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Query: UNIPROT:P06889 (Mol)
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Protein phosphorylation systems regulated by cyclic adenosine 3',5'-monophosphate (cyclic AMP), or calcium in conjunction with calmodulin or phospholipid/diacylglycerol, have been studied by phosphorylation in vitro of particulate and soluble fractions from human postmortem brain samples. One-dimensional or two-dimensional gel electrophoretic protein separations were used for analysis. Protein phosphorylation catalyzed by cyclic AMP-dependent protein kinase was found to be highly active in both particulate and soluble preparations throughout the human CNS, with groups of both widely distributed and region-specific substrates being observed in different brain nuclei. Dopamine-innervated parts of the basal ganglia and cerebral cortex contained the phosphoproteins previously observed in rodent basal ganglia. In contrast, calcium/phospholipid-dependent and calcium/calmodulin-dependent protein phosphorylation systems were less prominent in human postmortem brain than in rodent brain, and only a few widely distributed substrates for these protein kinases were found. Protein staining indicated that postmortem proteolysis, particularly of high-molecular-mass proteins, was prominent in deeply located, subcortical regions in the human brain. Our results indicate that it is feasible to use human postmortem brain samples, when obtained under carefully controlled conditions, for qualitative studies on brain protein phosphorylation. Such studies should be of value in studies on human neurological and/or psychiatric disorders.
J Mol Neurosci 1989
PMID:Protein phosphorylation systems in postmortem human brain. 264 Dec 77

Dopamine D1 receptors can be covalently labeled with the photo-affinity ligand (+-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetrah yd ro-1H-3-benzazepine ([125I]IMAB) and visualized following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. In brain membranes, [125I]IMAB labels a polypeptide of apparent Mr approximately equal to 74,000 as the major ligand binding subunit of D1 receptors and two minor polypeptides of Mr approximately equal to 64,000 and 52,000. In contrast, [125I]IMAB labels a single polypeptide of apparent Mr approximately equal to 64,000 in bovine parathyroid glands. In this study, the carbohydrate nature of dopamine D1 receptors from the brain and parathyroid gland were examined using specific exo- and endoglycosidases and lectin affinity chromatography. [125I]IMAB-labeled brain and parathyroid D1 receptors were sensitive to treatment with the exoglycosidases neuraminidase or alpha-mannosidase, suggestive of the existence of terminal sialic acid and oligomannose residues. Photolabeled D1 receptor polypeptides are not however, associated with distinct populations of complex-type or high mannose-containing carbohydrate chains because 1) wheat germ agglutinin and concanavalin A lectin chromatography of solubilized and photolabeled neuronal D1 receptors followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed no differences in the electrophoretic mobility of column pass-through and specifically eluted [125I]IMAB-labeled polypeptides, and 2) [125I]IMAB-labeled D1 receptors specifically bound to and eluted from concanavalin A-Sepharose were neuraminidase sensitive, indicative of the colocalization of oligomannose- and complex-type glycans. Removal of these terminal glycan residues did not affect the binding of [3H]SCH 23390 to dopamine D1 receptors. Complete N-linked deglycosylation of photolabeled D1 receptors from both the brain and parathyroid with peptide N-glycosidase F resulted in the migration of a single major labeled polypeptide of apparent Mr approximately equal to 46,000. These data suggest that, despite differences observed in the electrophoretic mobility and glycosylation patterns of brain and parathyroid D1 receptor polypeptides, the protein backbones of central and peripheral dopamine D1 receptors display similar if not identical molecular weights.
Mol Pharmacol 1989 Oct
PMID:Glycoprotein nature of dopamine D1 receptors in the brain and parathyroid gland. 268 4

In vitro and in vivo receptor-binding properties of the new serotonin antagonist, ritanserin, are reported. In in vitro binding assays, ritanserin shows high affinity binding to serotonin-S2 sites in rat frontal cortex tissue: IC50 = 0.9 nM without drug preincubation and 0.3 nM with 30-min drug preincubation; IC50 values for histamine-H1, dopamine-D2, and adrenergic-alpha 1 and -alpha 2 sites were 39-, 77-, 107-, and 166-fold higher, and at up to 1 microM, the drug did not bind to serotonin-S1 sites. In in vitro assays, ritanserin dissociated very slowly from serotonin-S2 (t1/2 = 160 min) and histamine-H1 sites (t1/2 = 77 min) and rapidly from dopamine-D2 sites (t1/2 = 11 min). Half-times of dissociation from adrenergic-alpha 1 and -alpha 2 sites were 18 and 26 min. The inhibition by ritanserin of [3H]ketanserin binding was found to be partially noncompetitive and the inhibitory potency increased with drug preincubation. Due to the slow dissociation of ritanserin from the serotonin-S2 sites, the drug cannot be displaced completely by [3H]ketanserin. In contrast, inhibition by ritanserin of [3H]haloperidol binding to dopamine-D2 sites in rat striatum was fully competitive, in agreement with the rapid dissociation of the drug from the latter sites. In ex vivo binding assays using brain areas of rats and guinea pigs treated subcutaneously with ritanserin, occupation of serotonin-S2 sites was observed at very low dosage (50% occupation at 0.08-0.1 mg/kg) and sites remained occupied during a prolonged time period (greater than 70% occupation up to 48 hr after 2.5 mg/kg ritanserin). Histamine-H1 receptor sites in guinea pig cerebellum became occupied at dosages 25-fold higher than the dosage producing occupation of frontal cortical serotonin-S2 sites. Dopamine-D2 sites in rat striatum and cortical adrenergic-alpha 1 sites became only slightly occupied (less than 20%) at higher dosages and the effect was not dose-dependent. Adrenergic-alpha 2 sites were not occupied up to doses of 160 mg/kg given subcutaneously. In vivo binding assays using [3H]spiperone confirmed the occupation of frontal cortical serotonin-S2 sites following low dosage of ritanserin and a minor occupation of striatal dopamine-D2 sites. Levels of dopamine and serotonin and their metabolites remained unchanged in brain areas of rats orally treated with ritanserin up to dosages of 40 mg/kg. At 160 mg/kg, there seemed to be a slight reduction in dopamine and serotonin content.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1985 Jun
PMID:Receptor-binding properties in vitro and in vivo of ritanserin: A very potent and long acting serotonin-S2 antagonist. 286 May 58

Forskolin, an activator of adenylate cyclase, has been used to investigate the effects of raising pituitary cell cyclic AMP concentrations on prolactin and growth hormone secretion and to examine the role of cyclic AMP in the inhibitory actions of dopamine and somatostatin. Incubation of cultured ovine pituitary cells with forskolin (0.1-10 microM; 30 min) produced a modest dose-related increase in prolactin release (120-140% of basal) but a much greater stimulation of growth hormone secretion (170-420% of basal). Cellular cyclic AMP concentrations were only increased in the presence of 1 and 10 microM forskolin (2-5.5 times basal). A study of the time course for forskolin (10 microM) action showed that stimulation of prolactin (1.5-fold) and growth hormone (4.7-fold) secretion occurred over 15 min; subsequently (15-60 min) the rate of prolactin secretion from forskolin-treated cells was equivalent to that measured in controls, while growth hormone release remained elevated. Cellular cyclic AMP concentrations were also rapidly stimulated by forskolin (10 microM); they reached a maximum (12 times control) within 15 min, and then declined (15-60 min) but remained elevated relative to those in untreated cells (4.9 times control at 60 min). Dopamine (0.1 microM) inhibited basal secretion of both prolactin and growth hormone. In the presence of forskolin (0.1-10 microM), dopamine (0.1 microM) inhibited prolactin secretion to below the basal level and considerably attenuated the stimulation of growth hormone secretion. Similarly, somatostatin suppressed both basal and forskolin-induced prolactin and growth hormone secretion. However, neither dopamine nor somatostatin significantly decreased the stimulatory effect of forskolin on cellular cyclic AMP accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1986 May
PMID:Dopamine and somatostatin inhibit forskolin-stimulated prolactin and growth hormone secretion but not stimulated cyclic AMP levels in sheep anterior pituitary cell cultures. 287 92

PC12 cells on extracellular matrix (ECM) or plastic were incubated with 3H-tyrosine (3H-TY) in the presence and absence of serum or cold tyrosine. 3H-Dopamine (3H-DA) was determined in medium and cells from 1 to 48 h later with Dowex cation exchange chromatography. In serum-free and tyrosine-free medium, PC12 cells on ECM released significantly more 3H-DA, whereas cells on plastic had a significantly higher cellular content of 3H-DA, but total 3H-DA (medium plus cells) was equal in ECM and plastic cultures. When 3H-TY was added to tyrosine-containing medium, there was a significant decrease in the levels of 3H-DA detected and the differences between ECM and plastic cultures were attenuated, but the patterns of secretion and storage were similar to those observed with tyrosine-free medium and total synthesis did not decline at 48 h. Serum decreased the efficiency of the resin to retain 3H-DA from culture medium, attenuated the difference in dopamine release between ECM and plastic cultures, and contributed to variations in 3H-TY uptake. The morphometric relationship between the cell membrane and the internal compartment in PC12 cells of different shapes was also characterized. The perimeter length and area of the midsection of cells were determined with a modular system for quantitative digital analysis. The perimeter length of cells on ECM was significantly greater than cells on plastic, whereas the internal areas were similar. The ratio of perimeter length to area (P/A) for all cells on ECM was 30% higher than the P/A ratio for cells on plastic. The ratio of P/A for a subpopulation of very flat cells on ECM was 70% higher than the ratio for round cells on plastic. Immunocytochemistry for tyrosine hydroxylase revealed a more diffuse distribution of this enzyme in cells on ECM. These data suggest that there is an increase in the ratio of cell surface area to cell volume as PC12 cells spread on ECM which could facilitate secretory vesicle fusion with the cell membrane, and hence, exocytosis. Although there is a concomitant increase in the secretion of dopamine and a decrease in the storage of dopamine, the change in cell shape does not appear to immediately alter the synthesis of dopamine.
Mol Cell Endocrinol 1987 Nov
PMID:Extracellular matrix changes PC12 cell shape and processing of newly synthesized dopamine. 289 May 44

Forskolin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate stimulate prolactin and GH release from ovine anterior pituitary cells cultured in vitro. Dopamine and somatostatin inhibit release of prolactin and GH respectively, after stimulation by these agents, but without effects on intracellular cyclic AMP concentrations. In each case the inhibitory effects were reversed by pretreatment of cells with pertussis toxin, in a dose-related fashion (1-100 ng/ml), again without affecting cyclic AMP levels. The results suggest that the inhibitory effects of dopamine and somatostatin in this system are mediated by one or more pertussis toxin-sensitive G proteins, and that these act by a mechanism which does not involve inhibition of adenylate cyclase.
J Mol Endocrinol 1988 Nov
PMID:Actions of pertussis toxin on the inhibitory effects of dopamine and somatostatin on prolactin and growth hormone release from ovine anterior pituitary cells. 290 33

The ability of dopamine agonists and antagonists to compete with [3H]spiperone binding to rat striatal membrane preparations at 4, 15, 26, and 37 degrees varied markedly with temperature. Dopamine and the dopamine agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (ADTN) were more potent at lower temperatures. The ability of the dopamine antagonists, haloperidol, cis-flupenthixol, cis-N-(1-benzyl-1-methypyrrolidin-3-yl)-5-chloro-2-methoxy-9- methylaminobenzamide (YM 09151-2), raclopride, and clozapine, and of the agonists apomorphine and pergolide, to compete with [3H]spiperone binding was little altered by temperature. (+)-Butaclamol was more potent at higher temperatures. In contrast, the antagonists sulpiride, metoclopramide, clebopride, sultopride, tiapride, piquindone, and zetidoline were more potent at lower temperatures. The interaction of the agonists dopamine and ADTN was driven by a decrease in enthalpy, allowing an energetically unfavorable decrease in entropy. The binding of the antagonists, haloperidol, cis-flupenthixol, YM 09151-2, raclopride, (+)-butaclamol, and clozapine, and also of the agonists, apomorphine and pergolide, was entropy driven. The interaction of the antagonists sulpiride, metoclopramide, clebopride, alizapride, sultopride, tiapride, piquindone, and zetidoline differed from that of other antagonists in being enthalpy driven. The observed entropy changes correlated with the lipophilicity of the displacing drugs and not with their intrinsic activity.
Mol Pharmacol 1986 Sep
PMID:The thermodynamics of agonist and antagonist binding to dopamine D-2 receptors. 294 80

Dopamine (DA) regulation of intracellular cyclic AMP formation in purified, intact striatal neurons in primary culture was examined. DA (EC50, 3 microM) and vasoactive intestinal polypeptide (VIP; EC50, 10 nM) stimulated cyclic AMP formation by 2- and 5-fold, respectively. In the presence of 0.1 microM forskolin (which was virtually ineffective alone), neurohormone efficacy was augmented; potency was unaffected. In the presence of 0.1 microM SCH 23390, a selective D1 antagonist, the DA dose-response curve was shifted rightward in a competitive manner. At low concentrations (0.01-1.0 microM), however, DA inhibited basal cyclic AMP formation. The inhibitory effect, but not the shift of the dose-response curve, was blocked by 5 microM l-sulpiride, a selective D2 antagonist. At saturating concentrations of VIP (0.1-1.0 microM), no other neurohormone can further augment cyclic AMP formation. Under these conditions, increasing concentrations of DA resulted in a dose-dependent (IC50, 0.5 microM) inhibition of VIP-stimulated cyclic AMP synthesis. This effect was augmented in the presence of 0.1 microM SCH 23390 and blocked by 5 microM l-sulpiride. Sulpiride antagonism was stereospecific, with the l-isomer being 30-fold more potent than the d-isomer. The rank order of potency for a series of dopaminergic agonists and antagonists at the receptor mediating attenuation of cyclic AMP formation suggests that it is of the D2 type. Furthermore, both DA and Met-enkephalin inhibition of cyclic AMP formation is lost after exposure of striatal neurons to islet activator protein. These findings suggest that a D2 receptor mediates the inhibition of intracellular cyclic AMP formation by DA in striatal neurons in primary culture, and may do so by an interaction with the inhibitory guanine nucleotide regulatory protein of adenylate cyclase.
Mol Pharmacol 1985 Jun
PMID:D2-dopamine receptor-mediated inhibition of cyclic AMP formation in striatal neurons in primary culture. 298 58

Opioid receptors and enkephalinergic neurons in the central nervous system of Mytilus edulis have been reported. Also known is that the lateral epithelium of the gill is innervated by serotonergic, cilioexcitatory neurons and dopaminergic, cilioinhibitory neurons. The aim of the present report is to look for an effect of opioid agonists on the nervous control of the lateral cilia. Dopamine applied to the cerebral ganglion inhibited the activity of lateral cilia in the gill. This effect was blocked by the application of several opioids to the visceral ganglion. The block was reversed by the application of naloxone to the visceral ganglion. Dopamine applied to the visceral ganglion also inhibited lateral ciliary activity as shown earlier. Opioids applied to the visceral ganglion partially blocked this effect but this was overcome by higher concentrations of dopamine. Preparations with low endogenous rates of ciliary beating were stimulated by the application of opioids to the visceral ganglion. Naloxone blocked this effect. Preparations with high endogenous rates of ciliary beating were inhibited by the application of naloxone to the visceral ganglion. Electrical stimulation of the cerebrovisceral connective produced excitatory and inhibitory effects depending on the rate of stimulation. Morphine applied to the visceral ganglion diminished the cilioinhibitory effects and enhanced the cilioexcitatory effects of electrical stimulation. Morphine applied to the gill had no effect on the cilioinhibitory action of dopamine applied to the visceral ganglion. There was no observable effect of opioids applied to the gill and no alteration in the cilioinhibitory effect of dopamine or the cilioexcitatory effect of serotonin applied directly to the gill in the presence of opioids. Specific opioid binding sites were found in the visceral ganglion but were not found in gill, palp, mantle, or visceral mass tissue. A dopamine-stimulated adenylate cyclase activity was again found in the visceral ganglion and the gill. Etorphine reduced the dopamine stimulation of cyclase in the ganglion but not in the gill. It is postulated that a cilioinhibitory, dopaminergic mechanism includes nerves running from the cerebral ganglion to the gill with synaptic transmission in the visceral ganglion that can be modulated by opioids.
Cell Mol Neurobiol 1986 Mar
PMID:An opioid mechanism modulates central and not peripheral dopaminergic control of ciliary activity in the marine mussel Mytilus edulis. 301 5

PC12 cells on plastic grow as a single-layered lawn of cells which synthesize, store and secrete dopamine. In contrast, PC12 cells cultured on Englebreth-Holm-Swarm (EHS) tumor-derived extracellular matrix grow into multicellular aggregates. Matrix dissolution and cell migration appear to follow aggregate formation. PC12 cell plating efficiency is decreased on EHS-matrix but the doubling time of cells on EHS-matrix is comparable to plastic. Dopamine secretion and cellular content determined with a radioenzymatic assay as well as dopamine synthesis determined with cation-exchange chromatography are similar on a per cell basis in cultures of PC12 cells on plastic and EHS-matrix.
Mol Cell Endocrinol 1988 Aug
PMID:PC12 cell aggregation and dopamine production on EHS-derived extracellular matrix. 306 56


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