Forskolin-induced up-regulation and functional supersensitivity of dopamine D2long receptors expressed by Ltk− cells

ejp ELSEVIER

molecularpharmacology

European Journal of Pharmacology Molecular Pharmacology Section 269 (1994) 149-155

Forskolin-induced up-regulation and functional supersensitivity of dopamine Dzlong receptors expressed by Ltk- cells Maria H. J o h a n s s o n b A n i t a Westlind-Danielsson

a,,

a Department of Neuropharmacology, CNS Preclinical R&D, Astra Arcus AB, S-151 85 S6dertiilje, Sweden b Department of Neurochemistry and Neurotoxicology, University of Stockholm, S-106 91 Stockholm, Sweden Received 6 April 1994; revised MS received 7 June 1994; accepted 28 June 1994

Abstract

Mouse fibroblast Ltk- cells, stably expressing the human dopamine D21ong receptor, were grown in the presence of forskolin (100/xM) for 4 or 16 h. The 16 h treatment resulted in a significant up-regulation of the dopamine D21ong receptors by 43-96% as measured with [3H]raclopride with no change in the K d value. A significant increase in the maximal inhibition of acute forskolin-stimulated cAMP accumulation by dopamine (0.1 nM-3/~M) was found both at 4 and 16 h. No such Dzlong-receptorcoupled response to dopamine could be detected in wild-type, untransfected, Ltk- cells with or without forskolin treatment. Furthermore, basal cAMP levels as well as the maximal response to acute forskolin stimulation decreased in the D21ong receptor expressing cells with the treatment, by 33% and 23% respectively. The results indicate that persistent maintenance of high cAMP levels in transfected Ltk- cells may lead to adaptive quantitative and functional changes of the dopamine D21ong receptor reminiscent of receptor supersensitivity induced by chronic antagonist treatment in vivo where the receptor targeted is inhibitorily coupled to adenylyl cyclase, as is the D21ong receptor. This may provide a model for studying mechanisms underlying dopamine D 2 receptor up-regulation and receptor supersensitivity, not readily induced in cell lines.

Keywords: Forskolin treatment (chronic); Dopamine D 2 Receptor, human; Dopamine; Up-regulation; Supersensitivity; Adenylyl cyclase

1. Introduction

The antipsychotic effect of neuroleptic drugs is believed to be a result of central dopamine D 2 receptor blockade (Creese et al., 1976; Seeman, 1981). In experimental animals chronic dopamine D 2 receptor blockade is well known to lead to up-regulation of dopamine D z receptors in striatum (Muller and Seeman, 1977). Chronic treatment of schizophrenic patients with neuroleptic drugs can thus be expected to lead to an up-regulation of central dopamine D e receptors as well. This has been confirmed in a n u m b e r of studies measuring dopamine D 2 receptor density in the basal ganglia of p o s t m o r t e m tissue (Owen et al., 1978; MacKay et al., 1982; Seeman et al., 1984) and in vivo

* Corresponding author. Tel.: +46 8 55 32 80 66; Fax: +46 8 55 32 88 90. 0922-4106/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 9 2 2 - 4 1 0 6 ( 9 4 ) 0 0 1 0 7 - A

using positron emission tomography (PET) (Wong et al., 1986). However, several reports, based on studies of drug-naive or drug-free schizophrenics, also point to the possibility that such a change in receptor density might be a function of the sickness per se (Lee et al., 1978; Owen et al., 1978; VCong et al., 1986; Crawley et al., 1986). This latter notion supports the dopamine overactivity hypothesis of schizophrenia as postsynaptic dopamine D 2 receptor supersensitivity at the dopaminoceptive cells. Although the relationship between central dopamine D E receptor supersensitivity and schizophrenia still needs further clarification, it seems clear that a deeper knowledge of this relation can lead to a better understanding of schizophrenia, effects of neuroleptic drug treatment and neuroleptic-induced side-effects. G i - p r o t e i n - c o u p l e d receptors, o t h e r than the dopamine D 2 receptor, are also known to be up-regulated with chronic antagonist treatment. In some cases these effects have been shown to be related to changes

150

M.H. Johansson, A. Westfind-Danielsson / European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 149-155

in second messenger systems to which the targeted receptor is coupled. Muscarinic cholinergic receptor up-regulation in rat submandibular salivary gland after chronic atropine treatment, for example, has been shown to lead to a decrease in cAMP levels and a decline in adenylyl cyclase activity (Westlind-Danielsson et al., 1990). This finding suggests that adenylyl cyclase activity is regulated in the opposite direction to the targeted receptor when an inhibitory input to the enzyme is chronically blocked. If there is a causal relationship between up-regulation of receptors, which are coupled in an inhibitory fashion to adenylyl cyclase, and a decline in adenylyl cyclase activity the question arises whether it is possible to up-regulate receptor numbers by persistent activation of the adenylyl cyclase? The present study shows the result of an attempt to induce up-regulation of the human D21ong (alternative nomenclature D2443, D2A, D2L , D2L-A) receptor cloned into Ltk- cells by elevating the cell cAMP level persistently with forskolin. Consequences of this treatment have been measured at the level of the D21ong receptors and, in addition, at the level of their inhibitory action on acute forskolin-induced cAMP formation. The forskolin treated cells may provide a model for dopamine D 2 receptor supersensitivity otherwise not readily produced in cell lines. This model could be useful for enhancing the understanding of mechanisms underlying dopamine D 2 receptor up-regulation and consequences for its signal transduction coupling.

2.2. Cell culture

Mouse fibroblast (Ltk-) cells transfected with human D21ong receptors (inserted into a pZem3 expression vector) were obtained from Dr. O. Civelli (Vollum Institute, Oregon, USA). Cells expressing the receptor clones were in addition cotransfected with a plasmid containing the neo gene (pRSVNeo) conferring geneticin resistance to these ceils (Bunzow et al., 1988; Grandy et al., 1989). The cells were grown according to Malmberg et al. (1993) in DMEM supplemented with the following (denoted DMEM(+)); 10 mM Hepes (pH 7.4), 10% (v/v) heat-inactivated fetal calf serum, PeSt (14 /zg/ml benzylpenicillin K and 20 /xg/ml streptomycin sulphate) and 0.11 mg/ml sodium pyruvate in 175 cm 2 flasks with ventilated caps (Costar) in 5% (v/v) CO 2 at 37°C. 2.3 Membrane preparation

Cell membranes were prepared according to Maimberg et al. (1993). When the cells had grown to confluence they were detached by treatment with 0.05% (w/v) trypsin and 0.02% (w/v) EDTA in PBS (pH 7.4). Cells were harvested and then centrifuged at 300 x g for 10 min, washed in DMEM buffered with 20 mM Hepes (pH 7.4) two additional times and then homogenized with a Dounce homogenizer (tight fitting) in 10 mM Tris-HCl (pH 7.4) with 5 mM MgSO 4. The homogenate was centrifuged at 43 500 x g for 10 rain, the supernatant discarded and the membrane pellet saved and stored at -70°C.

2. Materials and methods

2.4. Receptor binding assay 2.1. Chemicals

Raclopride was synthesized and tritium labeled at Astra Arcus (46.0 Ci/mmol). [3H]cAMP, [5',83H]adenosine 3',5'-cyclic phosphate ammonium salt, (43.0 Ci/mmol) was purchased from Amersham (Arlington Heights, IL, USA). Fetal calf serum, DMEM (Dulbecco's modified Eagle's medium), PBS (phosphate-buffered saline), penicillin-streptomycin and trypsin-EDTA were purchased from Gibco (Paisley, Scotland, UK). Sodium-pyruvate was from Flow Laboratories (Irvine, Scotland, UK). Geneticin, forskolin, IBMX (3-isobutyl-l-mexanthine), EDTA (N, N'-l,2-ethylenediylbis[N-(carboxymethyl)glycine] and Hepes (4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid) and bovine serum albumin were from Sigma Chemical (St. Louis, MO, USA). (+)-Butaclamol was purchased from Research Biochemical (Natick, MA, USA) and quinpirole (or LY 171555) was from Lilly Laboratories (Indianapolis, Indiana, USA). All other chemicals were of analytical grade.

The receptor binding filtration assay was performed according to Malmberg et al. (1993). Briefly the frozen cell membranes were thawed, sonicated and suspended in binding buffer (50 mM Tris-HC1, pH 7.4, 1 mM EDTA, 5 mM KC1, 1.5 mM CaCI2, 4 mM MgC12, 120 mM NaCI). Twelve different concentrations (0.28-12 nM) of [3H]raclopride were used for the saturation binding curve and nonspecific binding was determined in the presence of 1 /~M (+)-butaclamol. 2.5. Forskolin treatment

Both chronic and acute experiments were carried out on immobilized ceils grown on 6-well cluster plates. The cells were detached from the culture flasks in the same way as described in section 2.3 above. Approximately 0.4 x 105 cells in 5 ml DMEM(+) were seeded into each 9.6 cm 2 well of 6-well cluster plates (Costar). After 5 days, when the ceils had grown to confluence, they received 3 ml of fresh medium per well before initiating 'chronic' forskolin (100 ~M) treatment. The 4

M.H. Johansson, A. Westlind-Danielsson / European Journal of Pharmacology

or 16 h forskolin treatment period was terminated by washing the cells with 8 ml D M E M for 3 × 10 min.

2.6. cAMP accumulation The acute c A M P experiments were carried out in an incubator at 37°C. The cells were preincubated for 20 min in 1.2 ml H e p e s (20 mM, p H 7.4) buffered D M E M supplemented with 1 m M IBMX. The acute experiment was initiated by adding forskolin plus various substances or the vehicle. After a 10-min incubation period the acute experiment was terminated by the addition of 10 /xl of 10 m M E D T A (pH 7.4) and 10 /xl of 12 M HCI to each well after which the plates were immediately placed and subsequently frozen between two blocks of dry-ice. Cells were detached from the plates with a cell scraper. The cell suspensions were sonicated and then centrifuged at 1 500 × g for 10 min at 4°C. The supernatant was removed and stored at - 2 0 ° C until c A M P levels were determined while cell pellets were used for protein determination according to Lowry et al. (1951) using bovine serum albumin as a standard. The c A M P levels were determined according to Brown et al. (1972) as modified by Nordstedt and Fredholm (1990).

2. 7. Statistics Statistical analysis was carried out using A N O V A followed by Dunnett's post hoc comparisons or an F-test according to Motulsky and Ransnas (1987).

-

Molecular Pharmacology Section 269 (1994) 149-155

160,-. 140 o 120 -~ P~ > ~100

~

80

-

60

" •40

"

&

2O

T

I

'

1

-6.0

'

1

-5.5

'

1

'

1

'

-5.0

1

-4.5

-4.0

-3.5

Log [forskolin] (M) Fig. 1. The curve shows the concentration-dependent incr6ase in cAMP formation by forskolin (2-100 ~M) in D21ong-Ltk- cells. The curve is the result of one experiment (mean + S.D.) representative of two with each point carried out in triplicate.

(n = 10) respectively. Fig. 2 shows one concentrationresponse curve representative of ten.

3.2. Chronic treatment with forskolin-cAMP studies Initially cells were grown in the presence of 100/xM forskolin for 1, 4 and 16 h. After each treatment period cells were monitored for their dopamine D 2 receptorcoupled cAMP response. The results indicated that the maximal inhibition for dopamine or quinpirole was

3. Results

140

3.1. Acute experiments

120

Forskolin ( 2 - 1 0 0 / x M ) stimulated c A M P production acutely in a concentration-dependent fashion in the D21ong-Ltk- cells. The highest concentration of forskolin used here, 100/zM, did not produce a maximal response. Higher concentrations of forskolin were not used due to its limited solubility above 1 0 0 / z M in water solution (cf. Seamon and Daly, 1986). However, a rough estimate of the EC50 value for forskolin can be approximated from the curve in Fig. 1 to a value > 50 /zM. A forskolin concentration of 100/.~M was used in both the acute and the chronic experiments. Acutely, 100 ~ M forskolin causes a 9-fold increase in intracellular c A M P in the presence of IBMX. The IC50 and maximal inhibition value for dopamine were estimated from concentration-response curves generated by using 1 n M - 1 0 p~M dopamine and were found to be 5.54_+ 2.6 nM (n = 10) and 73.5_+ 11%

151

-

. • %

"~ ~ 1 0 0

~ &

60

T 0

~

[

0

I

-9

'

I

-8

'

I

-7

'

I

'

-6

I

-5

'

I

-4

Log [dopamine] (M) Fig. 2. Concentration-response curve for dopamine-mediated inhibition (1 nM-10 p.M) of forskolin-stimulated (100 /zM) formation of cAMP in D21ong-Ltk- cells. The curve is from one experiment (mean _+S.D.) representative of ten with each point carried out in triplicate.

M.H. Johansson, A. Westlind-Danielsson / European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 149-155

152

Table 1 Basal and forskolin-stimulated (100 ~M) cAMP levels and maximum DA-mediated inhibition of forskolin-stimulated cAMP formation of control and 4 h forskolin-treated (100/zM) D21ong-Ltk- cells from four acute cAMP experiments (means ± S.E.M.) Experi-

D21ong-

Basal cAMP levels

ment no.

Ltk cells

pmol cAMP/mg protein

% of control

pmol cAMP/mg protein

% of control

inhibition (% of forskolin simulation)

l

Control Treated Control Treated Control Treated Control Treated

18.9 _+ 3.1 13.5 _+ 0.7 27.8_+ 3.5 20.8 _+ 1.1 15.1 ± 2.9 ~ 8.4 ± 0.7 a 28.8 ± 0.64 19.3 _+ 0.64

100 71 100 75 100 56 100 67

142 b 85.3 + 1.3 153 _+ 27 128 _+ 1.4 141 b 114 + 11 159 ± 1.4 131 ± 7.1

100 60 100 84 100 81 100 82

68 84 65 87 67 79 68 -

Control

25.2 ± 3.2 (n = 3) 17.9 ± 2.3

100

149 ± 4.4 (n = 4) 115 ± 1 1 "

100

67 + 0.7 (n = 4) 83_+2.3**

(n = 3)

(n = 4)

(n = 4)

(n = 4)

2 3 4

Means ± S.E.M.

Treated

Forskolin stimulation

67 ± 4.1

Maximal

77+5.5

(n = 3)

Asterisk indicates significant difference from control. *P < 0.05 and **P < 0.01. Statistical analysis was carried out using A N O V A followed by Dunnett's post hoc comparisons or an F-test according to Motulsky et al. (1987). a Not included in the mean value. b Estimated from the concentration-response curve.

augmented after either 1, 4 and 16 h. However, the inhibition was more robust at the longer time periods why these were chosen for further experiments. Results of the 4 h forskolin treatment period is depicted in Fig. 3. The maximal inhibition for dopamine was larger for the treated cells by about 24%. No significant shift in the IC50 value for dopamine could be detected. Basal c A M P levels as well as the maximal response to acute 100 /xM forskolin stimulation decreased significantly with the treatment, by 33% and 23% respectively (Table 1). 120 0 •

~" 100 o

m 0~

E

Control Treated

The L t k - ceils have initially been chosen as dopamine D 2 receptor transfection targets due to the fact that they do not express endogenous dopamine D2-1ike (D2, D 3 or D 4) receptors. Expression of endogenous dopamine D 2 receptors by the cells could confuse the interpretation of the results in this study. Wild-type L t k - cells reportedly do not express endogenous dopamine D 2 receptors (Bunzow et al. 1988; Neve et al. 1989). In order to confirm these results in our hands and to further establish whether forskolin treatment can induce expression of dopamine D 2 like receptors, acute cAMP experiments were carried out on both forskolin treated and untreated wild-type L t k cells. Non-transfected wild-type Ltk cells were grown with or without forskolin for 4 h. After treatment the dopamine mediated inhibition of forskolin-stimulated cAMP formation was monitored. No dopamine medi-

80

-~

.~

Table 2 Measurement of binding parameters with [3H]raclopride to membranes from D21ong-Ltk- cells of control and forskolin treated cells (100/xM for 16 h)

60 -

4o

"

"d

g

2o 0

i

i

,

i

-10 -9

,

i

-8

,

i

-7

,

i

-6

,

I

-5

,

i

-4

,

I

Experiment no.

Binding parameters

1

Kd

2

-3 3

Log [dopamine] (M) Fig. 3. Concentration-response curves for dopamine-mediated inhibition of forskolin-stimulated (100 IzM) cAMP formation in D21ongL t k - cells, controls and cells treated with 100 txM forskolin for 4 h. The curves are the mean-t- S.E.M. of three separate experiments.

Bmax Kd Bmax Kd Bmax

Control cells 1.6

2000 0.66 605 0.77 475

Forskolin treated cells 1.8

2980 0.75 865 0.82 929

% Increase in Bmax 49 43 96

Binding parameters, K d (nM) and Bmax ( f m o l / m g protein), were obtained from Scatchard analysis of saturation binding experiments performed as described in Materials and methods. The data are from three separate experiments.

M.H. Johansson, A. Westlind-Danielsson /European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 149-155

0,07 ]

0

Control

0,06

reated

0,05 0,04 0,03 0,02 0,01 0,00 0

200

400

600

800

1000

Bound (fmol/mg) Fig. 4. Scatchard plots showing the concentration dependent binding of [3H]raclopride (0.3-12 nM) to membranes from control or forskolin-treated(100/zM for 16 h) D21ong-Ltk- cells. The plots are the result of one experiment representative of three. Bmax and Kd values are listed in Table 2.

ated inhibition could be detected in either control or treated cells (data not shown). 3.3. Chronic treatment with forskolin-receptor binding studies

Table 2 shows the binding parameters obtained from Scatchard analysis of saturation binding experiments with [3H]raclopride + ( + ) - b u t a c l a m o l in membranes from transfected cells that have been grown in the absence or presence of forskolin. The Bmax value is consistently higher in the forskolin treated cells ranging from a 43% to a 96% increase. The K j values do not, however, differ significantly. Fig. 4 gives an example of a Scatchard plot obtained from data from the 3rd experiment (see Table 2). The data were also analyzed with non-linear regression analysis using the L I G A N D program (Munson and Rodbard, 1980). These values were in good agreement with those obtained from the Scatchard analysis (data not shown). Binding studies were also carried out on non-transfected wild-type Ltk cells that had been treated with forskolin for 16 h. No specific [3H]raclopride binding sites could be detected in these cells as those in the controls (data not shown).

4. Discussion

The strategy of using persistent forskolin treatment in attempting to induce receptor up-regulation was

153

based on the following findings. Treatment of cultured cells with agonists of G~ protein coupled receptors has been shown to lead to an increase in basal adenylyl cyclase activity (Nathanson et al., 1978; WestlindDanielsson et al., 1987; Thomas and Hoffman, 1987). In contrast long-term treatment with a G i protein-coupled receptor antagonist in vivo has been shown to lead to a change in basal adenylyl cyclase activity in the opposite direction paralleled by up-regulation of the R i targeted by the antagonist administered chronically to the animals (Westlind-Danielsson et al., 1990). Blocking a major inhibitory pathway to a cell can be thought to lead to an enhancement of incoming stimulatory signals (the 'break' has been removed), which could lead to a rise in the cAMP levels in the cell. Many studies have confirmed that the components which are known to comprise the adenylyl cyclase complex, R S and Gs (stimulatory receptor and G protein respectively), R i and Gi (inhibitory receptor and G protein respectively) and the enzyme itself seem to function interdependently (Hadcock et al., 1990, 1991; Thomas and Hoffman, 1987). This suggests that induced alteration of one of the components may lead to adaptive changes of the others presumably so that the cell can maintain a balance of incoming stimulatory and inhibitory signal input. Such cross-regulation of the inhibitory and stimulatory adenylyl cyclase pathways has been reported in $49 mouse lymphoma cells as an increase and decrease, respectively, in Gia and Gs~ proteins as well as m R N A levels with persistent forskolin stimulation (Hadcock et al., 1990). In addition, stimulation of an inhibitory pathway in hamster smooth muscle DDT~ MF-2 cells through the adenosine A1 receptor has been reported to increase /32adrenoceptor protein and m R N A levels (Hadcock et al., 1991) and enhance /32-adrenoceptor coupled response to stimulation of adenylyl cyclase activity with isoproterenol (Hadcock et al., 1991; Port et al., 1992). Interestingly and in contrast to these changes m R N A levels for G~ subunits do not seem to change with persistent adenylyl cyclase activation (Hadcock et al., 1989). Although the Ltk--D21ong cell line is genetically engineered, it is apparent that regulation of receptor and receptor-coupled effects is possible and, moreover, comparable to results of studies in non-transfected cell lines and tissue. The results of a decreased basal and forskolin activated cAMP production in the L t k - - D 2 long cells chronically treated with forskolin are suggestive of a forskolin-induced desensitization of adenylyl cyclase. (Had there been forskolin left in the cells after the chronic forskolin treatment, despite the rigorous washing protocol, the levels would be expected to be misleadingly high.) These results parallel our earlier findings of decreased basal tissue cAMP levels and adenylyl cyclase activity in submandibular salivary

154

M.H. Johansson, A. Westlind-Danielsson / European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 149-155

glands from rats which had been chronically treated with a muscarinic cholinergic receptor antagonist, thus blocking an important inhibitory adenylyl cyclase pathway in the salivary gland (Westlind-Danielsson et al., 1990). Also, Dix et al. (1984) found a similar desensitization of cAMP production stimulated with 100 /zM forskolin after Leydig tumour cells had been treated with 1 0 0 / z M forskolin for 4 h. The opposite effect on adenylyl cyclase, sensitization, has been documented in a number of tissues and cell lines after treatment with compounds which inhibit adenylyl cyclase activity (Sharma et al., 1975; Traber and Hampbrecht, 1975; Nathanson et al., 1978; Sabol and Nirenberg, 1979; Thomas and Hoffman, 1987). We have shown that forskolin treatment per se does not induce expression of dopamine D 2 receptors in non-transfected wild-type L t k - cells on the basis of both binding and functional studies. It is therefore likely that the D21ong receptor protein in the transfected cells, which is up-regulated with forskolin treatment, emanates from the incorporated cDNA. From the binding studies the additional receptors also appear identical to the original receptors. However, if, for any reason dopamine D 3 receptor expression is induced by the forskolin-treatment in the transfected ceils, we would not be able to detect these receptors using the [3H]raclopride binding assay, since the affinities of D21ong and dopamine D 3 are so similar for this ligand (Malmberg et al., 1993). We can therefore not rule out this possibility. The question of whether it is the high and persistently maintained cAMP levels that cause the D21ong receptor up-regulation remains to be determined. Treatment studies using non-hydrolyzable cAMP analogues and 1, 9-dideoxyforskolin, a forskolin analogue that does not activate adenylyl cyclase, could shed light on this issue. Such studies are now in progress. If we suppose that it is the high cAMP levels which cause the receptor up-regulation, how can this be thought to come about? cAMP is known to initiate a cascade of biochemical reactions which couples the ligandactivated plasma membrane R s signal to changes in the pattern of cellular gene regulation. Protein kinase A, once activated by cAMP, initiates the cAMP dependent process by catalyzing the phosphorylation of transcription factors such as the cAMP responsive element binding proteins or cAMP responsive element binding protein-like proteins (Montminy et al., 1990). cAMP responsive element binding proteins are then able in turn to bind to specific control elements on the DNA, so called cAMP responsive elements (cf. Meyer and Habener, 1993), which may thus enhance transcription. Since it has been discovered that the 87 bases which differentiate the D21ong receptor c D N A from the shorter D2short receptor cDNA, have a high intrinsic cAMP responsive element binding protein binding

property (Civelli et al., 1993 and personal communication), it is possible that cAMP may enhance the transcription of Dzlong through a cAMP responsive element like region inherent to its own DNA. Moreover, such up-regulation of the Dzshort receptor cDNA cloned into the same cell line would not be expected to occur since it is devoid of the 87 base sequence, if there are no other cAMP responsive elements in the plasmids with which the cells were transfected. No cAMP responsive element sequences are known to exist in the mouse metallothionein promotor coupled to the Dzlong-cDNA with which the L t k - cells were transfected (Bunzow et al., 1988) or in other regions of the pZem3-D21ong or pRSVNeo plasmids. Experiments are in progress in order to determine whether forskolin treatment causes up-regulation of the D2short receptor cloned into Ltk cells. Whether the type of regulation of the D21ong receptor discovered in this work has a physiological parallel remains to be determined. It would be interesting if regulation of the two receptor isomers is somehow critically different due to their differential regulation by intracellular cAMP levels at, for example, the 87 base sequence. Chronic treatment with antipsychotics is thought to lead to up-regulation of dopamine D 2 receptors in human brain (Seeman and Niznik, 1990). This treatment effect has been thought by many to be the underlying cause for the development of tardive dyskinesia, the major side-effect of long-term neuroleptic drug treatment (cf. Seeman, 1992). Others have presented data that could support that receptor up-regulation is related to schizophrenia itself (Wong et al., 1986). There is still much to be done when it comes to increasing the understanding of compensatory changes in the density of dopamine D 2 receptors and the additional components of the adenylyl cyclase complex with which dopamine D 2 receptors interact. With the use of cell lines transfected with the various dopamine receptor subtypes which have recently become available these studies will be significantly facilitated. The present studies have confirmed that it is possible to increase D21ong receptor levels in a unique fashion by manipulating a step in a signal transduction pathway indirectly activated by the dopamine D2-receptoragonist complex. This work introduces a means of manipulating a dopamine D 2 receptor expressing cell line which may be of use as a model for studying various aspects of dopamine D 2 receptor up-regulation and supersensitivity.

Acknowledgements We would like to thank Dr. Tamas Barffai and Dr. Olivier Civelli for valuable comments.

M.H. Johansson, A. Westlind-Danielsson / European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 149-155

References Brown, B.L., R.P. Ekins and J.D.M. Albano, 1972, Saturation assay for cyclic AMP using endogenous binding protein, Adv. Cyclic Nucleotide Res. 2, 25. Bunzow, J.R., H.H.M. Van Tol, D.K. Grandy, P. Albert, J. Salon, M. Christie, C.A. Machida, K.A. Neve and O. Civelli, 1988, Cloning and expression of a rat D 2 dopamine receptor cDNA, Nature 336, 783. Civelli, O., J.R. Bunzow and D.K. Grandy, 1993, Molecular diversity of dopamine receptors, Annu. Rev. Pharmacol. Toxicol. 32, 281. Crawley, J.C.W., T.J. Crow, E.C. Johnstone, S.R.D. Oldland, F. Owen, D.G.C. Owens, M. Poulter, T. Smith, N. Veall and G.D. Zanelli, 1986, Dopamine D 2 receptors in schizophrenia studied in vivo, Lancet 2, 224. Creese, I., D.R. Burt and S.H. Snyder, 1976, Dopamine receptor binding predicts clinical and pharmacological potencies of antischizophrenic drugs, Science 192, 481. Dix, C.J., A.D. Habberfield and B.A. Cooke, 1984, Characterization of the homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase, Biochem. J. 220, 803. Grandy, D.K., M.A. Marchionni, H. Makam, R.E. Stofko, M. AIfano, L. Frothingham, J.B. Fischer, K.J. Burke-Howie, J.R. Bunzow, A.C. Server and O. Civelli, 1989, Cloning of the cDNA and gene for a human D 2 dopamine receptor, Proc. Natl. Acad. Sci. USA. 86, 9762. Hadcock, J.R., J.D. Port and C.C. Malbon, 1991, Cross-regulation between G-protein-mediated pathways: Activation of the inhibitory pathway of adenylyl cyclase increases the expression of /32-adrenergic receptors, J. Biol. Chem. 266, 11915. Hadcock, J.R., M. Ros and C.C. Malbon, 1989, Agonist regulation of /3-adrenergic receptor mRNA: analysis in $49 mouse lymphoma mutants, J. Biol. Chem. 264, 13956. Hadcock, J.R., M. Ros, D.C. Watkins and C.C. Malbon, 1990, Cross-regulation between G-protein-mediated pathways: stimulation of adenylyl cyclase increases expression of the inhibitory G-protein, Gia2, J. Biol. Chem. 265, 14784. Lee, T., P. Seeman, W.W. Tourtellotte, I.J. Farley and O. Hornykeiwicz, 1978, Binding of 3 H-neuroleptlcs and 3H-apomorphine in schizophrenic brains, Nature 274, 897. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193, 265. MacKay, A.V.P., L.L. Iversen, M. Rossor, E. Spokes, E. Bird, A. Arreggui, I. Creese and S.H. Snyder, 1982, Increased brain dopamine and dopamine receptors in schizophrenia, Arch. Gen. Psychiatr. 39, 991. Malmberg, ,~., D.M. Jackson, A.M. Eriksson and N. Mohell, 1993, Unique binding characteristics of antipsycotic agents interacting with human dopamine DZA, D2B and D 3 receptors, Mol. Pharm. 43, 749. Meyer, T.E. and J.F. Habener, 1993, Cyclic adenosine 3',5'-monophosphate response element binding protein (CREB) and related transcription-activation deoxyribonucleic acid binding proteins, Endocrine Rev. 14, 269. Montminy, M.R., G.A. Gonzalez and K.K. Yamamoto, 1990, Regulation of cAMP-inducible genes by CREB, Trends Neurosci. 13, 184. Motulsky, H.J. and L.A. Ransnas, 1987, Fitting curves to data using nonlinear regression: a practical and nonmathematical review, FASEB J. 1,365. Muller, P. and P. Seeman, 1977, Brain neurotransmitter receptors

155

after long-term haloperidol: dopamine, acetylcholine, serotonin, alpha-noradrenergic and naloxone receptors, Life Sci. 21, 1751. Munson, P.J. and D. Rodbard, 1980, LIGAND: a versatile computerized approach for characterization of ligand-binding systems, Anal. Biochem. 107, 220. Nathanson, N.M., W.L. Klein and M. Nirenberg, 1978, Regulation of adenylate cyclase activity mediated by muscarinic acetylcholine, Proc. Natl. Acad. Sci. USA 75, 1788. Neve, K.A., R.A. Henningsen, J.R. Bunzow and O. Civelli, 1989, Functional characterization of a rat dopamine D-2 receptor cDNA expressed in a mammalian cell line, Mol. Pharm. 36, 446. Nordstedt, C. and B.B. Fredholm, 1990, A modification of a protein binding method for rapid quantification of cAMP in cell-culture supernatants and body fluid, Anal. Biochem. 89, 231. Owen, F., T.J. Crow and M. Poulter, 1978, Increased dopamine receptor sensitivity in schizophrenia, Lancet 2, 223. Port, J.D., J.R. Hadcock and C.C. Malbon, 1992, Cross-regulation between G-protein-mediated pathways: acute activation of the inhibitory pathway of adenylyl cyclase reduces /32-adrenergic receptor phosphorylation and increases /3-adrenergic responsiveness, J. Biol. Chem. 267, 8468. Sabol, S.L. and M. Nirenberg, 1979, Regulation of adenylate cyclase of neuroblastoma×glioma hybrid cells by alpha-adrenergic receptors. 2. Long lived increase of adenylate cyclase activity mediated by alpha receptors, J. Biol. Chem. 254, 1921. Seamon, K.B. and J.W. Daly, 1986, Forskolin: Its biological and chemical properties, Adv. Cyclic Nucleotide Res. 20, 1. Seeman, P., 1981, Brain dopamine receptors, Pharmacological Rev. 32, 229. Seeman, P., 1992, Dopamine receptor sequences: therapeutic levels of neuroeleptics occupy D 2 receptors, clozapine occupies D4, Neuropsychopharmacology 7, 261. Seeman, P., C. Ulpian, C. Bergeron, P. Riederer, K. Jellinger, E. Gabriel, G.P. Reynolds and W.W. Tourtellotte, 1984, Bimodal distribution of dopamine receptor densities in brains of schizophrenics, Science 225, 728. Seeman, P. and H.B. Niznik, 1990, Dopamine receptors and transporters in parkinson's disease and schizophrenia, FASEB J. 4, 2737. Sharma, S.K., W.A. Klee and M. Nirenberg, 1975, Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance, Proc. Natl. Acad. Sci. USA 72, 3092. Thomas, J.M. and B.B. Hoffman, 1987, Adenylate cyclase supersensivivity: a general means of cellular adaption to inhibitory agonists?, Trends Pharmacol. Sci. 8, 308. Traber, J., R. Gullis and B. Hamprecht, 1975, Influence of opiates on the levels of adenosine 3:5-cyclic monophosphate in neuroblastoma × glioma hybrid cells, Life Sci. 16, 1863. Westlind-Danielsson, A., P. Gillenius, P. Askel6f and T. Bartfai, 1987, Chronic exposure to pertussis toxin alters muscarinic receptor-mediated regulation of cAMP metabolism in neuroblastoma x glioma NGI08-15 hybrid cells, J. Neurochem. 51, 38. Westlind-Danielsson, A., R.M. Muller and T. Bartfai, 1990, Atropine treatment induced cholinergic supersensitivity at receptor and second messenger levels in the rat salivary gland, Acta Physiol. Scand. 138, 431. Wong, D.F., H.N. Wagner, Jr., L.E. Tune, R.F. Dannals, G.D. Pearlson, J.M. Links, C.A. Tamminga, E.P. Broussolle, H.T. Ravert, A.A. Wilson, J.K.T. Toung, J. Malat, J.A. Williams, L.A. O'Tuama, S.H. Snyder, M.J. Kuhar and A. Gjedde, 1986, Positron emission tomography reveals elevated D 2 dopamine receptors in drug-naive schizophrenics, Science 234, 1558.