左旋千金藤啶碱D₁激动作用
增加6-OHDA损毁大鼠的DARPP-32磷酸化效应^*

刘健　郭翔　王博成　金国章

　　摘要　为了进一步阐明SPD对大鼠纹状体突触后D₁受体的激动作用特性,本文应用反磷酸化在体内测定及放射配体结合方法,分别观察SPD对6-OHDA损毁大鼠纹状体DARPP-32体内磷酸化作用及突触后D₁受体密度的影响。结果表明: 皮下给予SPD(20,40　mg/kg,21 d),损毁侧纹状体DARPP-32体外［³²P］的掺入量较健侧下降50％(P<0.01)。换言之,损毁侧纹状体内DARPP-32的磷酸化程度增加了。然而,SPD使损毁导致D₁受体上调的作用减弱(B_max 从385.0±26.1 fmol/mg 降至319.7±20.1 fmol/mg水平)。因此,SPD激动D₁受体,使6-OHDA损毁大鼠纹状体内DARPP-32磷酸化作用加强,而受体密度减少。这是SPD调节脑内D₁受体信号转导功能的重要机制。
　　关键词:左旋千金藤啶碱；DARPP-32；反磷酸化测定;多巴胺D₁受体；放射配体测定;纹状体；6-OHDA
　　学科分类号:Q507

INCREASED PHOSPHORYLATION OF DARPP-32 BY D₁
AGONISTIC ACTION OF l-STEPHOLIDINE IN
THE 6-OHDA-LESIONED RAT STRIATUM^*

LIU　JIAN^1,2　GUO　XIANG¹,WANG　BO-CHENG²,JIN　GUO-ZHANG^1，3
(¹Shanghai Institute of Materia Medica,Chinese Academy of Sciences,Shanghai 200031;
²State Key Laboratory of Nuclear Medicine,Wuxi 214063)

　　ABSTRACT　In order to explore the characteristics of l-stepholidine (SPD) activating the postsynaptic D₁ receptors,the effects of SPD on DARPP-32 phosphorylation in vivo with back-phosphorylation assay and on the postsynaptic D₁ receptor densities with radioligand assay were observed in the striatum of 6-OHDA-lesioned rat.The results showed that following subcutaneously administration of 20 or 40 mg/kg SPD for 21 d,［³²P］phosphate incorporation into the DARPP-32 protein in the denervated striatum showed a 50% reduction (P<0.01) vs the intact striatum,indicating an increase of DARPP-32 phosphorylation in vivo in the denervated striatum.However,the D₁ receptor B_max was decreased from 385.0±26.1 to 319.7±20.1 fmol/mg protein.It is suggested that D₁ agonist action of SPD decreases the D₁ receptor density but increases the phosphorylation of DARPP-32 in the striatum of 6-OHDA-lesioned rat,which may be responsible for the regulation of D₁ receptor signal transduction in brain neurons.
　　Key words:stepholidine;DARPP-32;back-phosphorylation;dopamine D₁ receptors;radioligand assay;striatum;6-OHDA

　　Phosphorylation-dephosphorylation is one of the key mechanisms in cellular regulatory function.Dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32,MW=32 kD) has been regarded as a specific marker of D₁ receptors,which is regulated by dopamine (DA) and cAMP^［1］.DARPP-32 is enriched in the neurons with D₁ DA receptors in the striatum^［1,2］.According to the current view of cellular signal transduction,DA activates adenylate cyclase through D₁ receptors,and sequentially increases the level of cAMP.The latter stimulates the activity of cAMP-dependent protein kinase (PKA) which phosphorylates DARPP-32.Phosphorylated DARPP-32 is a potent inhibitor of the protein phosphatase-I^［3］ and thus enhances the physiological responses.Regulation of the postsyanptic D₁ receptors function by DARPP-32 is mediated only by positive feedback mechanism.Neuropharmacological studies proved that the selective D₁ receptor agonists,such as SKF38393,increase phosphorylation of DARPP-32 in vivo.Neither selective D₂ receptor agonist nor D₁/D₂ receptor antagonisthas any effect on the DARPP-32 phosphorylation^［4］.Therefore,DARPP-32 serves as the “third messenger” of specific neurons with D₁ receptors.The phosphorylation state of DARPP-32 is an excellent marker of the activity of D₁ receptors regulated by transduction mechanism.
　　Evidence has proved that l-stepholidine (SPD) behaves as D₁ receptor agonist in 6-OHDA-lesioned rats,such as inducing contralateral turning^［5］,increasing cAMP level in brain^［6］,and enhancing intrinsic activity only to D₁ but not to D₂ receptors.The present work has further studied the agonistic effect of SPD on DARPP-32 phosphorylation and B_max of D₁ receprors in the striata of 6-OHDA-lesioned rats in order to further elucidate the relationship between D₁ receptor supersensitivity and D₁ agonistic action induced by SPD.

　　1　MATERIALS AND METHODS

　　1.1　Reagents　　SPD (mp 161～162℃ ［α］_D-440° in pyridine,Shanghai Institute of Materia Medica) was dissolved in H₂SO₄ 0.1 mol/L,and then neutralized with NaOH 0.1 mol/L to pH 5.Saline control was also adjusted to pH 5.［³H］SCH23390 (2.66 TBq/mmol) was from Amersham.SCH23390 and ketanserine were from RBI.Acrylamide,bis-acrylamide,N,N,N′-,N′-tetramethyl ethylene diamine (TEMED) and ammonium persulfate were from Pharmacia Co.N-2-hydroxyethylpiperazine-N′-2-ethane sulphonic acid (HEPES),phenyl methyl sulphonyl fluoride (PMSF) and Triton X-100 were from MERCK Co.2-Mercapto-ethanol (β-ME) and Coomassie Brilliant blue R250 were from Fluka Co.Sodium dodecyl sulphate (SDS) was from Serva Co.Catalytic subunit of PKA was from Sigma Co.［γ-³²P］ATP was from Beijing Ya-Hui Biomedical Technical Co.Ethylene-glycol-bis-tetra-acetate (EGTA),ethylene diamine tetra-acetate (EDTA) and other reagents were all analytic grade.
　　1.2　Rats　　Sprague-Dawley rats (clean,male,n=70,187±22 g,Shanghai Experimental Animal Center,Chinese Academy of Sciences,China;Certification No 005 conferred by Animal Manage～ment Committee,Chinese Academy of Sciences) were housed in an air-conditioned room and fed with standard rat chow and water ad lib.
　　Rats were anesthetized with choral hydrate (400 mg/kg) and then placed in a stereotaxic frame (David Kopf,Tujunga,CA).6-OHDA (8 μg/4 μl dissolved in 0.9% saline with 0.2 mg/ml ascorbate,66 rats) or vehicle (4 μl,4 rats) was injected into the right substantia nigra par compacta (SNc) at a rate of 1 μl/min,according to the brain atlas of Paxinos and Watson (1982)^［7］ (coordinates: A=3.8,L=2.0,V=7.9 from dura).The needle was left in the place for additional 5 min to minimize the diffusion of toxin along the needle track.Fourteen days after the lesion,the animals were challenged with apomorphine (APO,0.2 mg/kg,ip).
　　1.3　Preparation of the striatal protein extracts　　The phosphorylation of DARPP-32 in the tissue was evaluated by back-phosphorylation of acid-extracted tissues according to Walaas et al^［2］.Briefly,frozen striata were immersed in ice-cold homogenization buffer (1∶20,W/V,containing 10 mmol/L Tris-HCl,pH 7.4,2 mmol/L EDTA and 0.1 mmol/L PMSF).Within 1 min the tissue was homo～genized with 10 strokes in a teflon-glass Potter homo～genizer.Immediately after homo～genization,the proteins were precipitated by adding 5 mmol/L zinc acetate (1∶15,v/v) and pelleted by centrifugation at 2000×g for 15 min.The pellet was resuspended in 10 mmol/L citric acid (pH 2.8) (1∶20,w/v,containing 0.1% Triton X-100) and the suspension was centri～fuged at 27000×g for 15 min.The supernatant was collected,adjusted to pH 6.5 by adding 0.5 mol/L Na₂HPO₄ and left on ice for 10 min.After centri～fugation at 16000×g for 15 min,the final supernatant was collected and kept at －80℃.
　　1.4　Back-phosphorylation assay　　To determine the amount of DARPP-32 (as phosphorylation form) present in the tissue,the back-phosphorylation assay in vitro^［9］ was applied.Briefly,phosphorylation reaction was carried out for 60 min at 30℃ in a final volume of 100 μl containing 50 mmol/L HEPES (pH 7.4),10 mmol/L MgCl₂,1 mmol/L EGTA,1 mmol/L EDTA,10 nmol/L catalytic subunit of protein kinase A,10 mmol/L ［γ-³²P］ATP (0.74 TBq/mmol) and 40 μg of striatal proteins.Phosphory～lation was started by addition of ［γ-³²P］ATP,and stopped by adding 50 μl of stopping solution (0.12 mol/L,pH 6.8,containing 3% SDS,5% β-ME,10% glycerol and 0.002% bromophenol blue).
　　One dimensional SDS-polyacrylamide gel electrophoresis (10% acrylamide,0.3% bis-acrylamide) was carried out according to Laemmli^［9］.
　　1.5　Preparation of synaptosomal membrane from rat striatum　　The procedure was performed according to Hess^［10］.The striatum was homogenized in ice-cold Tris-HCl 50 mmol/L (pH 7.4) (1∶10,w/v).The homogenate was centrifuged at 500×g at 4℃ for 10 min.The supernatant was collected and centrifuged at 20000×g at 4℃ for 20 min.The pellet was rinsed once with Tris-HCl buffer,and suspended in the same amount of D₁ assay buffer ［containing: Tris-HCl 50 mmol/L (pH 7.4),MgSO₄ 5 mmol/L,EDTA 0.5 mmol/L and 0.02% ascorbate］.After centrifugation again at 20000×g at 4℃ for 20 min,the pellet was resuspended in 1 ml D₁ assay buffer.
　　1.6　D₁ receptor binding assay　　Incubation was initiated by adding tissue (0.4 mg protein/tube) to tubes containing labeled ligand and unlabeled competitor (with or without GTP) to yield a 1.0 ml final assay volume.［³H］SCH23390 was used as radioligands to label D₁ receptors.Nonspecific binding was defined by SCH23390 100 nmol/L.After incubation at 37℃ for 30 min,the reaction was stopped by ice-cold Tris-HCl buffer.Rapid filtration was done through Whatman glass fiber filters (GF/C) with reduced pressure.The filters were rinsed with Tris-HCl buffer thrice,dried at 80℃,and counted by LKB scintillation spectroscopy (dmp).
　　Protein of striatal extracts was determined according to the method by Bradford^［11］.Bovine serum albumin was used as a standard.
　　1.7　Data analysis and statistical test　　The data of DARPP-32 phosphorylation (dpm of ［γ-³²P］ incorporated) are expressed as ±s and calculated with two-tailed t test.The data of D₁ binding assay (dmp) are pesented as ±s and analyzed with the GraphPAD InplotVS3 program.

　　2　RESULTS

　　The 6-OHDA-lesioned 48 rats displayed continuous contralateral rotation (>300 circles per h).Thirty-two out of the 48 rats were used for DARPP-32 phosphorylation experiment.The rats were radomly divided into 8 groups (4 rats per group),and injected (sc) with saline (adjusted pH to 5.0) or SPD 2.5,5,10,20,and 40 mg/kg once daily for 21 d,respectively.
　　The remaining 16 rats were used for D₁ binding assay.The animals were radomly divided into 4 groups (4 rats per group).Together with the 4 unlesioned rats,the rats were injected (sc) with saline (pH 5.0) or SPD 10,20,and 40 mg/kg once daily for 21 d,respectively.The rats were decapitated at 24 h after the last injection.
　　2.1　DARPP-32 protein in the rat striatum
　　SDS-PAGE of DARPP-32 extracted from rat striatum is shown in Fig 1.The gel was stained with Coomassie Brilliant Blue R250,destained,and dried.The apparent molecular weight of DARPP-32 was 32 kD.The portion of DARPP-32 was about 5% of the total extracted protein.Furthermore,administration of SPD did not cause significant alteration of the total amount of DARPP-32 protein in the rat denervated striatum,even if 40 mg/kg SPD had been used for 21 d.

Fig.1　SDS-PAGE of DARPP-32 extracted from rat striatum
The apparent molecular weight of DARPP-32 was 32 KD.The portion of DARPP-32 was about 5% of total extracted protein.A.Maker.B.Lesioned+vehicle.C.Lesioned+SPD 40 mg/kg,21 d,(sc).

　　2.2　SPD decreasing DARPP-32 phosphorylation in vitro in striata of 6-OHDA-lesioned rats
　　According to the back-phosphorylation assay technique,the decrease of ［³²P］ phosphate tissue incorporation following in vitro incubation reflects an increase of phosphorylation state of DARPP-32 in vivo.Conversely,the increase of ［³²P］ phosphate incorporation indicates a reduction of DARPP-32 phosphorylation in vivo.SDS-PAGE of DARPP-32 was autoradiographed on Kodak XAR 5 films.The amount of ［³²P］phosphate incorporated in the DARPP-32 band was determined by excision of the band from the gel followed by solubilization and liquid scintillation counting.
　　The results showed that only in the denervated striatum was there a significant reduction (P<0.01) in ［³²P］phosphate incorporation compared with the intact striatum.After chronic injection of SPD at 20 and 40 mg/kg for 21 d,58% and 54% of DARPP-32 phosphorylation were reduced in denervated striata,but the lower doses of 2.5,5,and 10 mg/kg SPD for 21 d could not induce significant reduction in DARPP-32 phosphorylation in denervated stiata (Table 1).Such a reduction reflects the decrease of dephospho-DARPP-32 available in vitro for the back-phosphorylation,and conversely indicates the increase of phospho-DARPP-32 in vivo.However,6-OHDA denervation did not modify DARPP-32 phosphorylation vs the intact striatum in the saline group.

Table1　Incorporation of ［γ-³²P］phosphate in DARPP-32 following administration of either vehicle or SPD in 6-OHDA-lesioned rats

Group		Intact side	Denervated side
Vehicle	21 d,sc	652.47±101.2	634.59±123.1
SPD 2.5 mg/kg	21 d,sc	634.77±133.3	551.24±88.2
SPD 5.0 mg/kg	21 d,sc	705.88±98.8	604.22±106.9
SPD 10.0 mg/kg	21 d,sc	674.34±131.4	454.62±100.0
SPD 20.0 mg/kg	21 d,sc	806.22±129.0	340.25±51.0^a,b
SPD 40.0 mg/kg	21 d,sc	732.49±109.9	338.11±77.5^a,b
SKF38393 3.0mg/kg	30 min,ip	671.73±87.3	222.14±45.7^a,b
SPD 40.0 mg/kg	30 min,ip	693.51±117.9	350.82±50.2^a,b

Data were presented as ±s of dpm of ［γ-³²P］phosphate incorporated in triplicate experiments performed on a pool tissue (n=4,in each group).^aP<0.01 vs intact side,^bP<0.01 vs vehicle (denervated side).

　　Similarly,the selective D₁ agonist SKF38393 reduced the DARPP-32 phosphorylation only in the denervated ipsilateral striatum,and more potently than SPD.
　　2.3　SPD causing down-regulation of the postsynaptic D₁ receptors in the striata of 6-OHDA-lesioned rats
　　6-OHDA-lesion induced an marked increase of postsynaptic D₁ receptors B_max (385.0±26.1 fmol/mg protein,P<0.01 vs non-lesioned group).But there was no significant difference in their K_D′s.
　　Interestingly,after chronic SPD treatment subcutaneously 10,20,40 mg/kg,the B_max of D₁ receptors gradually decreased.Particularly,the B_max in the group treated with SPD 40 mg/kg decreased to 319.7±20.1 fmol/mg protein (P<0.05 vs lesioned+vehicle group).However,no significant alteration of K_D was induced after administration of SPD (Table 2).

Table2　Effects of SPD treatment (once daily×21 d,sc) on postsynaptic D₁ receptor in the striatum of 6-OHDA-lesioned rats

Group	Bmax／fmol^.mg^－1 protein	KD／nmol^.L^－1
Non-lesioned+vehicle	274.4±22.4	2.93±0.4
Lesioned+vehicle	385.0±26.1^a	2.75±0.5
Lesioned+SPD 10 (mg/kg)	361.0±47.2^b	2.88±0.6
Lesioned+SPD 20 (mg/kg)	333.2±30.8^b	2.93±0.5
Lesioned+SPD 40 (mg/kg)	319.7±20.1^b,c	3.02±0.4

Data are presented as ±s (n=4 in each group).^aP<0.01 vs non-lesioned+vehicle,^bP<0.05 vs non-lesioned+vehicle,^cP<0.05 vs lesioned+vehicle.

　　3 DISCUSSION

　　The quantitative determination of DARPP-32 in tissue with back-phosphorylation assay is dependent on the content of its dephospho-form in the tested sample^［9］.During incubation in vitro experiment,the decrease of ［³²P］-phosphate incorporation in tissue just reflects the increase of DARPP-32 in phosphorylation state in vivo.In other words,the more ［³²P］ phosphate incorporation in vitro is reduced,the more active DARPP-32 phosphorylation in vivo is increased.On the basis of phosphorylation DARPP-32,the supersensitivity of D₁ receptors induced by 6-OHDA-lesioned might be partly explained.
　　The present results showed that ［³²P］ phosphate incorporation in DARPP-32 was significantly in～creased in the denervated striata by SPD and SKF38393.Because both SPD and SKF38393 are the D₁ receptor agonists and stimulate D₁ receptors coupled to Gs protein,they produce a larger amount of DARPP-32 phosphorylation.However,in accordance with an other report^［13］,only the 6-OHDA-lesion could not significantly modify the total amount of DARPP-32 phosphorylation.It is well-known that the DARPP-32 protein is a specific marker of D₁ receptors in brain.It is one of the target protein of cAMP-dependant protein kinase (PKA) in the signal transduction of D₁ receptors.Furthermore,the phospho-DARPP-32,an active inhibitor of phosphatase I,enhances the physiological responses.
　　The intrinsic activity of SPD to D₁ receptors is lower than that of DA (endogenous ligand),thus SPD is a partial agonist to D₁ receptors^{［6,13,14］}.Nevertheless,SPD displays its D₁ agonistic action in rotation with 6-OHDA-lesioned rats.Therefore,we have enough reason to believe that the SPD-induced rotational behavior is dependent on the cascade effect of D₁ receptor signal transduction(Fig.2).

Fig.2　DARPP-32 participates in the regulation of D₁ signal transduction

　　Our previous work has demonstrated that chronic treatment with SPD up-regulates the D₁ and D₂ receptors (B_max) by 41.5% and 43.7%,respectively,indicating that SPD is a DA antagonist in unlesioned rats^［15］.The present study,however,has demonstrated that up-regulation of D₁ receptors caused by 6-OHDA-lesion was reduced by SPD treatment (Table 2).This fact strongly suggests that SPD exerts its agonistic effect on D₁ receptors in 6-OHDA-lesioned rats.SPD increased the phosphorylation of DARPP-32 in vivo and sequentially enhanced the efficacy of D₁ signal transduction by reducing the number of the involved D₁ receptors.
　　In summary,the present results provide direct biochemical evidence for that SPD activates the D₁ receptor-dependent signal transduction in the striatum of 6-OHDA-lesioned rat by increasing the DARPP-32 phosphorylation in vivo.Thus it can be conclude that SPD processes definite agonistic effect on postsynaptic D₁ receptors in the D₁ signal transduction mechanism.
　　ACKNOWLEDGMENTS: To Miss SHAO Yue and Miss DAI Hu for their kind technical assistance.

^*国家自然科学基金资助项目 (No.39600179)
^*Project supported by the National Natural Science Foundation of China (No.39600179)

³联系作者。电话： (086)-21-64311833-402;电传：(086)-21-64370269
³Correspondence to Prof.JIN Guo-Zhang.Tel: 086-21-64311833×402;Fax: 086-21-64370269Acta Physiologica Sinica

作者单位：刘健　郭翔　金国章：中国科学院上海药物研究所,上海 200031;
　　　　　刘健　王博成：核医学国家重点实验室,无锡 214063

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Received 1997-12-09　　Revised 1998-04-02