D2‐like dopamine receptors mediate regulation of pupal diapause in Chinese oak silkmoth Antheraea pernyi

The present study investigated the pharmacological properties of dopamine receptors that functioned in the termination of pupal diapause in the Chinese oak silkmoth, Antheraea pernyi (Lepidoptera: Saturniidae). Dopamine receptors are classified according to their structure and function into two subfamilies as D1‐ and D2‐like receptors. D1‐like receptors activate, whereas D2‐like receptors inhibit, adenylate cyclase. We examined the effects of agonists and antagonists selective for D1‐ and D2‐like receptors on the diapause state. As A. pernyi is a long‐day species, pupal diapause is maintained during short days and can be terminated by exposure to a long‐day photoperiod. The D2‐like receptor‐selective agonist quinpirole delayed the timing of adult emergence under long days, and the D2‐receptor‐selective antagonist sulpiride terminated pupal diapause even under a short‐day photoperiod. The D1‐like receptor‐selective agonist and antagonist, SKF‐38393 and SCH‐23390, respectively, caused no significant effects on diapause pupae. These results suggest that not D1‐ but D2‐like receptors mediated diapause regulation in A. pernyi. This dopamine pathway appeared to block the termination of pupal diapause. Furthermore, the actions of the cAMP analog 8‐CPT‐cAMP and dopamine receptor antagonists upon diapause pupae were similar, which supports the notion that D2‐like receptors involved in diapause of this insect prevent adenylate cyclase from producing cAMP like vertebrate D2‐like receptors. Taken together, our findings suggest that dopamine blocked diapause termination through D2‐like receptors that inhibited adenylate cyclase in A. pernyi. During short days under which diapause was maintained in pupae, the dopaminergic mechanism might be stimulated to suppress cAMP levels in cells regulating diapause.     

Downregulation of the dopamine D2-like receptor in corpus allatum affects juvenile hormone synthesis in Drosophila melanogaster females

In Drosophila, juvenile hormone (JH) is synthesized de novo in the specialized endocrine gland, corpusallatum (CA). Dopamine (DA) controls JH levels by either stimulating or inhibiting its synthesis and degradation depending on the developmental stage. The present study focuses on the role of D2-like receptors in the regulation of JH synthesis by dopamine. We show that D2-like receptors (DD2R) are expressed in CA cells of Drosophila melanogaster females. In addition, the level of JH production was analyzed in D. melanogaster females with decreased DD2R expression in CA (vs. corresponding control flies) by assessing multiple indices of JH synthesis (JH-hydrolyzing activity and stress reactivity of the system of JH metabolism, activity and stress reactivity of the alkaline phosphatase, activity and stress reactivity of the tyrosine decarboxylase). The differential value obtained for each index suggests increased JH production in female flies that downregulate DD2R. Based on these findings, we postulate that the DA inhibiting effect on the JH synthesis in D. melanogaster is mediated at least in part via D2-like receptors.     

An antagonistic interaction between A2B adenosine and D2 dopamine receptors modulates the function of rat carotid body chemoreceptor cells

We have previously demonstrated that adenosine controls the release of catecholamines (CA) from carotid body (CB) acting on A2B receptors. Here, we have tested the hypothesis that the control is exerted via an interaction between adenosine A2B and dopamine D2 receptors present in chemoreceptor cells. Experiments were performed in vitro in CB from 3 months rats. The effect of A2B adenosine and D2 dopamine agonists and antagonists applied alone or in combination were studied on basal (20%O2) and hypoxia (10%O2)-evoked release of CA and cAMP content of CB. We have found that adenosine A2 agonists and D2 antagonists dose-dependently increased basal and evoked release CA from the CB while A2 antagonists and D2 agonists had an inhibitory action. The existence of A2B-D2 receptor interaction was established because the inhibitory action of A2 antagonists was abolished by D2 antagonists, and the stimulatory action of A2 agonists was abolished by D2 agonists. Further, A2 agonists increased and D2 agonist decreased cAMP content in the CB; their co-application eliminated the response. The present results provide direct pharmacological evidence that an antagonistic interaction between A2B adenosine and D2 dopamine receptors exist in rat CB and would explain the dopamine-adenosine interactions on ventilation previously observed.     

Short neuropeptide F (sNPF) is a stage-specific suppressor for juvenile hormone biosynthesis by corpora allata,and a critical factor for the initiation of insect metamorphosis

Molting and metamorphosis are essential events for arthropod development, and juvenile hormone (JH) and its precursors play critical roles for these events. We examined the regulation of JH biosynthesis by the corpora allata (CA) in Bombyx mori, and found that intact brain-corpora cardiaca (CC)–CA complexes produced a smaller amount of JH than that in CC–CA complexes and CA alone throughout the 4th and 5th (last) instar stadium. The smaller amount of synthesis was due to allatostatin-C (AST-C) produced by the brain. The CC synthesized short neuropeptide F (sNPF) that also suppressed the JH synthesis, but only in day 3 4th stadium and after the last larval ecdysis. For the suppression, both peptides prevented the expression of some of the distinct JH biosynthetic enzymes in the mevalonate pathway. Allatotropin (AT) stimulated sNPF expression in the CC of day 1 5th instar stadium, not of day 3 4th; therefore the stage-specific inhibition of JH synthesis by sNPF was partly due to the stimulative action of AT on the sNPF expression besides the stage-specific expression of the sNPF receptors in the CA, the level of which was high in day 2 4th and day 0 5th instar larvae. The cessation of JH biosynthesis in the last instar larvae is a key event to initiate pupal metamorphosis, and both sNPF and AST-C are key factors in shutting down JH synthesis, along with the decline of ecdysone titer and dopamine.     

昆虫多巴胺及其受体的研究进展及展望

多巴胺(dopamine,DA)是一种重要的神经递质,通过特异地结合其相关的多巴胺受体(dopamine receptors,DARs)发挥作用。昆虫DARs可分为D1-like DARs,D2-like DARs和多巴胺/蜕皮激素受体(dopamine/ecdysteroid receptor,DopEcR)。D1-like DARs包含两种亚型即DOP1和DOP2,都能偶联G s蛋白引起胞内cAMP上升,且DOP2还能偶联G q蛋白引起胞内Ca 2+浓度升高;D2-like DARs只有一种亚型DOP3,偶联G i蛋白导致胞内cAMP下降;DopEcR可以同时被DA和蜕皮激素激活。本文综述了近年来关于昆虫DA的调控、多巴胺神经元、DARs的药理学特性及生理功能等方面的研究进展。DA合成、转运和降解过程中的基因调控昆虫的多种表型,如表皮黑化、翅的颜色和图案等。DA在多巴胺神经元中合成和释放,不同类型的多巴胺神经元参与调控不同的功能。随着近年来单细胞测序和DA实时成像技术的兴起,这将有利于进一步探讨特异神经元的功能。不同昆虫DARs的激动剂和拮抗剂活性存在很大异同,这些药理学差异将为以昆虫DARs为作用靶标开发高效选择性杀虫剂提供重要依据。DARs参与调控昆虫的多种生理与行为过程,如取食、学习、记忆、遗忘、求偶、交配、睡眠及觉醒等。随着CRISPR/Cas9技术在不同昆虫中成功地应用,以及结合模式昆虫黑腹果蝇中丰富的遗传学操作手段,这些都将有利于精准解析DARs的功能。     

Effects of starvation and sucrose feeding on corpora allata of last instar larvae of Manduca sexta: in vitro activity, size, and cell number

Abstract Larvae of the tobacco hornworm moth Manduca sexta starved for the first 3 days of the last (fifth) stadium undergo a supernumerary moult. If they are provided with sucrose during the starvation period, they develop into normal pupae although pupation is delayed. The activities of the corpora allata (CA) from normal, starved, and sucrose fed larvae were followed through the fifth stadium with a radiochemical assay for Juvenile Hormone (JH) biosynthesis. An attempt was made to correlate CA-activity with CA cell number, size, and protein content.
In CA of normally fed larvae the rate of JH synthesis declined to undetectable levels by day 4 which was also the time of exposure of the dorsal vessel. In CA of starved larvae, the rate of JH synthesis at first decreased but began to increase on day 3 and reached a peak value by day 7 , at which time head capsule slippage occurred. In CA of sucrose fed larvae, the rate of biosynthesis declined as in normal larvae but the decline was extended over a longer period. Exposure of the dorsal vessel was delayed in the same manner and occurred on days 7–9. The major JH in all cases was JH-II.
The CA comprise c. 150 cells in the early fifth stadium, and this number remained constant during the fifth stadium in all three feeding regimens. In normal larvae, CA size and protein content increased several-fold during the stadium whereas in starved and sucrose-fed larvae they increased slowly and in agreement with the altered timing of developmental events. In none of the groups was the CA activity pattern correlated with morphometric changes of the CA. The rates of JH biosynthesis were not closely correlated with published JH titre curves. The in vivo mechanisms for regulation of JH production remain to be elucidated.     

Juvenile hormone biosynthesis by corpora allata of larval tomato moth, Lacanobia oleracea, and regulation by Manduca sexta allatostatin and allatotropin

Juvenile hormone (JH) biosynthesis and the effects of synthetic Manduca sexta allatostatin (Mas-AS) and M. sexta allatotropin (Mas-AT) were investigated in isolated corpora allata (CA) of Vth stadium larvae of the tomato moth, Lacanobia oleracea. Reversed-phase high-performance liquid chromatography (RP-HPLC) of JH extracted from CA shows that larvae produce predominantly JH II and its corresponding acid. It appears that the acid homologue is a result of JH esterase activity in the CA (and other tissues) rather than the lack of JH acid methyltransferase. Mean rates of synthesis (100-200fmol/pr/h) were inhibited ca. 70% by Mas-AS and stimulated in a dose-dependent manner up to three times by Mas-AT. However, Mas-AS had no significant effect on Mas-AT-stimulated rates of JH biosynthesis. Using RP-HPLC and an enzyme-linked immunosorbent assay (ELISA) to Mas-AT, a peak of Mas-AT-like immunoreactivity was detected in larval L. oleracea brain homogenates. Co-elution of this immunoreactive peak with synthetic Mas-AT suggests that this neuropeptide is also present in L. oleracea.     

多巴胺D3受体(D3R)的神经科学新进展

多巴胺(DA)是脑内一种重要的神经递质,通过不同DA受体亚型调控运动功能、认知活动和药物成瘾等生理、病理过程。多巴胺D3受体(D3R)属于D2样受体,但其功能长期不明。近年来,人们对它在神经科学中的意义有了新的认识。首先,D3R的信号通路独特,它被激活后显示细胞增殖效应,但cAMP信号传导途径不明显。其次,D3R基因敲除小鼠研究提示,正常生理状态下D3R仅表现辅助功能：在特定病理条件下,D3R显示出重要的“平衡缓冲作用”,在精神分裂症、帕金森病(PD)治疗中运动障碍副作用LID的发生和毒品复吸等病理过程扮演了重要角色。因此,D3R是一个重要的药物靶标。D3R拮抗剂在精神分裂症治疗中显示了临床前景,D3R激动剂则对PD治疗和毒品复吸防治展示了应用价值。     

High Fat Diet Increases [3H] Flunitrazepam Binding in the Mouse Brain that is Dependent on the Expression of the Dopamine D2 Gene

Neurochemical Research - Dopamine is an important neuromodulator in the brain that binds to dopamine D1-like receptors (D1, D5) as well as dopamine D2-like receptors (D2, D3, D4). The D2 receptor...     

Identification of D1-like dopamine receptors on human blood platelets

Dopamine is able to inhibit the epinephrine-induced aggregation of human blood platelets, but the mechanism of action has not been elucidated. In this study we report that membranes from human blood platelets possess high affinity, saturable and stereoselective binding sites for the D1 dopamine receptor antagonist (3H) SCH 23390. (3H) SCH 23390 appeared to label a single class of binding sites with a Bmax of 18.6 +/- 1.6 fmol/mg protein and a KD of 0.8 nM. The potencies of different dopaminergic antagonists and agonists in displacing (3H) SCH 23390 from blood platelet membranes were similar to those obtained for striatal membranes. Unlike the classically defined D1 receptors, e.g. those in striatum, the D1 receptor sites on platelets appeared not to be coupled to the adenylate cyclase system, hence the term "D1-like". The D1 agonist SKF 38393 was more potent than dopamine in inhibiting platelet aggregation induced by epinephrine, and the effects of dopamine and SKF 38393 were prevented by SCH 23390. These results suggest that the inhibitory action of dopamine on the epinephrine-induced platelet aggregation is mediated through these D1-like receptors.     

Comparative pharmacology of two D1-like dopamine receptors cloned from the silkworm Bombyx mori

Dopamine (DA) is a physiologically important biogenic amine in insect peripheral and nervous tissues. We recently cloned two DA receptors (BmDopR1 and BmDopR2) from the silkworm Bombyx mori and identified them as D1-like receptors, which activate adenylate cyclase to increase intracellular cAMP levels. In this study, these two receptors were stably expressed in HEK-293 cells, and the dose-responsiveness to DA and their pharmacological properties were examined using cAMP assays. BmDopR1 showed a dose-dependent increase in cAMP levels at DA concentrations up to 10^?7 M with EC₅₀ of 3.30 nM, while BmDopR2 required 10^?6 M DA for activation. In BmDopR1-expressing cells, DA at 10^?6–10^?4 M induced 30–50% lower cAMP production than 10^?7 M DA. BmDopR2-expressing cells showed a standard sigmoidal dose–response, with maximum cAMP levels attained with 10^?5–10^?4 M DA and EC₅₀ of 1.30 μM. Both receptors had similar agonist profiles, and the typical vertebrate D1-like receptor agonist SKF-38393 was ineffective. Experiments with antagonists revealed that BmDopR1 exhibits D1-like features. However, the pharmacology of BmDopR2 was distinct from D1-like receptors; the typical vertebrate D1-like receptor antagonist SCH-23390 was less potent than the nonselective antagonist flupenthixol and the D2-like receptor antagonist chlorpromazine. The rank order of activities of several antagonists for BmDopR1 and BmDopR2 was more similar to that of Drosophila melanogaster DA receptors than Apis mellifera DA receptors. These data suggest that DA receptors could be potential targets for specific insecticides or insectistatics.     

Characterization of a D2-like dopamine receptor (AmDOP3) in honey bee, Apis mellifera

Dopamine is an important neurotransmitter in vertebrate and invertebrate nervous systems and is widely distributed in the brain of the honey bee, Apis mellifera. We report here the functional characterization and cellular localization of the putative dopamine receptor gene, Amdop3, a cDNA clone isolated and identified in previous studies as AmBAR3 (Apis mellifera Biogenic Amine Receptor 3). The Amdop3 cDNA encodes a 694 amino acid protein, AmDOP3. Comparison of AmDOP3 to Drosophila melanogaster sequences indicates that it is orthologous to the D2-like dopamine receptor, DD2R. Using AmDOP3 receptors expressed in HEK293 cells we show that of the endogenous biogenic amines, dopamine is the most potent AmDOP3 agonist, and that activation of AmDOP3 receptors results in down regulation of intracellular levels of cAMP, a property characteristic of D2-like dopamine receptors. In situ hybridization reveals that Amdop3 is widely expressed in the brain but shows a pattern of expression that differs from that of either Amdop1 or Amdop2, both of which encode D1-like dopamine receptors. Nonetheless, overlaps in the distribution of cells expressing Amdop1, Amdop2 and Amdop3 mRNAs suggest the likelihood of D1:D2 receptor interactions in some cells, including subpopulations of mushroom body neurons.     

Cloning and characterization of a Caenorhabditis elegans D2-like dopamine receptor

The neurotransmitter dopamine plays an important role in the regulation of behavior in both vertebrates and invertebrates. In mammals, dopamine binds and activates two classes of dopamine receptors, D1-like and D2-like receptors. However, D2-like dopamine receptors in Caenorhabditis elegans have not yet been characterized. We have cloned a cDNA encoding a putative C. elegans D2-like dopamine receptor. The deduced amino acid sequence of the cloned cDNA shows higher sequence similarities to vertebrate D2-like dopamine receptors than to D1-like receptors. Two splice variants that differ in the length of their predicted third intracellular loops were identified. The receptor heterologously expressed in cultured cells showed high affinity binding to [125I]iodo-lysergic acid diethylamide. Dopamine showed the highest affinity for this receptor among several amine neurotransmitters tested. Activation of the heterologously expressed receptor led to the inhibition of cyclic AMP production, confirming that this receptor has the functional property of a D2-like receptor. We have also analyzed the expression pattern of this receptor and found that the receptor is expressed in several neurons including all the dopaminergic neurons in C. elegans.     

Norepinephrine acts as D1-dopaminergic agonist in the embryonic avian retina: late expression of beta1-adrenergic receptor shifts norepinephrine specificity in the adult tissue

Dopamine is the main catecholamine found in the chick retina whereas norepinephrine is only found in trace amounts. We compared the effectiveness of dopamine and norepinephrine in promoting cyclic AMP accumulation in retinas at embryonic day 13 (E13) and from post-hatched chicken (P15). Dopamine (EC(50)=10microM) and norepinephrine (EC(50)=30microM), but not the beta(1)-adrenergic agonist isoproterenol, stimulated over seven-fold the production of cyclic AMP in E13 retina. The cyclic AMP accumulation induced by both catecholamines in embryonic tissue was entirely blocked by 2microM SCH23390, a D(1) receptor antagonist, but not by alprenolol (beta-adrenoceptor antagonist). In P15 retinas, 100microM isoproterenol stimulated five-fold the accumulation of cAMP. This effect was blocked by propanolol (10microM), but not by 2microM SCH23390. Embryonic and adult retina display beta(1) adrenergic receptor mRNA as detected by RT-PCR, but the beta(1) adrenergic receptor protein was detected only in post-hatched tissue. We conclude that norepinephrine cross-reacts with D(1) dopaminergic receptor with affinity similar to that of dopamine in the embryonic retina. In the mature retina, however, D(1) receptors become restricted to activation by dopamine. Moreover, as opposed to the embryonic tissue, norepinephrine seems to stimulate cAMP accumulation via beta(1)-like adrenergic receptors in the mature tissue.     

Characterization of a novel D2-like dopamine receptor with a truncated splice variant and a D1-like dopamine receptor unique to invertebrates from Caenorhabditis elegans

We have cloned two novel Caenorhabditis elegans dopamine receptors, DOP-3 and DOP-4. DOP-3 shows high sequence homology with other D2-like dopamine receptors. As a result of alternative splicing, a truncated splice variant of DOP-3, DOP-3nf, was produced. Because of the in-frame insertion of a stop codon in the third intracellular loop, DOP-3nf lacks the sixth and seventh transmembrane domains that are found in the full-length DOP-3 receptor. Reporter gene assay showed that DOP-3 attenuates forskolin-stimulated cAMP formation in response to dopamine stimulation, whereas DOP-3nf does not. When DOP-3 was coexpressed with DOP-3nf, the ability to inhibit forskolin-stimulated cAMP formation was reduced. DOP-4 shows high sequence homology with D1-like dopamine receptors unique to invertebrates, which are distinct from mammalian D1-like dopamine receptors. Reporter gene assay showed that DOP-4 stimulates cAMP accumulation in response to dopamine stimulation. These two receptors provide new opportunities to understand dopaminergic signaling at the molecular level.     

昆虫多巴胺及其受体的研究进展

多巴胺（dopamine, DA）是脊椎动物和无脊椎动物体内一种重要的生物胺, 其参与调控了昆虫的多种生理反应和行为过程, 如学习与记忆、 认知、 性取向、 抉择、 运动以及型变等。多巴胺主要通过结合特异性的G蛋白偶联受体, 即多巴胺受体（dopamine receptors, DARs）来发挥生理作用。本文综述了多巴胺在昆虫中的调控、 分布及所参与的生理功能, 如多巴胺调控昆虫的交配、 发育、 嗅觉以及运动行为等, 特别对DARs的信号转导、 生理功能以及药理学等方面进行了详细评述。昆虫的DARs大致可分为两大类： D1-like DARs和D2-like DARs。D1-like DARs包含有2种亚型, 分别为DOP1和DOP2。DOP1仅能偶联胞内cAMP的上升, 而DOP2不仅可以起胞内cAMP的上升, 还可偶联胞内Ca²⁺的释放。 D2-like DARs仅包含有1种亚型DOP3, 其被激活后引起胞内cAMP的降低。DA通过激活不同的DARs可偶联不同的第二信使系统, 所产生的下游细胞反应则与昆虫的各种行为相关, 而对昆虫DARs的药理学研究将有助于我们开发特异性的杀虫剂用于害虫防治。     

Coupling of a cloned rat dopamine-D2 receptor to inhibition of adenylyl cyclase and prolactin secretion.

We have previously described a cDNA which encodes a binding site with the pharmacology of the D2-dopamine receptor (Bunzow, J. R., VanTol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M., Machida, C., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787). We demonstrate here that this protein is a functional receptor, i.e. it couples to G-proteins to inhibit cAMP generation and hormone secretion. The cDNA was expressed in GH4C1 cells, a rat somatomammotrophic cell strain which lacks dopamine receptors. Stable transfectants were isolated and one clone, GH4ZR7, which had the highest levels of D2-dopamine receptor mRNA on Northern blot, was studied in detail. Binding of D2-dopamine antagonist [3H]spiperone to membranes isolated from GH4ZR7 cells was saturable, with KD = 96 pM, and Bmax = 2300 fmol/mg protein. Addition of GTP/NaCl increased the IC50 value for dopamine competition for [3H]spiperone binding by 2-fold, indicating that the D2-dopamine receptor interacts with one or more G-proteins. To assess the function of the dopamine-binding site, acute biological actions of dopamine were characterized in GH4ZR7 cells. Dopamine, at concentrations found in vivo, decreased resting intra- and extracellular cAMP levels (EC50 = 8 +/- 2 nM) by 50-70% and blocked completely vasoactive intestinal peptide (VIP) induced enhancement of cAMP levels (EC50 = 6 +/- 1 nM). Antagonism of dopamine-induced inhibition of VIP-enhanced cAMP levels by spiperone, (+)-butaclamol, (-)-sulpiride, and SCH23390 occurred at concentrations expected from KI values for these antagonists at the D2-receptor and was stereoselective. Dopamine (as well as several D2-selective agonists) inhibited forskolin-stimulated adenylate cyclase activity by 45 +/- 6%, with EC50 of 500-800 nM in GH4ZR7 membranes. Dopaminergic inhibition of cellular cAMP levels and of adenylyl cyclase activity in membrane preparations was abolished by pretreatment with pertussis toxin (50 ng/ml, 16 h). Dopamine (200 nM) abolished VIP- and thyrotropin-releasing hormone-induced acute prolactin release. These data show conclusively that the cDNA clone encodes a functional dopamine-D2 receptor which couples to G-proteins to inhibit adenylyl cyclase and both cAMP-dependent and cAMP-independent hormone secretion.     

Dopamine-stimulated adenylate cyclase in human term placenta

The effect of dopamine on adenylate cyclase activity was investigated in slices of human term placentas. Dopamine elicited a dose-dependent stimulation of cAMP formation with a ED50 value of about 1 X 10(-6)M dopamine and an increase of 110% over the control with 1 X 10(-4)M dopamine. (-)-Epinephrine and (-)-norepinephrine also increased placental cAMP formation. Apomorphine displayed a slight but non-significant stimulatory effect while bromocriptine was not effective. SCH 23390, a selective antagonist of dopamine D1 receptors caused a dose-dependent decrease of the dopamine activation. In contrast, the dopamine increase of cAMP was unaffected by beta- and alpha-adrenergic blocking drugs and by the D2 selective antagonist, (-)-sulpiride. These data indicate that dopamine stimulates cAMP formation in human term placenta through a specific mechanism via D1 dopaminergic receptors positively coupled to adenylate cyclase.     

多巴胺D2受体在肿瘤治疗中的研究进展

多巴胺(Dopamine)(C6H3(OH)2-CH2-CH2-NH2)是人类中枢神经系统的重要儿茶酚胺类神经递质,通过其相应的膜受体而发挥情绪、饮食、运动、认知及外周血等的调节作用。多巴胺受体属于膜G蛋白偶联受体家族。目前发现的多巴胺受体有五种,其中D2受体基因主要分布于脑部。近年来的研究表明,多巴胺D2受体对肿瘤细胞具有抑制作用,对肿瘤的药物治疗具有重要意义。目前,D2受体激动剂已经成为大多数泌乳素瘤的首选治疗药物。本文通过文献回顾,对多巴胺受体在肿瘤的预后和治疗中的作用进行综述。