Receptor-mediated Phosphoinositide Hydrolysis Research Articles

The cannabinoid CB2 receptor positive allosteric modulator EC21a exhibits complicated pharmacology in vitro

Schizophrenia is a complex disease involving the dysregulation of numerous brain circuits and patients exhibit positive symptoms (hallucinations, delusions), negative symptoms (anhedonia), and cognitive impairments. We have shown that the antipsychotic efficacy of positive allosteric modulators (PAMs) of both the M4 muscarinic receptor and metabotropic glutamate receptor 1 (mGlu1) involve the retrograde activation of the presynaptic cannabinoid type-2 (CB2) receptor, indicating that CB2 activation or potentiation could result in a novel therapeutic strategy for schizophrenia. We used two complementary assays, receptor-mediated phosphoinositide hydrolysis and GIRK channel activation, to characterize a CB2 PAM scaffold, represented by the compound EC21a, to explore its potential as a starting point to optimize therapeutics for schizophrenia. These studies revealed that EC21a acts as an allosteric inverse agonist at CB2 in both assays and exhibits a mixed allosteric agonist/negative allosteric modulator profile at CB1 depending upon the assay used for profiling. A series of compounds related to EC21a also functioned as CB2 inverse agonists. Overall, these results suggest that EC21a exhibits complicated and potentially assay-dependent pharmacology, which may impact interpretation of in vivo studies.

Kinin-Stimulated B1 Receptor Signaling Depends on Receptor Endocytosis Whereas B2 Receptor Signaling Does Not

Kinins are potent pro-inflammatory peptides that act through two G protein-coupled receptor subtypes, B1 (B1R) and B2 (B2R). Kinin-stimulated B2R signaling is often transient, whereas B1R signaling is sustained. This was confirmed by monitoring agonist-stimulated intracellular Ca(2+) mobilization in A10 smooth muscle cells expressing human wild-type B2R and B1R. We further studied the role of receptor membrane trafficking in receptor-mediated phosphoinositide (PI) hydrolysis in model HEK293 cell lines stably expressing the receptors. Treatment of cells with brefeldin A, to inhibit maturation of de novo synthesized receptors, or hypertonic sucrose, to inhibit receptor endocytosis, showed that the basal cell surface receptor turnover was considerably faster for B1R than for B2R. Inhibition of endocytosis, which stabilized B1R on the cell surface, inhibited B1R signaling, whereas B2R signaling was not perturbed. Signaling by a B1R construct in which the entire C-terminal domain was deleted remained sensitive to inhibition of receptor endocytosis, whereas signaling by a B1R construct in which this domain was substituted with the corresponding domain in B2R was not sensitive. B2R and B1R co-expression, which also appeared to stabilize B1R on the cell surface, presumably by receptor hetero-dimerization, also inhibited B1R signaling, whereas B2R signaling was slightly enhanced. Furthermore, the B2R-specific agonist bradykinin (BK) directed both receptors through a common endocytic pathway, whereas the B1R-specific agonist Lys-desArg(9)-BK was unable to do so. These results suggest that B1R-mediated PI hydrolysis depends on a step in receptor endocytosis, whereas B2R-mediated PI hydrolysis does not. We propose that B1R uses at least part of the endocytic machinery to sustain agonist-promoted signaling.

P2Y2 Receptor-Gq/11 Signaling at Lipid Rafts Is Required for UTP-Induced Cell Migration in NG 108-15 Cells

Lipid rafts, formed by sphingolipids and cholesterol within the membrane bilayer, are believed to have a critical role in signal transduction. P2Y(2) receptors are known to couple with G(q) family G proteins, causing the activation of phospholipase C (PLC) and an increase in intracellular Ca(2+) ([Ca(2+)](i)) levels. In the present study, we investigated the involvement of lipid rafts in P2Y(2) receptor-mediated signaling and cell migration in NG 108-15 cells. When NG 108-15 cell lysates were fractionated by sucrose density gradient centrifugation, Galpha(q/11) and a part of P2Y(2) receptors were distributed in a fraction where the lipid raft markers, cholesterol, flotillin-1, and ganglioside GM1 were abundant. Methyl-beta-cyclodextrin (CD) disrupted not only lipid raft markers but also Galpha(q/11) and P2Y(2) receptors in this fraction. In the presence of CD, P2Y(2) receptor-mediated phosphoinositide hydrolysis and [Ca(2+)](i) elevation were inhibited. It is noteworthy that UTP-induced cell migration was inhibited by CD or the G(q/11)-selective inhibitor YM254890 [(1R)-1-{(3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-[(1R)-1-methoxyethyl]-4,9,10,12,16, 22-hexamethyl-15-methylene-2,5,8,11,14,17,-20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl}-2-methylpropyl rel-(2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate]. Moreover CD and YM254890 completely inhibited Rho-A activation. Downstream of Rho-A signaling, stress fiber formation and phosphorylation of cofilin were also inhibited by CD or YM254890. However, UTP-induced phosphorylation of cofilin was not affected by the expression of p115-regulator of G protein signaling, which inhibits the G(12/13) signaling pathway. This implies that UTP-induced Rho-A activation was relatively regulated by the G(q/11) signaling pathway. These results suggest that lipid rafts are critical for P2Y(2) receptor-mediated G(q/11)-PLC-Ca(2+) signaling and this cascade is important for cell migration in NG 108-15 cells.

Combined Treatment with Citalopram and Buspirone: Effects on Serotonin 5-HT2Aand 5-HT2CReceptors in the Rat Brain

We wanted to elucidate whether the proposed advantages of citalopram-buspirone combination treatment are related to changes in 5-HT(2A/C) receptor-mediated neurotransmission. The affinity of buspirone to 5-HT2A and 5-HT2C receptors was measured in vitro, and the influence of buspirone on 5-HT2C receptor-mediated phosphoinositide hydrolysis was estimated. Four groups of rats received citalopram (10 mg/kg), buspirone (6 mg/kg), citalopram-buspirone combination, or saline once a day s.c. for 14 days. Treatment effects on 5-HT2A and 5-HT2C receptors were investigated by receptor autoradiography with antagonist and agonist radioligands. Buspirone was found to be a weak 5-HT2C receptor antagonist, with a low affinity for 5-HT2A and 5-HT2C receptors. Repeated buspirone-citalopram combination treatment markedly decreased [3H]ketanserin and [125I]DOI binding to 5-HT2A receptors. Repeated administration of buspirone and buspirone-citalopram combination increased the affinity of [3H]mesulergine toward 5-HT2C receptors, and buspirone-citalopram combination also decreased [125I]DOI binding to 5-HT2C receptors. We suggest that downregulation of brain 5-HT2A receptors and possibly of 5-HT2C receptor agonist sites is involved in the beneficial clinical effects of buspirone-SSRI augmentation treatment. Furthermore, a conversion of brain 5-HT2C receptors from high- to low-affinity state may provide an additional mechanism for the anti-anxiety effects of buspirone.

Serotonin 5-HT 2C receptor-mediated phosphoinositide hydrolysis in rat choroid plexus after fluoxetine and citalopram treatments

Selective serotonin reuptake inhibitors (SSRIs) bind directly to various neurotransmitter receptors. The clinical effects of SSRIs appear gradually during weeks of treatment, suggesting a role for adaptive changes in neurotransmitter receptors. Most clinically used antidepressants, e.g. fluoxetine, bind to 5-HT 2C receptors. When administered chronically, many antidepressants elicit adaptive regulation of 5-HT 2C receptors. The present study was conducted in order to determine the effects of acute and chronic fluoxetine and citalopram treatments on the density and function of 5-HT 2C receptors in the rat choroid plexus. Acute and chronic treatments followed by phosphoinositide (PI) hydrolysis assays and quantitative receptor autoradiography were performed. Acute (single-dose) treatment with neither drug significantly affected basal or 5-HT-stimulated PI hydrolysis, but acute citalopram (20 mg/kg) treatment increased both agonist and antagonist binding to 5-HT 2C receptors. Chronic (14 days) citalopram treatment (20 mg/kg) increased the maximal PI hydrolysis response by 40%, but fluoxetine lacked this effect. The present data suggest that sensitisation of 5-HT 2C receptor-mediated intracellular signal transduction may play a role in the effects of citalopram. In contrast, fluoxetine treatment does not functionally sensitise 5-HT 2C receptors. Thus, functional 5-HT 2C receptor sensitisation is not a common effect of antidepressants, but the differential effects may explain some of the pharmacodynamic differences seen with these drugs, especially upon repeated administration.

Decrease in thromboxane A 2 receptor expression by differentiation with dibutyryl cyclic AMP in 1321N1 human astrocytoma cells☆

Thromboxane A 2 (TXA 2) receptor expression with its signaling was investigated in 1321N1 human astrocytoma cells differentiated with dibutyryl cyclic AMP (dbcAMP). The cells cultured in 0.5% fetal calf serum containing 0.5 mM dbcAMP for 3 days showed the star-shaped morphology, accompanied with the reduction of a TXA 2 mimetic U46619-induced phosphoinositide hydrolysis and Ca 2+ mobilization. Immunoblotting analysis revealed that human astrocytoma cells expressed phospholipase C (PLC)-β1 and -β3, but not PLC-β2. The contents of PLC-β1 and β3 were not changed by the differentiation. The α subunit of G q/11 bound to TXA 2-receptor was reduced by the differentiation, determined by immunoblotting after immunoprecipitation with an anti-TXA 2-receptor antibody. Scatchard analysis of the binding of [ 3H]SQ29548, a TXA 2 receptor antagonist, to the membranes revealed that the maximum binding site was reduced by the differentiation. The expression of TXA 2 receptor mRNA also was reduced by the differentiation, determined by reverse-transcribed-polymerase chain reaction. Although placental type of TXA 2 receptor mRNA expression increased after the differentiation, endothelial type of TXA 2 receptor mRNA expression slightly decreased. The results suggest that 1321N1 human astrocytoma cells differentiated with dbcAMP show impaired TXA 2 receptor-mediated phosphoinositide hydrolysis and Ca 2+ mobilization, due to the decrease in TXA 2 receptor number.

Nonselective effects of the putative phospholipase C inhibitor, U73122, on adenosine A 1 receptor-mediated signal transduction events in Chinese hamster ovary cells

Adenosine A 1 receptors can signal, through G i/o proteins, to inhibit adenylyl cyclase activity and also to stimulate phosphoinositide hydrolysis and the subsequent release of intracellular Ca 2+ stores. The aminosteroid U73122 (1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione) has been widely used as an inhibitor of phospholipase C, the enzyme mediating phosphoinositide hydrolysis. Using U73122, we sought to selectively block signalling through the phospholipase C pathway, in Chinese hamster ovary (CHO-K1) cells heterologously expressing human adenosine A 1 receptors. U73122 inhibited A 1 receptor-mediated phosphoinositide hydrolysis, as measured by total inositol phosphate accumulation, over the concentration range 1–15 μM. However, over the same concentration range, it also appeared to inhibit A 1 receptor-mediated inhibition of forskolin-stimulated cyclic AMP accumulation, A 1 receptor agonist-promoted [ 35S]GTPγS binding, and at the higher concentrations (10–15 μM) produced marked morphological changes, leading to cytolysis. The structural analogue of U73122, U73343 (1-[6-[[17β-3-methoxyestra-1,3,5(10-trien-17-yl]amino]hexyl]-2,5-pyrrolidone-dione), typically used as an inactive control compound, had little effect on these events. The data suggest that U73122 is not a selective inhibitor of phospholipase C activity, interfering with adenosine A 1 receptor signalling generally, either at the pre-effector level involving G i/o proteins, or as a consequence of the morphological changes it induces.

Protein kinase and Ca2+ modulation of myo-inositol transport in cultured retinal pigment epithelial cells.

The acute regulation of inwardly directed Na(+)-myo-inositol (MI) cotransporter activity and basal and volume-sensitive MI efflux by protein kinases C (PKC) and A (PKA), cytosolic Ca2+, and phosphoinositide (PI) turnover were characterized in cultured human retinal pigment epithelial cells using 2-[3H]MI and liquid scintillation spectrometry. Kinetic analysis revealed two distinct Na(+)-MI cotransporter components differing in apparent Michaelis constant and maximal velocity. Composite Na(+)-MI cotransport activity was stimulated by PKA activation, the muscarinic agonist carbachol, and the Ca2+ ionophore A-23187 and was inhibited by PKC activation. PKC activation also increased MI efflux, but only the volume-sensitive component, whereas PKA activation increased both basal and volume-sensitive MI efflux. These studies implicate PKC as a negative modulator of MI content through Na(+)-MI cotransport inhibition and potentiation of volume-sensitive MI efflux. PKA is a positive modulator of both Na(+)-MI cotransport and basal and volume-sensitive MI efflux. Cytosolic Ca2+ release through receptor-mediated PI hydrolysis may facilitate Na(+)-MI cotransport activity.

Inhibition of 5-hydroxytryptamine-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine cultured tracheal smooth muscle cells by phorbol ester.

1. Regulation of the increase in inositol-1,4,5-trisphosphate (IP3) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in canine cultured tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by 5-hydroxytryptamine (5-HT) caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. 2. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min blocked the 5-HT-induced IP3 formation and Ca2+ mobilization. This inhibition was reduced after the cells had been incubated with PMA for 8 h, and within 48 h the 5-HT-induced Ca2+ mobilization reached the same extent as control cells. 3. The concentration of PMA that gave half-maximal inhibition of 5-HT-induced increase in [Ca2+]i was 4 nM. Pretreatment of TSMCs with staurosporine (1 microM) of GF109203X (0.1 microM), PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. 4. In parallel with the effect of PMA on 5-HT-induced IP3 formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blot analysis in TSMCs. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TSMCs expressed PKC-alpha, beta I, beta II, delta, epsilon, theta and zeta. With PMA treatment of the cells for various times, translocation of PKC-alpha, beta I, beta II, delta, epsilon, and theta from the cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 24 h treatment caused a partial down-regulation of these PKC isozymes PKC-zeta was not significantly translocated and down-regulated at any of the times tested. 5. In conclusion, these results suggest that activation of PKC may inhibit the receptor-mediated phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit both responses independently. The translocation of PKC-alpha, beta I, beta II, delta, epsilon, and theta induced by PMA caused an attenuation of 5-HT-stimulated IP3 accumulation and Ca2+ mobilization in TSMCs.

Thrombin Receptor-Dependent Prostaglandin E2Synthesis in Hamster Fibroblasts: Synergistic Interactions with Interleukin-1β

Cellular responses to α-thrombin are mediated through a G-protein-coupled receptor that undergoes proteolytic cleavage, unveiling a tethered peptide ligand with the amino-terminal sequence SFLLRN. The synthetic peptide SFLLRN can mimic many of thrombin's actions via directly stimulating the thrombin receptor. Thrombin has been implicated in several cellular events associated with tissue injury, including fibroblast growth, matrix deposition, and inflammatory responses. The role of the thrombin receptor in fibroblast-dependent release of the inflammatory mediator prostaglandin E2was evaluated and compared to its well-characterized effect on cell proliferation. Both thrombin and SFLLRN stimulated [3H]thymidine incorporation into DNA but failed to induce prostaglandin E2release from CCL39 cells. The inflammatory cytokine interleukin-1β synergized with thrombin and SFLLRN to induce the release of prostaglandin E2, whereas it had no effect on thrombin receptor-mediated DNA synthesis. Interleukin-1β had no direct effects on thrombin receptor-mediated phosphoinositide hydrolysis, suggesting that its effects were downstream from early signal transduction events. Thrombin and interleukin-1β together significantly increased the expression of prostaglandin H synthase-2 in accordance with the prostaglandin E2results. These studies indicate that the fibroblast thrombin receptor differentially couples to intracellular signaling pathways leading to distinct functional responses and that thrombin receptor–effector interactions could be modulated by interleukin-1β.

Serotonin2C receptors and serotonin2C receptor-mediated phosphoinositide hydrolysis in the brain of alcohol-preferring and alcohol-nonpreferring rats.

To examine the role of serotonin2C (5HT2C) receptors in alcohol drinking behavior, the binding indices of 5HT2C receptors were determined in various brain regions of alcohol-preferring (P) and alcohol-nonpreferring (NP) rats. 5HT2C receptor-mediated phosphoinositide hydrolysis in the choroid plexus of P and NP rats was also determined. It was observed that the densities of 5HT2C receptors are significantly higher in the hippocampus, the amygdala, and the choroid plexus, but not in the cortex of P rats compared with NP rats. The Kd values of [3H]mesulergine binding to 5HT2C receptors were not different in these brain regions of P rats compared with NP rats. It was also observed that 5HT-stimulated [3H]inositol 1-phosphate formation was significantly higher in the choroid plexus of P rats compared with NP rats. The results of this study indicate that the numbers of 5HT2C receptors are higher in the hippocampus, the amygdala, and the choroid plexus, and that 5HT2C receptor-mediated phosphoinositide hydrolysis is more elevated in the choroid plexus of P rats compared with NP rats. Thus, it seems from these results that increased 5HT2C receptors may be involved in the genetic vulnerability to alcohol drinking behavior.

Inhibition of muscarinic stimulated phosphoinositide hydrolysis in the rat parotid gland by cAMP

The ability of agents that increase or mimic cAMP to affect muscarinic receptor mediated phosphoinositide hydrolysis was investigated in the rat parotid gland. Forskolin (10 μM) and isoproterenol (10 μM) elevated cAMP in the parotid gland by 2-fold and 7-fold, respectively, and these agents also inhibited oxotremorine-M (3 H-M) mediated phosphoinositide hydrolysis by 14% and 26%, respectively. Forskolin (1,4.3, 18, and 75 μM) increased cAMP accumulation and inhibited PIP 2 hydrolysis in a concentration-dependent manner. Forskolin (75 μM) shifted the concentration-response curve for the full agonist oxotremorine-M rightward by 4.2-fold. Pre-treatment with the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) reduced the maximum effect of oxotremorine-M by 31%. The inhibitory effect of isoproterenol and forskolin on muscarinic receptor-mediated phosphoinositide hydrolysis was unaffected by the removal of extracellular Ca 2+. Moreover, isoproterenol and forskolin dampened sodium fluoride and oxotremorine-M mediated phosphoinositide hydrolysis to the same extent suggesting that the inhibitory effect of cAMP is downstream from the muscarinic receptor.

Increases in cyclic AMP levels couple to H1 receptors in atria from autoimmune myocarditis mice

We have previously shown that myocardium from experimental autoimmune myocarditis expresses H1 receptors not present in normal mice heart. ThEA acting via H1 receptors, augments cyclic AMP production in atria from autoimmune myocarditis mice without any effect on atria from control mice. Addition of mepyramine before ThEA caused cyclic AMP levels to fall to a level similar to basal, confirming the H1 receptor participation. Histamine at low concentrations mimicked the ThEA action on H1 receptor-stimulation of cyclic AMP production by autoimmune myocardium. The fact that the inhibition of phospholipase C blocked the cyclic AMP stimulation by ThEA, supports the assumption that this action is secondary to receptor-mediated hydrolysis of phosphoinositides, generating some oxidative metabolites (IP3-DAG), which in turn may be responsible for the cyclic AMP effect. So, the inhibition of protein kinase C and calcium/ calmodulin partially prevented the stimulatory action of ThEA on cyclic AMP levels in autoimmune myocardium, suggesting that both pathways are implicated in this effect. Data shows that the stimulation of H1 receptors by specific agonist in atria from autoimmune myocarditis mice, augments the cyclic AMP, requiring the hydrolysis of phosphoinositide cycle. The role of this cyclic AMP augmentation in myocardium from autoimmune myocarditis mice, will provide a basis to assess the role of this second messenger as an important factor in the regulation and/or modulation of the physiological behaviour of the heart in the course of autoimmune myocarditis.

The role of the 5-HT2A and 5-HT2C receptors in the stimulus effects of hallucinogenic drugs. III: The mechanistic basis for supersensitivity to the LSD stimulus following serotonin depletion.

The present study was designed to determine the effects of p-chlorophenylalanine (PCPA) and p-chloroamphetamine (PCA) administration on (1) the levels of serotonin (5-hydroxytryptamine, 5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in rat brain, (2) the sensitivity of LSD-trained rats to the stimulus effects of LSD, and (3) the maximal levels of 5-HT2A and 5-HT2C receptor mediated phosphoinositide (PI) hydrolysis in rat brain. PCA and PCPA both produced a significant depletion of whole brain 5-HT and 5-HIAA concentrations. The depletion of serotonin with PCPA, but not PCA, resulted in supersensitivity of LSD-trained subjects to the stimulus effects of LSD. Neither PCPA nor PCA treatment altered the maximal level of 5-HT2A receptor-mediated PI hydrolysis. However, PCPA, but not PCA, treatment resulted in a significant upregulation (46%, P < 0.05) of the maximal level of 5-HT2C receptor mediated PI hydrolysis. These data suggest that upregulation of the 5-HT2C receptor mediates the supersensitivity to LSD discriminative stimulus which follows the depletion of central nervous system serotonin by PCPA.

Pharmacological evidence for the existance of different subtypes of muscarinic acetylcholine receptors for phosphoinositide hydrolysis in neonatal versus adult rat atria

The negative inotropic effect of carbachol, as well as phosphoinositide hydrolysis, was measured in atria from neonatal and adult rats. Carbachol increased phosphoinositide hydrolysis and decreased dF dt of both neonatal and adult atria; however, the neonatal atria showed hyperactivity to carbachol as compared with adult atria. Inhibition of phospholipase C reduced the supersensitivity to carbachol upon contractility in neonatal atria producing values similar to those of the adult atria, suggesting that muscarinic acetylcholine receptor (mAchR) stimulation is secondary to receptor-mediated hydrolysis of phosphoinositides. Pharmacological analysis with mAchR antagonists tends to support the idea that ml and m2 subtypes are the most important mediators of the response to carbachol in neonatal atria. In adult atria, the effect of carbachol is coupled only to mAchR m2 subtypes.

Distinct Pathways of Gi- and Gq-mediated Mitogen-activated Protein Kinase Activation

Receptors that couple to the heterotrimeric G proteins, Gi or Gq, can stimulate phosphoinositide (PI) hydrolysis and mitogen-activated protein kinase (MAPK) activation. PI hydrolysis produces inositol 1,4,5-trisphosphate and diacylglycerol, leading to activation of protein kinase C (PKC), which can stimulate increased MAPK activity. However, the relationship between PI hydrolysis and MAPK activation in Gi and Gq signaling has not been clearly defined and is the subject of this study. The effects of several signaling inhibitors are assessed including expression of a peptide derived from the carboxyl terminus of the beta adrenergic receptor kinase 1 (beta ARKct), which specifically blocks signaling mediated by the beta gamma subunits of G proteins (G beta gamma), expression of dominant negative mutants of p21ras (RasN17) and p74raf-1 (N delta Raf), protein-tyrosine kinase (PTK) inhibitors and cellular depletion of PKC. The Gi-coupled alpha 2A adrenergic receptor (AR) stimulates MAPK activation which is blocked by expression of beta ARKct, RasN17, or N delta Raf, or by PTK inhibitors, but unaffected by cellular depletion of PKC. In contrast, MAPK activation stimulated by the Gq-coupled alpha 1B AR or M1 muscarinic cholinergic receptor is unaffected by expression of beta ARKct or RasN17 expression or by PTK inhibitors, but is blocked by expression of N delta Raf or by PKC depletion. These data demonstrate that Gi- and Gq-coupled receptors stimulate MAPK activation via distinct signaling pathways. G beta gamma is responsible for mediating Gi-coupled receptor-stimulated MAPK activation through a mechanism utilizing p21ras and p74raf independent of PKC. In contrast, G alpha mediates Gq-coupled receptor-stimulated MAPK activation using a p21ras-independent mechanism employing PKC and p74raf. To define the role of G beta gamma in Gi-coupled receptor-mediated PI hydrolysis and MAPK activation, direct stimulation with G beta gamma was used. Expression of G beta gamma resulted in MAPK activation that was sensitive to inhibition by expression of beta ARKct, RasN17, or N delta Raf or by PTK inhibitors, but insensitive to PKC depletion. By comparison, G beta gamma-mediated PI hydrolysis was not affected by beta ARKct, RasN17, or N delta Raf expression or by PTK inhibitors. Together, these results demonstrate that G beta gamma mediates MAPK activation and PI hydrolysis via independent signaling pathways.

Tunicamycin inhibits prostaglandin F2 alpha receptor-mediated phosphoinositide hydrolysis in cultured rat astrocytes.

Effect of tunicamycin, an inhibitor of N-linked glycosylation, on prostaglandin (PG) F2 alpha-stimulated phosphoinositide (PI) hydrolysis was examined in cultured rat astrocytes. Pretreatment of cultured astrocytes with tunicamycin (25-250 ng/ml) inhibited subsequent PGF2 alpha (1 microM)-stimulated PI-hydrolysis in concentration- and time-dependent manners. The inhibition completely recovered after removal of tunicamycin and re-incubation for 12 h. Tunicamycin pretreatment (100 ng/ml for 12 h) significantly blocked [35S]methionine incorporation into cultured astrocytes, but cell viability was not affected under the condition. Inhibitors of processing of N-linked sugar chains such as bromoconduritol, 1-deoxymannojirimycin, and swainsonine had no effect on PI response to PGF2 alpha. These observations suggest that PGF2 alpha receptor is N-linked glycosylated.

Evidence that tolerance to the anxiogenic-like effects of mCPP does not involve alteration in the function of 5-HT(2C) receptors in the rat choroid plexus.

The mechanisms by which 1-(3-chlorophenyl) piperazine (mCPP) causes anxiety are unclear, but it has been suggested that the serotonin 5-HT(2C) receptor subtype may be involved in this effect. We have therefore studied the effect of chronic treatment (3 weeks) with mCPP in two animal models of anxiety (light/dark choice task in mice and elevated plus-maze test in rats) and subsequently assessed the function of 5-HT(2C) receptors (measured by maximal stimulation of 5-HT(2C) receptor-mediated phosphoinositide hydrolysis) in rat choroid plexus, where the receptor is present at very high levels. mCPP treatment regimens led to a tolerance to the anxiogenic-like action of the drug, but failed to alter the second messenger coupling of the 5-HT(2C) receptors in the choroid plexus, thereby suggesting the involvement of different mechanisms in this behavioral effect.

Differential effects of polychlorinated biphenyl congeners on phosphoinositide hydrolysis and protein kinase C translocation in rat cerebellar granule cells

Previous reports from our laboratory have suggested that the neuroactivity of some polychlorinated biphenyl (PCB) congeners is associated with perturbations in cellular Ca 2+-homeostasis. We have characterized further the neurochemical effects of PCBs on signal transduction in primary cultures of cerebellar granule cells. The present experiments found that neither 2,2′-dichlorobiphenyl (DCBP), an ortho-substituted congener, nor 3,3′,4,4′,5-pentachlorobiphenyl (PCBP), a non- ortho-substituted congener, affected basal phosphoinositide (PI) hydrolysis in cerebellar granule cells. However, at concentrations up to 50 μM, DCBP potentiated carbachol-stimulated PI hydrolysis, while decreasing it at 100 μM. PCBP, on the other hand, had no effect on carbachol-stimulated PI hydrolysis in concentrations up to 100 μM. [ 3H]Phorbol ester ([ 3H]PDBu) binding was used to determine protein kinase C (PKC) translocation. DCBP increased [ 3H]PDBu binding in a concentration-dependent manner and a twofold increase was observed at 100 μM in cerebellar granule cells. PCBP had no effect on [ 3H]PDBu binding at concentrations up to 100 μM. The effect of DCBP on [ 3H]PDBu binding was time-dependent and was also dependent on the presence of external Ca 2+ in the medium. To test the hypothesis that DCBP increases [ 3H]PDBu binding by acting on receptor-activated calcium channels, the effects of DCBP were compared to those of l-glutamate. The effects of DCBP (50 μM) and glutamate (20 μM) were additive. MK-801, a non-competitive NMDA antagonist, blocked the effects of glutamate, but had no effect on the DCBP-induced increase in [ 3H]PDBu binding. Other pharmacological pretreatments such as incubations with 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP; competitive NMDA antagonist), 6-cyano-7-nitro quinoxaline-2,3-dione (CNQX; AMPA antagonist), verapamil (Ca 2+-channel antagonist) or tetrodotoxin (Na + channel antagonist) also had no effect on DCBP-induced increased in [ 3H]PDBu binding. These studies indicate that DCBP, a putative neuroactive PCB congener, has a biphasic effect on receptor-mediated PI hydrolysis and causes translocation of PKC in cerebellar granule cells.

Regulation of serotonin 5-HT 2C receptors in the rat choroid plexus after acute clozapine treatment

We studied the effects of acute clozapine and haloperidol treatments on 5-HT 2C receptor binding characteristics and 5-HT 2C receptor-mediated phosphoinositide hydrolysis in the rat choroid plexus. Scatchard analysis (with [ 3H]mesulergine) showed that acute clozapine treatment (10 and 25 mg/kg) decreased the density ( B max) of 5-HT 2C receptors by 20–25% with no marked change in the affinity ( K d). Quantitative autoradiography was in accordance with homogenate binding studies showing that acute clozapine treatment, unlike haloperidol (0.5 mg/kg), decreased the number of both agonist ([ 125I](±)-1-(2,5-dimethoxy-4-iodophenyl)- -2-aminopropane, [ 125I]DOI) and antagonist ([ 3H]mesulergine) labeled 5-HT 2C receptor binding sites. The decrease was more robust with the higher dose of clozapine. For comparison, both doses of clozepine, unlike haloperidol, decreased equally the density of 5-HT 2A receptors in the frontal cortex by about 45%, whereas none of the treatments altered dopamine D 2 receptor characteristics in the striatum. The K d value of 5-HT 2A receptors was significantly increased after the dose of 25 mg/kg of clozapine. These clozapine treatments failed to decrease the maximal 5-HT 2C receptor-mediated phosphoinositide hydrolysis response. The higher dose of clozapine increased 5-HT-induced phosphoinositide hydrolysis response, but also decreased significantly the basal levels of phosphoinositide hydrolysis. Haloperidol did not significantly affect the 5-HT 2C receptor-mediated phosphoinositide hydrolysis. To summarize, the present data show that a single injection of clozapine is able to reduce the density of 5-HT 2C receptors but fails to cause functional desensitization of 5-HT 2C receptors in the rat choroid plexus.