Endomorphin-Stimulated [35S]GTPγS Binding in Rat Brain: Evidence for Partial Agonist Activity at μ-Opioid Receptors

Abstract: Endomorphin-1 is a peptide whose binding selectivity suggests a role as an endogenous ligand at μ-opioid receptors. In the present study, the effect of endomorphin-1 on μ receptor-coupled G proteins was compared with that of the μ agonist DAMGO by using agonist-stimulated [³⁵S]GTPγS binding in rat brain. [³⁵S]GTPγS autoradiography revealed a similar localization of endomorphin-1 and DAMGO-stimulated [³⁵S]GTPγS binding in areas including thalamus, caudate-putamen, amygdala, periaqueductal gray, parabrachial nucleus, and nucleus tractus solitarius. Naloxone blocked endomorphin-1-stimulated labeling in all regions examined. Although the distribution of endomorphin-1-stimulated [³⁵S]GTPγS binding resembled that of DAMGO, the magnitude of endomorphin-1-stimulated binding was significantly lower than that produced by DAMGO. Concentration-effect curves of endomorphin-1 and DAMGO in thalamic membranes confirmed that endomorphin-1 produced only 70% of DAMGO-stimulated [³⁵S]GTPγS binding. Differences in maximal stimulation of [³⁵S]GTPγS binding between DAMGO and endomorphin-1 were magnified by increasing GDP concentrations, and saturation analysis of net endomorphin-1-stimulated [³⁵S]GTPγS binding revealed a lower apparent B _max value than that obtained with DAMGO. Endomorphin-1 also partially antagonized DAMGO stimulation of [³⁵S]GTPγS binding. These results demonstrate that endomorphin-1 is a partial agonist for G protein activation at the μ-opioid receptor in brain.     

Characterization of the Functional Activity of Dopamine Ligands at Human Recombinant Dopamine D4 Receptors

Abstract: The human D₄ dopamine receptor has been expressed in Sf9 insect cells where it appears to couple to endogenous G proteins. Increased guanine nucleotide exchange to G proteins is a reflection of receptor activation and can be followed using a [³⁵S]GTPγS binding assay. By measuring D₄ receptor stimulation of [³⁵S]-GTPγS binding we have been able to characterize several dopaminergic compounds for their functional activity at this receptor. In Sf9 cells expressing the D₄ receptor, dopamine, quinpirole, and dp -2-aminodihydroxy-1,2,3,4-tetrahydronaphthalene were all full agonists, whereas (−)-apomorphine appeared to be a partial agonist. No increase in [³⁵S]GTPγS binding was observed for noninfected cells or cells infected with an unrelated sequence. The quinpirole-stimulated [³⁵S]GTPγS binding could be inhibited by the antagonists clozapine, eticlopride, and haloperidol, and a Schild analysis of these data showed that all three compounds were acting as competitive antagonists of D₄ receptors. The rank order of affinities derived from the Schild analysis correlated with that obtained from [³H]spiperone competition binding assays. In conclusion, we have shown that, using this assay system, it is possible to investigate functionally the pharmacology of a recombinant G protein-coupled receptor in the absence of any information regarding the eventual second messenger pathways involved.     

Activation of Opioid and Muscarinic Receptors Stimulates Basal Adenylyl Cyclase but Inhibits Ca2+/Calmodulin- and Forskolin-Stimulated Enzyme Activities in Rat Olfactory Bulb

Abstract: In rat olfactory bulb, muscarinic and opioid receptor agonists stimulate basal adenylyl cyclase activity in a GTP-dependent and pertussis toxin-sensitive manner. However, in the present study, we show that in the same brain area activation of these receptors causes inhibition of adenylyl cyclase activity stimulated by Ca²⁺ and calmodulin (CaM) and by forskolin (FSK), two direct activators of the catalytic unit of the enzyme. The opioid and muscarinic inhibitions consist of a decrease of the maximal stimulation elicited by either CaM or FSK, without a change in the potency of these agents. [Leu⁵]Enkephalin and selective δ- and μ-, but not κ-, opioid receptors agonists inhibit the FSK stimulation of adenylyl cyclase activity with the same potencies displayed in stimulating basal enzyme activity. Similarly, the muscarinic inhibition of FSK-stimulated adenylyl cyclase activity shows agonist and antagonist sensitivities similar to those characterizing the muscarinic stimulation of basal enzyme activity. Fluoride stimulation of adenylyl cyclase is not affected by either carbachol or [Leu⁵]enkephalin. In vivo treatment of olfactory bulb with pertussis toxin prevents both opioid and muscarinic inhibition of Ca²⁺/CaM- and FSK-stimulated enzyme activities. These results indicate that in rat olfactory bulb δ- and μ-opioid receptors and muscarinic receptors, likely of the M4 subtype, can exert a dual effect on cyclic AMP formation by interacting with pertussis toxin-sensitive GTP-binding protein(s) and possibly by affecting different molecular forms of adenylyl cyclase.     

Role of G Protein βγ Subunits in Muscarinic Receptor-Induced Stimulation and Inhibition of Adenylyl Cyclase Activity in Rat Olfactory Bulb

Abstract: In the olfactory bulb, muscarinic receptors exert a bimodal control on cyclic AMP, enhancing basal and G_s-stimulated adenylyl cyclase activities and inhibiting the Ca²⁺/calmodulin- and forskolin-stimulated enzyme activities. In the present study, we investigated the involvement of G protein βγ subunits by examining whether the muscarinic responses were reproduced by the addition of βγ subunits of transducin (βγ_t) and blocked by putative βγ scavengers. Membrane incubation with βγ_t caused a stimulation of basal adenylyl cyclase activity that was not additive with that produced by carbachol. Like carbachol, βγ_t potentiated the enzyme stimulations elicited by vasoactive intestinal peptide and corticotropin-releasing hormone. RT-PCR analysis revealed the expression of mRNAs encoding both type II and type IV adenylyl cyclase, two isoforms stimulated by βγ synergistically with activated G_s. In addition, βγ_t inhibited the Ca²⁺/calmodulin- and forskolin-stimulated enzyme activities, and this effect was not additive with that elicited by carbachol. Membrane incubation with either one of two βγ scavengers, the GDP-bound form of the α subunit of transducin and the QEHA fragment of type II adenylyl cyclase, reduced both the stimulatory and inhibitory effects of carbachol. These data provide evidence that in rat olfactory bulb the dual regulation of cyclic AMP by muscarinic receptors is mediated by βγ subunits likely acting on distinct isoforms of adenylyl cyclase.     

Influence of positive allosteric modulators on GABA(B) receptor coupling in rat brain: a scintillation proximity assay characterisation of G protein subtypes

Little is known concerning coupling of cerebral GABA_B receptors to G protein subtypes, and the influence of positive allosteric modulators (PAMs) has not been evaluated. These questions were addressed by an antibody-capture/scintillation proximity assay strategy. GABA concentration-dependently enhanced the magnitude of [³⁵S]GTPγS binding to Gαo and, less markedly, Gαi_1/3 in cortex, whereas Gq and Gs/olf were unaffected. ( R )-baclofen and SKF97581 likewise activated Gαo and Gαi_1/3, expressing their actions more potently than GABA. Similar findings were acquired in hippocampus and cerebellum, and the GABA_B antagonist, CGP55845A, abolished agonist-induced activation of Gαo and Gαi_1/3 in all structures. The PAMs, GS39783, CGP7930 and CGP13501, inactive alone, enhanced efficacy and potency of agonist-induced [³⁵S]GTPγS binding to Gαo in all regions, actions abolished by CGP55845A. In contrast, they did not modify efficacies at Gαi_1/3. Similarly, in human embryonic kidney cells expressing GABA_B(1a+2) or GABA_B(1b+2) receptors, allosteric modulators did not detectably enhance efficacy of GABA at Gαi_1/3, though they increased its potency. To summarise, GABA_B receptors coupled both to Gαo and to Gαi, but not Gq and Gs/olf, in rat brain. PAMs more markedly enhanced efficacy of coupling to Go versus Gi_1/3. It will be of interest to confirm these observations employing complementary techniques and to evaluate their potential therapeutic significance.     

Alteration of Tubulin-Gi Protein Interaction in Rat Cerebral Cortex with Aging

Abstract: The ability of the tubulin dimer to interact with and to modulate the G_i function inhibiting adenylyl cyclase was examined in cerebral cortex membranes from 2-month-old and 24-month-old rats. The hydrolysis-resistant GTP analogue 5'-guanylylimidodiphosphate (GppNHp)-dependent inhibition of adenylyl cyclase was significantly decreased in cerebral cortex membranes from 24-month-old rats. Tubulin, prepared from rat brains by polymerization with GppNHp, caused inhibition of adenylyl cyclase (∼28%) in 2-month-old rats. Tubulin-GppNHp-dependent inhibition of adenylyl cyclase in 24-month-old rats was significantly attenuated (∼15%). In 2-month-old rats, when tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analogue [³²P] P ³(4-azidoanilido)- P ¹-5'-GTP ([³²P]AAGTP), was incubated with cerebral cortex membranes, AAGTP was transferred from tubulin to G_iα. Transfer of AAGTP from tubulin to G_iα was reduced in 24-month-old rats. Furthermore, photoaffinity labeling of [³²P]AAGTP to G_iα in cortex membranes was significantly decreased in 24-month-old rats. No differences were observed in the amounts of G_sα, G_iα, or G_β subunits and tubulin, estimated by immunoblotting, in cortex membranes from 2-month-old and 24-month-old rats. These results suggest that the ability of tubulin to interact with G_i and thereby modulate the inhibitory regulation of adenylyl cyclase is reduced in the cerebral cortex of 24-month-old rats.     

Altered CB1 receptor-signaling in prefrontal cortex from an animal model of depression is reversed by chronic fluoxetine

Bilateral olfactory bulbectomy in the rat (OBX) induces behavioral, neurochemical, and structural abnormalities similar to those observed in human depression that are normalized after chronic, but not acute, treatment with antidepressants. In our study, OBX animals exhibited significant increases in both CB₁ receptor density ([³H]CP55490 binding) and functionality (stimulation of [³⁵S]GTPγS binding by the cannabinoid (CB) agonist WIN 55212-2) at the prefrontal cortex (PFC). After chronic treatment with fluoxetine (10 mg/kg/day, 14 days, s.c.), OBX-induced hyperactivity in the open-field test was fully abolished. Interestingly, chronic fluoxetine fully reversed the enhanced CB₁-receptor signaling in PFC observed following OBX. The CB agonist Δ⁹-tetrahydrocannabinol (5 mg/kg, i.p., 1 day) did not produce any behavioral effect in sham-operated animals but returned locomotor activity to control values in OBX rats. As both acute administration of Δ⁹-tetrahydrocannabinol and chronic fluoxetine elicited a similar behavioral effect in the OBX rat, it is not unlikely that the regionally selective enhancement of CB₁ receptor-signaling in the PFC could be related with the altered OBX behavior. Our findings reinforce the utility of this animal model to further investigating the implication of the endocannabinoid system in the modulation of emotional processes and its potential role in the adaptive responses to chronic antidepressants.     

Characterization, Distribution, and Protein Kinase C-Mediated Regulation of [35S]Glutathione Binding Sites in Mouse and Human Spinal Cord

Abstract: We have characterized a high-affinity [³⁵S]-glutathione ([³⁵S]GSH) binding site in mouse and human spinal cord. [³⁵S]GSH binding sites in mouse and human spinal cord were observed largely within the gray matter in both the dorsal and ventral horns of spinal cord at cervical, thoracic, and lumbosacral segments. High-affinity [³⁵S]GSH binding was saturable, showing a B _max of 72 fmol/mg of protein and a K _D of 3.0 n M for mouse spinal cord and a B _max of 52 fmol/mg of protein and a K _D of 1.6 n M for human spinal cord. [³⁵S]GSH binding was displaceable by GSH, l -cysteine, and S -hexyl-GSH, but not by glutamate, glycine, or NMDA. These [³⁵S]GSH binding sites exhibited kinetic and saturation characteristics similar to GSH binding sites in rat brain astrocytes. To determine whether [³⁵S]GSH binding sites could be regulated by protein kinase C, we exposed human spinal cord sections to phorbol 12,13-diacetate for 1 h before ligand binding. Phorbol ester treatment increased [³⁵S]GSH binding by ∼60%, an effect that could be blocked by exposure of spinal cord sections to 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a general protein kinase inhibitor. [³⁵S]GSH binding sites in the spinal cord of both species exhibited many of the characteristics of a receptor including saturable binding, high affinity, ligand specificity, and modulation by kinase activity. These data suggest that GSH is a neurotransmitter in the CNS.     

Participation of Tubulin in the Stimulatory Regulation of Adenylyl Cyclase in Rat Cerebral Cortex Membranes

Abstract: This study examined effects of tubulin on the activation of adenylyl cyclase in rat cerebral cortex membranes. Tubulin, prepared from rat brain by polymerization with the hydrolysis-resistant GTP analogue 5'-guanylylimidodiphosphate (GppNHp) caused significant activation of the enzyme by ∼156% under conditions in which stimulation rather than inhibition of the enzyme was favored. Tubulin-GppNHp activated isoproterenol-sensitive adenylyl cyclase, potentiated forskolin-stimulated activity of the enzyme, and reduced agonist binding affinity for β-adrenergic receptors. When tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analogue [³²P] P ³(4-azidoanilido)- P ¹-5'-GTP ([³²P]AAGTP), was incubated with cerebral cortex membranes, AAGTP was transferred from tubulin to G_sα as well as G_iα. These results suggest that, in rat cerebral cortex membranes, the tubulin dimer participates in the stimulatory regulation of adenylyl cyclase by transferring guanine nucleotide to G_sα, as well as affecting the G_i-mediated inhibitory pathway.     

Modulation of GABAA Receptor tert-[35S]Butylbicyclophosphorothionate Binding by Antagonists: Relationship to Patterns of Subunit Expression

Abstract: The multisubunit γ-aminobutyric acid type A (GABA_A) receptor is heterogeneous in molecular and pharmacological aspects. We used quantitative autoradiographic techniques to generate detailed pharmacological profiles for the binding of the GABA_A-receptor ionophore ligand tert -[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) and its modulation by GABA and the GABA_A antagonists bicuculline and 2'-(3'-carboxy-2',3'-propyl)-3-amino-6- p -methoxyphenylpyrazinium bromide (SR 95531). Regional differences in the actions of bicuculline and SR 95531 were correlated with the expression of 13 GABA_A subunits in brain as reported previously. In some brain regions SR 95531 reduced [³⁵S]TBPS binding much more than bicuculline, as illustrated by high ratios of bicuculline- to SR 95531-modulated [³⁵S]TBPS binding. This ratio correlated positively with α2-subunit mRNA levels. Binding that was equally affected by SR 95531 and bicuculline occurred prominently in regions with abundant α1 mRNA expression. The present findings thus reveal a novel pharmacological heterogeneity based on differences between α1 and α2 subunit-containing GABA_A receptors. The data aid in developing GABA_A-receptor subtype-specific antagonists and in establishing receptor domains critical for the actions of GABA_A antagonists.     

Differential Effects of 4'-Chlorodiazepam on Expressed Human GABAA Receptors

Abstract: The interactions of the atypical benzodiazepine 4'-chlorodiazepam (Ro 5-4864) with functionally expressed human GABA_A receptor cDNAs were determined. Cotransfection of human α₂, β₁, and γ₂ subunits was capable of reconstituting a 4'-chlorodiazepam recognition site as revealed by a dose-dependent potentiation of t -[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding to the GABA-activated chloride channel. This site is found on GABA_A receptor complexes containing sites for GABA agonist-like benzodiazepines and neuroactive steroids. The importance of the α subunit was further demonstrated as substitution of either α₁ or α₃ for the α₂ subunit did not reconstitute a 4'-chlorodiazepam recognition site that was capable of modulating [³⁵S]TBPS binding under the same experimental conditions. The 4'-chlorodiazepam modulatory site was shown to be distinct from the benzodiazepine site, but the phenylquinolines PK 8165 and PK 9084 produced effects similar to 4'-chlorodiazepam, consistent with the previous analysis of the 4'-chlorodiazepam site in brain homogenates. Further analysis of the subunit requirements revealed that coexpression of α₂ and β₁ alone reconstituted a 4'-chlorodiazepam recognition site. It is interesting, however, that the 4'-chlorodiazepam site was found to inhibit [³⁵S]TBPS binding to the GABA-activated chloride channel. Thus, the 4'-chlorodiazepam site may be reconstituted with only the α and β polypeptides.     

Dopamine D1 and D2 Receptors and Their Signal System Present in Coated Vesicles Prepared from Bovine Striatal Tissue

Abstract: Coated vesicles (CVs) isolated from bovine striatal tissue were examined to determine whether they are associated with dopamine signal systems consisting of dopamine D₁ and D₂ receptors, G proteins, and adenylate cyclase. Dopamine receptors in CVs were characterized by a dopamine D₁ receptor antagonist, [³H]SCH 23390, and a dopamine D₂ receptor antagonist, [³H]-spiroperidol. The bindings of both ligands were specifically saturable and reversible with a dissociation constant ( K _D) of 0.65 and 0.5 n M , respectively. Dopaminergic antagonists and agonists inhibited the specific bindings of [³H]SCH 23390 and [³H]spiroperidol in a stereoselective and concentration-dependent manner with an appropriate rank order potency for dopamine D₁ or D₂ receptors. The regulations of the agonist binding by guanyl-5-ylimidodiphosphate were observed. ADP ribosylation of the CVs with [³²P]NAD demonstrated predominant labeling of bands of M_r 47,000–52,000, 42,000–45,000, and 40,000-39,000, which corresponded to the known molecular weights of the α subunits of G_s and G_i proteins. The presence of α and β subunits of G proteins in the CVs was also confirmed by immunoblotting assay. Adenylate cyclase activity, which was stimulated by SKF 38393 and inhibited by dopamine D₂ receptor agonists, was present in the CVs. These findings suggest that the dopamine D₁ and D₂ receptors in the CVs couple with adenylate cyclase via G_s/G_i protein.     

Residues Specifically Involved in Down-Regulation but Not Internalization of the m1 Muscarinic Acetylcholine Receptor

Abstract: Human m1 muscarinic acetylcholine receptor mutants were screened to determine receptor domains and cellular pathways relevant to down-regulation. Mutations in the second intracellular loop and the junctions of the third intracellular loop of the receptor, where a role for receptor activation or internalization had been previously demonstrated in HEK293 cells, were selected for this study. To assess receptor down-regulation, the m1 receptor mutants were transfected into Chinese hamster ovary cells. Because receptor internalization is expected to precede down-regulation, mutants displaying intact internalization were selected to permit interpretation of mutational effects on down-regulation alone. Four mutations were identified that specifically impaired down-regulation without altering receptor internalization: V127A, I211A, E360A, and K362A. The results define new receptor domains in the second intracellular loop and the junctions of the third intracellular loop that are involved in down-regulation. These same four mutants were also defective in signaling via the phospholipase C and the adenylyl cyclase pathways and in G protein activation, as measured by [³⁵S]GTPγS binding. However, the level of second messenger stimulation correlated poorly with the extent of down-regulation. In summary, several mutations of the m1 receptor selectively affect down-regulation, demonstrating that internalization and down-regulation represent distinct events driven by different cellular mechanisms.     

Opposing Actions of D1 and D2-Dopamine Receptors on Arachidonic Acid Release and Cyclic AMP Production in Striatal Neurons

Abstract: D₁-and D₂-dopamine receptors exert important physiological actions on striatal neurons, but the intracellular second messenger pathways activated by these receptors are still incompletely understood. Using primary cultures of rat striatal cells, we have examined the effects of activating D₁ or D₂ receptors on arachidonic acid (AA) release and cyclic AMP accumulation. In striatal neurons labeled by incubation with [³H]AA, D₂-receptor stimulation enhanced release of [³H]AA produced by application of the Ca²⁺ ionophore A23187 or of the purinergic agonist ATP. By contrast, D₁-receptor stimulation inhibited [³H]AA release. This inhibitory effect of D₁ receptors was accompanied by stimulation of adenylyl cyclase activity, measured as accumulation of cyclic AMP, and was mimicked by application of the adenylyl cyclase activator forskolin. The results indicate the existence of a novel signaling pathway for D₂ and D₁ receptors in striatum, potentiation and inhibition, respectively, of Ca²⁺-evoked AA release.     

Lindane Administration to the Rat Induces Modifications in the Regional Cerebral Binding of [3H]Muscimol, [3H]-Flunitrazepam, and t-[35S]Butylbicyclophosphorothionate: An Autoradiographic Study

Abstract: The effect of lindane administration on the specific binding of ligands to different sites on the GABA_A receptor-ionophore complex was studied in the rat brain by receptor mapping autoradiography. [³H]Muscimol (Mus), [³H]flunitrazepam (Flu), and t -[³⁵S]butylbicyclophosphorothionate (TBPS) were used as specific ligands of GABA, benzodiazepine, and picrotoxinin binding sites, respectively. Rats received a single oral dose of 30 mg/kg lindane and they were classified into two groups according to the absence or presence of convulsions. Vehicle-treated groups acted as controls. The effect of the xenobiotic on ligand binding was measured in different brain areas and nuclei 12 min or 5 h after its administration. Lindane induced a generalized decrease in [³⁵S]TBPS binding, which was present shortly after dosing. In addition, [³H]Flu binding was increased in lindane-treated animals, this modification also appearing shortly after administration but diminishing during the studied time. Finally, lindane induced a decrease in [³H]Mus binding, which became more evident over time. These modifications were observed both in the presence and in the absence of convulsions. However, an increase in [³H]-Mus binding was detected shortly after lindane-induced convulsions. The observed decrease in [³⁵S]TBPS binding is in agreement with the postulated action of lindane at the picrotoxinin binding site of the GABA_A receptor chloride channel. The effects observed on the binding of [³H]Flu and [³H]Mus may be secondary to the action of lindane as an allosteric antagonist of the GABA_A receptor.     

Specificity of Muscarinic Acetylcholine Receptor Regulation by Receptor Activity

Abstract— Regulation of muscarinic acetylcholine receptor concentration by receptor activity in neuron-like NG108-15 hybrid cells is a highly specific process. Receptor levels, monitored by binding of [³H]quinuclidinyl benzilate ([³H]QNB), decreased 50-75% following 24-h incubation of cells with muscarinic agonists, but none of the following cellular processes was altered by this chronic receptor stimulation: (1) glycolytic energy metabolism, measured by [³H]deoxy- d -glucose ([³H]DG) uptake and retention; (2) rate of cell division; (3) transport, measured by [³H]valine and [³H]uridine uptake; (4) RNA biosynthesis, measured by [³H]uridine incorporation; (5) protein biosynthesis, measured by [³H]valine and [³⁵S]methionine incorporation into total protein and into protein fractions obtained by polyacrylamide gel electrophoresis. In contrast, chronic stimulation did cause a threefold decrease in the capacity of carbachol to stimulate phosphatidylinositol (PI) turnover, a receptor-mediated response. In addition to cholinomimetics, the neuroeffector adenosine (1 m m for 24 h) also caused a decrease in [³H]QNB binding levels, but chronic stimulation of α -adrenergic, opiate, prostaglandin E₁, and prostaglandin F_2α receptors found on NG108-15 cells caused no changes. The data indicate that loss of muscarinic receptors caused by receptor stimulation is not a consequence of fundamental changes evoked in overall cellular physiology but reflects a specific regulation of cholinoceptive cell responsiveness.     

Interaction of Guanine Nucleotides with [3H]Kainate and 6-[3H]Cyano-7-Nitroquinoxaline-2,3-dione Binding in Goldfish Brain

Abstract— Recent reports have suggested that a major proportion of [³H]kainate binding in goldfish brain is to a novel form of G-protein-linked glutamate receptor. Here we confirm that guanine nucleotides decrease [³H]kainate binding in goldfish brain membranes, but that binding is also reduced to a similar extent under conditions where G-protein modulation should be minimised. Inclusion of GTPγS resulted in an approximately twofold decrease in the affinity of [³H]kainate binding and a 50% reduction in the apparent B _max values in both Mg²⁺/Na⁺ and Mg²⁺/Na⁺-free buffer when assayed at 0°c. The pharmacology of [³H]kainate binding is similar to that of well-characterised ionotropic kainate receptors but unlike that of known me-tabotropic glutamate receptors, with neither 1 S ,3 R -amino-1,3-cyclopentanedicarboxylic acid (1 S ,3 R -ACPD) nor ibo-tenic acid being effective competitors. The molecular mass of the [³H]kainate binding protein, as determined by radiation inactivation, was 40 kDa, similar to the subunit sizes of other lower vertebrate kainate binding proteins that are believed to comprise ligand-gated ion channels. Furthermore, GTP-γS also inhibited the binding of the non-NMDA receptor-selective antagonist 6-[³H]cyano-7-ni-troquinoxaline-2,3-dione. These data strongly suggest that the regulatory interaction between guanine nucleotides and [³H]kainate and 6-[³H]cyano-7-nitroquinoxaline-2,3-dione binding is complex and involves competition at the agonist/antagonist binding site in addition to any G-protein-mediated modulation.     

Formation and Utilization of the Active Sulfate Donor [35S]3'-Phosphoadenosine 5'-Phosphosulfate in Brain Slices: Effects of Depolarizing Agents

Abstract: The accumulation and utilization of [³⁵S]3'-phos-phoadenosine 5'-phosphosulfate (PAPS) were studied in slices from rat cerebral cortex incubated in the presence of inorganic [³⁵S]sulfate. [³⁵S]PAPS levels were directly evaluated after either isolation by ion-exchange chromatography or quantitative enzymatic transfer of its active [³⁵S]sulfate group to an acceptor phenol under the action of added phenolsulfotransferase activity. [³⁵S]PAPS formation was also indirectly followed by incubating slices in the presence of β-naphthol and measuring the levels of [³⁵S]β-naphthyl sulfate ([³⁵S]β-NS). Whereas [³⁵S]PAPS levels rapidly reached a plateau, [³⁵S]β-NS formation proceeded linearly with time for at least 1h, an observation indicating that the nucleotide was continuously synthesized and utilized for endogenous sulfation reactions. [³⁵S]PAPS formation in ices was completely and rather potently blocked by 2,6-dichloro-4-nitrophenol (IC₅₀= .10 μM), an inhibitor of the PAPS-synthesizing enzyme system in a cytosolic preparation. [³⁵S]PAPS accumulation and [³⁵S]β-NS'formation were strongly reduced by depolarizing agents such as potassium or veratridine. At millimolar concentrations, various excitatory amino acids (glutamate, aspartate, cysteate, quisqualate, and homocysteate) also elicited similar effects, whereas kainate and N -methyl-D-aspartate were inactive. This suggests that PAPS synthesis is turned off when cerebral cells are strongly depolarized.     

Synergistic Interaction of Muscarinic and Opioid Receptors with Gs-Linked Neurotransmitter Receptors to Stimulate Adenylyl Cyclase Activity of Rat Olfactory Bulb

We reported previously that in homogenates of rat olfactory bulb muscarinic and opioid receptor agonists stimulate adenylyl cyclase activity. In the present study we show that carbachol (CCh) and Leu-Enkephalin act synergistically with vasoactive intestinal peptide (VIP) and corticotropin-releasing hormone (CRH), but not with /-isoproterenol, in increasing cyclic AMP formation. The synergistic interaction consists of an increase in the maximal a0denylyl cyclase activation without a significant change in the potency of each agonist. CCh also fails to affect ¹²⁵ICRH binding to olfactory bulb membranes. The synergism requires micromolar concentrations of GTP. Substitution of the stable GTP analog guanosine 5′-O-(3′-thiotriphosphate) for GTP allows the CRH stimulation, but abolishes the CCh enhancement of both basal and CRH-stimulated enzyme activities. Moreover, in vivo treatment of olfactory bulbs with pertussis toxin completely prevents the muscarinic and opioid effects. Thus, the synergistic interaction appears to result from opioid- and muscarinic-induced activation of a pertussis toxin-sensitive GTP-binding protein which may potentiate the adenylyl cyclase stimulation by the stimulatory GTP-binding protein activated by either VIP or CRH receptors.