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Am J Physiol Heart Circ Physiol 289: H1873-H1880, 2005. First published June 3, 2005; doi:10.1152/ajpheart.00357.2005
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Enhanced oxidative stress impairs cAMP-mediated dilation by reducing Kv channel function in small coronary arteries of diabetic rats

Aaron H. Bubolz,1,2 Hongwei Li,3 Qingping Wu,1,2 and Yanping Liu1,2

1Department of Medicine and the 2Cardiovascular Center, The Medical College of Wisconsin, Milwaukee, Wisconsin; and 3Heart and Vessel Diseases Center, Beijing Friendship Hospital, Affiliate of Capital University of Medical Sciences, People's Republic of China

Submitted 12 April 2005 ; accepted in final form 31 May 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have shown that short-term exposure of rat small coronary arteries (RSCAs) to high glucose enhances superoxide (O2–·) formation and impairs cAMP-mediated dilation by reducing voltage-gated K+ (Kv) channel function. However, it is not clear whether the impairment also occurs in diabetes mellitus (DM), where alternate mechanisms could mask or aggravate vasodilator dysfunction. RSCAs were isolated from control and streptozotocin-induced diabetic rats. Reduced constriction to 4-aminopyridine (4-AP) was observed in RSCAs from DM rats, indicating Kv channel impairment. Forskolin increased 4-AP-inhibitable K+ channel open-state probability and whole cell K+ current density in coronary myocytes from non-DM rats but had little effect on K+ current density in cells from DM rats. Diminished dilation to 8-bromo-cAMP, forskolin, or isoproterenol was observed in DM RSCAs. The attenuated dilation to forskolin or isoproterenol in DM RSCAs was partially restored by application of the superoxide dismutase mimetic manganese[III] tetrakis (4-benzoic acid) porphyrin. Histofluorescence studies using hydroethidine revealed a blockage of O2–· generation by the NADPH oxidase inhibitor apocynin in DM RSCAs. Sepiapterin, a precursor of tetrahydrobiopterin, had little effect on hyperglycemia-induced O2–· formation. Consistent with the findings from the concurrent fluorescence study, apocynin also partially restored the reduced dilator response to forskolin in DM RSCAs. Forskolin-induced cAMP production was unaltered in DM. We conclude that in diabetes, enhanced O2–· formation by activation of NADPH oxidase impairs cAMP-medicated dilation in RSCAs by inhibiting Kv channel activity.

hyperglycemia; superoxide; coronary circulation

CARDIOVASCULAR DISEASE has continued to be the principal cause of death among individuals afflicted with diabetes mellitus (DM). To this end, DM has been associated with widespread vascular dysfunction (6, 11, 14). There has been substantial evidence illustrating enhanced reactive oxygen species (ROS) as crucial pathological factors responsible for the impaired vasomotor function in DM patients (20, 27, 28) and animals (15, 18, 19). It has been well characterized that in conduit arteries, the decreased vasodilator response is largely due to the reduced nitric oxide (NO) bioavailability resulting from overproduction of superoxide (O2–·) that quenches NO (7, 9). In contrast to the conduit arteries' reliance on NO, the microcirculation partakes in hyperpolarization-mediated dilation via opening of K+ channels, which play a predominant role in regulating vasomotor function. We have shown that one of the prominent K+ channels expressed in rat small coronary arteries (RSCAs) is the voltage-gated K+ (Kv) channel. Kv channels play a critical role in regulating resting membrane potential and participate in a variety of physiological responses, including cAMP-mediated dilation (1, 2), vasomotor responses to changes in pH (4), and H1-receptor mediated coronary constriction (17). Enhanced ROS generation is a common feature found in diabetes and likely accounts for the impaired dilator responses. Despite the importance of Kv channels in modulating vascular tone, there has been no systematic evaluation of the effect of increased ROS in diabetes on Kv channel function in the coronary microcirculation where tissue perfusion is regulated.

Although we have previously demonstrated that exposure of RSCAs to culture media containing high glucose (23 mM, HG) for 24 h enhances O2–· generation and reduces cAMP-mediated dilation resulting from the impaired Kv channel function, short-term exposure to HG may not produce the same complexity or degree of vascular dysfunction as in DM, where alternate mechanisms could exaggerate or compensate for the impaired vasodilation. Thus it is important to determine if impaired Kv channel function and activity also occurs in diabetic animals as observed in in vitro HG incubation.

In addition, several pathways have been proposed as mechanisms for hyperglycemia-induced superoxide overproduction. These include activation of the polyol pathway, activation of the diacylglycerol-protein kinase C pathway, upregulation of NADPH oxidase, and mitochondrial generation of ROS. However, which pathway(s) contribute to the impaired Kv channel function of the coronary microcirculation in DM is not clearly defined. The present study tested the hypothesis that, in type 1 diabetes, enzymatic increase of O2–· by NADPH oxidase impairs cAMP-mediated vasodilation due to reduced Kv channel function in RSCAs. This study, for the first time, systematically evaluated Kv channel function in diabetes, providing new insight into the therapeutic treatment of vascular disease in DM.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemical induction of DM rats. Male Sprague-Dawley (SD) rats (300 g) received an intraperitoneal injection of streptozotocin (STZ, 60 mg/kg). Age-matched control rats were injected with saline. Blood glucose and body weight were monitored daily. DM rats were treated with 1–3 U/day of ultralente insulin (adjusted to the degree of hyperglycemia) to prevent ketoacidosis (32) and severe weight loss (13, 31). Animals were studied between 4 and 6 wk after developing frank diabetes (serum fasting glucose >250 mg/dl before insulin treatment).

All rat protocols were approved by the American Association for the Accreditation of Laboratory Animal Care as well as the Animal Care Committee at the Medical College of Wisconsin.

Preparation of RSCAs. DM or non-DM rats were anesthetized with pentobarbital sodium (60 mg/kg ip), and hearts were removed. RSCAs (100–200 µm) were dissected from the left ventricle and prepared for videomicroscopic, patch-clamp, histofluorescent, and cAMP meaurements.

Videomicroscopy. RSCAs were cannulated on glass micropipettes in an organ chamber filled with physiological salt solution (PSS) containing (in mmol/l) 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 20 NaHCO3, 0.026 Na2EDTA, and 11 dextrose, pH 7.4 (26). The PSS was warmed to 37°C, continuously circulated, and bubbled with 21% O2-5% CO2-74% N2. Most vessels equilibrated for 1 h at an intraluminal pressure of 60 mmHg developed spontaneous tone averaging 70% of the initial diameter. RSCAs that did not develop this degree of spontaneous tone were constricted with U-46619 (10 nmol/l) to 70% of the initial diameter. Kv channel function was also determined by contractile response to 4-aminopyridine (4-AP, 0.1–3 mmol/l, half-log unit). Dilation to 8-bromo-cAMP (8-Br-cAMP, 10–9 to 10–5 mol/l) was compared between RSCAs from non-DM and DM rats. Isoproterenol (Iso, 10–10 to 10–6 mol/l) and forskolin (10–10 to 10–6 mol/l) dose-response curves were performed in the absence and presence of manganese(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP, 300 µmol/l) or apocynin (5 mmol/l) in small coronary arteries from DM and non-DM rats. At the end of each experiment, vessels were maximally dilated with papaverine (10–4 mol/l), and the percent dilation to agonists was normalized to this diameter.

Patch-clamp recording of Kv currents. Enzymatic isolation of single VSMCs was performed according to published methods(23). Unitary Kv currents were obtained in cell-attached membrane patches of VSMCs from RSCAs, which were bathed in symmetrical 145 mmol/l K+ solutions. To eliminate interfering currents from Ca2+-activated (KCa) and ATP-sensitive K+ (KATP) channels, 100 nmol/l iberiotoxin, 1 µmol/l apamin, and 1 µmol/l glibenclamide were added to the bath solution. The effect of forskolin on unitary Kv currents was evaluated by 2-min recording intervals before and after application of 3 mmol/l 4-AP.

To minimize dialyzing of intracellular components, the perforated whole cell recording was applied by adding amphotericin B (2.5 µg/ml) in the pipette solution. Recordings were obtained using standard pulse protocols and instrumentation as previously described (26). Briefly, families of K+ currents were generated by stepwise 10-mV depolarizing pulses (400-ms duration, 5-s intervals) from a holding potential of –60 mV in cells dialyzed with 100 nmol/l ionized Ca2+. Seal resistance was 2–10 G{Omega}. Peak current elicited at a single membrane potential was defined as the average of 500 sample points encompassing the maximal current point. In a single cell, Kv currents were defined as the difference between outward current recorded in a drug-free bath solution and outward current recorded after superfusion with 10 µmol/l forskolin. Trials were performed in triplicate and averaged to estimate peak current amplitudes (pA/pF) to normalize for cellular membrane area (24). For each cell, 10-mV hyperpolarizing steps were averaged to account for capacitance and leak compensation values.

Histofluorescent detection of O2–·. The cell-permeable dye hydroethidine (HE) was used to evaluate the production of O2–· as described previously (26). RSCAs were incubated in HE (5 µmol/l) for 10 min, washed, and examined under a fluorescence microscope by exciting the oxidized nucleic product of HE, ethidium bromide, at a wavelength of 585 nm. RSCAs from DM and non-DM rats were examined in parallel by recording images using the same computer-specified gain, intensity settings, and amount of exposure time. Fluorescence intensities were measured using NIH image software.

cAMP measurement. RSCAs isolated from non-DM or DM rats were placed into Krebs solution containing 100 µmol/l 3-isobutyl-1-methylxanthine for 20 min at 37°C to inhibit degradation by cAMP phosphodiesterase. Both non-DM and DM vessels were then incubated with the direct adenylate cyclase agonist, forskolin (1 µmol/l), or Iso (10 or 100 nmol/l) for an additional 30 min at 37°C. One control group was precluded from the forskolin treatment for a cAMP baseline control. All RSCAs were placed in iced 6% (wt/vol) trichloroacetic acid (TCA), snap-frozen with liquid nitrogen, and stored at –80°C. The samples were then thoroughly homogenized in TCA on ice (2–8°C), and the homogenates were subsequently centrifuged at 2,000 g for 15 min at 4°C. Supernatants were collected and extracted with 5 vol of ethyl ether four times, after which, the organic components were lyophilized for 4 h and reconstituted in 200 µl of assay buffer. cAMP levels were determined using an acetylation enzyme immunoassay system purchased from Amersham Biosciences. Optical densities were read at a wavelength of 450 nm, and all cAMP raw values were normalized to the specific total protein content of each sample (µg).

Statistical analysis. All data are expressed as means ± SE. Percent dilation was calculated as the change from control internal diameter to maximal diameter measured in the presence of papaverine. Percent constriction was defined as the percent reduction from the diameter before contractile agents. Data from videomicroscopy, fluorescence intensities, patch-clamp, and cAMP measurements were compared using ANOVA two-way repeated measurements to detect differences between non-DM and DM rats. Dose-response curves before and after agonists or antagonists treatment were compared using one-way ANOVA. When test statistics for the main ANOVA test were significant (P value < 0.05), a corollary Newman-Keuls test was performed to detect differences between individual doses. All differences were judged to be significant at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Blood glucose and body weight. The blood glucose, hemoglobin-A1C (HbA1C), and body weights averaged from 42 DM and 42 non-DM rats are summarized in Table 1. Blood glucose and HbA1C were fourfold and twofold increased, respectively, in DM compared with non-DM. Body weights were significantly reduced in DM rats.


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  		Table 1. Summary of body weight, serum glucose, and HbA1C in non-DM and DM rats

 
Contractile response to 4-AP. Resting diameters of RSCAs from DM and non-DM rats were not different: 145 ± 8 and 133 ± 6 µm, respectively (n = 26, each group). Figure 1A shows diameter changes in response to four increasing concentrations of 4-AP. 4-AP elicited dose-dependent constriction in non-DM RSCAs (maximum constriction: 33 ± 5%, n = 6). In contrast, the maximal constriction to 4-AP in arteries from DM rats was lower (18 ± 3%, n = 6, P < 0.05 vs. non-DM), indicating decreased Kv channel function. To eliminate the possibility that the reduced constriction to 4-AP in DM rats was due to a nonspecific overall reduction of contractile response, constrictions of RSCAs to U-46619 (10–8 or 10–7 mol/l) and KCl (20 to 40 mmol/l) were also compared between DM and non-DM rats. As shown in Fig. 1, B and C, a similar contractile response to U-46619 or KCl was observed in small coronary arteries from DM and non-DM rats [n = 6, each group; P = not significant (NS)], suggesting that a nonspecific effect to the impaired coronary arteriolar Kv channel function in DM is not probable.



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  		Fig. 1. A: dose-dependent constriction of rat small coronary arteries (RSCAs) in response to the voltage-gated K+ (Kv) channel blocker 4-aminopyridine (4-AP). Constriction of RSCAs from diabetic mellitus (DM) rats is reduced signifying a loss in Kv channel function. B and C: similar contractile responses to graded doses of U-46619 and KCl were observed in arteries from non-DM and DM rats, respectively (n = 6, each group; P = not significant).

 
Forskolin-induced Kv current in diabetic vascular smooth muscle cells. To confirm the functional observations, coronary arteriolar Kv channel activity was further evaluated by patch-clamp methods. The contribution of Kv channels to cAMP-mediated vascular response was examined using cell-attached patches. As shown in the sample traces (Fig. 2A) and summarized data (Fig. 2B), forskolin (10 µmol/l) profoundly increased the open-state probabilities (NPo) of Kv channels (control vs. forskolin, 0.001 ± 0.0002 vs. 0.0039 ± 0.0016) in cell-attached patches. 4-AP inhibited forskolin-induced K+ channel activities (0.001 ± 0.0004), confirming the role of forskolin in regulating Kv channel activity. Subsequent experiments comparing Kv current in response to forskolin in vascular smooth muscle cells (VSMCs) from DM and non-DM rats were performed in perforated whole cell patches. Figure 3A illustrates that in the presence of iberiotoxin, forskolin enhanced whole cell currents in freshly isolated VSMC of non-DM rats. In contrast, the basal K+ currents were less in DM and did not respond to forskolin. Current-voltage relations (Fig. 3, B and C) averaged from six cells verified the reduced forskolin-induced Kv currents (peak current density: control vs. forskolin, 11.5 ± 0.9 vs. 12.3 ± 1.2 pA/pF) in coronary VSMCs from DM (Fig. 3C) compared with non-DM rats, (Fig. 3B, control vs. forskolin, 14 ± 0.8 vs. 21 ± 1.6 pA/pF), suggesting the impairment of Kv channels.



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  		Fig. 2. A: sample recording of unitary Kv currents from cell-attached patch. Membrane potential (MP) was held at +40 mV. Kv channel activity was greatly increased by forskolin (10 µmol/l) and reduced when 4-AP was applied in the presence of forskolin. B: average open-state probability (NPo) in response to forskolin and forskolin + 4-AP. Enhanced NPo by forskolin was markedly inhibited by 4-AP.

 


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  		Fig. 3. A: sample traces of whole cell currents in response to 10 µM forskolin recorded from perforated patches. In the presence of iberiotoxin, whole cell K+ currents elicited by 10-mV depolarizing steps from a constant holding potential of –60 mV to +40 mV. The basal K+ current was reduced in cell from DM rat compared with non-DM rat. Forskolin increased K+ current in cell from non-DM rat but had little effect on K+ current in cell from DM rat. Capacitances for non-DM and DM cells were 10 and 9 pF, respectively. B: current-voltage relationships comparing K+ current density between coronary smooth muscle cells from non-DM and DM rats. Forskolin increased peak K+ current densities in cells from non-DM rats but had little effect on K+ current density in cells from DM rats.

 
cAMP-induced dilation in diabetes. To determine whether the failure of the cAMP activator, forskolin, to increase the K+ current in diabetes also translated into an attenuation of cAMP-induced dilation, dilator response to the cAMP analog 8-Br-cAMP (10–9 to 10–5 mol/l) was compared in RSCAs from non-DM and DM rats (Fig. 4). Dilation to 8-Br-cAMP was markedly reduced in arteries from DM rats [maximal (max) dilation, DM vs. non-DM: 19 ± 4% vs. 86 ± 8%, n = 5; P < 0.05 vs. non-DM].



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  		Fig. 4. Comparison of dose-dependent dilation to 8-bromo-cAMP (8-Br-cAMP) in RSCAs between non-DM and DM rats. The dilator response to 8-Br-cAMP was significantly decreased in DM arteries compared with vessels from non-DM rats.

 
Effect of O2–· on cAMP-mediated dilation in diabetes. The contribution of O2–· to the impaired Kv channel function was examined by comparing the dilator response to forskolin or Iso before and after treatment with the O2–· scavenger MnTBAP. In the absence of MnTBAP, vasodilation to forskolin was markedly diminished in RSCAs from DM compared with non-DM (max dilation, DM vs. non-DM: 39.5 ± 2 vs. 77.3 ± 2.5%, n = 12, P < 0.05 vs. non-DM) (Fig. 5A). MnTBAP partially restored the reduced dilator response to forskolin (max dilation, DM vs. non-DM: 66 ± 8% vs. 78.3 ± 5%, n = 6, P = NS) (Fig. 5B). Similar to forskolin, Iso-induced dilator response was also decreased in diabetic RSCAs and was improved by treatment with MnTBAP (Fig. 5, C and D).



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  		Fig. 5. Effect of the superoxide dismutase (SOD) mimetic manganese[III] tetrakis (4-benzoic acid) porphyrin (MnTBAP) on forskolin- or isoproterenol (Iso)-induced dilation of small coronary arteries from non-DM or DM rats. In the absence of MnTBAP, dilation in response to forskolin or Iso was reduced in DM RSCAs. MnTBAP partially restored the impaired dilator response to forskolin or Iso in DM RSCAs but had little effect on both dilator responses of non-DM RSCAs.

 
Source for generating O2–· in diabetic rat small coronary arteries. The role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the production of O2–· in RSCAs of DM rats was examined by comparing HE-produced fluorescence intensity in RSCAs with and without a 2-h treatment of apocynin (3 mmol/l), a specific inhibitor of NADPH oxidase. As indicated in Fig. 6, without apocynin treatment, an enhanced O2–· level was observed in DM RSCAs (1.3 ± 0.1, n = 4, P < 0.05 vs. non-DM) relative to non-DM RSCAs (1 ± 0). Apocynin markedly decreased the level of O2–· in DM RSCAs (0.7 ± 0.1), indicating a significant role for the NADPH oxidase in generating O2–· in small coronary arteries of DM rats.



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  		Fig. 6. A: fluorescence intensity of DM RSCAs exposed to apocynin (3 mM). Both non-DM and DM arteries showed a reduction in the levels of superoxide when exposed to the NADPH oxidase inhibitor apocynin. B: summary of fluorescence intensities from non-DM and DM RSCAs normalized to non-DM. C: application of sepiapterin (Sepi, 1 µmol/l) did not change fluorescence intensity in RSCAs from non-DM or DM.

 
Because the reduced bioavailability of tetrahydrobiopterin (BH4), a co-factor of nitric oxide synthase (NOS), has been proposed as one of the mechanisms for producing O2–· in diabetic conduit arteries (3) and cultured aortic endothelial cells with high glucose (5), it is important to determine whether this is also true in small coronary arteries. As shown in Fig. 6C, sepiapterin (1 µmol/l), a precursor of BH4, tended to decrease O2–· generation in DM RSCAs but did not reach a difference of statistical relevance.

Effect of antioxidant with an NADPH oxidase inhibitor on Kv channel function. Whether O2–· generated by NADPH oxidase was responsible for the impaired Kv channel function was further determined by comparing the vasodilator response to forskolin before and after application of apocynin. In six experiments, apocynin greatly improved the dilation of DM RSCAs to forskolin (max dilation, before vs. after: 39 ± 1.5% vs. 60.3 ± 2.8; P < 0.05 vs. before treatment) but had little effect on the dilator response in non-DM RSCAs (Fig. 7).



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  		Fig. 7. Effect of apocynin, a NADPH oxidase inhibitor, on forskolin-induced dilation in RSCAs. Impaired dilation in response to forskolin in small coronary arteries from DM rats was partially restored by apocynin.

 
cAMP production in diabetes. To explore the possibility that impaired dilation to cAMP-mediated dilation is due to blunted cAMP production in diabetes, we measured cAMP levels, stimulated by forskolin (1 µmol/l) or Iso at two submaximal doses (10 or 100 nmol/l), in small coronary arteries from DM and non-DM rats. As illustrated in Fig. 8, A and B, cAMP production induced by forskolin or Iso was similar in DM and non-DM RSCAs (n = 4, each group; P = NS). These data suggest that the reduced cAMP-mediated dilation of RSCAs in diabetes is not due to the reduced formation of cAMP. The impairment appears to be downstream from cAMP.



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  		Fig. 8. Production of cAMP by RSCAs in response to forskolin (1 µM) (A) and Iso at concentration of 10 or 100 nmol/l (B). There was no difference in cAMP levels between non-DM and DM RSCAs stimulated by forskolin or Iso.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Three novel findings have originated from this study. First, cAMP-mediated dilation is impaired in RSCAs in diabetes due to reduced Kv channel function in coronary smooth muscle cells. This conclusion is supported by complementary studies where 4-AP-inhibitable dilation and K+ current to cAMP activators (21) are diminished in small coronary arteries and VSMCs of DM rats. Second, O2–· generated by activation of NADPH oxidase significantly contributes to the decreased cAMP-mediated dilation, because impairment was improved by the addition of an NADPH oxidase inhibitor. Finally, the level of cAMP is not altered in diabetes. Thus the defect in the signaling pathway is localized downstream from cAMP.

Impaired Kv channel function and cAMP-mediated dilation in diabetes.

The goal of this study was to determine whether Kv channel function is altered in diabetes as we observed previously in in vitro studies. Our results demonstrated that Kv channel activity is also reduced in diabetes where arteries are exposed to a variety of hormonal and neuronal factors in addition to hyperglycemia. This conclusion is supported by several lines of evidence. In cannulated and pressurized RSCAs, constriction to the Kv channel blocker, 4-AP, was attenuated in DM rats, indicating the diminished role of Kv channels at the resting state. The reduced constriction to 4-AP is not due to nonspecific reduction of vascular contractile response in DM, because similar constriction to U-46619 or KCl was observed in small coronary arteries from DM and non-DM rats. Additionally, the dilation to forskolin-associated Kv channel opening (2, 21) was impaired in DM RSCAs compared with non-DM RSCAs. Finally, patch-clamp studies revealed a failure of forskolin to augment Kv currents in VSMCs of DM rats, which is consistent with the observed functional data.

The signaling pathway for cAMP-mediated dilation includes multiple components in addition to Kv channels. Receptors, G proteins, and proteins required for phosphorylation also play fundamental roles in pathway conduction. Our results suggest that the defect of coronary dilator response to Iso or forskolin in DM is localized downstream of cAMP, based on the facts that 1) similar impairment was observed when cAMP analog was used or adenylate cyclase was directly activated with forskolin, bypassing receptors and G proteins; and 2) cAMP levels were not altered in DM RSCAs. Although the main focus of this study is Kv channel function, which appears to be downregulated, in turn attenuating cAMP-mediated dilation in DM, we could not completely exclude the possibility that decreased cAMP-mediated dilation in DM may be, in part, due to the alteration of other signaling components, which remains to be further investigated. Viewed collectively, these findings, together with our previous observations, suggest that hyperglycemia-induced oxidative stress plays a critical role in reducing Kv channel activity in diabetes.

Enhanced oxidative stress and Kv channel function in diabetes.

Enhanced production of ROS in diabetes has been observed in animal models as well as humans. One of the prominent ROS in diabetes is O2–·. O2–· quenches NO and reduces endothelium-dependent NO-mediated dilation (12, 29, 30). O2–· can also serve as a substrate in the generation of peroxynitrite (ONOO–), thereby conferring indirect inhibition of K+ channel function (22, 25). In the present study we determined whether increased O2–· production is responsible for the impaired Kv channel function in diabetes. MnTBAP, a scavenger of O2–·, partially restored the decreased dilation to forskolin and Iso, indicating the contribution of O2–· to the impaired Kv channel function.

The source of O2–· generation was also examined. Inhibition of NADPH oxidase with apocynin markedly decreased O2–· production and improved dilation to forskolin in small coronary arteries of diabetic rats. In contrast, supplementation with the precursor for BH4, sepiapterin, had little effect on generation of O2–· in diabetic RSCAs. We have also screened the role of xanthine oxidase (XO) and NOS in mediating O2–· generation in RSCAs exposed to HG by blocking their activity using allopurinol and NG-nitro-L-arginine methyl ester, respectively. Neither the blockade of XO or NOS had any effect on HG-induced O2–· formation (data not shown). These data suggest that over production of O2–· in diabetic RSCAs is most likely via activation of NADPH oxidase.

Study limitations.

In patch-clamp studies, we observed a greater reduction in baseline as well as forskolin-induced K+ current compared with the reduction in forskolin-induced vasodilation in isolated vessel experiments. The precise mechanism for this discrepancy is unclear. There are two possible explanations. First, we have previously reported that Kv channels play a predominant role in mediating dilation to forskolin in normal situations (21). We also know that forskolin-induced 4-AP-inhibitable dilation is impaired in coronary arteries exposed to HG (21). However, in diabetes, reduced dilator response may trigger other K+ channels, such as large-conductance Ca2+-activated K+ channel (BKCa), that are more resistant to O2–· (25), to compensate for the loss of Kv channel function. In our pilot study, we found an enhanced constriction to iberiotoxin, a specific BKCa channel blocker, in diabetic coronary arteries compared with the arteries from normal controls, indicating a possible increased BKCa channel function in diabetes. Second, the reduced dilation to 8-Br-cAMP in diabetic arteries was more profound compared with that which occurred in forskolin- or Iso-induced dilation in DM, suggesting that a cAMP-independent dilator mechanism exists that is not altered in DM, which may also compensate for the reduced Kv channel function. However, systematic examination of compensatory mechanism(s) in diabetes is beyond the scope of this study, which will be further investigated in later projects. To eliminate the confounding influence from BKCa channels in patch-clamp experiments, Kv channel activity was examined in the presence of iberiotoxin. Thus the K+ current recorded from whole cell patch-clamp techniques was mainly Kv current, while the dilation to forskolin observed during isolated vessel studies may have been due to multiple mechanisms. This proposal may account for the discrepancy seen in Kv channel current reduction compared with the much more modest reduction in dilation of RSCAs to forskolin in diabetes.

Enhanced oxidative stress is a common pathological feature in diabetes, and O2–· is not the only ROS produced in hyperglycemia. Other radicals, such as ONOO– formed by reaction of O2–· and NO, are also increased in diabetes. As we have reported previously, ONOO– has an inhibitory effect on Kv channel function as well. The restoration of attenuated dilator response by O2–· scavengers in this study may be the result of eliminating a substrate for producing ONOO–. However, we have previously demonstrated that exogenous generation of O2–· by the reaction of xanthine with XO blocks whole cell Kv current and NPo in inside-out patch, suggesting a direct inhibitory effect of O2–· on Kv channels. Thus the impaired Kv channel function in diabetes may result from both O2–· and ONOO–. The relative contribution of O2–· and ONOO– to the impairment of Kv channel function in chronic diabetes is not fully defined in this study and remains to be investigated.

As suggested by others, the levels of protein kinase C (16) and intracellular Ca2+ are elevated in diabetes (28), both of which have an inhibitory effect on Kv channels (8, 10). Although we observed the direct influence of oxidative stress on Kv channel activity as discussed above, other factors associated with hyperglycemia should also be taken into consideration when coronary Kv channel function is evaluated in diabetes. Future study that specifically investigates the interactions of multiple factors with Kv channel function in diabetes is needed.

In summary, we have previously shown that short-term elevated levels of glucose impair Kv channel function in RSCAs due to enhanced oxidative stress. However, whether the similar impairment also exists in a chronically diabetic situation is unknown. In this study, we determined the effect of oxidative stress on Kv channel function in diabetes using videomicroscopic, patch-clamp, and histofluorescence techniques. Reduced constriction to 4-AP was seen in RSCAs from DM rat, suggesting the impairment of Kv channels. Direct measurement of K+ currents in freshly isolated VSMCs using cell-attached and whole cell patches further confirmed the defect of Kv channels in DM. The reduced dilation to the cAMP analog 8-Br-cAMP, as well as cAMP activators forskolin and Iso, were found in DM RSCAs compared with small coronary arteries from non-DM rats. Treatment with MnTBAP or apocynin partially restored the decreased dilator response to forskolin or Iso, indicating the contribution of O2–·, generated by the activation of NADPH oxidase, to the impaired Kv channel activity. Altered cAMP production was not responsible for the attenuated dilator response observed in DM rats. Thus we conclude that in diabetes, enhanced O2–· formation by activation of NADPH oxidase impairs cAMP-mediated dilation in RSCAs by inhibiting Kv channel activity. The results derived from this study provide important implications with regard to the mechanisms of coronary vasomotor regulation in diabetes. The results of this study may suggest new therapeutic approaches for the treatment of coronary microvascular dysfunction in diabetes.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-067948 and a Scientist Development Grant from the American Heart Association.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Liu, Cardiovascular Center, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (e-mail: ypliu{at}mcw.edu)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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