Homologous Desensitization of Serotonin 5-HT2 Receptor-Stimulated Intracellular Calcium Mobilization in C6BU-1 Glioma Cells via a Mechanism Involving a Calmodulin Pathway

Abstract: Serotonin 5-HT₂ receptor-mediated intracellular Ca²⁺ mobilization was investigated in rat glioma C6BU-1 cells. The receptors became desensitized after previous exposure to 5-HT in a time-and concentration-dependent manner. The desensitization of 5-HT₂ receptor-mediated intracellular signaling appeared to be homologous because previous exposure to 5-HT did not alter the response to other transmitters such as thrombin or isoproterenol and because previous exposure to thrombin or isoproterenol did not diminish the response to 5-HT. The desensitization induced by pretreatment with 5-HT was potently prevented by the naphthalenesulfonamide derivative W-7, a calmodulin antagonist, when it was cosupplied with 5-HT. Furthermore, the preventive effect of W-7 was greater than that of W-5, a weak analogue of W-7, and than that of H-7, a nonselective inhibitor of protein kinases. These results suggest that 5-HT₂ receptor-mediated Ca²⁺ mobilization can be desensitized homologously after prolonged exposure to 5-HT in a calmodulin-dependent manner in rat glioma C6BU-1 cells.     

Chronic Imipramine Administration Amplifies the Serotonin2A Receptor-Induced Intracellular Ca2+ Mobilization in C6 Glioma Cells Through a Calmodulin-Dependent Pathway

Abstract: In the present study, we examined whether chronic exposure of C6BU-1 cells to 100 n M of several different types of antidepressants directly influences serotonin_2A (5-HT_2A) receptor-stimulated intracellular Ca²⁺ mobilization. Imipramine, desipramine, clomipramine, and maprotiline amplified the 5-HT response at 48, but not at 2, h. Imipramine increased the maximum response to 5-HT without altering the EC₅₀ of the dose-response curve. This effect was time dependent and cycloheximide blocked the maximal induction, suggesting an essential role for protein synthesis in this process. Previous exposure of the cells to thrombin or isoproterenol did not influence 5-HT_2A receptor function and pretreatment with imipramine did not alter the thrombin- or bradykinin-induced Ca²⁺ mobilization, which indicates that the effects of imipramine appear to be specific to the 5-HT_2A receptor. The effect of imipramine was potently suppressed by a calmodulin antagonist, W-13, in a dose-dependent manner. Furthermore, this amplified 5-HT response was blocked by KN-93, but not by H-7. Taken together, these results suggest that imipramine has a modulatory effect on the 5-HT_2A receptor-coupled intracellular Ca²⁺ in C6 cells through a calmodulin-dependent pathway, possibly involving Ca²⁺/calmodulin kinase activation.     

Contribution of Metabotropic Glutamate Receptor mGluR4 to L-2-[3H]Amino-4-Phosphonobutyrate Binding in Mouse Brain

Abstract : The binding of L-2-[³H]amino-4-phosphonobutyrate ([³H]L-AP4) was examined in brain sections of wild-type mice and mice lacking the mGluR4 subtype of metabotropic glutamate receptors (mGluRs). Very high relative densities of [³H]L-AP4 binding were observed in the molecular layer of the cerebellar cortex, the nucleus basalis, the outer layer of the superior colliculus, and the substantia nigra. In mGluR4 knock-out mice, very low levels of binding were observed in these regions. The moderate levels of binding observed with wild-type mice in the molecular layer of the hippocampal dentate gyrus and in the thalamus were absent in mGluR4 knock-out mice. In contrast, the moderate levels observed in most of the cerebral cortex, caudate putamen, and globus pallidus were not different in mGluR4 knock-out mice compared with wild-type. In these regions, mGluR8 is likely to be labeled by [³H]L-AP4 because mGluR8 is expressed in such brain regions and, like mGluR4, has high affinity for L-AP4. We conclude that mGluR4 contributes substantially to the high-affinity binding site for [³H]L-AP4 in several regions of mouse brain, including cerebellar cortex, nucleus basalis, thalamus, superior colliculus, substantia nigra, and hippocampal dentate gyrus.     

Possible Involvement of Pertussis Toxin-Sensitive G Proteins and D2 Dopamine Receptors in the A1 Adenosine Receptor-Adenylate Cyclase System in Rat Cerebral Cortex

To identify the involvement of dopamine receptors in the transmembrane signaling of the adenosine receptor-G protein-adenylate cyclase system in the CNS, we examined the effects of pertussis toxin (islet-activating protein, IAP) and apomorphine on A1 adenosine agonist (-)N6-R-[3H]phenylisopropyladenosine ([3H]PIA) and antagonist [3H]xanthine amine congener ([3H]XAC) binding activity and adenylate cyclase activity in cerebral cortex membranes of the rat brain. Specific binding to a single class of sites for [3H]XAC with a dissociation constant (KD) of 6.0 +/- 1.3 nM was observed. The number of maximal binding sites (Bmax) was 1.21 +/- 0.13 pmol/mg protein. Studies of the inhibition of [3H]XAC binding by PIA revealed the presence of two classes of PIA binding states, a high-affinity state (KD = 2.30 +/- 1.16 nM) and a low-affinity state (KD = 1.220 +/- 230 nM). Guanosine 5'-(3-O-thio)triphosphate or IAP treatment reduced the number of the high-affinity state binding sites without altering the KD for PIA. Apomorphine (100 microM) increased the KD value 10-fold and decreased Bmax by approximately 20% for [3H]PIA. The effect of apomorphine on the KD value increase was irreversible and due to a conversion from high-affinity to low-affinity states for PIA. The effect was dose dependent and was mediated via D2 dopamine receptors, since the D2 antagonist sulpiride blocked the phenomenon. The inhibitory effect of PIA on adenylate cyclase activity was abolished by apomorphine treatment. There was no effect of apomorphine on displacement of [3H]quinuclidinyl benzilate (muscarinic ligand) binding by carbachol. These data suggest that A1 adenosine receptor binding and function are selectively modified by D2 dopaminergic agents.     

Effects of Human Type 1α Metabotropic Glutamate Receptor Expression Level on Phosphoinositide and Ca2+ Signalling in an Inducible Cell Expression System

Abstract: Stable expression of the human type 1α metabotropic glutamate (mGlu1α) receptor was achieved in Chinese hamster ovary cells using an isopropyl-β- d -thiogalactoside (IPTG)-repressible expression system. Treatment of the cells with IPTG resulted in a time- and concentration-dependent induction of receptor expression. Maximal expression was obtained after treatment of the cells with 100 µ M IPTG for 20 h, leading to a marked increase in receptor immunoreactivity detected by western blot, >30-fold stimulation of ³H-labelled inositol phosphate (³H-InsP) production, and a robust increase in intracellular calcium concentration in single cells after stimulation with 20 µ M quisqualate. The basal level of ³H-InsP accumulation in cells induced with IPTG was increased by two- to threefold as compared with control cells; however, this basal activity was found to be dependent on glutamate released by the cells into the incubation medium. Following IPTG treatment, stable expression of the mGlu1α receptor was maintained for at least 1 week. Taken together, these results clearly indicate the advantages of working with an inducible expression system when studying the biochemical and pharmacological properties of the human mGlu1α receptor in transfected cells.     

Receptor-Mediated Desensitisation of Histamine H1 Receptor-Stimulated Inositol Phosphate Production and Calcium Mobilisation in GT1-7 Neuronal Cells Is Independent of Protein Kinase C

Abstract: GT1-7 cells, a clonal line derived from specific tumours of gonadotropin-releasing hormone-secreting neurons from mouse hypothalamus, were used as a model system to investigate the cellular mechanisms underlying the histamine H₁ receptor-mediated desensitisation. GT1-7 cells contain H₁ receptors, acute stimulation of which leads to the desensitisation of histamine-mediated calcium mobilisation and is manifest as a concurrent reduction in both the magnitude of the calcium transient and of the sustained phase. Acute pretreatment of the cells with the phorbol ester, phorbol 12-myristate 13-acetate, can also ablate the histamine-stimulated calcium mobilisation. In addition, acute H₁-receptor stimulation and acute phorbol ester treatment result in the attenuation of histamine-mediated inositol phosphate production. Receptor desensitisation resulting from acute stimulation with histamine is not affected by inhibiting protein kinase C (PKC) activity with Ro 31-7549 or staurosporine. In contrast, the desensitisation of H₁-receptor responses induced by direct activation of protein kinase C is preventable by PKC inhibitors. Thus, these results imply that a PKC-dependent mechanism and PKC-independent mechanism are involved in the H₁-receptor desensitisation cascade in GT1-7 cells and do not support the involvement of PKC in the receptor-mediated desensitisation of H₁ receptor-stimulated calcium and inositol phosphate responses.     

Guinea Pig Histamine H1 Receptor. II. Stable Expression in Chinese Hamster Ovary Cells Reveals the Interaction with Three Major Signal Transduction Pathways

Abstract: A cDNA encoding a guinea pig histamine H₁ receptor was stably expressed in Chinese hamster ovary (CHO) cells. In one resulting clone, named CHO(H₁), the H₁ receptor was found to be coupled to several major signal transduction pathways. In each case the involvement of a G_i/G_o protein with pertussis toxin (PTX) was assessed, as well as the influence of extracellular Ca²⁺ and of protein kinase C activation by phorbol 12-myristate 13-acetate (PMA). Histamine induced, in a PTX- and PMA-insensitive manner, a biphasic increase in the intracellular Ca²⁺ level of which only the second sustained phase was dependent on the extracellular Ca²⁺ level. Histamine also caused a threefold elevation of inositol phosphate production, which was PTX-insensitive, but slightly inhibited by PMA and reduced by 75% in the absence of extracellular Ca²⁺. Histamine also caused a massive release of arachidonic acid, which occurred in a Ca²⁺- and PMA-sensitive manner, probably through the activation of a cytosolic phospholipase A₂, which partly involves coupling to a PTX-sensitive G protein. In comparison, in HeLa cells endowed with a native H₁ receptor, the histamine-induced arachidonic acid release was also Ca²⁺- and PMA-sensitive, but totally PTX-insensitive. Finally, in CHO(H₁) cells, histamine in very low concentrations potentiated the cyclic AMP accumulation induced by forskolin. This response appeared to be insensitive to PTX, extracellular Ca²⁺, and PMA. These various observations show that stimulation of a single receptor subtype, the guinea pig H₁ receptor, can trigger four major intracellular signals through coupling to several G proteins that are variously modulated by extracellular Ca²⁺ and protein kinase C activation.     

Rapid Stimulation of EAAC1-Mediated Na+-Dependent l-Glutamate Transport Activity in C6 Glioma Cells by Phorbol Ester

Abstract: C6 glioma cells were used as a model system to study the regulation of EAAC1-mediated Na⁺-dependent l -[³H]glutamate transport. Although a 30-min preincubation with forskolin had no effect on transport activity, preincubation with phorbol 12-myristate 13-acetate (PMA) increased transport activity two- to threefold. PMA caused a time-dependent and concentration-dependent increase in EAAC1-mediated l -[³H]glutamate transport activity. A 2-min preincubation with PMA was sufficient to cause more than a twofold increase in transport activity and the protein synthesis inhibitor cycloheximide had no effect on the increase. These data suggest that this increase is independent of protein synthesis. The EC₅₀ value of PMA for stimulation of transport activity was 80 nM. Kinetic analyses demonstrated that the increase in transport activity was due to a 2.5-fold increase in V_max with no change in K_m. PMA also increased the transport of the nonmetabolizable analogue, d -[³H]aspartate to the same extent. In parallel assays, PMA did not, however, increase Na⁺-dependent glycine transport activity in C6 glioma. The inactive phorbol ester 4α-phorbol 12,13-didecanoate, did not stimulate l -[³H]glutamate transport activity, and the protein kinase C inhibitor chelerythrine blocked the stimulation caused by PMA. Okadaic acid and cyclosporin A, which are phosphatase inhibitors, had no effect on the stimulation of transport activity caused by PMA. The Ca²⁺ ionophore A23187 did not act synergistically to increase PMA stimulation. In previous studies, PMA caused a rapid increase in amiloride-sensitive Na⁺/H⁺ transport activity in C6 glioma. In the present study, pre- and coincubation with amiloride had no effect on the stimulation of transport activity caused by PMA. These studies suggest that activation of protein kinase C causes a rapid increase in EAAC1-mediated transport activity. This rapid increase in Na⁺-dependent l -[³H]-glutamate transport activity may provide a novel mechanism for protection against acute insults to the CNS.     

The Inhibitory Effect of Trifluoromethylphenylpiperazine on [3H]Acetylcholine Release in Guinea Pig Hippocampal Synaptosomes Is Mediated by a 5-Hydroxytryptamine1 Receptor Distinct from 1A, 1B, and 1C Subtypes

The effect of the serotonergic receptor agonist 1-(m-trifluoromethylphenyl)piperazine (TFMPP) was studied on the K(+)-evoked [3H]acetylcholine [( 3H]ACh) release from guinea pig hippocampal synaptosomes loaded with [3H]choline. TFMPP (5-1,000 microM) inhibited the evoked ACh release in a dose-dependent manner (IC50 = 81.8 microM). The inhibitory effect of TFMPP was mimicked by CGS-12066B (10, 30, and 100 microM), a 5-hydroxytryptamine1B (5-HT1B)/5-HT1D receptor agonist; 1-(m-chlorophenyl)piperazine (100 microM), a 5-HT1C/5-HT1B receptor agonist; and 5-carboxamidotryptamine (10 microM), a nonselective 5-HT1 receptor agonist. 8-Hydroxy-2-(di-n-propylamino)tetralin (10 and 100 microM), a 5-HT1A receptor agonist, and quipazine (10 and 100 microM), a 5-HT2 receptor agonist, did not have any significant effect. Serotonergic antagonists, such as dihydroergotamine (0.1 and 1 microM), metergoline (0.1 microM), methysergide (0.5 and 1 microM), or yohimbine (1 and 10 microM), blocked the TFMPP effect dose-dependently. In contrast, methiotepine (0.3 and 1 microM), propranolol (1 microM), ketanserin (0.1 microM), mesulergine (0.1 microM), ICS 205930 (0.1 and 1 microM), and spiroperidol (1 and 7 microM) did not affect the TFMPP-induced inhibition of the evoked ACh release. These data suggest that, in guinea pig hippocampus, the K(+)-evoked ACh release is modulated by a 5-HT1 receptor distinct from the 5-HT1A, 5-HT1B, and 5-HT1C subtypes.     

The C Terminal Tail of the Histamine H2 Receptor Contains Positive and Negative Signals Important for Signal Transduction and Receptor Down-Regulation

Abstract: To examine the role of the C terminal tail in H₂ receptor regulation, three cDNAs, encoding truncated histamine H₂ receptor mutants (H₂T295, H₂T307, and H₂T341), were constructed and stably transfected in Chinese hamster ovary (CHO) cells. The amino acids before position 307 appear to be necessary for proper receptor transport or folding, as no detectable H₂ receptor binding of the H₂T295 was observed after transfection. Truncation of the C terminal tail by 51 amino acids (H₂T307) did not affect the binding properties of H₂ antagonists and histamine or histamine-induced signaling. Yet, removal of 17 amino acids generated a mutant receptor (H₂T341), which was able to form a ternary complex but was unable to fully activate the G_s protein on histamine exposure. Agonist-induced but not the cyclic AMP-dependent H₂ receptor down-regulation was more profound for the H₂T307 receptor, indicating that different structural elements of the H₂ receptor protein are involved in the cyclic AMP-dependent and independent pathways of H₂ receptor down-regulation. Taken together, in this study we identified regions in the C terminal tail of the H₂ receptor that act as positive and/or negative signals in H₂ receptor signaling and down-regulation.     

Characterization of a Human NK1 Tachykinin Receptor in the Astrocytoma Cell Line U 373 MG

Abstract: The human NK₁ tachykinin receptor in the astrocytoma cell line U 373 MG was characterized using selective agonists and antagonists described for this receptor in the rat. Specific [³H]substance P binding sites were present on cell homogenates, whereas [³H]neurokinin A or [³H]-senktide binding sites were absent. The binding was saturable and reversible. The binding of [³H]substance P was inhibited by very low concentrations of [L-Pro⁹]substance P and [Sar⁹,Met(O₂)¹¹]substance P; septide was ∼ 1,000-fold less potent. The most potent peptide antagonist was trans -4-hydroxy-1-(1 H -indol-3-ylcarbonyl)-L-prolyl- N -methyl- N -(phenylmethyl)-L-tyrosineamide. The rank order of potency for the nonpeptide antagonists was ( S , S )-CP 96,345 > (±)-CP 96,345 > (±)-2-chlorobenzylquinuclidinone > ( R , R )-CP 96,345 > RP 67580 > RP 68651. In [³H]-inositol-labeled cells, substance P stimulated phosphatidylinositol turnover. A good correlation was found when the abilities of NK₁ receptor agonists for stimulating inositol phosphate production and for inhibiting [³H]substance P binding were compared. Similarly, the binding and functional assays were well correlated for the antagonists. As a result of its high sensitivity and selectivity, the U 373 MG cell line thus appears an excellent tool for investigating the pharmacology of the human NK₁ receptor.     

Lan-1: A Human Neuroblastoma Cell Line With M1 and M3 Muscarinic Receptor Subtypes Coupled to Intracellular Ca2+ Elevation and Lacking Ca2+ Channels Activated by Membrane Depolarization

The LAN-1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1-1,000 microM) caused a dose-dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to approximately 70% but completely abolished the plateau phase. The muscarinic-activated Ca2+ channel was gadolinium (Gd3+) blockade and nimodipine and omega-conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh-induced [Ca2+]i elevation was concentration-dependently inhibited by pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitive intracellular store. In addition, ryanodine did not prevent CCh-induced [Ca2+]i mobilization, and, finally, LAN-1 cells appear to lack caffeine-sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 microM), or after thapsigargin.     

The GTP-Insensitive Component of High-Affinity [3H]8-Hydroxy-2-(Di-n-Propylamino)tetralin Binding in the Rat Hippocampus Corresponds to an Oxidized State of the 5-Hydroxytryptamine1A Receptor

Previous studies on central 5-hydroxytryptamine1A (5-HT1A) receptors have consistently shown the existence of a GTP-insensitive component of agonist binding, i.e., binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) that persists in the presence of 0.1 mM GTP or guanylylimidodiphosphate (GppNHp). The molecular basis for this apparent heterogeneity was investigated pharmacologically and biochemically in the present study. The GppNHp-insensitive component of [3H]8-OH-DPAT binding increased spontaneously by exposure of rat hippocampal membranes or their 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate-soluble extracts to air; it was reduced by preincubation of solubilized 5-HT1A binding sites in the presence of dithiothreitol and, in contrast, reversibly increased by preincubation in the presence of various oxidizing reagents like sodium tetrathionate or hydrogen peroxide. In addition, exposure of hippocampal soluble extracts to short-cross-linking reagents specific for thiols produced an irreversible increase in the proportion of GppNHp-insensitive over total [3H]8-OH-DPAT binding. The pharmacological properties of this GppNHp-insensitive component of [3H]8-OH-DPAT binding were similar to those of 5-HT1A sites in the absence of nucleotide. Sucrose gradient sedimentation of solubilized 5-HT1A binding sites treated by dithiothreitol or sodium tetrathionate showed that oxidation prevented the dissociation by GTP of the complex formed by the 5-HT1A receptor binding subunit (R[5-HT1A]) and a guanine nucleotide-binding protein (G protein). Moreover, the oxidation of -SH groups by sodium tetrathionate did not prevent the inactivation of [3H]8-OH-DPAT specific binding by N-ethylmaleimide, in contrast to that expected from an interaction of both reagents with the same -SH groups on the R[5-HT1A]-G protein complex. These data suggest that the appearance of GTP-insensitive [3H]8-OH-DPAT specific binding occurs as a result of the (spontaneous) oxidation of essential -SH groups (different from those preferentially inactivated by N-ethylmaleimide) on the R[5-HT1A]-G protein complex.     

Endogenous serotonin and serotonin2C receptors are involved in the ability of M100907 to suppress cortical glutamate release induced by NMDA receptor blockade

Blockade of NMDA receptors by intracortical infusion of 3-( R )-2-carboxypiperazin-4-propyl-1-phosphonic acid (CPP) increases glutamate (GLU) and serotonin (5-HT) release in the medial prefrontal cortex and impairs attentional performance in the 5-choice serial reaction time task. These effects are prevented by the 5-HT_2A receptor antagonist, ( R )-(+)-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidine methanol (M100907). We explored the roles of endogenous 5-HT and 5-HT_1A and 5-HT_2C receptors in the mechanisms by which M100907 suppresses CPP-induced release of cortical GLU and 5-HT using in vivo microdialysis. CPP raised extracellular GLU and 5-HT by about 250% and 170% respectively. The 5-HT synthesis inhibitor, p -chlorophenylalanine (300 mg/kg), prevented M100907 suppressing CPP-induced GLU release. The effect of M100907 on these rises of GLU and 5-HT and attentional performance deficit was mimicked by the 5-HT_2C receptor agonist, ( S )-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine fumarate, (Ro60-0175, 30 μg/kg) while intra-mPFC (SB242084, 6-chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-indoline, 0.1 μM), a 5-HT_2C receptor antagonist, prevented the effect of M100907 on extracellular GLU. The 5-HT_1A receptor antagonist, N -[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]- N -(2-pyridinyl)cyclohexane carboxenide trihydrochloride (100 μM) abolished the effect of M100907 on the CPP-induced 5-HT release. The data show that blockade of 5-HT_2A receptors is not sufficient to suppress the CPP-induced rise of extracellular GLU and 5-HT and suggest that M100907 suppresses GLU release induced by CPP by enhancing the action of endogenous 5-HT on 5-HT_2C receptors.     

Neonatal 6-Hydroxydopamine and Adult SKF 38393 Treatments Alter Dopamine D1 Receptor mRNA Levels: Absence of Other Neurochemical Associations with the Enhanced Behavioral Responses of Lesioned Rats

Abstract: To study potential biochemical correlates of dopamine (DA) and serotonin receptor supersensitivity, rats were lesioned at 3 days after birth with 6-hydroxydopamine (6-OHDA; 67 µg in each lateral ventricle; desipramine pretreatment, 20 mg/kg i.p., 1 h) and then sensitized with the DA D₁ agonist, SKF 38393 HCl (3.0 mg/kg i.p. per day) either ontogenetically (daily, for 28 consecutive days from birth) and/or in adulthood (four weekly injections, 6–9 weeks from birth). Controls received vehicle in place of 6-OHDA or SKF 38393. Enhanced locomotor responses were observed after SKF 38393 at 6 weeks, only in rats that received SKF 38393 + 6-OHDA in ontogeny. Locomotor responses were further enhanced in this group after the last of four weekly SKF 38393 injections at the 9th week. These weekly SKF 38393 treatments also produced enhanced responses in 6-OHDA rats that did not receive SKF 38393 in ontogeny. When striata were studied at 11 weeks, the percentages of high and low affinity DA D₁ binding sites were not altered. Basal as well as DA-, NaF-, and forskolin-stimulated adenylyl cyclase activities also were not changed. Dot blot analysis showed that there was a reduction of mRNA levels for DA D₁, but not serotonin_1C, receptors in the 6-OHDA groups. However, SKF 38393 at 6–9 weeks eliminated this alteration. Based on these findings it can be proposed that supersensitization may be a consequence of altered neuronal cross talk rather than an imbalance of receptor elements per se.     

Induction of Myelin Components: Cyclic AMP Increases the Synthesis Rate of 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase in C6 Glioma Cells

In an effort to determine the factors that stimulate myelin synthesis, we investigated the mechanism by which dibutyryl cyclic AMP induces the activity of the myelin enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; EC 3.1.4.37), in C6 glioma cells. Immunotitration experiments and measurements of the accumulation of [35S]methionine-labeled CNP showed that dibutyryl cyclic AMP increased the amount of CNP in the cells but not the catalytic activity per molecule of the enzyme. Moreover, inhibition of protein synthesis with cycloheximide abolished induction of enzyme activity. Dibutyryl cyclic AMP doubled the rate of CNP synthesis but had no effect on the half-life of the enzyme (approximately 33 h). The induction was partially blocked by the inhibitors of mRNA synthesis, cordycepin or alpha-amanitin. Thus, cyclic AMP induces the synthesis of CNP.     

Lasting Increase in Serotonin 5-HT1A but Not 5-HT4 Receptor Subtypes in the Kindled Rat Dentate Gyrus: Dissociation from Local Presynaptic Effects

Abstract: We examined the effect of kindling on serotonergic neurotransmission in the hippocampus by measuring serotonin (5-HT) release and uptake in hippocampal synaptosomes and 5-HT_1A and 5-HT₄ receptor subtypes during and at different times after electrical kindling of the dentate gyrus. Using quantitative receptor autoradiography, we found that binding of 8-[³H]hydroxy-2-(di- n -propylamino)tetralin ([³H]8-OH-DPAT) to 5-HT_1A receptors was selectively increased by 20% on average ( p < 0.05) in the dentate gyrus of the stimulated and contralateral hippocampus 2 days after stage 2 (stereotypes and occasional retraction of a forelimb) and by 100% on average ( p < 0.05) 1 week after stage 5 (tonic-clonic seizures) compared with sham-stimulated rats. A 20% increase ( p < 0.05) was observed 1 month after the last generalized seizure. No changes were found after a single afterdischarge. 5-HT₄ receptors, which colocalize with 5-HT_1A receptors on hippocampal neurons, were not modified in kindled tissue. [³H]5-HT uptake and its release as well as the 5-HT_1B autoreceptor function did not differ from shams in hippocampal synaptosomes at stages 2 and 5. Systemic administration of 100 and 1,000 µg kg⁻¹ 8-OH-DPAT or 1,000 µg kg⁻¹ WAY-100,635, 30 min before each electrical stimulation, did not significantly alter kindling progression or the occurrence of stage 5 seizures in fully kindled rats. The changes in 5-HT_1A receptor density in the dentate gyrus are part of the plastic modifications occurring during kindling and may contribute to modulating tissue hyperexcitability.     

Identification of the 5-HT1A Receptor Binding Subunit in Rat Brain Membranes Using the Photoaffinity Probe [3H]8-Methoxy-2-[N-n-Propyl, N-3-(2-Nitro-4-Azidophenyl)Aminopropyl]Aminotetralin

The synthesis of a tritiated derivative of the 5-HT1A photoaffinity probe 8-methoxy-2-[N-n-propyl, N-3-(2-nitro-4-azidophenyl)aminopropyl]aminotetralin ([3H]8-methoxy-3'-NAP-amino-PAT) allowed the use of this probe for attempting the irreversible labeling of specific binding sites in rat brain membranes. Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis of proteins solubilized from hippocampal microsomal membranes that had been incubated with 20 nM [3H]8-methoxy-3'-NAP-amino-PAT under UV light revealed a marked incorporation of 3H label into a 63-kilodalton protein termed PI. As expected of a possible correspondence between PI and 5-HT1A receptor binding sites, 3H labeling by the photoaffinity probe could be prevented by selective 5-HT1A ligands such as 8-hydroxy-2-(di-n-propylamino)tetralin, ipsapirone, buspirone, and gepirone and by N-ethylmaleimide, but not by the 5-HT2 antagonist ketanserin, noradrenaline- and dopamine-related drugs, monoamine oxidase inhibitors, and chlorimipramine. Furthermore, the regional and subcellular distributions of PI were identical to those of specific 5-HT1A binding sites. These results indicated that the binding subunit of the 5-HT1A receptor is a 63-kilodalton protein with a functionally important sulfhydryl group(s).     

In Vitro Evaluation of 11C-Labeled (S)-Nicotine, (S)-3-Methyl-5-(1-Methyl-2-Pyrrolidinyl)isoxazole, and (R,S)-1-Methyl-2-(3-Pyridyl)azetidine as Nicotinic Receptor Ligands for Positron Emission Tomography Studies

Abstract: The binding characteristics of the novel ¹¹C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[¹¹C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [¹¹C]MPA in comparison with (S)-[¹¹C]nicotine and [¹¹C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K_D values) of 2.4 and 560 nM and binding site densities (B_max values) of 65.5 and 223 fmol/mg of protein for (S)-[¹¹C]nicotine, K_D values of 0.011 and 2.2 nM and B_max values of 4.4 and 70.7 fmol/mg of protein for [¹¹C]MPA, and K_D values of 1.3 and 33.4 nM and B_max values of 8.8 and 69.2 fmol/mg of protein for [¹¹C]ABT-418. In competing with the ¹¹C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (?)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the ¹¹C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [¹¹C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three ¹¹C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [¹¹C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [¹¹C]MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[¹¹C]nicotine and [¹¹C]ABT-418 raise the level of interest in [¹¹C]MPA for application in positron emission tomography.