Coronary microvessels generate reactive oxygen species in response to high glucose (HG), resulting in vasodilator defects involving an impaired function of vascular K+ channels. Inhibition of voltage-gated K+ (Kv) channels by peroxynitrite (ONOO−), formed by the interaction of superoxide and nitric oxide, may contribute to impaired dilation. The present study investigated whether HG induces ONOO− formation to mediate nitration and impairment of Kv channels in rat small coronary arteries (RSCAs). Exposure to ONOO− reduced the dilator influence of Kv channels in RSCAs. Patch-clamp studies revealed that ONOO− diminished whole-cell and unitary Kv currents attributable to the Kv1 gene family in smooth muscle cells. Subsequently, immunohistochemically detected enhancement of nitrotyrosine residues in RSCAs that were cultured in HG (23 mmol/l) compared with normal glucose (5.5 mmol/l) for 24 h correlated with the nitration of Kv1.2 channel α-subunits. HG-induced nitrotyrosine formation was partially reversed by scavenging ONOO−. Finally, RSCAs that were exposed to HG for 24 h showed a loss of Kv channel dilator influence that also was partially restored by the ONOO− scavengers urate and ebselen. We conclude that ONOO− generated by HG impairs Kv channel function in coronary microvessels, possibly by nitrating tyrosine residues in the pore-forming region of the Kv channel protein.
In type 2 diabetes, endothelium-dependent vasodilator responses are impaired in both the macro- and the microvasculature, possibly related to the formation of reactive oxygen species (ROS). The presence of hyperglycemia per se fosters the local formation of superoxide (O2˙−) (1,2), which may interact with nitric oxide (NO) to form the highly reactive oxidant peroxynitrite (ONOO−). Indeed, recent evidence has emphasized that NO derived from inducible NO synthase under conditions of high glucose (HG) may contribute to vasodilator dysfunction by driving the generation of ONOO− (3). In turn, ONOO− may promote oxidative and nitrosative tissue damage (4), in part by nitration of tyrosine residues to impair the function of multiple proteins, including K+ channels that mediate vasodilation (5,6).
In this regard, most studies have focused on ONOO−-induced inhibition of high-conductance Ca2+-activated K+ (KCa) channels as a mechanism of vasodilator impairment (5,6). However, a targeted inhibition of KCa channels by ONOO− cannot account for some of the profound vasodilator defects that are induced by HG and seem to relate more closely to an impaired function of voltage-gated K+ (Kv) channels. For example, vasodilator responses to forskolin and isoproterenol rely on cAMP-induced activation of Kv channels to mediate relaxation, but these responses are markedly blunted in rat small coronary arteries (RSCAs) that are exposed to HG for 24 h (7). In addition, the activity and expression levels of Kv channels are sensitive to oxidant stress (8), and although Kv channels emanate from at least 11 gene families (Kv1–Kv11), the Kv1 “Shaker-type” family channels that are densely expressed in vascular smooth muscle cells (VSMCs) may be particularly susceptible to open-channel block by redox agents (9,10). Collectively, these findings provide a compelling reason to investigate whether ROS associated with HG alter Kv channel function. The present study tested the hypothesis that HG induces endogenous ONOO− formation in RSCAs, resulting in nitration and impairment of Kv channels and vasodilator function.
RESEARCH DESIGN AND METHODS
Preparation of RSCAs.
Seven-week-old male Sprague-Dawley rats (Harlan, Madison, WI) were anesthetized with sodium pentobarbital (60 mg/kg i.p.). RSCAs (internal diameter 150–200 μm) were dissected from the left ventricle. Some RSCAs were incubated in culture media supplemented with either 5.5 mmol/l d-glucose (normal glucose [NG]) or 23 mmol/l d-glucose (high glucose [HG]), or 5.5 mmol/l d-glucose plus 17.5 mmol/l l-glucose (LG) for osmotic control as described previously (11). All rats were housed in the Association for Assessment and Accreditation of Laboratory Animal Care–approved Biomedical Resource Center at the Medical College of Wisconsin, and all protocols were approved by the Animal Care Committee.
Formation of ONOO−.
Authentic ONOO− was synthesized according to published methods (5,6). The amount of ONOO− in the stock solution was determined spectrophotometrically using the reported extinction coefficient for ONOO− (1,670 mol · l−1 · cm−1). Before each application, an aliquot of the stock solution was diluted in 1 mmol/l NaOH and rapidly added to the vessel chamber to achieve a final concentration of 5 μmol/l. Decomposed ONOO− (DC-ONOO−) was made by leaving ONOO− at room temperature for at least 2 h. The decay of ONOO− was confirmed spectrophotometrically.
RSCAs were cannulated on glass micropipettes in an organ chamber filled with physiological salt solution as described previously (11). The physiological salt solution was warmed to 37°C, continuously circulated, and bubbled with 21% O2, 5% CO2, and 74% N2. Most vessels that were equilibrated for 1 h at an intraluminal pressure of 60 mmHg developed spontaneous tone averaging 70% of the passive diameter, which was defined as the maximal diameter assessed in Ca2+-free solution at the end of the experiment. RSCAs that did not develop this degree of spontaneous tone were constricted by U46619 (10 nmol/l) to 70% of the passive diameter.
Patch-clamp recording of Kv current.
Enzymatic isolation of single VSMCs was performed according to published methods (12). Whole-cell patch-clamp recordings were obtained using standard pulse protocols and instrumentation (5). 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 to 50 mV in cells that were dialyzed with 10 nmol/l calcium. Trials were performed in triplicate, and peak current amplitudes were divided by membrane capacitance to obtain K+ current density (12).
Unitary Kv currents were obtained in inside-out membrane patches of VSMCs from RSCAs, which were bathed in symmetrical 145 mmol/l K+ solutions. For eliminating interfering currents from KCa and ATP-sensitive K+ channels, 100 nmol/l iberiotoxin was added to the pipette solution and 1 μmol/l apamin and glibenclamide was added to the bath solution. The effect of authentic ONOO− on unitary Kv currents was evaluated by 2-min recording intervals before and after drug application. Averaged current amplitudes were obtained at stepwise patch potentials for calculation of single-channel conductance.
Immunohistochemical detection of nitrotyrosine.
For determining the effect of HG on nitrotyrosine formation, RSCAs were exposed to NG or HG for 24 h. In some arteries, urate (100 μmol/l) was applied every 8 h to scavenge ONOO−. The joint effect of the O2˙− scavenger manganese [III] tetrakis 4-benzoic acid porphyrin (MnTBAP; 300 μmol/l) and the NO synthase inhibitor Nω-nitro-l-arginine methyl ester (l-NAME; 100 μmol/l) on nitrotyrosine formation was examined in other arteries. Freshly isolated RSCAs were used as controls. All RSCAs were prepared as frozen sections (5 μm thick). The immunodetection of nitrotyrosine residues has been described (5,6).
Immunoprecipitation and Western blot.
For obtaining adequate vascular tissue for immunoanalysis, VSMCs were enzymatically dissociated from RSCAs and cultured in RPMI-1640 medium that contained 20% FBS, 100 units/ml penicillin G, and 100 μg/ml streptomycin. The first passage of VSMCs was divided into six plates and permitted to reach 80% confluence. In other plates, the purity of VSMCs was verified by immunostaining with α-smooth muscle actin. Subsequently, cells were treated for 24 h with DMEM that contained NG, HG, or LG. Urate (100 μmol/l) was added to some HG plates to scavenge ONOO−. After incubation, total or membrane proteins were collected for immunoprecipitation and Western blot analysis, respectively (13).
Proteins (100–200 μg/reaction) were immunoprecipitated by incubations at 4°C with antinitrotyrosine (4 μl/reaction) for 2 h, and then incubated in protein A for 1 h. The immunoprecipitates were centrifuged and washed three times with ice-cold lysis buffer, and Western blots were performed using antibodies directed against Kv1.2 and Kv1.5 channel α-subunits (Upstate Biotechnology, Lake Placid, NY) (13). Anti-Kv1.2 and anti-Kv1.5 were also used to compare Kv1.2 and Kv1.5 expression at different glucose levels. β-Actin was used as an internal standard to ensure equal lane loading.
All chemicals were purchased from Sigma. Correolide was provided by Drs. Maria Garcia and Gregory Kaczorowski (Merck Research Laboratories, Rahway, NJ). Correolide was prepared as a 20-mmol/l stock in DMSO and stored at 4°C.
All data are expressed as mean ± SE. Percentage of constriction was defined as the percentage of reduction from control internal diameter. Data from videomicroscopy, patch-clamp, and Western blot studies were compared using one-way ANOVA, with repeated measures for dose and condition. Differences between individual means were determined by Newman-Keuls test. All differences were judged to be significant at P < 0.05.
ONOO− blunts the vasodilator influence of Kv channels.
Initial experiments were designed to determine whether ONOO− inhibited Kv channel function in RSCAs. Graded concentrations of 4-aminopyridine (4-AP), a broad-spectrum Kv channel blocker, constricted RSCAs in control conditions (Fig. 1; n = 5). These findings suggested that K+ efflux through Kv channels contributed significantly to resting diameter. Notably, decomposed ONOO− (DC-ONOO−) had no effect on the constriction of RSCAs to 4-AP (Fig. 1A). However, 4-AP–induced contractions were attenuated by incubation of RSCAs in authentic ONOO− (5 μmol/l), indicating a loss of Kv channel dilator function (Fig. 1B). Figure 2A shows similar U46619-induced constriction (10−9 to 10−7 mol/l) in RSCAs that were exposed to DC-ONOO− or ONOO−. Arteries that were incubated in DC-ONOO− or ONOO− showed similar dilation to the l-type Ca2+ channel antagonist nifedipine (10−8 to 10−6 mol/l; Fig. 2B). These results indicate that ONOO− selectively impairs vasoactive responses that are dependent on functional Kv channels.
ONOO− inhibits whole-cell and unitary Kv1 currents.
Kv1 “Shaker-type” gene family members are redox targets in some VSMCs (8). To determine whether ONOO− specifically impairs Kv1 channels in RSCAs, we used the specific Kv1 channel blocker correolide (COR) (13). DC-ONOO− did not significantly affect Kv current in freshly isolated VSMCs, but COR (1 μmol/l) blocked a large component, indicating the presence of Kv1 family channels (Fig. 3A). In contrast, authentic ONOO− (5 μmol/l) reduced total Kv current amplitude, and COR-sensitive Kv1 current was not detected after exposure to ONOO− (Fig. 3B). Current-voltage relations averaged from five cells verified that DC-ONOO− did not significantly alter Kv current density in coronary VSMCs (Fig. 3C), whereas authentic ONOO− fully eliminated the COR-sensitive Kv1 current component (Fig. 3D).
Subsequent studies determined whether ONOO− directly inhibited Kv channels in cell-free, inside-out patches of coronary VSMCs. A prominent Kv channel that progressively activated in response to stepwise patch depolarization was detected (Fig. 4A) and showed a unitary conductance of ∼75 pS (Fig. 4B). Exposing the cytosolic patch surface to ONOO− (5 μmol/l) profoundly inhibited Kv channel activity, whereas the unitary amplitude was unaffected (Fig. 4C). In five different cells, ONOO− reduced the open-state probability of single Kv channels by 40 ± 8% (Fig. 4D).
HG enhances ONOO− production.
On the basis of initial findings indicating that authentic ONOO− impaired Kv1 channel function, new studies investigated whether HG promotes ONOO− formation in RSCAs. Nitrotyrosine, a footprint of ONOO−, was used to assess ONOO− production in RSCAs that were exposed to NG or HG (5). Sample images from five representative experiments indicate that staining was more prominent in RSCAs that were incubated in HG media (Figs. 5A and B). Nitrotyrosine levels were reduced after treatment with 100 μmol/l urate, a scavenger of ONOO− (Figs. 5C and D). The nitrotyrosine signal also was decreased after dual incubation with MnTBAP (300 μmol/l) and l-NAME (100 μmol/l), drugs that scavenge O2˙− and inhibit NO synthesis, respectively (Figs. 5E and F). A low level of nitrotyrosine reactivity was observed in freshly isolated RSCAs (Fig. 5G), and elimination of the primary antinitrotyrosine antibody resulted in signal loss (Fig. 5H). These studies suggest that HG increases tyrosine nitration via endogenous ONOO− that is formed from the reaction of O2˙− with NO.
HG enhances nitration of Kv channels.
The Kv1 channels in VSMCs represent tetrameric structures composed of Kv1.2 and Kv1.5 α-subunits (13,14). Western blots indicated that the expression of Kv1.2 and Kv1.5 α-subunits was similar between RSCAs that were incubated for 24 h in NG, LG, or HG media (Figs. 6A and B). Averaged densitometric values from five studies confirmed these findings in which the expression of β-actin, a 42-kDa structural protein, provided an internal standard (Fig. 6C). However, nitrotyrosine immunoprecipitates revealed an elevated nitration of Kv1.2 α-subunits in RSCAs that were incubated in HG for 24 h, a response that was partially normalized by urate (Fig. 6D). In contrast, HG did not promote nitration of Kv1.5 α-subunits, and urate had little effect on basal nitration of this protein (Fig. 6E). Averaged data from five samples of immunoprecipitates indicated that HG enhanced nitration of Kv1.2 but not Kv1.5 α-subunits (Fig. 6F). Increased nitration of Kv1.2 α-subunits was also observed in Kv1.2 immunoprecipitates that were probed with nitrotyrosine antibody (data not shown).
Impaired Kv channel function is partially restored by scavengers of ONOO−.
A final set of experiments investigated whether incubation in HG for 24 h impairs Kv channel dilator function via generation of ONOO−. Compared with RSCAs that were exposed to NG levels (Fig. 7A), those that were exposed to HG constricted less to 4-AP (Fig. 7B), indicating a reduced dilator influence of Kv channels. Scavenging of ONOO− by urate (100 μmol/l) restored 30% constriction to 4-AP in HG but not NG arteries. Ebselen (100 μmol/l), a chemically distinct ONOO− scavenger, also partially (25%) and selectively restored the impaired constrictor response to 4-AP observed in RSCAs that were incubated in HG (Figs. 7C and D).
The present study provides several new findings. First, our data demonstrate that authentic ONOO− attenuates the dilator function of Kv channels in RSCAs and reduces macroscopic and single-channel Kv1 current. Second, short-term exposure of RSCAs to HG promotes ONOO− formation, an event that is associated with the nitration of Kv1.2 but not Kv1.5 pore-forming α-subunits. Third, RSCAs that are incubated in HG demonstrate impaired dilator function of Kv channels, and this defect is partially restored by ONOO− scavengers. Overall, these findings suggest potentially important roles for ONOO− production directly or through elevations in ambient glucose in the control of coronary vasomotor tone.
ONOO− attenuates Kv channel function.
The formation of ONOO−, driven by the interaction of O2˙− and NO, has been proposed as a contributing factor in the pathogenesis of vasodilator dysfunction (5). Recently, our laboratory reported that vasodilator responses that are dependent on cAMP activation of Kv channels were attenuated in RSCAs that were exposed to HG for 24 h (7). The findings of the present study directly demonstrate that authentic ONOO− reduces Kv channel dilator function in cannulated and pressurized RSCAs. In addition, patch-clamp studies revealed that ONOO− blocks a COR-sensitive component of Kv1 current in single VSMCs from RSCAs, suggesting that Kv1 family channels are susceptible to ONOO− inhibition. Finally, our data demonstrate that the open-state probability of Kv channels in cytosol-free membrane patches of RSCAs is reduced in response to authentic ONOO−, suggesting that ONOO− may directly inhibit Kv1 channels in VSMC membranes independent of intracellular signaling mechanisms. Thus, it seems that ONOO− may powerfully inhibit coronary Kv1 channel activity.
In this study, urate effectively prevented formation of ONOO− in RSCAs that were exposed to HG (Fig. 5) but partially restored the impaired 4-AP constriction in HG vessels. This may be due to a higher threshold for detection of ONOO− by immunohistochemistry than for its inhibition of Kv channels, which may occur at concentrations not detected by immunohistochemistry. Alternatively, it may be that factors other than ONOO− contribute to impaired Kv channel function during hyperglycemia. We previously demonstrated that superoxide dismutase, a O2˙− scavenger, partially restored impaired Kv channel activation in RSCAs that were exposed to HG, indicating an inhibitory role of O2˙− on Kv channels (11). The relative contribution of O2˙− and ONOO− in the reduction of Kv current in HG is not known. On the basis of our previous and present studies, we believe that both O2˙− and ONOO− contribute to the impaired Kv channel function during hyperglycemia.
HG-induced ONOO− formation is associated with nitration and inhibition of Kv1 channels.
The present study also provides initial evidence that HG promotes ONOO− formation in RSCAs, an event associated with nitration of tyrosine residues in VSMCs. In support of this hypothesis, scavenging of ONOO− by urate reduced the immunoreactivity correlating to nitrotyrosine residues in RSCAs that were exposed to HG for 24 h. Similarly, the use of MnTBAP and l-NAME jointly to reduce the availability of O2˙− and NO to form ONOO−-attenuated nitrotyrosine formation. Subsequent findings revealed that elevation of ONOO− by HG selectively increased the number of nitrotyrosine residues in Kv1.2 but not Kv1.5 α-subunits, an event that was partially reversed by the ONOO− scavenger urate. These findings suggest that ONOO− may specifically nitrate particular proteins that compose the Kv1 channel pore, providing initial insight into the molecular basis of ONOO−-induced inhibition of coronary Kv channels. Notably, short-term exposure to HG has been linked to an increased ONOO− production in the endothelial cells of human aorta and rat retinal arteries (15–17), and ONOO− may inhibit prostacyclin synthase in these endothelial cells (15). Thus, several lines of evidence suggest that the elevation of ONOO− in response to HG may impair the function of at least several vasodilator mechanisms in the arterial wall, including Kv channels in VSMCs.
Several limitations of the present study should be acknowledged. First, our findings have not established a causal relationship between ONOO−-induced nitration of Kv1.2 α-subunits and impaired Kv1 channel function. Mutagenesis will be required to confirm the molecular basis of inhibition, which may involve single or multiple nitrations of the 17 tyrosine residues that reside in the Kv1.2 α-subunit. Second, although nitrotyrosine is widely used as a marker for ONOO− formation, other mechanisms of tyrosine nitration exist, including H2O2-NO2-hemeperoxidase and NO2-mediated nitrosylation (18). However, because H2O2-NO2-hemeperoxidase occurs primarily in inflammatory cells (19) and nitrotyrosine formation by NO2 is a limited reaction (20), ONOO− most likely was responsible for Kv channel nitration in our study. Finally, short-term exposure to HG may not produce the same complexity or degree of vascular dysfunction as in diabetes, where alternative mechanisms could mask or aggravate vasodilator dysfunction. However, the advantage of this study is the ability to directly examine Kv channel function in response to HG without confounding influences, such as circulating plasma constituents and neurohumoral factors. Future studies will examine the overall effect of ONOO− in diabetes-induced K-channel–mediated vascular dysfunction.
Kv channels are a major contributor to the resting tone of small coronary arteries and represent a powerful dilator influence in the coronary circulation in vivo (for review, see 21). These channels participate in coronary vasodilator responses to acidosis, cAMP-dependent agonists such as β-adrenergic activation, and endothelial factors. Under these conditions, in which resting vascular tone relies on Kv channel function, the generation of ONOO− during hyperglycemia impairs channel availability and may compromise coronary blood flow and myocardial perfusion.
This work was supported by P50 HL65203, P01 HL68769, and a VA Merit Grant (to D.D.G.); NIH R01 HL59238 (to N.J.R.); NIH RO1 HL067948; and a Scientist Development Grant from the American Heart Association (to Y.L.).