TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
l&t,
19 I-
199 ( i 990)
Endothelium-Dependent Effects of Cigarette Smoke Components on Tone of Porcine lntrapulmonary Arteries in Vitro’ WILLIAM
E. HOLDEN,SHIRLEYS.JCISHIYAMA,STEVEN
P. DONG,ANDMOLLYL.
OSBORNE
Department of Medicine, Portland VA Medical Center, and Oregon Health Sciences University, Portland, Oregon 97207
Received May 5, 1989; accepted March 1,199t.I Endothelium-Dependent Effects of Cigarette Smoke Components on Tone of Porcine Intrapulmonary Arteries in Vitro. HOLDEN, W. E., KISHIYAMA, S. S., DONG, S. P., AND OSBORNE, M. L. ( 1990). Toxicol. Appl. Pharmacol. 104, 19 l-l 99. The acute effects of cigarette smoking on the pulmonary vasculature are poorly understood-both vasodilatory and vasoconstrictive effects have been described. To investigate the mechanisms involved, strips of pig intrapulmonary arteries with and without intact endothelium were exposed to an extract of cigarette smoke made by bubbling smoke through phosphate-buffered saline. After contraction with norepinephrine (2.5 X lo-’ M), smoke extract (concentration range 0.001 to 0.5%) caused a biphasic response in strips with intact endothelium-relaxation at lower concentrations and contraction at higher concentrations. Both relaxation and contraction responses were absent in strips without endothelium. Blockade of muscarinic, j3 adrenergic, serotonergic, and histamine type 1 and 2 receptors did not alter the effects. Indomethacin (5 X 10e6 M) or acetylsalicylic acid (IO-“ M) blocked the relaxation but not contraction effects of smoke extract, suggesting that relaxation was due to cyclooxygenase products of arachidonic acid. Nicotine caused endotheliumdependent contraction of intrapulmonary arteries and the contractile effects of both nicotine and smoke extract were blocked by hexamethonium ( 10m6M). However, the contractile effects of cigarette smoke components are more potent than those of nicotine. These findings help explain previously described acute effectsof smoking on the pulmonary vasculature and provide insight into the mechanisms involved. 0 1990 Academic p, hc.
The acute effects of cigarette smoking on the pulmonary vascular bed are poorly understood. Samenek and Aviado (1966) found an acute increase in pulmonary arterial pressure during exposure to cigarette smoke in dogs, but the effect was poorly reproducible in individual animals. In isolated pig lungs, cigarette smoke causes acute vasodilation when baseline vascular pressures are high, but has no effect when baseline pressure is low (Gilman et al., 198 1). In contrast, an infusion of nicotine (Larson et al., 1965) or an extract of cigarette smoke (Kitamura, 1987) causes an in’ Presented in part at the American Thoracic Society Annual Meeting, Cincinnati, OH, May 14-l 7, 1989.
crease in pulmonary vascular pressures and resistance. In this study the effects of an extract of cigarette smoke on the tone of porcine intrapulmonary arteries were analyzed in vitro in order to determine if the endothelium plays a role in the response of lung vessels to exposure to cigarette smoke, as is the case for other agents which influence vascular tone (Furchgott and Zawadski, 1980). Endothelium-dependent contractions of canine cerebral vessels in response to nicotine have recently been reported (Shirahase et al., 1988). Since components of cigarette smoke (e.g., nicotine) are rapidly taken up into the blood during smoking (Koch et al., 1980) and gas ex191
004 1008x/90
$3.00
Copyright 0 1990 by Academic Ress, Inc. All tights OfrEprodUaiOn in any fOIm Rserved.
192
HOLDEN
change occurs in intrapulmonary arteries as large as 3 mm id. (Sobel et al., 1963), it is reasonable to suspect that cigarette smoke components may directly affect the endothelium of the lung vasculature, including the smaller pulmonary arteries. The results show that cigarette smoke components have endothelium-dependent effects which vary with exposure concentration. At lower concentrations, smoke components cause endothehumdependent relaxation which is mediated through release of cyclooxygenase products. At higher concentrations, endothelium-dependent contractions were found, in part related to the effects of nicotine. These findings suggest that the endothelium may be involved in the acute effects of cigarette smoking on the pulmonary vasculature. METHODS An extract of cigarette smoke in phosphate-buffered saline (PBS) was prepared fresh for each day’s experiment and used within 4 hr of preparation. Smoke from research cigarettes (Kentucky 2R1, University of Kentucky) was bubbled through PBS ( 1cigarette/ml PBS) using a simple three-way stopcock with a cigarette inserted into a holder attached to one arm and the other two arms going to a 35-ml syringe and a 5-ml glass pipet with 1 ml PBS. The cigarettes were smoked (1 puff/min) using standardized puff size and draw time of 35 ml and 2 set, respectively (Guerin, 1980). All reagents were obtained from Sigma Chemical Co. (St. Louis, MO) except as indicated. Left lower lobes of lungs removed from pigs at a local slaughterhouse were transported to the laboratory in chilled Krebs-Ringer-bicarbonate buffer (KRBB) (NaCl. 118 mM; KCI, 4.8 mM; CaC12, 2.5 mM; MgSO,,, 1.2 mM; KHzPO,, 1.2 mM; NaHC02, 24 mM; glucose, 11 mM). The lobar pulmonary artery was identified and dissected down to branches l-3 mm in diameter. Excess adventitial tissue was trimmed and two transverse strips, approximately .2-mm wide, were cut from each artery and mounted, with care to avoid damage to the endothelium, in separate 50-ml baths between an anchor point and a force transducer (Grass, FT.03) for measurement of isometric contractions. The baths contained KRBB at 37.5’C bubbled continuously with a gas mixture containing 5% COZ in air (02 = 19.95%), pH 7.35-7.45. Strip tension was set at 2 g and continuously recorded on a polygraph (Grass, Model 7). During equilibration, the tension gradually decreased to near I .2 g where experiments were performed.
ET AL. After a 2-hr equilibration period the strips were stimulated with norepinephrine, 2.5 X 1O-‘M, a concentration which causes IO- 15% maximal contractions in similar vessels with and without intact endothelium (Holden and McCall, 1984). All concentrations refer to final bath concentration. The presence of endothelium was verified by serial addition of acetylcholine in increasing concentrations ( 1Oe9to IOe6M) and demonstration of endotheliurndependent relaxation (Furchgott and Zawadski. 1980). Both strips were then removed from the baths and the luminal surface of one gently rubbed with a scalpel using short strokes covering the total intimal surface three times in each direction lengthwise and then replaced in the baths at the same tension found at time of removal. The presence (or absence) of endotheliumdependent relaxation to acetylcholine was again tested (as above). Similar rubbing of the adventitial side of the intact endothelium strip did not affect endothelial responses to acetylcholine. In previous studies the contractile effects of norepinephrine were not changed by removal of endothelium from similar strips of porcine pulmonary arteries in vitro (Holden and McCall, 1984). To assesseffectsof endothelium removal on relaxation of the strips, the effects of adenosine or nitroglycerin on norepinephrine-induced contractions were tested in six strips from three animals. Adenosine was exposed to four of the strips and nitroglycerin to two strips before and after removal of the endothelium. Relaxation effects of adenosine and nitroglycerin are thought to be independent of the endothelium (Rapoportand Murad, 1983; Furchgott, 1983). The concentration of adenosine or nitroglycerin required to cause half-maximal relaxation (ED50) in unrubbed strips was compared with the ED50 in strips without endothelium. In 14 pairs of strips from individual animals, effects of cigarette smoke extract and vehicle (PBS) on tension of norepinephrine-stimulated contractions were tested over a concentration range of 0.00 1 to 0.5%. In four of these pairs, effects of cigarette smoke extract were repeated in the presence (simultaneously) ofatropine (3.5 X IO-’ M), cimetidine (5 X lo-‘M), diphenhydramine (5 X IO-'M), methysergide (5 X lo-’ M), and propranolol(6.5 X 10m6 M) in the muscle baths. The doses used blocked contractions (histamine, 10e6 M; acetylcholine, 1O-5 M; serotonin, 10m6M) or relaxations (acetylcholine, IO-’ M; isoproterenol, 10e6 M) in strips of pulmonary artery with intact endothelium. The effects of cigarette smoke on tone of pulmonary artery strips with intact endothelium were also tested before and during exposure to indomethacin (5 X 10m6M) or acetylsahcylic acid ( 10m4M) to block cyclooxygenase activity. Strips from eight animals were used for indomethacin and six animals for acetylsalicylic acid studies. The concentrations of indomethacin and acetylsalicylic acid used blocked cyclooxygenase activity in cultured porcine pulmonary artery endothelial cells as shown by
LUNG
VASCULAR
EFFECTS OF CIGARETTE
(a) bioassay (contractions of rat stomach strip in vitro)prostanoid bioactivity in supematants of cultures exposed to arachidonic acid ( 1Oe6 M) was abolished by either drug; and (b) radioimmunoassay of selected prostanoids-unstimulated release of prostaglandin Fz, (80% decrease), 6-keto prostaglandin F,, (66% decrease), and prostaglandin E2 (85% decrease) was decreased by indomethacin ( 10m6M). In strips from an additional six animals, cigarette smoke effects were tested before and during exposure to hexamethonium ( 10e6 M) to block ganglia and nicotinic receptors (Young et al., 1988). Methods similar to those reported by Hariharan et al. ( 1988) were used to measure the nicotine content of five, separately prepared cigarette smoke extracts. A Hewlett Packard HPLC with C-18, reverse-phase column was used with a mobile phase of citric acid-phosphate buffer (each 0.03 M) and 16% acetonitrile with an added ionsplitting agent, sodium pentanesulfonate (200 mg/liter), and 2-phenylimidazole as internal standard. Ultraviolet detection was done over 200 to 360 nm. Finally, using the measured concentrations (by HPLC) of nicotine in our smoke extracts as a guide, we prepared similar solutions of nicotine in PBS and tested their effects on pulmonary artery tone. In pulmonary artery strips from 12 animals, the effects of nicotine on muscle tone during norepinephrine contractions were analyzed. Paired strips with endothelium removed were studied from 5 of the animals. All data are expressed as means f SE. ED50 for relaxation effects of adenosine and nitroglycerin were determined using a probit-log cumulative dose analysis (Colquhoun, 1971). We analyzed the effects of cigarette smoke extract on strips with and without endothelium using two-way analysis of variance with replication (Zar, 1974) where the variables examined were presence (or absence) of endothelium and dose of cigarette smoke extract, followed by Newman-Keuls test for significance of differences between positive and negative endothelium conditions. Paired t tests were used for comparison of blocking experiments and ED50 values of relaxation agonists. A p value < 0.05 was considered significant.
RESULTS Efects of Endothelium ation Ability
’
* Removal on Relax-
In previous studies, removal of endothelium from similar porcine pulmonary arteries did not alter the contractile effects of norepinephrine (Holden and McCall, 1984). In the studies herein, the ED50 for relaxation effects of adenosine was similar in strips with and without endothelium (4.7 f 0.8 X lop5 M vs
Aceiylcholine
193
SMOKE Smoke
Extract
j50mg 2 FIG. 1. Effects of cigarette smoke extract (in PBS) on tone of intrapulmonary arteries contracted by norepinephrine (2.5 X lo-’ M, asterisk). Upper panel is a strip with intact endothelium (+endo) and lower panel is a paired strip from the same artery scraped on the intimal surface (-endo). Muscle tension (vertical axis) is recorded continuously over time (horizontal axis). During initial contractions (at left), acetylcholine (IO-* and lo-’ M, graphs are labeled with the negative log of concentration) was added. +Endo, strip relaxed; whereas -endo, strip contracted slightly demonstrating presence and absence of endothelium, respectively. During the next contraction (at right), increasing concentrations of cigarette smoke extract (graphs are labeled with negative log of the percentage extract) were added. At lower concentrations, the strip with intact endothelium relaxed, and at higher concentrations, the strip contracted. Both relaxation and contraction effectswere absent in the -endo strip.
3.8 + 1.6 X lop5 M, n = 4 of each). Also, the ED50 for relaxation effects of nitroglycerin was similar in strips with and without endothelium (5.6 X lo-’ M vs 5.6 X 10M8M, mean of two of each). Eflects of Cigarette Smoke Extract Cigarette smoke extract over the range of 0.00 1 to 0.5% had concentration- and endotheliumdependent effects on intrapulmonary arteries contracted by norepinephrine (2.5 X 10e7 M) (Figs. 1 and 2). The vehicle, (PBS), added at intervals or as a bolus of the total volume used in testing cigarette smoke, had no effects on muscle tension. In 12 of 14
194
HOLDEN Acetylcholine
I
50mg
Smoke Extract
‘min
FIG. 2. Effects of cigarette smoke extract on tone of intrapulmonary arteries contracted by norepinephrine (2.5 X lo-’ M, asterisk). Axis and labels are same as in Fig. 1. Some strips showed only relaxation to cigarette smoke extract (i.e., no contraction at higher concentrations as in Fig. 1). Contraction at top left shows relaxation to acetylchohne (lo-& and IO-’ M), demonstrating intact endothelium. During contraction at top right, smoke extract was added and strip relaxed without contraction at higher concentration of extract. Strip was removed from the bath and the intimal surface scraped to remove endothelium (S). Contraction at lower left demonstrates loss of relaxation to acetylcholine verifying removal of endothelium. Smoke extract was again added in the contraction at lower right and relaxation effects were abolished.
strips with intact endothelium (+endo), smoke extract decreased muscle tension at lower concentrations and increased tension at higher concentrations (Fig. 1). Some strips with intact endothelium (2 of 14 strips) showed only relaxation when exposed to smoke extract (Fig. 2). Both relaxation and contraction effects of the extract were greatly diminished in strips with endothelium removed. When applied to strips not contracted by norepinephrine, only contraction effects of cigarette smoke extract were seen, hence the relaxation effects of smoke extract were seen only in the presence of increased baseline tension. A summary of experiments of the type shown in Fig. 1 using paired strips from 14 animals is shown in Fig. 3.
ET AL.
Cigarette Smoke Efects after Blockade ofSelected Agonists and Antagonists of Vascular Smooth Muscle Tone Blockade of several common agonists or antagonists of smooth muscle tone did not alter the effects of cigarette smoke. In four strips with intact endothelium, blockade of cholinergic (atropine, 3.5 X low7 M), histamine types 1 and 2 (diphenhydramine, 5 X 10e7 M; or cimetidine 5 X 10m7 M), serotonergic (methysergide 5 X lop7 M), and /3-adrenergic (propranolo16.5 X 1O-6 M) receptors did not alter relaxation or contraction effects of the smoke extract (Table 1). These blocking agents were simultaneously present in the muscle baths during addition of the cigarette smoke extract. The cyclooxygenase inhibitors, indomethacin (5 X 1Oe6M) or acetylsalicylic acid ( 1Oe4 M), added to the muscle baths 30 min prior to norepinephrine, did not affect the contractile
40 -I
---*--.
+ END0 END0
T
20 -
0
,001 CIGARETTE
.Ol SMOKE w
1
1
EXTRACT
FIG. 3. Summary of experiments of the type shown in Fig. 1 in 14 pairs of porcine intrapulmonary arteries with (+endo) and without (-endo) endothelium. The horizontal axis is percentage cigarette smoke extract. Both dose and tension changes are cumulative. Zero points represent effect of PBS in the total volume used to deliver smoke extract. +Endo strips relaxed (negative change in tension) at lower extract concentrations and contracted (positive change in tension) at higher concentrations. -Endo strips showed little or no change in tension during the addition of smoke extract. Each point represents the mean + SE. *Significant difference between +endo and -endo groups by analysis of variance.
LUNG VASCULAR
EFFECTS OF CIGARETTE
195
SMOKE
TABLE 1 EFFECYSOFCIGAREITESM~KEEXTRACT.DURINGBLOCKADEOF ANDRELAXATIONAGONISTS
SELEC~ZD~ONTRACTION
Concentration of smoke extract (%)
Control” + Blockers’
0.01
0.05
0.075
0.1
0.15
-30 + 6b -20+7
-58 + 15 -40 + 10
0 -5+-3
+23 + 12 +15-+ 5
+45 + 18 +40+ 13
a Absence of blockers b Change in tension (mg) (+ or - sign indicates contraction or relaxation, respectively). ‘Simultaneous presence of blockers: atropine, 3.5 X lo-’ M; diphenhydramine, 5 X lo-’ M; cimetidine, 5 X lo-’ M; methysergide, 5 X IO-’ M; and propranolo16.5 X 10m6M.
effects of cigarette smoke extract, but the relaxation effect of smoke extract was inhibited (Fig. 4, Table 2). The effect of indomethacin was transient-relaxation effects of cigarette smoke extract reappeared 90 min after indomethacin was washed out of the muscle baths. However, the effects of acetylsalicylic acid were reversible within the time of our experiments (4-6 hr) (Fig. 4).
Are Efects of Cigarette Smoke Extract Due to Nicotine? In order to determine if nicotine might be causing the contractile effects seen at higher concentrations of cigarette smoke extract, the concentration of nicotine was measured in representative extracts using high-perfor-
TABLE 2 5.2
5rl
EFFE~S OF INWMETHACIN OR ACETYLSALICYLIC ACIDONRELAXATION OFPLJLMONARYARTERYSTRIPS BYCIGARETTESMOKEEXTRACT Concentration of smoke extract (%)
I
50mg
3mm
FIG. 4. Example of effect of indomethacin (5 X lO-6 M) on response of porcine intrapulmonary artery with intact endothelium contracted by norepinephrine (2.5 X IO-’ M, asterisk) to an extract of cigarette smoke. Cigarette smoke concentration is expressed as the negative log of the final bath percentage (v/v) of extract. Contraction at left shows dilation response to cigarette smoke extract (0.001%). Then, indomethacin (5 X 10m6M) was added to the bath (at indo 30’) and after 30 min the response to smoke extract was again tested (middle contraction). No relaxation was seen at lower concentrations of smoke extract, but contractions occurred at higher concentration of smoke extract. The bath was cleansed to remove indomethacin and, after 90 min, the effects of smoke extract were again tested (contraction at right) and the relaxation response to cigarette smoke was again found.
Control b
N”
0.01
0.05
9
-27 + 6’ p < 0.05 -6-c3
-55 + 10 D
AND
APPLIED
PHARMACOLOGY
l&t,
19 I-
199 ( i 990)
Endothelium-Dependent Effects of Cigarette Smoke Components on Tone of Porcine lntrapulmonary Arteries in Vitro’ WILLIAM
E. HOLDEN,SHIRLEYS.JCISHIYAMA,STEVEN
P. DONG,ANDMOLLYL.
OSBORNE
Department of Medicine, Portland VA Medical Center, and Oregon Health Sciences University, Portland, Oregon 97207
Received May 5, 1989; accepted March 1,199t.I Endothelium-Dependent Effects of Cigarette Smoke Components on Tone of Porcine Intrapulmonary Arteries in Vitro. HOLDEN, W. E., KISHIYAMA, S. S., DONG, S. P., AND OSBORNE, M. L. ( 1990). Toxicol. Appl. Pharmacol. 104, 19 l-l 99. The acute effects of cigarette smoking on the pulmonary vasculature are poorly understood-both vasodilatory and vasoconstrictive effects have been described. To investigate the mechanisms involved, strips of pig intrapulmonary arteries with and without intact endothelium were exposed to an extract of cigarette smoke made by bubbling smoke through phosphate-buffered saline. After contraction with norepinephrine (2.5 X lo-’ M), smoke extract (concentration range 0.001 to 0.5%) caused a biphasic response in strips with intact endothelium-relaxation at lower concentrations and contraction at higher concentrations. Both relaxation and contraction responses were absent in strips without endothelium. Blockade of muscarinic, j3 adrenergic, serotonergic, and histamine type 1 and 2 receptors did not alter the effects. Indomethacin (5 X 10e6 M) or acetylsalicylic acid (IO-“ M) blocked the relaxation but not contraction effects of smoke extract, suggesting that relaxation was due to cyclooxygenase products of arachidonic acid. Nicotine caused endotheliumdependent contraction of intrapulmonary arteries and the contractile effects of both nicotine and smoke extract were blocked by hexamethonium ( 10m6M). However, the contractile effects of cigarette smoke components are more potent than those of nicotine. These findings help explain previously described acute effectsof smoking on the pulmonary vasculature and provide insight into the mechanisms involved. 0 1990 Academic p, hc.
The acute effects of cigarette smoking on the pulmonary vascular bed are poorly understood. Samenek and Aviado (1966) found an acute increase in pulmonary arterial pressure during exposure to cigarette smoke in dogs, but the effect was poorly reproducible in individual animals. In isolated pig lungs, cigarette smoke causes acute vasodilation when baseline vascular pressures are high, but has no effect when baseline pressure is low (Gilman et al., 198 1). In contrast, an infusion of nicotine (Larson et al., 1965) or an extract of cigarette smoke (Kitamura, 1987) causes an in’ Presented in part at the American Thoracic Society Annual Meeting, Cincinnati, OH, May 14-l 7, 1989.
crease in pulmonary vascular pressures and resistance. In this study the effects of an extract of cigarette smoke on the tone of porcine intrapulmonary arteries were analyzed in vitro in order to determine if the endothelium plays a role in the response of lung vessels to exposure to cigarette smoke, as is the case for other agents which influence vascular tone (Furchgott and Zawadski, 1980). Endothelium-dependent contractions of canine cerebral vessels in response to nicotine have recently been reported (Shirahase et al., 1988). Since components of cigarette smoke (e.g., nicotine) are rapidly taken up into the blood during smoking (Koch et al., 1980) and gas ex191
004 1008x/90
$3.00
Copyright 0 1990 by Academic Ress, Inc. All tights OfrEprodUaiOn in any fOIm Rserved.
192
HOLDEN
change occurs in intrapulmonary arteries as large as 3 mm id. (Sobel et al., 1963), it is reasonable to suspect that cigarette smoke components may directly affect the endothelium of the lung vasculature, including the smaller pulmonary arteries. The results show that cigarette smoke components have endothelium-dependent effects which vary with exposure concentration. At lower concentrations, smoke components cause endothehumdependent relaxation which is mediated through release of cyclooxygenase products. At higher concentrations, endothelium-dependent contractions were found, in part related to the effects of nicotine. These findings suggest that the endothelium may be involved in the acute effects of cigarette smoking on the pulmonary vasculature. METHODS An extract of cigarette smoke in phosphate-buffered saline (PBS) was prepared fresh for each day’s experiment and used within 4 hr of preparation. Smoke from research cigarettes (Kentucky 2R1, University of Kentucky) was bubbled through PBS ( 1cigarette/ml PBS) using a simple three-way stopcock with a cigarette inserted into a holder attached to one arm and the other two arms going to a 35-ml syringe and a 5-ml glass pipet with 1 ml PBS. The cigarettes were smoked (1 puff/min) using standardized puff size and draw time of 35 ml and 2 set, respectively (Guerin, 1980). All reagents were obtained from Sigma Chemical Co. (St. Louis, MO) except as indicated. Left lower lobes of lungs removed from pigs at a local slaughterhouse were transported to the laboratory in chilled Krebs-Ringer-bicarbonate buffer (KRBB) (NaCl. 118 mM; KCI, 4.8 mM; CaC12, 2.5 mM; MgSO,,, 1.2 mM; KHzPO,, 1.2 mM; NaHC02, 24 mM; glucose, 11 mM). The lobar pulmonary artery was identified and dissected down to branches l-3 mm in diameter. Excess adventitial tissue was trimmed and two transverse strips, approximately .2-mm wide, were cut from each artery and mounted, with care to avoid damage to the endothelium, in separate 50-ml baths between an anchor point and a force transducer (Grass, FT.03) for measurement of isometric contractions. The baths contained KRBB at 37.5’C bubbled continuously with a gas mixture containing 5% COZ in air (02 = 19.95%), pH 7.35-7.45. Strip tension was set at 2 g and continuously recorded on a polygraph (Grass, Model 7). During equilibration, the tension gradually decreased to near I .2 g where experiments were performed.
ET AL. After a 2-hr equilibration period the strips were stimulated with norepinephrine, 2.5 X 1O-‘M, a concentration which causes IO- 15% maximal contractions in similar vessels with and without intact endothelium (Holden and McCall, 1984). All concentrations refer to final bath concentration. The presence of endothelium was verified by serial addition of acetylcholine in increasing concentrations ( 1Oe9to IOe6M) and demonstration of endotheliurndependent relaxation (Furchgott and Zawadski. 1980). Both strips were then removed from the baths and the luminal surface of one gently rubbed with a scalpel using short strokes covering the total intimal surface three times in each direction lengthwise and then replaced in the baths at the same tension found at time of removal. The presence (or absence) of endotheliumdependent relaxation to acetylcholine was again tested (as above). Similar rubbing of the adventitial side of the intact endothelium strip did not affect endothelial responses to acetylcholine. In previous studies the contractile effects of norepinephrine were not changed by removal of endothelium from similar strips of porcine pulmonary arteries in vitro (Holden and McCall, 1984). To assesseffectsof endothelium removal on relaxation of the strips, the effects of adenosine or nitroglycerin on norepinephrine-induced contractions were tested in six strips from three animals. Adenosine was exposed to four of the strips and nitroglycerin to two strips before and after removal of the endothelium. Relaxation effects of adenosine and nitroglycerin are thought to be independent of the endothelium (Rapoportand Murad, 1983; Furchgott, 1983). The concentration of adenosine or nitroglycerin required to cause half-maximal relaxation (ED50) in unrubbed strips was compared with the ED50 in strips without endothelium. In 14 pairs of strips from individual animals, effects of cigarette smoke extract and vehicle (PBS) on tension of norepinephrine-stimulated contractions were tested over a concentration range of 0.00 1 to 0.5%. In four of these pairs, effects of cigarette smoke extract were repeated in the presence (simultaneously) ofatropine (3.5 X IO-’ M), cimetidine (5 X lo-‘M), diphenhydramine (5 X IO-'M), methysergide (5 X lo-’ M), and propranolol(6.5 X 10m6 M) in the muscle baths. The doses used blocked contractions (histamine, 10e6 M; acetylcholine, 1O-5 M; serotonin, 10m6M) or relaxations (acetylcholine, IO-’ M; isoproterenol, 10e6 M) in strips of pulmonary artery with intact endothelium. The effects of cigarette smoke on tone of pulmonary artery strips with intact endothelium were also tested before and during exposure to indomethacin (5 X 10m6M) or acetylsahcylic acid ( 10m4M) to block cyclooxygenase activity. Strips from eight animals were used for indomethacin and six animals for acetylsalicylic acid studies. The concentrations of indomethacin and acetylsalicylic acid used blocked cyclooxygenase activity in cultured porcine pulmonary artery endothelial cells as shown by
LUNG
VASCULAR
EFFECTS OF CIGARETTE
(a) bioassay (contractions of rat stomach strip in vitro)prostanoid bioactivity in supematants of cultures exposed to arachidonic acid ( 1Oe6 M) was abolished by either drug; and (b) radioimmunoassay of selected prostanoids-unstimulated release of prostaglandin Fz, (80% decrease), 6-keto prostaglandin F,, (66% decrease), and prostaglandin E2 (85% decrease) was decreased by indomethacin ( 10m6M). In strips from an additional six animals, cigarette smoke effects were tested before and during exposure to hexamethonium ( 10e6 M) to block ganglia and nicotinic receptors (Young et al., 1988). Methods similar to those reported by Hariharan et al. ( 1988) were used to measure the nicotine content of five, separately prepared cigarette smoke extracts. A Hewlett Packard HPLC with C-18, reverse-phase column was used with a mobile phase of citric acid-phosphate buffer (each 0.03 M) and 16% acetonitrile with an added ionsplitting agent, sodium pentanesulfonate (200 mg/liter), and 2-phenylimidazole as internal standard. Ultraviolet detection was done over 200 to 360 nm. Finally, using the measured concentrations (by HPLC) of nicotine in our smoke extracts as a guide, we prepared similar solutions of nicotine in PBS and tested their effects on pulmonary artery tone. In pulmonary artery strips from 12 animals, the effects of nicotine on muscle tone during norepinephrine contractions were analyzed. Paired strips with endothelium removed were studied from 5 of the animals. All data are expressed as means f SE. ED50 for relaxation effects of adenosine and nitroglycerin were determined using a probit-log cumulative dose analysis (Colquhoun, 1971). We analyzed the effects of cigarette smoke extract on strips with and without endothelium using two-way analysis of variance with replication (Zar, 1974) where the variables examined were presence (or absence) of endothelium and dose of cigarette smoke extract, followed by Newman-Keuls test for significance of differences between positive and negative endothelium conditions. Paired t tests were used for comparison of blocking experiments and ED50 values of relaxation agonists. A p value < 0.05 was considered significant.
RESULTS Efects of Endothelium ation Ability
’
* Removal on Relax-
In previous studies, removal of endothelium from similar porcine pulmonary arteries did not alter the contractile effects of norepinephrine (Holden and McCall, 1984). In the studies herein, the ED50 for relaxation effects of adenosine was similar in strips with and without endothelium (4.7 f 0.8 X lop5 M vs
Aceiylcholine
193
SMOKE Smoke
Extract
j50mg 2 FIG. 1. Effects of cigarette smoke extract (in PBS) on tone of intrapulmonary arteries contracted by norepinephrine (2.5 X lo-’ M, asterisk). Upper panel is a strip with intact endothelium (+endo) and lower panel is a paired strip from the same artery scraped on the intimal surface (-endo). Muscle tension (vertical axis) is recorded continuously over time (horizontal axis). During initial contractions (at left), acetylcholine (IO-* and lo-’ M, graphs are labeled with the negative log of concentration) was added. +Endo, strip relaxed; whereas -endo, strip contracted slightly demonstrating presence and absence of endothelium, respectively. During the next contraction (at right), increasing concentrations of cigarette smoke extract (graphs are labeled with negative log of the percentage extract) were added. At lower concentrations, the strip with intact endothelium relaxed, and at higher concentrations, the strip contracted. Both relaxation and contraction effectswere absent in the -endo strip.
3.8 + 1.6 X lop5 M, n = 4 of each). Also, the ED50 for relaxation effects of nitroglycerin was similar in strips with and without endothelium (5.6 X lo-’ M vs 5.6 X 10M8M, mean of two of each). Eflects of Cigarette Smoke Extract Cigarette smoke extract over the range of 0.00 1 to 0.5% had concentration- and endotheliumdependent effects on intrapulmonary arteries contracted by norepinephrine (2.5 X 10e7 M) (Figs. 1 and 2). The vehicle, (PBS), added at intervals or as a bolus of the total volume used in testing cigarette smoke, had no effects on muscle tension. In 12 of 14
194
HOLDEN Acetylcholine
I
50mg
Smoke Extract
‘min
FIG. 2. Effects of cigarette smoke extract on tone of intrapulmonary arteries contracted by norepinephrine (2.5 X lo-’ M, asterisk). Axis and labels are same as in Fig. 1. Some strips showed only relaxation to cigarette smoke extract (i.e., no contraction at higher concentrations as in Fig. 1). Contraction at top left shows relaxation to acetylchohne (lo-& and IO-’ M), demonstrating intact endothelium. During contraction at top right, smoke extract was added and strip relaxed without contraction at higher concentration of extract. Strip was removed from the bath and the intimal surface scraped to remove endothelium (S). Contraction at lower left demonstrates loss of relaxation to acetylcholine verifying removal of endothelium. Smoke extract was again added in the contraction at lower right and relaxation effects were abolished.
strips with intact endothelium (+endo), smoke extract decreased muscle tension at lower concentrations and increased tension at higher concentrations (Fig. 1). Some strips with intact endothelium (2 of 14 strips) showed only relaxation when exposed to smoke extract (Fig. 2). Both relaxation and contraction effects of the extract were greatly diminished in strips with endothelium removed. When applied to strips not contracted by norepinephrine, only contraction effects of cigarette smoke extract were seen, hence the relaxation effects of smoke extract were seen only in the presence of increased baseline tension. A summary of experiments of the type shown in Fig. 1 using paired strips from 14 animals is shown in Fig. 3.
ET AL.
Cigarette Smoke Efects after Blockade ofSelected Agonists and Antagonists of Vascular Smooth Muscle Tone Blockade of several common agonists or antagonists of smooth muscle tone did not alter the effects of cigarette smoke. In four strips with intact endothelium, blockade of cholinergic (atropine, 3.5 X low7 M), histamine types 1 and 2 (diphenhydramine, 5 X 10e7 M; or cimetidine 5 X 10m7 M), serotonergic (methysergide 5 X lop7 M), and /3-adrenergic (propranolo16.5 X 1O-6 M) receptors did not alter relaxation or contraction effects of the smoke extract (Table 1). These blocking agents were simultaneously present in the muscle baths during addition of the cigarette smoke extract. The cyclooxygenase inhibitors, indomethacin (5 X 1Oe6M) or acetylsalicylic acid ( 1Oe4 M), added to the muscle baths 30 min prior to norepinephrine, did not affect the contractile
40 -I
---*--.
+ END0 END0
T
20 -
0
,001 CIGARETTE
.Ol SMOKE w
1
1
EXTRACT
FIG. 3. Summary of experiments of the type shown in Fig. 1 in 14 pairs of porcine intrapulmonary arteries with (+endo) and without (-endo) endothelium. The horizontal axis is percentage cigarette smoke extract. Both dose and tension changes are cumulative. Zero points represent effect of PBS in the total volume used to deliver smoke extract. +Endo strips relaxed (negative change in tension) at lower extract concentrations and contracted (positive change in tension) at higher concentrations. -Endo strips showed little or no change in tension during the addition of smoke extract. Each point represents the mean + SE. *Significant difference between +endo and -endo groups by analysis of variance.
LUNG VASCULAR
EFFECTS OF CIGARETTE
195
SMOKE
TABLE 1 EFFECYSOFCIGAREITESM~KEEXTRACT.DURINGBLOCKADEOF ANDRELAXATIONAGONISTS
SELEC~ZD~ONTRACTION
Concentration of smoke extract (%)
Control” + Blockers’
0.01
0.05
0.075
0.1
0.15
-30 + 6b -20+7
-58 + 15 -40 + 10
0 -5+-3
+23 + 12 +15-+ 5
+45 + 18 +40+ 13
a Absence of blockers b Change in tension (mg) (+ or - sign indicates contraction or relaxation, respectively). ‘Simultaneous presence of blockers: atropine, 3.5 X lo-’ M; diphenhydramine, 5 X lo-’ M; cimetidine, 5 X lo-’ M; methysergide, 5 X IO-’ M; and propranolo16.5 X 10m6M.
effects of cigarette smoke extract, but the relaxation effect of smoke extract was inhibited (Fig. 4, Table 2). The effect of indomethacin was transient-relaxation effects of cigarette smoke extract reappeared 90 min after indomethacin was washed out of the muscle baths. However, the effects of acetylsalicylic acid were reversible within the time of our experiments (4-6 hr) (Fig. 4).
Are Efects of Cigarette Smoke Extract Due to Nicotine? In order to determine if nicotine might be causing the contractile effects seen at higher concentrations of cigarette smoke extract, the concentration of nicotine was measured in representative extracts using high-perfor-
TABLE 2 5.2
5rl
EFFE~S OF INWMETHACIN OR ACETYLSALICYLIC ACIDONRELAXATION OFPLJLMONARYARTERYSTRIPS BYCIGARETTESMOKEEXTRACT Concentration of smoke extract (%)
I
50mg
3mm
FIG. 4. Example of effect of indomethacin (5 X lO-6 M) on response of porcine intrapulmonary artery with intact endothelium contracted by norepinephrine (2.5 X IO-’ M, asterisk) to an extract of cigarette smoke. Cigarette smoke concentration is expressed as the negative log of the final bath percentage (v/v) of extract. Contraction at left shows dilation response to cigarette smoke extract (0.001%). Then, indomethacin (5 X 10m6M) was added to the bath (at indo 30’) and after 30 min the response to smoke extract was again tested (middle contraction). No relaxation was seen at lower concentrations of smoke extract, but contractions occurred at higher concentration of smoke extract. The bath was cleansed to remove indomethacin and, after 90 min, the effects of smoke extract were again tested (contraction at right) and the relaxation response to cigarette smoke was again found.
Control b
N”
0.01
0.05
9
-27 + 6’ p < 0.05 -6-c3
-55 + 10 D
