AB1787P Sigma-AldrichAnti-Dopamine D4 Receptor Antibody, extracellular domain

Pharmacological studies suggest that dopamine release from lateral olivocochlear efferent neurons suppresses spontaneous and sound-evoked activity in cochlear nerve fibers and helps control noise-induced excitotoxicity; however, the literature on cochlear expression and localization of dopamine receptors is contradictory. To better characterize cochlear dopaminergic signaling, we studied receptor localization using immunohistochemistry or reverse transcriptase PCR and assessed histopathology, cochlear responses and olivocochlear function in mice with targeted deletion of each of the five receptor subtypes. In normal ears, D1, D2, and D5 receptors were detected in microdissected immature (postnatal days 10-13) spiral ganglion cells and outer hair cells but not inner hair cells. D4 was detected in spiral ganglion cells only. In whole cochlea samples from adults, transcripts for D1, D2, D4, and D5 were present, whereas D3 mRNA was never detected. D1 and D2 immunolabeling was localized to cochlear nerve fibers, near the first nodes of Ranvier (D2) and in the inner spiral bundle region (D1 and D2) where presynaptic olivocochlear terminals are found. No other receptor labeling was consistent. Cochlear function was normal in D3, D4, and D5 knock-outs. D1 and D2 knock-outs showed slight, but significant enhancement and suppression, respectively, of cochlear responses, both in the neural output [auditory brainstem response (ABR) wave 1] and in outer hair cell function [distortion product otoacoustic emissions (DPOAEs)]. Vulnerability to acoustic injury was significantly increased in D2, D4 and D5 lines: D1 could not be tested, and no differences were seen in D3 mutants, consistent with a lack of receptor expression. The increased vulnerability in D2 knock-outs was seen in DPOAEs, suggesting a role for dopamine in the outer hair cell area. In D4 and D5 knock-outs, the increased noise vulnerability was seen only in ABRs, consistent with a role for dopaminergic signaling in minimizing neural damage.

We examined the role of endogenous dopamine (DA) in regulating the number of intrinsic tyrosine hydroxylase-positive (TH(+)) striatal neurons using mice at postnatal day (PND) 4 to 8, a period that corresponds to the developmental peak in the number of these neurons. We adopted the strategy of depleting endogenous DA by a 2-day treatment with α-methyl-p-tyrosine (αMpT, 150 mg/kg, i.p.). This treatment markedly increased the number of striatal TH(+) neurons, assessed by stereological counting, and the increase was highly correlated to the extent of DA loss. Interestingly, TH(+) neurons were found closer to the clusters of DA fibers after DA depletion, indicating that the concentration gradient of extracellular DA critically regulates the distribution of striatal TH(+) neurons. A single i.p. injection of the D1 receptor antagonist, SCH23390 (0.1 mg/kg), the D2/D3 receptor antagonist, raclopride (0.1 mg/kg), or the D4 receptor antagonist, L-745,870 (5 mg/kg) in mice at PND4 also increased the number of TH(+) neurons after 4 days. Treatment with the D1-like receptor agonist SKF38393 (10 mg/kg) or with the D2-like receptor agonist, quinpirole (1 mg/kg) did not change the number of TH(+) neurons. At least the effects of SCH23390 were prevented by a combined treatment with SKF38393. Immunohistochemical analysis indicated that striatal TH(+) neurons expressed D2 and D4 receptors, but not D1 receptors. Moreover, treatment with the α4β2 receptor antagonist dihydro-β-erythroidine (DHβE) (3.2 mg/kg) also increased the number of TH(+) neurons. The evidence that DHβE mimicked the action of SCH23390 in increasing the number of TH(+) neurons supports the hypothesis that activation of D1 receptors controls the number of striatal TH(+) neurons by enhancing the release of acetylcholine. These data demonstrate for the first time that endogenous DA negatively regulates the number of striatal TH(+) neurons by direct and indirect mechanisms mediated by multiple DA receptor subtypes.

Dopaminergic pathways that influence mood and behaviour are severely affected in cerebral hypoxia. In contrast, hypoxia promotes the differentiation of dopaminergic neurons. In order to clarify the hypoxic sensitivity of key dopaminergic genes, we aimed to study their transcriptional regulation in the context of neuroblastoma and astrocytoma cell lines exposed to 1% hypoxia.

The murine D4 dopamine receptor was isolated from a murine genomic DNA library. The receptor's entire coding region was contained within a 6 kb EcoRI genomic fragment, indicating that the murine D4 receptor gene is significantly smaller than the corresponding D2 and D3 receptor genes, the coding regions of which each stretch over 30 kb. The murine D4 receptor gene has three introns and four exons, in common with the rat and human D4 receptor genes. RT-PCR on mRNA from different brain regions shows that the D4 receptor mRNA is expressed in various areas of the brain, with some differences from the rat and human receptor homologues.

Because antipsychotic drugs selectively block dopamine receptors and since dopamine D4 receptors are elevated sixfold in postmortem schizophrenia brain, we searched for possible abnormalities in the coding region of the genomic DNA sequence for the dopamine D4 receptor in control and schizophrenia tissues. The DNA sequence for the first 250 bases of exon 3 of this receptor was examined in the genomic DNA from 296 control individuals and 58 schizophrenics. Twenty-three out of 183 control blacks (12.6%) and 3 out of 24 (12.5%) schizophrenic blacks revealed a replacement of T by G, predicting a substitution of valine by glycine at amino acid position 194. The identical prevalence of 12.5% indicates that the variant is not associated with schizophrenia. The amino acid replacement occurs one amino acid away from a serine amino acid which is critical for the attachment of dopamine. None of the 147 Caucasians (113 controls; 34 schizophrenics) revealed this variant, termed D4GLYCINE194.

Dopamine receptors have been implicated in a variety of neurological and neuropsychiatric disorders. Here we describe the use of the polymerase chain reaction and low stringency library screening to isolate a rat genomic clone encoding a novel dopamine receptor. Sequence data and pharmacological analysis reveal this clone to be the rat analog of the human D4 receptor, which exhibits a high affinity for the antipsychotic drug clozapine. The mRNA for this receptor shows a restricted pattern of expression in the central nervous system. Significant levels of expression were found in the hypothalamus, thalamus, olfactory bulb, and frontal cortex. However, 20-fold higher levels of D4 mRNA expression were observed in the cardiovascular system. Thus, this receptor appears to mediate dopamine function in the cardiovascular system as well as the central nervous system.

The dopamine D4 receptor structurally and pharmacologically resembles the dopamine D2 and D3 receptors. Clozapine, an atypical antipsychotic that is relatively free of the adverse effects of drug-induced parkinsonism and tardive dyskinesia, binds to the D4 receptor with an affinity 10 times higher than to the D2 and D3 receptors. This may explain clozapine's atypical properties. Here we report the existence of at least three polymorphic variations in the coding sequence of the human D4 receptor. A 48-base-pair sequence in the putative third cytoplasmic loop of this receptor exists either as a direct-repeat sequence (D4.2), as a fourfold repeat (D4.4) or as a sevenfold repeat (D4.7). Two more variant alleles were detected in humans. Expression of the complementary DNA for the three cloned receptor variants showed different properties for the long form (D4.7) and the shorter forms (D4.2, D4.4) with respect to clozapine and spiperone binding. To our knowledge, this is the first report of a receptor in the catecholamine receptor family that displays polymorphic variation in the human population. Such variation among humans may underlie individual differences in susceptibility to neuropsychiatric disease and in responsiveness to antipsychotic medication.

Dopamine receptors belong to the family of G protein-coupled receptors. On the basis of the homology between these receptors, three different dopamine receptors (D1, D2, D3) have been cloned. Dopamine receptors are primary targets for drugs used in the treatment of psychomotor disorders such as Parkinson's disease and schizophrenia. In the management of socially withdrawn and treatment-resistant schizophrenics, clozapine is one of the most favoured antipsychotics because it does not cause tardive dyskinesia. Clozapine, however, has dissociation constants for binding to D2 and D3 that are 4 to 30 times the therapeutic free concentration of clozapine in plasma water. This observation suggests the existence of other types of dopamine receptors which are more sensitive to clozapine. Here we report the cloning of a gene that encodes such a receptor (D4). The D4 receptor gene has high homology to the human dopamine D2 and D3 receptor genes. The pharmacological characteristics of this receptor resembles that of the D2 and D3 receptors, but its affinity for clozapine is one order of magnitude higher. Recognition and characterization of this clozapine neuroleptic site may prove useful in the design of new types of drugs.