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J MacDermot

Publications and source records attributed to J MacDermot.

At least 19 recordsLinked to original sources
PubMed1992

Gs alpha is a substrate for mono(ADP-ribosyl)transferase of NG108-15 cells. ADP-ribosylation regulates Gs alpha activity and abundance.

NG108-15 neuroblastoma x glioma somatic hybrid cells were permeabilized in the presence of [32P]NAD+ and then cultured for 18 h. Resolution of the cell proteins on polyacrylamide gels revealed [32P]ADP-ribosylation of five major protein species with molecular mass values of 52 kDa, 44 kDa, 35 kDa, 30 kDa and 25 kDa. A similar pattern of labelling was also seen when NG108-15 cell membranes were incubated with [32P]NAD+ and hydrolysis of the product revealed mono(ADP-ribosyl)ation. Immunoprecipitation of these products with anti-Gs alpha antiserum revealed a single band identical to cholera toxin substrate. Culture of [32P]NAD(+)-loaded cells for 18 h in the presence of 50 mM-nicotinamide inhibited the eukaryotic mono(ADP-ribosyl)transferase activity. Inhibition of the eukaryotic enzyme was also accompanied by an increase in the abundance of Gs alpha, whether measured by Western blotting with anti-Gs alpha antibody (two separate antisera) or by cholera toxin-dependent [32P]ADP-ribosylation. There was no accompanying change in the abundance of G beta. The increase in Gs alpha abundance in nicotinamide-treated NG108-15 cells was accompanied by a 2-fold increase in basal adenylate cyclase activity (measured in the presence of GTP), and by a smaller but significant increase in iloprost-dependent activation of adenylate cyclase. Receptor number or affinity was not affected by nicotinamide, since this treatment did not alter the binding parameters of [3H]iloprost to NG108-15 cell membranes. Short-term exposure of cells to nicotinamide for 1 h revealed no significant difference in either basal or agonist-stimulated adenylate cyclase activity. These results reveal that mono(ADP-ribosyl)ation of Gs alpha by eukaryotic ADP-ribosyltransferase modifies the abundance and activity of Gs alpha in NG108-15 cells, and hence may play a role in the hormonal regulation of cell function.

ADP Ribose Transferases↗
PubMed1992

Thrombin promotes actin polymerization in U937 human monocyte-macrophage cells. Analysis of the signalling mechanisms mediating actin polymerization.

The U937 human monocyte-macrophage cell line was used to examine the effect of thrombin, an ill-defined chemoattractant, on the polymerization of actin, a process essential for cell motility. In differentiated macrophage-like U937 cells, thrombin (0.5-50 units/ml) caused a rapid dose-dependent increase in the formation of filamentous (F-) actin, detected by the staining of F-actin with the fluorescent toxin, 7-nitrobenz-2-oxa-1,3-diazole-phallacidin. In contrast with other chemoattractants such as N-formylmethionyl-leucylphenylalanine or C5a, actin polymerization in response to thrombin occurred via a pertussis-toxin-insensitive G1-(inhibitory G-protein) independent signalling pathway. Further, this response was not affected by the Ca2+ chelator EGTA or by the specific protein kinase C (PKC) inhibitor RO-31-8220. The response to thrombin was not mimicked by the Ca2+ ionophore ionomycin or by the direct PKC activator phorbol 12-myristate 13-acetate. The thrombin response was, however, inhibited by the non-specific protein kinase inhibitor staurosporine. The present results suggest that in U937 cells thrombin stimulates the formation of F-actin via a signalling pathway independent of (i) the activation of PKC, (ii) the mobilization of intracellular Ca2+ and (iii) the activation of Ca(2+)-dependent protein kinases, but dependent on the activation of an undefined staurosporine-sensitive protein kinase.

Actins↗
PubMed1992

Characterization of the thromboxane receptor mediating prostacyclin release from cultured endothelial cells.

The thromboxane A2 (TXA2) mimetic, 9,11-dideoxy-11,9-epoxymethano-prostaglandin F 2 alpha (U46619), mobilized calcium in the bovine aortic endothelial cell line AG4762 and stimulated release of prostacyclin from these cells. The U46619-stimulated release of prostacyclin could be inhibited by TXA2 antagonists with the order of potency [Is-[1 less than a, 2 less than b(5z), 3 less than b, 4 less than a]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo- [2.2.1]hept-2-yl]-5- heptenoic acid (SQ29548) greater than 4-[2-(4-chlorobenzene-sulphonamido) ethyl]phenylacetic acid (BM13505) greater than 4-[2-(phenylsulphonamido)-ethyl]phenoxyacetic acid (BM13177), which was consistent with release being mediated by a TXA2 (TP) receptor. The TP receptor ligands, [3H]SQ29548 and 9,11-dimethylmethano-16(3-[125I]iodo-4-hydroxyphenyl)-13,14-dih ydr o-13-aza- 15-omega-o-tetranor-thromboxane ([125I]-PTA-OH), both appeared to bind to a homogenous population of sites in AG4762 cell membranes. The affinities of [3H]SQ29548 and [125I]PTA-OH were approximately 10 nM and approximately 0.3 nM, respectively, and the density of sites labelled by either ligand was approximately 25 fmol/mg protein. Under conditions where equilibrium was approached, the specific binding of [3H] SQ29548 or [125I]PTA-OH was displaced by SQ29548, BM13505 and BM13177 with the same order of potency and similar apparent affinities as in the functional assay, suggesting that these binding sites represent bona fide TP receptors.

Bradykinin↗
PubMed1992

Cyclic AMP produces desensitization of prostacyclin and adenosine A2 receptors in hybrid cell lines but does not affect Gs function.

Prostacyclin and adenosine A2 receptors stimulate adenylate cyclase activity in the related somatic hybrid cell lines NG108-15 and NCB20. The role of cAMP in the desensitization of these receptors has been examined. Pretreatment for 17 h with forskolin or 8-bromo-cAMP had the same effect in both cell lines. There was no change in the response to sodium fluoride or forskolin, suggesting that the function of Gs and adenylate cyclase were unaffected by increased levels of cAMP. Receptor responses were affected however; the maximum response to N-ethylcarboxamidoadenosine (an A2 receptor agonist) was reduced by 30-40%, there was a small but consistent shift to the right of the dose-response curve for iloprost (a stable analogue of prostacyclin) and [3H]iloprost binding studies revealed a loss of prostacyclin receptors. However, the loss of receptor responsiveness was much smaller than that which occurs following pretreatment with prostacyclin or adenosine A2 receptor agonists (Keen et al. (1989) Biochem. Pharmacol. 38, 3827-3833; Kelly et al. (1990) Br. J. Pharmacol. 99, 309-316) suggesting that cAMP may not play a major role in agonist mediated desensitization.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

The identity of IgE receptors (Fc epsilon RII) that mediate cellular activation of human macrophages: evidence against a role for CD23.

There is now compelling evidence that macrophages bind IgE, and are involved in several IgE-dependent responses. The CD23 antigen mediates a mitogenic response in "primed" B-lymphocytes, although its expression is not confined to B cells, and CD23 is inducably expressed in many cells including macrophages. CD23 is also known to bind IgE, a property that leads to inhibition of the mitogenic response in B cells. In the present review, the possibility that CD23 mediates IgE-dependent responses in macrophages has been re-examined, and it is proposed that the functional receptor for IgE on macrophages may be quite separate from the CD23 antigen.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1992

Activation of bovine endothelial thromboxane receptors triggers release of prostacyclin but not EDRF.

OBJECTIVE: The aim was to examine the capacity of U46619 (a stable thromboxane A2 mimetic) to mediate release of endothelium derived relaxing factor (EDRF) from bovine aortic endothelial cells, and compare the response to the U46619 dependent release of prostacyclin (PGI2). METHODS: Bovine aortic endothelial cells (AG4762) were cultured in vitro on microcarrier beads, which were then loaded onto a column and perfused. The cells were challenged with U46619, bradykinin, or the Ca2+ ionophore ionomycin in the perfusate, and measurements made of the release of 6-oxo-PGF1 alpha (the stable hydrolysis product of PGI2, measured by radioimmunoassay) and EDRF (bioassay). Cells were also cultured on glass cover slips, loaded with Fura 2-AM, and measurements made of the rise in intracellular Ca2+ after challenge with U46619, bradykinin or ionomycin. RESULTS: U46619 triggered release of 6-oxo-PGF1 alpha but not EDRF from AG4762 cells, contrasting with bradykinin which released both 6-oxo-PGF1 alpha and EDRF. Ionomycin had little or no capacity to mimic and trigger release of 6-oxo-PGF1 alpha, although ionomycin mediated large increases in intracellular Ca2+. In contrast, staurosporine (a putative inhibitor of protein kinase C) substantially inhibited the U46619 and bradykinin dependent release of 6-oxo-PGF1 alpha. CONCLUSIONS: In contrast to bradykinin linked receptors on AG4762 endothelial cells, which are coupled to the release of both prostacyclin and EDRF, activation of thromboxane A2 receptors on these cells selectively triggers release of PGI2 but not EDRF. Further, based on the distinct effects of ionomycin and staurosporine, it appears that agonist stimulated PGI2 release from these cells is mediated predominantly by protein kinase C, rather than by rises in intracellular Ca2+. This observation contrasts with previously described mechanisms of PGI2 release from endothelium obtained from other sources.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1992

Opiate-dependent changes in the sensitivity of adenylate cyclase to stimulatory agonists and 5'-guanylylimidodiphosphate are independent of G protein abundance and eukaryotic ADP-ribosyltransferase activity in NG108-15 cells.

NG108-15 cells were exposed in culture to 1 microM [D-Ala2,D-Leu5]enkaphalin (DADLE) for 17 h. This treatment increased the maximum iloprost- and 5'-(N-ethylcarboxamido)adenosine-dependent activation of adenylate cyclase, as well as basal enzyme activity. In addition, there was an increase in the capacity of 5'-guanylylimidodiphosphate [Gpp(NH)p] to inhibit adenylate cyclase activity by direct interaction with the alpha-subunit of the Gi regulatory protein. A similar effect was observed if the cells were exposed to 10 microM carbachol. These treatments of NG108-15 cells did not alter the capacity of NaF to activate adenylate cyclase by direct interaction with Gs alpha. Exposure of NG108-15 cells to DADLE alone or DADLE plus carbachol had no effect on the capacity of pertussis toxin to ADP-ribosylate membrane proteins in these cells; neither was there any change in the activity of eukaryotic ADP-ribosyltransferase expressed in these cells. Under these conditions, the endogenous enzyme did not label any protein with a molecular mass similar to Gi alpha, 41 kDa. Treatment of the cells with DADLE or carbachol had no effect on the abundance of Gs alpha, Gi alpha, or G beta. The underlying mechanism for the changes in agonist-dependent stimulatory responses or Gpp(NH)p-dependent inhibition of adenylate cyclase remains obscure, but appears not to be mediated by eukaryotic ADP-ribosyltransferase activity or a change in the abundance of G proteins known to regulate adenylate cyclase.

Adenylyl Cyclase Inhibitors↗
PubMed1992

Differential sensitivities of the prostacyclin and nitric oxide biosynthetic pathways to cytosolic calcium in bovine aortic endothelial cells.

1. Bovine aortic endothelial cells were cultured in vitro, and shown to release both prostacyclin (PGI2; Kact = 24.1 nM) and endothelium-derived relaxing factor (EDRF, NO; Kact = 0.7 nM) in a concentration-dependent manner when exposed to bradykinin. 2. The bradykinin-dependent release of PGI2 (but not EDRF) was inhibited by 1 microM isoprenaline or 5 microM forskolin, and the inhibitory effect of isoprenaline could be reversed by the beta 2-adrenoceptor antagonist, ICI 118551. In contrast, isoprenaline had no capacity to inhibit PGI2 release stimulated by exogenous arachidonic acid. 3. Exposure of cells to bradykinin increased the cytosolic concentration of Ca2+ ions ([Ca2+]i; Kact = 4.8 nM), and the effect was inhibited by both 1 microM isoprenaline and 5 microM forskolin. 4. In similar experiments, exposure of cells to ionomycin also increased [Ca2+]i and the values of [Ca2+]i were calibrated in terms of the ionomycin concentration. In subsequent experiments involving exposure of endothelial cells to selected concentrations of ionomycin, it was possible to show that the biosynthesis of NO was triggered at ionomycin concentrations about one tenth of the required for PGI2 biosynthesis and that these corresponded to a [Ca2+]i threshold of 350 nM for PGI2 release while that for EDRF release was less than 200 nM. 5. These differences in Ca2+ ion sensitivity explain the selective inhibition of bradykinin-stimulated PGI2 biosynthesis (to the exclusion of NO biosynthesis) by isoprenaline or forskolin, both of which attenuate bradykinin-dependent increases in [Ca2+]i.

Adrenergic beta-Antagonists↗
PubMed1991

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.

Adenylate Cyclase Toxin↗
PubMed1991

Guanine nucleotide sensitivity of [3H]iloprost binding to prostacyclin receptors.

A component of the displaceable binding of the stable prostacyclin analogue, [3H]iloprost, to membranes from human platelets and the somatic hybrid cell lines NG108-15 and NCB20, was inhibited by guanine nucleotides. The order of potency of a range of nucleotides for this effect was GTP gamma S greater than GppNHp greater than GTP greater than GDP = GMP; ATP, UTP and CTP were ineffective at concentrations up to 1 mM. In the presence of 100 microM GppNHp, iloprost binding curves were displaced to the right of curves obtained in the absence of guanine nucleotide, and their Hill slopes were greater. This was consistent with a conversion of a minor population of high affinity agonist binding sites to lower affinity sites in the presence of guanine nucleotides. These effects of guanine nucleotides on the binding of the agonist ligand [3H]iloprost were consistent with an interaction with a G protein coupled receptor.

Cell Membrane↗
PubMed1991

Pituitary adenylate cyclase-activating polypeptide: a novel, long-lasting, endothelium-independent vasorelaxant.

The vasoactivity of the 27- and 38-amino acid forms of the novel peptide pituitary adenylate cyclase-activating polypeptide (PACAP) was tested in vitro. Both forms of PACAP caused endothelium-independent vasodilation (assayed by their vasodilator action on rabbit aorta). When superfused for 1 min the relaxation EC50 of PACAP27 was 23 +/- 8 nM and of PACAP38 was 152 +/- 66 nM. PACAP was 100-fold more potent than vasoactive intestinal polypeptide (VIP) (PACAP27 shows 68% amino acid sequence homology with VIP), and had a prolonged duration of action, a 1 min exposure to 1 microM PACAP27 lasting 135 +/- 7 min and to 1 microM PACAP38 108 +/- 3 min. Adenylate cyclase activity in homogenates of rabbit aortic smooth muscle cells was increased by PACAP27 and PACAP38 with EC50s of 4.4 and 0.73 nM, respectively. PACAP27 and PACAP38 are potent, long-lasting, endothelium-independent vasodilators.

Adenylyl Cyclases↗
PubMed1991

IgE and IgG are synergistic in antigen-mediated release of thromboxane from human lung macrophages.

The mechanism of IgE-mediated release of thromboxane A2 from human lung macrophages has been studied using a monoclonal chimeric human/mouse IgE antibody and its specific antigen. The cells could be sensitized at 37 degrees C but not at 4 degrees C by incubation with IgE, and released a significant amount of thromboxane A2 (TXA2), measured as the stable hydrolysis product TXB2, in response to an anti-chimeric IgE antibody. In contrast, stimulation of IgE-sensitized macrophages with the specific antigen produced less than 10% of this response. A similar time course for the release of TXB2 and the formation of inositol monophosphate in the presence of LiCl was observed. Cleavage of the Fc domain of the anti-chimeric IgE antibody substantially eliminated its capacity to stimulate IgE-sensitized cells. However, the weak or undetectable response to chimeric IgE plus specific antigen was substantially potentiated by an antigen-specific chimeric IgG antibody. IgG-sensitized macrophages did not respond to antigen challenge by the release of TXB2. Preincubation of the cells with a monoclonal antibody against the low affinity receptor for IgE (Fc epsilon RII/CD23) did not prevent IgE sensitization. We conclude that cell-bound IgE antibody cannot induce the release of TXB2 but has fixed antigen which then must interact with specific IgG antibody and IgG receptors to induce mediator release.

Antigen-Antibody Complex↗
PubMed1990

Thromboxane release by lymphokine-differentiated U937 human monocytic cells: response to platelet-activating factor (PAF) and chemotactic peptide (FMLP) but not to low affinity IGE-receptor (Fc epsilon RII/CD23) occupation.

The primary objective of this study was to explore if the CD23 antigen is a functional low affinity IgE receptor on macrophages for the release of thromboxane B2 (TXB2). The responsiveness of U937 monocytic cells and their macrophage-like inducible forms to platelet-activating factor (Paf), the chemotactic peptide fMLP, and low affinity IgE-receptor occupation was examined. Differentiation of U937 cells by phorbol myristate acetate (PMA) and a cancer cell line (HBT 5637) conditioned medium (5637-CM), but not INFg or IL4, resulted in a macrophage-like cell line which released TXB2. A high basal release of TXB2 with no significant response to Paf or fMLP challenge was seen following culture of cells with PMA. In 5637-CM-differentiated cells, Paf and fMLP induced a rapid release of TXB2, about 10 fold above basal activity. There was a slow Ca-independent response to short-term treatment with PMA and a rapid Ca-dependent response to the ionophore A23187. Both stimulants acted synergistically on TXB2 synthesis in 5637-CM differentiated cells. Although low affinity receptors for IgE (Fc epsilon RII/CD23) were induced by 5637-CM, no TXB2 was released in response to soluble or latex-bound IgE-antigen complexes or to anti-Fc epsilon RII/CD23-antibodies. IL4 and to a lesser extent INFg both induced Fc epsilon RII/CD23 receptor expression, but inhibited release of TXB2 in response to Paf, fMLP, or PMA. We conclude that the functional receptors for IgE on mature macrophages are most probably not Fc epsilon RII/CD23.

Antibodies, Anti-Idiotypic↗
PubMed1990

Segregation of discrete GS alpha-mediated responses that accompany homologous or heterologous desensitization in two related somatic hybrids.

1. Prostacyclin and adenosine A2 receptors activate adenylate cyclase in the neuroblastoma hybrid cell lines NG108-15 and NCB-20. Prolonged exposure of NG108-15 cells to iloprost (a stable analogue of prostacyclin) results in a subsequent reduction in the capacity for adenylate cyclase activation by iloprost, the adenosine analogue 5'-(N-ethyl)-carboxamidoadenosine (NECA) or NaF. In contrast prolonged exposure of NCB-20 cells to iloprost results only in the loss of iloprost responsiveness. 2. Iloprost pretreatment of NG108-15 cells also magnified the morphine-dependent inhibition of iloprost-stimulated adenylate cyclase activity from 36 to 48%. This change was not due to lower iloprost stimulation following desensitization, since the % inhibition of adenylate cyclase activity by morphine in control cells was constant irrespective of enzyme activity. 3. These heterologous effects observed in NG108-15 cells following iloprost pretreatment may involve changes in the GS alpha protein, since there was a reduction of about 30% in the cholera toxin-induced [32P]-ADP-ribosylation of a 45 kDa protein from cell membranes (corresponding to the extent of loss of NECA or NaF responsiveness). A similar reduction was not observed in NCB-20 cells. 4. These results indicate that iloprost pretreatment induces different forms of desensitization in NG108-15 and NCB-20 cell lines. The heterologous desensitization in the former may, like the human platelet, involve a functional loss of GS alpha from the cell membrane. Changes in the activity of GS alpha may also account for the heterologous effects on receptors that mediate inhibition of adenylate cyclase.

Adenosine↗
PubMed1990

NaF and guanine nucleotides modulate adenylate cyclase activity in NG108-15 cells by interacting with both Gs and Gi.

1. NaF (10 mM) produced a 2-3 fold increase in adenylate cyclase activity in homogenates of NG108-15 cells incubated in the presence of 1 microM GTP. Higher concentrations of NaF suppressed adenylate cyclase activity. 2. In the presence of the adenosine receptor agonist 5'-(N-ethyl)-carboxamidoadenosine (NECA; 100 microM) or the prostacyclin receptor agonist iloprost (10 nM), NaF produced a much smaller increase in adenylate cyclase activity, whereas in the presence of a saturating concentration of iloprost (1 microM), NaF only inhibited adenylate cyclase activity. 3. Similarly, Gpp(NH)p activated basal adenylate cyclase activity, and inhibited 1 microM iloprost-activated enzyme activity. In the presence of 10 microM forskolin, NaF or Gpp(NH)p increased adenylate cyclase activity synergistically. Analysis of concentration-effect curves indicated that NaF (2 mM) or Gpp(NH)p (100 microM) increased the potency with which forskolin activated adenylate cyclase, whilst reducing the maximum activation of adenylate cyclase by iloprost. 4. Opiate receptors mediate inhibition of adenylate cyclase, and the opiate agonist morphine (100 microM) reduced the capacity of NaF or Gpp(NH)p to inhibit iloprost-activated adenylate cyclase. Unexpectedly, pertussis toxin treatment enhanced the ability of NaF or Gpp(NH)p to inhibit iloprost-activated adenylate cyclase. 5. In the absence of GTP, NaF and Gpp(NH)p remained able both to activate basal adenylate cyclase and to be synergistic with forskolin in activating the enzyme. In contrast the ability of NaF and Gpp(NH)p to inhibit iloprost-activated adenylate cyclase was substantially lost in the absence of added GTP. These results suggest that NaF modulates adenylate cyclase activity in NG108-15 cell membranes by interacting with the alpha subunits of both G0 and Gi regulatory proteins. The effects of NaF and Gpp(NH)p are critically dependent on the prior mode and extent of activation or inhibition of this transmembrane signalling pathway. This simple system may be of use in assessing alterations in GSO-O interaction following manipulations such as hormone receptor desensitization.

Adenosine↗
PubMed1989

A selective binding site for 3H-NECA that is not an adenosine A2 receptor.

In homogenates of NG108-15 cells, adenosine analogues activate adenylate cyclase with the following order of potency: N-ethylcarboxamidoadenosine (NECA) greater than 2-chloroadenosine greater than N6-(L-phenylisopropyl)-adenosine (PIA) = cyclohexyladenosine = 2-phenylaminoadenosine. Adenosine receptor antagonists inhibit NECA-stimulated adenylate cyclase activity with the order of potency 3-isobutyl-1-methyl-xanthine (IBMX) greater than theophylline greater than caffeine. These data suggest that these ligands act at an adenosine A2 receptor. There is an apparently homogenous population of saturable 3H-NECA binding sites in homogenates of NG108-15 cells. These sites have an affinity for 3H-NECA of approximately 1 microM, and are present at a density of approximately 10 pmol/mg protein. Unlabelled NECA, 2-chloroadenosine, IBMX and theophylline displace 3H-NECA binding, with an order of potency that suggests that the 3H-NECA binding site may represent an adenosine A2 receptor. However, PIA, cyclohexyladenosine and 2-phenylaminoadenosine produce no detectable displacement of 3H-NECA binding at concentrations that produce a maximal stimulation of adenylate cyclase activity. Pretreatment of NG108-15 cells with either NECA or PIA produces a homologous desensitization of subsequent responses to all the adenosine analogues, with no effect on subsequent responses to a prostacyclin receptor agonist or NaF. This suggests that all the adenosine analogues examined activate an adenosine A2 receptor. Therefore, the 3H-NECA site at which PIA is inactive cannot represent this receptor.

Adenosine↗