Regulation of responsiveness at D2 dopamine receptors by receptor desensitization and adenylyl cyclase sensitization.

The regulation of cellular responsiveness to dopamine via the D2 dopamine receptor was investigated in mouse fibroblast Ltk-cells stably expressing the rat D2-short receptor [Nature (Lond.) 336:783-787 (1988)]. Dopamine inhibited forskolin-stimulated cAMP levels in these cells (half-maximal inhibition at 3.9 +/- 1.1 nM), and the inhibition by dopamine was blocked by D2 antagonists and was pertussis toxin sensitive. Treatment of these cells with the D2 agonist quinpirole (1 microM) resulted in desensitization of dopaminergic inhibition of forskolin-stimulated cAMP accumulation, with a approximately 4-fold decrease in the potency of dopamine after 1 hr of treatment. No significant changes in total cellular D2 receptor concentrations were observed, even after prolonged agonist treatment. At longer time points, basal and forskolin-stimulated cellular cAMP levels were increased in treated cells. The effect of D2 agonist treatment on membrane adenylyl cyclase (EC 4.6.1.1) activity was examined. Basal and forskolin- and prostaglandin E1-stimulated adenylyl cyclase activities were increased by quinpirole treatment for 24 hr. This sensitization of adenylyl cyclase was blocked by the presence of a D2 antagonist. Pertussis toxin pretreatment blocked the sensitization of adenylyl cyclase by quinpirole, although pertussis toxin also caused increased adenylyl cyclase activity on its own. Sensitization was not dependent upon dopaminergic inhibition of intracellular cAMP levels, because quinpirole treatment in the presence of membrane-permeable cAMP analogs or 3-isobutyl-1-methylxanthine (an inhibitor of cAMP phosphodiesterase) resulted in greater sensitization of adenylyl cyclase activity than quinpirole treatment alone. These results suggest that, in this model system, responsiveness to dopamine via the D2 receptor is regulated by both desensitization of receptor function and sensitization of the stimulatory adenylyl cyclase pathway.     

Characterization of determinants of ligand binding to the nicotinic acid receptor GPR109A (HM74A/PUMA-G)

The G-protein-coupled receptor GPR109A (HM74A/PUMA-G) has recently been shown to function as a receptor for nicotinic acid (niacin) and to mediate its antilipolytic effects. Nicotinic acid is able to strongly raise plasma levels of high-density lipoprotein cholesterol, a property that distinguishes nicotinic acid from other lipid-lowering drugs. To investigate the structural determinants of GPR109A ligand binding, we performed site-directed mutagenesis of putative ligand binding residues combined with generation of chimeric receptors consisting of GPR109A and its close relative GPR109B, which does not bind nicotinic acid. We could identify Asn86/Trp91 [transmembrane helix (TMH) 2/extracellular loop (ECL) 1], Arg111 (TMH3), Ser178 (ECL2), Phe276 (TMH7), and Tyr284 (TMH7) as amino acid residues critical for binding of nicotinic acid. Together with data from molecular modeling studies, our data suggest that the ligand binding pocket for nicotinic acid of GPR109A is distinct from that of most other group A receptors. Although Arg111 at TMH3 serves as the basic anchor point for the carboxylate ligands, the ring system of nicotinic acid is embedded between Trp91 at the junction TMH2/ECL1 and Phe276/Tyr284 at TMH7. The heterocyclic ring is also bound to Ser178 at ECL2 via an H-bond. These data will facilitate the design of new antidyslipidemic drugs acting via GPR109A.     

Role of a pertussis toxin sensitive G-protein in mediating the effects of phorbol esters on receptor activated cyclic AMP accumulation in Jurkat cells

Summary In the human T -cell line, Jurkat, the accumulation of cyclic AMP induced by adenosine is enhanced by tumor-promoting phorbol esters, whereas prostaglandin E₂ receptor-stimulated cAMP accumulation is antagonized (Nordstedt et al. 1989). In the present study we examine the involvement of pertussis toxin sensitive guanine nucleotide binding proteins (G-proteins) in producing the phorbol ester effects.Pertussis toxin pretreatment of the Jurkat cells invariably caused an ADP ribosylation of two G-proteins that inhibit adenylyl cyclase, tentatively identified as G_i2 and G_i3, using Western blots. Pertussis toxin treatment had little effect on basal cAMP accumulation, but sometimes inhibited, sometimes stimulated agonist and cholera toxin induced cAMP accumulation. The latter effect was not mimicked by the B-oligomer. Irrespective of whether pertussis toxin stimulated or inhibited NECA and cholera toxin-induced cAMP accumulation it could not block the effect of phorbol-12,13-dibutyrate (PDBu). The inhibitory effect of PDBu on prostaglandin E2-induced cAMP accumulation was, however, invariably eliminated by pertussis toxin treatment.In conclusion, activation of protein kinase C by phorbol esters reveals a G_i-mediated prostaglandin E receptor-induced inhibition of adenylate cyclase in addition to the prostaglandin E receptor-mediated stimulation of cAMP accumulation in Jurkat cells. The enhancement of adenosine A₂ receptor stimulated CAMP accumulation by PDBu, on the other hand, does not involve a PTX sensitive G_i-protein. Send offprint requests to I. van der Ploeg at the above address     

Short- and long-term regulation of adenylyl cyclase activity by delta-opioid receptor are mediated by Galphai2 in neuroblastoma N2A cells

Activation of the opioid receptor results in short-term inhibition of intracellular cAMP levels followed by receptor desensitization and subsequent increase of cAMP above the control level (adenylyl cyclase superactivation). Using adenovirus to deliver pertussis toxin-insensitive mutants of the alpha-subunits of G(i/o) that are expressed in neuroblastoma Neuro2A cells (Galpha(i2), Galpha(i3), and Galpha(o)), we examined the identities of the G proteins involved in the short- and long-term action of the delta-opioid receptor (DOR). Pertussis toxin pretreatment completely abolished the ability of [d-Pen(2), d-Pen(5)]-enkephalin (DPDPE) to inhibit forskolin-stimulated intracellular cAMP production. Expression of the C352L mutant of Galpha(i2), and not the C351L mutants of Galpha(i3) or Galpha(o), rescued the short-term effect of DPDPE after pertussis toxin treatment. The ability of Galpha(i2) in mediating DOR inhibition of adenylyl cyclase activity was also reflected in the ability of Galpha(i2), not Galpha(i3) or Galpha(o), to coimmunoprecipitate with DOR. Coincidently, after long-term DPDPE treatment, pertussis toxin treatment eliminated the antagonist naloxone-induced superactivation of adenylyl cyclase activity. Again, only the C352L mutant of Galpha(i2) restored the adenylyl cyclase superactivation after pertussis toxin treatment. More importantly, the C352L mutant of Galpha(i2) remained associated with DOR after long-term agonist and pertussis toxin treatment whereas the wild-type Galpha(i2) did not. These data suggest that Galpha(i2) serves as the signaling molecule in both DOR-mediated short- and long-term regulation of adenylyl cyclase activity.     

Epidermal growth factor activation of rat parotid gland adenylate cyclase and mediation by a GTP-binding regulatory protein.

Injection of rats with a single dose of epidermal growth factor (EGF) or isoproterenol increased parotid gland acinar cell levels of cyclic AMP (cAMP) significantly above control basal concentrations (34, 177 and 11.5 pmol/g tissue/100 g body weight, respectively). Following a chronic regimen of isoproterenol (3 days), EGF, bovine galactosyltransferase (Gal Tase, EC 2.4.1.22) and isoproterenol increased cAMP levels, albeit to a lower level than observed for the single dose (21, 17 and 51 pmol, respectively). Using isolated parotid gland membranes, EGF and bovine galactosyltransferase also stimulated adenylate cyclase (EC 2.7.4.3) activity in a concentration-dependent manner. Introduction of the beta-adrenergic receptor antagonist propranolol blocked isoproterenol-stimulated adenylate cyclase activity and cAMP accumulation, but not that observed with EGF or the transferase treatment. Growth factor-stimulated adenylate cyclase activity required the presence of the guanosine triphosphate (GTP) analogue, guanyl-5'-imidodiphosphate (p[NH]ppG), while cAMP accumulation could additionally be blocked by introducing the GDP analog, guanosine 5'[beta-thio]diphosphate (GDP[S]). The ability of EGF to activate adenylate cyclase was not affected by pretreatment of acinar cell membranes with pertussis toxin, whereas pretreatment with cholera toxin eliminated EGF-stimulated cyclase activity. The experimental results presented here expand to the parotid gland our knowledge of the ability of EGF to stimulate the cAMP second messenger signalling pathway via a G-binding regulatory protein, by a mechanism independent of beta-adrenergic receptor activation.     

Influence of receptor number on functional responses elicited by agonists acting at the human adenosine A(1) receptor: evidence for signaling pathway-dependent changes in agonist potency and relative intrinsic activity

Activation of A(1) adenosine receptors leads to the inhibition of cAMP accumulation and the stimulation of inositol phosphate accumulation via pertussis toxin-sensitive G-proteins. In this study we have investigated the signaling of the A(1) adenosine receptor in Chinese hamster ovary (CHO) cells, when expressed at approximately 203 fmol/mg (CHOA1L) and at approximately 3350 fmol/mg (CHOA1H). In CHOA1L cells, the agonists N(6)-cyclopentyladenosine (CPA), (R)-N(6)-(2-phenylisopropyl)adenosine, and 5'-(N-ethylcarboxamido)adenosine (NECA) inhibited cAMP production in a concentration-dependent manner. After pertussis toxin treatment, the agonist NECA produced a stimulation of cAMP production, whereas CPA and (R)-N(6)-(2-phenylisopropyl)adenosine were ineffective. In CHOAIH cells, however, all three agonists produced both an inhibition of adenylyl cyclase and a pertussis toxin-insensitive stimulation of adenylyl cyclase. All three agonists were more potent at inhibiting adenylyl cyclase in CHOA1H cells than in CHOA1L cells. In contrast, A(1) agonists (and particularly NECA) were less potent at stimulating inositol phosphate accumulation in CHOA1H cells than in CHOA1L cells. After pertussis toxin treatment, agonist-stimulated inositol phosphate accumulation was reduced in CHOA1H cells and abolished in CHOA1L cells. The relative intrinsic activity of NECA in stimulating inositol phosphate accumulation, compared to CPA (100%), was much greater in the presence of pertussis toxin (289.6%) than in the absence of pertussis toxin (155.2%). These data suggest that A(1) adenosine receptors can couple to both pertussis toxin-sensitive and -insensitive G-proteins in an expression level-dependent manner. These data also suggest that the ability of this receptor to activate different G-proteins is dependent on the agonist present.     

Effects of pertussis toxin on cAMP and cGMP responses to carbamylcholine in N1E-115 neuroblastoma cells

As noted previously, in N1E-115 neuroblastoma cells, carbamylcholine, a muscarinic cholinergic agonist, increased cGMP over 15-fold and decreased basal and prostaglandin E1 (PGE1)-stimulated cAMP content. In contrast to the stimulatory effects of PGE1 on cAMP, which were immediate, the carbamylcholine-induced decrease in basal and PGE1-stimulated cAMP exhibited a delay. The delay in carbamylcholine inhibition was independent of the extent of adenylate cyclase activation. Although basal cAMP content was suppressed within 30 sec after addition of carbamylcholine, inhibition was not maximal for at least 2 min following agonist addition; the delay was similar in cells exposed to PGE1 for 10 min prior to carbamylcholine but could be eliminated by incubation of the cells with muscarinic cholinergic agonist for 5 min prior to addition of prostaglandin. N1E-115 neuroblastoma cells possess a 41,000-Da membrane protein believed to be a component of the inhibitory GTP-binding protein of adenylate cyclase that is ADP ribosylated by pertussis toxin. Incubation of the cells with pertussis toxin prior to the addition of carbamylcholine reduced the maximal extent of inhibition of cAMP content and prevented the [32P]ADP-ribosylation of a 41,000-Da protein by toxin and [32P]NAD in membrane preparations from these cells. Incubation of cells with pertussis toxin, however, did not significantly alter the dose-response curve for carbamylcholine effects on cGMP. Even high concentrations of carbamylcholine, effective in stimulating cGMP, had minimal effects on cAMP content in toxin-treated cells; thus, ADP-ribosylation of Gi converts the adenylate cyclase but not the guanylate cyclase system to an agonist-insensitive state.     

Mechanism of enhanced sensitivity to bradykinin in pertussis toxin-treated fibroblasts: toxin increases bradykinin-stimulated prostaglandin formation

Exposure of animals to pertussis toxin results in increased sensitivity to agents such as bradykinin. To elucidate the molecular mechanisms underlying the effects of toxin, bradykinin responsiveness was examined in control and intoxicated human fibroblasts. Exposure of fibroblasts to toxin resulted in a loss of inhibitory agonist action on adenylate cyclase, elevation of basal cAMP, and ADP-ribosylation of a 41-kDa protein, which was identified as Gi alpha, a component of adenylate cyclase, by its pattern of immuno-cross-reactivity with a family of antibodies to guanyl nucleotide-binding proteins, which are pertussis toxin substrates, and by the presence of an mRNA species with characteristics of a form of Gi alpha. Bradykinin increased prostaglandin accumulation to a greater extent in toxin-treated than in control fibroblasts. Agents such as cholera toxin, which elevated cAMP, also increased bradykinin-induced prostaglandin production. These data are consistent with the hypothesis that the enhanced sensitivity to bradykinin after pertussis toxin treatment results from modification of Gi alpha and increased cAMP, leading to enhanced formation of prostaglandins in response to bradykinin.     

Pharmacology of 5-hydroxytryptamine-1A receptors which inhibit cAMP production in hippocampal and cortical neurons in primary culture

Serotonin (5-hydroxytryptamine, 5-HT) inhibited the formation of cAMP promoted by vasoactive intestinal polypeptide, plus forskolin, in mouse hippocampal and cortical neurons in primary culture. The rank order of potencies of classical 5-HT1 agonists in inhibiting cAMP formation in hippocampal neurons was 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) greater than 5-carboxamidotryptamine (5-CT) greater than d-lysergic acid diethylamide greater than 5-HT greater than 5-methoxy-N,N-dimethyltryptamine (5-MeO-N,N-DMT) greater than RU 24969 greater than ipsapirone greater than bufotenine greater than buspirone [half-maximal efficacy (EC50) = 7, 18, 30, 52, 90, 102, 100, 110, and 128 nM, respectively]. All the tryptamine derivatives substituted in position 5 of the indol were potent agonists [5-HT, 5-CT, 5-MeO-N,N-DMT, 5-methoxytryptamine, and bufotenine], whereas tryptamine, N-methyltryptamine, and N,N-dimethyltryptamine were poor agonists. The most potent antagonists tested were spiperone, (+/-)-pindolol, (+/-)-cyanopindolol, WB4101, and methiothepin, the affinity of spiperone for this receptor being 22 nM. In contrast, ketanserin, a specific 5-HT2 antagonist, and 5-HT3-selective drugs (ICS 205 930 and MDL 72222) were very weak in antagonizing the 5-HT-inhibited cAMP formation. The pharmacological profiles of 5-HT receptors mediating the inhibition of cAMP formation indicate that these receptors correspond to the 5-HT1A-binding site subtypes. Experiments with the Bordetella pertussis toxin indicate that the 5-HT1A receptor mediating inhibition of cAMP production involves a pertussis toxin-sensitive GTP-binding protein. In the absence of VIP, cAMP formation could be stimulated through a 5-HT receptor, but the specific 5-HT1A agonists, 8-OH-DPAT and RU 24969 did not stimulate cAMP production. These results suggest that in mouse embryonic hippocampal neurons, the 5-HT1A receptors, which are negatively coupled to adenylate cyclase, are distinct from the receptor positively coupled to this enzyme. The pharmacological characterization of the 5-HT receptor negatively coupled to adenylate cyclase in mouse embryonic cortical neurons indicates that it differs from the 5-HT1A receptor found in hippocampal neurons. Its main differences with the 5-HT1A receptor in hippocampal neurons are as follows: 1) 8-OH-DPAT was only a poor partial agonist in cortical neurons, whereas it was the best full agonist in hippocampal neurons; and 2) metergoline and methysergide as well as the anxiolytic drugs, ipsapirone and buspirone, which were potent agonists in hippocampal neurons, were competitive antagonists in cortical neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thrombin signalling in U937 human monocytic cells is coupled to inositol phosphate formation but not to thromboxane B2 synthesis nor to inhibition of adenylate cyclase: distinct differences in thrombin signalling between U937 cells and platelets.

The blood coagulation factor, human thrombin has been shown to have chemotactic and mitogenic effects on mononuclear phagocytic inflammatory cells. In the present study, we have used the U937 human monocytic cell line to explore the signal transduction mechanisms utilised by thrombin in these cells. In U937 cells differentiated into a macrophage-like phenotype, thrombin stimulated the formation of inositol trisphosphate (IP3) and the mobilisation of intracellular Ca2+ [( Ca2+]i) via a mechanism which was partially sensitive to pertussis toxin. Thrombin failed, however, to evoke thromboxane (Tx) B2 synthesis in the differentiated cells. In contrast, the chemotactic peptide N-formyl-L-methionylleucyl-L-phenylalanine (FMLP) stimulated TxB2 synthesis under conditions where it evoked increases in IP3 formation and [Ca2+]i mobilisation, via a pertussis toxin-sensitive mechanism, comparable in extent to those mediated by thrombin. Thrombin also failed to cause inhibitory guanine nucleotide binding protein (Gi)-mediated inhibition of adenylate cyclase activity in U937 cell membranes. These results indicate that U937 cells express receptors for thrombin which are in part coupled via a pertussis toxin-sensitive guanine nucleotide binding protein to phospholipase C activation, the formation of IP3 and the mobilisation of [Ca2+]i. However, the failure of thrombin to stimulate TxB2 synthesis or cause Gi-mediated inhibition of adenylate cyclase in U937 cells contrasts with its effects in human platelets and other thrombin-responsive cells. These results suggest that the thrombin receptor or receptor-effector coupling mechanism(s) in mononuclear cells is functionally distinct from the thrombin receptor or receptor-effector coupling mechanism(s) present in other thrombin-responsive cells.     

1-Alkyl-benzotriazole-5-carboxylic acids are highly selective agonists of the human orphan G-protein-coupled receptor GPR109b

1-Substituted benzotriazole carboxylic acids have been identified as the first reported examples of selective small-molecule agonists of the human orphan G-protein-coupled receptor GPR109b (HM74), a low-affinity receptor for the HDL-raising drug niacin. No activity was observed at the highly homologous high-affinity niacin receptor GPR109a (HM74A). The high degree of selectivity was attributed to a difference in the amino acid sequence adjacent to a key arginine-ligand interaction allowing somewhat larger ligands to be tolerated by GPR109b.     

Analysis of second messenger pathways stimulated by different chemokines acting at the chemokine receptor CCR5

The chemokine receptor, CCR5, responds to several chemokines leading to changes in activity in several signalling pathways. Here, we investigated the ability of different chemokines to provide differential activation of pathways. The effects of five CC chemokines acting at CCR5 were investigated for their ability to inhibit forskolin-stimulated 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation and to stimulate Ca(2+) mobilisation in Chinese hamster ovary (CHO) cells expressing CCR5. Macrophage inflammatory protein 1alpha (D26A) (MIP-1alpha (D26A), CCL3 (D26A)), regulated on activation, normal T-cell expressed and secreted (RANTES, CCL5), MIP-1beta (CCL4) and monocyte chemoattractant protein 2 (MCP-2, CCL8) were able to inhibit forskolin-stimulated cAMP accumulation, whilst MCP-4 (CCL13) could not elicit a response. CCL3 (D26A), CCL4, CCL5, CCL8 and CCL13 were able to stimulate Ca(2+) mobilisation through CCR5, although CCL3 (D26A) and CCL5 exhibited biphasic concentration-response curves. The Ca(2+) responses induced by CCL4, CCL5, CCL8 and CCL13 were abolished by pertussis toxin, whereas the response to CCL3 (D26A) was only partially inhibited by pertussis toxin, indicating G(i/o)-independent signalling induced by this chemokine. Although the rank order of potency of chemokines was similar between the two assays, certain chemokines displayed different pharmacological profiles in cAMP inhibition and Ca(2+) mobilisation assays. For instance, whilst CCL13 could not inhibit forskolin-stimulated cAMP accumulation, this chemokine was able to induce Ca(2+) mobilisation via CCR5. It is concluded that different chemokines acting at CCR5 can induce different pharmacological responses, which may account for the broad spectrum of chemokines that can act at CCR5.     

Effects of prostaglandins and cAMP levels on monocyte IL-1 production

The effects of prostaglandin E2 (PGE2), as well as other cAMP-elevating agents, on the in vitro production of interleukin-1 (IL-1) by human monocytes (HM) were examined. Exposure to E. coli lipopolysacharide (LPS) resulted in a dose- and time-dependent increase of IL-1 activity in monocytes culture supernatants. Maximal levels of secreted IL-1 in response to 10 ng LPS/ml were obtained at 18 h. PGE1, PGE2, cholera toxin (CT) and the phosphodiesterase inhibitor, isobutylmethylxanthin (IBMX), when added with LPS, resulted in a dose-dependent increase in cellular cAMP and in secreted IL-1. Maximal levels of secreted IL-1 were 2.5-5.0-fold over LPS alone. When CT or PGE2 was added with IBMX a further increase was observed. These agents exhibited marginal effect on cell-associated IL-1. Maximum cAMP levels was achieved at 10 min in response to either PGE1 or PGE2 and returned to near basal levels after 18 h. While PGE1 elevated cAMP to a larger extent than PGE2 (58- vs. 30-fold) the latter resulted in a higher levels of secreted IL-1. Elevated cAMP persisted throughout the entire culture period in response to CT (4-6-fold) or IBMX (7-fold). Thus, we conclude that in adherent HM, IL-1 production is potentiated and not inhibited by prostaglandins or agents that elevate cellular cAMP.     

Effector coupling of stably transfected human A3 adenosine receptors in CHO cells

CHO cells stably transfected with adenosine receptors are widely utilized models for binding and functional studies. The effector coupling of human A3 adenosine receptors expressed in such a cellular model was characterized. Inhibition of adenylyl cyclase via a pertussis toxin-sensitive G protein was confirmed and exhibited a pharmacological profile in accordance with agonist binding data. The agonist potency was dependent on the assay system utilized to measure cyclase inhibition. Agonists were more potent in a cell-based assay than in experiments where cyclase inhibition was measured in a membrane preparation suggesting that receptor-effector coupling might be more efficient in intact cells. In addition to the modulation of cyclase activity, stimulation of A3 receptors elicited a Ca2+ response in CHO cells with agonist potencies corresponding to the values for the whole cell cAMP assay. The Ca2+ signal was completely eliminated by pertussis toxin treatment suggesting that it is mediated via betagamma release from a heterotrimeric G protein of the Gi/o family. These results show that cAMP and Ca2+ signaling characteristics of the A3 adenosine receptor are comparable to the ones found for the A1 subtype.     

Pertussis toxin inhibits alpha 2-adrenoceptor-mediated inhibition of adenylate cyclase without affecting muscarinic regulation of [Ca2+]i or inositol phosphate generation in SH-SY5Y human neuroblastoma cells

The present study reports the differential effects of pertussis toxin on muscarinic regulation of intracellular Ca2+ and inositol phosphate generation and alpha 2-adrenoceptor-mediated inhibition of cAMP formation in SH-SY5Y human neuroblastoma cells. Carbachol caused a biphasic increase in intracellular Ca2+ (release of internal stores and Ca2+ entry) and a dose-dependent increase in inositol phosphate formation. Pertussis toxin pretreatment did not affect either of these components of the signal transduction pathway but did completely reverse the alpha 2-adrenoceptor-mediated inhibition of forskolin-stimulated cAMP formation. These data indicate that muscarinic regulation of inositol phosphate generation occurs via a pertussis toxin-insensitive G-protein and that Ca2+ entry in these cells may not occur via a G-protein.