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Cocaine-induced supersensitivity of striatal dopamine receptors: role of endogenous calmodulin
COCAINE-INDUCED SUPERSENSITIVITY OF STRIATAL DOPAMINE RECEPTORS: ROLE OF ENDOGENOUS CALMODUL~N M. MEMO, SIKTA PRADHAN* and I. HATKWX Section for Biochemical Pharmacology, National Heurr. Lun,k’and Blood National Institute of Health. Bethesda. MD 200. L’.S..A.
IIl>III11lc.
Summary--The effect Of cocaine on the binding of specific rddioligands to rat striatal dopaminc (DA) receptors and on the activation by DA of striatal adenyhile tyclase activit? was studied in Ritz and in ritro. As early as 60 min after an injection of cocaine (20 mg/kg. i.p.1 the apparent B,,,,, of [~H]spiroperidol and [3H]~-propy~norapomorphine binding was increased by 37”,,. In contrast. the atfinity of the receptor for the labelled ligands remained unchanged. The injection of cocaine (20 mg, kg. i.p.) also increased the content of membrane-bound calmodulin and the responsiveness of adenylate cyclase to DA stimulation, The activation of adenylate cyclase by sodium fluoride (NaF) or choleratoxin was similar in saline and cocaine-treated rats. indicating that cocaine did not modify the guanosine triphosphate (GTP)-binding protein which couples the cyclase with the DA recognition site. An increase in DA stimulation of adenylate cyclase and B,,, for the binding of radioligands was also obtained in rifro after a 60 min incubation of striatal slices with cocaine (IO -’ M). but not with lidocaine (IO- M). The present data suggest that cocaine may interact with post-synaptic membrane constituents and thereby increase the availability of membrane-bound calmodulin. This mobilization of calmodulin may be the cause for the supersensitivity of adenyiate cyciase to activation by DA.
Cocaine is an indirectly acting central metic agent which like d-amphetamine
sympathomi-
preferentially increases the release of dopamine (DA) from neuronal stores onto specific post-synaptic receptors. Simitar to other
receptors
for putative
neurotransmitters,
DA
receptors function as supramolecular entities consisting of a detector, a coupling device and an amplifying system. These membrane proteins, because of their great mobility within the lipid bilayers of the postsynaptic membranes (Singer and Nicholson, 1972), can interact with each other and thereby express their functional activity. The pattern and modalities of such interactions are controlled by conformational changes of the detector site. When this site is occupied by a specific agonist, the flow of information across the target membrane proceeds from the recognition site by way of a coupling system to the amplifier system (Lefkowitz, Mullikin and Caron, 1976). The coupling system of DA receptors includes GTP-binding protein (Creese, Presser and Snyder. 1978). and caimodulin (Gnegy, Uzunov and Costa, 1977), while the ampiifier system consists of adenylate cyclase (Kebabian, Petzoid and Greengard, 1972). It has been suggested for several receptors that the continuous occupancy of a detector site by an agonist leads to receptor desensitization (Su, Harden and Perkins, 1980). Hence, persistent stimulation of DA receptors with DA (Memo and Hanbauer, 1981}, broKey words: cocaine, ~lm~ulin, DA-sensitive adenylate cyclase. DA-receptor supersensitivity. * Present address: Howard University, 2400 6th St. N.W., Washington, DC 20001. U.S.A.
mocryptine (Quick and lversen (197X) or tl-amphetamine (Hanbauer. Pradhan and Yang. 1080). is associated with receptor desensitization. ~-Amphetamine C:III reverse h~tlo~ridol-intltlce~i supersensilivjty of DA receptors (H;tratz and Tscng. 1980). suggesting that this drug can clungc tlw malecular properties and the intcroctions of the receptor proteins. The existence of many prominent Jitycrcnccs in the actions of chrdnicnlly udministcrcd tl-amphetamine and cocaine (Post. lY77) prompted an examinination of the action of cocaine OII striatal DA receptors, including the action of coc:iinc on the B,,,.,, of agonist binding. stimulatiol~ of adenylate cyclase by DA and on the content of calmodulin bound to rat striatal membranes. The content of membrane-bound calmodulin is characteristically increased in supersensitive DA receptors (Hanbauer and Costa, 1980), whereas, sub-sensitivity triggered by persistent activation of dopamine receptors by DA or apomorphine, is associated with a reduced membrane-bound calmodulin content (Hanbauer, Gimhie and Lovenberg, 1979). METHODS Male Sprague-Dawley rats (15&200 g) were kept under standard conditions with 12 hr lightdark cycle and free access to water and food (Purina Chow). The rats were injected with saline or cocaine HCl (20 mg/kg,i.p.), killed by decapitation at various times thereafter and the nucleus caudatus and nucleus accumbens were dissected rapidly.
1146
M.
MEMO
Adenylate cyclase activity was measured as described by Clement-Cormier. Kebabian. Petzold and Greengard (1974) with minor modifications. In brief, the tissue was homogenized in 10 mM Tris-maleate buffer pH 7.5 containing 1.2 mM EGTA using a glassteflon homogenizer. The incubation medium contained 82.5 mM Tris-maleate buffer pH 7.5, 5 mM MgSOd, 2mM cyclic AMP, 5 mM theophylline, 0.3 mM EGTA (carried over from tissue preparation), 0.01 mM GTP. 2Okcg pyruvate kinase. 4mM phosphoenolpyruvate. I mM C3H]ATP (1 10h cpm) and homogenate equivalent to 1 mg of tissue. The mixtures were incubated in the absence or presence of various concentrations of dopamine or NaF in a final volume of 300~1 for 3 min at 30 C. The reaction was stopped by the addition of a solution containing 20 mM ATP and 0.7 mM cyclic AMP and by heating the tubes for 3 min at 90 C. The amount of [3H]cyclic AMP formed was measured as described by Salomon, I.ondos and Rodbell ( 1974) using subsequent chromatography on Dowex 50W X 4 (IO@-200 mesh. BioRad) and alumina oxide (alumina, Woelm neutral, Act. 1: ICN Nutritional Biochemicals). The recovery of cyclic AMP 65-75”,,. Cyclic AMP content was measured in striatal slices with a radioimmunoassay kit supplied by New England Nuclear. Striatal slices (230ltm thick) prepared with a Sorvall tissue sectioner (TC-2) were preincubated for 30 min in Krebs-bicarbonate solution pH 7.4 supplemented with 10 mM dextrose and 1.14mM ascorbic acid under constant oxygenation (95”,, O2 + 5”,, COz). The incubation was continued for 40min in the absence or presence of various concentrations of choleratoxin (Schwarz,‘Mann). Thereafter. the medium containing the slices was heated for 3 min at 90 C and centrifuged at 1OOOg for IOmin. The cyclic AMP content was measured in 50~1 aliquots of the supernatant fraction.
et (11.
thereafter rapidly filtered over Whatman GF/B filter. Specific C3H]spiroperidolor [3H]N-propylnorapomorphine-binding was determined by the difference in binding obtained in the presence or absence of dopamine (lo-4 M). In experiments designed to study in citro the effect of cocaine or lidocaine (xylocaine HCI, Astra Pharmaceutical Products, Worchester, MA), on specific [‘H]spiroperidolor [3H]N-propylnorapomorphine binding, the membrane suspension was preincubated for 60min in the presence of 10eh M cocaine or lidocaine and thereafter, the ligand binding was assayed as described above. The protein content was measured according to Lowry, Rosenbrough, Farr and Randall (195 I) using bovine serum albumin (Miles Laboratories) as a standard. En:_vme linked
[3H]N-propylnorapomorphine(58.5 Ci/mmol: New England Nuclear) and [3H]spiroperidol(35.9 Ciimmol. New England Nuclear) specific binding was determined as described by Creese. Padgett. Fazzini and Lopez (1979) and Burt. Creese and Snyder (1977). respectively. Rats were killed 60min after an injection of saline or cocaine (20mg,kg. i.p.) and striatal homogenates were prepared at 4-C in 50 mM Tris-HCI buffer. pH 7.6 using a Polytron homogenizer and centrifuged at 5O.OOOg for 10min. The pellet was washed in the same volume of buffer and centrifuged as described before. The pellet obtained was then resuspended in the same volume of 50 mM Tris-HCI buffer. pH 7.6 containing 120 mM NaCl. 5 mM KCI. 2 mM CaCI, and 1 mM MgCI,. The incubation mixture (1 ml) contained [3H]spiroperidol (0.2-1.2 nM) or [3H]N-propylnorapomorphine (0.2-1.4nM) and an aliquot of membrane suspension corresponding to 10 mg of tissue fresh weight. The mixtures were incubated for 10min at 37’C and
Assam (ELISA)
for
cal-
Caudate nuclei were homogenized (Glass-teflon homogenizer) in 0.32 M sucrose and centrifuged at 1OOOg for 1Omin. The supernatant fraction was removed and centrifuged at 100,000 g for 30 min. The resultant pellet was homogenized in 0.05 M Trisbuffer pH 7.4 containing O.l”, Lubrol WX (Sigma), sonicated for 0.5 min and centrifuged at 100,000 g for 30min. The calmodulin content was measured in the supernatant fraction obtained by the first high speed centrifugation (referred to as soluble) and in the pellet extract (referred to as membrane-bound) by microELISA using a specific antibody toward calmodulin as described recently (Hanbauer et al.. 1980).
RESULTS
Spehjic
binding of[‘H]spiroperido/
norapomorpkine
in srriutum
and [‘H]N-prop_vl-
qf‘rars trrrrted
with cocaine
injection of cocaine (20mgikg. i.p.) significantly increased the specific binding of [3H]spiroperidol-a DA receptor antagonist-and that of C3H]Npropylnorapomorphine-a DA receptor agonist. Figure 1 shows a Scatchard analysis of [3H]spiroperidol (A) and [3H]1V-propylnorapomorphine (B) specific binding to crude synaptic membranes prepared from the striata of saline- and cocaine-treated rats. The data indicate that cocaine increased the number of binding sites for both ligands by 37”,,. but the affinity of these ligands for their binding sites was not changed. When striatal slices were preincubated in presence of lo-” M cocaine. the binding capacity for [3H]spiroperidol and [-‘H]N-propylnorapomorphine was significantly increased (Table 1). These results indicate that cocaine may facilitate the specific binding of these ligands by a direct action on striatal membranes. In order to determine whether the changes in B,,, for both ligands was due to the local anesthetic action of cocaine. the effect of lidocaine on the specific’binding of [3H]spiroperidol was studied. Table 1 shows that incubation of striatal slices with One
Specjfic ligtrr~n binding to doptrmine receptors
immunosorhent
mod&n
DA receptor sensitivity and
Bmsx
415 f 12
KD
1.7 f 0.1
Bmsx
584 2 31
KD
1147
cocaine
so-
z
40-
E
1.4 f 0.1
2
30-
s $
20Saline /
10 t
_-
DOFAMINE
I
I
I
100
2&l
300
400
500
BOUND/FREE
lfmolhg
Saline
P ;
400-
\ \
u
w z Kz
\\
prot/nM)
Smsx
391 + 11
Ko
Cocaine
0.53 + 0.03
Bmsx
538 + 21
I
I 10-S
lo-’
I
_
r;/0-i I
01
I
0--O
Cocaine
10.’
CONCENTRATION
(MI
Fig. 2. Stimulation by dopamine of dopamine-sensitive adenylate cyclase in striatal homogenates measured in saline or cocaine-(20 mg/kg. Lp.) treated rats. The rats were killed 60 min after injection of the drug and the formation of c3H]cAMP, catalyzed by adenylate cyclase, was measured. The basal activity of adenylate cyclase in saline and cocaine-treated rats was 57 + 2.3 and 57 k 1.9 pmol/ mg/min, respectively. The results were plotted vs the respective dopamine concentrations mean + SE of CAMP increase (n = 6). The K, for dopamine was obtained by linear regression analysis.
0.59 2 0.04
az* Oh3oi)_..
lidocaine (10m6 M) did not modify the KI, or B,,, for [3H]spiroperidol.
5; qa g% i=-
200-
Dopamine-dependent t
0
adenylate
cyclase
in striatum
of
rats treated with cocaine
0” +
loo-
\
I
1000
500 BOUND/FREE
lfmollmp
proVnMI
Fig. 1. Scatchard plot of [3H]spiroperidol (A) and [‘H]Npropylnorapomorphine (3) binding to dopamine receptors in striatal membranes prepared from rats 60min after injection of saline or cocaine (20mg/kg, i.p.). The ratio of bound was plotted as a function of bound to free radioligand (in the range of 0.2-1.2nM). The results are expressed as mean of 4 experiments in triplicates. The slope of the line l/K,, was computed and the B,,, was determined from the intercept of the line on the ordinate.
The sus~ptibility of striatal adenyiate cyclase to stimulation by DA was significantly increased at 304Omin following an injection of cocaine (20 mg/kg). Figure 2 shows the stimulation of adenylate cyclase activity elicited by various doses of DA. The data indicate that 60min after the cocaine injection, activation by DA of adenylate cyclase was facilitated and resulted in a shift to the left of the doseresponse for DA. Table 2 reports the EDS0 and K, for activation by DA in saline and cocaine-treated rats. The supersensitivity of adenylate cyclase to activation by DA elicited by cocaine injections, was indicated by a decrease in the apparent K, and the ED,, for DA. In addition, when striatal slices were incubated for
Table 1. Changes in kinetic properties of specific ligand binding and dopamine-de~ndent cyclase elicited by cocaine in rat striatal slices Drug Saline Cocaine
Lidocaine
[3H]spiroperidol &nU. K1, 403 * 21 564 f 2s* 398 k 19
1.1 If 0.13 1.0 * 0.11 1.1 +_0.11
* P < 0.05. K, is expressed as nM & SE and B,,,
[3H]N-propyl-norapomorphine B mux
KI,
384 + 22 480 f 29* -
0.51 f 0.041 0.59 + 0.051 -
adenylate
Adenylate cyclase K,W
DA)
1.5 0.34* -
as fmol/mg prot. +_SE of three different experiments. After preincubation of striatal slices in Krebs-bicarbonate solution pH 7.4 for I5 min, the incubation was continued for 60 min in the presence or absence of cocaine or iidocaine (lOa M). The slices were removed and then homogenized and assayed as described in Methods section.
M. MEMO CI ul
1148
Table 2. The effect of cocaine on dopamine-stimulated enylate cyclase in rat striatal homogenates Drug (mg/kg. i.p.) Saline Cocaine
Adenylate ED&M)
Table 4. Effect of cholera toxin on cyclic AMP in striatal slices of saline and cocaine-treated
cyclase K&M)
46 + 5 IS i 2*
(20)
ad-
Cholera toxin (~gjm))
3.2 + 0.35 I.2 + _ 0.13*
0 IO 25 50 100
* P < 0.05 when compared with saline-injected rats. The rats were injected with cocaine 60 min before killing. The values are expresssed as mean k SE of 4 experiments, The EDso represents the dopamine concentration causing a half-maximal stimulation. The K, values were derived from a linear regression analysis of a concentration curve for dopamine in the range of 10~h-lO-J M.
ate
cyclase
to stimulation
by
NaF
or
choleratoxin.
that,
Cocaine treatment increased the striatal content calmodulin. A time course of the selective increase
of in
Table 3. Effect of NaF on adenylate cyclase activity in striatal homogenate of saline and cocaine-treated rats
NaF concentration (mM) 0
I 2.5 5 IO
cyclic AMP (pmol,mg/min) Saline Cocaine 56 86 I I9 142 157
+ f f f f
3.2 4.8 6.7 6.8 7.1
Animals were killed 1 hr after injection cocaine (20 mg/kg. i.p.). Adenylate cyclase activity was measured Salomon et al. (1974).
54 81 I19 136 172
f f f k f
0.35 0.41 0.63 I.1 I.8
f f f * +
0.028 0.031 0.058 0.091 0.1’
0.38 0.39 0.59 0.99 I.8
+ + + + +
0.018 0.021 0.045 0.10 0.13
content extracted from striatal membranes induced by cocaine is shown in Table 5. This increase could be observed at 30 min after injection of the drug and lasted longer than 1 hr. In contrast, the striatal content of soluble calmodulin, which represents about l/4 of the total content of calmodulin, failed to change as a result of cocaine treatment (Table 5). Repeated daily injections of cocaine (30mg/kg) over a period of 3 weeks increased the content of membrane-bound calmodulin without changing the amount of soluble calmodulin stored in the caudate nuclei (Table 6). the calmodulin
in the striatal homogenates of saline-treated rats. the half-maximal activation of adenylate cyclase was elicited by 2.5 mM NaF. In membranes prepared from rats injected with cocaine (20 mgikg. i.p.) 60 min before the experiment, the responsiveness of adenylate cyclase to the activation by NaF was similar. In Table 4. the activation of adenylate cyclase by various concentrations of choleratoxin is compared in striatals’membranes prepared from saline- and cocaine-treated rats. The results indicated that treatment with cocaine failed to modify the responsiveness of membrane-bound adenylate cyclase to choleratoxin activation. 3 shows
Cyclic AMP (pmoljmg + SE) Saline Cocaine
Rats were injected with cocaine (20mg/kg, i.p.) I hr before killing. Striatal slices were preincubated in Krebsbicarbonate solution pH 7.4 for 30 min and then incubated for 40min in the presence of cholera toxin, The cyclic AMP content in the incubation medium was determined by RIA (II = 6).
60min with cocaine (lo-(’ M), a similar decrease in the K, for the DA-elicited activation of adenylate cyclase was obtained (Table 1). In contrast. in the nucleus accumbens, the stimulation of DA-sensitive adenylate cyclase by DA was not modified 60min after the injection of cocaine (data not shown). In this tissue, the EDso for the adenylate cyclase activation by DA was 5 x 10e5 M in saline and cocaine-treated rats. Additional studies were carried out to determine whether cocaine could modify the response of adenylTable
formation rats
3.1 4.5 5.7 6.2 7.3
of saline
or
according
to
DISCUSSION
The data presented suggest that cocaine can influence dopaminergic receptor function by a direct action on the receptor proteins. Cocaine appeared to increase the number of recognition sites for DA and facilitated the responsiveness of adenylate cyclase located in striatal membranes to stimulation by DA. Under the influence of cocaine, the K, for activation by DA of adenylate cyclase was decreased, reflected by a shift to the left of the dose-response curve for
Table 5. Changes various
in calmodulin content of rat striatum times after injection of cocaine
Minutes after injection
j(g Calmodulin. Soluble
0 I5 30 60
I.13 + 0.09 ND 0.94 f 0.29 1.28 f 0.20
at
mg protein i SE Membrane-bound 4.3 4.9 6.4 9.9
+ 0.54 + I.0 + 0. I * If: 1.0*
*P < 0.01: a = 5. ND-not determined. Striatal homogenates were centrifuged at IOOOg for 10 min. The supernatant fraction was removed and centrifuged at 1OO.OOOg for 30 min. The resulting pellet was extracted with 0.05 M Tris-buffer pH 7.4 containing O.l”,, Lubrol WX:The calmodulin content of supernatant fraction (soluble) and pellet extract (membrane-bound) were measured by ELISA.
DA receptor
Table 6. Content
Number of cocaine injections Control 1 21
of calmodulin in rat caudate nucleus cocaine treatment
pg Calmodulin/mg Soluble 1.5 + 0.32 0.94 + 0.29 1.2 + 0.26
sensitivity
after
protein + SE Membrane-bound 4.03 * 0.70 6.38 + 0.3_5* 6.51 k 0.69*
* P < 0.05; n = 6. Rats were injected with cocaine (30 mg/kg, i.p.) once or once daily for 21 days and were killed 30 min after the last injection. Striatal homogenates in 0.32 M sucrose were centrifuged at IOOOg for IOmin. The supernatant was removed and centrifuged 100.000 g for 30 min. Calmodulin content in the supernatant fraction (soluble) and pellet extract (membrane-bound) was determined by microELISA (Hanbauer et al., 1980).
DA. This action of cocaine occurred within 60min after its administration. Since cocaine increased the amount of DA that is present in the synaptic cleft by inhibiting DA reuptake (Iversen, 1966; Ross and Renyi, 1966), and increasing the transmitter turnover rate (Costa, Groppetti and Naimzada, 1972), it was expected that the response would be a down-regulation of the DA receptor. However, unlike d-amphetamine, which down-regulates DA receptors (Hanbauer et crl., 1980), cocaine increased the receptor sensitivity to activation by DA. This unexpected action of cocaine on DA receptor regulation can be also elicited by incubating striatal slices with cocaine and appears to be due to the direct interaction between the drug and post-synaptic membranes. Similar in ritra studies, which were carried out with lidocaine. a local anesthetic with a greater potency than cocaine, failed to show a change in B,,,, for spiroperidol. Hence. cocaine appears to act specifically on postsynaptic DA receptors. At the present time. the exact molecular mechanism mediating this action is unclear. It appears unlikely that cocaine modifies the GTP-binding protein, which couples the DA recognition sites with the cyclase (Creese et ol., 1978). In fact, cocaine failed to modify the activation of adenylate cyclase by NaF or choleratoxin, which have been shown to require GTP and GTP-binding protein for their action (Pfeuffer, 1977; Gill and Meren. 1978: Ross, Howlett. Ferguson and Gilman, 1978). Cocaine appears to modify directly the mechanism that couples the regulation of receptor excitability with the amount of DA present in the synaptic cleft. It is possible that. due to a direct action of cocaine, the responsiveness of the DA receptor is never diminished even after repeated administration of the drug. In the present working model, it was proposed that cocaine changes the characteristics of the lipid bilayers allowing an uncovering of a greater number of DA recognition sites. Whether cocaine exerts its effect by changing the methylation of membrane phospholipids (Hirata. Strittmatter and Axelrod, 1979) remains to be
and cocaine
1149
ascertained by future experiments. An important molecular mechanism operative in the sensitization of DA receptors is the increase of calmodulin content that is bound to synaptic membranes (Gnegy et al., 1977: Lucchelli, Guidotti and Costa. 1978). In contrast, a decrease in membranebound calmodulin appears to be associated with desensitization of the dopamine receptor (.Memo and Hanbauer, 1981). Since cocaine failed to modify the soluble calmodulin content, it has to be considered whether. during sensitization of the receptor, the amount of extractable calmodulin is increased. This possibility leads to the speculation that calmodulin can be bound more or less tightly to specific membrane proteins by a CaZ+ -dependent mechanism and that. during desensitization, the amount of Ca2’ present in membranes may increase while, during supersensitivity, the Ca2+ content of membranes may be decreased. Cocaine could also act directly on the phospholipids in the membrane facilitating Ca2+ release from membrane constituents thereby increasing the mobility of membrane-bound calmodulin. It is important to emphasize that. although the total calmodulin content may be in excess of the amount required for enzyme activation. the availability of free calmodulin appears to be limited through its Ca2+dependent binding to specific proteins present in membrane and cytosol fractions. In regard to the action of cocaine, the increased availability of calmodulin could promote the supersensitivity of adenylate cyclase to activation by DA. Future experiments in this laboratory are planned to determine whether enzyme activation is dependent on the dissociation of calmodulin from its binding sites.
AcknoMl~dgrmmt-This grant of the Scottish NiMiTi. U.S.A.
work was supported Rite Schizophrenia
in part by a Foundation.
REFERENCES Burt. D.. Creese, 1. and Snyder. S. H. (1977). Antischizophrenic drugs: chronic treatment elevates dopamine receptor binding in brain. Science 1%: 326-328. Clement-Cormier, Y. C., Kebabian. J. W.. Petzold, G. L. and Greengard, P. (1974). Dopamine-sensitive adenylate cyclase in mammalian brain: a possible site of action of antipsychotic drugs. Proc. nafn. Acad. Sci. U.S.A. 71: 1113-1117. Costa, E., Groppetti. A. and Naimzada. M. K. (1972). Effect of amphetamine on the turnover rate of brain catecholamines and motor activity. Br. J. Phurmuc. 44: 742-751. Creese. I., Prosser, T. and Snyder, S. H. (1978). Dopamine receptor binding: specificity localization and regulation by ions and guanyl nucleotides. ,!$e Sci. 23: 495-500. Creese. I.. Padgett. L.. Fazzini, E. and Lopez, F. (1979). ‘H-N-n-Propylnorapomorphine: a novel agonist ligand for central dopaminergic receptors. Eur. J. Phurmuc. 56: 41 l-412. tiill. D. M. and Meren. R. (1978). ADP-ribosylation of membrane proteins catalyzed bv choleratoxin: Basis of the activation of adenylat; cyclaie. Proc. nurrt. Acud. Sci. U.S.A. 75: 3050-3054.
11.50
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Gnegy. M. E., Uzunov, P. and Costa, E. (1977). Participation of an endogenous Ca’+ binding protein activator in the development of drug-induced supersensitivity of striatal dopamine receptors. J. Pharmac. exp. Ther. 202: 558-564. Hanbauer, I. and Costa, E. (1980). Role of calmodulin in dopaminergic transmission. In: Calcium and Ceil Functiun (Cheung, W. Y., Ed.), Vol. I, pp. 253-271. Academic Press, New York. Hanbauer, I.. Gimble, J. and Lovenberg, W. (1979). Changes in soluble calcium-dependent regulator following activation of dopamine receptors in rat striatal slices, Neuropharmacology
18: 851-857.
Hanbauer, I.. Pradhan. S. and Yang, H.-Y. T. (1980). Role of calmodulin in dopaminergic transmission. Ann. N. Y. Acad. Sci. 356: 292-303. Haracz, J. L. and Tseng, L-F. (1980). Reduction of dopaminergic su~r~nsitivity by a single dose of amphetamine. N~unyn Schmiedebergs Arch. Phur~c. 313: 131-133.
Hirata, I., Strittmatter, W. J. and Axelrod, J. (1979). ,&Adrenergic receptor agonists increase phospholipid methylation, membrane fluidity and j-andrenergic receptor-adenylate cyclase coupling. Proc. nafn. Acad. Sci. U.S.A. 76: 368-372. Iversen, L. L. (1966). Accumulation of a-methyltryramine by the noradrenaline uptake process in the isolated rat heart. J. Pbarm. 18: 481484. Kebabian, J. W., Petzold, Cl. L. and Greengard, P. (1972). Dopamine-sensitive adenylate cyclase in caudate nucleus of rat brain and its similarity to the dopamine receptor. Proc. natn. Acad. Sci. U.S.A. 69: 2145-2149.
Lefkowitz, R. J., Mullikin, D. and Caron, M. Cl. (1976). Regulation of beta-adrenergic receptors by guanyl-S-ylimidophosphate and other purine nucleotides. J. biol. Chem. 251: 4686-4692.
Lowry, 0. H., Rosenbrough, M. J., Farr, A. C. and Randall. R. J. (1951). Protein measurement with the Folin phenol reagent. J. biol. Chem. 193: 265-275.
Lucchelli, A. Guidotti, A. and Costa, E. (1978). Striatal content of Ca*+ dependent regulator protein and dopaminergic receptor function. Brain Res. 155: 13&135. Memo, M. and Hanbauer, 1. (1981). In uitro subsensitivity of adenylate cyclase-coupled dopamine receptors in striatal slices. Role of calmodulin. Fed. Prof. 40: 653. Pfeutfer, T. (1977). GTP-binding proteins in membranes and the control of adenylate cyclase activity. J. biol. Chem. 252: 7224-7234.
Post, R. M. (1977). Progressive changes in behavior and seizures following chronic cocaine administration: Relationship to kindling and psychosis. In: Adcances in Behacioraf Biology (Ellinwood, E. H. and, Kilbey. M. M. Ed@. Vol. 21. pp. 353-374. Plenum Press. New York. Quick, M. and Ivetsen, L. L. (1978). Subsensitivity of the rat striatal douaminernic system after treatment with bromocriptine:- Effects-on f3H]Spiperone binding and dooamine-stimulated cvclic AMP formation. NaunvnSch’miedergs Arch. Pharkac. 304: 141-145.
Ross, S. B. and Renyi, A. L. (1966). Uptake of some tritiated sympathomimetic amines by mouse brain cortex in vitro. Acta pharmac. tax. 24: 297-309.
ROSS,E., Howlett, A., Ferguson. K. and Gilman, A. (1978). Reconstitution of hormone-sensitive adenylate cyclase activity with resolved components of the enzyme. J. bioi. Chem. 253: 64i31-6412.
Salomon, Y., Londos, C. and Rodbeil, M. (1974). A highly sensitive adenylate cyclase assay. Analpt. &o&em. 58: 541-548. Singer.S. L. and Nicholson, G. L. (1972). The fluid mosaic model of the structure of cell membranes. Science 175: 720-731. Su, Y-F., Harden, T. K. and Perkins, J. P. (1980). Catecholamine-specific desensitization of adenylate cyclase. Evidence for a multiple process. J. biof. Chem. 255: 7410-7419.
IIl>III11lc.
Summary--The effect Of cocaine on the binding of specific rddioligands to rat striatal dopaminc (DA) receptors and on the activation by DA of striatal adenyhile tyclase activit? was studied in Ritz and in ritro. As early as 60 min after an injection of cocaine (20 mg/kg. i.p.1 the apparent B,,,,, of [~H]spiroperidol and [3H]~-propy~norapomorphine binding was increased by 37”,,. In contrast. the atfinity of the receptor for the labelled ligands remained unchanged. The injection of cocaine (20 mg, kg. i.p.) also increased the content of membrane-bound calmodulin and the responsiveness of adenylate cyclase to DA stimulation, The activation of adenylate cyclase by sodium fluoride (NaF) or choleratoxin was similar in saline and cocaine-treated rats. indicating that cocaine did not modify the guanosine triphosphate (GTP)-binding protein which couples the cyclase with the DA recognition site. An increase in DA stimulation of adenylate cyclase and B,,, for the binding of radioligands was also obtained in rifro after a 60 min incubation of striatal slices with cocaine (IO -’ M). but not with lidocaine (IO- M). The present data suggest that cocaine may interact with post-synaptic membrane constituents and thereby increase the availability of membrane-bound calmodulin. This mobilization of calmodulin may be the cause for the supersensitivity of adenyiate cyciase to activation by DA.
Cocaine is an indirectly acting central metic agent which like d-amphetamine
sympathomi-
preferentially increases the release of dopamine (DA) from neuronal stores onto specific post-synaptic receptors. Simitar to other
receptors
for putative
neurotransmitters,
DA
receptors function as supramolecular entities consisting of a detector, a coupling device and an amplifying system. These membrane proteins, because of their great mobility within the lipid bilayers of the postsynaptic membranes (Singer and Nicholson, 1972), can interact with each other and thereby express their functional activity. The pattern and modalities of such interactions are controlled by conformational changes of the detector site. When this site is occupied by a specific agonist, the flow of information across the target membrane proceeds from the recognition site by way of a coupling system to the amplifier system (Lefkowitz, Mullikin and Caron, 1976). The coupling system of DA receptors includes GTP-binding protein (Creese, Presser and Snyder. 1978). and caimodulin (Gnegy, Uzunov and Costa, 1977), while the ampiifier system consists of adenylate cyclase (Kebabian, Petzoid and Greengard, 1972). It has been suggested for several receptors that the continuous occupancy of a detector site by an agonist leads to receptor desensitization (Su, Harden and Perkins, 1980). Hence, persistent stimulation of DA receptors with DA (Memo and Hanbauer, 1981}, broKey words: cocaine, ~lm~ulin, DA-sensitive adenylate cyclase. DA-receptor supersensitivity. * Present address: Howard University, 2400 6th St. N.W., Washington, DC 20001. U.S.A.
mocryptine (Quick and lversen (197X) or tl-amphetamine (Hanbauer. Pradhan and Yang. 1080). is associated with receptor desensitization. ~-Amphetamine C:III reverse h~tlo~ridol-intltlce~i supersensilivjty of DA receptors (H;tratz and Tscng. 1980). suggesting that this drug can clungc tlw malecular properties and the intcroctions of the receptor proteins. The existence of many prominent Jitycrcnccs in the actions of chrdnicnlly udministcrcd tl-amphetamine and cocaine (Post. lY77) prompted an examinination of the action of cocaine OII striatal DA receptors, including the action of coc:iinc on the B,,,.,, of agonist binding. stimulatiol~ of adenylate cyclase by DA and on the content of calmodulin bound to rat striatal membranes. The content of membrane-bound calmodulin is characteristically increased in supersensitive DA receptors (Hanbauer and Costa, 1980), whereas, sub-sensitivity triggered by persistent activation of dopamine receptors by DA or apomorphine, is associated with a reduced membrane-bound calmodulin content (Hanbauer, Gimhie and Lovenberg, 1979). METHODS Male Sprague-Dawley rats (15&200 g) were kept under standard conditions with 12 hr lightdark cycle and free access to water and food (Purina Chow). The rats were injected with saline or cocaine HCl (20 mg/kg,i.p.), killed by decapitation at various times thereafter and the nucleus caudatus and nucleus accumbens were dissected rapidly.
1146
M.
MEMO
Adenylate cyclase activity was measured as described by Clement-Cormier. Kebabian. Petzold and Greengard (1974) with minor modifications. In brief, the tissue was homogenized in 10 mM Tris-maleate buffer pH 7.5 containing 1.2 mM EGTA using a glassteflon homogenizer. The incubation medium contained 82.5 mM Tris-maleate buffer pH 7.5, 5 mM MgSOd, 2mM cyclic AMP, 5 mM theophylline, 0.3 mM EGTA (carried over from tissue preparation), 0.01 mM GTP. 2Okcg pyruvate kinase. 4mM phosphoenolpyruvate. I mM C3H]ATP (1 10h cpm) and homogenate equivalent to 1 mg of tissue. The mixtures were incubated in the absence or presence of various concentrations of dopamine or NaF in a final volume of 300~1 for 3 min at 30 C. The reaction was stopped by the addition of a solution containing 20 mM ATP and 0.7 mM cyclic AMP and by heating the tubes for 3 min at 90 C. The amount of [3H]cyclic AMP formed was measured as described by Salomon, I.ondos and Rodbell ( 1974) using subsequent chromatography on Dowex 50W X 4 (IO@-200 mesh. BioRad) and alumina oxide (alumina, Woelm neutral, Act. 1: ICN Nutritional Biochemicals). The recovery of cyclic AMP 65-75”,,. Cyclic AMP content was measured in striatal slices with a radioimmunoassay kit supplied by New England Nuclear. Striatal slices (230ltm thick) prepared with a Sorvall tissue sectioner (TC-2) were preincubated for 30 min in Krebs-bicarbonate solution pH 7.4 supplemented with 10 mM dextrose and 1.14mM ascorbic acid under constant oxygenation (95”,, O2 + 5”,, COz). The incubation was continued for 40min in the absence or presence of various concentrations of choleratoxin (Schwarz,‘Mann). Thereafter. the medium containing the slices was heated for 3 min at 90 C and centrifuged at 1OOOg for IOmin. The cyclic AMP content was measured in 50~1 aliquots of the supernatant fraction.
et (11.
thereafter rapidly filtered over Whatman GF/B filter. Specific C3H]spiroperidolor [3H]N-propylnorapomorphine-binding was determined by the difference in binding obtained in the presence or absence of dopamine (lo-4 M). In experiments designed to study in citro the effect of cocaine or lidocaine (xylocaine HCI, Astra Pharmaceutical Products, Worchester, MA), on specific [‘H]spiroperidolor [3H]N-propylnorapomorphine binding, the membrane suspension was preincubated for 60min in the presence of 10eh M cocaine or lidocaine and thereafter, the ligand binding was assayed as described above. The protein content was measured according to Lowry, Rosenbrough, Farr and Randall (195 I) using bovine serum albumin (Miles Laboratories) as a standard. En:_vme linked
[3H]N-propylnorapomorphine(58.5 Ci/mmol: New England Nuclear) and [3H]spiroperidol(35.9 Ciimmol. New England Nuclear) specific binding was determined as described by Creese. Padgett. Fazzini and Lopez (1979) and Burt. Creese and Snyder (1977). respectively. Rats were killed 60min after an injection of saline or cocaine (20mg,kg. i.p.) and striatal homogenates were prepared at 4-C in 50 mM Tris-HCI buffer. pH 7.6 using a Polytron homogenizer and centrifuged at 5O.OOOg for 10min. The pellet was washed in the same volume of buffer and centrifuged as described before. The pellet obtained was then resuspended in the same volume of 50 mM Tris-HCI buffer. pH 7.6 containing 120 mM NaCl. 5 mM KCI. 2 mM CaCI, and 1 mM MgCI,. The incubation mixture (1 ml) contained [3H]spiroperidol (0.2-1.2 nM) or [3H]N-propylnorapomorphine (0.2-1.4nM) and an aliquot of membrane suspension corresponding to 10 mg of tissue fresh weight. The mixtures were incubated for 10min at 37’C and
Assam (ELISA)
for
cal-
Caudate nuclei were homogenized (Glass-teflon homogenizer) in 0.32 M sucrose and centrifuged at 1OOOg for 1Omin. The supernatant fraction was removed and centrifuged at 100,000 g for 30 min. The resultant pellet was homogenized in 0.05 M Trisbuffer pH 7.4 containing O.l”, Lubrol WX (Sigma), sonicated for 0.5 min and centrifuged at 100,000 g for 30min. The calmodulin content was measured in the supernatant fraction obtained by the first high speed centrifugation (referred to as soluble) and in the pellet extract (referred to as membrane-bound) by microELISA using a specific antibody toward calmodulin as described recently (Hanbauer et al.. 1980).
RESULTS
Spehjic
binding of[‘H]spiroperido/
norapomorpkine
in srriutum
and [‘H]N-prop_vl-
qf‘rars trrrrted
with cocaine
injection of cocaine (20mgikg. i.p.) significantly increased the specific binding of [3H]spiroperidol-a DA receptor antagonist-and that of C3H]Npropylnorapomorphine-a DA receptor agonist. Figure 1 shows a Scatchard analysis of [3H]spiroperidol (A) and [3H]1V-propylnorapomorphine (B) specific binding to crude synaptic membranes prepared from the striata of saline- and cocaine-treated rats. The data indicate that cocaine increased the number of binding sites for both ligands by 37”,,. but the affinity of these ligands for their binding sites was not changed. When striatal slices were preincubated in presence of lo-” M cocaine. the binding capacity for [3H]spiroperidol and [-‘H]N-propylnorapomorphine was significantly increased (Table 1). These results indicate that cocaine may facilitate the specific binding of these ligands by a direct action on striatal membranes. In order to determine whether the changes in B,,, for both ligands was due to the local anesthetic action of cocaine. the effect of lidocaine on the specific’binding of [3H]spiroperidol was studied. Table 1 shows that incubation of striatal slices with One
Specjfic ligtrr~n binding to doptrmine receptors
immunosorhent
mod&n
DA receptor sensitivity and
Bmsx
415 f 12
KD
1.7 f 0.1
Bmsx
584 2 31
KD
1147
cocaine
so-
z
40-
E
1.4 f 0.1
2
30-
s $
20Saline /
10 t
_-
DOFAMINE
I
I
I
100
2&l
300
400
500
BOUND/FREE
lfmolhg
Saline
P ;
400-
\ \
u
w z Kz
\\
prot/nM)
Smsx
391 + 11
Ko
Cocaine
0.53 + 0.03
Bmsx
538 + 21
I
I 10-S
lo-’
I
_
r;/0-i I
01
I
0--O
Cocaine
10.’
CONCENTRATION
(MI
Fig. 2. Stimulation by dopamine of dopamine-sensitive adenylate cyclase in striatal homogenates measured in saline or cocaine-(20 mg/kg. Lp.) treated rats. The rats were killed 60 min after injection of the drug and the formation of c3H]cAMP, catalyzed by adenylate cyclase, was measured. The basal activity of adenylate cyclase in saline and cocaine-treated rats was 57 + 2.3 and 57 k 1.9 pmol/ mg/min, respectively. The results were plotted vs the respective dopamine concentrations mean + SE of CAMP increase (n = 6). The K, for dopamine was obtained by linear regression analysis.
0.59 2 0.04
az* Oh3oi)_..
lidocaine (10m6 M) did not modify the KI, or B,,, for [3H]spiroperidol.
5; qa g% i=-
200-
Dopamine-dependent t
0
adenylate
cyclase
in striatum
of
rats treated with cocaine
0” +
loo-
\
I
1000
500 BOUND/FREE
lfmollmp
proVnMI
Fig. 1. Scatchard plot of [3H]spiroperidol (A) and [‘H]Npropylnorapomorphine (3) binding to dopamine receptors in striatal membranes prepared from rats 60min after injection of saline or cocaine (20mg/kg, i.p.). The ratio of bound was plotted as a function of bound to free radioligand (in the range of 0.2-1.2nM). The results are expressed as mean of 4 experiments in triplicates. The slope of the line l/K,, was computed and the B,,, was determined from the intercept of the line on the ordinate.
The sus~ptibility of striatal adenyiate cyclase to stimulation by DA was significantly increased at 304Omin following an injection of cocaine (20 mg/kg). Figure 2 shows the stimulation of adenylate cyclase activity elicited by various doses of DA. The data indicate that 60min after the cocaine injection, activation by DA of adenylate cyclase was facilitated and resulted in a shift to the left of the doseresponse for DA. Table 2 reports the EDS0 and K, for activation by DA in saline and cocaine-treated rats. The supersensitivity of adenylate cyclase to activation by DA elicited by cocaine injections, was indicated by a decrease in the apparent K, and the ED,, for DA. In addition, when striatal slices were incubated for
Table 1. Changes in kinetic properties of specific ligand binding and dopamine-de~ndent cyclase elicited by cocaine in rat striatal slices Drug Saline Cocaine
Lidocaine
[3H]spiroperidol &nU. K1, 403 * 21 564 f 2s* 398 k 19
1.1 If 0.13 1.0 * 0.11 1.1 +_0.11
* P < 0.05. K, is expressed as nM & SE and B,,,
[3H]N-propyl-norapomorphine B mux
KI,
384 + 22 480 f 29* -
0.51 f 0.041 0.59 + 0.051 -
adenylate
Adenylate cyclase K,W
DA)
1.5 0.34* -
as fmol/mg prot. +_SE of three different experiments. After preincubation of striatal slices in Krebs-bicarbonate solution pH 7.4 for I5 min, the incubation was continued for 60 min in the presence or absence of cocaine or iidocaine (lOa M). The slices were removed and then homogenized and assayed as described in Methods section.
M. MEMO CI ul
1148
Table 2. The effect of cocaine on dopamine-stimulated enylate cyclase in rat striatal homogenates Drug (mg/kg. i.p.) Saline Cocaine
Adenylate ED&M)
Table 4. Effect of cholera toxin on cyclic AMP in striatal slices of saline and cocaine-treated
cyclase K&M)
46 + 5 IS i 2*
(20)
ad-
Cholera toxin (~gjm))
3.2 + 0.35 I.2 + _ 0.13*
0 IO 25 50 100
* P < 0.05 when compared with saline-injected rats. The rats were injected with cocaine 60 min before killing. The values are expresssed as mean k SE of 4 experiments, The EDso represents the dopamine concentration causing a half-maximal stimulation. The K, values were derived from a linear regression analysis of a concentration curve for dopamine in the range of 10~h-lO-J M.
ate
cyclase
to stimulation
by
NaF
or
choleratoxin.
that,
Cocaine treatment increased the striatal content calmodulin. A time course of the selective increase
of in
Table 3. Effect of NaF on adenylate cyclase activity in striatal homogenate of saline and cocaine-treated rats
NaF concentration (mM) 0
I 2.5 5 IO
cyclic AMP (pmol,mg/min) Saline Cocaine 56 86 I I9 142 157
+ f f f f
3.2 4.8 6.7 6.8 7.1
Animals were killed 1 hr after injection cocaine (20 mg/kg. i.p.). Adenylate cyclase activity was measured Salomon et al. (1974).
54 81 I19 136 172
f f f k f
0.35 0.41 0.63 I.1 I.8
f f f * +
0.028 0.031 0.058 0.091 0.1’
0.38 0.39 0.59 0.99 I.8
+ + + + +
0.018 0.021 0.045 0.10 0.13
content extracted from striatal membranes induced by cocaine is shown in Table 5. This increase could be observed at 30 min after injection of the drug and lasted longer than 1 hr. In contrast, the striatal content of soluble calmodulin, which represents about l/4 of the total content of calmodulin, failed to change as a result of cocaine treatment (Table 5). Repeated daily injections of cocaine (30mg/kg) over a period of 3 weeks increased the content of membrane-bound calmodulin without changing the amount of soluble calmodulin stored in the caudate nuclei (Table 6). the calmodulin
in the striatal homogenates of saline-treated rats. the half-maximal activation of adenylate cyclase was elicited by 2.5 mM NaF. In membranes prepared from rats injected with cocaine (20 mgikg. i.p.) 60 min before the experiment, the responsiveness of adenylate cyclase to the activation by NaF was similar. In Table 4. the activation of adenylate cyclase by various concentrations of choleratoxin is compared in striatals’membranes prepared from saline- and cocaine-treated rats. The results indicated that treatment with cocaine failed to modify the responsiveness of membrane-bound adenylate cyclase to choleratoxin activation. 3 shows
Cyclic AMP (pmoljmg + SE) Saline Cocaine
Rats were injected with cocaine (20mg/kg, i.p.) I hr before killing. Striatal slices were preincubated in Krebsbicarbonate solution pH 7.4 for 30 min and then incubated for 40min in the presence of cholera toxin, The cyclic AMP content in the incubation medium was determined by RIA (II = 6).
60min with cocaine (lo-(’ M), a similar decrease in the K, for the DA-elicited activation of adenylate cyclase was obtained (Table 1). In contrast. in the nucleus accumbens, the stimulation of DA-sensitive adenylate cyclase by DA was not modified 60min after the injection of cocaine (data not shown). In this tissue, the EDso for the adenylate cyclase activation by DA was 5 x 10e5 M in saline and cocaine-treated rats. Additional studies were carried out to determine whether cocaine could modify the response of adenylTable
formation rats
3.1 4.5 5.7 6.2 7.3
of saline
or
according
to
DISCUSSION
The data presented suggest that cocaine can influence dopaminergic receptor function by a direct action on the receptor proteins. Cocaine appeared to increase the number of recognition sites for DA and facilitated the responsiveness of adenylate cyclase located in striatal membranes to stimulation by DA. Under the influence of cocaine, the K, for activation by DA of adenylate cyclase was decreased, reflected by a shift to the left of the dose-response curve for
Table 5. Changes various
in calmodulin content of rat striatum times after injection of cocaine
Minutes after injection
j(g Calmodulin. Soluble
0 I5 30 60
I.13 + 0.09 ND 0.94 f 0.29 1.28 f 0.20
at
mg protein i SE Membrane-bound 4.3 4.9 6.4 9.9
+ 0.54 + I.0 + 0. I * If: 1.0*
*P < 0.01: a = 5. ND-not determined. Striatal homogenates were centrifuged at IOOOg for 10 min. The supernatant fraction was removed and centrifuged at 1OO.OOOg for 30 min. The resulting pellet was extracted with 0.05 M Tris-buffer pH 7.4 containing O.l”,, Lubrol WX:The calmodulin content of supernatant fraction (soluble) and pellet extract (membrane-bound) were measured by ELISA.
DA receptor
Table 6. Content
Number of cocaine injections Control 1 21
of calmodulin in rat caudate nucleus cocaine treatment
pg Calmodulin/mg Soluble 1.5 + 0.32 0.94 + 0.29 1.2 + 0.26
sensitivity
after
protein + SE Membrane-bound 4.03 * 0.70 6.38 + 0.3_5* 6.51 k 0.69*
* P < 0.05; n = 6. Rats were injected with cocaine (30 mg/kg, i.p.) once or once daily for 21 days and were killed 30 min after the last injection. Striatal homogenates in 0.32 M sucrose were centrifuged at IOOOg for IOmin. The supernatant was removed and centrifuged 100.000 g for 30 min. Calmodulin content in the supernatant fraction (soluble) and pellet extract (membrane-bound) was determined by microELISA (Hanbauer et al., 1980).
DA. This action of cocaine occurred within 60min after its administration. Since cocaine increased the amount of DA that is present in the synaptic cleft by inhibiting DA reuptake (Iversen, 1966; Ross and Renyi, 1966), and increasing the transmitter turnover rate (Costa, Groppetti and Naimzada, 1972), it was expected that the response would be a down-regulation of the DA receptor. However, unlike d-amphetamine, which down-regulates DA receptors (Hanbauer et crl., 1980), cocaine increased the receptor sensitivity to activation by DA. This unexpected action of cocaine on DA receptor regulation can be also elicited by incubating striatal slices with cocaine and appears to be due to the direct interaction between the drug and post-synaptic membranes. Similar in ritra studies, which were carried out with lidocaine. a local anesthetic with a greater potency than cocaine, failed to show a change in B,,,, for spiroperidol. Hence. cocaine appears to act specifically on postsynaptic DA receptors. At the present time. the exact molecular mechanism mediating this action is unclear. It appears unlikely that cocaine modifies the GTP-binding protein, which couples the DA recognition sites with the cyclase (Creese et ol., 1978). In fact, cocaine failed to modify the activation of adenylate cyclase by NaF or choleratoxin, which have been shown to require GTP and GTP-binding protein for their action (Pfeuffer, 1977; Gill and Meren. 1978: Ross, Howlett. Ferguson and Gilman, 1978). Cocaine appears to modify directly the mechanism that couples the regulation of receptor excitability with the amount of DA present in the synaptic cleft. It is possible that. due to a direct action of cocaine, the responsiveness of the DA receptor is never diminished even after repeated administration of the drug. In the present working model, it was proposed that cocaine changes the characteristics of the lipid bilayers allowing an uncovering of a greater number of DA recognition sites. Whether cocaine exerts its effect by changing the methylation of membrane phospholipids (Hirata. Strittmatter and Axelrod, 1979) remains to be
and cocaine
1149
ascertained by future experiments. An important molecular mechanism operative in the sensitization of DA receptors is the increase of calmodulin content that is bound to synaptic membranes (Gnegy et al., 1977: Lucchelli, Guidotti and Costa. 1978). In contrast, a decrease in membranebound calmodulin appears to be associated with desensitization of the dopamine receptor (.Memo and Hanbauer, 1981). Since cocaine failed to modify the soluble calmodulin content, it has to be considered whether. during sensitization of the receptor, the amount of extractable calmodulin is increased. This possibility leads to the speculation that calmodulin can be bound more or less tightly to specific membrane proteins by a CaZ+ -dependent mechanism and that. during desensitization, the amount of Ca2’ present in membranes may increase while, during supersensitivity, the Ca2+ content of membranes may be decreased. Cocaine could also act directly on the phospholipids in the membrane facilitating Ca2+ release from membrane constituents thereby increasing the mobility of membrane-bound calmodulin. It is important to emphasize that. although the total calmodulin content may be in excess of the amount required for enzyme activation. the availability of free calmodulin appears to be limited through its Ca2+dependent binding to specific proteins present in membrane and cytosol fractions. In regard to the action of cocaine, the increased availability of calmodulin could promote the supersensitivity of adenylate cyclase to activation by DA. Future experiments in this laboratory are planned to determine whether enzyme activation is dependent on the dissociation of calmodulin from its binding sites.
AcknoMl~dgrmmt-This grant of the Scottish NiMiTi. U.S.A.
work was supported Rite Schizophrenia
in part by a Foundation.
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11.50
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Gnegy. M. E., Uzunov, P. and Costa, E. (1977). Participation of an endogenous Ca’+ binding protein activator in the development of drug-induced supersensitivity of striatal dopamine receptors. J. Pharmac. exp. Ther. 202: 558-564. Hanbauer, I. and Costa, E. (1980). Role of calmodulin in dopaminergic transmission. In: Calcium and Ceil Functiun (Cheung, W. Y., Ed.), Vol. I, pp. 253-271. Academic Press, New York. Hanbauer, I.. Gimble, J. and Lovenberg, W. (1979). Changes in soluble calcium-dependent regulator following activation of dopamine receptors in rat striatal slices, Neuropharmacology
18: 851-857.
Hanbauer, I.. Pradhan. S. and Yang, H.-Y. T. (1980). Role of calmodulin in dopaminergic transmission. Ann. N. Y. Acad. Sci. 356: 292-303. Haracz, J. L. and Tseng, L-F. (1980). Reduction of dopaminergic su~r~nsitivity by a single dose of amphetamine. N~unyn Schmiedebergs Arch. Phur~c. 313: 131-133.
Hirata, I., Strittmatter, W. J. and Axelrod, J. (1979). ,&Adrenergic receptor agonists increase phospholipid methylation, membrane fluidity and j-andrenergic receptor-adenylate cyclase coupling. Proc. nafn. Acad. Sci. U.S.A. 76: 368-372. Iversen, L. L. (1966). Accumulation of a-methyltryramine by the noradrenaline uptake process in the isolated rat heart. J. Pbarm. 18: 481484. Kebabian, J. W., Petzold, Cl. L. and Greengard, P. (1972). Dopamine-sensitive adenylate cyclase in caudate nucleus of rat brain and its similarity to the dopamine receptor. Proc. natn. Acad. Sci. U.S.A. 69: 2145-2149.
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Lowry, 0. H., Rosenbrough, M. J., Farr, A. C. and Randall. R. J. (1951). Protein measurement with the Folin phenol reagent. J. biol. Chem. 193: 265-275.
Lucchelli, A. Guidotti, A. and Costa, E. (1978). Striatal content of Ca*+ dependent regulator protein and dopaminergic receptor function. Brain Res. 155: 13&135. Memo, M. and Hanbauer, 1. (1981). In uitro subsensitivity of adenylate cyclase-coupled dopamine receptors in striatal slices. Role of calmodulin. Fed. Prof. 40: 653. Pfeutfer, T. (1977). GTP-binding proteins in membranes and the control of adenylate cyclase activity. J. biol. Chem. 252: 7224-7234.
Post, R. M. (1977). Progressive changes in behavior and seizures following chronic cocaine administration: Relationship to kindling and psychosis. In: Adcances in Behacioraf Biology (Ellinwood, E. H. and, Kilbey. M. M. Ed@. Vol. 21. pp. 353-374. Plenum Press. New York. Quick, M. and Ivetsen, L. L. (1978). Subsensitivity of the rat striatal douaminernic system after treatment with bromocriptine:- Effects-on f3H]Spiperone binding and dooamine-stimulated cvclic AMP formation. NaunvnSch’miedergs Arch. Pharkac. 304: 141-145.
Ross, S. B. and Renyi, A. L. (1966). Uptake of some tritiated sympathomimetic amines by mouse brain cortex in vitro. Acta pharmac. tax. 24: 297-309.
ROSS,E., Howlett, A., Ferguson. K. and Gilman, A. (1978). Reconstitution of hormone-sensitive adenylate cyclase activity with resolved components of the enzyme. J. bioi. Chem. 253: 64i31-6412.
Salomon, Y., Londos, C. and Rodbeil, M. (1974). A highly sensitive adenylate cyclase assay. Analpt. &o&em. 58: 541-548. Singer.S. L. and Nicholson, G. L. (1972). The fluid mosaic model of the structure of cell membranes. Science 175: 720-731. Su, Y-F., Harden, T. K. and Perkins, J. P. (1980). Catecholamine-specific desensitization of adenylate cyclase. Evidence for a multiple process. J. biof. Chem. 255: 7410-7419.