p-ethynylphenylalanine: a potent inhibitor of tryptophan hydroxylase.

Tryptophan hydroxylase (TPH) is the initial and rate-limiting enzyme in serotonin biosynthesis. The enzyme activity is dependent on molecular oxygen, a tetrahydropterin cosubstrate, and ferrous iron. The present study demonstrates that TPH is inhibited by a novel compound, p-ethynylphenylalanine (pEPA), produced by the Heck reaction of trimethylsilylacetylene with N-tertbutyloxycarbonyl-4-iodo-L-phenylalanine methyl ester. pEPA is a more potent and specific inhibitor of TPH than p-chlorophenylalanine (pCPA). In the present study, pEPA was demonstrated to inhibit competitively and reversibly TPH in vitro (Ki = 32.6 +/- 6.2 microM vs. tryptophan). pEPA displayed little inhibitory activity toward tyrosine hydroxylase (EC 1.14.16.2), the initial and rate-limiting enzyme for catecholamine biosynthesis, and no inhibition of phenylalanine hydroxylase or tyrosinase. In addition, pEPA was a poor ligand for the serotonin transporter and several serotonin receptors. Administration of pEPA (30 mg/kg) to rats produced a 95 +/- 5% decrease in TPH activity in brain homogenates and a concomitant decrease in serotonin and 5-hydroxyindole-3-acetic acid levels (85%) at 24 h after injection. In contrast, pCPA produced a similar effect (87 +/- 5% decrease in TPH activity) only at 10 times the concentration (300 mg/kg). These results suggest that pEPA is a selective, reversible, and potent inhibitor of TPH both in vitro and in vivo. The potential for pEPA to inhibit selectively and reversibly the biosynthesis of serotonin may contribute to the characterization of the role of serotonin in behavioral and physiological activities.

Expression of the human 5-hydroxytryptamine1A receptor in Sf9 cells. Reconstitution of a coupled phenotype by co-expression of mammalian G protein subunits.

The possibility that Spodoptera frugiperda (Sf9) cells can provide an intact cell setting for reconstitution of the human 5-hydroxytryptamine1A (5-HT1A) receptor with mammalian G protein subunits was explored. The 5-HT1A receptor was found to assume an uncoupled phenotype when expressed alone in Sf9 cells at relatively high levels (5-34 pmol of receptor/mg of membrane protein), i.e. agonist-binding to the receptor was characterized by a relatively high Kd and an insensitivity to GTP. Co-expression of the receptor with members of the alpha i "family" together with various combinations of beta 1 and gamma subunits increased the affinity for agonists to that observed for the coupled form of receptor in mammalian cells, concomitant with conferrance of guanosine 5'-(beta,gamma-imino)triphosphate sensitivity. The agonists employed were [3H]8-hydroxy-N,N-dipropyl-2-aminotetralin ([3H]8-OH-DPAT) and [125I]R(+)-trans-8-hydroxy-2-[N-n-propyl-N-(3'-iodo-2'-propenyl) amino]tetralin ([125I]8-OH-PIPAT). The binding of an antagonist, [125I]4-(2'-methoxyphenyl)-1-[2'-[N-(2"- pyridinyl)-p-iodobenzamido]ethyl]piperazine ([125I]p-MPPI), was unaffected by co-expression of G protein subunits. Both alpha and beta gamma subunits were required for optimal coupling. No differences were evident among alpha i1, alpha i2, alpha i3, alpha o, and alpha z when expressed with beta 1 gamma 2 in this regard, nor among most permutations of beta 1 gamma subunits when expressed with alpha i1 (beta 1 gamma 2 approximately beta 1 gamma 3 approximately beta 1 gamma 5 > beta 1 gamma 1). Alpha s and alpha q expressed with beta 1 gamma 2 did not participate in coupling. These data support the conclusion that normal interactions between a mammalian receptor and a select array of G proteins can be established in intact Sf9 cells, and extend previous observations of 5-HT1A receptor coupling to G(o) and the pertussis toxin-insensitive G protein Gz.

The heterotrimeric G-protein Gi is localized to the insulin secretory granules of beta-cells and is involved in insulin exocytosis.

Mastoparan, a tetradecapeptide found in wasp venom that stimulates G-proteins, increases insulin secretion from beta-cells. In this study, we have examined the role of heterotrimeric G-proteins in mastoparan-induced insulin secretion from the insulin-secreting beta-cell line beta-TC3. Mastoparan stimulated insulin secretion in a dose-dependent manner from digitonin-permeabilized beta-TC3 cells. Active mastoparan analogues mastoparan 7, mastoparan 8, and mastoparan X also stimulated secretion. Mastoparan 17, an inactive analogue of mastoparan, did not increase insulin secretion from permeabilized beta-TC3 cells. Mastoparan-induced insulin secretion from permeabilized beta-TC3 cells was inhibited by pretreatment of the cells with pertussis toxin, suggesting that mastoparan-induced insulin secretion is mediated through a pertussis toxin-sensitive G-protein present distally in exocytosis. Enriched insulin secretory granules (ISG) were prepared by sucrose/nycodenz ultracentrifugation. Western immunoblotting performed on beta-TC3 homogenate and ISG demonstrated that G alpha i was dramatically enriched in ISG. Levels of G alpha o and G alpha q were comparable in homogenate and ISG. Mastoparan stimulated ISG GTPase activity in a pertussis toxin-sensitive manner. Mastoparan 7 and mastoparan 8 also stimulated GTPase activity in the ISG, while the inactive analogue mastoparan 17 had no effect. Selective localization of G alpha i to ISG was confirmed with electron microscopic immunocytochemistry in beta-TC3 cells and beta-cells from rat pancreas. In contrast to G alpha o and G alpha q, G alpha was clearly localized to the ISG. Together, these data suggest that mastoparan may act through the heterotrimeric G-protein G alpha i located in the ISG of beta-cells to stimulate insulin secretion.

Increases in guanine nucleotide binding to striatal G proteins is associated with dopamine receptor supersensitivity.

The role of the regulatory guanine nucleotide binding proteins (G proteins) in the development of dopamine (DA) receptor supersensitivity was studied in striatal membranes from reserpine treated rats. [alpha-32P]GTP labeled two striatal proteins with molecular masses of 45 and 40 kD. These proteins were previously identified as alpha subunits of Gs and Gi/Go, respectively. Seven days of reserpine treatment caused a 43% increase in steady-state basal [alpha-32P]GTP binding to striatal G alpha s. Basal [alpha-32P]GTP binding to the 40-kD protein band was unchanged by reserpine treatment. Incubation of membranes with DA stimulated [alpha-32P]GTP binding to both protein bands. Although 10 microM DA stimulated guanine nucleotide binding to G alpha s and G alpha i/o in control tissue by 317% and 236%, respectively, the increases in [alpha-32P]GTP binding in the reserpine-treated animals were 482% (P < .01) and 366% (P < .01), respectively. A single injection of reserpine did not alter basal or DA-stimulated [alpha-32P]GTP binding. Repeated reserpine treatment also enhanced serotonin-induced stimulation of [alpha-32P]GTP binding to striatal G alpha i/o but not to G alpha s. However, carbachol-stimulated binding was unaffected by the treatment Reserpine treatment did not change membrane G alpha s, G alpha i/1/2 or G alpha o levels, as assessed by immunoblotting or by toxin-catalyzed ADP ribosylation. These results suggest that increases in both basal and receptor-mediated activations of G proteins are associated with the development of reserpine-induced striatal DA receptor supersensitivity. (ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of receptor-stimulated and basal guanine nucleotide binding to membrane G proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A method to study [alpha-32P]GTP binding to the alpha subunit of GTP-binding proteins in rat brain membranes is described. This method measures receptor-stimulated GTP binding to individual alpha subunits. GTP binding is associated with two protein bands following sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The bands, 40- and 45-kDa in size, comigrate with the alpha subunits of Gi/Go and Gs, respectively. Binding of [alpha-32P]GTP is saturable and Mg(2+)-dependent. Nucleotides compete with [alpha-32P]GTP binding in the following order: GTP > GDP > Gpp(NH)p > App(NH)p. Dopamine stimulates [alpha-32P]GTP labeling of the 40- and 45-kDa bands. A binding increase of 300-400% is observed at 10 microM dopamine. Isoproterenol (10 microM) stimulates [alpha-32P]GTP binding only to the 45-kDa protein band. The effects of dopamine and isoproterenol are blocked by their respective receptor antagonists, fluphenazine and propranolol. The individual G proteins activated by dopamine are resolved by immunoprecipitation of stimulated [alpha-32P]GTP binding to G alpha s, G alpha i, and G alpha o with specific anti-G alpha antisera. Dopamine stimulates [alpha-32P]GTP binding to G alpha s and G alpha i while the labeling of G alpha o was not significantly changed. Pertussis toxin-mediated ADP ribosylation prevents the activation of G alpha i which is mediated by dopamine receptor stimulation. The methods described are useful in defining the coupling of specific neurotransmitter receptors to specific G proteins in native membranes. These procedures also allow measurements of receptor stimulation of individual G proteins in intact biological membranes.

Repeated reserpine increases striatal dopamine receptor and guanine nucleotide binding protein RNA.

In the present study the effects of repeated administration of reserpine on striatal dopamine receptor and guanine nucleotide binding protein mRNAs were determined. Twenty-four hours after seven consecutive daily injections of reserpine--a treatment that is known to produce functional sensitization of D1 and D2 dopamine receptors--the level of striatal D1 dopamine receptor mRNA was unchanged. However, the level of mRNA for the G protein Gs alpha was increased by 127%. After extended reserpine treatment for 14 days, levels of both striatal D1 DA receptor and Gs alpha mRNAs were elevated by 99 and 78%, respectively. Seven days of reserpine treatment also increased levels of mRNA of the striatal D2 dopamine receptor and of G proteins Gi2 alpha and Go alpha by 200, 79, and 32%, respectively. After 14 days of reserpine treatment the level of striatal D2 dopamine receptor mRNA was increased by twofold. In contrast, levels of the mRNAs coding for the G proteins Gi2 alpha and Go alpha were unchanged. These data suggest that dopamine receptors and their respective G proteins play important roles in the development of sensitization of striatal dopamine receptors during repeated reserpine treatment. Furthermore, the persistent increase in level of striatal Gs alpha mRNA suggests that this G protein is necessary to maintain supersensitivity of the striatal D1 dopamine receptor system following long-term dopamine depletion.

Decreased striatal release of acetylcholine following withdrawal from long-term treatment with haloperidol: modulation by cholinergic, dopamine-D1 and -D2 mechanisms.

The effect of chronic treatment with haloperidol (2.7-5.3 mumol/kg/day) on K(+)-evoked release of [3H]acetylcholine (ACh) from superfused slices of the striatum was assessed. Acute injections of haloperidol (0.7-13.3 mumol/kg) produced 5-54% increases in the release of [3H]ACh in the striatum. Chronic treatment with haloperidol for 2.5 and 5 months also resulted in enhanced release of [3H]ACh in the striatum (28-35%). However, withdrawal from 2.5 and 5 months of treatment produced 34 and 38% decreases in K(+)-evoked release of [3H]ACh in the striatum, respectively. The drug SKF 38393 (D1-agonist), produced concentration-dependent (0.1-10 microM) increases (24-59%) in the release of [3H]ACh in the striatum which were blocked by the selective D1-antagonist, SCH 23390. The effect of stimulation of D1-receptors was significantly reduced after 2.5 or 5 months of chronic treatment with haloperidol. Both LY171555 (D2-agonist) and carbachol (muscarinic agonist) produced concentration-dependent (0.1-10 microM) inhibitions of the release of [3H]ACh in the striatum (LY171555: 28-62%; carbachol: 23-63%). Long-term treatment with haloperidol (2.5 and 5 months) elicited increases in sensitivity to the effect of LY171555, while the effect of carbachol was diminished only after the 5-month treatment period. These findings demonstrate that withdrawal from chronic exposure to haloperidol in the rat results in a reduction in the release of acetylcholine in the striatum. This effect is accompanied by (1) attenuated dopaminergic D1 mechanisms which ordinarily facilitate evoked release of ACh, (2) enhanced D2 mechanism which elicits inhibition of the release of ACh in the striatum, and (3) diminished muscarinic inhibitory influence which regulates the release of ACh.

Scopolamine modulates apomorphine-induced behavior in rats treated with haloperidol or SCH 23390.

In acute experiments, scopolamine (1.0 mg/kg) potentiated apomorphine stereotypy and inhibited the antistereotypic effect of both haloperidol (0.5 mg/kg) and SCH 23390 (0.2 mg/kg). Daily administration of either haloperidol (0.5 mg/kg) or SCH 23390 (0.2 mg/kg) for 3 weeks produced enhanced stereotypic responses to apomorphine. Co-administration of scopolamine (1.0 mg/kg) with haloperidol or SCH 23390 significantly reduced the behavioral supersensitivity produced by haloperidol or SCH 23390 alone. It is suggested that both D-1 and D-2 dopamine receptors are linked to a cholinergic mechanism.