Differential coupling of dopaminergic D2 receptors expressed in different cell types. Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in LtK- fibroblasts, hyperpolarization, and cytosolic-free Ca2+ concentration decrease in GH4C1 cells.

Dopaminergic D2 receptors are widely regarded as typical inhibitory receptors, as they both inhibit adenylyl cyclase and decrease the cytosolic free Ca2+ concentration ([Ca2+]i) by activating K+ channels. A D2 receptor has recently been cloned (Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M. D., Machida, C. A., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787) and expressed in two different cell lines, pituitary GH4C1 cells and Ltk- fibroblasts, where it has been shown to induce inhibition of adenylyl cyclase. We have investigated the additional effector systems coupled to this receptor. The responses observed in the two cells lines, which express similar levels of receptors (0.5-1 x 10(5)/cell), were surprisingly different. In GH4C1 cells D2 receptors failed to affect phosphoinositide hydrolysis and induced a decrease of [Ca2+]i. This latter effect appears to be mediated by hyperpolarization, most likely due to the activation of K+ channels. In striking contrast, in Ltk- fibroblasts the D2 receptor induced a rapid stimulation of inositol(1,4,5)-trisphosphate (+73% at 15 s) followed by the other inositol phosphates, and an immediate increase of [Ca2+]i due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. In both GH4C1 and Ltk- cells, the D2 receptor response was mediated by G protein(s) sensitive to pertussis toxin. The increases of inositol trisphosphate and [Ca2+]i observed in Ltk- cells required dopamine concentrations only slightly higher than those inhibiting adenylyl cyclase (EG50 = 25, 29, and 11 nM, respectively) and were comparable in magnitude to the responses induced by the endogenous stimulatory receptor agonists, thrombin and ATP. The results demonstrate that in certain cells D2 receptors are efficiently coupled to the stimulation of phosphoinositide hydrolysis. The nature of receptor responses appears therefore to depend on the specific properties not only of the receptor molecule but also of the cell type in which it is expressed

Dopamine D 4 Receptor-Deficient Mice Display Cortical Hyperexcitability

The dopamine D(4) receptor (D(4)R) is predominantly expressed in the frontal cortex (FC), a brain region that receives dense input from midbrain dopamine (DA) neurons and is associated with cognitive and emotional processes. However, the physiological significance of this dopamine receptor subtype has been difficult to explore because of the slow development of D(4)R agonists and antagonists the selectivity and efficacy of which have been rigorously demonstrated in vivo. We have attempted to overcome this limitation by taking a multidimensional approach to the characterization of mice completely deficient in this receptor subtype. Electrophysiological current and voltage-clamp recordings were performed in cortical pyramidal neurons from wild-type and D(4)R-deficient mice. The frequency of spontaneous synaptic activity and the frequency and duration of paroxysmal discharges induced by epileptogenic agents were increased in mutant mice. Enhanced synaptic activity was also observed in brain slices of wild-type mice incubated in the presence of the selective D(4)R antagonist PNU-101387G. Consistent with greater electrophysiological activity, nerve terminal glutamate density associated with asymmetrical synaptic contacts within layer VI of the motor cortex was reduced in mutant neurons. Taken together, these results suggest that the D(4)R can function as an inhibitory modulator of glutamate activity in the FC.Fil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Cepeda, Carlos. University of California at Los Angeles; Estados UnidosFil: Hurst, Raymond S.. University of California at Los Angeles; Estados UnidosFil: Flores Hernandez, Jorge. University of California at Los Angeles; Estados UnidosFil: Ariano, Marjorie A.. The Chicago Medical School; Estados UnidosFil: Falzone, Tomas Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Kozell, Laura B.. Oregon Health Sciences University; Estados UnidosFil: Meshul, Charles K.. Oregon Health Sciences University; Estados UnidosFil: Bunzow, James R.. Oregon Health Sciences University; Estados UnidosFil: Low, Malcolm J.. Oregon Health Sciences University; Estados UnidosFil: Levine, Michael S.. University of California at Los Angeles; Estados UnidosFil: Grandy, David K.. Oregon Health Sciences University; Estados Unido

Distinct regulation of dopamine D2S and D2L autoreceptor signaling by calcium

D2 autoreceptors regulate dopamine release throughout the brain. Two isoforms of the D2 receptor, D2S and D2L, are expressed in midbrain dopamine neurons. Differential roles of these isoforms as autoreceptors are poorly understood. By virally expressing the isoforms in dopamine neurons of D2 receptor knockout mice, this study assessed the calcium-dependence and drug-induced plasticity of D2S and D2L receptor-dependent G protein-coupled inwardly rectifying potassium (GIRK) currents. The results reveal that D2S, but not D2L receptors, exhibited calcium-dependent desensitization similar to that exhibited by endogenous autoreceptors. Two pathways of calcium signaling that regulated D2 autoreceptor-dependent GIRK signaling were identified, which distinctly affected desensitization and the magnitude of D2S and D2L receptor-dependent GIRK currents. Previous in vivo cocaine exposure removed calcium-dependent D2 autoreceptor desensitization in wild type, but not D2S-only mice. Thus, expression of D2S as the exclusive autoreceptor was insufficient for cocaine-induced plasticity, implying a functional role for the co-expression of D2S and D2L autoreceptors

Molecular cloning and tissue distribution of a putative member of the rat opioid receptor gene family that is not a μ, δ or κ opioid receptor type

AbstractA novel G protein-coupled receptor was cloned by PCR and homology screening. Its deduced amino acid sequence is 47% identical overall to the μ, δ and κ opioid receptors and 64% identical in the putative transmembrane domains. When transiently expressed in COS-7 cells this receptor did not bind any of the typical μ, δ or κ opioid receptor ligands with high affinity. In situ hybridization analysis revealed that LC132 mRNA is highly expressed in several rat brain areas, including the cerebral cortex, thalamus, subfornical organ, habenula, hypothalamus, central gray, dorsal raphe, locus coeruleus and the dorsal horn of the spinal cord. Based on this distribution and its high homology with the μ, δ and κ opioid receptors, it is proposed that LC132 is a new member of the opioid receptor family that is involved in analgesia and the perception of pain

The distribution of dopamine D2 receptor heteronuclear RNA (hnRNA) in the rat brain

Conventional in situ hybridization methods have been useful in characterizing the anatomical distribution of cells in the central nervous system that express dopamine D2 receptor mRNA. However, due to the large size of the D2 mRNA pool, this method may be insensitive to changes in D2 gene transcription. We have developed a method of hybridizing a 35S-labelled cRNA probe to an intron in the D2 receptor gene in order to measure the amount of primary transcript or heteronuclear RNA (hnRNA) in D2-expressing cells. Introns are found uniquely in hnRNA and are thought to be short-lived intermediates. Thus, monitoring introns could represent a more direct measure of D2 gene transcription. The anatomical distribution of the D2 hnRNA is similar to the distribution of D2 mRNA in the rat brain. D2 heteronuclear RNA was found in the nuclei of cells in the caudate putamen, nucleus accumbens, hippocampus, olfactory tubercle, substantia nigra, ventral tegmental area, and zona incerta. Other regions that contain D2 mRNA, but do not demonstrate intronic signal, include the globus pallidus, prefrontal, cingulate, entorhinal, and piriform cortex, septum, and amygdala. However, these areas have low amounts of D2 mRNA and may contain levels of D2 hnRNA that are below detection. Heteronuclear RNA quantitation by solution hybridization followed by RNase protection was performed on striatum, substantia nigra, cerebral cortex, hippocampus, hypothalamus, and pituitary using a D2 intron 7/exon 8 border probe. These results corroborate the distribution of hnRNA revealed with intronic in situ hybridization. In addition, protection assays were able to detect hnRNA in areas that express low levels of D2 like the cortex, hippocampus and hypothalamus. hnRNA/mRNA ratios calculated from intron/exon border probe protection assays were not equivalent for all the tissue areas studied, indicating that transcription and/or hnRNA half lives may differ between tissues that express D2 receptors. The combined use of intronic in situ hybridization and intron/exon border protection assay as an index of D2 gene transcription and RNA processing provides more information than measuring the mRNA pool alone. It may also prove to be a more useful measure of gene regulation, allowing for evaluation of gene responses to acute treatments.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30480/1/0000108.pd