D1 and D2 dopamine receptor mRNA in rat brain.

Physiological and pharmacological criteria have divided dopamine receptors into D1 and D2 subtypes, and genes encoding these subtypes have recently been cloned. Based on the sequences of the cloned receptors, we prepared oligodeoxynucleotide probes to map the cellular expression of the corresponding mRNAs in rat brain by in situ hybridization histochemistry. These mRNAs showed largely overlapping yet distinct patterns of expression. The highest levels of expression for both mRNAs were observed in the caudate-putamen, nucleus accumbens, and olfactory tubercle. Within the caudate-putamen, 47 +/- 6% and 46 +/- 5% of the medium-sized neurons (10-15 microns) expressed the D1 and D2 mRNAs, respectively, and only the D2 mRNA was observed in the larger neurons (greater than 20 microns). The D1 and D2 mRNAs were expressed in most cortical regions, with the highest levels in the prefrontal and entorhinal cortices. Within neocortex, D1 mRNA was observed primarily in layer 6 and D2 mRNA in layers 4-5. Within the amygdala, D1 mRNA was observed in the intercalated nuclei, and D2 mRNA in the central nucleus. Within the hypothalamus, D1 mRNA was observed in the suprachiasmatic nucleus and D2 mRNA in many of the dopaminergic cell groups. Within the septum, globus pallidus, superior and inferior colliculi, mammillary bodies, and substantia nigra only D2 mRNA was detected. These data provide insight into the neuroanatomical basis of the differential effects of drugs that act on D1 or D2 receptors.     

Distribution of D2 dopamine receptor mRNA in rat brain.

The distribution of mRNA coding for the D2 dopamine receptor was studied in the rat brain by in situ hybridization. A cDNA probe corresponding to the putative third cytosolic loop and sixth and seventh transmembrane domains of the rat D2 receptor was used to generate an 35S-labeled riboprobe to hybridize to D2 receptor mRNA. D2 mRNA was found both in dopamine projection fields and in regions associated with dopamine-containing cell bodies, suggesting both postsynaptic and presynaptic autoreceptor localization. Highest concentrations of D2 mRNA were found in neostriatum, olfactory tubercle, substantia nigra, ventral tegmental area, and the nucleus accumbens. This distribution is consistent with those reported with D2 receptor autoradiography.     

Localization of interleukin-1 receptor antagonist mRNA in rat brain.

Interleukin-1 (IL-1) is an inflammatory peptide hormone, with potent neuroendocrine effects. IL-1 stimulates the central nervous system production of corticotropin-releasing hormone (CRH), growth hormone (GH), thyroid-stimulating hormone (TSH), and somatostatin, and inhibits the secretion of prolactin and luteinizing hormone (LH). Interleukin-1 receptor antagonist (IL-1ra) is a novel cytokine, recently purified, characterized, and cloned. IL-1ra is a pure endogenous antagonist of IL-1:IL-1 function is modulated not only by local levels of IL-1, but also by the levels of IL-1ra. We have localized by in situ hybridization histochemistry IL-1ra mRNA in rat brain, in areas of importance to neuroendocrine function, such as the paraventricular nucleus (PVN) of the hypothalamus, hippocampus, as well as cerebellum. These findings indicate that IL-1ra is produced in brain in areas of relevance to the regulation of neuroendocrine function and suggest that IL-1ra may modulate the neuroendocrine effects of IL-1.     

Localization of the mRNA for the dopamine D2 receptor in the rat brain by in situ hybridization histochemistry.

32P-labeled oligonucleotides derived from the coding region of rat dopamine D2 receptor cDNA were used as probes to localize cells in the rat brain that contain the mRNA coding for this receptor by using in situ hybridization histochemistry. The highest level of hybridization was found in the intermediate lobe of the pituitary gland. High mRNA content was observed in the anterior lobe of the pituitary gland, the nuclei caudate-putamen and accumbens, and the olfactory tubercle. Lower levels were seen in the substantia nigra pars compacta and the ventral tegmental area, as well as in the lateral mammillary body. In these areas the distribution was comparable to that of the dopamine D2 receptor binding sites as visualized by autoradiography using [3H]SDZ 205-502 as a ligand. However, in some areas such as the olfactory bulb, neocortex, hippocampus, superior colliculus, and cerebellum, D2 receptors have been visualized but no significant hybridization signal could be detected. The mRNA coding for these receptors in these areas could be contained in cells outside those brain regions, be different from the one recognized by our probes, or be present at levels below the detection limits of our procedure. The possibility of visualizing and quantifying the mRNA coding for dopamine D2 receptor at the microscopic level will yield more information about the in vivo regulation of the synthesis of these receptors and their alteration following selective lesions or drug treatments.     

Localization of D1 and D2 dopamine receptors in brain with subtype-specific antibodies.

Five or more dopamine receptor genes are expressed in brain. However, the pharmacological similarities of the encoded D1-D5 receptors have hindered studies of the localization and functions of the subtypes. To better understand the roles of the individual receptors, antibodies were raised against recombinant D1 and D2 proteins and were shown to bind to the receptor subtypes specifically in Western blot and immunoprecipitation studies. Each antibody reacted selectively with the respective receptor protein expressed both in cells transfected with the cDNAs and in brain. By immunocytochemistry, D1 and D2 had similar regional distributions in rat, monkey, and human brain, with the most intense staining in striatum, olfactory bulb, and substantia nigra. Within each region, however, the precise distributions of each subtype were distinct and often complementary. D1 and D2 were differentially enriched in striatal patch and matrix compartments, in selective layers of the olfactory bulb, and in either substantia nigra pars compacta or reticulata. Electron microscopy demonstrated that D1 and D2 also had highly selective subcellular distributions. In the rat neostriatum, the majority of D1 and D2 immunoreactivity was localized in postsynaptic sites in subsets of spiny dendrites and spine heads in rat neostriatum. Presynaptic D1 and D2 receptors were also observed, indicating both subtypes may regulate neurotransmitter release. D1 was also present in axon terminals in the substantia nigra. These results provide a morphological substrate for understanding the pre- and postsynaptic functions of the genetically defined D1 and D2 receptors in discrete neuronal circuits in mammalian brain.     

多巴胺受体亚型D4和D5在人胃、十二指肠的定位及表达

目的　探讨人体胃、十二指肠是否有多巴胺受体亚型D4和D5的表达及表达的部位。方法　人体胃、十二指肠标本液氮保存 ,低温固定 ,石蜡包埋 ,组织块连续切片 ;地高辛标记的寡核苷酸探针行原位杂交 ;病理图像分析系统进行分析并积分。结果　D4和D5在人体胃、十二指肠均有表达。在胃相对集中于腺体间质细胞及近黏膜肌层的黏膜固有层间质细胞上 ;而在十二指肠则较为弥散 ,除表达于十二指肠腺体间质细胞外 ,在黏膜肌层及布氏腺内亦有表达。D4和D5在胃和十二指肠的分布形态相似。D4在胃内的表达水平高于D5,阳性目标个数为 4 1.2 9± 5 .0 6比 2 6 .2 5± 5 .82 ;数密度为 4 .6 8± 0 .5 9比 2 .97± 0 .6 5 ,差异均有显著性 (P值均 <0 .0 1) ;在十二指肠黏膜内阳性目标个数为 30 .71± 10 .0 6比 2 2 .18± 4 .96 ,数密度为 3.4 8± 1.13比 2 .5 2± 0 .5 7,差异均有显著性 (P值均 <0 .0 1)。D4在胃内的表达高于十二指肠 ,D5表达无差异。结论　D4和D5在人体胃、十二指肠均有分布 ,主要位于腺体间质细胞上 ,十二指肠黏膜肌层及布氏腺亦有少量分布 ;D4的含量高于D5,且以胃内表达最高。     

Excitatory amino acid neurotransmission evidence for a role in neuroendocrine regulation.

There is compelling evidence that endogenous excitatory amino acid neurotransmission is an important component of the neuroendocrine transmission line that regulates anterior pituitary-hormone release and, thus, reproduction. Excitatory amino acids (EAAs), such as glutamate and aspartate, are found in large quantities in neuroendocrine tissues such as the hypothalamus, and neurons from a variety of hypothalamic nuclei respond with marked excitation to EAA application. Exogenous EAA administration rapidly increases the release of GnRH, LH, and prolactin secretion in vivo and in vitro. Antagonist studies demonstrate that EAA-receptor activation is involved in a number of reproductive-endocrine events, such as the induction of puberty, seasonal breeding, steroid-induced LH secretion, and the preovulatory surge of LH and prolactin in the female. EAA regulation of these neuroendocrine events appears to be achieved through modulation and regulation of hypothalamic GnRH secretion.     

多巴胺D1受体在帕金森脑黑质纹状体的表达

目的观察帕金森(PD)病人脑黑质纹状体多巴胺D1受体的表达变化。方法采用免疫放射自显影法观察PD标本(PD组)中黑质纹状体多巴胺D1受体的含量改变,并与非神经系统疾病脑标本(对照组)进行对照研究。结果 PD组多巴胺D1受体标记信号在壳及尾状核比对照组减弱,黑质多巴胺D1受体的标记信号比对照组增强;灰度值分析显示,壳及尾状核的灰度值比对照组分别增加了11.35%和10.52%,而黑质比对照组减了48.89%(P0.01)。结论多巴胺D1受体在PD人脑标本壳及尾状核的表达降低、黑质表达增加。     

Cloning, molecular characterization, and chromosomal assignment of a gene encoding a second D1 dopamine receptor subtype: differential expression pattern in rat brain compared with the D1A receptor.

Multiple D1 dopaminergic receptor subtypes have been postulated on the basis of pharmacological, biochemical, and genetic studies. We describe the isolation and characterization of a rat gene encoding a dopamine receptor that is structurally and functionally similar to the D1 dopamine receptor. The coding region, which is intronless, encodes a protein of 475 amino acids (Mr 52,834) with structural features that are consistent with receptors coupled to guanine nucleotide-binding regulatory proteins. The expressed protein binds dopaminergic ligands and mediates stimulation of adenylyl cyclase with pharmacological properties similar to those of the D1 dopamine receptor. The gene encoding the human homologue of this receptor subtype is located to the short arm of chromosome 4 (4p16.3), the same region as the Huntington disease gene. In striking contrast to the previously cloned D1 receptor, little or no mRNA for the receptor described here was observed in striatum, nucleus accumbens, olfactory tubercle, and frontal cortex. High levels of mRNA for this receptor were found in distinct layers of the hippocampus, the mammillary nuclei, and the anterior pretectal nuclei, brain regions that have been shown to exhibit little or no D1 dopamine receptor binding. On the basis of its properties we propose that this dopamine receptor subtype be called D1B.     

Localization of glucocorticoid receptor mRNA in the male rat brain by in situ hybridization.

The localization and distribution of mRNA encoding the glucocorticoid receptor (GR) was investigated in tissue sections of the adult male rat brain by in situ hybridization and RNA blot analysis. GR mRNA levels were measured by quantitative autoradiography with 35S- and 32P-labeled RNA probes, respectively. Strong labeling was observed within the pyramidal nerve cells of the CA1 and CA2 areas of the hippocampal formation, in the granular cells of the dentate gyrus, in the parvocellular nerve cells of the paraventricular hypothalamic nucleus, and in the cells of the arcuate nucleus, especially the parvocellular part. Moderate labeling of a large number of nerve cells was observed within layers II, III, and VI of the neocortex and in many thalamic nuclei, especially the anterior and ventral nuclear groups as well as several midline nuclei. Within the cerebellar cortex, strong labeling was observed all over the granular layer. In the lower brainstem, strong labeling was found within the entire locus coeruleus and within the mesencephalic raphe nuclei rich in noradrenaline and 5-hydroxytryptamine cell bodies, respectively. A close correlation was found between the distribution of GR mRNA and the distribution of previously described GR immunoreactivity. These studies open the possibility of obtaining additional information on in vivo regulation of GR synthesis and how the brain may alter its sensitivity to circulating glucocorticoids.     

Localization of insulin receptor mRNA in rat brain by in situ hybridization

Insulin receptor mRNA was demonstrated in rat brain slices by in situ hybridization with three 35S-oligonucleotide probes and contact film autoradiography. Specificity was confirmed by showing that (a) excess unlabeled probe abolished the signal, (b) an oligonucleotide probe for rat neuropeptide Y mRNA showed a different distribution of hybridization signal, and (c) the distribution of insulin receptor binding was consistent with the distribution of insulin receptor mRNA. Insulin receptor mRNA was most abundant in the granule cell layers of the olfactory bulb, cerebellum and dentate gyrus, in the pyramidal cell body layers of the pyriform cortex and hippocampus, in the choroid plexus and in the arcuate nucleus of the hypothalamus.     

Aberrant D1 and D3 dopamine receptor transregulation in hypertension

Dopamine plays a role in the regulation of blood pressure by inhibition of sodium transport in renal proximal tubules (RPTs) and relaxation of vascular smooth muscles. Because dopamine receptors can regulate and interact with each other, we studied the interaction of D(1) and D(3) receptors in immortalized RPT cells and mesenteric arteries from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs), and in human coronary artery smooth muscle cells (CASMCs). In WKY rats, the D(1)-like agonist, fenoldopam, increased D(3) receptor protein in a time-dependent and concentration-dependent manner (EC(50)=4.5x10(-9) M, t(1/2)=15.8 hours). In SHRs, fenoldopam (10(-5) M) actually decreased the expression of D(3) receptors. D(1) and D(3) receptor co-immunoprecipitation was increased by fenoldopam (10(-7) M/24 h) in WKY rats but not in SHRs. The effects of fenoldopam in CASMCs were similar as those in WKY RPT cells (ie, fenoldopam increased D(1) and D(3) receptor proteins). Both D(3) (PD128907, Emax=80%+/-6%, pED(50)=5+/-0.1) and D(1)-like receptor (fenoldopam, Emax=81%+/-8%, pED(50)=5+/-0.2, n=12) agonists relaxed mesenteric arterial rings. Co-stimulation of D(1) and D(3) receptors led to additive vasorelaxation in WKY rats, but not in SHRs. D(1) and D(3) receptors interact differently in WKY and SHRs. Altered interactions between D(1) and D(3) receptors may play a role in the pathogenesis of genetic hypertension, including human hypertension, because these receptors also interact in human vascular smooth muscle cells.     

Distribution of G-protein-coupled receptor (GPCR)135 binding sites and receptor mRNA in the rat brain suggests a role for relaxin-3 in neuroendocrine and sensory processing

G-protein-coupled receptor 135 (GPCR135), a former orphan GPCR also known as SALPR, has recently been shown to be modulated by relaxin-3 (R3). In addition to GPCR135, R3 has been shown to be an agonist for GPCR142 (which is a pseudogene in the rat) and to activate LGR7, which is primarily the receptor for relaxin-1/2. The interaction of R3 with LGR7 has confounded the autoradiographic study of the GPCR135 distribution in the rat CNS due to significant expression of LGR7 in the brain. R3/I5, a chimera of the B-chain of R3 bonded to the A-chain of INSL-5, is a specific GPCR135 agonist which is highly selective for GPCR135 over LGR7. [(125)I]R3/I5 specifically binds to sites on rat brain sections with a pharmacology matching results from membrane preparations of recombinant GPCR135 receptors. Autoradiographic studies show the GPCR135 receptor density is most prominent in areas such as the olfactory bulb, sensory cortex, amygdala, thalamus, paraventricular nucleus, supraoptic nucleus, inferior and superior colliculus. The GPCR135 mRNA distribution generally overlaps the pattern of GPCR135 binding sites shown by autoradiography using [(125)I]R3/I5. The nucleus incertus, which has been implicated in the extrapituitary actions of corticotropin-releasing hormone, is the primary source of R3 in the rat central nervous system and expresses GPCR135 receptors. These binding autoradiography and in situ hybridization data suggest that GPCR135 plays an important role in the central processing of sensory signals in rats, are consistent with a putative role for R3/GPCR135 as modulators of stress responses, and confirm the identity of R3 as the central nervous system ligand for GPCR135.     

Increased abundance of alternatively spliced forms of D2 dopamine receptor mRNA after denervation.

The existence of two molecular forms of D2 dopamine receptors suggests that differences in the distribution or regulation of the two forms could be exploited for the pharmacological treatment of disease. Using probes selective for each alternatively spliced variant of D2 receptor mRNA, we determined that both variants were widely distributed in rat brain and pituitary but that the ratio of the forms varied among regions. mRNA for the 444-amino acid-long variant, D2(444), was the most abundant form in pituitary and neostriatum. Intermediate levels of both D2(444) mRNA and the short form, D2(415), were detected in midbrain, and low levels of D2(444) and D2(415) mRNAs were detected in all other regions examined, including hippocampus, cerebellum, and cortex. The D2(444)/D2(415) ratio was generally lower in the regions of low expression than in pituitary and neostriatum. Dopamine-depleting lesions increased the density of D2 receptors in the denervated neostriatum by 29% without altering the affinity of the receptors for [3H]spiperone. The proliferation of receptors appeared to be due to a lesion-induced increase of up to 120% in the abundance of both variants of mRNA in the neostriatum.     

Expression and localization of human dopamine D2 and D4 receptor mRNA in the adrenal gland, aldosterone-producing adenoma, and pheochromocytoma

Aldosterone secretion is evidently regulated by a dopaminergic inhibitory mechanism. Pharmacological characterization and autoradiographic studies revealed D2-like receptors in the adrenal cortex, especially in the zona glomerulosa. However, the subtype of the dopamine receptors involving this regulation has not been elucidated. To investigate which subtype of receptors expresses in the adrenal cortex, we examined the messages of D2-like receptors, D2, D3, and D4, by RT-PCR and in situ hybridization of adrenal glands and adrenal neoplasm. Both D2 and D4 receptors were expressed in normal adrenal glands, pheochromocytoma, and aldosterone-producing adenoma. However, the D2 receptors were not universally expressed, in contrast with the D4 receptors that were detected in all cases of aldosterone-producing adenoma and adrenal remnant. No D3 receptor message was detected by RT-PCR in any adrenal sample. Both D2 and D4 receptors were expressed in significant amounts in the adrenal medulla and pheochromocytoma. In the adrenal cortex, the expression of the D2 receptors was in the zona glomerulosa and zona reticularis, with no different signal intensities between the two zones. D4 receptors were mainly localized in the zona glomerulosa and, to a lesser extent, in the zona reticularis. Both receptors were expressed at low levels in the zona fasciculata. In aldosterone-producing adenoma, the expression of D2 and D4 was especially found in nonzona fasciculata-like cells. To elucidate which dopamine receptor regulates aldosterone secretion, the effects of specific D2 and D4 antagonists, raclopride and clozapine, respectively, were examined in cultured NCI-H295 cells. Dopamine further increased angiotensin II-induced aldosterone secretion by 20%. In the presence of 1 microM dopamine and angiotensin II, 10(-5)-10(-7) M clozapine decreased aldosterone levels by 40-55%. The decrease in aldosterone secretion by clozapine was completely reversed when raclopride was added simultaneously. These data suggest that dopamine exerts dual effects on aldosterone secretion in NCI-H295 cells. Activation of D4 receptors can increase aldosterone secretion, whereas an inhibitory effect is mediated via D2 receptors. In summary, we demonstrated the existence of both D2 and D4 receptors in the human adrenal gland and adrenal neoplasm. Both receptors play significant roles in the modulation of aldosterone secretion, but in opposite directions.     

D1A, D1B, and D1C dopamine receptors from Xenopus laevis.

Three distinct genes encoding members of the D1 dopamine receptor family were isolated from Xenopus laevis. Based on the deduced amino acid sequence, two of the receptors (Xen D1A and Xen D1B) appear to be homologues of mammalian D1/D1A and D5/D1B receptors. The third receptor, termed Xen D1C, displays equal overall amino acid and nucleotide sequence identity (approximately 55%) with mammalian D1A and D1B/D5 receptors. In agreement with their structural similarities, Xen D1A and D1B receptors, when expressed in COS-7 cells, displayed pharmacological profiles that paralleled those of their mammalian counterparts, with dopamine and 2-amino-6,7-dihydroxytetralin exhibiting 10-fold higher affinity for D1B than for D1A. The Xen D1C receptor displayed an overall rank order of potency and pharmacological profile clearly indicative of a D1-like receptor, with individual affinities for most agonists higher than those for either Xen or mammalian D1/D1A and D5/D1B receptors, whereas antagonist Ki values were intermediate to those for the D1/D1A and D5/D1B receptors. All three receptors stimulated adenylate cyclase activity in response to dopamine or SKF-82526. Xen D1A, D1B, and D1C receptor mRNAs were differentially distributed, with all three receptors expressed in brain and only D1B and D1C receptors expressed in kidney. The existence of a receptor which lacks appreciable overall sequence similarity to, but displays pharmacological homology with, mammalian D1-like receptors lends strong support to the contention that additional mammalian D1-like receptor gene products may exist to allow for the expression of the full spectrum of D1-like dopamine receptor-mediated events.     

Localization and quantification of pro-opiomelanocortin mRNA and glucocorticoid receptor mRNA in pituitaries of suicide victims.

Suicidal behavior has been associated with hypothalamic-pituitary-adrenal overactivity in humans, as measured by increased corticosteroid secretion. To investigate whether this overactivity is reflected at the pituitary level, we have studied the localization of pro-opiomelanocortin (POMC) mRNA, and glucocorticoid receptor (GR) mRNA, in human anterior pituitaries, and quantified these messages relative to controls. Pituitaries from 7 suicide victims and 11 cardiac deaths were sectioned into 10-microns slides, stained with thionin and processed for in situ hybridization using a riboprobe complementary to human POMC mRNA. To correct for possible postmortem cell loss, hybridization with P1B15, a cDNA complementary to rat cyclophillin mRNA, was used in adjacent sections. POMC mRNA containing cells were found to be localized in clusters and were highly associated with corticotropin-releasing hormone (CRH) receptors. In contrast, GR mRNA containing cells were distributed through the pituitary, although areas of increased density were associated with POMC mRNA cells. Quantification with a computerized image analysis system revealed a 25% increase in POMC message in suicide victims. Analysis of the corticotrophic cell clumps showed that the suicide victims had higher POMC mRNA density per cell (p = 0.04) and larger corticotrophic cell size (p = 0.04) than the cardiac death victims. No differences in GR mRNA were detected between the two groups, although GR and POMC mRNA levels were highly and significantly correlated (r = 0.8, p < 0.001). There were no differences in P1B15 message between the two groups. We conclude that in situ hybridization is a useful tool to study gene regulation in human neuroendocrine tissue and that suicide victims show evidence of chronic hypothalamic-pituitary-adrenal axis activation.     

Link between D1 and D2 dopamine receptors is reduced in schizophrenia and Huntington diseased brain.

Dopamine receptor types D1 and D2 can oppose or enhance each other's actions for electrical, biochemical, and psychomotor effects. We report a D1-D2 interaction in homogenized tissue as revealed by ligand binding. D2 agonists lowered the binding of [3H]raclopride to D2 receptors in striatal and anterior pituitary tissues. Pretreating the tissue with the D1-selective antagonist SCH 23390 prevented the agonist-induced decrease in [3H]raclopride binding to D2 sites in the striatum but not in the anterior pituitary, which has no D1 receptors. Conversely, a dopamine-induced reduction in the binding of [3H]SCH 23390 to D1 receptors could be prevented by the D2-selective antagonist eticlopride. Receptor photolabeling experiments confirmed both these D1-D2 interactions. The blocking effect by SCH 23390 was similar to that produced by a nonhydrolyzable guanine nucleotide analogue, and SCH 23390 reduced the number of agonist-labeled D2 receptors in the high-affinity state. Thus, the D1-D2 link may be mediated by guanine nucleotide-binding protein components. The link may underlie D1-D2 interactions influencing behavior, since the link was missing in over half the postmortem striata from patients with schizophrenia and Huntington disease (both diseases that show some hyperdopamine signs) but was present in human control, Alzheimer, and Parkinson striata.