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Alteration of alpha and muscarinic receptors in rat brain and heart following chronic nicotine treatment
Brain Research, 348 (1985) 241-248 Elsevier
241
BRE 11156
Alteration of Alpha and Muscarinic Receptors in Rat Brain and Heart Following Chronic Nicotine Treatment KYOZO YAMANAKA, MASAFUMI OSHITA and IKUNOBU MURAMATSU
Department of Pharmacology, Fukui Medical School, Matsuoka, Fukui 910-11 (Japan) (Accepted February 12th, 1985)
Key words: nicotine - - chronic treatment - - a~-receptor - - Ctz-receptor- - muscarinic receptor - cerebral cortex - - hypothalamus/thalamus - - brainstem
Adrenergic and muscarinic binding sites in 4 brain regions (cerebral cortex, corpus striatum, hypothalamus/thalamus and brainstem) and in heart ventricles were measured in rats chronically treated with nicotine added to the drinking water in doses ranging from 6 to 8 mg/kg/day, for 4 weeks. Control rats received only tap water. The nicotine treatment led to increases in the specific binding of both [3H]prazosin and [3H]clonidine in the cerebral cortex. An increase in [3H]prazosin binding was also observed in the hypothalamus/thalamus of nicotine-treated rats. These changes were all due to an increase of about 23% in Bmax. In the brainstem and heart left ventricle, respectively, an increase and a decrease in the affinity of [3H]quinuclidinylbenzilate binding were observed. There were no changes of the binding parameters for the 3 radioligands in other regions tested, and no alteration of [3H]dihydroalprenolol binding was detected in any region examined. These results indicate that chronic administration of nicotine causes an increase in the density of a l- and a~-binding sites in some brain regions and reciprocal changes of the affinity of muscarinic binding sites in the brain and in the heart.
INTRODUCTION
peripheral adrenergic n e u r o n s has also been noted in rats receiving nicotine chronically 13. However, the
Acute administration of small doses of nicotine produces an activation of cortical electroencephalogram ( E E G ) and behavioral arousal in h u m a n s and animals~6,lv,3L Most of the effects may be due to ac-
long-term effects of nicotine on central adrenergic and muscarinic receptors is poorly understood. The present study was designed to assess binding sites changes in brain and heart following chronic nicotine treatment. Nicotine was added to the drinking water given to rats, for 4 weeks as reported by Hfig-
tions on central cholinergic neurons19.32 but catecholaminergic n e u r o n s may also be involved since nicotine causes an increased turnover and release of catecholamines in the hypothalamusl.2,12 and nigrostriatal system 20,21. On chronic administration, nicotine produces tolerance to the acute behavioral effects 16,24. Chronic nicotine treatment has been shown to induce an increased density of nicotine binding sites in brain24. The up-regulation of nicotinic binding sites after chronic nicotine treatment reflects adaptive change of the binding sites to a desensitized conformational form that is functionally inactive24~33. Thus, the sensitivity of central nicotine receptors decreases after chronic nicotine treatment. Decreased sensitivity of
gendal and H e n n i n g 13, and adrenergic (Ctl-, a2-, fl-) and muscarinic binding sites were measured in discrete brain regions (cerebral cortex, corpus striatum, hypothalamus/thalamus and brainstem) and heart ventricles, using a receptor binding technique. We found localized changes in ct- and muscarinic binding sites in the brain and heart following chronic nicotine treatment. MATERIALS AND METHODS
Animals and nicotine treatment Male Wistar rats (6 weeks of age: initial body
Correspondence: I. Muramatsu, Department of Pharmacology, Fukui Medical School, Matsuoka, Fukui 910-11, Japan. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
242 weight of 210-230 g) were housed in groups of 5 animals with a 12 h day/night cycle and free access to the usual chow diet. Three groups of the rats were given tap water for drinking, ad libitum, as the control group. An additional 3 groups were provided with tap water containing nicotine in a dose of 100 rag/liter (as nicotine base) for 4 weeks. The nicotine solution provided for drinking was freshly prepared daily by dissolving bitartrate in the drinking water, with an adjustment of pH to 5.5-6.(I. The average daily consumption of nicotine by each animal was calculated to be between 6-8 mg/kg. All rats were weighed once daily. Control rats gained in body weight throughout the 4 weeks to 383 _+ 7 g, whereas the nicotine-treated rats gained only to 333 _+ 6 g (significantly different from the control; P < (/.()l). The nicotine-induced weight loss was in agreement with reports by Schechter and Cook 29 and Hfiggendal and Henning 13.
Membrane preparation Both control and nicotine-treated rats were killed 4 weeks after the beginning of the nicotine treatment. Four brain regions (cerebral cortex, corpus striatum, hypothalamus/thalamus and brainstem) and the left and right heart ventricles of each rat were dissected on ice. Tissue from each brain region (except the cortex) and from heart ventricles was pooled from the 3-5 animals of each group. Tissues were homogenized twice for 15 s with a 30-s rest. using a Kinematika Polytron PT-10 (setting at 6.(i) in 40 vols. of ice-cold 50 mM Tris-HCl buffer (pH 7.7 at 25 '~C). The homogenates were incubated for 10 rain at 37 °C, and centrifuged at 62,700 g for 15 rain at 4 °C. The pellets were washed once by suspension in the original buffer and centrifugation under the same conditions. The resulting pellet was resuspended in the original volume of Tris-HC1 buffer and used for the binding assays.
Radioligand binding assays (~-, (z~-, fl- and muscarinic binding sites in the brain were measured from saturation experiments with a specific radioligand for each receptor; that is, [3H]prazosin, [3H]clonidine, [3H]dihydroalprenolol (DHA) and [3H]quinuclidinyl benzilate (QNB), respectively. The binding assays, except ¢]-receptor assay, were carried out according to the methods de-
scribed by Hornung et al.ls I,.'Prichard e~ a|. ~ and Yamamura and Snyder 3~, respectively, with mino~ modifications :~'>. In the binding assays with [-~H]prazosin, ! H]clonidine and. [3H]DHA, the tissue homogenates (5 or 7~5 mg of tissue wet wt.) were incubated with 6 or 8 different concentrations of each radioligand @H]prazosin, 0.03-2.35 nM; [3H]clonidme, /). 13.-,2.68 nM; [3H]DHA, 0.09-1.58 nM) for 30 min at 25 ~C, in a final 2-ml volume of 50 mM Tris-HC1 buffer (pH 7,7). In the [3H]QNB binding assay, the homogenates (0.5 mg of tissue wet wt.) were incubated with 8 different concentrations (9-350 pM) of the radioligand for 60 rain at 37 °C, in a total 2-ml volume of 50 mM Na +, K*-phosphate buffer (pH 7.4). Cardiac binding sites (a I-, [:•- and muscarinic receptors) were measured under the same assay conditions as brain binding sites. except for the use of 7.5 and 1.25 mg of tissue wet wt. for [3H]prazosin (0.09-2.33 nM)'and [-~H]QNB binding assays, respectively. All assays were performed in duplicate. The binding was terminated by rapid filtration over Whatmann GF/B filters, under reduced pressure, followed by 4 × 4 ml rinses with ice-cold incubation buffer. The filters were translerred into scintillation vials and dried overnight. The radioligand retained on the filters was extracted by 8 ml of scintillation fluid for at least 12 h. The radioactivity was counted using a Packard Tri-Carb Scintillation spectrometer at efficiences of 32-36%. Non-specific binding of each radioligand was defined as the binding in the presence of phentolamine (10/~M), l~-norepinephrine (10gM), propranolol (0.1 uM) and atropine (1 ~M) for the binding assays of czl-, ~--,/4- and muscarinic sites, respectively. The specific binding as percentage of total ranged 51-96% in the case of prazosin, 72-99% in the case of ctonidine, 45.7-90% in the case of D H A (except 25-50% for brainstem) and 93-98% in the case of QNB, respectively. Protein measurement was performed by the method of Lowry et al. 23, using bovine scrum albumin as a standard.
Data analysis Saturation isotherms for each radioligand binding constructed from saturation experiments were analyzed by estimation of the maximum number of binding sites (Bronx) and the apparent dissociation constant (K~t), according to the Scatchard analysis de-
243 scribed by B e n n e t 3. Hill coefficients (nil) w e r e also
RESULTS
e s t i m a t e d f r o m the d a t a by Hill analysis. A n a l y s e s of S c a t c h a r d and Hill plots w e r e c a r r i e d out using a
Regional variation o f radioligand binding in brain
m e t h o d of linear l e a s t - s q u a r e s r e g r e s s i o n analysis.
T h e p a r a m e t e r s for specific binding of r a d i o l i g a n d s
All results w e r e e x p r e s s e d as m e a n v a l u e + S . E .
([3H]prazosin,
f r o m 3 d i f f e r e n t groups. T h e statistical e v a l u a t i o n of the data was e x a m i n e d by u n p a i r e d ( t w o - t a i l e d ) Stud e n t ' s t-test.
[ 3 H ] O N B ) in discrete brain regions are s u m m a r i z e d
pH]clonidine,
[3H]DHA
and
in T a b l e I. All of the radioligands b o u n d to a single
Materials
gave slope factors (nil) of close to unity (1t.92 _+
p o p u l a t i o n of binding sites with a high affinity in the regions tested, since Hill analysis of s a t u r a t i o n d a t a
Radio[igands
([3H]prazosin,
17.4
Ci/mmol;
[3H]clonidine, 20.5 C i / m m o l ; [ 3 H ] D H A , retool; [ 3 H ] Q N B ,
0 . 0 7 - 1 . 0 6 _+ 0.06).
34.1 Ci/
In g e n e r a l , t h e r e was a regional v a r i a t i o n in the
33.2 C i / m m o l ) w e r e p u r c h a s e d
m a x i m u m n u m b e r of binding sites (B ...... ) for each ra-
from New England Nuclear (Boston, MA, U.S.A.)
dioligand.
and o t h e r agents used in this study w e r e o b t a i n e d
[3H]clonidine binding d e c r e a s e d in the rank o r d e r of
The
Bma x values for [3H]prazosin
f r o m c o m m e r c i a l sources; a t r o p i n e sulfate, D,L-pro-
c o r t e x => h y p o t h a l a m u s / t h a l a m u s
pranolol hydrochloride
striatum.
(Nakarai Chemicals,
Ltd.,
The
B .....
values
for
>
and
brainstem
[3H]DHA
> and
K y o t o , J a p a n ) , L - n o r e p i n e p h r i n e b i t a r t r a t e (Sigma C h e m i c a l s , St. L o u i s , M O , U . S . A . ) and p h e n t o l a -
o r d e r of s t r i a t u m > c o r t e x > > h y p o t h a l a m u s / t h a l a -
m i n e m e s y l a t e ( C i b a - G e i g y , Basel, S w i t z e r l a n d ) .
mus > b r a i n s t e m . T h e K,t v a l u e for each r a d i o l i g a n d
[3H]QNB binding, h o w e v e r , d e c r e a s e d in the rank
TABLE I
Parameters of specific -~H-ligand binding for discrete regions of the rat brain after chronic nicotine treatment Each brain region except the cerebral cortex isolated from 3-5 rats in one group was pooled, and the membrane fraction was used in binding study. The binding parameters were determined by saturation studies using 0.03-2.35 nM of [3H]prazosin. 0.13-2.68 nM of [3H]clonidine, 0.09-1.58 nM of [3H]DHA and 9-350 pM of [3H]QNB. The values given represent mean _+ S.E. from 3 groups of rats. but the values of cortex were obtained from 4 rats. Control, nicotine-untreated rats: nicotine treatment, rats in which nicotine was administered for 4 weeks. Hill analysis of all the data gave coefficients close to unity.
~H-ligand
Region
Control B,,a~ (fmol/mg protein)
Nicotine treatment K a (pM)
B ..... (fmol/mg protein)
[3H]Prazosin
Cerebral cortex Corpus striatum Hypothalamus/thalamus Brainstem
235.2 81.5 207.1 109.8
_+ 2.9 + 8.1 __+7.2 ± 9.1
99.0 +_ 14.1/ 117.7 + 13.5 100.9 ± 6.2 153.2 + 35.11
[3H]Clonidine
Cerebral cortex Corpus striatum Hypothalamus/thalamus Brainstem
110.3 51.0 115.9 81.5
_+ 7.8 _+ 7.7 + 0.6 _+ 5.9
715.8 + 147.0 704.8 _+ 208./) 1135.1 ± 150.8 1000.7 ± 203.8
[3HIDHA
Cerebralcortex Corpus striatum Hypothalamus/thalamus Brainstem
182.3 196.3 113.4 72.6
_+ 15.4 _+ 3.6 + 2.6 ± 3.2
422.4 468.8 617.7 509.1
[3HIQNB
Cerebral cortex Corpus striatum Hypothalamus/thalamus Brainstem
1577.5 1596.1 491.8 413.2
± 64.9 _+ 143.9 _+ 17.9 ± 12.9
73.7 52.4 41.9 44.0
* Significant difference from controls, P < 0.05.
± + + ±
14.4 14.9 55.9 36.9
± 6.7 + 7.1 _+ 7.7 +_ 3.9
290.5 _+ 13.9" 111.6 __+25.2 254.7 ± 8.8* 118.3 + 15.8 135.5 55.0 123.0 82.4
_+ 4.3* ± 1.7 + 4,4 + 2.3
180.8 202.9 117.2 68.4
± 10.1 +_ 5.3 _+ 6.3 ± 9.3
1519.8 ± 70.7 1657.6 _+ 1118.9 511.8 ± 39.0 376.6 ± 7.2
K~I(pM) 119.2 ±_ 15.0 148.0 +__29.0 111.9 + 9.9 132.1 ± 13.11 919.7 +_ 69.6 784.7 +_ 98.1 1152.4 ± 74.4 887.6 + 51/.1/ 424.6 462.8 612.3 417.3
+ 36.5 +_ 29.7 ± 47.0 _+ 67.7
53.9 50.2 42.2 29.9
+_ 6.7 +_ 7.4 _+ 4.4 ± 1.4"
244
[
e x h i b i t e d m u c h less regional variation.
50 I \ \ , 0
Central C~l-receptor T h e Bma x v a l u e s for specific [3H]prazosin b i n d i n g in the c o r t e x and h y p o t h a l a m u s / t h a l a m u s
i \ .... E \
.,2-" c:
w e r e in-
c r e a s e d f r o m 235.2 to 290.5 f m o l / m g p r o t e i n , and f r o m 207.1 to 254.7 f m o l / m g p r o t e i n , r e s p e c t i v e l y , or by a p p r o x i m a t e l y 2 3 % in e a c h case ( P < 0.05) ( T a b l e I). T y p i c a l d a t a for cortical s a m p l e s are s h o w n in Fig, 1; no c h a n g e in K a but a c h a n g e in Bma x was ob-
,oo
o
F
•
t ~\ ~
e
°
o
-x ~.
ta_ \
i
treatment
",, cX
•
50 i i
Control
:) Nicotine
s e r v e d b e t w e e n the two groups. T h e Hill coefficients w e r e also u n c h a n g e d , T h e s t r i a t u m and b r a i n s t e m i
0
s h o w e d n o d i f f e r e n c e s in any binding p a r a m e t e r for
5O
150
I00
[3HJprazosin b e t w e e n c o n t r o l and n i c o t i n e - t r e a t e d groups.
B (fmol/mg
Central a2-receptor T h e Brnax v a l u e for c o r t e x was i n c r e a s e d f r o m 110.3 to 135.5 f m o l / m g p r o t e i n , or by a b o u t 23% ( P < 0.05) ( T a b l e I) in the n i c o t i n e - t r e a t e d g r o u p large-
protein)
Fig. 2. Representative Scatchard plots of specific pH]clonidine binding for cerebral cortex from control and nicotine-treated rats. The binding parameters for control rat (@) are Bin,x = 112.6 fmol/mg protein, Ka = 1.01 nM and n H = 1.00, and those for nicotine-treated rat ((3) are BmaX = 141.3 fmol/mg protein, Kd = 0.92 nM and n H = 1.01, respectively.
ly b e c a u s e of an i n c r e a s e in total binding. Typical data are s h o w n in Fig. 2; again n o c h a n g e in K a was observed
between
control
and
nicotine-treated
groups. A l t e r a t i o n s o f the b i n d i n g p a r a m e t e r s w e r e
Bmax and K a v a l u e s for [ 3 H ] D H A binding in the 4
not o b s e r v e d in o t h e r r e g i o n s tested.
brain regions e x a m i n e d ( T a b l e I).
Central fl-receptor N i c o t i n e t r e a t m e n t p r o d u c e d no a l t e r a t i o n o f the
Central muscarinic receptor In the n i c o t i n e g r o u p , t h e r e was a significantly ( P
3.0
o
.=
20
•
Control
o
Nicotine
treatment
o. o.°
~ I0
• ~
Control
o Nicotine treatment
E
m
'°I
m
0
I O0
200
B (fmol/mg
300
protein)
Fig. 1. Representative Scatchard plots of specific [3H]prazosin binding for cerebral cortex from control and nicotine-treated rats. The binding parameters for control rat (@) a r e B m a x = 230.6 fmol/mg protein, Ka = 122.3 pM and n H = 0.97, and those for nicotine-treated rat ((3) a r e B m a x = 304.5 fmol/mg protein, Kj = 119.3 pM and n H = 0.87, respectively.
0
•
I O0
2 O0
B (fmol/mg
3 O0
400
protein)
Fig. 3. Representative Scatchard plots of specific [-~H]QNB binding for brainstem from control and nicotine-treated rats. The binding parameters for control rat (O) are Bmax = 388.8 fmol/mg protein, K a = 51.9 pM and n H = 0.96, and those for nicotine-treated rat ((3) are Bmax = 380.9 fmol/mg protein, Ka = 32.2 pM and n H = 0.98, respectively.
245 < 0.05) lower Kj value of [3H]QNB binding (29.9 + 1.4 pM) for the brainstem, as compared with the value (44.0 + 3.9 pM) in the control group (Table I). This indicates an increased affinity of the radioligand binding. A representative example is shown in Fig. 3, in which no difference in Bma x values were observed between the two groups. In other brain regions, neither Bma x n o r K a values differed between the control and nicotine groups.
Cardiac (at-, fl- and muscarinic) receptors All 3 ligands ([3H]prazosin, [3H]DHA, [3H]QNB) bound to single populations of sites in the heart ventricles, as evidenced by Hill coefficients of close to unity (0.97 + 0.02-1.16 + 0.02). Bmax and K a values in left and right ventricles are summarized in Table II. There were no alterations in binding parameters for either [3H]prazosin or [3H]DHA in the heart ventricles following chronic nicotine treatment. The nicotine group, however, showed a significantly (P < 0.05) higher K d value for [3H]QNB binding (58.7 + 7.9 pM) in the left ventricle, than the controls (31.9 + 6.5 pM) (Table II). This finding indicates a decreased affinity. A typical example is shown in Fig. 4, in which there was no significant change in the Bmax value. In the case of the right ventricle, no alteration in binding parameters was observed after chronic nicotine treatment.
40
30 Q.
Control
C oJ
o Nicotine
treatment
a.
20
E LI_
m
I0
°2. I
50
B (fmol/mg
tO0
protein)
Fig. 4. Representative Scatchard plots of specific P H I Q N B binding for heart left ventricle from control and nicotine-treated rats. The binding parameters for control rat ( 0 ) are BmaX = 82.2 fmol/mg protein, K d = 24.4 pM and n H = 0.97, and those for nicotine-treated rat (©) are Bma× = 93.3 fmol/mg protein, K d = 66.4 pM and n~ = 0.97, respectively.
TABLE II
Parameters of specific-~H-ligand binding for left and right ventricles of the rat heart after chronic nicotine treatment Each hcart region isolated from 3 rats in one group was pooled, and the membrane fraction was used in binding study. The binding parameters were determined by saturation studies using 0.09-2.33 nM of [3H]prazosin, 0.09-1.58 nM of [3H]DHA and 9-350 pM of [3H]QNB. The values given represent mean + S.E.M. from 3 groups of rats. Control, nicotine-untreated rats; nicotine treatment, rats in which nicotine was administered for 4 weeks. Hill analysis of all the data gave coefficients close to unity.
Region
3H-ligand
Control Bmax (fmol/mg protein)
Nicotine treatment Ka (pM)
B .... (fmol/mg protein)
Kj (pM)
Left ventricle
[3H]Prazosin [3H]DHA [3H]QNB
75.1 + 8.6 74.4 _+ 5.1 84.1 + 5.1
125.3 + 29.5 409.1 + 41.1 31.9 + 6.5
78.7 + 9.9 75.4 + 12.3 95.9 + 4.7
118.9 + 23.8 448.6 _+ 42.9 58.7 + 7.9*
Right ventricle
[3H]Prazosin [3H]DHA [3H]QNB
64.2 + 4.9 83.5 + 3.4 103.6+4.8
88.8 + 16.0 690.8 +_ 10.0 41.9+_ 1.0
64.8 + 3.9 89.2 + 12.7 116.2,+ 8.0
79.1 + 9.4 730.9 +_ 20.1 59.5_+ 13.5
* Significant difference from control, P < 0.05.
246 DISCUSSION Receptor binding assays have revealed changes of presumed central receptors which reflect alterations in neuronal activities in the brain after chronic administration of drugs to animals ~s,3(,,:<. We used such binding assays to assess the effects of chronic nicotine treatment on the rat brain and heart
Alteration of adrenergic receptors and a2-binding sites which were separately labeled by [3H]prazosin and [3H]clonidine, exhibited increased numbers in forebrain regions following nicotine treatment, a~-Sites increased in the cerebral cortex and hypothalamus/thalamus, and a2-sites increased in the cerebral cortex. Similar evidence has been reported for the same rat brain regions following reserpine and 6-hydroxydopamine treatment 31. Such phenomena have been considered as up-regulation of the receptors subsequent to a decreased activity of noradrenergic neurons induced by chemical denervation. The majority of al- and a2-binding sites in the cortex and hypothalamus/thalamus are postsynaptic to noradrenergic nerve terminals3~ which originate primarily from the locus coeruleus >. Thus, our finding may be indicative of depressed activities of noradrenergic neurons derived principally from the dorsal bundle 2e. However, the extent of increase in cortical (~ receptors obtained in this experiments was limited to only 23%, as compared with the much larger effects of chemical denervation of the dorsal (tegmental) bundle (74 and 80% increase in (zI and a 2 receptors, respectively) 31. Chemical denervation also induced a 62% increase in cortical/4 receptors~t. Concomitant changes in/3 and a receptors in the brain were also noted in rats given DSP-4, a selective noradrenergic neurotoxin 7, or a few chronic treatments (3-4 weeks) with antidepressants and lithium :s. No alteration of antagonist binding for fi receptors was seen. however, in the nicotine-treated rats. These findings indicate that long-term ingestion of nicotine produces slight changes in only the cz binding sites. a j-
Alteration of muscarinic receptors [3H]QNB binding exhibited an increased affinity in the brainstem and a decreased affinity in the heart left ventricle in the nicotine-treated rats. These find-
ings may indicate an alteration of the kinetics of mu~. carinic receptors for the binding of the antagonist. There are reports that [ql]QNB binding is mcreased by guanine nucleotides in the r:~'~ heart and brainstem (medulla-pons) ',i~ . Such an enhancement of antagonist binding is suggested to be dm ~to a conversion of low-affinity antagonist sites miv high-af° finity sites 11, probably regulated by the c,mpling of guanine nucleotide binding protein. Thu~, !he allerations in muscarinic receptors seen in our experiments might be ascribed to a conversion of antagonist affinity sites, perhaps at the level of coupling states of the receptor to the regulatory protein ~1. However, a1 present, the exact mechanism related *o affinity changes and the relation of reciprocal changes between central and peripheral muscarinic receptors remain unclear. Alterations in muscarinic receptors occur in animals receiving organophosphates chronically, flowever, such treatment induces a decreased density of muscarinic receptors in the corpus striatum and cerebral cortex, but not in brainstem and heart left ventricle 3('.37. This is in contrast to the results in the nicotine-treated rats. Such differences are probably related to the localized action of nicotine which is mediated through central nicotinic receptors exhibiting regional variation in distribution in the brain ~
Relation to the pharmacological long-term e{~ects ()/ nicotine Pharmacological long-term effects ot nicotine seem to be diverse, depending on the challenge doses. For example, low doses (1--2 mg/kg/day) of nicotine produce a pressor effect ~5or a biphasic prcssor-depressor effect 34 during chronic treatment (1012 weeks), whereas higher doses (more than 3 mg/kg/ day) produce only a depressor effect ~4 or no effect~:L In our study, the animals received relatively high doses of nicotine (6-8 mg/kg/day) for 4 ~accks. Thus, the few alterations in cardiac receptors, except for a slight change in muscarinic receptors, appear to be consistent with the slight effects of higher doses of nicotine on blood pressure. Bhagat a.5 observed an increased turnover of noradrenaline m the brain of rats chronically treated with relatively low doses of nicotine (1.5-2.5 mg/kg/day for 6 weeks). Our findings suggested depressed activities of noradrenergic neurons ascending from the lo-
247 cus coeruleus in the cortex and hypothalamus/thala-
noted in the case of cigarette smoking, including irri-
mus. Since nicotine readily leads to tolerance following repeated administration of higher doses 16,24, the
tability, restlessness, sleep disturbances and drowsiness 17.
chronic state in our experiments would correspond to
In conclusion, we have demonstrated that chronic treatment of rats with high doses of nicotine induces
the developed state of tolerance. The noradrenergic n e u r o n s ascending from the locus coeruleus (innervating the cerebral cortex and
an increased concentration of a receptors in the cortex and hypothalamus/thalamus, and reciprocal
hypothalamus/thalamus) participate in the regulation of wakefulness x8,25. Hilakivi and Lepp~ivuori 14
changes in muscarinic receptors in the brainstem and heart left ventricle. While the significance of the
suggested that the level of cerebral al-adrenergic transmission is high during aroused waking but is low
muscarinic receptor changes is unclear, our evidence
during drowsy waking, a2-Adrenergic transmission has been also implicated in the regulation of sedation and drowsinessa,27. Accordingly, the increased densi-
does suggest that chronic nicotine treatment produces alterations in neurotransmission through a and muscarinic receptors, in localized regions of the brain
ty of cortical a receptors found in our study may imply alteration in the aroused state of rats after chronic
and heart. These changes seem to be a specific feature of the long-term effects of nicotine and may be related to the state of developed tolerance.
nicotine treatment. The increased concentration of a receptors also
ACKNOWLEDGEMENTS
suggests the specific development of supersensitivity to n o r e p i n e p h r i n e following chronic nicotine administration. Therefore, a high level of neurotransmission through the a receptors can be expected after the cessation of nicotine treatment, and significant alterations in the aroused state would follow. This may be associated with the withdrawal symptoms
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We thank M. Ohara for reading the manuscript and Miss Naomi Y a m a d a for excellent secretarial assistance. This work was supported in part by a grant from the Nippon Tobacco and Salt Public Corporation.
beta- adrenergic receptors in the rat after DSP-4, a selective noradrenergic neurotoxin, Neuroscience, 9 (1983) 889898. 8 Drew, G.M., Gower, A.J. and Marriot, A.S., a~-adrenoceptors mediate clonidine-induced sedation in the rat, Br. J. Pharmacol., 67 (1979) 133-141. 9 Ehlert, F.J., Roeske, W.R. and Yamamura, H.I., Regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide, J. Supramol. Struct., 14 (1980) 149162. 10 Ehlert, F.J., Roeske, W.R. and Yamamura, H.I., Muscarinic receptor: regulation by guanine nucleotides, ions, and N-ethylmaleimide, Fed. Proc. Fed. Am. Soc. Exp. Biol., 40 (1981) 153-159. 11 Ehlert, F.J., Roeske, W.R. and Yamamura, H.I., Muscarinic cholinergic receptor heterogeneity, Trends" Neurosci., 5 (1982) 336-339. 12 Hall, G.H. and Turner, D.M.. Effects of nicotine on the release of 3H-noradrenaline from the hypothalamus, Biochem. Pharmacol., 21 (1972) 1829-1838. 13 H~iggendal,J. and Henning, M., Effect of chronically administered nicotine on axonal transport of dopamine-fl-hydroxylase in peripheral adrenergic neurons and on blood pressure and heart rate in the rat, Acta Physiol. Scan&. Suppl., 479 (198(/) 35 38. 14 Hilakivi, I. and Leppfivuori, A., Effects of methoxaminc, an alpha-I adrenoceptor agonist, and prazosin, an alpha-1
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241
BRE 11156
Alteration of Alpha and Muscarinic Receptors in Rat Brain and Heart Following Chronic Nicotine Treatment KYOZO YAMANAKA, MASAFUMI OSHITA and IKUNOBU MURAMATSU
Department of Pharmacology, Fukui Medical School, Matsuoka, Fukui 910-11 (Japan) (Accepted February 12th, 1985)
Key words: nicotine - - chronic treatment - - a~-receptor - - Ctz-receptor- - muscarinic receptor - cerebral cortex - - hypothalamus/thalamus - - brainstem
Adrenergic and muscarinic binding sites in 4 brain regions (cerebral cortex, corpus striatum, hypothalamus/thalamus and brainstem) and in heart ventricles were measured in rats chronically treated with nicotine added to the drinking water in doses ranging from 6 to 8 mg/kg/day, for 4 weeks. Control rats received only tap water. The nicotine treatment led to increases in the specific binding of both [3H]prazosin and [3H]clonidine in the cerebral cortex. An increase in [3H]prazosin binding was also observed in the hypothalamus/thalamus of nicotine-treated rats. These changes were all due to an increase of about 23% in Bmax. In the brainstem and heart left ventricle, respectively, an increase and a decrease in the affinity of [3H]quinuclidinylbenzilate binding were observed. There were no changes of the binding parameters for the 3 radioligands in other regions tested, and no alteration of [3H]dihydroalprenolol binding was detected in any region examined. These results indicate that chronic administration of nicotine causes an increase in the density of a l- and a~-binding sites in some brain regions and reciprocal changes of the affinity of muscarinic binding sites in the brain and in the heart.
INTRODUCTION
peripheral adrenergic n e u r o n s has also been noted in rats receiving nicotine chronically 13. However, the
Acute administration of small doses of nicotine produces an activation of cortical electroencephalogram ( E E G ) and behavioral arousal in h u m a n s and animals~6,lv,3L Most of the effects may be due to ac-
long-term effects of nicotine on central adrenergic and muscarinic receptors is poorly understood. The present study was designed to assess binding sites changes in brain and heart following chronic nicotine treatment. Nicotine was added to the drinking water given to rats, for 4 weeks as reported by Hfig-
tions on central cholinergic neurons19.32 but catecholaminergic n e u r o n s may also be involved since nicotine causes an increased turnover and release of catecholamines in the hypothalamusl.2,12 and nigrostriatal system 20,21. On chronic administration, nicotine produces tolerance to the acute behavioral effects 16,24. Chronic nicotine treatment has been shown to induce an increased density of nicotine binding sites in brain24. The up-regulation of nicotinic binding sites after chronic nicotine treatment reflects adaptive change of the binding sites to a desensitized conformational form that is functionally inactive24~33. Thus, the sensitivity of central nicotine receptors decreases after chronic nicotine treatment. Decreased sensitivity of
gendal and H e n n i n g 13, and adrenergic (Ctl-, a2-, fl-) and muscarinic binding sites were measured in discrete brain regions (cerebral cortex, corpus striatum, hypothalamus/thalamus and brainstem) and heart ventricles, using a receptor binding technique. We found localized changes in ct- and muscarinic binding sites in the brain and heart following chronic nicotine treatment. MATERIALS AND METHODS
Animals and nicotine treatment Male Wistar rats (6 weeks of age: initial body
Correspondence: I. Muramatsu, Department of Pharmacology, Fukui Medical School, Matsuoka, Fukui 910-11, Japan. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
242 weight of 210-230 g) were housed in groups of 5 animals with a 12 h day/night cycle and free access to the usual chow diet. Three groups of the rats were given tap water for drinking, ad libitum, as the control group. An additional 3 groups were provided with tap water containing nicotine in a dose of 100 rag/liter (as nicotine base) for 4 weeks. The nicotine solution provided for drinking was freshly prepared daily by dissolving bitartrate in the drinking water, with an adjustment of pH to 5.5-6.(I. The average daily consumption of nicotine by each animal was calculated to be between 6-8 mg/kg. All rats were weighed once daily. Control rats gained in body weight throughout the 4 weeks to 383 _+ 7 g, whereas the nicotine-treated rats gained only to 333 _+ 6 g (significantly different from the control; P < (/.()l). The nicotine-induced weight loss was in agreement with reports by Schechter and Cook 29 and Hfiggendal and Henning 13.
Membrane preparation Both control and nicotine-treated rats were killed 4 weeks after the beginning of the nicotine treatment. Four brain regions (cerebral cortex, corpus striatum, hypothalamus/thalamus and brainstem) and the left and right heart ventricles of each rat were dissected on ice. Tissue from each brain region (except the cortex) and from heart ventricles was pooled from the 3-5 animals of each group. Tissues were homogenized twice for 15 s with a 30-s rest. using a Kinematika Polytron PT-10 (setting at 6.(i) in 40 vols. of ice-cold 50 mM Tris-HCl buffer (pH 7.7 at 25 '~C). The homogenates were incubated for 10 rain at 37 °C, and centrifuged at 62,700 g for 15 rain at 4 °C. The pellets were washed once by suspension in the original buffer and centrifugation under the same conditions. The resulting pellet was resuspended in the original volume of Tris-HC1 buffer and used for the binding assays.
Radioligand binding assays (~-, (z~-, fl- and muscarinic binding sites in the brain were measured from saturation experiments with a specific radioligand for each receptor; that is, [3H]prazosin, [3H]clonidine, [3H]dihydroalprenolol (DHA) and [3H]quinuclidinyl benzilate (QNB), respectively. The binding assays, except ¢]-receptor assay, were carried out according to the methods de-
scribed by Hornung et al.ls I,.'Prichard e~ a|. ~ and Yamamura and Snyder 3~, respectively, with mino~ modifications :~'>. In the binding assays with [-~H]prazosin, ! H]clonidine and. [3H]DHA, the tissue homogenates (5 or 7~5 mg of tissue wet wt.) were incubated with 6 or 8 different concentrations of each radioligand @H]prazosin, 0.03-2.35 nM; [3H]clonidme, /). 13.-,2.68 nM; [3H]DHA, 0.09-1.58 nM) for 30 min at 25 ~C, in a final 2-ml volume of 50 mM Tris-HC1 buffer (pH 7,7). In the [3H]QNB binding assay, the homogenates (0.5 mg of tissue wet wt.) were incubated with 8 different concentrations (9-350 pM) of the radioligand for 60 rain at 37 °C, in a total 2-ml volume of 50 mM Na +, K*-phosphate buffer (pH 7.4). Cardiac binding sites (a I-, [:•- and muscarinic receptors) were measured under the same assay conditions as brain binding sites. except for the use of 7.5 and 1.25 mg of tissue wet wt. for [3H]prazosin (0.09-2.33 nM)'and [-~H]QNB binding assays, respectively. All assays were performed in duplicate. The binding was terminated by rapid filtration over Whatmann GF/B filters, under reduced pressure, followed by 4 × 4 ml rinses with ice-cold incubation buffer. The filters were translerred into scintillation vials and dried overnight. The radioligand retained on the filters was extracted by 8 ml of scintillation fluid for at least 12 h. The radioactivity was counted using a Packard Tri-Carb Scintillation spectrometer at efficiences of 32-36%. Non-specific binding of each radioligand was defined as the binding in the presence of phentolamine (10/~M), l~-norepinephrine (10gM), propranolol (0.1 uM) and atropine (1 ~M) for the binding assays of czl-, ~--,/4- and muscarinic sites, respectively. The specific binding as percentage of total ranged 51-96% in the case of prazosin, 72-99% in the case of ctonidine, 45.7-90% in the case of D H A (except 25-50% for brainstem) and 93-98% in the case of QNB, respectively. Protein measurement was performed by the method of Lowry et al. 23, using bovine scrum albumin as a standard.
Data analysis Saturation isotherms for each radioligand binding constructed from saturation experiments were analyzed by estimation of the maximum number of binding sites (Bronx) and the apparent dissociation constant (K~t), according to the Scatchard analysis de-
243 scribed by B e n n e t 3. Hill coefficients (nil) w e r e also
RESULTS
e s t i m a t e d f r o m the d a t a by Hill analysis. A n a l y s e s of S c a t c h a r d and Hill plots w e r e c a r r i e d out using a
Regional variation o f radioligand binding in brain
m e t h o d of linear l e a s t - s q u a r e s r e g r e s s i o n analysis.
T h e p a r a m e t e r s for specific binding of r a d i o l i g a n d s
All results w e r e e x p r e s s e d as m e a n v a l u e + S . E .
([3H]prazosin,
f r o m 3 d i f f e r e n t groups. T h e statistical e v a l u a t i o n of the data was e x a m i n e d by u n p a i r e d ( t w o - t a i l e d ) Stud e n t ' s t-test.
[ 3 H ] O N B ) in discrete brain regions are s u m m a r i z e d
pH]clonidine,
[3H]DHA
and
in T a b l e I. All of the radioligands b o u n d to a single
Materials
gave slope factors (nil) of close to unity (1t.92 _+
p o p u l a t i o n of binding sites with a high affinity in the regions tested, since Hill analysis of s a t u r a t i o n d a t a
Radio[igands
([3H]prazosin,
17.4
Ci/mmol;
[3H]clonidine, 20.5 C i / m m o l ; [ 3 H ] D H A , retool; [ 3 H ] Q N B ,
0 . 0 7 - 1 . 0 6 _+ 0.06).
34.1 Ci/
In g e n e r a l , t h e r e was a regional v a r i a t i o n in the
33.2 C i / m m o l ) w e r e p u r c h a s e d
m a x i m u m n u m b e r of binding sites (B ...... ) for each ra-
from New England Nuclear (Boston, MA, U.S.A.)
dioligand.
and o t h e r agents used in this study w e r e o b t a i n e d
[3H]clonidine binding d e c r e a s e d in the rank o r d e r of
The
Bma x values for [3H]prazosin
f r o m c o m m e r c i a l sources; a t r o p i n e sulfate, D,L-pro-
c o r t e x => h y p o t h a l a m u s / t h a l a m u s
pranolol hydrochloride
striatum.
(Nakarai Chemicals,
Ltd.,
The
B .....
values
for
>
and
brainstem
[3H]DHA
> and
K y o t o , J a p a n ) , L - n o r e p i n e p h r i n e b i t a r t r a t e (Sigma C h e m i c a l s , St. L o u i s , M O , U . S . A . ) and p h e n t o l a -
o r d e r of s t r i a t u m > c o r t e x > > h y p o t h a l a m u s / t h a l a -
m i n e m e s y l a t e ( C i b a - G e i g y , Basel, S w i t z e r l a n d ) .
mus > b r a i n s t e m . T h e K,t v a l u e for each r a d i o l i g a n d
[3H]QNB binding, h o w e v e r , d e c r e a s e d in the rank
TABLE I
Parameters of specific -~H-ligand binding for discrete regions of the rat brain after chronic nicotine treatment Each brain region except the cerebral cortex isolated from 3-5 rats in one group was pooled, and the membrane fraction was used in binding study. The binding parameters were determined by saturation studies using 0.03-2.35 nM of [3H]prazosin. 0.13-2.68 nM of [3H]clonidine, 0.09-1.58 nM of [3H]DHA and 9-350 pM of [3H]QNB. The values given represent mean _+ S.E. from 3 groups of rats. but the values of cortex were obtained from 4 rats. Control, nicotine-untreated rats: nicotine treatment, rats in which nicotine was administered for 4 weeks. Hill analysis of all the data gave coefficients close to unity.
~H-ligand
Region
Control B,,a~ (fmol/mg protein)
Nicotine treatment K a (pM)
B ..... (fmol/mg protein)
[3H]Prazosin
Cerebral cortex Corpus striatum Hypothalamus/thalamus Brainstem
235.2 81.5 207.1 109.8
_+ 2.9 + 8.1 __+7.2 ± 9.1
99.0 +_ 14.1/ 117.7 + 13.5 100.9 ± 6.2 153.2 + 35.11
[3H]Clonidine
Cerebral cortex Corpus striatum Hypothalamus/thalamus Brainstem
110.3 51.0 115.9 81.5
_+ 7.8 _+ 7.7 + 0.6 _+ 5.9
715.8 + 147.0 704.8 _+ 208./) 1135.1 ± 150.8 1000.7 ± 203.8
[3HIDHA
Cerebralcortex Corpus striatum Hypothalamus/thalamus Brainstem
182.3 196.3 113.4 72.6
_+ 15.4 _+ 3.6 + 2.6 ± 3.2
422.4 468.8 617.7 509.1
[3HIQNB
Cerebral cortex Corpus striatum Hypothalamus/thalamus Brainstem
1577.5 1596.1 491.8 413.2
± 64.9 _+ 143.9 _+ 17.9 ± 12.9
73.7 52.4 41.9 44.0
* Significant difference from controls, P < 0.05.
± + + ±
14.4 14.9 55.9 36.9
± 6.7 + 7.1 _+ 7.7 +_ 3.9
290.5 _+ 13.9" 111.6 __+25.2 254.7 ± 8.8* 118.3 + 15.8 135.5 55.0 123.0 82.4
_+ 4.3* ± 1.7 + 4,4 + 2.3
180.8 202.9 117.2 68.4
± 10.1 +_ 5.3 _+ 6.3 ± 9.3
1519.8 ± 70.7 1657.6 _+ 1118.9 511.8 ± 39.0 376.6 ± 7.2
K~I(pM) 119.2 ±_ 15.0 148.0 +__29.0 111.9 + 9.9 132.1 ± 13.11 919.7 +_ 69.6 784.7 +_ 98.1 1152.4 ± 74.4 887.6 + 51/.1/ 424.6 462.8 612.3 417.3
+ 36.5 +_ 29.7 ± 47.0 _+ 67.7
53.9 50.2 42.2 29.9
+_ 6.7 +_ 7.4 _+ 4.4 ± 1.4"
244
[
e x h i b i t e d m u c h less regional variation.
50 I \ \ , 0
Central C~l-receptor T h e Bma x v a l u e s for specific [3H]prazosin b i n d i n g in the c o r t e x and h y p o t h a l a m u s / t h a l a m u s
i \ .... E \
.,2-" c:
w e r e in-
c r e a s e d f r o m 235.2 to 290.5 f m o l / m g p r o t e i n , and f r o m 207.1 to 254.7 f m o l / m g p r o t e i n , r e s p e c t i v e l y , or by a p p r o x i m a t e l y 2 3 % in e a c h case ( P < 0.05) ( T a b l e I). T y p i c a l d a t a for cortical s a m p l e s are s h o w n in Fig, 1; no c h a n g e in K a but a c h a n g e in Bma x was ob-
,oo
o
F
•
t ~\ ~
e
°
o
-x ~.
ta_ \
i
treatment
",, cX
•
50 i i
Control
:) Nicotine
s e r v e d b e t w e e n the two groups. T h e Hill coefficients w e r e also u n c h a n g e d , T h e s t r i a t u m and b r a i n s t e m i
0
s h o w e d n o d i f f e r e n c e s in any binding p a r a m e t e r for
5O
150
I00
[3HJprazosin b e t w e e n c o n t r o l and n i c o t i n e - t r e a t e d groups.
B (fmol/mg
Central a2-receptor T h e Brnax v a l u e for c o r t e x was i n c r e a s e d f r o m 110.3 to 135.5 f m o l / m g p r o t e i n , or by a b o u t 23% ( P < 0.05) ( T a b l e I) in the n i c o t i n e - t r e a t e d g r o u p large-
protein)
Fig. 2. Representative Scatchard plots of specific pH]clonidine binding for cerebral cortex from control and nicotine-treated rats. The binding parameters for control rat (@) are Bin,x = 112.6 fmol/mg protein, Ka = 1.01 nM and n H = 1.00, and those for nicotine-treated rat ((3) are BmaX = 141.3 fmol/mg protein, Kd = 0.92 nM and n H = 1.01, respectively.
ly b e c a u s e of an i n c r e a s e in total binding. Typical data are s h o w n in Fig. 2; again n o c h a n g e in K a was observed
between
control
and
nicotine-treated
groups. A l t e r a t i o n s o f the b i n d i n g p a r a m e t e r s w e r e
Bmax and K a v a l u e s for [ 3 H ] D H A binding in the 4
not o b s e r v e d in o t h e r r e g i o n s tested.
brain regions e x a m i n e d ( T a b l e I).
Central fl-receptor N i c o t i n e t r e a t m e n t p r o d u c e d no a l t e r a t i o n o f the
Central muscarinic receptor In the n i c o t i n e g r o u p , t h e r e was a significantly ( P
3.0
o
.=
20
•
Control
o
Nicotine
treatment
o. o.°
~ I0
• ~
Control
o Nicotine treatment
E
m
'°I
m
0
I O0
200
B (fmol/mg
300
protein)
Fig. 1. Representative Scatchard plots of specific [3H]prazosin binding for cerebral cortex from control and nicotine-treated rats. The binding parameters for control rat (@) a r e B m a x = 230.6 fmol/mg protein, Ka = 122.3 pM and n H = 0.97, and those for nicotine-treated rat ((3) a r e B m a x = 304.5 fmol/mg protein, Kj = 119.3 pM and n H = 0.87, respectively.
0
•
I O0
2 O0
B (fmol/mg
3 O0
400
protein)
Fig. 3. Representative Scatchard plots of specific [-~H]QNB binding for brainstem from control and nicotine-treated rats. The binding parameters for control rat (O) are Bmax = 388.8 fmol/mg protein, K a = 51.9 pM and n H = 0.96, and those for nicotine-treated rat ((3) are Bmax = 380.9 fmol/mg protein, Ka = 32.2 pM and n H = 0.98, respectively.
245 < 0.05) lower Kj value of [3H]QNB binding (29.9 + 1.4 pM) for the brainstem, as compared with the value (44.0 + 3.9 pM) in the control group (Table I). This indicates an increased affinity of the radioligand binding. A representative example is shown in Fig. 3, in which no difference in Bma x values were observed between the two groups. In other brain regions, neither Bma x n o r K a values differed between the control and nicotine groups.
Cardiac (at-, fl- and muscarinic) receptors All 3 ligands ([3H]prazosin, [3H]DHA, [3H]QNB) bound to single populations of sites in the heart ventricles, as evidenced by Hill coefficients of close to unity (0.97 + 0.02-1.16 + 0.02). Bmax and K a values in left and right ventricles are summarized in Table II. There were no alterations in binding parameters for either [3H]prazosin or [3H]DHA in the heart ventricles following chronic nicotine treatment. The nicotine group, however, showed a significantly (P < 0.05) higher K d value for [3H]QNB binding (58.7 + 7.9 pM) in the left ventricle, than the controls (31.9 + 6.5 pM) (Table II). This finding indicates a decreased affinity. A typical example is shown in Fig. 4, in which there was no significant change in the Bmax value. In the case of the right ventricle, no alteration in binding parameters was observed after chronic nicotine treatment.
40
30 Q.
Control
C oJ
o Nicotine
treatment
a.
20
E LI_
m
I0
°2. I
50
B (fmol/mg
tO0
protein)
Fig. 4. Representative Scatchard plots of specific P H I Q N B binding for heart left ventricle from control and nicotine-treated rats. The binding parameters for control rat ( 0 ) are BmaX = 82.2 fmol/mg protein, K d = 24.4 pM and n H = 0.97, and those for nicotine-treated rat (©) are Bma× = 93.3 fmol/mg protein, K d = 66.4 pM and n~ = 0.97, respectively.
TABLE II
Parameters of specific-~H-ligand binding for left and right ventricles of the rat heart after chronic nicotine treatment Each hcart region isolated from 3 rats in one group was pooled, and the membrane fraction was used in binding study. The binding parameters were determined by saturation studies using 0.09-2.33 nM of [3H]prazosin, 0.09-1.58 nM of [3H]DHA and 9-350 pM of [3H]QNB. The values given represent mean + S.E.M. from 3 groups of rats. Control, nicotine-untreated rats; nicotine treatment, rats in which nicotine was administered for 4 weeks. Hill analysis of all the data gave coefficients close to unity.
Region
3H-ligand
Control Bmax (fmol/mg protein)
Nicotine treatment Ka (pM)
B .... (fmol/mg protein)
Kj (pM)
Left ventricle
[3H]Prazosin [3H]DHA [3H]QNB
75.1 + 8.6 74.4 _+ 5.1 84.1 + 5.1
125.3 + 29.5 409.1 + 41.1 31.9 + 6.5
78.7 + 9.9 75.4 + 12.3 95.9 + 4.7
118.9 + 23.8 448.6 _+ 42.9 58.7 + 7.9*
Right ventricle
[3H]Prazosin [3H]DHA [3H]QNB
64.2 + 4.9 83.5 + 3.4 103.6+4.8
88.8 + 16.0 690.8 +_ 10.0 41.9+_ 1.0
64.8 + 3.9 89.2 + 12.7 116.2,+ 8.0
79.1 + 9.4 730.9 +_ 20.1 59.5_+ 13.5
* Significant difference from control, P < 0.05.
246 DISCUSSION Receptor binding assays have revealed changes of presumed central receptors which reflect alterations in neuronal activities in the brain after chronic administration of drugs to animals ~s,3(,,:<. We used such binding assays to assess the effects of chronic nicotine treatment on the rat brain and heart
Alteration of adrenergic receptors and a2-binding sites which were separately labeled by [3H]prazosin and [3H]clonidine, exhibited increased numbers in forebrain regions following nicotine treatment, a~-Sites increased in the cerebral cortex and hypothalamus/thalamus, and a2-sites increased in the cerebral cortex. Similar evidence has been reported for the same rat brain regions following reserpine and 6-hydroxydopamine treatment 31. Such phenomena have been considered as up-regulation of the receptors subsequent to a decreased activity of noradrenergic neurons induced by chemical denervation. The majority of al- and a2-binding sites in the cortex and hypothalamus/thalamus are postsynaptic to noradrenergic nerve terminals3~ which originate primarily from the locus coeruleus >. Thus, our finding may be indicative of depressed activities of noradrenergic neurons derived principally from the dorsal bundle 2e. However, the extent of increase in cortical (~ receptors obtained in this experiments was limited to only 23%, as compared with the much larger effects of chemical denervation of the dorsal (tegmental) bundle (74 and 80% increase in (zI and a 2 receptors, respectively) 31. Chemical denervation also induced a 62% increase in cortical/4 receptors~t. Concomitant changes in/3 and a receptors in the brain were also noted in rats given DSP-4, a selective noradrenergic neurotoxin 7, or a few chronic treatments (3-4 weeks) with antidepressants and lithium :s. No alteration of antagonist binding for fi receptors was seen. however, in the nicotine-treated rats. These findings indicate that long-term ingestion of nicotine produces slight changes in only the cz binding sites. a j-
Alteration of muscarinic receptors [3H]QNB binding exhibited an increased affinity in the brainstem and a decreased affinity in the heart left ventricle in the nicotine-treated rats. These find-
ings may indicate an alteration of the kinetics of mu~. carinic receptors for the binding of the antagonist. There are reports that [ql]QNB binding is mcreased by guanine nucleotides in the r:~'~ heart and brainstem (medulla-pons) ',i~ . Such an enhancement of antagonist binding is suggested to be dm ~to a conversion of low-affinity antagonist sites miv high-af° finity sites 11, probably regulated by the c,mpling of guanine nucleotide binding protein. Thu~, !he allerations in muscarinic receptors seen in our experiments might be ascribed to a conversion of antagonist affinity sites, perhaps at the level of coupling states of the receptor to the regulatory protein ~1. However, a1 present, the exact mechanism related *o affinity changes and the relation of reciprocal changes between central and peripheral muscarinic receptors remain unclear. Alterations in muscarinic receptors occur in animals receiving organophosphates chronically, flowever, such treatment induces a decreased density of muscarinic receptors in the corpus striatum and cerebral cortex, but not in brainstem and heart left ventricle 3('.37. This is in contrast to the results in the nicotine-treated rats. Such differences are probably related to the localized action of nicotine which is mediated through central nicotinic receptors exhibiting regional variation in distribution in the brain ~
Relation to the pharmacological long-term e{~ects ()/ nicotine Pharmacological long-term effects ot nicotine seem to be diverse, depending on the challenge doses. For example, low doses (1--2 mg/kg/day) of nicotine produce a pressor effect ~5or a biphasic prcssor-depressor effect 34 during chronic treatment (1012 weeks), whereas higher doses (more than 3 mg/kg/ day) produce only a depressor effect ~4 or no effect~:L In our study, the animals received relatively high doses of nicotine (6-8 mg/kg/day) for 4 ~accks. Thus, the few alterations in cardiac receptors, except for a slight change in muscarinic receptors, appear to be consistent with the slight effects of higher doses of nicotine on blood pressure. Bhagat a.5 observed an increased turnover of noradrenaline m the brain of rats chronically treated with relatively low doses of nicotine (1.5-2.5 mg/kg/day for 6 weeks). Our findings suggested depressed activities of noradrenergic neurons ascending from the lo-
247 cus coeruleus in the cortex and hypothalamus/thala-
noted in the case of cigarette smoking, including irri-
mus. Since nicotine readily leads to tolerance following repeated administration of higher doses 16,24, the
tability, restlessness, sleep disturbances and drowsiness 17.
chronic state in our experiments would correspond to
In conclusion, we have demonstrated that chronic treatment of rats with high doses of nicotine induces
the developed state of tolerance. The noradrenergic n e u r o n s ascending from the locus coeruleus (innervating the cerebral cortex and
an increased concentration of a receptors in the cortex and hypothalamus/thalamus, and reciprocal
hypothalamus/thalamus) participate in the regulation of wakefulness x8,25. Hilakivi and Lepp~ivuori 14
changes in muscarinic receptors in the brainstem and heart left ventricle. While the significance of the
suggested that the level of cerebral al-adrenergic transmission is high during aroused waking but is low
muscarinic receptor changes is unclear, our evidence
during drowsy waking, a2-Adrenergic transmission has been also implicated in the regulation of sedation and drowsinessa,27. Accordingly, the increased densi-
does suggest that chronic nicotine treatment produces alterations in neurotransmission through a and muscarinic receptors, in localized regions of the brain
ty of cortical a receptors found in our study may imply alteration in the aroused state of rats after chronic
and heart. These changes seem to be a specific feature of the long-term effects of nicotine and may be related to the state of developed tolerance.
nicotine treatment. The increased concentration of a receptors also
ACKNOWLEDGEMENTS
suggests the specific development of supersensitivity to n o r e p i n e p h r i n e following chronic nicotine administration. Therefore, a high level of neurotransmission through the a receptors can be expected after the cessation of nicotine treatment, and significant alterations in the aroused state would follow. This may be associated with the withdrawal symptoms
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