During the 1980s, indications for the existence of intramembrane interactions between different G protein-coupled receptors, mainly between neuropeptide and monoamine receptors, were obtained in several brain areas (1, 2). It was later proposed that a possible molecular mechanism for this phenomenon was receptor heteromerization (3) and direct evidence for homo-and heteromerization of G protein-coupled receptors has been obtained by several groups. It was first shown that serotonin 5-HT-1B receptors exist as monomers and dimers (4). This was followed by demonstration of dimers and oligomers of dopamine D 1 and D 2 receptors (D 1 and D 2 R) in transfected Sf cells (5-7) and of adenosine A 1 receptors (A 1 Rs) in a natural cell line and in mammalian brain (8). It has recently been reported that a fully functional ␥-aminobutyric acid (GABA) type B receptor demands the heterodimerization of GABA B R1 and GABA B R2 receptors (9-12). Moreover, two functional opioid receptors, the and ␦ subtypes, can undergo heteromerization, which changes the pharmacology of the individual receptors and potentiates signal transduction (13). Finally, D 2 R and somatostatin receptor subtype 5 have been shown to physically interact by forming heterooligomers with enhanced functional activity (14). Direct protein-protein coupling can also exist between G proteincoupled anion channel receptors, as recently shown for dopamine D5 receptor and GABA A receptor, making possible bilateral inhibitory interactions between these receptors (15).Antagonistic adenosine͞dopamine interactions have been widely reported in the central nervous system in behavioral and biochemical studies. Furthermore, in animal models, adenosine agonists and antagonists are potent atypical neuroleptics and antiparkinsonian drugs, respectively (16-18). Thus, adenosine agonists inhibit and adenosine antagonists, such as caffeine, potentiate the behavioral effects induced by dopamine agonists. The evidence suggests that this antagonism is at least in part caused by an intramembrane interaction between specific subtypes of dopamine and adenosine receptors, namely, between

Abstract:The effects of depolarization by elevated potassium concentrations were studied in PC12 cells and in stably transfected AtT-20 cells expressing wild-type or [Leu19] -recombinant tyrosine hydroxylase (rTH). Changes in the phosphorylation states of Ser 19 and Ser4°i n tyrosine hydroxylase (TH) were determined immunochemically using antibodies specific for the phosphorylated state of each site and compared with changes in TH activity in PC12 cell lysates and with changes in L-DOPA biosynthesis rates in intact AtT-20 cells. Treatment of either PCi 2 cells or AtT-20 cells expressing wildtype rTH with elevated potassium produced a transient increase in the phosphorylation state of Ser19 (up to 0.7 mol of phosphate/mol of subunit) in concert with a more gradual and sustained increase in Ser4°phosphorylation. Elevated potassium treatment also increased TH activity in PC12 cell lysates, but these increases paralleled the temporal course of Ser40, as opposed to Ser19, phosphorylation. Similarly, increases in DOPA accumulation produced by elevated potassium in AtT-20 cells expressing wild-type rTH paralleled the increases in the phosphorylation state of Ser4°but not Ser19. Moreover, elevated potassium produced comparable increases in DOPA accumulation in AtT-20 cells expressing rTH in which Ser19 phosphorylation had been eliminated (by substitution of Leu for Ser19). Thus, depolarization-induced increases in the stoichiometry of Ser19 phosphorylation do not appear to influence directly the activity of TH in situ.

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Background Ara h 2 and Ara h 6, co-purified together in a 13-25 kD fraction (Ara h 2/6; 20 kD fraction) on gel filtration chromatography, account for the majority of effector activity in a crude peanut extract (CPE) when assayed with RBL SX-38 cells sensitized with IgE from human peanut allergic sera. Objectives To determine if Ara h 2/6 are the primary peanut allergens responsible for allergic reactions in vivo and to determine if Ara h 2/6 would be sufficient to prevent allergic reactions to a complete CPE. Methods An oral sensitization mouse model of peanut allergy was used to assess the activity of Ara h 2/6 (20 kD) and CPE without the 20 kD fraction (CPE w/o 20 kD) for allergic provocation challenge and immunotherapy. The activity of these preparations was also tested in an assay of histamine release from human basophils in whole blood. Results Compared to mice challenged with control CPE, mice challenged with CPE w/o 20 kD experienced reduced symptoms (p<0.05) and a smaller decrease in body temperature (p<0.01). Results with the basophil histamine release assay corroborated these findings (p<0.01). The mouse model was also used to administer Ara h 2/6 (20 kD) in an immunotherapy protocol, in which peanut-allergic mice treated with the 20 kD fraction experienced significantly reduced symptoms, changes in body temperature, and mast cell protease (MMCP-1) release compared to placebo (p<0.01 for all parameters). Importantly, immunotherapy with the 20 kD fraction was just as effective as treatment with CPE, whereas CPE w/o 20 kD was significantly less effective for higher dose peanut challenges. Conclusions and Clinical Relevance Ara h 2/6 are the most potent peanut allergens in vivo and can be used to desensitize peanut-allergic mice. These results have potential implications for clinical research in the areas of diagnosis and immunotherapy for peanut allergy.

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Activation of striatal dopamine (DA) neurons by neuroleptic treatment or by electrical stimulation of the nigrostriatal pathway increases the activity of tyrosine hydroxylase (TH). The increase is mediated by phosphorylation of the enzyme. However, abolition of DA neuronal activity [by gamma-butyrolactone (GBL) treatment or transection of the nigrostriatal pathway] also increases TH activity. Quantitative blot immunolabeling experiments using site- and phosphorylation state-specific antibodies to TH demonstrated that GBL treatment (750 mg/kg, 35 min) significantly increased phosphorylation at Ser19 (+40%) and Ser40 (+217%) without altering Ser31 phosphorylation. Concomitantly, GBL treatment [along with the 3,4-dihydroxyphenylalanine (dopa) decarboxylase inhibitor NSD-1015, 100 mg/kg, 30 min] increased in vivo striatal dopa accumulation and in vitro TH activity 3-fold. Likewise, cerebral hemitransection of the nigrostriatal pathway significantly increased phosphorylation of TH at Ser19 (+89%) and Ser40 (+158%) but not at Ser31; dopa levels were increased accordingly (+191%). Kinetic analysis of TH activity established that GBL treatment and hemitransection primarily decreased the Km for the cofactor tetrahydrobiopterin (3-fold). The effects of GBL and hemitransection were abolished or attenuated by pretreatment with the DA agonist R-(-)-N-n-propylnorapomorphine (NPA; 30 microgram/kg, 40 min), presumably via stimulation of inhibitory presynaptic DA autoreceptors. NPA dose-response curves for reversal of GBL-induced dopa accumulation and Ser40 phosphorylation were identical; however, only the highest dose of NPA reversed the small and variable increase in Ser19 phosphorylation. Thus, TH activity seems to be regulated by phosphorylation in both hyper- and hypoactive striatal DA neurons; in the latter case, activation seems to be caused by selective phosphorylation of Ser40.

The activity of pergolide, an N-propylergoline derivative, has been tested for stimulation of central dopaminergic receptors. Binding to dopamine receptors shows that pergolide acts as an agonist with respect to these receptors. GTP decreases the potencies of dopamine agonists and of pergolide, It is now established that the major abnormality in parkinsonism is the degeneration of the nigrostriatal 3,4-dihydroxyphenylethylamine (dopamine) neurons. The effectiveness of 3,4-dihydroxyphenylalanine (L-dopa) in parklnsonism is dependent on the capacity of the remaining nigrostriatal dopamine neurons to synthesize dopamine from administered L-dopa. It has become apparent that therapeutic response diminishes after prolonged treatment with L-dopa (1, 2). This decrease might be due to progressive degeneration of the nigrostriatal dopamine neurons or to a decreased sensitivity of dopamine receptors in the striatum. It was therefore of interest to investigate the effectiveness of drugs that directly stimulate dopamine receptors in the brain. Among various dopamine agonists tested, certain ergot alkaloids were found to stimulate dopamine receptors (3, 4), and their therapeutic effectiveness in parkinsonism was investigated (5, 6).We now describe dopamine agonist properties of a semisynthetic ergoline derivative, pergolide (8fl-[8-(methylthio)-methyl]-6-propylergoline), tested both in vitro and in two animal models that simulate certain features of parkinsonism. The potency of pergolide was compared with that of bromocriptine (2-bromo-a-ergocriptine), another ergot derivative currently used in the treatment of parkinsonism (7,8). The long duration of action of pergolide as an agonist (9, 10) prompted us to investigate its therapeutic effectiveness in parkinsonism patients. Preliminary reports on this study have been presented (9,11,12). MATERIALS AND METHODSNPA) (75 Ci/nmol) were purchased from New England Nuclear (1 Ci = 3.7 X 1010 becquerels). Pergolide was a gift from Eli Lilly, and bromocriptine from Sandoz Pharmaceutical.Binding Assay. Preparation of bovine or rat membranes and the ensuing binding assay were carried out as described (13). For routine assay each tube contained 1.8 ml. of membrane suspension (5 mg of wet tissue), 0. Values for the mean inhibitory concentration, ICso, were derived by log probit analysis. Values for the dissociation constant, Kd, for each radioactive ligand were determined from the corresponding Scatchard plots, and values for the inhibitor constant, Ki, were determined according to the equation Ki =