic (non-Ovx) females. In the present experiments, Ovx female rats were made diabetic by STZ treatment 2–3 wk after removal of the ovaries. Cells were isolated 7–14 days later, and similar protocols to those described were repeated.

The first set of experiments demonstrated that 8-bromo-cAMP (100 µM for 5–9 h) significantly augmented I_peak and I_sus as in males. In this group, I_peak at +50 mV was increased from 16.6 ± 1.2 (n = 21) to 24.0 ± 1.9 pA/pF (n = 13; P < 0.002). I_sus was increased from 5.7 ± 0.3 to 7.6 ± 0.5 pA/pF (P < 0.004). This result is shown in Fig. 9.

View larger version (37K): [in this window] [in a new window]   Fig. 9. Effects of 8-bromo-cAMP on outward currents in cells from Ovx diabetic female rats. A: sample current traces in the absence of drug and after 7-h incubation in 100 µM 8-bromo-cAMP. B: mean current densities at +50 mV in the absence of or after 5–9 h of incubation with 8-bromo-cAMP. Both currents are significantly augmented as in diabetic males but unlike non-Ovx diabetic females. **P < 0.005.

View larger version (8K): [in this window] [in a new window]   Fig. 10. Comparison of the effects of genistein on Isus in diabetic males (left), females (middle), and Ovx females (right). Mean current densities (at +50 mV) are shown in the absence of drugs or after 5–9 h of incubation in 100 µM genistein. Isus is significantly augmented in males and Ovx females but not in diabetic females. The results suggest that (in females) estrogen prevents ANG II activation of tyrosine kinases. Thus no attenuation of currents occurs, so that current augmentation by genistein is also absent. *P < 0.05; **P < 0.005; n.s., not significant.

	DISCUSSION

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Earlier work (3, 14) showed inhibition of the PKA pathway by ANG II. The present results show that under diabetic conditions, activation of PKA by 8-bromo-cAMP augments I_peak and I_sus in cells from males but not females (see Figs. 2 and 6). This suggests that PKA is inhibited in males but not in females, presumably due to the gender differences in ANG II elevation (see Fig. 1). Support for the involvement of ANG II in mediating PKA involvement was obtained by experiments showing that the augmentation of currents by the ACE inhibitor quinapril was blocked when PKA inhibition was maintained (see Fig. 3). That 8-bromo-cAMP was without effect on either current in cells from control males also suggests that only after ANG II levels rise is there inhibition of PKA, which contributes to current attenuation.

The inhibition of tyrosine kinases by genistein also augmented K⁺ currents in cells from diabetic males but not females (see Figs. 4 and 7). This is in concordance with the known activation of tyrosine kinases by ANG II (37), which would not occur in females in the absence of ANG II elevation. Again, the lack of effect of genistein on I_peak and I_sus in cells from control males supports the suggestion that tyrosine kinases are activated only after ANG II is elevated under diabetic conditions. This link between tyrosine kinases and K⁺ currents in diabetes has not been reported before. It is possible that the high concentration of genistein used led to effects on other kinases. However, no effects on currents were found in control myocytes, which indicates that the drug target had changed in the diabetic state. In addition, there are gender differences in the effects of genistein on diabetic cells that indicate differences in the target of genistein action.

The activation of PKC by ANG II has been amply demonstrated before (20, 37). A link between PKC activation and cardiac K⁺ current inhibition has also been shown (22, 31). Interestingly, the effects of the PKC inhibitor on I_peak and I_sus were still present (see Fig. 8) in cells from diabetic females (although to a smaller degree than in males). This suggests that some PKC activation occurs in diabetic females independently of ANG II. This PKC activation leads to current attenuation as it does in males. An alternative mechanism for PKC activation could be the de novo synthesis of the PKC activator diacylglycerol, which occurs during hyperglycemic conditions (15, 38). The larger augmentation of I_peak and I_sus by PKC inhibition in males may be due to a larger activation of PKC (and enhanced attenuation of currents). This would reflect the combined contribution of ANG II and de novo synthesis of diacylglycerol in diabetic males with only the latter mechanism present in females.

The effects described here, whereby K⁺ currents are augmented, presumably reflect the synthesis of new channel proteins. We showed this in earlier work (34) using Western blotting with antibodies directed against Kv4.2 and Kv1.2, which (partially) underlie I_peak and I_sus. In that work, ACE inhibition increased (after >5 h) channel-protein abundance, which suggests that the increase in ANG II associated with the diabetic state inhibits channel-protein synthesis. This will attenuate both transient and sustained K⁺ currents. In the present work, it is hypothesized that activation of PKA or inhibition of tyrosine kinases also blocks the action of ANG II and thereby allows enhanced channel synthesis rather than direct modulation of the currents.

The suppressing role of estrogen on the renin-angiotensin system has been implicated in previous studies (6, 11, 23). Our results suggest that estrogen plays a major role in preventing K⁺ current changes as well, because the activation of PKA and the inhibition of tyrosine kinases by genistein augmented I_peak and I_sus only in Ovx females (see Figs. 9 and 10).

The results presented here illustrate the presence of important gender differences in an autocrine regulation of major repolarizing currents in cardiac cells. These differences may underlie the differential propensity for development of some cardiac arrhythmias in males and females (4, 17, 25). ANG II is increasingly recognized as a central mediator of cardiac pathophysiology (7). Upregulation of the renin-angiotensin system occurs during heart failure as well as during diabetes (5) and has been shown to accelerate cell death in human diabetes (10). Our earlier work (32, 34) showed that blocking ANG II receptors also enhances K⁺ currents as does the inhibition of ANG II formation. In addition, the enhancement of current magnitude by the receptor blocker saralasin was shown to be absent when PKC translocation was prevented (34). The present work shows gender-specific differences in both ANG II formation and in downstream mediators of ANG II action. An understanding of the mechanisms underlying gender differences is obviously of great importance for the design of better strategies for prevention and treatment of cardiac disease and arrhythmias in diabetes and other cardiac pathologies.

	GRANTS

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This work was supported by grants from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Alberta.

	FOOTNOTES

 

Address for reprint requests and other correspondence: Y. Shimoni, Health Sciences Centre, 3330 Hospital Dr. N.W., Calgary, Alberta, Canada T2N 4N1 (E-mail: shimoni{at}ucalgary.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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