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Fusion transfer of dopamine DA1 receptors to friend erythroleukemia cells
Neuroscience Letters, 53 (1985) 209-214
209
Elsevier Scientific Publishers Ireland Ltd.
NSL 03108
F U S I O N T R A N S F E R OF D O P A M I N E D A t R E C E P T O R S TO FRIEND
ERYTHROLEUKEMIA CELLS
MAR JUT OLASMAA* and LARS TERENIUS
Department of Pharmacology, Uppsala University, Uppsala (Sweden) (Received July 19th, 1984; Revised version received and accepted October 23rd, 1984)
Key words: adenylate cyclase - dopamine DA~ receptor - membrane fusion transfer - receptor coupling - striatum
Dopamine DAI receptors were transferred from rat striatal membranes to Friend erythroleukemia cells (Fc) by membrane fusion. The Fc cells lack DA~ receptors but have a functional adenylate cyclase system. The striatal membranes bearing DA1 receptors were treated with N-ethylmaleimide (NEM) prior to fusion to inactivate their intrinsic adenylate cyclase activity. Fusion of the NEM-treated membranes and the Fc cells was induced by polyethylene glycol treatment to form a functional system de novo, which could be demonstrated by measuring the increase in cAMP production after addition of dopamine. This method provides a possibility to study the functional competence of receptors in neural tissue in more detail.
A hormone receptor-coupled adenylate cyclase (AC) system is believed to be composed of three components: the receptor, a regulatory guanosine 5'-triphosphate (GTP)-binding protein (termed G or N) and the catalytic moiety [7]. The generation of cAMP in such a system will consequently be a function of the amounts and the functional integrity of all its components. It has been shown by Schramm et al. [9] that there is a functional homology between the N-protein and the catalytic moiety of the AC system of several cell types from different species and that it is possible to exchange different recognition units by fusion. Studies on reconstitution have also been performed, where for instance purified N-protein has successfully been implanted into cells lacking this regulatory protein [2, 10]. Several neurotransmitter receptors are coupled to AC and the biological characteristics of these receptors have been studied extensively [6]. However, the estimation of the enzyme activity in neural tissue is somewhat problematic because of the high basal activity. We decided to apply the fusion method as an analytical tool for analysis of the functional competence of receptors and of receptor-AC coupling avoiding the interference from basal AC activity. It was investigated whether the dopamine DA1 receptor could be transferred to Friend erythroleukemia cells (Fc). *Author for correspondence at: Box 591, S-75124 Uppsala, Sweden. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
210 The brain membranes bearing receptors were prepared from corpus striatum of male Sprague-Dawley rats (b.wt. 125 g). The tissue was homogenized in the cold in 0.32 M sucrose including 20 mM Tris-HC1, pH 7.4, and 2 mM ethyteneglycoltetraacetic acid (EGTA) and subjected to differential centrifugation. The crude mitochondrial pellet was recovered and subjected to osmotic shock. The following centrifugation (11,000 g, 20 min) sedimented mitochondria leaving brain membranes in the supernatant, which were then spun down and resuspended in sucrose for freezing at - 8 0 ° C [11]. The protein concentration was measured according to Lowry [4]. Prior to fusion the membranes were treated with N-ethylmaleimide (NEM) to destroy their AC activity. The membranes were suspended in 10 mM MOPS (morpholinopropanesulfonic acid) buffer, pH 7.4 (3 mg protein per ml) and incubated with 5 mM NEM for 25 min at 4°C. The reaction was stopped by the addition of 5 vols. of 10 mM MOPS buffer with 3 mM mercaptoethanol, followed by centrifugation (30,000 g, 15 min). The pellet was exposed to a solution of phospholipid (L-ct-phosphatidylcholine (type II-s; Sigma), 0.3 mg per mg protein) for 5 min at 4°C. To solubilize the phospholipid it was suspended in 10 mM Tris-HC1, pH 7.4, 1 mM ethylenediaminetetraacetic acid (EDTA) and sonicated 3 × 5 min under N2, or until an opalescent solution was obtained. To the phospholipid-treated membranes, 1 M MgC12 was added to a final concentration of 10 mM, which was followed by additional incubation for 15 min at 4°C, whereafter 2 vols. of 10 mM MOPS, pH 7.4, were added. The membrane suspension was then divided into aliquots (200 #g protein), pelleted and frozen on liquid N2. The Fc cells derive from a culture used by Schramm [9]. The cells were grown in H a m ' s F-10 culture medium (GIBCO, New York) supplemented with 10o70 newborn calf se~um and antibiotics (100 IU penicillin/ml, 50/~g streptomycin/ml), in a humidified 5°70 CO2 in air atmosphere at 37°C. They were harvested when the concentration reached 3 × 105 per ml, washed in Na ÷ salt medium (mM: 135 NaC1, 5 KC1, 0.8 MgCl2, 20 Tris-HC1, pH 7.4) and finally suspended in the same medium at a concentration o f 1 x 107 cells per ml. For fusion, 10 7 cells were sedimented on top of a pellet of NEM-treated brain membranes corresponding to 200 #g protein. From then on the fusion procedure was carried out a~ 37°C. The tubes were transferred to a 37°C incubator and 0.5 ml 52°7o polyethylene glycol (PEG 6000; Kebo AB, Stockholm), dissolved in supplemented Na ÷ salt medium [mM: 2 adenosine triphosphate (ATP), 0, l EDTA, 4 MgC12, 6 glucose, titrated to p H 7.5 with NaOH], was added under strong vortex mixing. The cell m e m b r a n e - P E G mixture was incubated for 2 min, 1 ml of Na ÷ salt medium was added and after another 2 min, 9 ml of the same buffer. Then the suspension was pelleted and resuspended in 600/~l o f hypotonic buffer (1 mM TrisHCI, pH 7.5, 0.2 mM MgCI2, l0 #M E G T A and 1 mM mercaptoethanol) for the AC assay. The AC activity was measured in a reaction mixture containing 50/A of AC assay
211 solution, 50 #1 of membrane suspension and 10 #1 of test agent to give a final volume of 110 #l. The AC assay solution consisted of 39 mM creatine phosphate, 50 units/ml creatine phosphokinase, 2.4 mM cAMP, 0.12 M MOPS (pH 7.5), 14.4 mM MgCI2, 0.5 mM EGTA, 10 mM mercaptoethanol, 0.3 mM ATP, 10 mM theophylline and 44 nM [ct-32p]ATP (410 Ci/mmol; Amersham Radiochemical Centre). The reaction was initiated by adding the membrane suspension (equivalent to 16 #g protein) to the AC assay solution with test agent, incubated at 37°C for 10 min and terminated by addition of 0.1 ml stop solution (0.5 mM cAMP, 4 mM ATP and 2500 cpm [3H]cAMP (36 Ci/mmol; Amersham) to obtain the cAMP recovery. The tubes were heated to boiling for 3 min, cooled and then centrifuged. cAMP was isolated according to Salomon et al. [8]. The standard assay setting comprised 10 tubes with duplicates of one of the following agents: 50 /zM dopamine, 1 /tM spiroperidol, 12 mM NaF, 10 #M GppNHp (guanyl-5-ylimidodiphosphate), a non-hydrolyzable analogue of GTP and 30/zM PGE1 (prostaglandin El). As controls, NEM-treated brain membranes and Fc cells, fused with themselves (Fcx Fc) were exposed to these agents. Dopamine (DA) was used as a ligand to show the DA1 receptor-induced stimulation of the AC activity and the basal level indicated by spiroperidol, which even blocks the possible endogenous DA activity. NaF and GppNHp, which are direct activators of the AC system, were used to confirm that the AC system of the Fc cells could be activated even after fusion. Prostaglandin E1 (PGEI) was used to determine the receptor coupled AC activity in the recipient cells. A critical prerequisite for our study was that the F~ cells lacked DAI receptors. This could be demonstrated (Table I) by showing that the cells did not respond to DA. On the other hand, the response to fluoride was substantial and GppNHp also gave considerable elevation of the cAMP level. As the Fc cells carry endogenous
TABLE I R E C O V E R Y OF DAI R E C E P T O R F U N C T I O N A L C A P A C I T Y A F T E R F U S I O N OF NEMT R E A T E D BRAIN M E M B R A N E S W I T H Fc CELLS L A C K I N G DA1 R E C E P T O R S Effects of 50 ~M DA, 12 m M NaF, 10 #M G p p N H p and 30/zM PGEI on Friend erythroleukemia cells fused with themselves (Fcx Fc), Fc cells fused with NEM-treated brain m e m b r a n e s (Fc x Bm NEM), Bm N E M or Bm only. The effects are expressed as production o f c A M P in pmol per min, and values are m e a n s ± S.E.M. o f duplicates from 3 independent experiments. Treatment
Bm (n = 6)
Bm N E M (n = 3)
F¢ x Fc (n = 6)
Fc × Bm N E M (n -= 6)
DA Spiroperidol NaF GppNHp PGE1
1.46 ± 0.34 1.03 + 0.27 6.35 ± 1.25 6.09 ± 0.56 1.44+0.35
0.08 +_0.01 0.04 ± 0.02 0.13 ± 0.05 0.05 + 0.05 0.02±0.02
0.33 + 0.03 0.28 + 0.02 14.4 ± 0.76 1.75 ± 0.08 1.66+0.17
0.92 + 0.05 0.45 _+0.01 9.67 _+0.28 4.70 ± 0.22 0.94±0.11
212
PGE1 receptors, the PGEI stimulation, which was of the same magnitude as that induced by G p p N H p , indicated the functional capacity of the F~ ceils. The N E M treatment inactivated the brain m e m b r a n e AC system, as indicated by incapability of DA, N a F and G p p N H p to produce c A M P formation in these membranes (Table I). When the two components, the Fc cells with a functional AC system and the NEM-treated brain membranes with the receptors, but no functional AC, were fused together and subjected to DA stimulation, a two-fold increase in c A M P production could be observed (Table I). This can be compared with a corresponding increase of 1.4 obtained f r o m the stimulation of untreated brain membranes without fusion. The time course of the reaction was studied over the time range 2.5 to 22.5 min. Within this time interval linearity was observed (Fig. 1). Because a 10-min incubation time was found to be convenient and in the linear range, it was chosen. The ratio between DA and spiroperidol treatment was found to be stable in at least 10 independent assays (2.04_+ 0.11, mean + S.E.M.). Butyrophenones, like spiroperidol, are known to antagonize DA effects. Spiroperidol has micromolar affinity to DA1 sites and exhibits nanomolar affinity only to DAz sites. To indicate that the fusion transferred receptor is of the DA~ type, we studied the effects of two different concentrations of spiroperidol against stimulation obtained with 50/~M D A in the Fc ×Bm N E M system (Bm = brain membrane). Spiroperidol at 1 #M gave 45.2 + 2.1070 inhibition (mean + S.E.M.; n = 7, P < 0 . 0 0 1 ) whereas at 1 nM there was no effect 1.0+7.1°70 ( n = 7 , P > 0 . 0 5 ) . A
DA (Bm)
40 o O
S P I R e (Bm)
E ¢z V
a.
:E <
DA ( F c x ~ M )
°~
o >,. o
10 .0
0
&
5
10
S P I R e (F c x ~ ~ )
15
20
25
Time (rain) Fig. 1. Time course of dopamine-stimulated adenylate cyclase activity as determined by linear regression analysis. The activity is shown in brain membranes (Bm) stimulated directly (o) and in NEM-treated brain membranes fused with Friend leukemia cells (Fc ×Bm NEM) (A). Membrane protein concentration in the assay was 145 t~g per ml. Values are means o f duplicate determinations.
213 specific DA1 antagonist, SCH 23390 (Schering, N J, U.S.A.) [3] at 0.1 #M, gave 63.0___7.6°-/o ( n = 5, P < 0 . 0 1 ) inhibition of 50 #M D A stimulation. The agonists a p o m o r p h i n e and N P A (N-propylnorapomorphine; Research Biochemicals Inc., MD, U.S.A.), at concentrations o f 10 /~M gave 6 6 . 7 + 5 . 6 % ( n = 5 , P < 0 . 0 1 ) increase, and 67.4 + 5.2% (n = 5, P < 0.001) respectively, whereas 50 #M D A stimulation caused 108 + 5.6% (n = 8, P < 0 . 0 0 1 ) i n c r e a s e in AC activity. Pilot experiments have also been performed with human nucleus caudatus tissue, prepared according to the same procedure. Here the increase with 50 ttM dopamine was 52% for the fused material and 22% for the untreated m e m b r a n e s (mean values o f 3 experiments). P o s t m o r t e m studies have shown that the functional capacity of an AC-coupled receptor declines more rapidly with postmortem interval [12] than receptor binding capacity, the variable c o m m o n l y studied by most investigators [5]. The fusion method m a y allow estimations not only of binding capacity o f receptors but also o f their biological functionality after longer postmortem periods. This may be of interest in clinical studies. It is notable that by fusing together membranes from different species it is still possible to form a functional unity o f the system, where molecular interaction a m o n g the three separate components is needed. Consequently, this enables each individual c o m p o n e n t of AC-coupled receptors to be studied. The initial phases of this study were carried out at the laboratory of Professor Michael Schramm, Department of Biological Chemistry, Hebrew University, Jerusalem, Israel, We are most grateful to him and to Ms. Mira Korner and Dr. Yoav Citri for patiently guiding us through the secrecies of m e m b r a n e fusion transfer. We thank Professor Kenneth Nilsson and Ms. Helena Jernberg for housing o f the cells. W o r k in Israel was supported by Grant AM-10451 f r o m the National Institute o f Health. L.T. was supported by the Swedish Medical Research Council (Grants 04X-3766 and 04R-6624,6724).
1 Carenzi, A., Gillin, J.C., Guidotti, A., Schwartz, M.A., Trabucchi, M. and Wyatt, R.J., Dopaminesensitive adenylyl cyclase in human caudate nucleus, Arch. Gen. Psychiat., 32 (1975) 1056-1059. 2 Citri, Y. and Schramm, M., Resolution, reconstitution and kinetics of the primary action of a hormone receptor, Nature (Lond.), 287 (1980) 297-300. 3 lorio, L.C., Barnett, A., Leitz, F.H., Houser, V.P. and Korduba, C.A., SCH 23390, a potential benzazepine antipsychotic with unique interactions on dopaminergic systems, J. Pharmacol. Exp. Ther., 226 (1983) 462-468. 4 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 5 Owen, F., Cross, A.J. and Crow, T.J., Ligand binding studies in brains of schizophrenics. In P.G. Strange (Ed.), Cell Surface Receptors, Ellis Horwood, Chichester, U.K. 1983. 6 Palmer, G.C., Effects of psychoactivedrugs on cyclicnucleotides in the central nervous system, Prog. Neurobiol., 21 (1983) 1-133. 7 Rodbell, M., The role of hormone receptors and GTP-regulatory proteins in membrane transduction, Nature (Lond.), 284 (1980) 17-22.
214 8 Salomon, Y., Londos, C. and Rodbell, M., A highly sensitive adenylate cyclase assay, Anal. Biochem., 58 (1974) 541-548. 9 Schramm, M., Transfer of glucagon receptor from liver membranes to a foreign adenylate cyclase by a membrane fusion procedure, Proc. Nat. Acad. Sci. USA, 76 (1979) 1174-1178. 10 Stiles, G.L. and Lefkowitz, R.J., A reconstitution assay for the guanine nucleotide regulatory protein of the adenylate cyclase system using turkey erythrocyte membranes as the acceptor preparation, Arch. Biochem. Biophys., 217 (1982) 368-375. 11 Terenius, L., Stereospecific interaction between narcotic analgesics and a synaptic plasma membrane fraction of rat cerebral cortex, Acta Pharmacol. Toxicol., 32 (1973) 317-319. 12 Witte, P. and Matthaei, H., Post mortem changes in adenylate cyclase activity in rat brain striatum, Experientia, 35 (1979) 1421-1422.
209
Elsevier Scientific Publishers Ireland Ltd.
NSL 03108
F U S I O N T R A N S F E R OF D O P A M I N E D A t R E C E P T O R S TO FRIEND
ERYTHROLEUKEMIA CELLS
MAR JUT OLASMAA* and LARS TERENIUS
Department of Pharmacology, Uppsala University, Uppsala (Sweden) (Received July 19th, 1984; Revised version received and accepted October 23rd, 1984)
Key words: adenylate cyclase - dopamine DA~ receptor - membrane fusion transfer - receptor coupling - striatum
Dopamine DAI receptors were transferred from rat striatal membranes to Friend erythroleukemia cells (Fc) by membrane fusion. The Fc cells lack DA~ receptors but have a functional adenylate cyclase system. The striatal membranes bearing DA1 receptors were treated with N-ethylmaleimide (NEM) prior to fusion to inactivate their intrinsic adenylate cyclase activity. Fusion of the NEM-treated membranes and the Fc cells was induced by polyethylene glycol treatment to form a functional system de novo, which could be demonstrated by measuring the increase in cAMP production after addition of dopamine. This method provides a possibility to study the functional competence of receptors in neural tissue in more detail.
A hormone receptor-coupled adenylate cyclase (AC) system is believed to be composed of three components: the receptor, a regulatory guanosine 5'-triphosphate (GTP)-binding protein (termed G or N) and the catalytic moiety [7]. The generation of cAMP in such a system will consequently be a function of the amounts and the functional integrity of all its components. It has been shown by Schramm et al. [9] that there is a functional homology between the N-protein and the catalytic moiety of the AC system of several cell types from different species and that it is possible to exchange different recognition units by fusion. Studies on reconstitution have also been performed, where for instance purified N-protein has successfully been implanted into cells lacking this regulatory protein [2, 10]. Several neurotransmitter receptors are coupled to AC and the biological characteristics of these receptors have been studied extensively [6]. However, the estimation of the enzyme activity in neural tissue is somewhat problematic because of the high basal activity. We decided to apply the fusion method as an analytical tool for analysis of the functional competence of receptors and of receptor-AC coupling avoiding the interference from basal AC activity. It was investigated whether the dopamine DA1 receptor could be transferred to Friend erythroleukemia cells (Fc). *Author for correspondence at: Box 591, S-75124 Uppsala, Sweden. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
210 The brain membranes bearing receptors were prepared from corpus striatum of male Sprague-Dawley rats (b.wt. 125 g). The tissue was homogenized in the cold in 0.32 M sucrose including 20 mM Tris-HC1, pH 7.4, and 2 mM ethyteneglycoltetraacetic acid (EGTA) and subjected to differential centrifugation. The crude mitochondrial pellet was recovered and subjected to osmotic shock. The following centrifugation (11,000 g, 20 min) sedimented mitochondria leaving brain membranes in the supernatant, which were then spun down and resuspended in sucrose for freezing at - 8 0 ° C [11]. The protein concentration was measured according to Lowry [4]. Prior to fusion the membranes were treated with N-ethylmaleimide (NEM) to destroy their AC activity. The membranes were suspended in 10 mM MOPS (morpholinopropanesulfonic acid) buffer, pH 7.4 (3 mg protein per ml) and incubated with 5 mM NEM for 25 min at 4°C. The reaction was stopped by the addition of 5 vols. of 10 mM MOPS buffer with 3 mM mercaptoethanol, followed by centrifugation (30,000 g, 15 min). The pellet was exposed to a solution of phospholipid (L-ct-phosphatidylcholine (type II-s; Sigma), 0.3 mg per mg protein) for 5 min at 4°C. To solubilize the phospholipid it was suspended in 10 mM Tris-HC1, pH 7.4, 1 mM ethylenediaminetetraacetic acid (EDTA) and sonicated 3 × 5 min under N2, or until an opalescent solution was obtained. To the phospholipid-treated membranes, 1 M MgC12 was added to a final concentration of 10 mM, which was followed by additional incubation for 15 min at 4°C, whereafter 2 vols. of 10 mM MOPS, pH 7.4, were added. The membrane suspension was then divided into aliquots (200 #g protein), pelleted and frozen on liquid N2. The Fc cells derive from a culture used by Schramm [9]. The cells were grown in H a m ' s F-10 culture medium (GIBCO, New York) supplemented with 10o70 newborn calf se~um and antibiotics (100 IU penicillin/ml, 50/~g streptomycin/ml), in a humidified 5°70 CO2 in air atmosphere at 37°C. They were harvested when the concentration reached 3 × 105 per ml, washed in Na ÷ salt medium (mM: 135 NaC1, 5 KC1, 0.8 MgCl2, 20 Tris-HC1, pH 7.4) and finally suspended in the same medium at a concentration o f 1 x 107 cells per ml. For fusion, 10 7 cells were sedimented on top of a pellet of NEM-treated brain membranes corresponding to 200 #g protein. From then on the fusion procedure was carried out a~ 37°C. The tubes were transferred to a 37°C incubator and 0.5 ml 52°7o polyethylene glycol (PEG 6000; Kebo AB, Stockholm), dissolved in supplemented Na ÷ salt medium [mM: 2 adenosine triphosphate (ATP), 0, l EDTA, 4 MgC12, 6 glucose, titrated to p H 7.5 with NaOH], was added under strong vortex mixing. The cell m e m b r a n e - P E G mixture was incubated for 2 min, 1 ml of Na ÷ salt medium was added and after another 2 min, 9 ml of the same buffer. Then the suspension was pelleted and resuspended in 600/~l o f hypotonic buffer (1 mM TrisHCI, pH 7.5, 0.2 mM MgCI2, l0 #M E G T A and 1 mM mercaptoethanol) for the AC assay. The AC activity was measured in a reaction mixture containing 50/A of AC assay
211 solution, 50 #1 of membrane suspension and 10 #1 of test agent to give a final volume of 110 #l. The AC assay solution consisted of 39 mM creatine phosphate, 50 units/ml creatine phosphokinase, 2.4 mM cAMP, 0.12 M MOPS (pH 7.5), 14.4 mM MgCI2, 0.5 mM EGTA, 10 mM mercaptoethanol, 0.3 mM ATP, 10 mM theophylline and 44 nM [ct-32p]ATP (410 Ci/mmol; Amersham Radiochemical Centre). The reaction was initiated by adding the membrane suspension (equivalent to 16 #g protein) to the AC assay solution with test agent, incubated at 37°C for 10 min and terminated by addition of 0.1 ml stop solution (0.5 mM cAMP, 4 mM ATP and 2500 cpm [3H]cAMP (36 Ci/mmol; Amersham) to obtain the cAMP recovery. The tubes were heated to boiling for 3 min, cooled and then centrifuged. cAMP was isolated according to Salomon et al. [8]. The standard assay setting comprised 10 tubes with duplicates of one of the following agents: 50 /zM dopamine, 1 /tM spiroperidol, 12 mM NaF, 10 #M GppNHp (guanyl-5-ylimidodiphosphate), a non-hydrolyzable analogue of GTP and 30/zM PGE1 (prostaglandin El). As controls, NEM-treated brain membranes and Fc cells, fused with themselves (Fcx Fc) were exposed to these agents. Dopamine (DA) was used as a ligand to show the DA1 receptor-induced stimulation of the AC activity and the basal level indicated by spiroperidol, which even blocks the possible endogenous DA activity. NaF and GppNHp, which are direct activators of the AC system, were used to confirm that the AC system of the Fc cells could be activated even after fusion. Prostaglandin E1 (PGEI) was used to determine the receptor coupled AC activity in the recipient cells. A critical prerequisite for our study was that the F~ cells lacked DAI receptors. This could be demonstrated (Table I) by showing that the cells did not respond to DA. On the other hand, the response to fluoride was substantial and GppNHp also gave considerable elevation of the cAMP level. As the Fc cells carry endogenous
TABLE I R E C O V E R Y OF DAI R E C E P T O R F U N C T I O N A L C A P A C I T Y A F T E R F U S I O N OF NEMT R E A T E D BRAIN M E M B R A N E S W I T H Fc CELLS L A C K I N G DA1 R E C E P T O R S Effects of 50 ~M DA, 12 m M NaF, 10 #M G p p N H p and 30/zM PGEI on Friend erythroleukemia cells fused with themselves (Fcx Fc), Fc cells fused with NEM-treated brain m e m b r a n e s (Fc x Bm NEM), Bm N E M or Bm only. The effects are expressed as production o f c A M P in pmol per min, and values are m e a n s ± S.E.M. o f duplicates from 3 independent experiments. Treatment
Bm (n = 6)
Bm N E M (n = 3)
F¢ x Fc (n = 6)
Fc × Bm N E M (n -= 6)
DA Spiroperidol NaF GppNHp PGE1
1.46 ± 0.34 1.03 + 0.27 6.35 ± 1.25 6.09 ± 0.56 1.44+0.35
0.08 +_0.01 0.04 ± 0.02 0.13 ± 0.05 0.05 + 0.05 0.02±0.02
0.33 + 0.03 0.28 + 0.02 14.4 ± 0.76 1.75 ± 0.08 1.66+0.17
0.92 + 0.05 0.45 _+0.01 9.67 _+0.28 4.70 ± 0.22 0.94±0.11
212
PGE1 receptors, the PGEI stimulation, which was of the same magnitude as that induced by G p p N H p , indicated the functional capacity of the F~ ceils. The N E M treatment inactivated the brain m e m b r a n e AC system, as indicated by incapability of DA, N a F and G p p N H p to produce c A M P formation in these membranes (Table I). When the two components, the Fc cells with a functional AC system and the NEM-treated brain membranes with the receptors, but no functional AC, were fused together and subjected to DA stimulation, a two-fold increase in c A M P production could be observed (Table I). This can be compared with a corresponding increase of 1.4 obtained f r o m the stimulation of untreated brain membranes without fusion. The time course of the reaction was studied over the time range 2.5 to 22.5 min. Within this time interval linearity was observed (Fig. 1). Because a 10-min incubation time was found to be convenient and in the linear range, it was chosen. The ratio between DA and spiroperidol treatment was found to be stable in at least 10 independent assays (2.04_+ 0.11, mean + S.E.M.). Butyrophenones, like spiroperidol, are known to antagonize DA effects. Spiroperidol has micromolar affinity to DA1 sites and exhibits nanomolar affinity only to DAz sites. To indicate that the fusion transferred receptor is of the DA~ type, we studied the effects of two different concentrations of spiroperidol against stimulation obtained with 50/~M D A in the Fc ×Bm N E M system (Bm = brain membrane). Spiroperidol at 1 #M gave 45.2 + 2.1070 inhibition (mean + S.E.M.; n = 7, P < 0 . 0 0 1 ) whereas at 1 nM there was no effect 1.0+7.1°70 ( n = 7 , P > 0 . 0 5 ) . A
DA (Bm)
40 o O
S P I R e (Bm)
E ¢z V
a.
:E <
DA ( F c x ~ M )
°~
o >,. o
10 .0
0
&
5
10
S P I R e (F c x ~ ~ )
15
20
25
Time (rain) Fig. 1. Time course of dopamine-stimulated adenylate cyclase activity as determined by linear regression analysis. The activity is shown in brain membranes (Bm) stimulated directly (o) and in NEM-treated brain membranes fused with Friend leukemia cells (Fc ×Bm NEM) (A). Membrane protein concentration in the assay was 145 t~g per ml. Values are means o f duplicate determinations.
213 specific DA1 antagonist, SCH 23390 (Schering, N J, U.S.A.) [3] at 0.1 #M, gave 63.0___7.6°-/o ( n = 5, P < 0 . 0 1 ) inhibition of 50 #M D A stimulation. The agonists a p o m o r p h i n e and N P A (N-propylnorapomorphine; Research Biochemicals Inc., MD, U.S.A.), at concentrations o f 10 /~M gave 6 6 . 7 + 5 . 6 % ( n = 5 , P < 0 . 0 1 ) increase, and 67.4 + 5.2% (n = 5, P < 0.001) respectively, whereas 50 #M D A stimulation caused 108 + 5.6% (n = 8, P < 0 . 0 0 1 ) i n c r e a s e in AC activity. Pilot experiments have also been performed with human nucleus caudatus tissue, prepared according to the same procedure. Here the increase with 50 ttM dopamine was 52% for the fused material and 22% for the untreated m e m b r a n e s (mean values o f 3 experiments). P o s t m o r t e m studies have shown that the functional capacity of an AC-coupled receptor declines more rapidly with postmortem interval [12] than receptor binding capacity, the variable c o m m o n l y studied by most investigators [5]. The fusion method m a y allow estimations not only of binding capacity o f receptors but also o f their biological functionality after longer postmortem periods. This may be of interest in clinical studies. It is notable that by fusing together membranes from different species it is still possible to form a functional unity o f the system, where molecular interaction a m o n g the three separate components is needed. Consequently, this enables each individual c o m p o n e n t of AC-coupled receptors to be studied. The initial phases of this study were carried out at the laboratory of Professor Michael Schramm, Department of Biological Chemistry, Hebrew University, Jerusalem, Israel, We are most grateful to him and to Ms. Mira Korner and Dr. Yoav Citri for patiently guiding us through the secrecies of m e m b r a n e fusion transfer. We thank Professor Kenneth Nilsson and Ms. Helena Jernberg for housing o f the cells. W o r k in Israel was supported by Grant AM-10451 f r o m the National Institute o f Health. L.T. was supported by the Swedish Medical Research Council (Grants 04X-3766 and 04R-6624,6724).
1 Carenzi, A., Gillin, J.C., Guidotti, A., Schwartz, M.A., Trabucchi, M. and Wyatt, R.J., Dopaminesensitive adenylyl cyclase in human caudate nucleus, Arch. Gen. Psychiat., 32 (1975) 1056-1059. 2 Citri, Y. and Schramm, M., Resolution, reconstitution and kinetics of the primary action of a hormone receptor, Nature (Lond.), 287 (1980) 297-300. 3 lorio, L.C., Barnett, A., Leitz, F.H., Houser, V.P. and Korduba, C.A., SCH 23390, a potential benzazepine antipsychotic with unique interactions on dopaminergic systems, J. Pharmacol. Exp. Ther., 226 (1983) 462-468. 4 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 5 Owen, F., Cross, A.J. and Crow, T.J., Ligand binding studies in brains of schizophrenics. In P.G. Strange (Ed.), Cell Surface Receptors, Ellis Horwood, Chichester, U.K. 1983. 6 Palmer, G.C., Effects of psychoactivedrugs on cyclicnucleotides in the central nervous system, Prog. Neurobiol., 21 (1983) 1-133. 7 Rodbell, M., The role of hormone receptors and GTP-regulatory proteins in membrane transduction, Nature (Lond.), 284 (1980) 17-22.
214 8 Salomon, Y., Londos, C. and Rodbell, M., A highly sensitive adenylate cyclase assay, Anal. Biochem., 58 (1974) 541-548. 9 Schramm, M., Transfer of glucagon receptor from liver membranes to a foreign adenylate cyclase by a membrane fusion procedure, Proc. Nat. Acad. Sci. USA, 76 (1979) 1174-1178. 10 Stiles, G.L. and Lefkowitz, R.J., A reconstitution assay for the guanine nucleotide regulatory protein of the adenylate cyclase system using turkey erythrocyte membranes as the acceptor preparation, Arch. Biochem. Biophys., 217 (1982) 368-375. 11 Terenius, L., Stereospecific interaction between narcotic analgesics and a synaptic plasma membrane fraction of rat cerebral cortex, Acta Pharmacol. Toxicol., 32 (1973) 317-319. 12 Witte, P. and Matthaei, H., Post mortem changes in adenylate cyclase activity in rat brain striatum, Experientia, 35 (1979) 1421-1422.