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Am J Physiol Heart Circ Physiol 289: H2244-H2250, 2005; doi:10.1152/ajpheart.00254.2005
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Peroxynitrite hyperpolarizes smooth muscle and relaxes internal carotid artery in rabbit via ATP-sensitive K+ channels

Masuo Ohashi,1 Frank Faraci,1,2 and Donald Heistad1,2,3

1Cardiovascular Center and Department of Internal Medicine and 2Pharmacology, The University of Iowa and Roy J. and Lucille A. Carver College of Medicine; and 3Veterans Affairs Medical Center, Iowa City, Iowa

Submitted 16 March 2005 ; accepted in final form 8 July 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The goal of this study was to determine the effects of peroxynitrite (ONOO–) on smooth muscle membrane potential and vasomotor function in rabbit carotid arteries. ONOO– is known to affect vascular tone by several mechanisms, including effects on K+ channels. Xanthine (X, 0.1 mM), xanthine oxidase (XO, 0.01 U/ml), and a low concentration of sodium nitroprusside (SNP, 10 nM) were used to generate ONOO–. In the common carotid artery, X and XO (X/XO) in the presence of SNP tended to increase tension. In contrast, in the internal carotid artery, X/XO in the presence of SNP transiently hyperpolarized the membrane (–8.5 ± 1.8 mV, mean ± SE) and decreased tension (by 85 ± 5.6%). In internal carotid arteries, in the absence of SNP, X/XO did not hyperpolarize the membrane and produced much less relaxation (by 23 ± 5.6%) than X/XO and SNP. Ebselen (50 µM) inhibited both hyperpolarization and relaxation to X/XO and SNP, and uric acid (100 µM) inhibited relaxation. Glibenclamide (1 µM) abolished hyperpolarization and inhibited relaxation during X/XO and SNP. Charybdotoxin (100 nM) or tetraethylammonium (1 mM) did not affect hyperpolarization or relaxation, respectively. These results suggest that ONOO– hyperpolarizes and relaxes smooth muscle in rabbit internal carotid artery but not in common carotid artery through activation of KATP channels.

hyperpolarization; membrane potential; vasomotor function; reactive oxygen species

REACTIVE OXYGEN SPECIES (ROS) and nitrogen species play an important role in the maintenance of vascular homeostasis and after injury. Pathophysiological conditions such as ischemia and reperfusion increase production of both nitric oxide (NO) and superoxide (28). Superoxide reacts with NO and forms peroxynitrite (ONOO–), which is a strong oxidant (21). There is evidence that, in some cardiovascular diseases, nitrotyrosine accumulates in vascular tissue, which reflects an increase in the production of ONOO– (10).

Activity of K+ channels, which is an important determinant of membrane potential in smooth muscle (5), may be altered by ROS and thus modulate vascular resistance and arterial pressure (16). ONOO– produces dilatation in several vascular beds, including coronary (15), mesenteric (1), and cerebral (25, 26). Electrophysiological mechanisms responsible for vasodilatation, however, are not clear. In human coronary artery, ONOO– inhibits Ca2+-activated potassium channel (KCa) (17), whereas it activates ATP-sensitive potassium channel (KATP) in cat cerebral arteries (25).

In the present study, we used xanthine (X) and xanthine oxidase (XO) to generate superoxide and added sodium nitroprusside (SNP) to generate ONOO–. This reaction mimics pathophysiological situations in which ONOO– is formed by the reaction of superoxide with NO. We also measured vasomotor responses and membrane potential of smooth muscle cells with a microelectrode.

The first goal of this study was to examine the effects of X, XO, and SNP (X/XO+SNP) on vasomotor tone and membrane potential in rabbit carotid arteries. The second goal was to examine the role of several ROS in this response. We used ebselen or uric acid to scavenge ONOO–, SOD to dismutate superoxide, and catalase to degrade hydrogen peroxide. The third goal was to examine K+ channels by which ROS alter vasomotor function and membrane potential. We used glibenclamide to inhibit KATP channel, charybdotoxin to inhibit large conductance Ca2+ activated K+ channel, and tetraethylammonium (TEA) to inhibit KCa channel nonselectively.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male New Zealand White rabbits (2.5 to 3.5 kg body wt, n = 32) were euthanized with pentobarbital sodium (50 mg/kg) into the marginal ear vein followed by exsanguination. The common and internal carotid arteries were quickly removed and placed in cold (4°C) oxygenated Krebs solution containing (in mM) 118.3 NaCl, 4.7 KCl, 1.2 NaH2PO4, 25 NaHCO3, 1.2 MgSO4, 11 glucose, and 2.5 CaCl2. Loose connective tissue was removed. All procedures followed institutional guidelines as approved by the Animal Care and Use Committee of the University of Iowa.

Membrane potential. Membrane potential was measured by using a conventional microelectrode technique. The internal carotid artery was cut along its long axis with small scissors, and circularly cut strips were carefully prepared. The strip was mounted horizontally in a chamber of 0.3-ml volume placed on the stage of an inverted microscope (CK40, Olympus) and superfused with oxygenated Krebs solution (3 ml/min) containing N{omega}-nitro-L-arginine methyl ester hydrochloride (L-NAME, 300 µM). Glass microelectrodes made from borosilicate tubing (1.2 mm, outer diameter) with glass filament inside (Heilgenberg) were filled with 1 M KCl.

The resistance of the electrode was 120–180 M{Omega}. The electrode was inserted into the artery from the internal surface using a micromanipulator (PatchMan Eppendolf). Criteria for acceptable impalement were stable membrane potential for at least 5 min before every experimental procedure and sudden changes on penetration and withdrawal. Successful recordings could usually be obtained from the internal carotid artery for at least 30 min and for up to 2 h. Membrane potentials were recorded by using an amplifier (model 8100–1, Dagan) and displayed on a cathode-ray oscilloscope (DSO420, Gould). Data relating to membrane potential were stored at an acquisition rate of 200 Hz using AxoScope 7/Digidata 1200 data acquisition system (Axon Instrument) on a personal computer (Gateway 2000).

Changes in membrane potential induced by ONOO– were obtained by application of X (0.1 mM) with XO (0.01 U/ml) in the presence of SNP (10 nM). Responses to X/XO were measured 5 min after application of SNP. To examine the role of ROS that contribute to hyperpolarization induced by X/XO+SNP, effects of ebselen, catalase or polyethylene glycol-SOD (PEG-SOD) were examined. For these experiments, the inhibitor of ROS was applied 5 min before application of SNP, and the effects of X/XO were studied in the presence of SNP together with the inhibitor.

To determine whether K+ channels are involved in hyperpolarization induced by X/XO+SNP, the effects of several types of K+ channel blockers were examined by the protocol described above, using glibenclamide or charybdotoxin instead of a scavenger.

Changes in membrane potential by hydrogen peroxide were measured during cumulative application of increasing doses of hydrogen peroxide (10–100 µM) for 4 min at each dose.

Vascular function. Isometric tension was measured in common and internal carotid arteries. Common and internal carotid arteries were cut into two segments each (4 mm and 2 mm in length, respectively). Vascular rings were mounted on stainless steel hooks at optimal resting tension (3 g in common carotid artery and 1 g in internal carotid artery) in organ baths with Krebs bicarbonate solution at 37°C and bubbled with 95% O2-5% CO2.

Tension was periodically adjusted to the desired level during a 45-min equilibration period. Vascular rings were then contracted twice with 60 mM KCl and rinsed three times after each contraction. The effects of X (0.1 mM) with XO (0.01 U/ml) and/or SNP (10 nM) were observed after precontraction with phenylephrine (10 µM or 1–3 µM, in the presence or absence of SNP, respectively). In other vessels, inhibitory effects of several ONOO– scavengers or K+ channel inhibitor were studied by pretreatment with a scavenger or an inhibitor before and after precontraction by phenylephrine. Because glibenclamide can inhibit Cl– channels, bumetanide (which inhibits Na+-K+-Cl– cotransporter) and HEPES-buffered physiological salt solution (PSS) (which inhibits HCO3–-Cl– transporter) were given to determine whether Cl– channels modulate the response to X/XO+SNP (12). HEPES-buffered PSS was prepared by substituting NaHCO3 with 10 mM HEPES. All sets of vessels were studied in the presence of L-NAME (300 µM) to inhibit NO production.

Chemicals. X, XO, SNP, PEG-SOD, l-phenylephrine hydrochloride, L-NAME, uric acid, ebselen, catalase, TEA, charybdotoxin, bumetanide, HEPES, and glibenclamide were obtained from Sigma Chemical. Hydrogen peroxide was obtained from Fisher Scientific. Uric acid and X were dissolved in NaOH. Ebselen was dissolved in ethanol. Glibenclamide was dissolved in DMSO (Fisher). Other drugs were dissolved in normal saline. Final concentration of ethanol, NaOH, or DMSO had no effect on the membrane potentials and vasomotor function.

Statistical analysis. All values are expressed as means ± SE, with n denoting the number of animals. Statistical significance was determined using a one-way ANOVA followed by Student's unpaired t-test. Differences were considered to be significant at a value of P < 0.05. Relative force represents the force recorded relative to contraction to 10 µM phenylephrine in the same ring.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Membrane potential. Resting membrane potential of smooth muscle cells in rabbit internal carotid artery was –48.8 ± 1.5 mV (n = 24 animals).

SNP (10 nM) did not significantly change the resting membrane potential (0.9 ± 0.7 mV, n = 24 animals). In the presence of SNP, X (0.1 mM) with XO (0.01 U/ml), which together were used to generate ONOO–, transiently hyperpolarized the membrane by –8.5 ± 1.8 mV (Fig. 1A). In the absence of SNP, X/XO did not hyperpolarize the membrane (Fig. 1B).



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  		Fig. 1. Effect of xanthine (X, 0.1 mM), xanthine oxidase (XO, 0.01 U/ml), and sodium nitroprusside (SNP, 10 nM) (X/XO+SNP) on membrane potential in internal carotid artery. In presence of SNP, X/XO hyperpolarized the membrane (A, actual trace). SNP or X/XO alone did not affect the membrane potential (B). Arrow indicates withdrawal of the electrode. Values are means ± SE from 3 to 6 animals. *P < 0.05 vs. SNP.

 
In the presence of ebselen (50 µM), a scavenger of both hydrogen peroxide and ONOO–, hyperpolarization induced by X/XO+SNP was inhibited (Fig. 2). PEG-SOD (50 U/ml) tended to reduce (not significant, P = 0.3) hyperpolarization induced by X/XO+SNP (Fig. 2). Hyperpolarization produced by X/XO+SNP was inhibited by catalase (800 U/ml, Fig. 2). A high concentration of exogenous hydrogen peroxide is required to produce hyperpolarization in internal carotid arteries (Fig. 3A). These data suggest that ONOO–, but not superoxide, produced by X/XO+SNP account for hyperpolarization of smooth muscle.



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  		Fig. 2. Effects of ebselen (50 µM), polyethylene glycol-SOD (50 U/ml, PEG-SOD), or catalase (800 U/ml) on hyperpolarization induced by X/XO+SNP in internal carotid artery. Values are means ± SE from 4 to 6 animals. Ebselen or catalase inhibited hyperpolarization. *P < 0.05 vs. hyperpolarization induced by X/XO+SNP.

 


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  		Fig. 3. Effect of hydrogen peroxide (H2O2) on membrane potential (A) and tension (B) in internal carotid artery. Values are means ± SE from 4 animals. H2O2 hyperpolarized membrane and relaxed artery in doses from 10 to 100 µM. Hyperpolarization was inhibited by glibenclamide (1 µM). *P < 0.05 vs. 100 µM H2O2.

 
In the presence of glibenclamide (1 µM), hyperpolarization induced by X/XO+SNP was abolished. Charybdotoxin (100 nM) did not alter hyperpolarization induced by X/XO+SNP (Fig. 4). These results suggest that ONOO– hyperpolarized the membrane via KATP channel.



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  		Fig. 4. Effect of glibenclamide (1 µM) or charybdotoxin (ChTx, 100 nM) on hyperpolarization by X/XO+SNP in internal carotid artery. Actual trace shows that glibenclamide abolished hyperpolarization induced by X/XO+SNP (A). In contrast, ChTx (100 nM) did not affect hyperpolarization (B). Values are means ± SE from 4 animals. Arrow in A indicates withdrawal of electrode. *P < 0.01 vs. X/XO+SNP.

 
Vasomotor tone. In internal carotid artery precontracted by phenylephrine, SNP (10 nM) decreased vascular tension (85 ± 5.6%). In the presence of SNP, X (0.1 mM) with XO (0.01 U/ml) further decreased the tension (Figs. 5 and 6A). These data indicate that X/XO+SNP relax smooth muscle, as well as producing hyperpolarization of smooth muscle cells.



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  		Fig. 5. Effect of X/XO+SNP on tension in internal (A) and common (B) carotid artery. Addition of X/XO to SNP produced relaxation in internal, but not common, carotid artery. Phe, phenylephrine.

 


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  		Fig. 6. Effect of X/XO+SNP on tension in internal (A) and common (B) carotid artery. Values are means ± SE from 4 to 5 animals. {dagger}P < 0.05 vs. common carotid artery, *P < 0.0001 vs. +SNP.

 
In contrast, in common carotid artery, X/XO did not augment relaxation to SNP (Figs. 5 and 6). The findings indicate that effects of X/XO+SNP differ in internal and common carotid arteries.

In the absence of SNP, X/XO tended to reduce tension in common (–0.4 ± 0.2 g, P > 0.1, n = 5 animals) and internal carotid arteries (–1.7 ± 0.6 g, P > 0.1, n = 7 animals), but the effect was modest compared with effects of X/XO+SNP (Fig. 6). A high concentration of exogenous hydrogen peroxide is required to produce relaxation (Fig. 3B), as well as hyperpolarization (Fig. 3A) in internal carotid arteries. Ebselen (50 µM) inhibited relaxation induced by X/XO+SNP (Fig. 7A). Uric acid (100 µM, a scavenger of ONOO–) also inhibited vascular relaxation to X/XO+SNP (Fig. 7B).



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  		Fig. 7. Effect of ebselen (50 µM, A) or uric acid (100 µM, UA, B) on relaxation in internal carotid artery. Values are means ± SE from 4 animals. *P < 0.05 vs. in the absence of ebselen or UA.

 
Glibenclamide (1 µM) inhibited about 50% of relaxation to X/XO+SNP in the internal carotid artery (Fig. 8A). TEA (1 mM) did not affect this relaxation (Fig. 8B). Glibenclamide or TEA did not inhibit the effects of X/XO in the absence of SNP (–1.3 ± 0.1 g, P < 0.05, n = 5 animals; 0.9 ± 0.3 g, P > 0.1, n = 5 animals, respectively). Modulation of intracellular [Cl–] by HEPES-buffered PSS and bumetanide did not affect responses to X/XO+SNP (83 ± 4% and 87 ± 10%, in the absence and presence of HEPES-buffered PSS and bumetanide, respectively, n = 4 animals).



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  		Fig. 8. Effect of glibenclamide (1 µM, A) or tetraethylammonium (1 mM, TEA; B) on relaxation in internal carotid artery. Values are means ± SE from 7 animals. *P < 0.001 vs. in absence of glibenclamide.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
There are three major novel findings in this study. First, ROS generated by X/XO+SNP hyperpolarize and relax smooth muscle in rabbit carotid artery. X/XO+SNP produced relaxation of the internal carotid artery but not the common carotid artery. Second, hyperpolarization and relaxation to X/XO+SNP in the internal carotid artery were inhibited by ebselen or uric acid. Third, the response was inhibited by glibenclamide but not by charybdotoxin or TEA. Taken together, these findings suggest that ONOO– generated by X/XO+SNP hyperpolarizes and relaxes smooth muscle via KATP channel in rabbit internal carotid artery but not in the common carotid artery.

Mechanisms by which ROS hyperpolarize and relax smooth muscle. The combination of X/XO that we used in this study produces superoxide (18). Superoxide rapidly reacts with NO to produce ONOO– (21). We used SNP to release NO in our study. This combination of X/XO+SNP applied to the organ chamber generates ONOO– within vascular tissue (17).

In our study, smooth muscle of rabbit internal carotid artery hyperpolarized and relaxed during X/XO+SNP. Hyperpolarization and relaxation were inhibited by ebselen or uric acid, both of which are scavengers of ONOO– (13). These results suggest that ONOO– produced by X/XO+SNP hyperpolarized the membrane of smooth muscle cells, leading to relaxation of smooth muscle. In contrast, hyperpolarization was not induced and relaxation was much less by X/XO without SNP. These results suggest that superoxide itself does not affect membrane potential or contraction in rabbit internal carotid artery.

Superoxide produced by X/XO spontaneously dismutates to hydrogen peroxide in the presence of endogenous SOD (9). Hydrogen peroxide hyperpolarizes (8) and relaxes (6) smooth muscle. Thus hyperpolarization and relaxation induced by X/XO+SNP in the present study might involve hydrogen peroxide produced from superoxide by endogenous SOD. In fact, X/XO without SNP tended to relax the internal carotid artery, and hyperpolarization induced by X/XO+SNP was inhibited by catalase in this study. It is possible, therefore, that hyperpolarization and relaxation after X/XO+SNP are due in part to the generation of hydrogen peroxide. It seems likely, however, that hydrogen peroxide plays a relatively minor role because 1) hyperpolarization and relaxation were not induced by X/XO, in the absence of SNP, and one would expect that X/XO would produce superoxide and hydrogen peroxide; 2) PEG-SOD, which produces hydrogen peroxide from superoxide, tended to decrease hyperpolarization in this study; 3) a high concentration of exogenous hydrogen peroxide is required to produce hyperpolarization in internal carotid arteries, and the concentration of hydrogen peroxide produced by X/XO (in concentrations that we used in this study) is <8 µM (22); and 4) the effects of X/XO without SNP were far less than the effects of X/XO+SNP and not affected by glibenclamide or TEA.

Hyperpolarization and relaxation from ONOO–. ONOO– produced hyperpolarization and relaxation in this study. Hyperpolarization was abolished by glibenclamide but not affected by charybdotoxin. In addition, relaxation was inhibited by glibenclamide but not affected by TEA. These results suggest that hyperpolarization and relaxation induced by ONOO– open KATP channels in rabbit internal carotid artery.

In several vascular beds, including mesenteric (1) and cerebral (25, 26), ONOO– can elicit vasodilatation. Electrophysiological mechanisms responsible for the vasodilatation to ONOO– are not clear. In cerebral arteries, dilatation to ONOO– is blocked by glibenclamide (25, 26). ONOO– reduces dilatation to prostacyclin through a mechanism involving KATP channel in mesenteric artery (1), which suggests that ONOO– may inhibit the KATP channel. In addition, in human coronary artery, ONOO– inhibits KCa channels, as demonstrated with the patch-clamp technique (17). In the present study, using microelectrodes, ONOO– increased resting membrane potential and relaxed the internal carotid artery by opening the KATP channel. KCa channel may not be involved with this action of ONOO–, because charybdotoxin or TEA did not affect hyperpolarization or relaxation by ONOO–, respectively. Our results are consistent with previous findings in cat cerebral artery (25, 26) but differ from findings in other vascular beds (1, 17). Differences among species or vessels may contribute to difference findings.

In our study, glibenclamide abolished ONOO–-induced hyperpolarization, whereas half of the relaxation produced by ONOO– remained even in the presence of glibenclamide. This finding suggests that X/XO+SNP produce relaxation by changes in membrane potential and, in part, independently of changes in membrane potential. We speculate that ONOO– may alter vasomotor tone by an effect on cGMP levels, myosin light chain phosphatase activity, or cellular membrane Ca2+ entry in this preparation (14, 26), as well as by changing membrane potential.

Glibenclamide can inhibit Cl– channels as well as KATP channels (27). It seems unlikely, however, that Cl– channels contribute to hyperpolarization induced by X/XO+SNP, because of the following observations. 1) The equilibrium potential of Cl– in vascular tissue is between –11 and –50 mV (3, 11, 24). Resting membrane potential of rabbit internal carotid artery in this study was –48.8 mV, so opening of Cl– channels would be expected to depolarize, not hyperpolarize, the vascular membrane. 2) The IC50 of glibenclamide to Cl– channel is about 11–12 µM (27). Thus the concentration of glibenclamide that we used (1 µM) would not be expected to have a large effect on Cl– channels. 3) Finally, we found that relaxation due to X/XO+SNP was unchanged in the presence of bumetanide and absence of HCO3– (HEPES buffer), which inhibit the Cl– transporter and modulate intracellular [Cl–].

DMSO (the vehicle for glibenclamide) or ethanol (the vehicle for ebselen) has been reported to inhibit an opening of a KATP channel in rat pial arterioles (19). The concentration of DMSO that we used (0.001%), however, was much lower than concentrations that inhibit KATP channels (0.01–0.2%). The concentration of ethanol (0.1%) that we used did not affect dilatation by a KATP channel opener (19). Thus it is unlikely that DMSO or ethanol contributed to inhibitory effects of glibenclamide or ebselen on hyperpolarization and relaxation by X/XO+SNP.

Effects of ONOO– in internal and common carotid artery. In this study, ONOO– elicited relaxation in internal carotid artery. In common carotid artery, however, ONOO– did not relax the artery. Acetylcholine has been reported to produce hyperpolarization, and relaxation mediated by NO has been reported in common carotid artery (19) as well as internal carotid artery (2). It is unlikely that the common carotid artery is less sensitive to the relaxation produced by hyperpolarization. We cannot exclude the possibility that a higher concentration of ONOO– would hyperpolarize and relax the common carotid artery.

Large arteries to the brain contribute importantly to cerebral vascular resistance (4). Because the internal carotid artery is a muscular artery, vasomotor regulation of the internal carotid artery may affect the cerebral circulation.

Some limitations of the study. X/XO+SNP were used to mimic the in vivo situation in which ONOO– is formed by a similar reaction of superoxide with NO. The concentration of SNP (10 nM) that we used in this study is sufficient to generate ONOO– (17), and a submaximal concentration of SNP was used to avoid producing maximal relaxation from SNP alone (7). The interpretation of the results using X/XO+SNP is complicated, because the reaction generates superoxide and hydrogen peroxide as well as ONOO–. In addition, the concentration of ONOO– generated by X/XO+SNP may vary depending on the time course. Several studies have used authentic ONOO– to examine vasomotor function. The use of authentic ONOO–, however, does not seem to be a better approach, because ONOO– rapidly decomposes at physiological pH and may not reach the tissue consistently at a sufficiently high concentration to alter K+ channel function. In addition, high local concentration after application of authentic ONOO– may produce damage to the tissue from nonhomogeneous mixing. Real-time measurement of ONOO– concentration would be helpful, but it is extremely difficult to estimate the local concentration of this radical species (23). Thus our data and others must be interpreted with caution, because it is not certain that we have mimicked pathophysiological conditions in which ONOO– is generated.

In conclusion, ONOO– hyperpolarizes and relaxes smooth muscle in rabbit internal carotid artery but not in common carotid artery. Hyperpolarization and relaxation from ONOO– appear to be mediated by the opening of KATP channels.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants HL-16066, HL-62984, NS-24621, HL-38901, DK-54759, DK-15843, DK-52617, HL-55006, funds provided by the Veterans Affairs Medical Service, and a Carver Trust Research Program of Excellence.


   ACKNOWLEDGMENTS
 
We thank Dr. Fred S. Lamb for advice about Cl– channels, Leonard Brooks for technical assistance with microelectrodes, and Arlinda LaRose for typing the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Heistad, Dept. of Internal Medicine, Univ. of Iowa, 200 Hawkins Dr., Iowa City, IA 52242-1801 (e-mail: donald-heistad{at}uiowa.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.


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