Differential effects of ascorbate and EDTA on high-affinity binding of 3H-apomorphine and 3H-ADTN to calf caudate membranes

Recent reports describe effects of ascorbate on binding of dopaminergic agonists to membrane preparations of brain tissue. We now report that with calf caudate membranes, in the absence of EDTA, ascorbate (up to 10 mM) lowered the binding of [3H] (-)apomorphine (3H-APO) by up to 34%, and the proportion "specific," from 69% to 36%, and lowered binding of [3H] (+/-)2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (3H-ADTN) by up to 38%, with little effect on the proportion of its specific binding. EDTA (5 mM), alone, also reduced binding of both ligands by 40-50% but had no effect on the proportion specific. With ascorbate plus EDTA, although total binding was lowered, the proportion of specific binding of 3H-APO rose to a maximum of 82% at 2.5 mM ascorbate, whereas that of 3H-ADTN was only slightly increased. Moreover, TLC revealed that the antioxidants were required during incubation to prevent temperature- and time-dependent degradation of APO much more than ADTN. Thus, while each additive, alone, lowered binding of 3H-APO and of 3H-ADTN, ascorbate plus EDTA increased the proportion of specific binding, especially of 3H-APO, and protected both from degradation.     

Effect of 3-PPP,a putative dopamine autoreceptor agonist,on [3H]ligand binding to dopamine receptors of calf and rat striatum

3-(3-Hydroxyphenyl)-N-n-propyliperidine (3-PPP) is most effective in inhibiting [³H]apomorphine binding in rat striatal membranes, with Ki values of 63 nM. 3-PPP was six to 27 times less effective when it competed with the binding of [³H]dopamine or [³H]spiperone in calf and rat striatal membranes. At concentrations up to 10 μM, 3-PPP failed to substitute for dopamine in the activation of adenylate cyclase in rat striatal membranes. 3-PPP at 4.8-5 μM caused 50% inhibition of catecholamine uptake in synaptosomes of corpus striatum and hypothalamus, therefore appearing to be a relatively weak uptake inhibitor. The higher affinity of 3-PPP for [³H]apomorphine binding sites is consistent with its binding to a subset of dopamine receptors which are characterized by a high affinity for both the agonist and antagonist of dopamine.     

Stimulation of 3H-apomorphine binding by dopamine and bromocriptine

Pretreatment of spontaneously hypertensive rats (SHR) with the converting enzyme inhibitor captoril (10 or 100 mg/kg p.o.) had no effect on pressor responses to angiotensin II or norepinephrine whereas the response to angiotensin I was markedly inhibited. In contrast, pressor responses to symphathetic stimulation in pitched SHR were inhibited by captoril whereas the positive chronotropic responses to stimulation were unaltered. These results suggest that captoril causes a prejunctional inhibition of norepinephrine release to sympathetic nerve stimulation which is selective for the vasculature. This is probably due to inhibition of angiotensin II formation in the vasculature.     

四氢原小檗碱类对小牛纹状体D_1和D_2多巴胺受体结合的特性(英文)

目的:研究四氢原小檗碱类(THPB)对脑内多巴胺受体D_1和D_2亚型的结合特性,并阐明它们之间的构效关系.方法:放射配位体测定结合双位点模型分析.结果:4个THPB与D_1受体以R_H和R_L双位点结合,它们在C_2和C_9或C_2和C_(10)位有两个羟基,另外11个THPB与D_1受体以单位点结合.对于D_2受体,11个被检测的化合物均以单位点结合,其中,在C_2位有羟基的THPB亲和力最强.结论:在C_2和C_9或C_2和C_(10)位有双羟基的THPB具有D_1受体激动剂的内在活性,其它THPB则无此活性.11个THPB均为D_2受体拮抗剂.     

Characterization of dopamine autoreceptor and [3H]spiperone binding sites in vitro with classical and novel dopamine receptor agonists

The specific D2 receptor agonist, LY 141865, but not the specific D1-receptor agonist, SK&F 38393, potently inhibited electrically evoked [3H]dopamine release from slices of the cat caudate. Similarly, LY 141865, but not SK&F 38393, inhibited [3H]spiperone binding to membranes of the cat caudate. The inhibition by dopamine receptor agonists of electrically evoked [3H]dopamine release was antagonized by the specific D2-receptor antagonist S-sulpiride. The inhibition of the electrically evoked release of [3H]dopamine by apomorphine was not, however, antagonized by the specific D1-receptor antagonist, bulbocapnine. Similarly, S-sulpiride but not bulbocapnine potently inhibited [3H]spiperone binding to membranes of the cat caudate. These results suggest that the dopamine autoreceptor modulating the depolarization-evoked release of [3H]dopamine, and the binding site of [3H]spiperone, are valid in vitro models for D2-dopamine receptors. Contrary to some previous reports, DPI was inactive in both in vitro dopamine receptor models. The IC50 values of a series of dopamine receptor agonists correlated very well in the two in vitro dopamine receptor models. One exception to this correlation was bromocriptine, which was more potent at [3H]spiperone binding sites than at the dopamine autoreceptor. With the exception of bromocriptine, all dopamine receptor agonists had one-hundred fold higher potency at the dopamine autoreceptor than at [3H]spiperone binding sites. [3H]Spiperone binding sites are localized primarily postsynaptic to dopamine terminals. Possible differences between the pharmacological properties of pre- and postsynaptic dopamine receptors should become apparent in the comparison of the two in vitro dopamine receptor models. However, the order of potency of dopamine receptor agonists with both in vitro models, dopamine autoreceptor and [3H]spiperone binding, was the same: N-n-propylnorapomorphine greater than TL-99 = 7-HAT greater than M-7 greater than Apomorphine greater than LY 141865.     

Presynaptic dopamine receptors

Summary An in vivo model for investigating the interaction of drugs with presynaptic receptors was used to study the effect of chronic treatment of rats with fluphenazine decanoate on the presynaptic dopamine receptors in the neostriatum and olfactory tubercle. In this model system dopaminergic impulse flow is inhibited pharmacologically with -butyrolactone (GBL). This results in an increase of apparent tyrosine hydroxylase activity, estimated by the accumulation of a dopa during the 30-min interval following administration of a dopa decarboxylase inhibitor. Dopamine agonists produce a dose-dependent blockade of the effect of GBL on dopa accumulation.On days 12 through 21 after rats were injected with fluphenazine decanoate, 2 dopamine agonists-apomorphine (tested on days 12, 15, 17, and 21) and trivastal (ET 495; piribidel; tested on days 12, 14, and 19)-were more potent in blocking the GBL-induced increase of dopa accumulation in the striata of treated animals than in controls. Thirty days after the fluphenazine injection the effect of the dopamine agonists on dopa accumulation in the neostriatum returned to control levels. On Day 14 after fluphenazine decanoate injection the dose-response curve of apomorphine's inhibitory effect on the GBL-induced increase of dopa accumulation in the neostriatum was shifted to the left.In control animals apomorphine was approximately 10-fold more potent in depressing the GBL-induced increase of dopa accumulation in the olfactory tubercles than in the neostriatum. Apomorphine was even more potent in the olfactory tubercle on day 14 after a single injection of fluphenazine decanoate. These results suggest that following chronic treatment of rats with a phenothiazine, presynaptic receptors on dopamine nerve terminals in the striatum and limbic system become supersensitive to dopamine agonists.A preliminary account of this work was presented at the Seventh Annual Meeting of the American Neurochemical Society held in Vancouver, British Columbia, March 15–19, 1976     

Chronic phencyclidine treatment decreases phencyclidine and dopamine receptors in rat brain

Chronic phencyclidine treatment (10 mg/kg/day, SC for 14 days) significantly decreased the number of [³H]phencyclidine and [³H]spiperone binding sites in rat brain. [³H]Dihydromorphine binding was not affected by the same treatment. An acute treatment with phencyclidine (10 mg/kg, SC) did not modify any of the binding sites under study. These results suggest that a chronic phencyclidine treatment induces a down-regulation of phencyclidine and dopamine receptors without affecting opiate receptors. These reductions in the number of phencyclidine and dopamine binding sites might be related to the development of tolerance and/or dependence to phencyclidine.     

Multiple [3H]-nemonapride binding sites in calf brain

[³H]-Nemonapride has been the ligand of choice to label D₄ dopamine receptors. Its specificity was questioned when it was discovered that sigma (σ) sites were also labeled by [³H]-nemonapride. To further characterize the binding of [³H]-nemonapride, three areas of calf brain (striatum, frontal cortex and cerebellum) were examined. In all three areas, [³H]-nemonapride labeled multiple sites. Dopaminergic and σ sites were the most prominent. The σ binding profile was σ-1 like with a K_i binding profile as follows (in order of decreasing potency): haloperidol, PPAP, pentazocine, DTG, U-50488, R(+)-3-PPP. Experiments using sulpiride and pentazocine to block striatal dopaminergic and σ sites, respectively, revealed additional, not previously characterized binding sites for [³H]-nemonapride. One component which was present in striatum but not in frontal cortex or cerebellum, had affinity for some neuroleptics and WB-4101, but not for typical serotonergic agents. Thus, [³H]-nemonapride has no selectivity for dopamine receptors unless stringent experimental conditions are met. Received: 19 September 1996 / Accepted: 12 March 1997     

3H-Apomorphine interactions with dopamine receptors in calf brain

3H-apomorphine binds to membranes from areas of the corpus striatum and limbic system of calf brain saturable and with a drug specificity indicating that it labels dopamine receptors. In terms of drug specificity, log-logit displacement curve slopes and number of binding sites, 3H-apomorphine interacts with receptors in a manner more like 3H-dopamine than 3H-haloperidol. These properties of 3H-apomorphine binding are those of an apparently "pure" agonist in contrast to the partial agonist effects of apomorphine upon the dopamine-sensitive adenylate cyclase.     

铅对发育脑纹状体毒蕈碱和多巴胺受体的影响

母鼠从受孕d0至分娩后d21连续饮用含300和1000ppm的铅水。新生大鼠出生后d21处死。结果表明,血和纹状体铅含量明显增加;在1000ppm组,DNA,RNA降低和蛋白质/DNA比值升高;纹状体DA含量亦减少。铅不影响[~3H]spiperone与纹状体DA受体的特异结合。但影响[~3H]QNB与MACh受体的特异结合,受体数目增加,亲和力未变,提示脑纹状体MACh受体功能状态与铅含量有关。铅影响发育脑纹状体的这两种受体的相互平衡,可能是铅对仔代神经毒性的原因之一。     

Changes in soluble calmodulin following activation of dopamine receptors in rat striatal slices.

Various molecular forms of cyclic nucleotide phosphodiesterase (PDE) are present in the striatum of rats. While Ca²⁺ by itself cannot modulate striatal PDE, this ion is essential for the activation of striatal PDE by calmodulin (CaM). Incubation of striatal slices with apomorphine (10^?7 M) for 30 min increased the total CaM content of the supernatant fraction. Also the amount of CaM associated with PDE was increased and the K_m of PDE for cAMP was lowered. A shorter incubation with dopamine or apomorphine (10 min) failed to increase CaM and to lower the K_m of PDE.Haloperidol (10^?7 M), a dopamine receptor antagonist, prevented the change in the kinetic profile of PDE elicited by dopamine (2 × 10^?7M). Transection of the nigra-striatal fibre bundle by itself did not change the kinetic profile of striatal PDE, but in slices prepared from deafferented striata, a 30 min activation of dopamine receptors still elicited a decrease in the K_m of PDE for cAMP. These findings suggest that following a persistent stimulation of dopamine receptors, the CaM content increases in the cytosol because it is mobilized from a pool located in post-synaptic membranes. This mobilization of CaM regulates PDE; thus, regulation of PDE through a translocation of CaM may participate in reducing the functional output of dopamine receptors following persistent stimulation.     

Effects of cations on high-affinity binding of 3H-ADTN to dopaminergic sites in calf caudate tissue

Effects of cations on binding of 0.1-10 nM 3H-ADTN to calf caudate membranes included decreased apparent Bmax by [Na+] greater than or equal to 100 mM, little effect on Kd or on affinity of other dopamine (DA) agonists (DA and apomorphine), decreased slopes of inhibition curves produced by agonists, but increased affinity of the antagonists (+)butaclamol; in contrast, low (10-20 mM) [Na+] did not decrease Bmax, increased ligand and agonist affinity and specific binding, and gave steep monophasic inhibition curves for DA agonists. K+, Li+, and Rb+ had little effect at a wide range of concentrations. Mg++ and Ca++, in physiologic concentrations, moderately increased binding of 3H-ADTN, as did microM Mn++ or Co++; the latter ions inhibited binding at greater than or equal to 10 mM, as did Cu++ (IC50 = 10 microM). The results extend impressions that physiologic [Na+] favors binding of DA antagonists and diminishes binding of agonists, but optimal agonist binding occurred at low [Na+] (10-20 mM), while divalent cations had complex actions.     

多巴胺D_2/血管紧张素AT_1嵌合受体:D_2受体第三细胞内环影响受体与配基结合以及与G蛋白偶联的特性(英文)

目的:研究受体第三细胞内环(IL_3)的长度对受体与配基结合及与G蛋白偶联特性的影响.方法:用目前已知的G蛋白偶联受体中IL_3最短的血管紧张素Ⅱ AT_1受体的IL_3替换野生型D_2受体较长的IL_3,组成D_2/AT,嵌合受体.结果:与野生型D_2受体相比,D_2/AT_1嵌合受体与拮抗剂的亲和性均降低,与激动剂的亲和性有的增高,有的降低.嵌合受体失去与G蛋白偶联的能力,也不能产生磷酸肌醇水解.结论:受体的IL_3对受体配基结合位点和空间构象有一定影响;受体与G蛋白的偶联不仅与IL_3有关,而且还受非IL_3区域的影响,而IL_3的长度是决定这两方面影响的因素之一.     

Contributions of cysteine 114 of the human D3 dopamine receptor to ligand binding and sensitivity to external oxidizing agents

1. Cysteine 114 (C114) of the human dopamine D3 receptor is located at the helical face of transmembrane segment III (TMIII) near aspartate 110, a counterion for the amine group of catecholamines. The contributions of C114 to receptor function were investigated here using site-directed mutagenetis of C114 to serine.
2. The C114S mutant, as expressed in Sf-9 cells, bound aminotetralin antagonists (UH-232 and AJ-76) and several agonists ((−)3-PPP, apomorphine, pramipexole and quinpirole) with markedly lower affinities as compared to the wild type D3 receptor, but bound other structurally diverse dopaminergic ligands with only minor changes in affinity. Because an N-propyl substituent is the only common structural feature among most affected ligands, we propose that the mutation alters `a propyl cleft'' on the receptor. The mutation hardly affected quinpirole-dependent [³⁵S]-GTPγS binding, suggesting C114 plays a minimal role in receptor-G-protein coupling.
3. N-Ethylmaleimide(NEM), a sulfhydryl modifying agent, blocked ligand binding to the D3 receptor, but not to the C114S mutant. We infer that C114 is the primary residue on the D3 receptor vulnerable to external oxidizing agents. Dopamine D2long and D4₂ receptors contain highly homologous TMIII sequences including an equivalent cysteine residue. However, only the D2long receptor, not the D4₂ receptor, displayed NEM sensitivity similar to that of the D3 receptor.
4. We conclude that C114 is critical for high affinity interactions between the D3 receptor and ligands containing an N-propyl substituent, and unlike its counterpart in the D4₂ receptor, is highly susceptible to external oxidizing agents.

     

Identification of human dopamine receptors agonists from Chinese herbs

AIM: To find human dopamine receptors, especially D1-like receptor specific agonists from Chinese herbs as potential antihypertension drug leads. METHODS: Two D1-like receptor cell lines carrying a beta-lactamase reporter gene, and a D2 receptor cell line coexpressing a promiscuous G protein G15 were constructed using HEK293 cells. A natural compound library made from fractionated samples of herbal extracts was used for high-throughput screening (HTS) against one of the cell lines, HEK/D5R/CRE-blax. The interested hits were evaluated for their activities against various dopamine receptors. RESULTS: Fourteen hits were identified from primary screening, of which 2 of the better hit samples, HD0522 and HD0059, were selected for further material and activity analysis, and to obtain 2 compounds that appeared as 2 single peaks in HPLC, HD0522H01 and HD0059H01. HD0059H01 could activate D1, D2, and D5 receptors, with EC(50 ) values of 2.28 microg/mL, 0.85 microg/mL, and 1.41 microg/mL, respectively. HD0522H01 could only activate D1R and D5R with EC(50 ) values of 2.95 microg/mL and 8.38 microg/mL. CONCLUSION: We established cellbased assays for 3 different human dopamine receptors and identified specific agonists HD0522H01 and HD0059H01 through HTS. The specific agonist to D1-like receptors, HD0522H01, may become a new natural product-based drug lead for antihypertension treatment.     

Effect of toxaphene on the binding of 3H-labeled ouabain and dopamine to rat brain synaptosomes

The effects of toxaphene, a chlorinated hydrocarbon pesticide, on the binding of ouabain and dopamine to rat brain synaptosomes enriched with Na⁺-K⁺ ATPase were investigated. For in vitro assessment of the effects of toxaphene, the synaptosomes prepared from normal rats were used. For in vivo effects the rats were fed on 0, 50, 100, 150 and 200 ppm toxaphene mixed in their daily ration for 8 weeks. At the end of treatment the rats were killed and synaptosomes were prepared. Toxaphene inhibited Na⁺-K⁺ and Mg²⁺ ATPases of synaptosomes in vitro and the inhibition was significant and concentration-dependent. The IC₅₀ values were about 30 and 12 μM toxaphene for Na⁺-K⁺ and Mg²⁺ ATPases, respectively. However, much higher concentrations of toxaphene were required to inhibit the binding of [³H]ouabain and [³H]dopamine to synaptosomes. A 50% inhibition of ouabain and dopamine binding was obtained at 150 and 200 μM of toxaphene. The enzyme activities of synaptosomes in toxaphene-pretreated rats were decreased significantly. However, a dose-dependent decrease was not observed. The rats receiving dosages of 100 ppm and above showed a 30–40% decrease in enzyme activities. The binding of ouabain and dopamine to synaptosomes of toxaphene-pretreated rats showed no significant changes as compared to controls. The present in vitro results suggest that toxaphene may be an effective inhibitor of ATPases with substantial effects on the binding of ouabain and dopamine to rat brain synaptosomes. However, data obtained through in vivo studies do not support this contention. The reason for this discrepancy may be that the toxaphene is being rapidly metabolized or might not have reached the site of action.     

In vivo [3H]spiperone binding to the rat hippocampal formation: Involvement of dopamine receptors

The existence of DA receptors in the rat hippocampus was demonstrated with an in vivo [³H]spiperone radio-receptor assay. Kinetic studies revealed that maximum binding of [³H]spiperone in hippocampus was much smaller than in striatum and frontal cortex but much higher than in cerebellum. In inhibition studies of [³H]spiperone binding, all neuroleptics tested were active in hippocampus as well as in striatum. In contrast, 5HT antagonists were definetely less potent in these two brain regions than in frontal cortex. Finally, even when 5HT receptors were blocked, dipropyl-ATN and haloperidol remained fully effective in hippocampus, striatum, but also in frontal cortex although to a lesser degree. From these results it was concluded that [³H]spiperone binds mainly to DA receptors in hippocampus as well as in striamtum, whereas both 5HT and DA receptors are present in frontal cortex.     

Nigrostriatal lesions enhance striatal 3H-apomorphine and 3H-spiroperidol binding.

Following unilateral 6-hydroxydopamine nigrostriatal lesions in rats, the binding of both 3H-apomorphine and 3H-spiroperidol in the striatum is increased. In rats with incomplete lesions or at early time points after lesion, binding is not significantly different from control levels.