He Y et al / Acta Pharmacol Sin 2003 Mar; 24 (3): 225-229

Expression of dopamine D1 receptor in Sf9 insect cells and agonism of l-12-chloroscoulerine on recombinant D1 receptor1

HE You, JIN Wen-Qiao2, SHEN Qing-Xiang3, CHEN Xin-Jian, JIN Guo-Zhang

Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031; 3Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China

1 Project supported by the National Natural Science Foundation of China (No 30070866) and Grant G (1998) 051126 from the Ministry of Science and Technology of China.

2 Correspondence to Prof JIN Wen-Qiao. Phn 86-21-6431-1833, ext 510. Fax 86-21-6437-0269. E-mail wqjin@mail.shcnc.ac.cn

Received 2002-07-12 Accepted 2002-12-12

KEY WORDS recombinant proteins; dopamine D1 receptors; insects; baculoviridae; l-12-chloroscoulerine; cyclic AMP

ABSTRACT

AIM: To express dopamine D1 receptor in baculovirus-Sf9 cell system, and to investigate the effects of l-12-chloroscoulerine (l-CSL) on the recombinant D1 receptor (D1R). METHODS: The recombinant baculovirus, Autographa californica nuclear polyhedrosis virus bearing D1R (AcNPV- D1R) was generated, and then was used to produce recombinant D1R in Sf9 insect cells. Expression of D1R in Sf9 cells was monitored by [3H]SCH23390 binding assay. The effects of l-CSL on recombinant D1R were investigated by [3H]SCH23390 binding assay and cAMP assay. RESULTS: The recombinant baculovirus AcNPV bearing D1R cDNA was generated, and was successfully expressed in Sf9 insect cells. The expression level of (Bmax) was (0.94±0.06) nmol/g protein. The Kd value of [3H]SCH23390 was (1.9±0.3) nmol/L, which was consistent with the previous results from calf striutam tissues. l-CSL had a high affinity to recombinant D1R with Ki value of (6.3±1.4) nmol/L, and increased the intracellular cAMP level in a concentration-dependent manner with EC50 value of 0.72 mol/L and 95 % confidence limit was 0.67-0.77 mol/L. Thus l-CSL has the D1 receptor agonism. CONCLUSION: An efficient baculovirus-Sf9 insect cell system for dopamine D1 receptor was constructed and l-CSL presented the D1 receptor agonism on cellular-molecular level directly.

INTRODUCTION

Dopamine (DA) receptor, with seven transmembrane regions, is one of the members in G protein-coupled receptor family. Five DA receptor subtypes have been identified by molecular cloning, which are nominated D1, D2, D3, D4, and D5 . The D1 and D5 receptors are grouped into the pharmacological D1-like receptor subtype, which activate the second message cAMP level via Gs protein in intracellular signal transduction. While D2, D3, and D4 receptors belong to the pharmacological D2-like receptor subtype, which decrease the second message cAMP level via Gi/o protein.

12-Chloroscoulerine (CSL) is a synthetic compound of tetrahydroprotoberberines. Due to presence of one asymmetric carbon atom, the effects of CSL enantiomers were studied. The results showed that l-12-chloroscoulerine (l-CSL) is really more potent one in the CSL enantiomers[1-4]. In biochemical determination, l-CSL has high affinities for both D1 and D2 receptors[4]. In the electrophysiological recording, l-CSL reversed the apomorphine (APO)-induced inhibition of firing activity on nigral DA neurons[2]. In the behavioral assay, l-CSL antagonized the APO-induced stereotype in rats and induced the catalepsy[4]. All these results indicate that l-CSL is a D2 antagonist.

Chemical structure of 12-chloroscoulerine

In the 6-OHDA unilateral lesioned rats, however, l-CSL induced contralateral rotation[4], which was similar to the D1-selective agonist SKF38393. It has reported that the D1 receptors were supersensitive in the 6-OHDA unilateral lesioned rats[5-7]. The results suggested that l-CSL showed the D1 agonism in the lesioned rats.

To clarify the D1 agonism of l-CSL, the recombinant D1 receptor was expressed in Sf9 insect cells to overcome the interference between DA receptor subtypes. In this study, the effects of l-CSL on D1 receptor were evaluated at the molecular and cellular level directly. For comparison, the D1-selective agonist SKF38393 was included in the experiments.

MATERIALS AND METHODS

Drugs and reagents [3H]SCH23390 (3.1 GBq/mol) and [3H]spiperone (2.8 GBq/mol) were purchased from Amersham Pharmacia Co (USA). D1 agonist SKF38393, non-selective antagonist (+)-butaclamol and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma (USA). l-12-Chloroscoulerine (l-CSL) was synthesized in Shanghai Institute of Materia Medica (China). cAMP radioimmunoassay kit was purchased from Shanghai Second Medical University (China).

Generation of recombinant baculovirus and expression of D1 receptor The plasmid containing D1R cDNA was a generous gift from Prof Kim NEVE(Oregon Health Sciences University, USA). The cDNA was digested from the plasmid with EcoR I and Not I, and then subcloned into the comparable sites of the transfer vector pVL1393. The recombinant baculovirus was generated by co-infection of Sf9 insect cells with pVL1393-D1R and BaculoGoldTM linearized baculovirus genomic DNA (Pharmingen, USA) and plaque purification. Sf9 insect cells were routinely cultured in monolayer at 27 ºC in TNM-FH medium (Gibco BRL, USA) supplemented with 10 % heat-inactived fetal bovine serum (Gibco BRL, USA).

Receptor binding assay In saturation binding experiments, cells containing 20 g protein were incubated with increasing concentration of [3H]SCH23390 (0.1-9.0 nmol/L) in a final volume 200 L binding buffer containing (in mmol/L) Tris-HCl 50, NaCl 120, KCl 5, MgCl2 5, CaCl2 1.5, edetic acid 5, pH7.4, at 30 ºC for 40 min. For the competition binding experiments, the cells were incubated with [3H]SCH23390 0.8 nmol/L in the presence of increasing amount of competing drugs. The nonspecific binding was determined by the addition of (+)-butaclamol 6 mol/L.

CAMP assay Cells expressing D1R were challenged with indicated agonists in the presence of 3-isobutyl-1-methylxanthine (IBMX) 500 mol/L at 27 ºC for 15 min. The reactions were terminated with perchloric acid 1 mol/L and neutralized with KOH 1 mol/L. The cAMP level of each sample was determined with radioimmunoassay kit as the protocol.

Data analysis Data were analyzed using Origin and Prism programs, and expressed as mean±SD obtained from 3 independent experiments.

RESULTS

Expression of D1 receptor in the Sf9 insect cells After recombinant baculovirus AcNPV-D1R infection, the morphologic change of Sf9 insect cells that cell body became bulgy, was observed as an index for expression of recombinant receptor (Fig 1). Expression of D1 receptor was further monitored using the radioactive antagonist [3H]SCH23390. Receptor expression approximately peaked at 72 h (Fig 2). [3H]SCH23390 binding to D1 receptor expressed in Sf9 cells was saturable and of high affinity, with Scatchard analysis indicating the presence of a homogenous binding site. The maximum binding amount (Bmax) was (0.94±0.06) nmol/g protein. The equilibrium dissociation constant (Kd) of [3H]SCH23390 was (1.9±0.3) nmol/L (Fig 3). The Sf9 cells expressing the D1 receptor did not show the specific [3H]spiperone binding to D2-like receptors.

Fig 1. Morphologic change of Sf9 cells. ×400. A) normal cells; B) infected cells.

Fig 2. Time course of expression of D1R in Sf9 cells. n=3. Mean±SD.

Fig 3. Saturation curve for the specific binding of [3H]SCH23390 to Sf9 cells expressing D1R. Inset, Scatchard analysis.

Affinity of l-CSL to recombinant D1 receptor In receptor binding assay, l-CSL inhibited the [3H]SCH23390 binding competitively to recombinant D1 receptor with Ki (6.3±1.4) nmol/L, while D1 agonist SKF38393 displaced the [3H]SCH23390 binding with Ki (0.53±0.04) mol/L (Fig 4).

Fig 4. Competitive inhibition of binding of [3H]SCH23390 to D1R expressed in Sf9 cells by indicated drugs. The radioligand was used at a concentration of 0.8 nmol/L and non-specific binding was defined using (+)-butaclamol 6 mol/L.

Effect of l-CSL on cAMP level After a 15-min incubation with Sf9 cells expressing D1 receptor, l-CSL challenged the increase of intracellular cAMP level in a dose-dependent manner with EC50 0.72 mol/L and 95 % confidence limit was 0.67-0.77 mol/L. SKF38893 increased intracellular cAMP level with EC50 0.84 mol/L and 95 % confidence limit was 0.51-1.40 mol/L (Fig 5).

Fig 5. cAMP accumulation induced by l-CSL and SKF38393 in Sf9 cells expressing D1R.

DISCUSSION

l-CSL showed high affinities for both D1 and D2 receptors in the binding assay with the calf striatum, and possessed D1 agonistic-D2 antagonistic dualactions[4]. In order to clarify its D1 agonism, it is necessary to look for some tissue or cell line bearing D1R only. Neither specific [3H]SCH23390 binding to D1-like receptors, nor specific [3H]spiperone binding to D2-like receptors was found in uninfected Sf9 insect cells in our preliminary experimental detection. Moreover, there were no endogenous 5-HT receptor[8], -adrenergic receptor[9], and-adrenergic receptor[10] in Sf9 insect cells. All of these indicated that the heterogenous baculovirus expression system met the need, and it could exclude the potential interference between the dopamine receptor subtypes or from other catecholamine receptors. Therefore, the baculovirus expression system was adopted here, which seemed an ideal model to assess the interaction between drug and dopamine receptor subtypes.

O'Dowd BF and his colleagues have expressed the dopamine D1 receptor in Sf9 cell[11]. In order to detect the effect of l-CSL, we independently expressed the D1R. The results of our recombinant D1R binding assay were consistent with those of membrane preparation from calf striatum[1]. Compared with the results from calf striatum, the affinity of [3H]SCH23390 to the recombinant D1 receptor had no significant changes. The Kd value was (1.9±0.3) nmol/L in Sf9 cells, while the Kd value was 1.65 nmol/L in calf striatum. So, our recombinant D1R maintained a native property, at least in our experiment.

D1 receptor couples with G protein and adenylyl cyclase. D1 receptor activation induces the cAMP level increase. As no other catecholamine receptors were found in Sf9 cells[8-10], the intracellular cAMP increase challenged by l-CSL was just induced by the recombinant D1 receptor expressed in Sf9 cells.

This study showed the agonism of l-CSL on recombinant D1 receptor directly, which was consistent with the previous observation of D1 agonism of l-CSL in the 6-OHDA unilateral lesioned rats[4]. The data, taken with the previous results[1-4], confirmed that l-CSL possessed the D1 agonistic-D2 antagonistic dual actions. Both l-CSL and l-stepholidine (l-SPD) share the com mon active structural elements, dihydroxy groups, in the tetrahydroprotoberberines (so called DH-THPB), which result in the unique D1 agonistic-D2 antagonistic dual actions. The animal studies and clinical observations have indicated that dual effects of l-SPD may represent a novel antipsychotic drug[12]. In our study, l-CSL showed a more preferential affinity to recombinant D1 receptor than l-SPD. The more potent dual actions were found compared to SPD in previous studies[4]. All data implicated that l-CSL would be a potential antipsychotic drug.

ACKNOWLEDGEMENT Authors thank Prof Kim NEVE (Oregon Health Sciences University, USA) for the generous gift of the plasmid containing D1 receptor cDNA.

REFERENCES