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Am J Physiol Heart Circ Physiol 284: H528-H534, 2003. First published October 10, 2002; doi:10.1152/ajpheart.00752.2002
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Vol. 284, Issue 2, H528-H534, February 2003

Impairment of nitric oxide synthase-dependent dilatation of cerebral arterioles during infusion of nicotine

Qin Fang, Hong Sun, and William G. Mayhan

Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4575


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of nicotine on nitric oxide synthase (NOS)-dependent reactivity of cerebral arterioles remain uncertain. Our first goal was to examine whether infusion of nicotine alters NOS-dependent reactivity of cerebral arterioles. Our second goal was to examine the mechanisms that may account for the effects of nicotine on cerebral arterioles. We measured the diameter of pial arterioles to NOS-dependent (ADP and acetylcholine) and NOS-independent (nitroglycerin) agonists before and after the infusion of nicotine (2 µg · kg-1 · min-1 iv for 30 min, followed by a maintenance dose of 0.35 µg · kg-1 · min-1). ADP- and acetylcholine-induced vasodilatation was impaired after the infusion of nicotine. In contrast, nicotine did not alter vasodilatation to nitroglycerin. Next, we examined whether the impaired responses of pial arterioles during infusion of nicotine may be related to oxygen radicals. We found that application of superoxide dismutase or tetrahydrobiopterin during infusion of nicotine could prevent impaired NOS-dependent vasodilatation. Thus acute exposure of cerebral vessels to nicotine specifically impairs NOS-dependent dilatation via the production of oxygen radicals possibly related to an alteration in the utilization of tetrahydrobiopterin.

vasoreactivity; acetylcholine; ADP; nitroglycerin; oxygen radicals; tetrahydrobiopterin; superoxide dismutase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ONE OF THE MOST DEVASTATING health problems of cigarette smoking or smokeless tobacco use in human subjects is vascular damage to vital organs such as the lung, heart, peripheral circulation, and brain (1, 20, 27, 29, 61). Several studies have shown morphological abnormalities in endothelial cells of peripheral vascular tissue in humans and animals exposed to tobacco products. These abnormalities consist of endothelial cell swelling, extensive endothelial edema, subendothelial blebs, and an increased number of macrophages in the arterial wall (3, 4, 10). In addition, others have shown that the use of tobacco products produces functional alterations in endothelium, i.e., impaired endothelium-dependent dilatation of large peripheral arteries (5, 19, 21, 23, 47). Furthermore, we (36) and others (57) have shown that nitric oxide (NO) synthase (NOS)-dependent dilatation of peripheral resistance arterioles is impaired during exposure to cigarette smoke extract and/or smokeless tobacco.

The precise component of cigarette smoke and smokeless tobacco that accounts for vascular abnormalities/vascular dysfunction is not certain, but it appears that (-)-1 methyl-2-(3-pyridyl)-pyrrolidine (nicotine) may be a candidate. Investigators have shown that nicotine has toxic effects on endothelium (24, 29), and we (35, 37, 39) have shown that NOS-dependent responses of peripheral arterioles are impaired during acute and chronic exposure to nicotine. The mechanism for altered responses of peripheral arterioles during exposure to nicotine appeared to be related to the production of oxygen radicals because treatment with superoxide dismutase significantly restored impaired vasodilatation (37, 39). However, the precise cellular pathway that accounts for the formation of oxygen radicals remains unclear.

Although many studies have shown that cigarette smoking is a risk factor for the pathogenesis of cerebrovascular abnormalities, including stroke (20, 29, 41), no studies that we are aware of have examined the effects of the products of cigarette smoke/nicotine on NOS-dependent reactivity of cerebral arterioles in vivo. Thus the first goal of the present study was to examine the effects of acute infusion of nicotine on NOS-dependent and -independent reactivity of cerebral arterioles. Our second goal was to study possible mechanisms for the effects of nicotine on cerebral arterioles. First, we examined whether treatment with a conventional inhibitor of oxidants, superoxide dismutase, influenced vascular reactivity during exposure to nicotine. Second, we examined a potential pathway for the formation of oxygen radicals during infusion of nicotine. Previous studies have suggested that, under various conditions, activation of endothelial NOS (eNOS) can produce both NO and superoxide anion (55, 59, 60). It appears that the metabolism of an important cofactor for eNOS, tetrahydrobiopterin (BH4), may play a key role in the control of production of NO and superoxide anion by eNOS (55, 59, 60, 63). Thus we examined the effects of exogenous application of BH4 on NOS-dependent reactivity of cerebral arterioles during infusion of nicotine.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of animals. All procedures were carried out after institutional approval and are in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Adult male Sprague-Dawley rats were anesthetized with thiobutabarbital sodium (Inactin; 100 mg/kg body wt ip). Supplemental doses (10-20 mg/kg) of the anesthetic agent were given intravenously as needed. The animals were ventilated mechanically with room air and supplemental oxygen. A catheter was placed in a femoral vein for injection of nicotine and supplemental anesthetic, and a femoral artery was cannulated to measure arterial blood pressure. At the end of the experiment, all anesthetized animals were euthanized with an intravenous injection of KCl (5 ml of a saturated solution) or an overdose of anesthetic (250 mg/kg).

To visualize the pial microcirculation, a window was prepared over the parietal cortex. The rat was placed in a head holder, and an incision was made in the skin to expose the skull. The skin was retracted with sutures and served as a "well" for the suffusion fluid. A small window of skull was removed over the parietal cortex using a dental drill. The dura was then incised to expose the pial microcirculation. The cranial window was continuously suffused with artificial cerebral spinal fluid, which was bubbled with 95% N2-5% CO2. The temperature of the suffusate was maintained at 37 ± 1°C. The cranial window was connected via a three-way valve to a pump that allowed for infusion of agents into the suffusate without altering temperature, pH, PCO2, and PO2 (pH = 7.31 ± 0.01, PCO2 = 45 ± 1 mmHg, and PO2 = 59 ± 2 mmHg). Arterial blood gases were monitored and maintained within normal limits throughout the experiment.

We measured the diameter of pial arterioles using a video image-shearing device coupled to a videomonitor (model 908, Instrumentation for Physiology and Medicine; San Diego, CA). The diameter of pial arterioles was measured before the application of agonists and at 1-min intervals for 5 min during the application of agonists. During the infusion of nicotine, the diameter of pial arterioles was measured at 1-min intervals for the first 5 min and then at 5-min intervals for the next 25 min. All agonists were prepared using artificial cerebral spinal fluid, and application of agonists was randomized.

Experimental protocol. Cerebral vessels were superfused with artificial cerebral spinal fluid for 30 min before the responses of arterioles to the agonists were tested. In the first groups of rats (n = 8), we examined the effects of acute infusion of nicotine on reactivity of cerebral arterioles. Thus, in these studies, we initially examined the responses of arterioles to NOS-dependent agonists [ADP (10 and 100 µM) and acetylcholine (1.0 and 10 µM)] and to a NOS-independent agonist [nitroglycerin (1.0 and 10 µM)]. We (33) have shown previously that dilatation of pial arterioles in response to ADP and acetylcholine, but not to nitroglycerin, is related to activation of NOS. Thirty minutes after this initial examination of vascular reactivity, we then started an intravenous infusion of nicotine (2.0 µg · kg-1 · min-1 for 30 min, followed by a maintenance dose of 0.35 µg · kg-1 · min-1 for the remainder of the experimental protocol). We have shown previously that this methodology produces a plasma level of nicotine that is similar to that observed in smokers (15-20 ng/ml) (35, 38) and that infusion of nicotine does not alter arterial blood pressure (35). Thirty minutes after the infusion of nicotine was started, we again examined the responses of arterioles to ADP, acetylcholine, and nitroglycerin.

In a second group of rats (n = 6), we examined the reproducibility of responses of arterioles to the agonists. Thus we initially measured the responses of cerebral arterioles to ADP, acetylcholine, and nitroglycerin. We then started an intravenous infusion of vehicle (saline). Thirty minutes after the infusion of vehicle was started, we again examined the responses of the arterioles to ADP, acetylcholine, and nitroglycerin.

In a third group of rats (n = 7), we examined the effects of superoxide dismutase on nicotine-induced impairment in NOS-dependent reactivity of cerebral arterioles. Thus, in these studies, we initially measured the responses of cerebral arterioles to ADP, acetylcholine, and nitroglycerin. We then started a continuous topical application of superoxide dismutase (150 U/ml) over the pial circulation. We (34, 39) have shown previously that this concentration of superoxide dismutase is efficacious. Thirty minutes after the application of superoxide dismutase was started, we started an intravenous infusion of nicotine (2.0 µg · kg-1 · min-1 for 30 min, followed by a maintenance dose of 0.35 µg · kg-1 · min-1 for the remainder of the experimental protocol). Thirty minutes after the infusion of nicotine was started, we again examined the responses of arterioles to ADP, acetylcholine, and nitroglycerin. Control studies (n = 4) using the vehicle instead of nicotine were conducted to determine the effect of superoxide dismutase on the reactivity of cerebral arterioles to the agonists.

In a fourth group of rats (n = 7), we examined the effects of BH4 on nicotine-induced impairment in NOS-dependent reactivity of cerebral arterioles. Thus, in these studies, we initially measured the responses of cerebral arterioles to ADP, acetylcholine, and nitroglycerin. We then started a continuous topical application of BH4 (0.1 µM) over the pial circulation. We (56) have shown previously that this concentration of BH4 is efficacious. Thirty minutes after the application of BH4 was started, we started an intravenous infusion of nicotine (2.0 µg · kg-1 · min-1 for 30 min, followed by a maintenance dose of 0.35 µg · kg-1 · min-1 for the remainder of the experimental protocol). Thirty minutes after the infusion of nicotine was started, we again examined the responses of arterioles to ADP, acetylcholine, and nitroglycerin. Control studies (n = 6) using the vehicle instead of nicotine were conducted to determine the effect of BH4 on the reactivity of cerebral arterioles to the agonists.

Drugs. ADP, acetylcholine, nicotine, and superoxide dismutase were purchased from Sigma (St. Louis, MO). Nitroglycerin was purchased from SoloPak Laboratories (Elk Grove Village, IL). BH4 was purchased from Calbiochem (San Diego, CA). All stock solutions of agents were prepared with saline and then diluted with artificial cerebral spinal fluid.

Statistical analysis. A paired t-test was used to compare the responses of pial arterioles to the agonists before and after the intravenous infusion of the vehicle or nicotine in the absence or presence of superoxide dismutase or BH4. Values are means ± SE. A P value of <= 0.05 was considered to be significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of nicotine on reactivity of pial arterioles. The baseline diameter of pial arterioles before the infusion of nicotine was 39 ± 1 µm. Topical application of ADP, acetylcholine, and nitroglycerin produced dose-related dilatation of pial arterioles before the infusion of nicotine (Fig. 1). Intravenous infusion of nicotine did not alter the baseline diameter of pial arterioles (39 ± 1 µm before the infusion of nicotine vs. 40 ± 1 µm during the infusion of nicotine, P > 0.05). The responses of pial arterioles to ADP and acetylcholine were profoundly impaired during infusion of nicotine (Fig. 1). ADP (10 and 100 µM) dilated pial arterioles by 12 ± 1% and 21 ± 2%, respectively, before the infusion of nicotine but only 3 ± 1% and 8 ± 1%, respectively, after the infusion of nicotine (P < 0.05). Acetylcholine (1.0 and 10 µM) dilated pial arterioles by 8 ± 1% and 13 ± 2%, respectively, before the infusion of nicotine but only 3 ± 1% and 4 ± 1%, respectively, after the infusion of nicotine (P < 0.05). In contrast, infusion of nicotine did not alter the dilatation of pial arterioles in response to nitroglycerin (Fig. 1).


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  		Fig. 1.   Response of pial arterioles to acetylcholine (A), ADP (B), and nitroglycerin (C) before (open bars) and during (solid bars) infusion of nicotine. Values are means ± SE. *P < 0.05 vs. the response before infusion of nicotine.

To determine whether responses to the agonists were reproducible, we examined the dilatation of pial arterioles in response to the agonists before and after the infusion of vehicle. The baseline diameter of pial arterioles before suffusion of agonists and infusion of vehicle was 42 ± 1 µm. Topical application of ADP, acetylcholine, and nitroglycerin produced dose-related dilatation of pial arterioles before infusion of vehicle (Fig. 2). Intravenous infusion of vehicle did not alter the baseline diameter of pial arterioles (42 ± 1 µm before infusion of vehicle vs. 42 ± 2 µm during infusion of vehicle, P > 0.05). In contrast to that observed during infusion of nicotine, intravenous infusion of vehicle did not impair dilatation of pial arterioles to ADP and acetylcholine (Fig. 2). In addition, infusion of vehicle did not alter vasodilatation in response to nitroglycerin (Fig. 2). Thus infusion of nicotine produces selective impairment in NOS-dependent, but not NOS-independent, dilatation of pial arterioles that cannot be explained by an alteration in the reproducibility in responsiveness to the agonists.


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  		Fig. 2.   Response of pial arterioles to acetylcholine (A), ADP (B), and nitroglycerin (C) before (open bars) and during (solid bars) infusion of vehicle. Values are means ± SE.

Effect of superoxide dismutase. The baseline diameter of pial arterioles before suffusion of agonists, application of superoxide dismutase, and infusion of nicotine was 41 ± 4 µm. Topical application of ADP, acetylcholine, and nitroglycerin produced dose-related dilatation of pial arterioles before suffusion of superoxide dismutase and infusion of nicotine (Fig. 3). Topical application of superoxide dismutase and infusion of nicotine did not alter the baseline diameter of pial arterioles (41 ± 4 vs. 40 ± 4 µm, P > 0.05). In contrast to that observed after the infusion of nicotine in the absence of superoxide dismutase (Fig. 1), intravenous infusion of nicotine did not impair NOS-dependent dilatation of pial arterioles in the presence of superoxide dismutase (Fig. 3). Superoxide dismutase did not alter the dilatation of pial arterioles in response to nitroglycerin (Fig. 3). Thus it seems that superoxide dismutase can inhibit the effects of nicotine on NOS-dependent reactivity of pial arterioles.


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  		Fig. 3.   Response of pial arterioles to acetylcholine (A), ADP (B), and nitroglycerin (C) before (open bars) and during (solid bars) infusion of nicotine in the presence of superoxide dismutase (150 U/ml). Values are means ± SE.

We also examined whether topical application of superoxide dismutase could influence the responses of pial arterioles to the agonists in the absence of infusion of nicotine. The baseline diameter of pial arterioles before suffusion of agonists, application of superoxide dismutase, and infusion of vehicle was 40 ± 1 µm. Topical application of superoxide dismutase and infusion of vehicle did not alter the baseline diameter of pial arterioles (40 ± 2 vs. 41 ± 1 µm, P > 0.05). Similarly, topical application of superoxide dismutase and infusion of vehicle did not alter the reactivity of pial arterioles to ADP, acetylcholine, and nitroglycerin. ADP (10 and 100 µM) dilated pial arterioles by 12 ± 1% and 18 ± 1%, respectively, before the application of superoxide dismutase and infusion of vehicle and by 11 ± 2% and 18 ± 2%, respectively, during the application of superoxide dismutase and infusion of vehicle (P > 0.05). Acetylcholine (1 and 10 µM) dilated pial arterioles by 7 ± 1% and 12 ± 1%, respectively, before the application of superoxide dismutase and infusion of vehicle and by 7 ± 1% and 11 ± 1%, respectively, during the application of superoxide dismutase and infusion of vehicle (P > 0.05). Nitroglycerin (1 and 10 µM) dilated pial arterioles by 22 ± 4% and 29 ± 5%, respectively, before the application of superoxide dismutase and infusion of vehicle and by 23 ± 1% and 31 ± 3%, respectively, during the application of superoxide dismutase and infusion of vehicle (P > 0.05). Thus it appears that superoxide dismutase does not alter the reactivity of pial arterioles to the agonists during infusion of vehicle.

Responses after BH4. The baseline diameter of pial arterioles before suffusion of agonists, application of BH4, and infusion of nicotine was 41 ± 1 µm. Topical application of ADP, acetylcholine, and nitroglycerin produced dose-related dilatation of pial arterioles before suffusion of BH4 and infusion of nicotine (Fig. 4). Topical application of BH4 and infusion of nicotine did not alter the baseline diameter of pial arterioles (41 ± 1 vs. 42 ± 2 µm, P > 0.05). During topical application of BH4, intravenous infusion of nicotine did not impair the NOS-dependent dilatation of pial arterioles (Fig. 4). In addition, topical application of BH4 did not alter the dilatation of pial arterioles in response to nitroglycerin (Fig. 4). Thus it seems that BH4 can inhibit the effects of nicotine on NOS-dependent reactivity of pial arterioles.


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  		Fig. 4.   Response of pial arterioles to acetylcholine (A), ADP (B), and nitroglycerin (C) before (open bars) and during (solid bars) infusion of nicotine in the presence of tetrahydrobiopterin (0.1 µM). Values are means ± SE.

We also examined whether topical application of BH4 could influence the responses of pial arterioles to the agonists in the absence of infusion of nicotine. The baseline diameter of pial arterioles before the suffusion of agonists, application of BH4, and infusion of vehicle was 44 ± 3 µm. Topical application of BH4 and infusion of vehicle did not alter the baseline diameter of pial arterioles (43 ± 3 vs. 44 ± 3 µm, P > 0.05). Similarly, topical application of BH4 and infusion of vehicle did not influence the reactivity of pial arterioles to ADP, acetylcholine, and nitroglycerin. ADP (10 and 100 µM) dilated pial arterioles by 12 ± 1% and 20 ± 1%, respectively, before the application of BH4 and infusion of vehicle and by 14 ± 2% and 23 ± 3%, respectively, during the application of BH4 and infusion of vehicle (P > 0.05). Acetylcholine (1 and 10 µM) dilated pial arterioles by 7 ± 1% and 13 ± 1%, respectively, before the application of BH4 and infusion of vehicle and by 8 ± 1% and 13 ± 1%, respectively, during the application of BH4 and infusion of vehicle (P > 0.05). Nitroglycerin (1 and 10 µM) dilated pial arterioles by 20 ± 2% and 30 ± 3%, respectively, before the application of BH4 and infusion of vehicle and by 20 ± 2% and 30 ± 2%, respectively, during the application of BH4 and infusion of vehicle (P > 0.05). Thus it appears that BH4 does not alter the reactivity of pial arterioles to agonists during infusion of vehicle.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

There are three major new findings in this study. First, acute infusion of nicotine, at a level seen in chronic smokers, selectively impairs NOS-dependent dilatation of cerebral arterioles. Second, treatment of the cerebral microcirculation with an inhibitor of the formation of superoxide anion, i.e., superoxide dismutase, prevents the effects of nicotine on NOS-dependent vasodilatation. Thus it appears that the formation of oxygen radicals is important in the impaired responses of pial arterioles during infusion of nicotine. Third, treatment of the cerebral microcirculation with an important cofactor for eNOS, BH4, prevented the effects of nicotine on NOS-dependent vasodilatation. On the basis of these findings, we speculate that an alteration in the availability/utilization BH4 by nicotine results in an imbalance in the production of NO and superoxide anion by eNOS and contributes to the impaired NOS-dependent dilatation of pial arterioles.

The plasma concentration of nicotine is elevated in habitual smokers throughout the course of the day (2, 26, 46). In fact, Isaac and Rand (26) reported that the plasma level of nicotine in human subjects was 25 ± 6 mg/ml 6.5 h after ad libitum smoking. We (35, 38) have shown previously that infusion of nicotine at the concentration used in the present study produces a plasma level similar to that observed in chronic smokers (2, 26, 46). Thus, although we are examining the acute effects of infusion of nicotine on the reactivity of pial arterioles, we believe that our study has important implications for the chronic effects of cigarette smoking/use of tobacco products on vascular dysfunction.

Many previous studies have examined the effects of cigarette smoking/cigarette smoke extract on the reactivity of large peripheral blood vessels and cerebral blood vessels in animal models and human subjects. For the most part, these previous studies have shown that cigarette smoking/cigarette smoke extract impairs the NOS-dependent reactivity of large peripheral blood vessels in animal models (40, 45, 47, 49) and human subjects (5, 13, 21, 42). In addition, investigators have shown that cigarette smoking impairs the reactivity of cerebral blood vessels in response to hypercapnia (54). Furthermore, we (51) have shown that cigarette smoke extract impairs the NOS-dependent reactivity of resistance arterioles. Thus cigarette smoking and/or exposure of blood vessels to the products of cigarette smoke produces selective impairment in the reactivity of large and small peripheral and large cerebral blood vessels to NOS-dependent agonists.

The mechanisms by which cigarette smoking/cigarette smoke extract impairs the NOS-dependent reactivity of blood vessels are less clear and have been reported to involve a direct effect of cigarette smoke on NOS (22, 62, 64), an increase in the formation of endothelin (15, 16, 28), and/or an increase in the local and systemic formation of oxygen-derived free radicals (9, 12, 31, 44, 45). Murohara et al. (45) have reported that cigarette smoke extract contracts porcine coronary arteries via the production of superoxide anion to presumably inactivate NO. Ota et al. (47) report that the impairment in endothelium-dependent relaxation of the rabbit aorta by cigarette smoke extract could be alleviated by treatment with inhibitors of oxygen radical formation (superoxide dismutase, dimethylsulfoxide, and captopril). Mays et al. (40) report that the smoking-induced impairment in endothelium-dependent reactivity of femoral arteries in rabbits could be reversed by treatment with an inhibitor of oxygen radical formation (ascorbic acid). Clinical studies have also implicated an important role for oxygen radicals in vascular dysfunction in smokers. Several studies have shown that treatment of smokers with vitamin C protects against endothelial dysfunction (13, 17, 65). Thus it appears that the formation of oxygen radicals plays an important role in vascular dysfunction after exposure to cigarette smoke/cigarette smoke extract. However, the precise cellular pathway for the formation of oxygen radicals in cigarette smoke-induced vascular dysfunction remains unclear.

While a number of studies have examined the effects of cigarette smoking/cigarette smoke extract on vascular reactivity, few studies have examined the direct effects of a major component of cigarette smoke/cigarette smoke extract, i.e., nicotine, on vascular reactivity. Investigators (6, 52) found that treatment of humans with nicotine impaired endothelium-dependent reactivity of large peripheral blood vessels. In addition, Miller et al. (43) report that treatment of dogs with nicotine produced a time- and dose-dependent alteration in endothelium-dependent reactivity of coronary arteries that appeared to be related to the bioavailability of NO. Furthermore, we found that acute treatment of hamsters with nicotine produced selective impairment in the NOS-dependent reactivity of resistance arterioles that was attributed to the formation of oxygen radicals (39).

In addition to studies of the peripheral circulation, investigators also have examined the effects of nicotine on the cerebral circulation. However, these studies for the most part have only examined the effects of nicotine on the baseline diameter of cerebral arteries. Schilling et al. (53) found that topical application of nicotine did not alter the baseline diameter of cerebral arterioles in cats. In contrast, Iida et al. (25) report that intravenous infusion of nicotine in rats produced a marked dilatation of pial arterioles. In the present study, we did not find a significant effect of nicotine on the baseline diameter of pial arterioles in vivo. The discrepancy between the present study and the study of Schilling et al. (53) with that of Iida et al. (25) may be related to the concentration of nicotine. Iida et al. (25) injected a high concentration of nicotine (0.05 mg · ml-1 · rat-1) in a relatively short period of time (1 min). This methodology not only produced a significant increase in pial arteriolar diameter, but also produced a profound increase in mean arterial pressure (~40 mmHg). This increase in blood pressure could have produced an increase in pial arteriolar diameter independent of the effect of nicotine.

One study has examined the effects of nicotine on reactivity of cerebral blood vessels. In this previous study (14), the investigators report that chronic (15-22 days) treatment of rats with nicotine impaired cerebrovasodilation (measured by laser Doppler) in response to a NO donor [S-nitroso-N-acetyl penicillamine (SNAP)]. The mechanism for the effect of nicotine on changes in cerebral blood flow in response to SNAP was speculated to be related to effects on calcium and potassium channels. In the present study, we found that acute infusion of nicotine specifically impaired the NOS-dependent reactivity of pial arterioles. In contrast to the previous study (14), however, we did not find an effect of acute infusion of nicotine on the dilatation of pial arterioles in response to nitroglycerin, a NO donor. The discrepancy between the present study and the previous study (14) may be related to the acute versus chronic effects of nicotine on cerebral blood vessels.

To determine a potential role of oxygen radicals in the impaired NOS-dependent responses of pial arterioles during acute infusion of nicotine, we treated cerebral arterioles with superoxide dismutase. We found that treatment of pial arterioles with superoxide dismutase prevented impaired NOS-dependent reactivity after infusion of nicotine. This finding is similar to that we (39) reported for the peripheral microcirculation. Thus it appears that the formation of oxygen radicals, presumably superoxide anion, contributes to the impaired NOS-dependent reactivity of cerebral arterioles during acute infusion of nicotine.

Many recent studies have begun to examine the role of BH4 in the impaired responses of peripheral blood vessels during disease states. A number of studies have shown that treatment of diabetic human subjects (18) or diabetic animals (30, 48) with BH4 ameliorates impaired the NOS-dependent vasoreactivity of large peripheral blood vessels. In addition, recent studies have shown that BH4 can prevent the impaired NOS-dependent dilatation of coronary blood vessels in human subjects (32, 58) and pigs (58) with atherosclerosis. Furthermore, we (56) have shown that impaired NOS-dependent responses of pial arterioles in rats fed an alcohol-containing diet could be returned toward that observed in non-alcohol-fed rats by treatment of the cerebral microcirculation with BH4. Finally, Higman et al. (22) found that impaired NOS-dependent dilatation of the saphenous vein from smokers could be prevented by treatment with BH4. These investigators (22) concluded that impaired NOS-dependent relaxation of the saphenous vein in chronic smokers was related to a reduction in the activity of eNOS that was attributed to an inadequate supply of BH4. Thus it appears that an alteration in BH4 can contribute to impaired responses of cerebral and peripheral blood vessels in humans and animal models during many disease states, including smoking. In the present study, we found that treatment of pial arterioles with BH4 could prevent the impaired NOS-dependent reactivity of cerebral arterioles during infusion of nicotine. Thus the findings of the present study complement and extend that reported by others by examining the effects of BH4 on cerebral resistance blood vessels in vivo and by examining the effects of BH4 during acute infusion of a major component of cigarette smoke, i.e., nicotine.

The mechanism by which BH4 can prevent the impaired NOS-dependent reactivity of blood vessels during disease states is not entirely clear but may relate to the uncoupling of eNOS due to an insufficiency of, or impaired metabolism of, BH4. Many investigators have suggested that, under various conditions, activation of eNOS can produce both NO and superoxide anion (55, 60). In addition, it appears that the metabolism of BH4 may play a key role in the control of the production of NO and superoxide anion (50, 59, 63). Thus, in the absence of or impaired metabolism of BH4, there is an uncoupling of the L-arginine-NO pathway, resulting in an increased formation of superoxide anion and a reduced formation of NO (7, 8, 11, 63). The findings of the present study appear to indicate that acute infusion of nicotine alters the balance of NO and superoxide anion production during receptor-mediated stimulation of NO release.

In summary, we examined the role of oxygen radicals in nicotine-induced impairment of NO-dependent reactivity of pial arterioles. We found that infusion of nicotine impaired the reactivity of pial arterioles to acetylcholine and ADP but not nitroglycerin. In addition, treatment with superoxide dismutase and BH4 prevented the nicotine-induced impairment of NOS-dependent arteriolar reactivity. We suggest that the formation of oxygen radicals, possibly via an alteration in the utilization of BH4, contributes to the impairment of NOS-dependent dilatation of pial arterioles during infusion of nicotine. We speculate that our findings have important implications for the pathogenesis of cerebrovascular abnormalities, including stroke, observed in smokers and users of tobacco products.


    ACKNOWLEDGEMENTS

This study was supported by National Institutes of Health Grants HL-40781, AA-11288, and DA-14258, a Pre-Doctoral Fellowship from the American Heart Association, Heartland Affiliate, and funds from the University of Nebraska Medical Center.


    FOOTNOTES

Address for reprint requests and other correspondence: W. G. Mayhan, Dept. of Physiology and Biophysics, 984575 Nebraska Medical Center, Omaha, NE 68198-4575 (E-mail: wgmayhan{at}unmc.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.

First published October 10, 2002;10.1152/ajpheart.00752.2002

Received 29 August 2002; accepted in final form 3 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 284(2):H528-H534
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