Effect of Cigarette Smoke Extract on Arteriolar Dilatation in Vivo

Effect of cigarette smoke extract on arteriolar dilatation in vivo

WILLIAM G. MAYHAN AND GLENDA M. SHARPE Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4575

Mayhan, William G., and Glenda M. Sharpe. Effect of In addition to activation of guanylate cyclase via the cigarette smoke extract on arteriolar dilatation in vivo. J. synthesis and/or release of nitric oxide, relaxation of Appl. Physiol. 81(5): 1996–2003, 1996.—The goal of this vascular smooth muscle can be influenced by activation study was to determine whether cigarette smoke extract of ATP-sensitive potassium channels and activation of alters dilatation of arterioles in vivo in response to agonists adenylate cyclase. However, no studies have examined that produce activation of ATP-sensitive potassium channels and activation of adenylate cyclase. By using intravital the effects of cigarette smoking on these important microscopy, we measured diameter of arterioles contained cellular dilator pathways. Thus the goal of this study within the microcirculation of the hamster cheek pouch was to examine the effects of cigarette smoke extract on during suffusion with agonists in the absence and presence of dilatation of resistance arterioles in vivo in response to cigarette smoke extract (0.1, 0.5, and 1.0%). Before treatment activation of ATP-sensitive potassium channels and in with cigarette smoke extract, activation of ATP-sensitive response to activation of adenylate cyclase. potassium channels with aprikalim and cromakalim produced dose-related dilatation of cheek pouch arterioles. Simi- METHODS larly, activation of adenylate cyclase with isoproterenol and forskolin produced dose-related dilatation of cheek pouch Preparation of animals. Adult male hamsters weighing arterioles before treatment with cigarette smoke extract. between 120 and 140 g were anesthetized with pentobarbital Superfusion of 0.1% cigarette smoke extract did not change sodium (6 mg/100 g body wt ip). A tracheotomy was performed baseline diameter of arterioles and did not alter responses of to facilitate spontaneous breathing. A catheter was placed in a femoral vein for the purpose of infusing supplemental cheek pouch arterioles to activation of ATP-sensitive potas- 21 21 sium channels and adenylate cyclase. Superfusion of 0.5 and anesthesia (2–4 mg·100 g ·h ). A femoral artery was 1.0% cigarette smoke extract also did not alter baseline cannulated to measure arterial blood pressure, which re- diameter of arterioles but did impair dilatation of arterioles mained constant during the experiment (data not shown). At in response to activation of ATP-sensitive potassium channels the end of the experiment, all animals were killed with an and adenylate cyclase. These findings suggest that cigarette intravenous infusion of anesthesia (60 mg/100 g body wt). All smoke extract impairs dilatation of resistance arterioles in procedures were carried out after institutional approval and response to activation of important cellular dilator pathways. within institutional guidelines. To visualize the microcirculation of the cheek pouch, we aprikalim; cromakalim; isoproterenol; forskolin; microcircula- used a method described previously (35, 36, 48). Briefly, the tion; adenosine 58-triphosphate-sensitive potassium chan- left cheek pouch was spread gently over a small plastic nels; adenylate cyclase; cheek pouch; hamsters baseplate, and an incision was made in the skin to expose the cheek pouch membrane. An upper chamber was positioned over the baseplate and was used to maintain the suffusion fluid. This arrangement forms a triple-layered complex: the CIGARETTE SMOKING in human subjects produces diffuse baseplate, the upper chamber, and the cheek pouch mem- vascular injury in many organ systems (8, 16, 27, 51). brane exposed between these two plates (35, 36, 48). Although the morphological and functional effects of After these initial procedures, the hamster was transferred cigarette smoke and/or cigarette smoke extract on to a heated microscope stage. Body temperature was moni- isolated blood vessels and endothelial cells in vitro have tored and maintained constant via a feedback controller and been examined, the pathogenesis of cigarette smoking- heating pad. The cheek pouch chamber was connected to a induced vascular damage in vivo is still unclear. Morpho- reservoir that allowed continuous suffusion of the cheek logical abnormalities, i.e., endothelial cell swelling, pouch microcirculation with warm (36–38°C) bicarbonated buffer (pH 7.40) bubbled continuously with 95% N2-5% CO2. extensive subendothelial edema, and subendothelial The chamber was also connected via a three-way valve to an blebs, and an increased number of subendothelial infusion pump that allowed for the controlled administration macrophages in the arterial wall have been observed in of cigarette smoke extract and vasoactive agonists. human subjects exposed to cigarette smoke for pro- The cheek pouch microcirculation was epi-illuminated longed periods of time (2, 4, 32). Functionally, exposure with a fiber-optic light source and viewed through an Olym- of animals and endothelial cell monolayers to cigarette pus microscope. The image of the cheek pouch microcircula- smoke has been shown to increase permeability to tion was projected through the microscope and into a closed- plasma proteins (3, 20, 23, 32) and to decrease nitric circuit television system, which consists of television camera oxide synthase-mediated relaxation of blood vessels (Panasonic WV-1500), monitor (Panasonic TR-124 MA), and from human subjects (17, 18). In addition, recent videotape recorder (Panasonic AG-1230). The inner diameter of cheek pouch arterioles (2nd-order arterioles) was measured studies from our laboratory suggest that cigarette on-line by using a video image-shearing monitor (model 908, smoke extract potentiates agonist-induced increases in Instrumentation for Physiology and Medicine, San Diego, microvascular permeability (36) and impairs nitric CA). oxide synthase-mediated dilatation of resistance arteri- Agonists were mixed in the bicarbonate buffer and then oles in vivo (48). superfused over the cheek pouch microcirculation. Applica-

1996 0161-7567/96 $5.00 Copyright r 1996 the American Physiological Society CIGARETTE SMOKING AND ARTERIOLAR REACTIVITY 1997 tion of vehicle did not affect vessel diameter, and application oles by 23 and 44%, respectively, before and by 20 and 41%, of agonists was randomized. The diameter of cheek pouch respectively, after application of freshly made cigarette smoke arterioles was measured immediately before application of extract. Isoproterenol (1.0 and 10 µM) dilated arterioles by 16 agonists and every 1 min during a 5-min application period. and 25%, respectively, before and by 12 and 21%, respectively, Steady-state responses to agonists were reached within 2 min after application of freshly made cigarette smoke extract. after the application was started, and the diameter of arteri- Forskolin (0.1 and 1.0 µM) dilated arterioles by 13 and 28%, oles returned to control within 3 min after application of respectively, before and by 13 and 27%, respectively, after agonist was stopped. This time course was similar in all application of freshly made cigarette smoke extract. The groups of hamsters. We used steady-state responses of arteri- conclusions drawn from these findings of studies using freshly oles to the agonists to compare the effects of the various made cigarette smoke extract are similar to those drawn from concentrations of cigarette smoke extract on vascular reactiv- studies using frozen and thawed cigarette smoke extract ity. We examined responses of second-order arterioles to (0.1%). consecutive application of different doses of the same agonist, In a second group of hamsters (n 5 8), a similar protocol and application of different agonists was separated by a was followed with the exception that we examined the effect period of 15 min. We chose to examine reactivity of second- of 0.5% cigarette smoke extract. Thus we initially examined order arterioles because they are important resistance ves- responses of cheek pouch arterioles to activation of ATP-sensi- sels in the hamster cheek pouch (6, 7, 25), and thus changes in tive potassium channels by using aprikalim (0.1 and 1.0 µM) diameter of these vessels directly regulates tissue perfusion. and cromakalim (0.1 and 1.0 µM) and to activation of adenyl- We examined reactivity of one arteriole per hamster. In ate cyclase by using isoproterenol (1.0 and 10 µM) and forskolin preliminary experiments, we determined that repeated appli- (0.1 and 1.0 µM). Then, we suffused cigarette smoke extract cations of all agonists onto cheek pouch arterioles produced (0.5%) over the cheek pouch microcirculation. Thirty minutes reproducible vasodilatation (unpublished observations). after starting a continuous superfusion of cigarette smoke Preparation of cigarette smoke extract. A water-soluble extract, we again examined responses of arterioles to aprika- extract of cigarette smoke (vapor phase) was prepared by lim, cromakalim, isoproterenol, and forskolin. The concentra- using methods previously (19, 20, 36, 48) described. Briefly, tions of agonists used in these studies were based on previous smoke from eight reference cigarettes without filters (3A1, studies (34) that indicated that these concentrations pro- Tobacco and Health Research Institute, University of Ken- duced marked dilatation of cheek pouch resistance arterioles. tucky, Lexington, KY) was collected with a modified 10-ml In a third group of hamsters (n 5 8), a similar protocol was polypropylene syringe-driven apparatus. The smoke from followed with the exception that we examined the effect of each cigarette was bubbled slowly through 100 ml of phos- 1.0% cigarette smoke extract. Thus we initially examined phate-buffered saline (pH 7.4). The resulting suspension was responses of cheek pouch arterioles to activation of ATP- filtered through a 0.20-µm filter (Acrodisc) to remove particu- sensitive potassium channels by using aprikalim (0.1 and 1.0 late material and bacteria and was stored in aliquots at µM) and cromakalim (0.1 and 1.0 µM) and to activation of 270°C until use. On the day of the experiment, one aliquot of adenylate cyclase by using isoproterenol (1.0 and 10 µM) and the stock solution (considered 100% cigarette smoke extract) forskolin (0.1 and 1.0 µM). Then, we suffused cigarette smoke was thawed and diluted in buffer to the appropriate concentra- extract (1.0%) over the cheek pouch microcirculation. Thirty tions (0.1, 0.5, and 1.0%). In one study, we determined minutes after starting a continuous superfusion of cigarette whether freshly made vs. frozen cigarette smoke extract smoke extract, we again examined responses of arterioles to affected responses of arterioles to the agonists. It is difficult to aprikalim, cromakalim, isoproterenol, and forskolin. precisely determine the concentration of cigarette smoke In a fourth group of hamsters (n 5 5), a similar protocol products in blood and tissue in habitual smokers at rest and was followed with the exception that we examined the effect during cigarette smoking. The concentrations of cigarette of vehicle on reactivity of arterioles to the agonists. Thus we smoke extract that we chose in the present study are based on initially examined responses of cheek pouch arterioles to previous studies from our laboratory (36, 48) and studies from activation of ATP-sensitive potassium channels by using other investigators (19, 20, 41) that postulate that concentra- aprikalim (0.1 and 1.0 µM) and cromakalim (0.1 and 1.0 µM) tions of cigarette smoke products in tissue and/or blood in and to activation of adenylate cyclase by using isoproterenol habitual smokers are within the range of concentrations used (1.0 and 10 µM) and forskolin (0.1 and 1.0 µM). Then, we in the present study. suffused vehicle (bicarbonated buffer) over the cheek pouch Experimental protocol. The cheek pouch microcirculation microcirculation. Thirty minutes after starting a continuous was superfused for 30 min before responses of arterioles to superfusion of vehicle, we again examined responses of arte- the agonists were tested. In the first group of hamsters (n 5 rioles to aprikalim, cromakalim, isoproterenol, and forskolin. 7), we initially examined responses of cheek pouch arterioles Drugs. Aprikalim was a gift from Rhone-Poulenc Rorer. to activation of ATP-sensitive potassium channels by using Aprikalim was dissolved in dimethylsulfoxide (DMSO) to aprikalim (0.1 and 1.0 µM) and cromakalim (0.1 and 1.0 µM) make a stock solution of 1.0 mM. Dilutions from the stock and to activation of adenylate cyclase by using isoproterenol were made in saline on the day of the experiment. Cromaka- (1.0 and 10 µM) and forskolin (0.1 and 1.0 µM). Then, we lim was a gift from SmithKline Beecham Pharmaceutical. suffused cigarette smoke extract (0.1%) over the cheek pouch Cromakalim was dissolved in ethanol to make a stock solu- microcirculation. Thirty minutes after starting a continuous tion of 1.0 mM. Dilutions from the stock were made in saline superfusion of cigarette smoke extract, we again examined on the day of the experiment. Isoproterenol was purchased responses of arterioles to aprikalim, cromakalim, isoproter- from Sanofi Winthrop Pharmaceuticals and mixed in saline enol, and forskolin. In one study, we were able to determine on the day of the experiment. Forskolin was purchased from whether freshly made cigarette smoke extract produced Calbiochem. Forskolin was dissolved in DMSO to make a similar effects on reactivity of arterioles as did frozen ciga- stock solution of 0.1 mM. Dilutions from the stock were made rette smoke extract. Aprikalim (0.1 and 1.0 µM) dilated in saline on the day of the experiment. arterioles by 21 and 36%, respectively, before and by 20 and Statistical analysis. Repeated-measures analysis of vari- 34%, respectively, after application of freshly made cigarette ance with Student-Newman-Keuls test was used to compare smoke extract. Cromakalim (0.1 and 1.0 µM) dilated arteri- responses of cheek pouch arterioles with the agonists before 1998 CIGARETTE SMOKING AND ARTERIOLAR REACTIVITY and after exposure to a specific concentration of cigarette nels by using aprikalim and cromakalim produced smoke extract. A P value of ,0.05 was considered to be dose-related dilatation of cheek pouch arterioles (Fig. significant. Data are expressed as means 6 SE. 1). In addition, activation of adenylate cyclase by using RESULTS isoproterenol and forskolin produced dose-related dilatation of cheek pouch arterioles under control condi- Control conditions. Under control conditions (before tions (Fig. 1; P , 0.05 vs. baseline diameter). superfusion with vehicle), activation of ATP-sensitive Topical application of 0.1% cigarette smoke extract potassium channels by using aprikalim and cromaka- did not alter baseline diameter of cheek pouch arteri- lim produced dose-related dilatation of cheek pouch oles. Diameter of cheek pouch arterioles was 50 6 2 arterioles. Aprikalim (0.1 and 1.0 µM) dilated arterioles (SE) µm before application of cigarette smoke extract by 13 6 1 and 21 6 2%, respectively. Cromakalim (0.1 and 48 6 2 µm after application of cigarette smoke and 1.0 µM) dilated arterioles by 13 6 3 and 22 6 3%, extract (P . 0.05). Furthermore, application of 0.1% respectively. In addition, activation of adenylate cy- cigarette smoke extract did not alter dilatation of cheek clase by using isoproterenol and forskolin produced pouch arterioles in response to activation of ATP- dose-related dilatation of cheek pouch arterioles before sensitive potassium channels and activation of adenyl- superfusion with vehicle. Isoproterenol (1.0 and 10 µM) ate cyclase (P . 0.05; Fig. 1). dilated arterioles by 10 6 1 and 18 6 1%, respectively. Effect of 0.5% cigarette smoke extract. Under control Forskolin (0.1 and 1.0 µM) dilated arterioles by 11 6 1 conditions, activation of ATP-sensitive potassium chan- and 17 6 1%, respectively. nels by using aprikalim and cromakalim and activation Topical application of vehicle did not alter baseline of adenylate cyclase by using isoproterenol and forsko- diameter of cheek pouch arterioles. Diameter of cheek lin produced dose-related dilatation of cheek pouch pouch arterioles was 55 6 2 (SE) µm before application arterioles (Fig. 2; P , 0.05 vs. baseline diameter). of vehicle and 54 6 4 µm after application of vehicle Topical application of 0.5% cigarette smoke extract (P . 0.05). Furthermore, application of vehicle did not did not alter baseline diameter of cheek pouch arteri- alter dilatation of cheek pouch arterioles in response to oles. Diameter of cheek pouch arterioles was 51 6 2 activation of ATP-sensitive potassium channels and (SE) µm before application of cigarette smoke extract activation of adenylate cyclase (P . 0.05 vs. response and 52 6 2 µm after application of cigarette smoke before application of vehicle). Aprikalim (0.1 and 1.0 extract (P . 0.05). µM) dilated arterioles by 12 6 2 and 18 6 2%, respec- In contrast to the effect observed with 0.1% cigarette tively. Cromakalim (0.1 and 1.0 µM) dilated arterioles smoke extract, application of 0.5% cigarette smoke by 12 6 2 and 20 6 3%, respectively. Isoproterenol (1.0 impaired dilatation of cheek pouch arterioles in re- and 10 µM) dilated arterioles by 9 6 1 and 16 6 2%, sponse to aprikalim, the high dose (1.0 µM) of cromaka- respectively. Forskolin (0.1 and 1.0 µM) dilated arteri- lim, the high dose (10 µM) of isoproterenol, and the oles by 11 6 1 and 17 6 1%, respectively. Thus repeated high dose (1.0 µM) of forskolin (P , 0.05; Fig. 2). application of agonists yields reproducible responses. Effect of 1.0% cigarette smoke extract. Under control Effect of 0.1% cigarette smoke extract. Under control conditions, activation of ATP-sensitive potassium chan- conditions, activation of ATP-sensitive potassium channels by using aprikalim and cromakalim and activation

Fig. 1. Response of cheek pouch arterioles to aprikalim (A), cromakalim (B), isoproterenol (C), and forskolin (D) before (open bars) and after (solid bars) exposure to cigarette smoke extract (0.1%). Values are means 6 SE; n 5 7. CIGARETTE SMOKING AND ARTERIOLAR REACTIVITY 1999

Fig. 2. Response of cheek pouch arterioles to aprikalim (A), cromakalim (B), isoproterenol (C), and forskolin (D) before (open bars) and after (solid bars) exposure to cigarette smoke extract (0.5%). Values are means 6 SE; n 5 8. *P , 0.05 vs. response before application of cigarette smoke extract.

of adenylate cyclase by using isoproterenol and forsko- DISCUSSION lin produced dose-related dilatation of cheek pouch arterioles (Fig. 3; P , 0.05 vs. baseline diameter). The present study is the ﬁrst to examine the effects of Topical application of 1.0% cigarette smoke extract cigarette smoke extract dilatation of resistance arteri- did not alter baseline diameter of cheek pouch arterioles in vivo in response to activation of ATP-sensitive oles. Diameter of cheek pouch arterioles was 49 6 2 potassium channels and adenylate cyclase. There are (SE) µm before application of cigarette smoke extract two new ﬁndings of this study. First, a low concentra- and 50 6 1 µm after application of cigarette smoke tion of cigarette smoke extract (0.1%) did not alter extract (P . 0.05). dilatation of cheek pouch arterioles in response to In addition, application of 1.0% cigarette smoke activation of ATP-sensitive potassium channels and impaired dilatation of cheek pouch arterioles in re- adenylate cyclase. Second, higher concentrations of sponse to activation of ATP-sensitive potassium channels cigarette smoke extract (0.5 and 1.0%) impaired dilata- and activation of adenylate cyclase (P , 0.05; Fig. 3). tion of cheek pouch in response to activation of ATP-

Fig. 3. Response of cheek pouch arterioles to aprikalim (A), cromakalim (B), isoproterenol (C), and forskolin (D) before (open bars) and after (solid bars) exposure to cigarette smoke extract (1.0%). Values are means 6 SE; n 5 7. *P , 0.05 vs. response before application of cigarette smoke extract. 2000 CIGARETTE SMOKING AND ARTERIOLAR REACTIVITY sensitive potassium channels and adenylate cyclase. ATP-sensitive potassium channels (21). Thus it ap- The findings of the present study suggest that exposure pears that cigarette smoke extract impairs reactivity of of resistance arterioles to components of cigarette arterioles in response to activation of ATP-sensitive smoke alters important cellular vasodilator pathways. potassium channels and in response to activation of Consideration of methods. Many previous studies adenylate cyclase. have examined the effects of activation of ATP-sensitive We examined the effects of various concentrations of potassium channels on large peripheral and cerebral cigarette smoke extract on baseline diameter of cheek blood vessels in vitro (26, 28, 33, 38–40, 43) and in vivo pouch arterioles. We found that cigarette smoke extract (9, 11, 52, 55). In addition, Mayhan (34) and other did not alter baseline diameter of cheek pouch arteri- investigators (52) have examined the effects of activa- oles. Two other studies examined the effect of cigarette tion of ATP-sensitive potassium channels on peripheral smoke extract on diameter of arteries in vitro. Holden resistance arterioles. In general, activation of ATP-sensi- et al. (19) found that cigarette smoke extract produced tive potassium channels with cromakalim, pinacidil, biphasic responses of intrapulmonary arteries. Relax- nicorandil, and aprikalim produces marked relaxation ation of intrapulmonary arteries occurred at low concen- and/or dilatation of arteries and arterioles. Relaxation trations of cigarette smoke extract (0.001–0.01%), and and/or dilatation of arteries and arterioles in response constriction of arteries occurred at higher concentra- to activation of ATP-sensitive potassium channels in tions (0.1–1.0%) of cigarette smoke extract. In other vitro and in vivo appears to be specific because glibencl- studies, investigators found that the gaseous phase of amide inhibits responses to activation of ATP-sensitive cigarette smoke produced relaxation of coronary arter- potassium channels (9, 33, 34, 38, 44, 50). Furthermore, ies in vitro (12). The mechanism of relaxation appeared Mayhan has shown that dilatation of cheek pouch to be related to the activation of guanylate cyclase, arterioles in response to activation of ATP-sensitive presumably by production of nitric oxide. The results of potassium channels with aprikalim and cromakalim is the present study differ from previous studies (12, 19). not related to the synthesis/release of nitric oxide or a We did not find a direct effect of cigarette smoke extract nitric oxide-containing compound. The results of the on baseline diameter of cheek pouch arterioles. There present study support findings of previous studies (9, are, however, significant differences between the pres- 11, 34, 55) that suggest an important role for activation ent study and previous studies (12, 19). of ATP-sensitive potassium channels in dilatation of We examined responses of small arterioles contained blood vessels in vivo. In addition, the results of the within the hamster cheek pouch in vivo, and the present experiments extend findings of previous stud- previous studies examined responses of large bovine ies by examining responses of resistance arterioles in coronary and pulmonary arteries in vitro (12, 19). Thus vivo to activation of ATP-sensitive potassium channels the discrepancy between the present study and previ- during exposure to cigarette smoke extract. ous studies may be related to size of blood vessels Relaxation of vascular smooth muscle also can occur studied, the tissue from which the blood vessels were via activation of adenylate cyclase via stimulation of derived, and/or a species difference in response to b-adrenergic receptors and/or direct activation of ad- cigarette smoke extract. We examined the effects of enylate cyclase by forskolin (15, 49). Although many topical application of cigarette smoke extract, made by previous studies have examined responses of arteries bubbling cigarette smoke through a buffer solution, on and arterioles to isoproterenol and forskolin, a recent arteriolar reactivity. There may be several limitations study (21) has examined responses of hamster cheek of this methodology. First, during cigarette smoking the pouch arterioles to isoproterenol and forskolin. This products of inhaled smoke that interact with the endo- previous study (21) found that isoproterenol and forsko- thelium and/or vascular smooth muscle are filtered by lin produced pronounced dose-related dilatation of cheek the lungs before entering the blood. In the present pouch arterioles. Surprisingly, dilatation of cheek pouch study, we were not able to examine the effects of inhaled arterioles in response to isoproterenol was partially smoke vs. smoke extract on reactivity of resistance inhibited by application of glibenclamide (21). This arterioles, and thus we cannot exclude the possibility finding suggests a role for activation of ATP-sensitive that reactivity of arterioles may be affected differently potassium channels in isoproterenol-induced dilatation by inhaled smoke. Second, there may be complex inter- of cheek pouch arterioles. Dilatation of cheek pouch actions between the components of blood and/or vascu- arterioles in response to forskolin, however, was not lar wall and the products produced during cigarette altered by treatment with glibenclamide. Although we smoking that may attenuate or exacerbate the effects of did not examine a specific role for activation of ATP- cigarette smoke. In the present study we were not able sensitive potassium channels in dilatation of cheek to examine these potential interactions and thus can- pouch arterioles in response to isoproterenol in the not draw conclusions with regard to the role of these present study, it is possible that inhibition of isoproter- potentially important interactions to impaired reactiv- enol-induced vasodilatation by cigarette smoke is par- ity of arterioles. Although there may be limitations to tially related to an effect on ATP-sensitive potassium the present study, we suggest that our findings provide channels. However, we also observed that cigarette new insights into the potential effects of cigarette smoke extract (0.5 and 1.0%) inhibited vasodilatation smoking on important cellular vasodilator pathways. in response to forskolin, which presumably dilates Consideration of previous studies. No studies have cheek pouch arterioles independent of the activation of examined the effect of cigarette smoke extract on CIGARETTE SMOKING AND ARTERIOLAR REACTIVITY 2001 dilator responses of arterioles to activation of ATP- rette smoke extract-induced vascular injury. Nicotine, sensitive potassium channels and activation of adenyl- a major component of cigarette smoke, has been shown ate cyclase. For the most part, previous studies have to increase the frequency of endothelial cell death and concentrated on an examination of the effects of ciga- thus could be considered a mediator of vascular injury rette smoking on endothelium-dependent, i.e., nitric (29, 31). However, several studies failed to demonstrate oxide synthase-mediated, responses of peripheral blood consistent and significant functional and/or pathologi- vessels. Holden et al. (19) found that reactivity of cal alterations in vascular wall properties during expo- intrapulmonary arteries in vitro in response to ciga- sure to nicotine (1, 24, 42), and no studies have rette smoke extract (0.001–0.01%) could be inhibited examined the effects of nicotine on reactivity of resis- by removal of the endothelium. Thus it appears that tance arterioles in response to activation of ATP- the endothelium may participate in the vascular re- sensitive potassium channels and adenylate cyclase. sponse of intrapulmonary arteries during exposure to Thus we cannot determine whether the impaired reac- cigarette smoke. These investigators (19), however, did tivity of arterioles observed in the present study during not examine whether cigarette smoke extract altered exposure to cigarette smoke extract is related to eleva- responses of intrapulmonary arteries to nitric oxide tions in nicotine. synthase-mediated agonists. Other studies using hu- Cigarette smoke extract contains many toxic sub- man subjects have shown that cigarette smoking im- stances, in addition to nicotine, that may contribute to pairs nitric oxide synthase-mediated relaxation of large impaired vascular reactivity. Acrolein and acetalde- peripheral blood vessels (17, 18). However, other stud- hyde are highly soluble components of cigarette smoke ies using human subjects have shown either no alter- extract and have been shown to cause irritation of ation in nitric oxide synthase-mediated vasorelaxation airway and ocular mucosa in humans, impair mucocili- (22) or increased sensitivity of vascular smooth to nitric ary clearance in the upper airway, produce pulmonary oxide synthase-mediated vasodilators in smokers (47). edema in animals, and damage epithelial cells (13, 14, Rubinstein et al. (48) have previously examined nitric 30, 45). In addition, Holden et al. (20) have shown that oxide synthase-mediated dilatation of cheek pouch cigarette smoke extract increases albumin flux across arterioles after exposure to cigarette smoke extract cultured porcine pulmonary endothelium, and this (1.0%). They found that dilatation of cheek pouch effect was predominantly due to the vapor phase of arterioles in vivo in response to acetylcholine was cigarette smoke. Furthermore, cigarette smoke extract profoundly impaired after exposure to cigarette smoke contains considerable amounts of oxygen radicals such extract (48). Dilatation of arterioles in response to as superoxide anion and hydrogen peroxide that may nitroglycerin, however, was not altered by exposure to contribute to vascular dysfunction observed in ciga- cigarette smoke extract, suggesting that the effects of rette smokers (41). Thus, although the precise contribu- cigarette smoke extract are specific for nitric oxide tion of the various components of cigarette smoke synthase-mediated responses. Although there appears extract to vascular reactivity cannot be determined to be some discrepancy in results concerning the effects from the present study, we speculate that it involves of cigarette smoking on nitric oxide synthase-mediated complex interactions between various components of vasoreactivity, the results from the previous study of cigarette smoke extract. Rubinstein et al. (48) and studies by other investigators Summary and implications. We found that cigarette (17, 18) suggest an important effect of cigarette smok- smoke extract produced a concentration-related impair- ing on nitric oxide synthase-mediated responses of ment in dilatation of resistance arterioles in response blood vessels. to activation of ATP-sensitive potassium channels and The present study is the first to examine the effects of activation of adenylate cyclase. We suggest that these cigarette smoke extract on other important cellular findings may have important implications for chronic dilator pathways, i.e., activation of ATP-sensitive potas- tobacco smokers. Studies have shown that cigarette sium channels and activation of adenylate cyclase. We smoking contributes to the development of many cardio- found that low concentrations of cigarette smoke ex- vascular abnormalities, including tissue ischemia, ath- tract do not alter dilatation of arterioles in response to erosclerosis, stroke, and coronary artery disease (29, activation of ATP-sensitive potassium channels and 37, 53, 54, 56). Several cellular mechanisms, including activation of adenylate cyclase. Modest-to-moderate synthesis and/or release of nitric oxide, synthesis and/ concentrations of cigarette smoke extract, however, or release of endothelium-derived hyperpolarizing fac- produced profound impairment in dilatation of cheek tor to activate ATP-sensitive potassium channels, and/or pouch arterioles in response to activation of ATP- synthesis and/or release of prostaglandins to activate sensitive potassium channels and adenylate cyclase. of adenylate cyclase (5, 10, 46), attempt to maintain Thus findings from the present series of experiments adequate tissue blood flow during increases in demand. extend previous findings (17, 18, 48) by examining the Although it is clear that cigarette smoking may alter effects of cigarette smoke extract on important cellular the nitric oxide synthase-mediated pathway, the find- dilator pathways of resistance arterioles. ings of the present study suggest that cigarette smok- Mechanism of cigarette smoke extract-induced altering also may contribute to vascular dysfunction, in ations in vascular reactivity. The chemical composition part, by altering the ability of resistance arterioles to of cigarette smoke is complex, and it is difficult to dilate in response to activation of ATP-sensitive potas- determine which compound(s) may be involved in ciga- sium channels and activation of adenylate cyclase. 2002 CIGARETTE SMOKING AND ARTERIOLAR REACTIVITY

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