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5-HT1A receptors have been hypothesized to mediate some of the neuronal plasticity and behavioral responses stimulated by serotonin selective reuptake inhibitors. Although the cellular signaling pathways required for inducing these actions have not yet been determined, roles for the neuroprotective extracellular-regulated kinase (ERK) mitogen-activated protein (MAP) kinase and Akt pathways have been suggested. In the current studies we have utilized primary cultures to directly determine whether hippocampal 5-HT1A receptors couple to activation of Akt and ERK. We found that E18 hippocampal neurons exhibit a twofold activation of Akt when exposed to nanomolar concentrations of 5-HT. The 5-HT1/7 receptor-selective agonist 5-carboxamidotryptamine maleate (5-CT) and the 5-HT1A/7 receptor-selective agonist 8-hydroxy-N,N-dipropyl-aminotetralin (8-OH-DPAT) maleate were found to activate Akt with equal efficacy, and similar potency, to 5-HT. p-MPPI and WAY-100635, antagonists selective for 5-HT1A receptors, completely inhibited 5-CT- stimulated Akt activation. Activation of Akt was also inhibited by pretreatment with pertussis toxin as well as the phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002. In contrast, the 5-HT selective antagonist, SB269970, caused no inhibition. Although the density of 5-HT1A receptors expressed by cultured neurons was sufficient to activate Akt, no activation of ERK was observed. These findings suggest that Akt, and not ERK, may be relevant to previous reports of hippocampal 5-HT1A receptors mediating neurotrophic responses. [ABSTRACT FROM AUTHOR]

Mesangial cell apoptosis has been proposed as a means of resolution of glomerular hypercellularity in proliferative forms of glomerular disease. We previously demonstrated that adenosine causes mesangial cell apoptosis by stimulating the A3-type adenosine receptor. This is a G protein-coupled receptor shown to activate kinases involved in apoptotic signaling. In this work, we assessed changes in phosphorylation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)1/2 and in levels of specific pro- and antiapoptotic proteins following exposure of mesangial cells to the A3 adenosine receptor agonist N(6)-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA). Cultured mesangial cells were incubated with IB-MECA for 30 minutes and 6, 24, and 48 hours. IB-MECA was used at a concentration (30 μM) that induces a reproducible degree of mesangial cell apoptosis. Changes in ERK1/2 phosphorylation and in protein levels of Bcl-2, Bax, and caspase 3 were assessed by Western blot analysis. IB-MECA markedly increased phosphorylation of ERK1/2. This effect peaked at 5 minutes, dissipated by 20 minutes, and was abolished by the inhibitor of ERK phosphorylation, compound U0126, in a dose-dependent manner. This inhibitor had no effect on the extent of IB-MECA-induced apoptosis. Bcl-2 levels progressively declined, whereas those of Bax and activated caspase 3 increased. These observations indicate that stimulation of the A3-type adenosine receptor causes mesangial cell apoptosis via mechanisms independent of ERK activation. The observations also point to an imbalance in the expression of antiapoptotic (Bcl-2) and proapoptotic (Bax, caspase 3) proteins as a potential mechanism underlying adenosine-induced mesangial cell apoptosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The roles of 3′,5′-cyclic adenosine monophosphate (cAMP) and protein kinase A in 5-hydroxytryptamine (5-HT)7 receptor-mediated activation of extracellular-regulated kinase (ERK) were studied in cultured hippocampal neurons and transfected PC12 cells. Activation of ERK by neuronal Gs -coupled receptors has been thought to proceed through a protein kinase A-dependent pathway. In fact we identified coupling of 5-HT7 receptors to activation of adenylyl cyclase and protein kinase A. However, no inhibition of agonist-stimulated ERK activation was found when cells were treated with H-89 and KT5720 at concentrations sufficient to completely inhibit activation of protein kinase A. However, activation of ERK was found to be sensitive to the adenylyl cyclase inhibitor 9-(tetrahydrofuryl)-adenine, suggesting a possible role for a cAMP-guanine nucleotide exchange factor (cAMP-GEF). Co-treatment of cells with 8-(4-chlorophenylthio)-2′- O -methyladenosine 3′,5′-cyclic monophosphate, a direct activator of the cAMP-GEFs Epac1 and 2, reversed the inhibition of agonist-stimulated ERK activation induced by adenylyl cyclase inhibition. Additionally, over-expression of Epac1 enhanced 5-HT7 receptor-mediated activation of ERK. These results demonstrate that the activation of ERK mediated by neuronal Gs -coupled receptors can proceed through cAMP-dependent pathways that utilize cAMP-GEFs rather than protein kinase A. [ABSTRACT FROM AUTHOR]

1: Since all 5-HT(1) receptors couple to G(i)-type G proteins and inhibit adenylyl cyclase, the functional significance of five distinct subtypes of 5-HT(1) receptors has been unclear. 2: In previous studies we have used transfected cells to demonstrate that 5-HT(1B) receptors can couple more efficiently than 5-HT(1A) receptors to activation of extracellular signal-regulated kinase (ERK) and to inhibition of adenylyl cyclase. These findings suggested the possibility that individual 5-HT(1) receptors differentially couple to isoforms of G(ialpha). 3: In the present study we utilized a model system in which pertussis toxin resistant forms of human G(ialpha1), G(ialpha2), and G(ialpha3) were used to directly compare the coupling of human 5-HT(1A), 5-HT(1B), and 5-HT(1D) receptors to each G(ialpha) in transfected human HeLa cells. 4: 5-HT(1A) receptors displayed a preference for G(ialpha1) and G(ialpha2), relative to G(ialpha3). Pertussis toxin resistant forms of G(ialpha1), G(ialpha2), and G(ialpha3) rescued 73%, 76%, and 44%, respectively, of the ERK activation stimulated by 5-HT in the absence of pertussis toxin. 5: In contrast, pertussis toxin resistant forms of G(ialpha1), G(ialpha2), and G(ialpha3) rescued 32%, 118%, and 35% of 5-HT(1B) receptor-stimulated activity, respectively, indicating that 5-HT(1B) receptors coupled primarily through G(ialpha2). A similar preference for G(ialpha2) was found in studies of the 5-HT(1D) receptor, where toxin resistant G(ialpha1), G(ialpha2), and G(ialpha3) rescued 30%, 70%, and 40% of activity, respectively. 6: In conclusion, the observed differential coupling of 5-HT(1) receptors to isoforms of G(ialpha), provides additional evidence for our previous findings that the subtypes of 5-HT(1) receptors exhibit similar, but distinct, functions. [ABSTRACT FROM AUTHOR]

Abstract: Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins for heterotrimeric G proteins. One of the best-studied RGS proteins, RGS4, accelerates the rate of GTP hydrolysis by all Gi and Gq α subunits yet has been shown to exhibit receptor selectivity. Although RGS4 is expressed primarily in brain, its effect on modulating the activity of serotonergic receptors has not yet been reported. In the present study, transfected BE(2)-C human neuroblastoma cells expressing human 5-HT1B receptors were used to demonstrate that RGS4 can inhibit the coupling of 5-HT1B receptors to cellular signals. Serotonin and sumatriptan were found to stimulate activation of extracellular signal-regulated kinase. This activation was attenuated, but not completely inhibited, by RGS4. Similar inhibition by RGS4 of the protein kinase Akt was also observed. As RGS4 is expressed at high levels in brain, these results suggest that it may play a role in regulating serotonergic pathways. [ABSTRACT FROM AUTHOR]

Although the subtypes of serotonin 5-HT1 receptors have distinct structure and pharmacology, it has not been clear if they also exhibit differences in coupling to cellular signals. We have sought to compare directly the coupling of 5-HT1A and 5-HT1B receptors to adenylyl cyclase and to the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase-2). We found that 5-HT1B receptors couple better to activation of ERK2 and inhibition of adenylyl cyclase than do 5-HT1A receptors. 5-HT stimulated a maximal fourfold increase in ERK2 activity in nontransfected cells that express endogenous 5-HT1B receptors at a very low density and a maximal 13-fold increase in transfected cells expressing 230 fmol of 5-HT1B receptor/mg of membrane protein. In contrast, activation of 5-HT1A receptors stimulated only a 2.8-fold maximal activation of ERK2 in transfected cells expressing receptors at 300 fmol/mg of membrane protein but did stimulate a 12-fold increase in activity in cells expressing receptors at 3,000 fmol/mg of membrane protein. Similarly, 5-HT1A, but not 5-HT1B, receptors were found to cause significant inhibition of forskolin-stimulated cyclic AMP accumulation only when expressed at high densities. These findings demonstrate that although both 5-HT1A and 5-HT1B receptors have been shown to couple to G proteins of the Gi class, they exhibit differences in coupling to ERK2 and adenylyl cyclase. [ABSTRACT FROM AUTHOR]

5-Hydroxytryptamine (5-HT)(2A) receptors have been implicated to play a role in both the treatment and pathophysiology of a number of psychiatric disorders. Therefore, the coupling of this receptor to signals, such as extracellular signal-regulated kinase (ERK), that elicit long-term neuronal changes may be relevant. In the present study we examined the coupling of the G(q)-coupled receptor to ERK in PC12 cells, a cell line commonly used as a neuronal model system. Activation of ERK occurred through a pathway different than the protein kinase C-dependent pathways described previously in studies of non-neuronal cells. Activation of ERK, in PC12 cells, was inhibited by both chelation of extracellular Ca(2+) and by depletion of intracellular Ca(2+) stores. Surprisingly, activation was not inhibited, but actually potentiated, by a variety of protein kinase C inhibitors covering all known protein kinase C isoforms. In contrast, the coupling of receptor to activation of ERK was found to be sensitive to N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7) and N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide (W13), inhibitors of calmodulin, but not to 1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine (KN62) and 2-[N-(2-hydroxyethyl)]-N-4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) (KN93), inhibitors of calmodulin-dependent protein kinase. Additionally, the general tyrosine kinase inhibitor genistein, as well as the Src inhibitor PP1 and the epidermal growth factor receptor kinase inhibitor 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG 1478), inhibited receptor-mediated activation of ERK, suggesting a role for tyrosine kinases. In fact, 5-HT was found to stimulate tyrosine phosphorylation of a number of proteins, and this phosphorylation was inhibited by W7. 5-HT(2A) receptor-activation of ERK through a protein kinase C-independent pathway requiring Ca(2+)/calmodulin/tyrosine kinases represents a pathway distinct from those described in studies of non-neuronal cells.