adenosine-5--(n-ethylcarboxamide) and Pheochromocytoma

Tyrosine hydroxylase (TH) gene transcription rate is increased in rat adrenal medulla after administration of muscarinic agonists. In order to study this muscarinic regulation of TH gene expression in more detail, we have generated a rat pheochromocytoma PC18 cell line that stably expresses the mouse m1 muscarinic acetylcholine receptor. Treatment of this cell line, designated PC18/m1-13, with carbachol leads to rapid increases in phosphatidylinositol turnover and intracellular [Ca2+]i; these increases are totally blocked by the muscarinic antagonist atropine. Carbachol produces no changes in cAMP levels or protein kinase A activity in PC18/m1-13 cells. TH mRNA levels in PC18/m1-13 cells increase approximately 3-fold after 6 h of treatment with carbachol. This induction of TH mRNA is also completely inhibited by simultaneous treatment with atropine. Transient transfection assays using a TH gene promoter-chloramphenicol acetyltransferase (TH-CAT) construct demonstrate that sequences within the most proximal 272 bp of the TH gene 5'-flanking region are responsive to carbachol in PC18/m1-13 cells. Studies using TH-CAT constructs with site-directed mutations within the TH gene promoter indicate that the responsiveness of the promoter to carbachol is mediated primarily by the cAMP response element; however, the AP1 site also participates to a lesser extent in this response. The carbachol-mediated stimulation of TH gene promoter activity is partially inhibited by down-regulation of protein kinase C (PKC) or by treatment with the Ca2+/calmodulin-dependent protein kinase inhibitor, KN62. These results are consistent with the hypothesis that agonist occupation of m1 muscarinic receptors stimulates the TH gene via signal transduction pathways that are initiated by activation of PKC and Ca2+/calmodulin-dependent protein kinase, leading to activation of transcription factors that interact with the TH CRE and AP1 sites.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adrenal Gland Neoplasms; Animals; Atropine; Calcium; Carbachol; Cell Membrane; Chloramphenicol O-Acetyltransferase; Colforsin; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Mice; Mutagenesis, Site-Directed; Pheochromocytoma; Phosphatidylinositols; Quinuclidinyl Benzilate; Rats; Receptor, Muscarinic M1; Receptors, Muscarinic; Recombinant Fusion Proteins; Transcription, Genetic; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

The adenosine (ADO) receptor in NIH 3T3 fibroblasts was characterized using a series of adenosine agonists and selected xanthine and non-xanthine antagonists. The ADO receptor elicited accumulations of cyclic AMP in intact NIH 3T3 fibroblasts and caused activation of adenylate cyclase in membrane preparations. The receptor had characteristics of the A2b subtype of adenosine receptor. ADO analogs had relatively high EC50 values at the receptor and were antagonized competitively by xanthines. The rank order of potency for adenosine analogs in NIH 3T3 fibroblasts for cyclic AMP accumulation was: NECA > 2-ClADO > R-PIA >> CV1808, CGS 21680. The EC50 for 2-ClADO was 4.3 microM in intact cells and 15 microM in membrane preparations. All ADO analogs were more potent at the A2a receptor of pheochromocytoma PC12 membranes than at the A2b receptor of fibroblast NIH 3T3 membranes. Structure-activity relationships suggested that the regions of interaction with 5'- and N6-substituents of ADO were similar for both the PC12 A2a and NIH 3T3 A2b receptor. However, ADO analogs with large substituents in the 2'-position, such as 2-cyclohexylethoxy ADO and CGS 21680, were highly selective for the A2a receptor. All ADO analogs tested were stimulatory to adenylate cyclase at the NIH 3T3 A2b receptor, including 5'-methylthioADO, which was a weak partial agonist. A series of xanthine antagonists were not selective for the NIH 3T3 A2b versus the PC12 A2a receptor. In all cases, xanthines were more potent as antagonists in the intact NIH 3T3 cells than in NIH 3T3 membranes. In a series of non-xanthine antagonists, most compounds were equipotent or slightly more potent at the A2a receptor except for alloxazine, which was approximately 9-fold selective for the A2b receptor.

Topics: 3T3 Cells; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclases; Animals; Cell Membrane; Cyclic AMP; Deoxyadenosines; Mice; PC12 Cells; Pheochromocytoma; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Thionucleosides; Xanthines

The relationship of adenotin, a low-affinity adenosine-binding protein, to adenosine receptors was examined in two human tissues and two mammalian cultured cell lines. An adenosine A2 receptor exists in the membranes from platelets, PC-12 cells, and JAR cells as shown by a stimulation of adenylate cyclase related to 5'-N-ethylcarboxamidoadenosine (NECA) or a NECA-related increase in intracellular cAMP levels. In contrast, binding studies with tritiated NECA revealed typical adenotin-like low-affinity binding sites on the membranes from the sources studied with agonist potencies as follows: NECA greater than 2-chloroadenosine greater than R-PIA. No evidence was found of coupling to a guanine nucleotide regulatory protein. Solubilization of platelet and placental membranes and precipitation with polyethylene glycol separated adenotin or the adenotin-like protein from a second adenosine binding site in each tissue. The pharmacologic properties of the precipitated binding sites were compatible with an adenosine A2 receptor in platelets and an adenosine A1 receptor in placenta. Our observations indicate that adenotin-like proteins exist outside the placenta. In addition, adenotin and adenotin-like proteins coexist with the adenosine A1 or A2 receptor in a number of cells and tissues and do not couple to a guanine nucleotide regulatory protein and stimulate adenylate cyclase. Therefore, adenotin is pharmacologically distinct from adenosine receptors, and its function remains to be discovered.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclases; Adrenal Gland Neoplasms; Animals; Blood Platelets; Carrier Proteins; Cell Membrane; Choriocarcinoma; Chromatography, High Pressure Liquid; Cyclic AMP; Female; Humans; Pheochromocytoma; Placenta; Polyethylene Glycols; Pregnancy; Radioimmunoassay; Receptors, Purinergic; Tumor Cells, Cultured; Uterine Neoplasms; Vasodilator Agents

Examination of the binding characteristics of the adenosine agonist radioligands [3H]N6-cyclohexyladenosine [( 3H]CHA), [3H]cyclopentyladenosine [( 3H]CPA), and [3H]5'-N-ethylcarboxamido adenosine [( 3H]NECA) to membranes prepared from PC12 cells showed that the A-1-selective ligands (CHA and CPA) had minimal binding, which was not amenable to analysis using curve-fitting programs. However, [3H]NECA, a nonselective A-1/A-2 agonist, gave reproducible binding, which was enhanced by removal of endogenous adenosine, using the catabolic enzyme adenosine deaminase. This binding was of high affinity (KD = 4.7 nM) with limited capacity (263 fmol/mg of protein). Specific binding of [3H]NECA was unaffected by the presence of either CPA (50 nM) or MgCl2 (10 mM) but was sensitive to guanylylimidodiphosphate (100 microM), a finding suggesting involvement of an N-protein mechanism in the coupling of the adenosine receptor labeled by [3H]NECA to other components of the receptor complex. Binding of [3H]NECA to PC12 cell membranes was stereo-selective, with the R isomer of N6-phenylisopropyladenosine (PIA) being approximately 12 times more active than S-PIA. The A-1-selective agonist CPA was a weak inhibitor of [3H]NECA binding (Ki = 251 nM). The rank order of activity of adenosine agonists in displacing specific [3H]NECA binding was NECA greater than or equal to 2-chloroadenosine greater than CHA greater than or equal to 5'-N-methylcarboxamido adenosine greater than or equal to R-PIA greater than CPA greater than S-PIA. Binding was also displaced by the marine adenosine agonist 1-methylisoguanosine and by a series of xanthine antagonists with the activity order being 1,3-dipropyl-8-(2-amino-4-chloro)phenylxanthine greater than 8-phenyltheophylline greater than 8-p-sulfophenyltheophylline.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adrenal Gland Neoplasms; Animals; Cell Line; Guanylyl Imidodiphosphate; Magnesium; Magnesium Chloride; Pheochromocytoma; Radioligand Assay; Receptors, Purinergic; Substrate Specificity

The coronary vasoactivity of N-ethyl-1'-deoxy-1'-(6-amino-9H-purin-9-yl)-beta-D-ribofuranuronamide (NECA, 1) is over 2 orders of magnitude greater than that of adenosine, and the vasoactivity of certain N6-substituted adenosines is as much as 1 order of magnitude greater. Such results suggest that a combination of appropriate modifications at N6 and C-5' might additively augment the agonist potency of adenosine. At low temperatures 1-deoxy-1-(6-chloro-9H-purin-9-yl)-2',3'-O-isopropylidene- beta-D-ribofuranosyl chloride (5), obtained in three steps from inosine, reacts with amines to yield uronamides. The subsequent reaction of such uronamides with amines at elevated temperatures displaces the purine 6-chloro group to yield, after deblocking, N-alkyl(or aryl)-N6-alk(ar)yl-adenosine-5'-uronamides. At the coronary artery A2 receptor the potency of N6-modified analogues of 1 is similar to that of the N6-substituted adenosine, rather than equal to or greater than 1. As agonists in the A2 receptor-mediated stimulation of adenylate cyclase in plasma membranes of PC12 pheochromocytoma cells or human platelets, N6-substituted analogues of 1 are intermediate between the high potency of 1 and the lower potency of the N6-substituted adenosines. At the A1 receptor of rat brain the potency of an N6-substituted analogue of 1 is often greater than that of the corresponding N6-substituted adenosine. At all four receptors, replacing the ethyl group of N-ethyl-N6-3-pentyladenosine-5'-uronamide by larger alkyl groups reduces potency; amides of secondary amines are inactive or have only marginal activity. Analogues of 1 containing a chiral center in the N6 substituent retain the stereoselectivity characteristic of each of the four receptors. Thus, at either A1 or A2 adenosine receptors, adenosine analogues interact with both the N6 and the C-5' receptor regions. However, the effects of N6 and C-5' modifications on potency are less than additive, evidence that the interaction of a substituent with its receptor region influences the interaction of other substituents with their respective receptor regions.

Topics: Adenosine; Adenylyl Cyclases; Adrenal Gland Neoplasms; Animals; Binding, Competitive; Biological Assay; Blood Platelets; Cell Membrane; Cerebral Cortex; Chemical Phenomena; Chemistry; Coronary Circulation; Dogs; Humans; Ligands; Pheochromocytoma; Rats; Receptors, Cell Surface; Receptors, Purinergic; Structure-Activity Relationship

Several analogs of caffeine have been investigated as antagonists at A2 adenosine receptors stimulatory to adenylate cyclase in membranes from rat pheochromocytoma PC12 cells and human platelets and at A1 adenosine receptors inhibitory to adenylate cyclase from rat fat cells. Among these analogs, 1-propargyl-3,7-dimethylxanthine was about 4- to 7-fold and 7-propyl-1,3-dimethylxanthine about 3- to 4-fold more potent than caffeine at A2 receptors of PC12 cells and platelets. At A1 receptors of fat cells, both compounds were about 2-fold less potent than caffeine. These caffeine analogs have an A1/A2 selectivity ratio of about 10-20 and are the first selective A2 receptor antagonists yet reported. The results may provide the basis for the further development of highly potent and highly selective A2 adenosine receptor antagonists.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclases; Adipose Tissue; Adrenal Gland Neoplasms; Animals; Blood Platelets; Caffeine; Humans; Phenylisopropyladenosine; Pheochromocytoma; Rats; Receptors, Cell Surface; Receptors, Purinergic; Structure-Activity Relationship

Incubation of cell-free extracts from PC12 cells with [32P]ATP leads to the phosphorylation of a 100,000-dalton protein. In extracts from cells treated with nerve growth factor, the labeling of the 100,000-dalton protein is substantially and selectively reduced. Direct quantitation indicates that the reduction is a minimum of 30-50% in the various experiments. The decrease is evident after as little as 15 min of nerve growth factor treatment, and disappears within 2 h after the removal of nerve growth factor. The decrease is dose dependent; a complete response is seen after treatment with 10 ng of nerve growth factor/ml. Some decrease in phosphorylation is also seen after treatment of the cells with epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, or 5'-N-ethylcarboxamideadenosine, a potent adenosine receptor agonist, but not after treatment with insulin. The phosphorylation of the 100,000-dalton protein, in extracts from either control or nerve growth factor-treated cells, leads almost exclusively to the formation of phosphothreonine. The addition of equal amounts of extract from untreated cells and extract from nerve growth factor-treated cells produces a level of phosphorylation exactly intermediate between those of the two extracts used separately, indicating the absence of a soluble kinase inhibitor. The data suggest that nerve growth factor treatment produces either a covalent inhibition or a physical removal of the kinase for the 100,000-dalton protein.

Topics: Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Animals; Cell Line; Cell-Free System; Cyclic AMP; Epidermal Growth Factor; Ganglia, Sympathetic; Nerve Growth Factors; Nerve Tissue Proteins; Pheochromocytoma; Phosphorylation; Protein Kinase Inhibitors; Rats; S100 Proteins

Nerve growth factor treatment produces a large increase in the activity of ornithine decarboxylase and a moderate decrease in the activity of S-adenosylmethionine decarboxylase in PC12 cells. These changes are reflected weakly, if at all, in the levels of putrescine, spermidine, and spermine in the cells. The rates of polyamine synthesis are increased somewhat more than the overall levels, but still are not comparable in extent to the increase in the ornithine decarboxylase activity. Inhibitors of ornithine decarboxylase and S-adenosylmethionine decarboxylase have their expected effects on the induction of ornithine decarboxylase and on the activities of both enzymes. Neither inhibitor alone, nor a combination of inhibitors, altered the rate or extent of nerve growth factor-induced neurite outgrowth in the cells.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenosylmethionine Decarboxylase; Adrenal Gland Neoplasms; Animals; Carboxy-Lyases; Cell Line; Epidermal Growth Factor; Kinetics; Neoplasms, Experimental; Nerve Growth Factors; Ornithine Decarboxylase; Pheochromocytoma; Polyamines; Rats; Vasodilator Agents

PC12 cells, a nerve growth factor-responsive clone of rat pheochromocytoma, contain a membrane-bound adenylate cyclase, which can be activated by adenosine analogs. The characteristics of the cyclase response indicate the presence of stimulatory adenosine receptors. Adenosine analogs also produce a marked increase in the ornithine decarboxylase levels of the cells, and the characteristics of this response suggest that it is linked to the adenylate cyclase-stimulatory adenosine receptors. The ornithine decarboxylase response elicited by 5'-N-ethylcarboxamideadenosine (NECA), a potent stimulatory adenosine analog, is synergistic with that produced by nerve growth factor. Differentiation of the cells with nerve growth factor, however, does not substantially alter either the response of cyclase to the adenosine analog or the magnitude of the adenosine-evoked ornithine decarboxylase response. Treatment of the cells with NECA produces an increase in the phosphorylation of a specific non-histone nuclear protein. While causing little or no morphological alteration by itself, NECA is synergistic with nerve growth factor in producing neurite outgrowth in PC12 cells. NECA does not cause an induction of acetylcholinesterase in the cells. NECA does not cause an induction of acetylcholinesterase in the cells, nor does it appear to affect the induction of this enzyme by nerve growth factor.

Topics: Acetylcholinesterase; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclases; Adrenal Gland Neoplasms; Animals; Carboxy-Lyases; Cell Line; Cell Membrane; Clone Cells; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Nerve Growth Factors; Ornithine Decarboxylase; Pheochromocytoma; Rats