AMP Content Of Cells Research Articles

The 6H1 Orphan Receptor, Claimed to Be the p2y5 Receptor, Does Not Mediate Nucleotide-Promoted Second Messenger Responses

An orphan G protein-coupled receptor, termed 6H1, with ∼30% sequence identity to P2Y receptors has been proposed to be a P2Y receptor (p2y5) based solely on a radioligand binding assay with [35S]dATPαS [Webb et al. (1997) Biochem. Biophys. Res. Commun. 219:105-110]. Previous work in our laboratory has shown that [35S]-dATPαS is not a general ligand for P2Y receptors, and thus inclusion of the p2y5 receptor in the family of P2Y receptors is questionable. To define unambiguously whether the p2y5 receptor is a P2Y receptor, we have cloned the turkey homologue of the chick p2y5 receptor. Sequence analysis indicated that the turkey receptor contains an additional 32 amino acids at its carboxy terminus compared to the published chick sequence. HA epitope-tagged turkey p2y5 receptors were stably expressed in 1321N1 human astrocytoma cells, and cells shown to express the HA-tagged p2y5 receptor by an intact cell-based ELISA were used to determine whether changes in second messenger levels occurred in response to a series of nucleotides. ATP, ADP, UTP, UDP, dATPαS, and A2P4had no effect on either inositol phosphate or cyclic AMP concentrations in cells expressing the p2y5 receptor. Robust inositol phosphate and cyclic AMP responses occurred to other G protein-coupled receptors expressed in 1321N1 cells, which indicate that these cells contain all of the necessary signaling components to generate these second messenger responses. These data indicate that the 6H1/p2y5 receptor is not a member of the P2Y receptor family of signaling proteins.

Prostaglandin cytoprotection of galactosamine-incubated hepatocytes isolated from young and old rats.

The aim of the study was to investigate the effect of aging on cytoprotective properties of prostaglandins. Hepatocytes were obtained by collagenase perfusion of livers of young (4-6 mo) and old (24-28 mo) male Wistar rats. Cells were incubated for 1.5 h in Krebs-Ringer-bicarbonate buffer containing glucose and 3H-leucine in the presence of galactosamine (2.5-100 mM), PGE1, or two prostacyclin analogues: 9 beta-methylcarbacyclin and TRK-100. Cell damage was assessed by decrease in the rate of protein synthesis measured as 3H-leucine incorporation into acid precipitable material, and by increase in lactate dehydrogenase release into the medium. Hepatocytes from old rats were more susceptible to suppression of protein synthesis by GalN than cells of young ones. Preincubation of cells for 15 min with 9MC (41-560 nM) or PGE1 (10-100 nM), but not with TRK-100, before adding 10 mM GalN, led to a partial recovery of protein synthesis in both age groups. GalN increased LDH release and decreased ATP/ADP ratio to a similar extent in hepatocytes of young and old rats; both parameters were not altered by preincubation of cells with PGs. PGE1 and 9MC, but not TRK-100, elevated cyclic AMP content in hepatocytes of young but not old rats. Glucagon and forskolin similarly increased cyclic AMP content in cells of both young and old animals. These in vitro results suggest that PGE1 and some prostacyclin analogues might protect hepatocytes of both young and old rats from chemical damage, and stress the necessity for further research on cyto- and hepato-protection in the elderly.

Site-directed Mutagenesis of β-Adrenergic Receptors: Identification of Conserved Cysteine Residues that Independently Affect Ligand Binding and Receptor Activation

Abstract Using site-directed mutagenesis of the human beta 2-adrenergic receptor and continuous expression in B-82 cells, the role of 3 conserved cysteines in transmembrane domains and 2 conserved cysteines in the third extracellular domain in receptor function was examined. Cysteine was replaced with serine in each mutant receptor as this amino acid is similar to cysteine in size but it cannot form disulfide linkages. Replacement of cysteine residues 77 and 327, in the second and seventh transmembrane-spanning domains, respectively, had no effect on ligand binding or the ability of the receptor to mediate isoproterenol stimulation of adenylate cyclase. Substitution of cysteine 285, in the sixth transmembrane domain of the receptor, produced a mutant receptor with normal ligand-binding properties but a significantly attenuated ability to mediate stimulation of adenylate cyclase. Mutation of cysteine residues 190 and 191, in the third extracellular loop of the beta 2 receptor, had qualitatively similar effects on ligand binding and isoproterenol-mediated stimulation of adenylate cyclase. Replacement of either of these residues with serine produced mutant receptors that displayed a marked loss in affinity for both beta-adrenergic agonists and antagonists. Replacement of both cysteine 190 and 191 with serine had an even greater effect on the ability of the receptor to bind ligands. Consistent with the loss of Ser190 and/or Ser191 mutant receptor affinity for agonists was a corresponding shift to the right in the dose-response curve for isoproterenol-induced increases in intracellular cyclic AMP concentrations in cells expressing the mutant receptors. These data implicate one of the conserved transmembrane cysteine residues in the human beta 2-adrenergic receptor in receptor activation by agonists and also suggest that conserved cysteine residues in an extracellular domain of the receptor may be involved in ligand binding.

Comparative effects of forskolin and isoproterenol on the cyclic AMP content of human adipocytes

Alterations in adipocyte cyclic AMP concentrations in response to 100 μM forskolin and 10 μM isoproterenol over a 4 hour period were found to be similar; with each agent, a peak response was noted within 30 minutes. In general, the greater the magnitude of peak response, the more rapid the decline of cyclic AMP concentration during the ensuing 3 1 2 hours. Alpha-2 adrenergic activation, achieved with 10 μM clonidine or 10 μM epinephrine, substantially reduced the cyclic AMP concentrations in cells stimulated by 100 μM forskolin or 10 μM isoproterenol. Isoproterenol-stimulated cells appeared to be more sensitive to alpha adrenergic inhibition than did forskolin-stimulated cells. Cells preincubated for 3 hours with 100 μM forskolin were markedly less responsive to a second exposure to the diterpine. Cells exposed to forskolin for 3 hourd also had a reduced response when incubated with isoproterenol; thus, desensitization to forskolin appears to be heterologous. Forskolin desensitization did not appear to be dependent on cellular ATP depletion since cells mildly stimulated during preincubation were as severely desensitized as those adipocytes strongly stimulated. Maximum desensitization required a preincubation time of 1–2 hours with either isoproterenol or forskolin.

Stimulatory effect of latex and zymosan particles on cyclic adenosine 3′,5′-monophosphate content in human granulocytes

Human polymorphonuclear leukocytes respond to latex beads and opsonized zymosan particles with increased cyclic AMP formation. The enhancement of cyclic AMP content in cells is related to particle concentration in the incubation medium and to the time of exposure. The temporary stimulation declines after 15 min of phagocytosis, that is in good relation to the uptake of latex particles which reaches saturation point 10 min after the addition of the beads. The cyclic AMP content in granulocytes decreases after between 15 and 30 min of incubation with latex particles. The decrease is not caused by the loss of the granulocyte viability for: (a) the incorporation of labeled methyldeoxythymidine and uridine into macromolecules continues, (b) the sensitivity of phagocytosing granulocytes to prostaglandin E 1 stimulation of cyclic AMP production persists, (c) less than 10% of cells absorbs trypan blue dye after phagocytosis of latex beads. The prostaglandin synthesis inhibitors, aspirin and indomethacin, have no effect on cyclic AMP content in phagocytosing granulocytes.

Stimulatory effect of ionophores on adenosine 3',5'-monophosphate content in human mononuclear leukocytes

Ionophores A23187 and bromo-lasalocid ethanolate enhanced the cyclic AMP content in human mononuclear leukocytes. The maximum effect of A23187 with a 10-min incubation was found with 0.3–1.0μM concentrations with or without l-isoproterenol (1 μM) or prostaglandin E 1 (pge 1) (0.3 μM). The maximum effect after 5 min of incubation at 37° was observed with 0.05, 0.2 and 1 μm A23187. The effect of ionophore A23187 was enhanced by both aminophylline (1 mM) and isobutyl-methylxanthine (1 mM). Calcium (1 mM). aspirin (1 mM) and indomethacin (100 μM) decreased the stimulatory action of A23187. Bromo-lasalocid ethanolate increased cyclic AMP content in cells maximally at a 3 μM concentration with or without 0.3 μM pge 1.

Cyclic AMP formation during phagocytosis

The content of cyclic adenosine-3′, 5′-monophosphate (AMP) in phagocytic macrophages was shown to be increased especially during phagocytosis of the living microbes. The cyclic AMP formed during phagocytosis could be detected in the incubation medium, but in the cells it remained at almost the same level. The cyclic AMP concentration in cells of the intestinal mucosa and in the blood serum of germfree guinea pigs also was increased after injection ofEscherichia coli 055 cells; this points to the participation of the adenylate cyclase system in interaction between microorganisms and the epithelium of the small intestine.