Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway

Regulation of intracellular cyclic adenosine 3 ',5 '-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed G(s)-dependent receptors for isoproterenol and prostaglandin E(2). Whereas two ligands, uridine 5 '-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via G(q)/calcium and G(i), the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for G(s)-coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P(2) receptor and the heterotrimeric G(13) protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP.     

Cross-Talk between cAMP and MAPK Pathways in HSD11B2 Induction by hCG in Placental Trophoblasts

Overexposure of the fetus to glucocorticoids in gestation is detrimental to fetal development. The passage of maternal glucocorticoids into the fetal circulation is governed by 11beta-Hydroxysteroid Dehydrogenase Type 2 (HSD11B2) in the placental syncytiotrophoblasts. Human chorionic gonadotropin (hCG) plays an important role in maintaining placental HSD11B2 expression via activation of the cAMP pathway. In this study, we investigated the relationship between the activation of the cAMP pathway by hCG and subsequent phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) or p38 mitogen-activated protein kinase (MAPK) pathways in the regulation of placental HSD11B2 expression in human placental syncytiotrophoblasts. We found that treatment of the placental syncytiotrophoblasts with either hCG or dibutyl cAMP (dbcAMP) could promote the phosphorylation of p38 and ERK1/2. Inhibition of p38 MAPK with SB203580 not only reduced the basal HSD11B2 mRNA and protein levels but also attenuated HSD11B2 levels induced by either hCG or dbcAMP. By contrast, inhibition of ERK1/2 with PD98059 increased the basal mRNA and protein levels of HSD11B2 and had no effect on HSD11B2 mRNA and protein levels induced by either hCG or dbcAMP. These data suggest that p38 MAPK is involved in both basal and hCG/cAMP-induced expression of HSD11B2, and ERK1/2 may play a role opposite to p38 MAPK at least in the basal expression of HSD11B2 in human placental syncytiotrophoblasts and that there is complicated cross-talk between hCG/cAMP and MAPK cascades in the regulation of placental HSD11B2 expression.     

Molecular mechanism of thromboxane A(2)-induced platelet aggregation. Essential role for p2t(ac) and alpha(2a) receptors.

Thromboxane A(2) is a positive feedback lipid mediator produced following platelet activation. The G(q)-coupled thromboxane A(2) receptor subtype, TPalpha, and G(i)-coupled TPbeta subtype have been shown in human platelets. ADP-induced platelet aggregation requires concomitant signaling from two P2 receptor subtypes, P2Y1 and P2T(AC), coupled to G(q) and G(i), respectively. We investigated whether the stable thromboxane A(2) mimetic, (15S)-hydroxy-9, 11-epoxymethanoprosta-5Z,13E-dienoic acid (U46619), also causes platelet aggregation by concomitant signaling through G(q) and G(i), through co-activation of TPalpha and TPbeta receptor subtypes. Here we report that secretion blockade with Ro 31-8220, a protein kinase C inhibitor, completely inhibited U46619-induced, but not ADP- or thrombin-induced, platelet aggregation. Ro 31-8220 had no effect on U46619-induced intracellular calcium mobilization or platelet shape change. Furthermore, U46619-induced intracellular calcium mobilization and shape change were unaffected by A3P5P, a P2Y1 receptor-selective antagonist, and/or cyproheptadine, a 5-hydroxytryptamine subtype 2A receptor antagonist. Either Ro 31-8220 or AR-C66096, a P2T(AC) receptor selective antagonist, abolished U46619-induced inhibition of adenylyl cyclase. In addition, AR-C66096 drastically inhibited U46619-mediated platelet aggregation, which was further inhibited by yohimbine, an alpha(2A)-adrenergic receptor antagonist. Furthermore, inhibition of U46619-induced platelet aggregation by Ro 31-8220 was relieved by activation of the G(i) pathway by selective activation of either the P2T(AC) receptor or the alpha(2A)-adrenergic receptor. We conclude that whereas thromboxane A(2) causes intracellular calcium mobilization and shape change independently, thromboxane A(2)-induced inhibition of adenylyl cyclase and platelet aggregation depends exclusively upon secretion of other agonists that stimulate G(i)-coupled receptors.     

S1P1 receptor localization confers selectivity for Gi-mediated cAMP and contractile responses

Adult mouse ventricular myocytes express S1P(1), S1P(2), and S1P(3) receptors. S1P activates Akt and ERK in adult mouse ventricular myocytes through a pertussis toxin-sensitive (G(i/o)-mediated) pathway. Akt and ERK activation by S1P are reduced approximately 30% in S1P(3) and 60% in S1P(2) receptor knock-out myocytes. With combined S1P(2,3) receptor deletion, activation of Akt is abolished and ERK activation is reduced by nearly 90%. Thus the S1P(1) receptor, while present in S1P(2,3) receptor knock-out myocytes, is unable to mediate Akt or ERK activation. In contrast, S1P induces pertussis toxin-sensitive inhibition of isoproterenol-stimulated cAMP accumulation in both WT and S1P(2,3) receptor knock-out myocytes demonstrating that the S1P(1) receptor can functionally couple to G(i). An S1P(1) receptor selective agonist, SEW2871, also decreased cAMP accumulation but failed to activate ERK or Akt. To determine whether localization of the S1P(1) receptor mediates this signaling specificity, methyl-beta-cyclodextrin (MbetaCD) treatment was used to disrupt caveolae. The S1P(1) receptor was concentrated in caveolar fractions, and associated with caveolin-3 and this localization was disrupted by MbetaCD. S1P-mediated activation of ERK or Akt was not diminished but inhibition of cAMP accumulation by S1P and SEW2871 was abolished by MbetaCD treatment. S1P inhibits the positive inotropic response to isoproterenol and this response is also mediated through the S1P(1) receptor and lost following caveolar disruption. Thus localization of S1P(1) receptors to caveolae is required for the ability of this receptor to inhibit adenylyl cyclase and contractility but compromises receptor coupling to Akt and ERK.     

Polarized release of matrix metalloproteinase-2 and -9 from cultured human placental syncytiotrophoblasts

The large increase in placental surface area and fetal villous vascular development in the third trimester of pregnancy requires degradation and reformation of the placental basal lamina. Degradation is carried out by matrix metalloproteinases (MMPs) secreted by adjacent cells. Although the gelatinases, MMP-2 and MMP-9, which are released by extravillous cytotrophoblasts (CTs) are believed to play crucial roles in early placental expansion, neither has been reported in third trimester villous trophoblasts nor has appropriate (basolateral) release of any MMP by the highly polarized syncytiotrophoblast (ST) been demonstrated. We demonstrated villous trophoblast expression of both MMP-2 and MMP-9 by in situ immunohistochemistry and by Western blot analysis and zymography of lysates and culture supernatants of highly purified villous CTs. We also found that epidermal growth factor (EGF)-stimulated CT differentiation into ST and stimulation by the phorbol diester, PMA, both increase MMP-9 secretion. The direction of MMP release was determined with confluent cultures of ST on porous membranes. We found that >90% of MMP-2 and MMP-9 were released from the basolateral surface. We conclude that villous STs express and release gelatinases from their basolateral surfaces in a regulated manner and suggest that such polarized release may be important to villous tissue remodeling.     

LH and hCG Action on the Same Receptor Results in Quantitatively and Qualitatively Different Intracellular Signalling

Human luteinizing hormone (hLH) and chorionic gonadotropin (hCG) act on the same receptor (LHCGR) but it is not known whether they elicit the same cellular and molecular response. This study compares for the first time the activation of cell-signalling pathways and gene expression in response to hLH and hCG. Using recombinant hLH and recombinant hCG we evaluated the kinetics of cAMP production in COS-7 and hGL5 cells permanently expressing LHCGR (COS-7/LHCGR, hGL5/LHCGR), as well as cAMP, ERK1/2, AKT activation and progesterone production in primary human granulosa cells (hGLC). The expression of selected target genes was measured in the presence or absence of ERK- or AKT-pathways inhibitors. In COS-7/LHCGR cells, hCG is 5-fold more potent than hLH (cAMP ED₅₀: 107.1±14.3 pM and 530.0±51.2 pM, respectively). hLH maximal effect was significantly faster (10 minutes by hLH; 1 hour by hCG). In hGLC continuous exposure to equipotent doses of gonadotropins up to 36 hours revealed that intracellular cAMP production is oscillating and significantly higher by hCG versus hLH. Conversely, phospho-ERK1/2 and -AKT activation was more potent and sustained by hLH versus hCG. ERK1/2 and AKT inhibition removed the inhibitory effect on NRG1 (neuregulin) expression by hLH but not by hCG; ERK1/2 inhibition significantly increased hLH- but not hCG-stimulated CYP19A1 (aromatase) expression. We conclude that: i) hCG is more potent on cAMP production, while hLH is more potent on ERK and AKT activation; ii) hGLC respond to equipotent, constant hLH or hCG stimulation with a fluctuating cAMP production and progressive progesterone secretion; and iii) the expression of hLH and hCG target genes partly involves the activation of different pathways depending on the ligand. Therefore, the LHCGR is able to differentiate the activity of hLH and hCG.     

The Alternative Epac/cAMP Pathway and the MAPK Pathway Mediate hCG Induction of Leptin in Placental Cells

Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it works as an autocrine hormone. In this work, we demonstrated that human chorionic gonadotropin (hCG) added to JEG-3 cell line or to placental explants induces endogenous leptin expression. We also found that hCG increased cAMP intracellular levels in BeWo cells in a dose-dependent manner, stimulated cAMP response element (CRE) activity and the cotransfection with an expression plasmid of a dominant negative mutant of CREB caused a significant inhibition of hCG stimulation of leptin promoter activity. These results demonstrate that hCG indeed activates cAMP/PKA pathway, and that this pathway is involved in leptin expression. Nevertheless, we found leptin induction by hCG is dependent on cAMP levels. Treatment with (Bu)₂cAMP in combination with low and non stimulatory hCG concentrations led to an increase in leptin expression, whereas stimulatory concentrations showed the opposite effect. We found that specific PKA inhibition by H89 caused a significant increase of hCG leptin induction, suggesting that probably high cAMP levels might inhibit hCG effect. It was found that hCG enhancement of leptin mRNA expression involved the MAPK pathway. In this work, we demonstrated that hCG leptin induction through the MAPK signaling pathway is inhibited by PKA. We observed that ERK1/2 phosphorylation increased when hCG treatment was combined with H89. In view of these results, the involvement of the alternative cAMP/Epac signaling pathway was studied. We observed that a cAMP analogue that specifically activates Epac (CPT-OMe) stimulated leptin expression by hCG. In addition, the overexpression of Epac and Rap1 proteins increased leptin promoter activity and enhanced hCG. In conclusion, we provide evidence suggesting that hCG induction of leptin gene expression in placenta is mediated not only by activation of the MAPK signaling pathway but also by the alternative cAMP/Epac signaling pathway.     

The role of prostaglandins in the mechanism of lipopolysaccharide-induced proMMP9 secretion from human placenta and fetal membrane cells

The abnormal degradation of the extracellular matrix by matrix metalloproteinases (MMPs) in the fetal membranes has been proposed as a central event in preterm premature rupture of the membranes (pPROM). Prostaglandins (PGs) are thought to increase the risk of preterm premature rupture of the fetal membranes by causing matrix degradation. The aim of this study was to assess the mediating role of PGs on lipopolysaccharide (LPS)-induced MMP9 secretion in vitro. ELISA, zymography, and Western blotting were performed on cells and medium from cultures of purified chorion trophoblasts (CTs) and syncytiotrophoblasts (STs) from the human placenta and fetal membranes treated with LPS, meloxicam, (a selective prostaglandin-endoperoxide synthase 2 [PTGS2, previously known as cyclooxygenase 2] inhibitor), or replacement PGE(2) or PGF(2alpha). LPS significantly (P < 0.01) increased proMMP9 secretion and prostaglandin E(2) (PGE(2)) output by cultured CTs and STs, but there was no effect on tissue inhibitor of matrix metalloproteinase 1 (TIMP1) secretion. In these cells, meloxicam significantly blocked LPS-induced proMMP9 secretion and PGE(2) output (P < 0.01). Exogenous PGE(2) and PGF(2alpha) significantly reversed the reduction in proMMP9 secretion caused by meloxicam in a dose-dependent manner (P < 0.01). The expression of PTGS2 protein in CTs and STs was increased dramatically after LPS treatment, but there was no significant effect on the expression of PTGS1 (previously known as cyclooxygenase 1), membrane-associated prostaglandin E synthases (membrane-associated PTGES, previously known as mPGES) 1 and 2, or cytosolic prostaglandin E synthase (cytosolic PTGES, previously knows as cPGES) proteins. Our results suggest that PGs may mediate the selective increase in MMP9 after exposure of trophoblast cells to LPS. There was no effect of LPS on TIMP1. Understanding this relationship may help in developing strategies for the prevention and management of pPROM and preterm labor.     

Lysophospholipid receptor-dependent and -independent calcium signaling

Changes in cellular Ca(2+) concentrations form a ubiquitous signal regulating numerous processes such as fertilization, differentiation, proliferation, contraction, and secretion. The Ca(2+) signal, highly organized in space and time, is generated by the cellular Ca(2+) signaling toolkit. Lysophospholipids, such as sphingosine-1-phosphate (S1P), sphingosylphosphorylcholine (SPC), or lysophosphatidic acid (LPA) use this toolkit in a specific manner to initiate their cellular responses. Acting as agonists at G protein-coupled receptors, S1P, SPC, and LPA increase the intracellular free Ca(2+) concentration ([Ca(2+)](i)) by using the classical, phospholipase C (PLC)-dependent pathway as well as PLC-independent pathways such as sphingosine kinase (SphK)/S1P. The S1P(1) receptor, via protein kinase C, inhibits the [Ca(2+)](i) transients caused by other receptors. Both S1P and SPC also act intracellularly to regulate [Ca(2+)](i). Intracellular S1P mobilizes Ca(2+) in intact cells independently of G protein-coupled S1P receptors, and Ca(2+) signaling by many agonists requires SphK-mediated S1P production. As shown for the FcepsilonRI receptor, PLC and SphK may contribute specific components to the overall [Ca(2+)](i) transient. Of the many open questions, identification of the intracellular S1P target site(s) appears to be of particular importance.     

Characterization and differentiation of human first trimester placenta trophoblastic cells in culture.

A preparation of highly enriched isolated cytotrophoblasts was obtained from first trimester placenta using dispase incubation of villous tissue at 4 degrees C, followed by a spontaneous cell release at 37 degrees C. After 24 h of culture, 90-95% of the cells were immunostained by anticytokeratin antibody, showing their epithelial characteristic. After 48 h of culture, these cells differentiated into syncytiotrophoblast, as shown by optic and electron microscopic study. The secretion of hCG, and of its free alpha and beta subunits, and the secretion of hPL were studied as a function of cell culture time. While the level of secreted hCG and its free subunits was stable during 72 h of culture, the hPL level was undetectable during the first 48 h of culture, increasing continuously afterwards. Addition of dibutyryl cAMP from the start or after 96 h of cell culture induced an increase of hCG production and of its free subunits and also stimulated the secretion of hPL. This suggests that these cells maintained the capacity to respond to stimuli which increased intracellular cAMP level. Such a cell culture is of interest in further determining the mechanisms of early gestation involved in the differentiation and growth of placental cytotrophoblasts, and in the regulation of their endocrine functions.     

Methotrexate induced differentiation in colon cancer cells is primarily due to purine deprivation

The folate antagonist methotrexate (MTX) inhibits synthesis of tetrahydrofolate (THF), pyrimidines and purines, and induces differentiation in several cell types. At 1 microM, MTX reduced proliferation and induced differentiation in HT29 colon cancer cells; the latter effect was augmented (P < 0.001) by thymidine (100 microM) but was reversed (P < 0.001) by the purines, hypoxanthine (Hx; 100 microM) and adenosine (100 microM). In contrast 5-fluoro-uracil (5-FU), a specific thymidylate synthase (TS) inhibitor, had no effect on differentiation, suggesting that MTX-induced differentiation is not due to a reduction in thymidine but to the inhibition of purine biosynthesis. Inhibition of cyclic AMP (cAMP) by RpcAMP (25 microM) further enhanced (P < 0.001) MTX induced differentiation, whereas the cAMP activator forskolin (10 microM) reversed (P < 0.001) MTX induced differentiation. These observations implicate a central role of adenosine and cAMP in MTX induced differentiation. By combining Western blot analysis with liquid chromatography-mass spectrometry (LC-MS)and HPLC analyses we also reveal both the expression and activity of key enzymes (i.e. methionine synthase (MS), s-adenosylhomocysteinase, cystathionine beta-synthase and ornithine decarboxylase) regulating methyl cycle, transsulfuration and polyamine pathways in HT29 colon cancer cells. At 1 microM, MTX induced differentiation was associated with a marked reduction in the intracellular concentrations of adenosine and, consequently, S-adenosylmethionine (SAM), S-adenosylhomocysteine, polyamines and glutathione (GSH). Importantly, the marked reduction in methionine that accompanied MS inhibition following MTX treatment was non-limiting with respect to SAM synthesis. Collectively, these findings indicate that the effects of MTX on cellular differentiation and single carbon metabolism are primarily due to the intracellular depletion of purines.     

Sphingosine 1-phosphate is a ligand of the human gpr3, gpr6 and gpr12 family of constitutively active G protein-coupled receptors

Five G protein-coupled receptors (GPCRs) for the lysophospholipid sphingosine 1-phosphate (S1P) have been cloned and characterized so far. We report here about the identification of gpr3, gpr6 and gpr12 as additional members of the S1P-GPCR family. When expressed transiently in HEK293 cells, gpr3, gpr6 and gpr12 confer constitutive activation of adenylate cyclase (AC) similar in amplitude to that seen with fully activated G(alpha)(s)-coupled receptors. Culturing the transfected cells in medium with charcoal-stripped serum (devoid of lipids) significantly reduces cyclic adenosine monophosphate (cAMP) levels, suggesting a lipid-like ligand. A library containing 200 bioactive lipids was applied in functional assays recording intracellular Ca(2+) mobilization. S1P and dihydrosphingosine 1-phosphate (DHS1P) were identified as functional activators exhibiting nanomolar EC(50) values. In the presence of the S1P and LPA receptor antagonist suramin, gpr3-, gpr6- and gpr12-mediated intracellular Ca(2+) mobilization via S1P is enhanced. Besides constitutive activation of G(alpha)(s) type of G proteins, all three receptors are capable of constitutively activating inhibitory G(alpha)(i/o) proteins: (i) in the presence of pertussis toxin, gpr3-, gpr6- and gpr12-mediated stimulation of AC is enhanced; and (ii) overexpression of G(alpha)(i) significantly reduces the stimulatory action on intracellular cAMP levels. Agonist (S1P)-mediated internalization can be visualized in intact HEK293 cells using a gpr6 green fluorescent protein (GFP) fusion protein. In summary, our data suggest that gpr3, gpr6 and gpr12 are a family of constitutively active receptors with dual coupling to G(alpha)(s) and G(alpha)(i) type of G proteins. Constitutive activation of AC and mobilization of [Ca(2+)](i) can be modulated by the sphingophospholipids S1P and DHS1P, adding three additional members to the family of S1P receptors.     

Inhibition of Ca(2+) signalling by the sphingosine 1-phosphate receptor S1P(1)

The lysophospholipid, sphingosine 1-phosphate (S1P), regulates a multitude of cellular functions by activating specific G protein-coupled receptors (GPCRs) (S1P(1-5), plus three newly identified S1P receptors). The G(i)-coupled S1P(1) receptor inhibits adenylyl cyclase, stimulates mitogen-activated protein kinases (MAP kinases) and cell migration, and is required for blood vessel maturation. Here, we report that S1P(1) inhibits Ca(2+) signalling in a number of cell types. In HEK-293 cells, which endogenously express S1P(1-3), overexpression of S1P(1) reduced intracellular free Ca(2+) concentration ([Ca(2+)](i)) increases induced by various receptor agonists as well as thapsigargin. The inhibitory Ca(2+) signalling of S1P(1) was blocked by pertussis toxin (PTX) and the protein kinase C (PKC) inhibitor, G?6976, and imitated by phorbol ester and overexpression of classical PKC isoforms. Activation of S1P(1) stably expressed in RH7777 cells, which endogenously do not express S1P receptors, also inhibited Ca(2+) signalling, without mediating Ca(2+) mobilization on its own. It is concluded that the widely expressed S1P receptor S1P(1) inhibits Ca(2+) signalling, most likely via G(i) proteins and classical PKC isoforms. Co-expression of S1P(1) with S1P(3), but not S1P(2), reversed the inhibitory effect of S1P(1), furthermore suggesting a specific interplay of S1P receptor subtypes usually found within a single cell type.     

IRS-1 mediates inhibition of Ca2+ mobilization by insulin via the inhibitory G-protein Gi

Platelet agonists initiate aggregation and secretion by activating receptors coupled to the G-protein G(q), thereby raising cytosolic Ca(2+), [Ca(2+)](i). The rise in [Ca(2+)](i) is facilitated via inhibition of cAMP formation by the inhibitory G-protein of adenylyl cyclase, G(i). Since insulin attenuates platelet activation, we investigated whether insulin interferes with cAMP regulation. Here we report that insulin (0.5-200 nmol/liter) interferes with agonist-induced increases in [Ca(2+)](i) (ADP, thrombin), cAMP suppression (thrombin), and aggregation (ADP). The effects of insulin are as follows: (i) independent of the P2Y(12) receptor, which mediates ADP-induced cAMP lowering; (ii) not observed during G(s)-mediated cAMP formation; (iii) unaffected by treatments that affect phosphodiesterases (3-isobutyl-1-methylxanthine); and (iv) not changed by interfering with NO-mediated regulation of cAMP degradation (N(G)-monomethyl-l-arginine). Hence, insulin might interfere with G(i). Indeed, insulin induces the following: (i) tyrosine phosphorylation of the insulin receptor, the insulin receptor substrate-1 (IRS-1) and G(i)alpha(2); (ii) co-precipitation of IRS-1 with G(i)alpha(2) but not with other G alpha subunits. Despite persistent receptor activation, the association of IRS-1 with G(i)alpha(2) is transient, being optimal at 5 min and 1 nmol/liter insulin, which is sufficient to suppress Ca(2+) signaling by ADP, and at 10 min and 100 nmol/liter insulin, which is required to suppress Ca(2+) signaling by thrombin. Epinephrine, a known platelet sensitizer and antagonist of insulin, abolishes the effect of insulin on [Ca(2+)](i), tyrosine phosphorylation of G(i)alpha(2), and aggregation by interfering with the phosphorylation of the insulin receptor beta subunit. We conclude that insulin attenuates platelet functions by interfering with cAMP suppression through IRS-1 and G(i).     

G protein preferences for dopamine D2 inhibition of prolactin secretion and DNA synthesis in GH4 pituitary cells

Dopamine is the primary inhibitory regulator of lactotroph proliferation and prolactin (PRL) secretion in vivo, acting via dopamine D2 receptors (short D2S and long D2L forms). In GH4C1 pituitary cells transfected with D2S or D2L receptor cDNA, dopamine inhibits PRL secretion and DNA synthesis. These actions were blocked by pertussis toxin, implicating G(i)/G(o) proteins. To address roles of specific G(i)/G(o)4 proteins in these actions a series of GH4C1 cell lines specifically depleted of individual Galpha subunits was examined. D2S-mediated inhibition of BayK8644-stimulated PRL secretion was primarily dependent on G(o) over G(i), as observed for BayK8644-induced calcium influx. By contrast, inhibitory coupling of the D2S receptor to TRH-induced PRL secretion was partially impaired by depletion of any single G protein, but especially G(i)3. Inhibitory coupling of D2L receptors to PRL secretion required G(o), but not G(i)2, muscarinic receptor coupling was resistant to depletion of any G(i)/G(o) protein, whereas the 5-HT1A and somatostatin receptors required G(i)2 or G(i)3 for coupling. The various receptors also demonstrated distinct G protein requirements for inhibition of DNA synthesis: depletion of any G(i)/G(o) subunit completely uncoupled the D2S receptor, the D2L receptor was uncoupled by depletion of G(i)2, and muscarinic and somatostatin receptors were resistant to depletion of G(i)2 only. These results demonstrate distinct receptor-G protein preferences for inhibition of TRH-induced PRL secretion and DNA synthesis.     

Nrg-1 belongs to the endothelial differentiation gene family of G protein-coupled sphingosine-1-phosphate receptors

The previously cloned rat nerve growth factor-regulated G protein-coupled receptor NRG-1 (Glickman, M., Malek, R. L., Kwitek-Black, A. E., Jacob, H. J., and Lee N. H. (1999) Mol. Cell. Neurosci. 14, 141-52), also known as EDG-8, binds sphingosine-1-phosphate (S1P) with high affinity and specificity. In this paper we examined the signal transduction pathways regulated by the binding of S1P to EDG-8. In Chinese hamster ovary cells heterologously expressing EDG-8, S1P inhibited forskolin-induced cAMP accumulation and activated c-Jun NH2-terminal kinase. Surprisingly, S1P inhibited serum-induced activation of extracellular regulated protein kinase 1 and 2 (ERK1/2). Treatment with pertussis toxin, which ADP-ribosylates and inactivates G(i), blocked S1P-mediated inhibition of cAMP accumulation, but had no effect on c-Jun NH2-terminal kinase activation or inhibition of ERK1/2. The inhibitory effect of S1P on ERK1/2 activity was abolished by treatment with orthovanadate, suggesting the involvement of a tyrosine phosphatase. A subunit selective [35S] guanosine 5'-3-O-(thio)triphosphate binding assay demonstrates that EDG-8 activated G(i/o) and G12 but not Gs and G(q/11) in response to S1P. In agreement, EDG-8 did not stimulate phosphoinositide turnover or cAMP accumulation. The ability of S1P to induce mitogenesis in cells expressing the EDG-1 subfamily of G protein-coupled receptors is well characterized. In contrast, S1P inhibited proliferation in Chinese hamster ovary cells expressing EDG-8 but not empty vector. The antiproliferative effect, like S1P-mediated ERK1/2 inhibition, was orthovanadate-sensitive and pertussis toxin-insensitive. Our results indicate that EDG-8, a member of the EDG-1 subfamily, couples to unique signaling pathways.     

The inhibition of progesterone secretion and the regulation of cyclic nucleotides by atrial natriuretic factor in gonadotropin responsive murine Leydig tumor cells

We have found that atrial natriuretic factor (ANF) has a profound effect on testicular cells in altering intracellular cyclic nucleotide levels as well as progesterone secretion. Using clonal cultured Leydig tumor cells we found that 1 X 10(-8)M ANF caused a two thousand-fold elevation in the accumulation of cellular cGMP and inhibited cAMP in treated cells by more than 90% as compared to the controls. ANF (1 X 10(-8)M) also significantly inhibited gonadotropin-stimulated accumulation of cAMP in response to bovine luteinizing hormone (bLH) or human chorionic gonadotropin (hCG). Gonadotropin-stimulated progesterone secretion was inhibited by ANF (1 X 10(-10) - 1 X 10(-9)M) in these cultured Leydig tumor cells. Approximately 50% inhibition of progesterone secretion was observed at the peptide concentration of 1 X 10(-9) M.     

The effect of cAMP on differentiation inducing factor (DIF)-mediated formation of stalk cells in low-cell-density monolayers of Dictyostelium discoideum

Abstract. The requirement for cyclic AMP (cAMP) for stalk-cell formation in low-density monolayers of Dictyoste-lium discoideum , is mediated through the cell-surface cAMP receptor. Stalk-cell formation is markedly inhibited by the cAMP relay inhibitor, caffeine. This inhibition is reversed by high concentrations of 8-Br-cAMP, a highly cell-perme-abie analog of cAMP. These results suggest that elevated intracellular concentrations of cAMP are necessary for stalk-cell formation. The induction of stalk-cell formation in monolayers can be divided into a cAMP-dependent period and a differentiation inducing factor (DIF)-dependent period [21]. Despite this clearcut separation of the cAMP and DIF induction periods, cAMP enhanced the effects of DIF at low DIF concentrations. Evidence is presented that this was not a direct cooperative effect. The effect of caffeine was inhibitory during both periods and was not reversed by a combination of DIF and cAMP. Ammonia inhibited stalk-cell formation at all stages of the differentiation process, but the inhibition engendered during the initial cAMP-induction period was reversed by a combination of DIF and cAMP.     

Cyclic-AMP inhibits cell growth and negatively interacts with platelet membrane glycoprotein expression on the Dami human megakaryoblastic cell line

Intracellular signaling processes by which hematopoietic growth factors regulate megakaryocytopoiesis remain uncompletely understood. Cyclic AMP (cAMP) has been shown to be implicated in the regulation of growth and differentiation in various normal and malignant cell types. Since a few studies have suggested the possible involvement of the cAMP pathway as one of the intracellular mechanisms whereby megakaryocytopoiesis may be regulated, we investigated the functional effects of cAMP on the human megakaryoblastic Dami cell line. We observed that exposure of Dami cells to cAMP analogs or to agents elevating intracellular cAMP levels yielded dose-dependent cell growth inhibition. Cell cycle progression analysis of cells predominantly synchronized at the G1/S boundary by prior treatment with hydroxyurea revealed that cAMP transiently accumulated cells in the G2/M phase, then slowing down cell cycle. On the other hand, immunofluorescence and Northern blot analysis of megakaryocytic differentiation marker expression showed that probes we have used significantly inhibited GPlb expression. Moreover, although these agents used alone did not affect GPllb/llla expression, they markedly reversed phorbol ester-induced GPllb/llla expression increase. These inhibitory cAMP actions on glycoprotein expression were not the result of cell cycle perturbation since we observed that GPlb and GPllb/llla expression were not cell cycle dependent. All these data may then be consistent with a potential negative regulatory role of the cAMP intracellular signaling pathway during megakaryocytopoiesis. © 1995 Wiley-Liss, Inc.