From the Department of Anesthesiology and Critical Care Medicine and The
2nd Department of Internal Medicine, Gifu University School of Medicine, Gifu,
Japan.
Correspondence to Hiroki Iida, MD, Department of Anesthesiology and Critical Care Medicine, Gifu University School of Medicine, 40 Tsukasamachi, Gifu-City, Gifu 500-8705, Japan. E-mail address iida{at}cc.gifu-u.ac.jp
Methods—In pentobarbital-anesthetized, mechanically
ventilated Sprague-Dawley rats, pial vessel diameters were measured
with the use of a cranial window preparation. We studied the effects of
(1) 60 puffs per minute of mainstream cigarette smoke from cigarettes
having 2 nicotine levels (0.1 and 1 mg per cigarette), (2)
administration of nicotine (0.05 mg per body IV), and (3) repeated
smoking (four 1 mg nicotine–containing cigarettes at 30-minute
intervals) (n=6 each).
Results—Inhalation of smoke from a 0.1 or 1 mg
nicotine–containing cigarette for 1 minute caused pial arterioles to
constrict at 30 seconds (7.2% and 7.3%, respectively) and then to
dilate (peak at 5 to 10 minutes; 4.6% and 17.9%, respectively).
Nicotine infusion caused pial vasodilation (35.7%) without an initial
vasoconstriction. Repeated smoking suppressed the pial vasodilation but
not the initial vasoconstriction. The vasodilation induced by a single
cigarette was greatly inhibited by pretreatment with mecamylamine or
glibenclamide and attenuated by propranolol or
N
Conclusions—Single-cigarette smoking had a significant biphasic
effect on cerebral arteriolar tone. The vasodilation was attenuated by
repeated smoking. The vasodilation is most likely an effect of
nicotine, at least in part mediated via sympathetic activation, NO
production, and K+ channel activation. The
vasoconstriction is partially due to thromboxane
A2 induced by cigarette smoke.
Mainstream cigarette smoke has been said to contain 4000 or more
constituents (eg, nicotine, tar, phenol, acetic acid, CO,
CO2, NO, and NO2).9 The
influence of cigarette smoking on cerebral vasculature seems likely to
be a net effect of not only nicotine but also the other constituents of
mainstream cigarette smoke. In isolated coronary arteries,
cigarette smoke extract produces a biphasic action on vascular tone
(contraction followed by relaxation).10 In
addition to the potential biophysiological
differences between coronary and cerebral arteries, in vivo
cigarette smoking is quite different from the in vitro situation. In
vitro studies bypass the airways and eliminate the filtering action of
the lungs. The aims of the present study were to investigate the
acute effects of cigarette smoking on cerebral vessels in rats in vivo
with the use of the cranial window technique and to elucidate the
mechanisms involved. We tested the hypothesis that the effects of
cigarette smoking on tone in the cerebral vasculature are not same as
the effects of nicotine and that these effects are not only by means of
nicotinic receptors. In addition, we investigated the effects of
multiple-cigarette smoking on the pial microcirculation by studying
repeated inhalation. We also tested the hypothesis that
multiple-cigarette inhalation attenuates the effects of single
inhalation on cerebral vasculature.
Each rat was anesthetized (pentobarbital sodium, 50 mg/kg body
weight IP) and mechanically ventilated through a tracheostomy tube by a
ventilator (60/min, KN-56, Natsume Seisakusho Co Ltd) using room air
supplemented with oxygen. Tidal volume was adjusted to maintain
PaCO2 between 35 and 40 mm Hg
at the beginning of each experiment by a pressure-limited system.
Supplemental pentobarbital was administered intravenously
by continuous infusion at 4 mg ·
kg-1 · h-1.
The femoral artery was cannulated for the continuous measurement of
arterial blood pressure and to provide blood samples for
the determination of arterial blood gas tensions, pH,
glucose, and serum electrolytes. The femoral vein was cannulated for
administration of fluid and drugs. Body temperature was maintained
between 37°C and 38°C by means of a heating blanket.
A closed cranial window was used for observation of the pial
microcirculation; the head was fixed in the sphinx position. The scalp
was retracted, a 3x2-mm-diameter hole was made in the bone over the
right parietal cortex, and the dura was opened carefully. A
polypropylene ring with a fitted glass coverslip was placed over the
hole and secured with dental acrylic. The space under the window was
filled with artificial cerebrospinal fluid. Two catheters were inserted
through the ring: one was attached to a reservoir bottle containing
artificial cerebrospinal fluid to maintain a constant intracranial
pressure; the other was for continuous monitoring of intracranial
pressure. The composition of the artificial cerebrospinal fluid was as
follows: Na+, 151 mEq/L;
K+, 4 mEq/L; Ca2+, 3 mEq/L;
Cl-, 110 mEq/L; and glucose, 100 mg/dL; the pH
was adjusted to 7.48. The solution was freshly prepared each day and
bubbled with 5% CO2 in air at 37.5°C.
The pial views obtained in these experiments were stored on videotape
(with a time record) for later playback and analysis. The
diameters of 3 pial arterioles and 3 pial venules (baseline diameter,
21 to 75 µm and 23 to 81 µm, respectively) were measured
using a videomicrometer (model VM-20, Olympus) on a
television monitor attached to a microscope (model SZH-10, Olympus).
The value of the percent changes in pial vessel diameter was used in
the statistical analysis. Mean arterial blood
pressure (MAP) and heart rate (HR) were continuously monitored.
Experimental Protocols
In protocol 2, we investigated the vasodilator mechanisms involved in
the effects of mainstream smoking on pial vessels. Four kinds of drugs
were used for pretreatment in 6 rats each. Nicotine receptors were
blocked with mecamylamine (0.7 mg/kg IV) (Sigma); sympathetic
ß-adrenoceptors, with propranolol (1 mg/kg IV) (Wako Pure
Chemical); ATP-sensitive K+ channels, with
glibenclamide (20 mg/kg IV) (Sigma); and NO synthase, with
N
In protocol 3, we investigated the vasoconstrictor mechanisms involved
in the effects of mainstream smoking on pial vessels. During the
blockade of thromboxane (Tx) A2
receptors with seratrodast (5 mg/kg IV) (Takeda Chemical), the effect
of smoking a 1 mg nicotine–containing cigarette on pial vessels was
investigated in 6 rats as in protocol 1. Seratrodast was administered 5
minutes before smoking. The arterial concentration of
TxB2 (a stable metabolite of
TxA2) was measured at baseline and just after the
smoking of a 1 mg nicotine–containing cigarette (n=6) or
intravenous nicotine administration (0.05 mg per rat,
n=6).
In protocol 4, the same preparation was used in 6 rats, and mainstream
smoke from four 1 mg nicotine–containing cigarettes was inhaled for 1
minute each time at 30-minute intervals (repeated smoking).
Measurements were made as in protocol 1 when the fourth dose was
inhaled.
In protocol 5, venous nicotine concentrations were measured at
baseline, immediately after, and 30 minutes after 1-minute inhalation
of smoke from a 1 mg nicotine–containing cigarette or
intravenous nicotine administration (0.05 mg per rat), as
in protocol 1 (n=4 each).
All drug solutions were freshly prepared on the day of the
experiment.
Data Analysis
Arterial blood gas tensions, pH, serum electrolytes, and
body temperature did not change significantly throughout the
experiment, although there were significant increases in blood glucose
at 15 minutes after smoking a 1 mg nicotine–containing cigarette and
nicotine infusion (Table 1
Effects of Antagonistic Drugs on the Vasodilator
Response to Smoking a 1 mg Nicotine–Containing Cigarette
(Protocol 2)
Propranolol attenuated the smoking-induced arteriolar
dilation but not the initial vasoconstriction observed during cigarette
smoking (Figure 2B
L-NAME partially attenuated the smoking-induced arteriolar dilation at
5 minutes but not the initial vasoconstriction observed during
cigarette smoking (Figure 2C
Glibenclamide completely inhibited the smoking-induced arteriolar
dilation but not the initial vasoconstriction (Figure 2D
Physiological variables, including
arterial pH, PaCO2,
PaO2, Na+,
K+, and body temperature, all remained stable in
the presence of the above antagonists, but there was a
decrease in pH (from 7.37 to 7.33) at 60 minutes in the presence of
L-NAME. However, blood glucose was increased by propranolol
and decreased by glibenclamide (Table 2
Effects of an Antagonistic Drug on the Pial
Vasoconstrictor Response to Smoking a 1 mg Nicotine–Containing
Cigarette and on Plasma TxB2 Concentration (Protocol
3)
Smoking a 1 mg nicotine–containing cigarette caused the
arterial TxB2 concentration to
increase significantly from 48.7±6.0 to 110.3±18.4 pg/mL
(P<0.01), but intravenous nicotine
administration did not affect the concentration (from 37.8±6.5 to
34.9±2.9 pg/mL) (Figure 4
Effects of Repeated Smoking (Protocol 4)
Plasma Nicotine Concentration (Protocol 5)
The effects of short-term smoking on the cardiovascular
system have been reported to be mediated by an immediate release of
catecholamines from local adrenergic terminals, followed by
a systemic release from the adrenal medulla, as evidenced by the early
rise primarily of norepinephrine, followed by a rise in
epinephrine level.11 12 13 Nicotine is
described as a ganglion-stimulating drug; in fact, it stimulates both
sympathetic ganglia and the adrenal medulla.14
Mecamylamine has been reported to block neuronal nicotinic receptors in
various locations, including in the brain,15
ganglia,14 and spinal
cord,16 and to exhibit both competitive and
noncompetitive properties in antagonizing the central effects of
nicotine.17 A wide range of doses given by
different routes (0.125 mg/kg PO to 5 mg/kg
IV)16 18 have been found to be effective at
antagonizing the effects of nicotine in previous rat studies. Since
with an intravenous dose of 1.0 mg/kg mecamylamine it was
difficult to keep MAP at 90 mm Hg or above, we used a dose of 0.7
mg/kg IV in the present study. The complete inhibition of the
smoking-induced vasodilation by mecamylamine would demonstrate that the
vasodilation is most likely initiated by nicotine via an activation of
nicotinic receptors.
In canine experiments, nicotine seems to cause predominantly
ß-adrenoceptor–mediated vasodilation in the cerebral cortex,
although it also activates
Recent in vivo studies have suggested that ATP-sensitive
K+ channels are present in cerebral
arterioles.22 23 The opening of such channels in
the vascular smooth muscle cell causes
vasorelaxation.24 The complete inhibition of
smoking-induced pial vasodilation by glibenclamide, a putative
ATP-sensitive K+ channel
blocker,25 suggests that this response is
probably induced via an activation of ATP-sensitive
K+ channels in the cerebral arterioles.
Glibenclamide has been shown to block vascular K+
channels directly and to competitively antagonize vasodilation evoked
by ATP-sensitive K+ channel openers such as
diazoxide and cromakalim.25 Indeed, we have
previously demonstrated that topical application of glibenclamide can
effectively block cromakalim-induced pial arteriolar and venular
dilation in the dog in vivo.22 In rat studies,
the following occurred: (1) 20 mg/kg of glibenclamide given
intravenously blunted the vasodilator effect of the
K+ channel opener, diazoxide, but not that of the
L-type Ca2+ channel blocker,
nicardipine,26 and (2) 20 to 30
mg/kg of glibenclamide given intravenously inhibited the
vasorelaxant effects of cromakalim and diazoxide in
vivo.25 The dose of glibenclamide used in the
present study was similar to the doses used on those previous
studies. Since a recent in vivo study suggested that ATP-sensitive
K+ channels play an important role in
metabolic coronary vasodilation associated with
ß1-adrenoceptor
activation,27 glibenclamide may have modulated
the effects of sympathetic activation induced by cigarette smoking.
In addition, since NO acts as a neurotransmitter in the vasodilator
nerves innervating the cerebral arterial
wall,28 we anticipated that NO might also play a
role in smoking-induced pial vasodilation. In fact, the release of
acetylcholine induces NO production by the
endothelial cells.29 In practice,
the smoking-induced pial vasodilation was only partially attenuated by
L-NAME pretreatment, and its duration was actually prolonged (from 30
to 60 minutes). Systemic administration of L-NAME (3 to 190 mg/kg IV)
is widely used in physiological experiments in vivo
to produce NO synthase inhibition.30 31 32 33 In a
recent report, intravenous administration of L-NAME
partially inhibited the catalytic activity of brain NO synthase
(assayed ex vivo) in a time- and dose-dependent fashion in the
rat.33 Thus, 5 or 10 to 40 mg/kg of L-NAME
administered intravenously resulted in NO synthase
inhibition by 26% and 40%, respectively, 30 minutes after a bolus
injection, and 20 mg/kg of L-NAME resulted in a stable 52% inhibition
within 2 hours. The continuous intravenous infusion of
L-NAME at 10 mg · kg-1 ·
h-1 used in the present study might not have
been enough to achieve complete inhibition of NO synthase, but the fact
that the systemic hypertension occurred after the infusion seems to
suggest that vascular endothelial NO synthase was
substantially inhibited. Furthermore, it is possible that the systemic
effects associated with L-NAME administration (a decrease in pH) might
have been responsible for such a sustained dilation of pial
arterioles.
The fact that attenuated responses are associated with repeated
cigarette smoke inhalation has been reported by
others.34 35 We found evidence that short-term
repeated smoking of 1 mg nicotine–containing cigarettes attenuated the
pial vasodilation. Since the MAP change was not affected by such
repeated smoking, a rapid adaptation of the response of the pial
arterioles might have occurred, perhaps because of central effects on
the sympathetic innervation caused by repeated smoking. In addition, it
has been demonstrated that endothelium-dependent
vasodilation of the brachial arteries is impaired in apparently healthy
young adult smokers36 and that long-term smoking
is associated with a diminution of the NO-dependent component of basal
vascular tone.37 Such effects could account for
our finding that repeated smoking (even in the short term) causes an
impairment of cerebral vasodilation; in that case, the cause would be
endothelial dysfunction. A significant increase of the
plasma concentration of HbCO after both single and repeated cigarette
smoking could also affect the vascular responses. Traystman and
colleagues38 39 have reported that elevated
levels of HbCO produce a leftward shift of the oxyhemoglobin
dissociation curve for the remaining O2-available
binding sites and that this is the factor responsible for the excessive
CBF response. It is possible that a mechanisms of the vasodilator
response observed after single cigarette smoking may be due to elevated
levels of HbCO. Nevertheless, baseline high concentration of HbCO and
repeated exposure to CO may attenuate the vasodilator response
associated with smoking. One more difference between single and
repeated cigarette smoking on the cerebral vasculature was glucose
concentration. It has been reported that acute hyperglycemia decreases
cerebrovascular resistance and increases CBF40 ;
thus, hyperglycemia after single-cigarette smoking may modulate the
vasodilator effect. The initial vasoconstriction remained in the
repeated inhalation experiment, so if we can extrapolate from these
results, the slight but significant initial vasoconstriction may be
present and have some physiological importance
in the chronic smoker.
Previous studies4 9 have demonstrated CBF changes
at only 1 or 2 time points during smoking, and in no study has the time
course of the cerebrovascular responses been examined during and after
smoking. The initial constriction of pial vessels during smoking might
not be entirely due to the pharmacological action of cigarette smoke.
However, some vasoconstrictors—such as airway smooth muscle
TxA2 in guinea pigs,41
plasma endothelin-1 in humans,42 43 and cerebral
cortex serotonin in anesthetized
rats44 —have been found to be elevated after
smoking or nicotine administration. In the present study, the
arterial TxB2 level, a product of
TxA2, was found to be significantly elevated
early on in the period under study, and a TxA2
receptor antagonist,
seratrodast,45 46 inhibited the initial pial
vasoconstriction. Seratrodast has been reported to competitively
inhibit contractions of guinea pig tracheal strips and saphenous vein
strips in response to the TxA2 mimic, U-46619,
but it has not been reported to inhibit the contractions of tracheal
strips induced by leukotriene D4,
platelet-activating factor, or histamine.45
Seratrodast also competitively inhibits the binding of
[3H]U-46619 to Chinese hamster ovary cells into
which the TxA2 receptor–coding gene has been
introduced and which stably express the human
TxA2 receptor.46 These
findings suggest that the pharmacological effects of seratrodast are
due to its antagonism of TxA2 receptors. Since
seratrodast at doses of 0.08 to 5 mg/kg suppressed the
bronchoconstriction caused by intravenous U-46619 in an in
vivo study,45 the intravenous dose of
5 mg/kg seratrodast used in the present study should block or at
least reduce a TxA2-induced vasoconstriction.
Therefore, at least in part, smoking-induced
TxA2, which can cross the blood-brain
barrier,47 would be able to cause the initial
pial vasoconstriction. Although we did not measure circulating
endothelin and serotonin concentrations (because they do
not cross the blood-brain barrier47 ), we cannot
exclude the possibility that endothelin or serotonin
locally induced by smoking might also cause pial vessels to
constrict.
In the present study, there might be several limitations that
should be considered in evaluating the cerebrovascular changes related
with smoking inhalation. The increase of MAP from a minimum of 97 to
109 mm Hg to a maximum of 126 to 163 mm Hg that was
observed for 1 minute during cigarette smoking should affect the
autoregulatory response in cerebral vessels. In the steady-state
responses to hypertension in a feline study,48
smaller (37 to 59 µm in diameter) and larger (117 to 174
µm in diameter) vessels did not show any significant change in
caliber until blood pressure was elevated to 170 and 190 mm Hg,
respectively. Considering the difference in size between rats and cats,
it is uncertain whether pial arterioles in rats (21 to 75 µm)
would respond similarly to the same-sized vessels in cats. In a
previous rat study,49 cortical arterioles with a
resting diameter of 20 to 70 µm responded by nearly equal
proportional changes in diameter over 65 to 155 mm Hg of systemic
MAP for constant CBF. Therefore, the early phase of the response until
MAP became stable during and after cigarette smoking or nicotine
infusion may be partially modulated by the autoregulatory response in
cerebral vessels. And pretreatment with mecamylamine or L-NAME caused
baseline MAP decrease or increase. Although an increase in MAP
associated with L-NAME did not change the baseline diameter of pial
arterioles, an 8.2% increase in the arteriolar diameter associated
with mecamylamine would be possible to partially change the pial
arteriolar response to cigarette smoke per se. Since the effects of
mainstream cigarette smoke on the venules would be mostly passive when
cardiovascular changes occur, it would not be
reasonable to consider the possible mechanism of such an effect on the
pial venule as a result of cigarette smoking.48
Perhaps the vasodilator stimuli can dilate vessels upstream, resulting
in an increase in downstream capillary and venular microvascular
pressure. Venular changes could appear to be the summation of the
direct and indirect (passive) influences of smoking. Thus, we only
showed the effect of cigarette smoking on cerebral pial venules.
Although we have clearly demonstrated that acute cigarette smoking
causes an initial constriction and a subsequent dilation of pial
arterioles and although we have provided evidence to help identify some
of the mechanisms involved, it would be speculative to extrapolate from
our results to humans, if only because of the species differences.
However, the plasma nicotine concentrations after 1 mg
nicotine–containing cigarette smoking measured in the present
study (35.0±9.3 ng/mL) were quite consistent with those
recorded in human studies50 51 after
single-cigarette smoking (33 ng/mL [on average] in 330
smokers,50 greater than 60 ng/mL when a cigarette
was "smoked deeply,"51 and 15±3 ng/mL in 12
smokers52 ). At least on this basis, the results
of the present study may be relevant to the human smokers.
In summary, cigarette smoking appears to have a significant biphasic
effect on tone in the cerebral vasculature. Nicotine itself induced
vasodilation of pial vessels in a dose-dependent manner. The mechanisms
underlying the vasodilation induced by cigarette smoke may be initiated
by nicotine by means of an activation of nicotinic receptors. This may
cause vasodilation, at least in part, by sympathetic activation, NO
production, and K+ channel activation.
The initial vasoconstriction seems to be caused by the other
constituents of cigarette smoke, which may induce
TxA2. In addition, the pial vasodilation caused
by smoking shows attenuation on repeated smoking. Thus, multiple
mechanisms could be involved in the response of pial arterioles to
cigarette smoking.
Received December 30, 1997;
revision received May 12, 1998;
accepted May 13, 1998.
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Department
of Anesthesiology/Critical Care Medicine,
Johns Hopkins University,
School of Medicine,
Baltimore, Maryland
The effect of cigarette smoke on human physiology and pathophysiology
has been a hotly debated issue for many decades, and while most
researchers believe that cigarette smoke in many ways is harmful to
physiological systems, absolute hard data have not been easy to come
by. In this study, the effects of cigarette smoke to result in cerebral
vasoconstriction followed by vasodilation seems clear. The critical
question, however, is this: What are the mechanisms for these
responses? One of the problems centers around the fact that cigarette
smoke contains a multitude of constituents, and thus the effects of
cigarette smoke on the cerebrovasculature may be a net effect of not
only nicotine but many, many other constituents in mainstream cigarette
smoke as well. In mainstream cigarette smoke, carbon monoxide,
acidaldehyde, nitric oxides, hydrogen cyanide, acrolian, ammonia,
tobacco alkaloids, pyrites, and particulate matter are but only a few
of cigarette smoke's constituents. Precisely which of these agents
have direct effects on the cerebrovasculature, or which of these agents
affect or modulate other responses, is completely unclear. There may be
additive and/or synergistic effects of all of these agents. Thus, the
absolute mechanisms are unclear. There are also issues related to acute
cigarette smoke, chronic cigarette smoke, and secondary cigarette
smoke, and how the constituents within cigarette smoke may affect the
cerebrovasculature differently under all of these conditions.
Certainly, cigarette smoking for 20 years at a high volume may have
completely different effects than cigarette smoking of only a few acute
cigarettes. These issues need to be considered when examining the
effects of cigarette smoke on physiological and pathophysiological
parameters. Merely because a specific agent such as nicotine causes an
effect that appears similar to the effect of cigarette smoking does not
necessarily mean that nicotine is the major culprit in altering the
physiological response. Coupled with all of the other agents in
cigarette smoke, one does not know the other modulating factors. There
may be an association, but it may not in fact be a cause-and-effect
relationship. Similarly, one of the major constituents of cigarette
smoke is carbon monoxide. When inhaled, the combination with hemoglobin
produces elevated levels of carboxyhemoglobin and results in hypoxia.
Under conditions of hypoxia, the effects of the other constituents of
cigarette smoke may be modified by virtue of the hypoxic response of
the cerebral vessels. Thus, the issues related to the effects of
cigarette smoke and the cerebrovasculature again remain unclear despite
the fact that it is almost a certainty that constituents in cigarette
smoke can modify the cerebrovasculature itself. And as shown in this
manuscript, nicotine, as one constituent of cigarette smoke, clearly
does alter cerebral blood vessels under these methodological
circumstances.
Received December 30, 1997;
revision received May 12, 1998;
accepted May 13, 1998.
© 1998 American Heart Association, Inc.
Original Contributions
Mechanisms Underlying Cerebrovascular Effects of Cigarette Smoking in Rats In Vivo
Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
Background and Purpose—The effects
of acute smoking on cerebral circulation are controversial. This study
was designed (1) to clarify any differences between the effects of
cigarette smoking and nicotine infusion and between the effects of
single- and multiple-cigarette smoking on cerebral vessels and (2) to
probe the mechanism(s) underlying the vascular responses. 
-nitro-L-arginine methyl
ester; the initial vasoconstriction was inhibited by seratrodast, a
thromboxane A2 receptor antagonist
(n=6 in each case).
Key Words: cerebral vessels • cigarette smoking • microcirculation • nicotine • rats
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
Cigarette smoking is
known to be associated with atherosclerosis and to be
an important risk factor for stroke.1 2 3 Although
chronic cigarette smoking has been reported to reduce cerebral blood
flow (CBF),4 5 acute inhalation of cigarette
smoke or administration of nicotine has been reported to
increase,6 to maintain,7 or
to decrease8 CBF levels in smokers. These
apparent discrepancies between the effects of
chronic4 5 and acute
smoking6 and among acute
studies6 7 8 could arise from many factors,
including the dose of cigarette smoke or nicotine, the individual's
smoking history, and different timing of the measurements. Since
chronic smokers in effect undergo repeated acute inhalation of
mainstream smoke, it is crucial to clarify the acute influence of
cigarette smoking on cerebral vasculature.
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
Experimental Animals
We studied 86 male Sprague-Dawley rats weighing 350 to 400
g. Experimental protocols were approved by our institution's animal
care committee.
In protocol 1, we tested whether the main effect of cigarette
smoking on pial vessels was due to nicotine. We evaluated the effects
of mainstream smoke on pial vessels and compared them with the effects
of intravenous nicotine. Two different commercial
cigarettes, a high nicotine–containing cigarette (nicotine, 1 mg; tar,
13 mg; Marlboro, Philip Morris) and a low nicotine–containing
cigarette (nicotine, 0.1 mg; tar, 1 mg; Next, Philip Morris) were used
to provide mainstream smoke in 6 rats each. Each animal inspired
mainstream smoke for 1 minute through its tracheal cannula (by way of
the ventilator). The gas inlet of the ventilator was divided into 2
tubes. A lighted cigarette was fitted into one of them, and oxygen
supply tube was connected to the other one. Therefore, a mixture of
mainstream cigarette smoke, air, and oxygen was inspired through the
ventilator. Nicotine (0.05 mg per rat in 0.5 mL saline) (Sigma Chemical
Co) was infused intravenously over 1 minute in 6 rats. We
chose 0.05 mg nicotine for infusion because in our pilot study this
dose induced an increase in MAP that was similar to that caused by
smoking a 1 mg nicotine–containing cigarette for 1 minute. -nitro-L-arginine methyl
ester (L-NAME, 10 mg · kg-1 ·
h-1, continuous intravenous
infusion) (Sigma). The effect of smoking a 1 mg nicotine–containing
cigarette on pial vessels was investigated as in protocol 1.
Mecamylamine, propranolol, and glibenclamide were given 5
minutes before smoking. L-NAME was started 30 minutes before
smoking.
All variables (time-dependent effects of smoking, of
nicotine injection, of repeated smoking, and of smoking after
pretreatment with mecamylamine, propranolol, L-NAME,
glibenclamide, or seratrodast) were examined by a 1-way ANOVA for
repeated measurements followed by the Scheffé F test for post hoc
comparison. The effects of mecamylamine, propranolol,
L-NAME, glibenclamide, seratrodast, or repeated smoking on the
smoking-induced pial vessel changes were compared with control values
(no pretreatment) by a 2-way ANOVA. An unpaired t test was
used to examine the differences between 2 groups. A paired t
test was used to determine the significance of changes in the plasma
TxB2 level induced by cigarette smoking or
intravenous nicotine administration. Significance was set
at P<0.05. All values are presented as
mean±SEM.
Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
Effects of Smoking or Nicotine Infusion on Pial Vessels and
Physiological Values (Protocol 1)
Acute single-cigarette smoking and nicotine infusion both caused
significant changes in pial arteriolar and venular diameter and MAP
(Figure 1
). Smoking (both 0.1 mg and 1 mg
nicotine–containing cigarettes) caused pial arterioles to constrict at
30 seconds; they then dilated, with the peak occurring at 5 to 10
minutes and being dose dependent (in terms of nicotine and tar).
Nicotine infusion caused a vasodilation of pial arterioles without an
initial vasoconstriction. Venular vasodilation was induced at 2 to 10
minutes after the smoking of a 1 mg nicotine–containing cigarette or
nicotine infusion. MAP increased significantly during smoking or
nicotine infusion but decreased to baseline immediately after the
inhalation or infusion was stopped. Thus, the peak vasodilation
associated with cigarette smoking occurred some 4 minutes or more after
the peak increase in MAP, at a time when MAP was at or below baseline
values. In terms of MAP, there was no significant difference between
these 2 groups throughout the experiment. There was no marked change in
HR after smoking or nicotine infusion, except for a decrease at 2
minutes after nicotine infusion (Figure 1).
View larger version (31K):
[in a new window]
Figure 1. Line graphs showing the effect of smoking or
nicotine infusion on pial arteriolar (A) and venular (B) diameter, mean
blood pressure (C), and heart rate (D) in rats. Inhalation of smoke
from low or high nicotine–containing cigarettes caused pial arterioles
to constrict at 30 seconds and then to dilate (peaking at 5 to 10
minutes). Intravenous infusion of nicotine caused
vasodilation of pial arterioles without the initial vasoconstriction
observed on cigarette smoking. Values are mean±SEM (low
nicotine–containing cigarette, n=6; high nicotine–containing
cigarette, n=6; and intravenous nicotine, n=6).
*P<0.05, 
P<0.01 compared with
baseline.).
View this table:
[in a new window]
Table 1. Changes in Physiological
Parameters During and After Smoking or Nicotine
Infusion
Blockade of autonomic ganglia (nicotine receptor blocking) with
mecamylamine completely inhibited the smoking-induced pial arteriolar
vasodilation (Figure 2A). The small, but
significant, initial vasoconstriction was not reduced by mecamylamine;
in fact, it was prolonged (it persisted for 30 minutes).
View larger version (24K):
[in a new window]
Figure 2. Line graphs showing the effects of mecamylamine
(A), propranolol (B), L-NAME (C), and glibenclamide (D) on
the responses to smoking a 1 mg nicotine–containing cigarette. The
initial vasoconstriction observed was not affected by mecamylamine,
propranolol, L-NAME, or glibenclamide. The smoking-induced
vasodilation was completely inhibited by mecamylamine, greatly
inhibited by glibenclamide, and reduced by propranolol or
L-NAME. Values are mean±SEM (control, n=6; mecamylamine, n=6;
propranolol, n=6; L-NAME, n=6; and glibenclamide, n=6).
*P<0.05, P<0.01 compared with
baseline.
). ). Pial arterioles showed a continuous
smoking-induced dilation from 2 to at least 60 minutes as a result of
L-NAME pretreatment. ). Baseline
pial vessel diameters did not change as a result of
propranolol, L-NAME, and glibenclamide administration,
although they were dilated 8.2% by mecamylamine pretreatment. ).
The changes in MAP and HR during protocol 2 are summarized in Table 3. MAP decreased with mecamylamine and
increased with L-NAME.
View this table:
[in a new window]
Table 2. Changes in Blood Glucose Concentration During and
After Smoking in the Presence of Antagonists
View this table:
[in a new window]
Table 3. MAP and HR Changes During Experiments With
Antagonists
The TxA2 receptor blocker, seratrodast,
completely inhibited the smoking-induced initial pial arteriolar
vasoconstriction. The subsequent vasodilation was not increased in
amplitude by seratrodast, but it was prolonged (it persisted for 60
minutes) (Figure 3). Baseline pial vessel
diameters did not change as a result of seratrodast administration.
Physiological parameters did not change
throughout the experiment, and the changes in MAP and HR are shown in
Table 3.
View larger version (20K):
[in a new window]
Figure 3. Line graph showing the effect of seratrodast on
the response to smoking a 1 mg nicotine–containing cigarette. The
initial vasoconstriction was completely inhibited by seratrodast, and
the subsequent vasodilation was prolonged (it persisted for at least 60
minutes). Groups are as follows: control (n=6) and seratrodast (n=6).
*P<0.05, P<0.01 compared with
baseline.). The second
measurement was made immediately after the cessation of the smoking or
nicotine infusion.
View larger version (22K):
[in a new window]
Figure 4. Plasma TxB2 concentration was
significantly elevated after smoking; after nicotine infusion, it was
not changed. Values are mean±SEM (smoking, n=6; nicotine infusion,
n=6). *P<0.01 compared with baseline.
Although the initial vasoconstriction was similar in size during
the smoking of the fourth cigarette in repeated smoking, the subsequent
vasodilation was attenuated from 17.9±3.7% to 4.2±1.2% (compared
with the effect of smoking a single cigarette). MAP increased
significantly during repeated smoking as it did in the single-dose
experiment, and again it returned to baseline immediately after the
cessation of smoking (Figure 5).
Arterial blood gas tensions, pH, serum electrolytes, and
body temperature did not change significantly throughout the
experiment. As shown in Table 4, in
contrast to single-cigarette smoking, there was not significant
increase in blood glucose after repeated smoking. Carboxyhemoglobin
(HbCO) was significantly increased after smoking a single cigarette
(P<0.01), and repeated smoking caused HbCO to rise
significantly higher than the level observed in the single-dose
experiment.
View larger version (26K):
[in a new window]
Figure 5. The initial vasoconstriction observed after
smoking was still present after repeated smoking, but the
smoking-induced vasodilation was attenuated. Values are mean±SEM
(single, n=6; repeated, n=6). *P<0.05,
P<0.01 compared with baseline.
View this table:
[in a new window]
Table 4. Changes in HbCO and Blood Glucose Concentration
During and After Single or Repeated Cigarette Smoking
Venous nicotine concentration rose from 5 ng/mL or less at
baseline to 35.0±9.3 ng/mL immediately after smoking a 1 mg
nicotine–containing cigarette and to 22.5±1.2 ng/mL after nicotine
infusion and then declined to 24.1±5.6 ng/mL and to 15.1±1.8 ng/mL at
30 minutes, respectively.
Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
The major finding of the present study was that acute
inhalation of mainstream cigarette smoke produced a significant
biphasic change in the diameter of cerebral arterioles. Smoke
inhalation caused pial arterioles to constrict at 30 seconds, followed
by a dose-related vasodilation that peaked at 5 to 10 minutes; MAP
increased only during the inhalation and returned to baseline 4 minutes
or more before arteriolar diameter reached its peak. Since the increase
in arteriolar diameter was not concomitant with the increase in MAP,
the changes in arteriolar diameter seem likely to be due to a direct
effect of smoking on vascular tissue. Nicotine infusion caused a
vasodilation of pial vessels without an initial vasoconstriction (in
spite of a similar increase in MAP). Thus, the initial vasoconstriction
caused by cigarette smoking seems likely to be due not to the airborne
nicotine in cigarette smoke but to some smoking-induced substance.
Since the vasoconstriction was blocked by seratrodast, the substance
responsible for vasoconstriction is presumably
TxA2. We also found that mecamylamine,
propranolol, L-NAME, and glibenclamide all reduced or
prevented the dilation of cerebral vessels induced by acute cigarette
smoking in the present rat model. The mechanism by which inhalation
of mainstream smoke from a low or high nicotine–containing cigarette
causes the vascular tone to decrease could be activated by
nicotine and involve, at least in part, sympathetic activation,
stimulation of NO production, and the opening of ATP-sensitive
K+ channels. 
-adrenoceptors and a
nonadrenergic, noncholinergic vasodilator
mechanism.19 The present attenuation of the
smoking-induced vasodilation by propranolol would indicate
that the vasodilation is, at least in part, achieved via ß-adrenergic
stimulation. The significant increase in blood glucose concentration
observed in the present study might be indirect evidence of
smoking-induced sympathetic nerve stimulation.20
The transient increase in MAP without a change in HR at the end of
smoke inhalation observed in the present study is presumably due to
a smoking-inducing sympathetic activation. Referring to the previous
studies,21 we used 1 mg/kg IV
propranolol for effective blocking of ß-adrenoceptors.
Although we cannot exclude the possibility that a complete inhibition
with propranolol could occur if we used a different timing
or dosage, we suspect that the pial vessel response to smoking cannot
be attributed entirely to sympathetic ß-adrenoceptor stimulation.
Acknowledgments
This work was supported by Grant-in-Aid for Scientific Research
No. 08457405 and No. 09671555 (Ministry of Education, Science, and
Culture, Japan).
References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
1.
Kannel WB. Current status of
epidemiology of brain infarction associated
with occlusive arterial disease. Stroke. 1971;2:295–318.
Editorial Comment
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
In this interesting article, the authors attempt to determine the
mechanisms underlying the cerebrovascular effects of cigarette smoking
in rats in vivo. The authors used a standard pial window technique to
examine the effects of cigarette smoke and nicotine and a variety of
blocking agents to determine the potential mechanisms of nicotine's
vasoconstrictory and vasodilatory effects on the pial vessels. The
authors found that cigarette smoking had a biphasic effect on
cerebrovascular tone, ie, vasoconstriction followed by vasodilation.
The authors used a variety of pharmacological agents to demonstrate
that the vasodilation is most likely an effect of nicotine, at least in
part mediated via sympathetic activation, nitric oxide production, and
K+ channel activation. They speculate that the
vasoconstriction is due partly to thromboxane A2 induced by
cigarette smoke.
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