A complex signaling cascade links the serotonin2A receptor to phospholipase A2 activation: the involvement of MAP kinases

Previous studies in our laboratory have shown that in NIH3T3-5HT2A cells, 5-HT-induced AA release is PLA2-coupled and independent of 5-HT2A receptor-mediated PLC activation. Although 5-HT2A receptor-mediated PLC activation is known to be Galphaq-coupled, much less is understood about 5-HT2A receptor-mediated PLA2 activation. Therefore, the studies presented here were aimed at elucidating the signal transduction pathway linking stimulation of the 5-HT2A receptor to PLA2 activation. By employing various selective inhibitors, toxins, and antagonistic peptide constructs, we propose that the 5-HT2A receptor can couple to PLA2 activation through two parallel signaling cascades. Initial experiments were designed to examine the role of pertussis toxin-sensitive G proteins, namely Galphai/o, as well as pertussis toxin-insensitive G proteins, namely Galpha12/13, in 5-HT-induced AA release. Furthermore, inactivation of both Gbetagamma heterodimers and Rho proteins resulted in decreased agonist-induced AA release, without having any effect on PLC-IP accumulation. We also demonstrated 5-HT2A receptor-mediated phosphorylation of ERK1,2 and p38. Moreover, pretreatment with selective ERK1,2 and p38 inhibitors resulted in decreased 5-HT-induced AA release. Taken together, these results suggest that the 5-HT2A receptor expressed in NIH3T3 cells can couple to PLA2 activation though a complex signaling mechanism involving both Galphai/o-associated Gbetagamma-mediated ERK1,2 activation and Galpha12/13-coupled, Rho-mediated p38 activation.     

Enhanced activation of Akt and extracellular-regulated kinase pathways by simultaneous occupancy of Gq-coupled 5-HT2A receptors and Gs-coupled 5-HT7A receptors in PC12 cells

The most commonly prescribed antidepressants, the serotonin (5-HT) selective reuptake inhibitors, increase 5-HT without targeting specific receptors. Yet, little is known about the interaction of multiple receptor subtypes expressed by individual neurons. Specifically, the effect of increases in cAMP induced by Gs-coupled 5-HT receptor subtypes on the signaling pathways modulated by other receptor subtypes has not been studied. We have, therefore, examined the activation of the extracellular-regulated kinase (ERK) and Akt pathways by Gs-coupled 5-HT7A receptors and Gq-coupled 5-HT2A receptors, which are co-expressed in discrete brain regions. Agonists for both receptors were found to activate ERK and Akt in transfected PC12 cells. 5-HT2A receptor-mediated activation of the two pathways was found to be Ca2+-dependent. In contrast, 5-HT7A receptor-mediated activation of Akt required increases in both [cAMP] and intracellular [Ca2+], while activation of ERK was inhibited by Ca2+. The activation of ERK and Akt stimulated by simultaneous treatment of cells with 5-HT2A and 5-HT7A receptor agonists was found to be at least additive. Cell-permeable cAMP analogs mimicked 5-HT7A receptor agonists in enhancing 5-HT2A receptor-mediated activation of ERK and Akt. A role was identified for the cAMP-guanine exchange factor, Epac, in this augmentation of ERK, but not Akt, activation. Our finding of enhanced activation of neuroprotective Akt and ERK pathways by simultaneous occupancy of 5-HT2A and 5-HT7A receptors may also be relevant to the interaction of other neuronally expressed Gq- and Gs-coupled receptors.     

Differential phospholipase C activation by phenylalkylamine serotonin 5-HT 2A receptor agonists

Experiments compared a series of phenethylamine hallucinogens with their phenylisopropylamine analogues for binding affinity and ability to stimulate serotonin 5-HT 2A receptor-mediated hydrolysis of phosphatidyl inositol in cells expressing cloned rat and human 5-HT 2A receptors. The (+/-)phenylisopropylamine analogues had significantly higher intrinsic activities for 5-HT 2A receptor-mediated hydrolysis of phosphatidyl inositol compared to their phenethylamine analogues. With respect to the effects of the stereochemistry of the phenylisopropylamines, those with the (R) absolute configuration at the alpha carbon had higher intrinsic activities for hydrolysis of phosphatidyl inositol in a cell line expressing the human 5-HT 2A receptor compared to those with the (S) absolute configuration. In virtual docking studies comparing the (R)- and (S)-phenylisopropylamines with their phenethylamine analogues, there were distinct differences in the orientations of key ligand binding domain residues that have been identified as important by previous mutagenesis studies. In conclusion, our data support the hypothesis that phenylisopropylamines have higher hallucinogenic potency than their phenethylamine analogues primarily because they have higher intrinsic activities at 5-HT 2A receptors. Although virtual ligand binding led to significant perturbations of certain key residues, our results emphasize the conclusion reached by others that overall three-dimensional structural microdomains within the receptor must be considered.     

CB1 Cannabinoid Receptors Couple to Focal Adhesion Kinase to Control Insulin Release

Endocannabinoid signaling has been implicated in modulating insulin release from β cells of the endocrine pancreas. β Cells express CB₁ cannabinoid receptors (CB₁Rs), and the enzymatic machinery regulating anandamide and 2-arachidonoylglycerol bioavailability. However, the molecular cascade coupling agonist-induced cannabinoid receptor activation to insulin release remains unknown. By combining molecular pharmacology and genetic tools in INS-1E cells and in vivo, we show that CB₁R activation by endocannabinoids (anandamide and 2-arachidonoylglycerol) or synthetic agonists acutely or after prolonged exposure induces insulin hypersecretion. In doing so, CB₁Rs recruit Akt/PKB and extracellular signal-regulated kinases 1/2 to phosphorylate focal adhesion kinase (FAK). FAK activation induces the formation of focal adhesion plaques, multimolecular platforms for second-phase insulin release. Inhibition of endocannabinoid synthesis or FAK activity precluded insulin release. We conclude that FAK downstream from CB₁Rs mediates endocannabinoid-induced insulin release by allowing cytoskeletal reorganization that is required for the exocytosis of secretory vesicles. These findings suggest a mechanistic link between increased circulating and tissue endocannabinoid levels and hyperinsulinemia in type 2 diabetes.     

Localization of an endocannabinoid system in the hypophysial pars tuberalis and pars distalis of man

The hypophysial pars tuberalis (PT) acts as an important interface between neuroendocrine brain centers (hypothalamus, pineal organ) and the pars distalis (PD) of the hypophysis. Recently, we have identified an endocannabinoid system in the PT of hamsters and provided evidence that 2-arachidonoylglycerol is a messenger molecule that appears to play an essential role in seasonal reproduction and prolactin release by acting on the cannabinoid receptors in the PD. We now demonstrate the enzymes involved in endocannabinoid synthesis and degradation, namely sn-1-selective diacylglycerol lipase α, N-acylphosphatidylethanolamine-specific phospholipase D, and monoacylglycerol lipase, in the PT of man by means of immunohistochemistry. High-performance liquid chromatography coupled with tandem mass spectrometry revealed 2-arachidonoylglycerol and other endocannabinoids in the human PT. Furthermore, we detected the expression of the cannabinoid receptor 1 (CB1), a primary receptor for endocannabinoids, in the PD. Double-immunofluorescence staining for CB1 and various hypophysial hormones or S-100, a marker for folliculostellate (FS) cells, revealed that CB1 immunoreactivity was mainly localized to corticotrophs and FS-cells. A limited number of lactotrophs and somatotrophs also showed CB1 immunoreactivity, which was however absent from gonadotrophs and thyrotrophs. Our data thus indicate that the human PT comprises an endocannabinoid system, and that corticotrophs and FS-cells are the main target cells for endocannabinoids. The functional significance of this newly discovered pathway remains to be elucidated in man; it might be related to the control of stress responses and/or reflect a remnant seasonal control of hypophysial hormonal secretion.     

Interaction of calmodulin with the serotonin 5-hydroxytryptamine2A receptor. A putative regulator of G protein coupling and receptor phosphorylation by protein kinase C

The 5-hydroxytryptamine2A (5-HT2A) receptor is a G(q/11)-coupled serotonin receptor that activates phospholipase C and increases diacylglycerol formation. In this report, we demonstrated that calmodulin (CaM) co-immunoprecipitates with the 5-HT2A receptor in NIH-3T3 fibroblasts in an agonist-dependent manner and that the receptor contains two putative CaM binding regions. The putative CaM binding regions of the 5-HT2A receptor are localized to the second intracellular loop and carboxyl terminus. In an in vitro binding assay peptides encompassing the putative second intracellular loop (i2) and carboxyl-terminal (ct) CaM binding regions bound CaM in a Ca2+-dependent manner. The i2 peptide bound with apparent higher affinity and shifted the mobility of CaM in a nondenaturing gel shift assay. Fluorescence emission spectral analyses of dansyl-CaM showed apparent K(D) values of 65 +/- 30 nM for the i2 peptide and 168 +/- 38 nM for the ct peptide. The ct CaM-binding domain overlaps with a putative protein kinase C (PKC) site, which was readily phosphorylated by PKC in vitro. CaM binding and phosphorylation of the ct peptide were found to be antagonistic, suggesting a putative role for CaM in the regulation of 5-HT2A receptor phosphorylation and desensitization. Finally, we showed that CaM decreases 5-HT2A receptor-mediated [35S]GTPgammaS binding to NIH-3T3 cell membranes, supporting a possible role for CaM in regulating receptor-G protein coupling. These data indicate that the serotonin 5-HT2A receptor contains two high affinity CaM-binding domains that may play important roles in signaling and function.     

Monoacylglycerols Activate TRPV1 – A Link between Phospholipase C and TRPV1

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H₁ receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H₁ receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous “entourage” compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.     

Molecular and functional characterization of proteins interacting with the C-terminal domains of 5-HT2 receptors: emergence of 5-HT2 "receptosomes"

Many cellular functions are carried out by multiprotein complexes. The last five years of research have revealed that many G-protein coupled receptor (GPCR) functions that are not mediated by G proteins involve protein networks, which interact with their intracellular domains. This review focuses on one family of GPCRs activated by serotonin, the 5-HT(2) receptor family, which comprises three closely related subtypes, the 5-HT(2A), the 5-HT(2B) and the 5-HT(2c) receptors. These receptors still raise particular interest, because a large number of psychoactive drugs including hallucinogens, anti-psychotics, anxiolytics and anti-depressants, mediate their action, at least in part, through activation of 5-HT(2) receptors. Recent studies based on two-hybrid screens, proteomic, biochemical and cell biology approaches, have shown that the C-terminal domains of 5-HT(2) receptors interact with intracellular proteins. To date, the protein network associated with the C-terminus of the 5-HT(2C) receptor has been the most extensively characterized, using a proteomic approach combining affinity chromatography, mass spectrometry and immunoblotting. It includes scaffolding proteins containing one or several PDZ domains, signalling proteins and proteins of the cytoskeleton. Data indicating that the protein complexes interacting with 5-HT(2) receptor C-termini tightly control receptor trafficking and receptor-mediated signalling will also be reviewed.     

Ca(2+)-assisted receptor-driven endocannabinoid release: mechanisms that associate presynaptic and postsynaptic activities

Endogenous cannabinoids (endocannabinoids) serve as retrograde messengers at synapses in various regions of the brain. They are released from postsynaptic neurons and cause transient and long-lasting reduction of neurotransmitter release through activation of presynaptic cannabinoid receptors. Endocannabinoid release is induced either by increased postsynaptic Ca(2+) levels or by activation of G(q/11)-coupled receptors. When these two stimuli coincide, endocannabinoid release is markedly enhanced, which is attributed to the Ca(2+) dependency of phospholipase Cbeta (PLCbeta). This Ca(2+)-assisted receptor-driven endocannabinoid release is suggested to participate in various forms of synaptic plasticity, including short-term associative plasticity in the cerebellum and spike-timing-dependent long-term depression in the somatosensory cortex. In these forms of plasticity, PLCbeta seems to function as a coincident detector of presynaptic and postsynaptic activities.     

Secretory phospholipase A(2) inhibits epidermal growth factor-induced receptor activation

Secretory phospholipase A(2) (sPLA(2)) plays important roles in mediating various cellular processes, including cell proliferation, differentiation, apoptosis, and inflammatory response. In this study, we demonstrated that a basic sPLA(2) inhibits epidermal growth factor (EGF)-induced EGF receptor activation, as determined by autophosphorylation of EGF receptor, EGF-activated phospholipase D (PLD) activity, and phospholipase C-gamma(1) (PLC-gamma(1)) tyrosine phosphorylation in a human epidermoid carcinoma cell line, A-431. Treatment of cells with exogenous neutral sphingomyelinase (SMase) or a cell permeable ceramide analog, C(2)-ceramide, also caused similar inhibitory effects on EGF-induced activation of EGF receptor, tyrosine phosphorylation of PLC-gamma(1), and the activation of PLD. sPLA(2)-induced inhibition of EGF receptor was associated with arachidonic acid release, which was followed by an increase in intracellular ceramide formation. Both sPLA(2) and exogenous C(2)-ceramide are able to inhibit the proliferation of A-431. The data presented indicate for the first time that sPLA(2) downregulates the EGF receptor-mediated intracellular signal transduction that may be mediated by arachidonic acid and/or ceramide.     

Involvement of phosphatidylcholine-specific phospholipase C in thromboxane A2-induced activation of mitogen-activated protein kinase in astrocytoma cells

Thromboxane A2 (TXA2) receptor-mediated signal transduction was investigated in 1321N1 human astrocytoma cells. 9,11-Epithio-11,12-methano-TXA2 (STA2), a TXA2 receptor agonist, induced Ca2+ mobilization and phosphoinositide hydrolysis in a concentration-dependent manner. These responses were inhibited by treatment with U73122, an inhibitor of phosphatidylinositol-specific phospholipase C, or by culturing in 0.5% fetal calf serum containing 0.5 mM dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP) for 2 days. However, the dbcAMP treatment augmented the TXA2 receptor-mediated phosphorylation of mitogen-activated protein kinase (MAPK). These results were confirmed by a functional MAPK assay measuring the incorporation of 32P into the MAPK substrate peptide. The TXA2 receptor-mediated MAPK activation was inhibited by SQ29548, a TXA2 receptor antagonist, and GF109203X, an inhibitor of protein kinase C. Although U73122 did not inhibit or only slightly inhibited the activation of MAPK, D-609, an inhibitor of phosphatidylcholine-specific phospholipase C, potently attenuated the activation in a concentration-dependent manner. Furthermore, STA2 accelerated the release of [3H]choline metabolites from the cells prelabeled with [3H]choline chloride. This release was inhibited by treatment with D-609. These results suggest that phosphatidylcholine-specific phospholipase C and protein kinase C, but not phosphatidylinositol-specific phospholipase C, are involved in TXA2 receptor-mediated MAPK activation in 1321N1 human astrocytoma cells.     

Interactions Between Phospholipase C-Coupled and N-Methyl-D-Aspartate Receptors in Cultured Cerebellar Granule Cells: Protein Kinase C Mediated Inhibition of N-Methyl-D-Aspartate Responses

The N-methyl-D-aspartate (NMDA) receptor of rat cerebellar granule cells in primary culture is inhibited by phospholipase C-coupled receptor activation. In the absence of ionotropic agonist, cells modulate their cytoplasmic free Ca2+, [Ca2+]c, in response to stimulation of M3 muscarinic receptors, metabotropic glutamate receptors, and endothelin receptors by the respective agonists carbachol, trans-1-amino-1,3-cyclopentanedicarboxylic acid, and endothelin-1. The response is consistent with the ability of phospholipase C-coupled receptors to release a pool of intracellular Ca2+ and induce a subsequent Ca2+ entry into the cell; both of these responses can be abolished by discharge of internal Ca2+ stores with low concentrations of ionomycin or thapsigargin. In the case of cells stimulated with NMDA, the [Ca2+]c response to the phospholipase C-coupled agonists is complex and agonist dependent; however, in the presence of ionomycin each agonist produces a partial inhibition of the NMDA component of the [Ca2+]c signal. This inhibition can be mimicked by the protein kinase C activator 4 beta-phorbol 12,13-dibutyrate. It is concluded that NMDA receptors on cerebellar granule cells are inhibited by phospholipase C-coupled muscarinic M3, glutamatergic, and endothelin receptors via activation of protein kinase C.     

Human adipose tissue binds and metabolizes the endocannabinoids anandamide and 2-arachidonoylglycerol

Endocannabinoids are a group of biologically active endogenous lipids that have recently emerged as important mediators in energy balance control. The two best studied endocannabinoids, anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) are the endogenous ligands of the central and peripheral cannabinoid receptors. Furthermore, AEA binds to the transient receptor potential vanilloid type-1 (TRPV1), a capsaicin-sensitive, non-selective cation channel. The synthesis of these endocannabinoids is catalyzed by the N-acylphosphatidylethanolamine-selective phospholipase D (NAPE-PLD) and the sn-1-selective diacylglycerol lipase (DAGL), whereas their degradation is accomplished by the fatty acid amide hydrolase (FAAH) and the monoglyceride lipase (MGL), respectively. We investigated the presence of a functional endocannabinoid system in human adipose tissue from seven healthy subjects. Subcutaneous abdominal adipose tissue underwent biochemical and molecular biology analyses, aimed at testing the expression of this system and its functional activity. AEA and 2-AG levels were detected and quantified by HPLC. Real time PCR analyzed the expression of the endocannabinoid system and immunofluorescence assays showed the distribution of its components in the adipose tissue. Furthermore, binding assay for the cannabinoid and vanilloid receptors and activity assay for each metabolic enzyme of the endocannabinoid system gave clear evidence of a fully operating system. The data presented herein show for the first time that the human adipose tissue is able to bind AEA and 2-AG and that it is endowed with the biochemical machinery to metabolize endocannabinoids.     

Endocannabinoids in the central nervous system

The major psychoactive component of cannabis derivatives, delta9-THC, activates two G-protein coupled receptors: CB1 and CB2. Soon after the discovery of these receptors, their endogenous ligands were identified: lipid metabolites of arachidonic acid, named endocannabinoids. The two major main and most studied endocannabinoids are anandamide and 2-arachidonyl-glycerol. The CB1 receptor is massively expressed through-out the central nervous system whereas CB2 expression seems restricted to immune cells. Following endocannabinoid binding, CB1 receptors modulate second messenger cascades (inhibition of adenylate cyclase, activation of mitogen-activated protein kinases and of focal-adhesion kinases) as well as ionic conductances (inhibition of voltage-dependent calcium channels, activation of several potassium channels). Endocannabinoids transiently silence synapses by decreasing neurotransmitter release, play major parts in various forms of synaptic plasticity because of their ability to behave as retrograde messengers and activate non-cannabinoid receptors (such as vanilloid receptor type-1), illustrating the complexity of the endocannabinoid system. The diverse cellular targets of endocannabinoids are at the origin of the promising therapeutic potentials of the endocannabinoid system.     

The evolution and comparative neurobiology of endocannabinoid signalling

CB₁- and CB₂-type cannabinoid receptors mediate effects of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide in mammals. In canonical endocannabinoid-mediated synaptic plasticity, 2-AG is generated postsynaptically by diacylglycerol lipase alpha and acts via presynaptic CB₁-type cannabinoid receptors to inhibit neurotransmitter release. Electrophysiological studies on lampreys indicate that this retrograde signalling mechanism occurs throughout the vertebrates, whereas system-level studies point to conserved roles for endocannabinoid signalling in neural mechanisms of learning and control of locomotor activity and feeding. CB₁/CB₂-type receptors originated in a common ancestor of extant chordates, and in the sea squirt Ciona intestinalis a CB₁/CB₂-type receptor is targeted to axons, indicative of an ancient role for cannabinoid receptors as axonal regulators of neuronal signalling. Although CB₁/CB₂-type receptors are unique to chordates, enzymes involved in biosynthesis/inactivation of endocannabinoids occur throughout the animal kingdom. Accordingly, non-CB₁/CB₂-mediated mechanisms of endocannabinoid signalling have been postulated. For example, there is evidence that 2-AG mediates retrograde signalling at synapses in the nervous system of the leech Hirudo medicinalis by activating presynaptic transient receptor potential vanilloid-type ion channels. Thus, postsynaptic synthesis of 2-AG or anandamide may be a phylogenetically widespread phenomenon, and a variety of proteins may have evolved as presynaptic (or postsynaptic) receptors for endocannabinoids.     

Mutagenesis analysis of the serotonin 5-HT2C receptor and a Caenorhabditis elegans 5-HT2 homologue: conserved residues of helix 4 and helix 7 contribute to agonist-dependent activation of 5-HT2 receptors

An alignment of serotonin [5-hydroxytryptamine (5-HT)] G protein-coupled receptors identified a lysine at position 4.45 (helix 4) and a small polar residue (serine or cysteine) at 7.45 (helix 7) that occur exclusively in the 5-HT2 receptor family. Other serotonin receptors have a hydrophobic amino acid, typically a methionine, at 4.45 and an invariant asparagine at 7.45. The functional significance of these class-specific substitutions was tested by site-directed mutagenesis of two distantly related 5-HT2 receptors, Caenorhabditis elegans 5-HT2ce and rat 5-HT2C. Residues 4.45 and 7.45 were each mutated to a methionine and asparagine, respectively, or an alanine and the resulting constructs were tested for activity. A K4.45M mutation decreased serotonin-dependent activity (Emax) of the rat 5-HT2C receptor by 60% and that of the C. elegans homologue by 40%, as determined by a fluorometric plate-based calcium assay. The rat mutant also exhibited nearly sixfold higher agonist binding affinity and significantly lower constitutive activity compared with wildtype. Mutagenesis of S7.45 in the C. elegans receptor increased serotonin binding affinity by up to 25-fold and decreased Emax by up to 65%. The same mutations of the cognate C7.45 in rat 5-HT2C produced a smaller fourfold change in the affinity for serotonin and decreased agonist efficacy by up to 50%. Substitutions of S/C7.45 did not produce a significant change in the basal activity of either receptor. All mutants tested exhibited levels of receptor expression similar to the corresponding wildtype based on measurements of specific [3H]-mesulergine binding or flow cytometry analyses. Taken together, these results suggest that K4.45 and S/C7.45 play an important role in the conformational rearrangements leading to agonist-induced activation of 5-HT2 receptors.     

Deletion of the serotonin 5-HT2C receptor PDZ recognition motif prevents receptor phosphorylation and delays resensitization of receptor responses

Phosphorylation-deficient serotonin 5-HT(2C) receptors were generated to determine whether phosphorylation promotes desensitization of receptor responses. Phosphorylation of mutant 5-HT(2C) receptors that lack the carboxyl-terminal PDZ recognition motif (Ser(458)-Ser-Val-COOH; DeltaPDZ) was not detectable based on a band-shift phosphorylation assay and incorporation of (32)P. Treatment of cells stably expressing DeltaPDZ or wild-type 5-HT(2C) receptors with serotonin produced identical maximal responses and EC(50) values for eliciting [(3)H]inositol phosphate formation. In calcium imaging studies, treatment of cells expressing DeltaPDZ or wild-type 5-HT(2C) receptors with 100 nm serotonin elicited initial maximal responses and decay rates that were indistinguishable. However, a second application of serotonin 2.5 min after washout caused maximal responses that were approximately 5-fold lower with DeltaPDZ receptors relative to wild-type 5-HT(2C) receptors. After 10 min, responses of DeltaPDZ receptors recovered to wild-type 5-HT(2C) receptor levels. Receptors with single mutations at Ser(458) (S458A) or Ser(459) (S459A) decreased serotonin-mediated phosphorylation to 50% of wild-type receptor levels. Furthermore, subsequent calcium responses of S459A receptors were diminished relative to S458A and wild-type receptors. These results establish that desensitization occurs in the absence of 5-HT(2C) receptor phosphorylation and suggest that receptor phosphorylation at Ser(459) enhances resensitization of 5-HT(2C) receptor responses.     

Pertussis toxin-sensitive GTP-binding proteins may regulate phospholipase A2 in response to thrombin in rabbit platelets

Incubation of rabbit platelets with thrombin resulted in rapid accumulations of inositol trisphosphate (IP3) in [3H]inositol-labeled platelets, increases of [3H]arachidonic acid [( 3H]AA) release, and [3H]serotonin secretion from the platelets prelabeled with these labeled compounds. The experiments using phospholipase A2 or C inhibitor suggested that not only phospholipase C but also phospholipase A2 activity plays an important role in serotonin secretion. We then studied the regulatory mechanisms of phospholipase A2 activity. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), guanyl-5'-(beta,gamma-iminio)triphosphate), or AlF4- caused a significant liberation of AA in digitonin-permeabilized platelets but not in intact platelets. Thrombin-stimulated AA release was not observed in permeabilized platelets, whereas thrombin acted synergistically with GTP or GTP analogs to stimulate AA release. GTP analog-stimulated AA release was inhibited by guanosine 5'-(2-O-thio)diphosphate) and was also inhibited by decreased Mg2+ concentrations. Thrombin-induced, GTP-dependent AA release, but not IP3 formation, was diminished by 100 ng/ml of pertussis toxin, associated with ADP-ribosylation of membrane 41-kDa protein(s). Thrombin-stimulated AA release from intact platelets and GTP gamma S-stimulated release from permeabilized platelets were both markedly dependent on Ca2+. However, Ca2+ addition could not enhance AA release without GTP gamma S even when Ca2+ was increased up to 10(-4) M in permeabilized platelets. The results show that thrombin-stimulated AA release from rabbit platelets is mainly mediated by phospholipase A2 activity, not by phospholipase C activity, and that Ca2+ is an important factor to the activation of phospholipase A2 but is not the sole factor to the regulation. GTP-binding protein(s) is involved in receptor-mediated activation of phospholipase A2.     

Enhancement of hippocampal cholinergic neurotransmission through 5-HT1A receptor-mediated pathways by repeated lithium treatment in rats

Hippocampal cholinergic neuronal activity is reported to be regulated, at least partly, through serotonin1A (5-HT1A) receptors. Chronic lithium treatment has been shown to alter both behavioral and neurochemical responses mediated by postsynaptic 5-HT1A receptors. We investigated whether long-term lithium treatment affects central cholinergic neurotransmission through 5-HT1A receptor-mediated pathways. Changes in acetylcholine (ACh) release induced by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT1A receptor agonist, in the rat hippocampus were measured using a microdialysis technique and a radioimmunoassay for ACh. Administration of lithium for 21 days resulted in a serum lithium concentration of 1.03 mM and caused little change in density or affinity of [3H]8-OH-DPAT binding sites in the hippocampus. The local application of 8-OH-DPAT into the hippocampus of lithium treated rats increased the ACh efflux in both the absence and the presence of physostigmine, a cholinesterase (ChE) inhibitor, in the perfusion fluid. The basal ACh efflux of lithium treated rats was not different from that of the control rats under normal conditions, but was significantly higher than that of the controls when ChE was inhibited. These results demonstrate that chronic lithium treatment increases spontaneous ACh release in the hippocampus under conditions of ChE inhibition, but not under normal conditions, and enhances cholinergic neurotransmission through 5-HT1A receptor-mediated pathways, and suggest that activation of 5-HT1A receptor function by lithium is related to the enhancement of hippocampal cholinergic neurotransmission.