1234
Decreased /3-Adrenergic Receptor
Density and Catecholamine Response in Male
Cigarette Smokers
A Study of Monozygotic Twin Pairs Discordant for Smoking
Kai E. Laustiola, MD, Riitta Lassila, MD,
Jaakko Kaprio, MD, and Markku Koskenvuo, MD
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The effect of long-term cigarette smoking on ,B-adrenoceptor density and catecholamine
response was studied in 10 monozygotic male twin-pairs discordant for smoking, with an
average discordance time for smoking of 23 years (range, 12-35 years). The density of
fl-adrenergic receptors was 40% lower in the lymphocytes of smoking twins compared with
their nonsmoking cotwins (fl-receptor density, 6.7 1.2 and 11.1±1.8 fmol/106 cells, respectively; p<0.05). The corresponding apparent Kd values were 31.7±5.5 and 26.7+±5.4 pM,
respectively. Stimulation of the lymphocyte fl-receptors resulted in significantly lower levels of
cyclic adenosine monophosphate in the smokers compared with the nonsmokers (16.2 3.3 vs.
29.2 6.5 pmol/106 cells, p<0.05). When subjected to submaximal exercise, the smokers had a
lower level of cyclic adenosine monophosphate in plasma (25.9 ± 1.2 vs. 28.6 ± 1.0, p<0.05) and
a net decrease was seen in plasma free fatty acids in the smokers compared with a net increase
in the nonsmokers ( - 15% vs. + 19%, p<0.01). The total plasma catecholamine level was, in the
basal state, significantly higher in smokers compared with nonsmokers (74.8%, p<0.05). The
intrapair difference in plasma norepinephrine predicted well the intrapair difference in
fl-receptor density (r = -0.84, p<0.001). We conclude that the autonomic neurohumoral
response evoked by cigarette smoking results in downregulation of P-adrenergic receptors in
long-term smokers. (Circulation 1988;78:1234-1240)
C igarette smoking is considered to be a major
risk factor of atherosclerotic disease and its
acute complications (e.g., sudden death,
stroke, and acute myocardial infarction).1-3 Smoking appears to induce increased sympathetic discharge and leads to an increase in plasma levels of
both the adrenomedullary hormone epinephrine and
the sympathetic neurotransmitter norepinephrine.4
The increments in pulse rate, blood pressure, and
lipolysis are probably due to this increased sympathetic discharge during smoking.45 However, the
long-term effects of smoking on the autonomic
nervous system have not been as evident. Surprisingly, epidemiological studies show smokers to have
a lower rather than a higher blood pressure comFrom the Wihuri Research Institute, Helsinki, and the Department of Public Health, University of Helsinki, Finland.
Supported in part by grants from the Council for Tobacco
Research-USA, Inc, and the Meilahti Foundation, Helsinki,
Finland.
Address for correspondence: Kai E. Laustiola, MD, Wihuri
Research Institute, Kalliolinnantie 4, SF-00140 Helsinki, Finland.
Received February 1, 1988; revision accepted June 16, 1988.
pared with nonsmokers.6 It has also recently been
shown that smoking may be associated with an
increased formation of prostacyclin, the antiaggregatory and vasodilatory eicosanoid, secreted by the
endothelium.7 These observations suggest the existence of a complicated set of compensatory mechanisms in response to the harmful short-term effects
of smoking. The present study was undertaken to
elucidate whether the continuous increments in
catecholamines in nerve endings and plasma are
reflected in changes in the density and sensitivity of
,B-adrenergic receptors.
Subjects and Methods
Characteristics of Study Population
Ten monozygotic male twin-pairs who were discordant with respect to cigarette smoking were
enrolled from the Finnish Twin Cohort Study, a
nationwide twin panel.8 Their mean age was 40
years (range, 31-53 years). The smoking cotwin had
a history of at least 10 years of uninterrupted
cigarette use, whereas the nonsmoking cotwin was
accepted for the study if he had never smoked
Laustiola et al Smoking and ,B-Adrenergic Receptors
1235
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TABLE 1. Baseline Characteristics of Study Population
Nonsmokers
Smokers
77 ± 5
76±4
Weight
177 ± 2
177 ± 2
Height (cm)
74 ± 3
79 ± 3
Heart rate
Blood pressure
121 ±2
118±2
Systolic
±
2
83 ± 4
82
Diastolic
(%)
47±0.2
45±2
Hematocrit
6.6 ± 0.7*
4.6 ± 0.3
Leukocytes ( x 10/ 1)
±
± 18
x
208
14
233
Platelets ( 109/ l)
12
180±
14
168±
Cholesterol (mg/dl)
42 ± 4
42 ± 4
HDL cholesterol (mg/dl)
2.9±0.4
LDL: HDL cholesterol ratio
3.0 ± 0.4
1.7±0.2
1.6±0.1
Creatinine clearance (ml/sec)
Values are mean ± SEM.
*p<0.05 (paired t test) smokers vs. nonsmokers.
TABLE 2. Cigarette Consumption Characteristics of
Smoking Cotwins
Starting age
Twin-pairs
Years of smoking Cigarettes/day
(yr)
1
20
22
24
2
5
19
30
3
24
17
32
4
20
22
35
5
13
21
7
6
14
17
17
7
17
22
15
8
20
12
32
9
20
16
12
10
14
22
22
regularly. Smoking history was obtained from two
questionnaire studies in 1975 and in 1981, and it was
checked by an interview in 1986. Zygosity was
determined by a highly accurate questionnaire
method validated by genetic markers.9 The excretion of nicotine metabolites was also tested in all
cases.10 The mean excretion of cotinine in smokers
was 1,406 + 300 ng/ml, whereas all of the nonsmokers had a secretion of less than 100 ng/ml. The mean
duration of discordance was 23 years (range, 12-35
years), and in all except one case smoking had been
uninterrupted during the whole discordance period.
The mean number of cigarettes consumed by the
smokers was 18 cigarettes/day (range, 5-32 cigarettes/day). The twins were apparently healthy
(except for one smoker who had signs of peripheral
atherosclerotic disease), and no other major differences in risk factors or life-style between the smokers and nonsmokers were detected.
No significant differences in dietary habits between
the nonsmokers and smokers were detected. Average coffee consumption for nonsmokers was 5.2 and
for smokers was 5.5 cups/day. Three of the smoking
cotwins used more alcohol than did their nonsmoking brothers, whereas one nonsmoking cotwin used
more alcohol than his smoking brother. The nonsmoking cotwin exercised more in three twin pairs,
whereas there were no differences in leisure time
physical activity in seven pairs. The socioeconomic
status (SES) was evaluated on the basis of educational level and occupational history. SES was very
similar in all pairs. In two pairs, the nonsmoker had
a slightly higher SES, whereas the converse was
true for one pair. Baseline physiological characteristics are given in Table 1, and individual smokinghabit characteristics are given in Table 2.
Study Design
Venous blood samples were taken at 9 AM through
an intravenous catheter inserted in an antecubital
vein. The smokers had refrained from smoking at
least for 2 hours before the tests and had ingested
no coffee on the morning of the tests. They had also
abstained from alcohol for the preceding 48 hours.
Subjects reclined in a supine position for 30 minutes
before the blood samples were taken for measurement of baseline values. After the initial blood
sampling, an exercise test was carried out with a
bicycle ergometer.
The workload was increased in steps of 50 W to
reach a heart rate corresponding to 85% of the ageadjusted maximum.'1 A second blood sample for
determination of exercise values of plasma cyclic
adenosine monophosphate, catecholamines, and free
fatty acids was drawn immediately after reaching the
peak heart rate.
Preparation of Lymphocytes
Lymphocytes were isolated by gradient separation
with the method of Boyum. 12 Sixty milliliters of blood
was drawn into Venoject tubes containing ethylenediaminetetraacetic acid (EDTA) (0.38 M). The lymphocytes were separated with Ficoll-Paque (Pharmacia, Uppsala, Sweden). Ten milliliters yielded
1-2 x 10' mononuclear cells, consisting of about 80%
lymphocytes and 20o monocytes. Trypan-blue exclusion showed a 98% viability. The lymphocytes
(1.2 x 106 cells/ml) were suspended in Dulbecco's
phosphate-buffered saline (pH 7.4) without calcium or
magnesium and were used within 1 hour for binding
and stimulation studies.
Ligand-Binding Assay
Binding studies were carried out on intact human
lymphocytes with a modification of the method
described by Halper et al. 13 One milliliter of the cell
suspension was used for the studies of ,-receptor
density.
['251]Iodocyanopindolol (New England Nuclear,
Boston, Massachusetts) was used as ligand in the
concentration range of 14-227 pM. At least eight
concentrations of the ligand were used in the bind-
Mean
Range
18
13-24
23
12-35
18
5-32
1236
Circulation Vol 78, No 5. November 1988
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ing studies. The reaction mixture was incubated for
60 minutes at 370 C. Bound and free ligand were
then separated by adding 10 ml of a mixture containing 0.9% NaCI 10 mM Tris-HCI and 12.5 mM
MgCI at 37° C followed by rapid filtration through
Whatman GF/C filters (Whatman, Clifton, New
Jersey). Each filter was then washed with an additional 10 ml buffer, and the retained radioactivity
was determined in a 1272 Clinigamma gamma counter (LKB-Wallac, Turku, Finland). Nonspecific binding was defined as the binding not competed for by
0.1 mM isoproterenol. /3-Receptor density (Bmax)
and affinity (Kd) for [1251]iodocyanopindolol binding
were determined from saturation curves of specific
binding analyzed by the method of Scatchard. l4
TABLE 3. Lymphocyte
Cyclic AMP Levels
Cvyclic AMP, Catecholamine, and
Free Fatty Acid Determinations
Aliquots of I ml (1.2 x 10 cells/mI) cell suspensions were taken for lymphocyte stimulation and
preincubated with methyl isobutylxanthine (final
concentration, 2 x 10- M) for 10 minutes at 37° C.
Incubation with buffer or L-isoproterenol (final concentration, 1 x 10-5 M) was performed for an additional 10 minutes at 37° C. The reaction was terminated by boiling for 3 minutes. After centrifugation
at 2,500g for 15 minutes, the supernatant was stored
at -20° C for subsequent determination of cyclic
AMP with a protein binding assay with a commercial [3H]cyclic AMP assay kit (Amersham, Buckinghamshire, UK) from deproteinized samples.
For plasma catecholamine determinations, 9 ml
blood was taken into Venoject tubes containing 1 ml
of a solution containing 90 mg ethyleneglycol-bis(,3-aminoethyl ether)-N,N,N' ,N'-tetraacetic acid
(EGTA) and 60 mg reduced glutathione. The blood
was centrifugated, and the supernatant was stored
at - 80° C. Plasma epinephrine and norepinephrine
were determined with high-performance liquid chromatography (HPLC) with electrochemical detection
and dihydroxybenzylamine as internal standard as
described by Goldstein et al. 15 The HPLC apparatus
consisted of a sample injector (Model U6K, Waters,
West Lafayette, Indiana), a solvent delivery system
(Model 6000A, Waters), Microbondapak C'8-column
(Waters), and an amperometric detector with glassy
carbon electrode (Model LC-4, Bioanalytical Systems, West Lafayette, Indiana).
Five milliliters of EDTA plasma was used for
the determination of free fatty acids. Lipids were
extracted from 0.50-ml aliquots of plasma and
determined as described previously.'6 Free fatty
acids were isolated by thin layer chromatography,
converted to their methyl esters, and analyzed
with gas chromatography (Model 5880 A, HewlettPackard, Avondale, Pennsylvania).
Statistical Analyses
Student's t test for paired data was used in the
statistical analyses. Linear regression analysis by
the least-squares method was performed to analyze
(stimulated/basal)
fl-Adrenergic Receptors and
Nonsmokers
Smokers
[r1I]Iodocyanopindolol
binding
/-Receptor density (fmol/105
cells)
Binding affinity (pM)
Cyclic AMP levels (pmol/106
cells)
Basal
Stimulated (isoproterenol.
10 gM)
Absolute increase
(stimulated minus basal)
Relative increase
ILl
+
1.8
6.7 ± 1.2*
31.7 5.5
26.7±5 4
3.3 +o0.9
29.2 6.5
3.4 --1.2
16.2w 3.3*
26.46.0
14. 7-3A*
11.8a 3.2
52 1 3
Values are mean ± SEM.
*p<0.05 (paired t test) smokers vs. nonsmokers.
the relation between catecholamines and ,3-receptor
density. p<0.05 was considered indicative of a
significant difference.
Results
The density of /-receptors in the lymphocytes of
the cigarette-smoking cotwins was reduced in nine
of the 10 pairs, being 40% lower in the smoking
cotwins compared with the nonsmokers (p<0.05,
Table 3 and Figure 1). The apparent Kd values did
not differ between the two groups (Table 3). The
basal level of cyclic AMP in the lymphocytes was
the same in nonsmokers and smokers (Table 3).
However, stimulation with 10 JLM isoproterenol
resulted in a significantly lower level of cyclic AMP
in the lymphocytes of smokers compared with nonsmokers (45%, p <0.05, Table 3). The absolute
increase (stimulated minus basal) in cyclic AMP
was 44% less (p <0.05, Table 3) in smokers compared with nonsmokers, and the relative increase
(stimulated/basal) was 56% less (p <0.05, Table 3).
The mean plasma level of epinephrine was more
in smokers at rest (49%, Table 4), but the difference
was not significant. The plasma level of norepinephrine was also more in smokers than in nonsmokers
at rest (66%, Table 4), but again this difference was
not significant.
However, the total catecholamine level, expressed
as epinephrine plus norepinephrine, was significantly elevated in smokers compared with nonsmokers at rest in the basal state (75%, p<O.05, Table 4).
At peak heart rate (85% of age-adjusted maximum),
the plasma level of norepinephrine increased further in smokers and nonsmokers, whereas the level
of epinephrine increased during exercise only in the
nonsmokers. However, the total catecholamine
level, expressed as epinephrine plus norepinephrine, was still slightly more at peak heart rate in
smokers than in nonsmokers (17%, NS, Table 4). A
significant reciprocal correlation (r= -0.84,
Laustiola
25r09
20k-
07
0
05
Oio
7
*5
_06
o3
10l
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82
@4
5
-
08
g10
0 1
2
* 1
L
Nonsmokers
al Smoking and ,B-Adrenergic Receptors
1237
mal exercise resulted in a significantly higher level
of cAMP in plasma in the nonsmokers compared
with the smokers (9%, p<0.05, Table 4). Exercise
resulted in a increase in plasma free fatty acids in
eight nonsmokers (mean increase, 19%), whereas
there was a decrease in plasma free fatty acids
during exercise in nine smokers (mean decrease,
15%; Figure 3). Thus, there was a net difference of
34% units (p<0.01) between the two groups.
Bmax
(fmol/106 cells)
15[-
et
&
A
Smokers
FIGURE 1. Plot of 3-receptor density in nonsmoking and
smoking cotwins. Numbers refer to twin-pairs. Values
are mean ±SEM.
p<0.001)
was found between the intrapair differin Bmax and the intrapair difference in norepinephrine (Figure 2). The correlation between the
intrapair difference in Bmax and the intrapair difference in epinephrine was nonsignificant (r= -0.28,
ence
Figure 2).
The plasma level of cyclic AMP in the basal state
was slightly lower in the smokers than in the
nonsmokers (6%, NS, Table 4). However, submaxi-
Discussion
The present study provides two sets of findings
that suggest downregulation of f3-adrenergic receptors in long-term cigarette smokers. Ligand binding
to the fl-receptors is decreased, and stimulation of
lymphocytes by isoproterenol results in decreased
formation of cyclic AMP. In addition to this direct
evidence, the significantly lower plasma level of
cyclic AMP and the decrease of the level of free
fatty acids in smokers during exercise are also
indications of an impaired catecholamine response,
especially, because there was a tendency toward
higher (17%) catecholamine levels in the plasma of
smokers compared with nonsmokers at peak heart
rate during exercise. These findings seem to imply
that the fl-receptors of other cell systems than the
lymphocytes (e.g., the f-receptors of the adipose
tissue) were also downregulated in smokers.
The level of free fatty acids in plasma during
exercise reflects both the rate of lipolysis and the
uptake rate by the working muscles. 17 Earlier studies indicate a decreased,'1819 an unchanged,20 and an
increased21,22 plasma level of free fatty acids during
exercise, depending on the load and the duration of
exercise applied. The most common finding, in
association with prolonged exercise, is an increase
in the plasma level of free fatty acids.23 However,
the consumption of fatty acids may, during vigorous
short-term exercise, be more than the amount provided by lipolysis. This is reflected as a decrease in
the plasma level of free fatty acids. The exercise
applied in the present study was short-term (mean,
10 minutes) during which no steady state was
reached. This led in the nonsmokers to a small
nonsignificant increase in the plasma level of free
fatty acids, whereas there was in the smokers a
TABLE 4. Plasma Levels of Catecholamines, Cyclic AMP, and Free Fatty Acids During Rest and Exercise
Smokers
Nonsmokers
Rest
Exercise
Exercise
Rest
Catecholamines
Epinephrine (pmol/ml)
Norepinephrine (pmol/ml)
Epinephrine and norepinephrine
0.43 ± 0.1
1.93 ±0.6
2.26±0.1
(pmol/ml)
26.2± 1.8
Cyclic AMP (pmol/ml)
Free fatty acids (gmol/L)
305+60
Values are mean ± SEM.
*p<0.05 (paired t test) smokers vs. nonsmokers.
0.57 ± 0.09
4.17±0.6
0.64 ± 0.09
3.20 ±0.6
0.52 ± 0.07
5.04± 0.90
4.74 ± 0.6
28.6± 1.0
362 + 78
3.95 ± 0.7*
24.6-+ 0.9
315 ± 42
5.55 ±0.87
25.9 ± 1.2*
267 ± 36
1238
Circulation Vol 78, No 5, November 1988
E
tpmol/ml)
NE
(pmoUlml)
7,t
15
60
5
10
4
3
0.5 _
2t
5
10
15
20
1
---
25
I1
I1
I
5
Bmax Ufmol/106 cells)
10
2
20
15
_
25
25-
Bmax (fmolJ106 cells)
AE
iNE
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r -0.84, p<0001
+ 1 ,otw
r =-0.28, n.s
+
+0.5
~~~~~~~~~~~0
0
0
4-
IBmax
15
-10
ABmax
-15
00
-10
+5
-5
-0.5r
-5k
10
FIGURE 2. Plots of relation between B,,,, and norepinephrine (upper left panel) in smoking (0) and nonsmoking (0)
cotwins, between Bm,a. and epinephrine (upper right panel) in smoking (o) and nonsmoking (o) cotwins, of the intrapair
difference (smoker-nonsmoker) in B,,,, to the intrapair difference (smoker-nonsmoker) in norepinephrine (left lower
panel), and of the intrapair difference (smoker-nonsmoker) in B,,,_, to the intrapair difference (smoker-nonsmoker) in
epinephrine (right lower panel).
small but consistent decrease in the plasma level of
free fatty acids during exercise (Figure 3). Because
lipolysis is /3-adrenergically regulated, we consider
that this finding reflects, in accordance with the
other results of the study, a desensitization of the
,8-adrenergic receptors mediating lipolysis. However, an increased smoking-induced uptake of free
fatty acids cannot be ruled out.
The present method of analysing lymphocyte
/3-receptor function can examine only /32-subtype
downregulation, although some effect on /3regulation might also be produced by cigarette
smoking because norepinephrine has a higher affinity
for ,3- than for ,32-receptors. This may be of importance because epinephrine and norepinephrine have
different affinities for ,8- and /32-receptors, and it has
been shown that differential regulation of /3adrenoceptor subtypes is possible.24 There is, however, evidence that cardiac sensitivity to isoproterenol, for example, can be predicted from leukocyte
,/-receptor density.25 This and other similar studies
imply that lymphocyte ,3-receptor function reflects
the function and regulation of the /3-receptors of the
cardiovascular system.
There are indications that /3-receptor density is
genetically regulated.26 In the present study, there
was a positive correlation of 0.41 (NS) between the
Bmax values of smokers and nonsmokers. Thus, the
correlation of the intrapair difference in Bmax to the
intrapair difference in catecholamines may best
describe the relation between /3-receptor density
and catecholamines because interpair genetic differences in Bmax are eliminated. The present study
shows that plasma norepinephrine predicts well the
density of /8-receptors if the genetic variance in
,/-receptor density is eliminated (r- - O.84,p<0.001.
Figure 2).
There is some evidence to suggest that /3adrenergic blockers are not as effective in smokers
as in nonsmokers. For example, it has been shown
that /3-blockade does not reduce the exerciseinduced increase in oxygen consumption in smokers as effectively as in nonsmokers.27 The same
study also showed that the heart rate for the same
Laustiola et al Smoking and P-Adrenergic Receptors
FFA
(per cent
it.35 Cigarette smoke- or nicotine-induced platelet
thrombus formation in the stenosed dog coronary
artery can be antagonized with the a-adrenergic
blocker phentolamine.36 These observations indicate that cigarette smoking may induce an increase
in a-adrenergic tone, with considerable impact on
cardiovascular regulation. The present results imply
that even if a-receptor function per se is unaltered
in cigarette smokers, the downregulation of ,Breceptors may result in a relative increase in aadrenergic tone at any given catecholamine concentration. This could explain the increased
vasoconstrictive tendency found in the coronary
vasculature of cigarette smokers.
In summary, the present study shows that longterm smoking induces downregulation of /3adrenergic receptors in genetically matched pairs.
This finding may explain why p-blockers tend, in
some studies, to be less effective in the treatment
of hypertension or angina pectoris in smokers
compared with nonsmokers.
change)
+60
+40 _
+2010
-20QH
-40_
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exercise
rest
Nonsmokers
1239
rest
exercise
Smokers
FIGURE 3. Plots of the exercise-induced change in plasma
level offree fatty acids in nonsmokers and smokers.
workload remained at a higher level in smokers
compared with nonsmokers despite ,B-adrenergic
blockade. The Medical Research Council's Mild
Hypertension Trial showed that the antihypertensive efficacy of propranolol was significantly
impaired in the treatment of mild hypertension
among smokers, whereas that of bendrofluazide, a
diuretic drug, was not.28 Furthermore, propranolol
appeared, in this trial, to be much less effective than
the diuretic drug in the prevention of stroke in
smokers, although both drugs were equally effective in nonsmokers.29 Trap-Jensen30 has also shown
that after long-term treatment, the beneficial bloodpressure lowering effect of both the 81-selective
blocker atenolol and the nonselective blocker propranolol is attenuated in hypertensive patients who
smoke. It has further been shown that propranolol
was not as effective as expected in reducing exerciseinduced changes in heart rate, blood pressure, the
cardiac oxygen demand, and ST segment depression on electrocardiography in smokers with
angina.31 The improvement of angina after patients
stopped smoking was more during nifedipine treatment than during the use of atenolol or propranolol
during a follow-up period of 1 month.32 These
observations suggest a change in ,B-adrenoceptor
function in smokers, which is explicable on the
basis of the present results.
Smoking induces coronary vasoconstriction,33 34
probably because of an increased a-adrenergic tone
in coronary arteries. a-Adrenergic blockers seem to
abolish the cigarette-induced vasoconstriction,
whereas ,3-adrenergic blockers may even aggravate
Acknowledgments
We are indebted to Dr. G.A. Ellard of the National
Institute for Medical Research, London, UK, for
determinations of urinary nicotine metabolites; to Dr.
Vesa Manninen for advice and constant support; to
Ms. T. Airaksinen for technical assistance; and to Ms.
U. Kulmala and E. Voipio for secreterial assistance.
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KEY WORDS
cigarette smoking
catecholamines * MZ twin pairs
*
3-adrenoceptors
Decreased beta-adrenergic receptor density and catecholamine response in male cigarette
smokers. A study of monozygotic twin pairs discordant for smoking.
K E Laustiola, R Lassila, J Kaprio and M Koskenvuo
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Circulation. 1988;78:1234-1240
doi: 10.1161/01.CIR.78.5.1234
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