Extracellular ATP-dependent suppression of proliferation and induction of differentiation of human HL-60 leukemia cells by distinct mechanisms

Extracellular ATP suppressed the growth of HL-60 leukemia cells and induced their differentiation as revealed by N-formyl-methionyl-leucyl-phenylalanine-induced beta-glucuronidase release. ATP degraded to ADP, AMP, and adenosine, and the effect of ATP on cell growth was mimicked by these metabolites added to the cultures. The stable analog alpha,beta-methylene ATP, however, had only a weak inhibitory effect on cell growth. Adenine nucleotide-induced growth suppression was reversed by uridine, suggesting the involvement of intracellular pyrimidine starvation secondary to adenosine accumulation. Consistent with this, ATP induced intracellular starvation of pyrimidine nucleotides, and this effect was also prevented by pretreatment of cells with uridine. The order of effectiveness of ATP-induced differentiation of HL-60 cells, unlike that for growth suppression, was ATP > ADP > AMP, and adenosine had no effect. Furthermore, uridine had no effect and the stable analog, alpha,beta-methylene ATP also induced HL-60 cell differentiation, suggesting that differentiation was due to ATP per se. We tested the hypothesis that ATP-induced differentiation arises from activation of adenylyl cyclase by the novel P2Y(11) receptor using the cell-permeable inhibitor of protein kinase A, Rp-CPT-cAMPS (8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate, Rp isomer). Rp-CPT-cAMPS (1-100 microM) prevented ATP-induced differentiation of HL-60 cells as assessed by fMLP-induced beta-glucuronidase release. However, Rp-CPT-cAMPS did not prevent ATP-induced growth suppression. Taken together, the data indicate that extracellular ATP suppresses HL-60 growth and induces their differentiation by distinct mechanisms. Growth suppression arises from adenosine generation and consequent pyrimidine starvation. Differentiation arises, at least in part, from a distinct mechanism involving the activation of cell surface P2 receptors coupled to cAMP generation and activation of protein kinase A.     

Studies on the receptor mediating cyclic AMP-independent enhancement by adenosine of IgE-dependent mediator release from rat mast cells.

Adenosine produced a concentration-related enhancement of antigen-induced 5-hydroxytryptamine (5-HT) release from rat serosal mast cells. This potentiation was maximal following the simultaneous addition of adenosine with antigen. Enhancement of 5-HT release was accompanied by potentiation of the adenosine 3':5'-cyclic monophosphate (cyclic AMP) response to challenge. The cyclic AMP response, which was antagonized by 8-phenyltheophylline, was characterized as an A2-purinoceptor-mediated effect by the use of 5'-N-ethylcarboxamideadenosine (NECA) and L-N6-phenylisopropyladenosine (L-PIA). Enhancement of 5-HT release, conversely, was not blocked by 8-phenyltheophylline suggesting it to be mediated by a cyclic AMP-independent mechanism. The effect of adenosine on 5-HT release was not reduced by the inhibition of the facilitated uptake of adenosine with dipyridamole, hexobendine or p-nitrobenzylthioguanosine, therefore, suggesting it to be mediated by a cell surface receptor. The receptor mediating enhancement of 5-HT does not appear to belong to the P2-purinoceptor subtype as adenosine was more potent than both adenosine monophosphate (AMP) and adenosine diphosphate (ADP) and alpha, beta-methylene ATP was inactive. Furthermore, the effects of AMP were blocked by alpha, beta-methylene ADP, which inhibits the conversion of AMP to adenosine. Adenosine, NECA, L- and D-PIA were all of equal potency in enhancing 5-HT release. Inosine and 3-deazaadenosine were also active. The rank order of potency of these adenosine analogues is not consistent with an effect at A1- or A2-purinoceptors. There appear to be two adenosine receptors on rat mast cells, an A2-purinoceptor which stimulates adenylate cyclase and a separate purinoceptor, stimulation of which produces enhancement of mediator release by an unknown mechanism. The effects mediated by these receptors appear to be independent of each other.     

P2 purinoceptor regulation of mucin release by airway goblet cells in primary culture.

1. The effects of adenine analogues on mucin release by airway goblet cells have been examined in a hamster primary tracheal epithelial cell culture. 2. Adenosine, a P1 receptor agonist, had no effect on mucin release even at 2 mM, while ATP, a P2 receptor agonist, stimulated mucin release in a dose-dependent fashion with an apparent EC50 of 20 microM. The relative potency order among adenine nucleotides was ATP greater than ADP greater than AMP = adenosine. 3. ATP gamma S, a non-hydrolyzable analogue of ATP, was equipotent with ATP in stimulating mucin release. The potency order among some ATP analogues was ATP greater than 2-methylthio ATP greater than alpha, beta-methylene ATP greater than beta, gamma-methylene ATP. Reactive blue 2, a putative P2 gamma-purinoceptor antagonist, did not block the ATP-induced mucin release. 4. The present results indicate that mucin release by airway goblet cells is stimulated by extracellular ATP via P2 receptor-mediated mechanism. We suggest that this mechanism may be important in the physiological regulation of airway goblet cell mucin release in vivo.     

Apoptosis induced by extracellular ATP in the mouse neuroblastoma cell line N1E-115: studies on involvement of P2 receptors and adenosine

Adenosine triphosphate (ATP) can be released in large amounts from (damaged) cells, leading to locally high concentrations. In this study, we investigated the effect of such high concentrations of ATP on neuroblastoma cells. ATP (>or=30 microM) induced apoptosis in the mouse neuroblastoma cell line N1E-115. Activation of the ATP receptor P2X(7) is one of the routes via which ATP has been shown to induce apoptosis. Although the P2X(7) receptor was present in N1E-115 cells, both at the protein and mRNA level, studies with the P2X(7) receptor agonist benzoyl-benzoyl ATP showed that this receptor was not involved in ATP-induced apoptosis. It has been shown previously that adenosine induces apoptosis in N1E-115 cells after transport inside the cell. In this study, both dipyridamole, a nucleoside transport protein blocker, and uridine, a substrate for this transporter, were able to block ATP-induced apoptosis. This indicated that ATP had to be broken down to adenosine to induce apoptosis. The ecto-nucleotidase inhibitors 6-N,N-diethyl-beta-dibromomethylene-D-adenosine-5'-triphosphate (ARL67156) and alpha,beta-methylene adenosine 5'-diphosphate (AOPCP) commonly used to slow breakdown of ATP did not inhibit ATP breakdown appreciably, while the ATP antagonist PPADS inhibited the breakdown of AMP to adenosine; PPADS was also the only compound capable of inhibiting ATP-induced apoptosis. We conclude that the main route of ATP-induced apoptosis in N1E-115 cells was via breakdown to adenosine.     

Stimulation of prostacyclin release from aortic smooth muscle cells by purine and pyrimidine nucleotides

ATP and ATP gamma S(10-100 microM) stimulated the release of prostacyclin (PGI2) from bovine aortic smooth muscle cells. This effect was reproduced by UTP, ITP and partially by GTP. ADP and ADP beta S, the P2X-selective agonist alpha, beta-methylene ATP (APCPP), AMP and adenosine were all inactive. This effect of ATP gamma S was not inhibited by Reactive Blue 2, an antagonist of P2Y receptors. The stimulation of PGI2 production in aortic smooth muscle cells by these nucleotides thus seems to involve receptors distinct from both P2X and P2Y subtypes, which are responsible for smooth muscle contraction and PGI2 release from endothelial cells, respectively.     

Functional expression of adenosine A2A and A3 receptors in the mouse dendritic cell line XS-106

There is increasing evidence to suggest that adenosine receptors can modulate the function of cells involved in the immune system. For example, human dendritic cells derived from blood monocytes have recently been described to express functional adenosine A1, A2A and A3 receptors. Therefore, in the present study, we have investigated whether the recently established murine dendritic cell line XS-106 expresses functional adenosine receptors. The selective adenosine A3 receptor agonist 1-[2-chloro-6[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-beta-D-ribofuranuronamide (2-Cl-IB-MECA) inhibited forskolin-mediated [3H]cyclic AMP accumulation and stimulated concentration-dependent increases in p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation. The selective adenosine A2A receptor agonist 4-[2-[[-6-amino-9-(N-ethyl-beta-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzene-propanoic acid (CGS 21680) stimulated a robust increase in [3H]cyclic AMP accumulation and p42/p44 MAPK phosphorylation. In contrast, the selective adenosine A1 receptor agonist CPA (N6-cyclopentyladenosine) did not inhibit forskolin-mediated [3H]cyclic AMP accumulation or stimulate increases in p42/p44 MAPK phosphorylation. These observations suggest that XS-106 cells express functional adenosine A2A and A3 receptors. The non-selective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) inhibited lipopolysaccharide-induced tumour necrosis factor-alpha (TNF-alpha) release from XS-106 cells in a concentration-dependent fashion. Furthermore, treatment with Cl-IB-MECA (1 microM) or CGS 21680 (1 microM) alone produced a partial inhibition of lipopolysaccharide-induced TNF-alpha release (when compared to NECA), whereas a combination of both agonists resulted in the inhibition of TNF-alpha release comparable to that observed with NECA alone. Treatment of cells with the adenosine A2A receptor selective antagonists 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5ylamino]ethyl)phenol (ZM 241385; 100 nM) and 5-amino-2-(2-furyl)-7-phenylethyl-pyrazolo[4,3-e]-1,2,4-triazolo[1,5c]pyrimidine (SCH 58261; 100 nM) and the adenosine A3 receptor selective antagonist N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide (MRS 1220; 100 nM) partially blocked the inhibitory effects of NECA on lipopolysaccharide-induced TNF-alpha release. Combined addition of MRS 1220 and SCH 58261 completely blocked the inhibitory effects of NECA on lipopolysaccharide-induced TNF-alpha release. In conclusion, we have shown that the mouse dendritic cell line XS-106 expresses functional adenosine A2A and A3 receptors, which are capable of modulating TNF-alpha release.     

Effects of IL-4 and IL-13 on adenosine receptor expression and responsiveness of the human mast cell line 1

BACKGROUND: Inhalation of adenosine-5'-monophosphate (AMP) causes bronchoconstriction in asthma but not in healthy subjects. Bronchoconstriction upon AMP inhalation is thought to occur by histamine release and subsequent binding to receptors on airway smooth muscle cells. METHODS: To explain enhanced sensitivity to AMP in asthma, mast cell expression of the adenosine A2A and A2B receptors and histamine release were measured after incubation of human mast cell line 1 (HMC-1) cells with AMP and the non-specific adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) for 1.5 and 6 h. To establish a Thelper-2 environment resembling the asthma phenotype, HMC-1 cells were additionally cultured with IL-4 and IL-13 alone or stimulated with the combination of both cytokines and AMP and NECA. To study effects of prolonged presence of the inflammatory environment, the cells were pre-incubated overnight (18 h) with IL-4 and IL-13 and additionally stimulated with AMP and NECA for 1.5 or 6 h. RESULTS: AMP and NECA hardly affected adenosine receptor expression but increased IL-8 secretion. Incubation with IL-4 and IL-13 for 6 h increased adenosine A2A receptor expression and histamine secretion, but decreased IL-8 secretion. The combination of IL-4, IL-13, and AMP/NECA for 6 h increased A2B receptor expression and IL-8 secretion. Overnight stimulation with IL-4, IL-13 and subsequent stimulation with AMP/NECA for 1.5 h decreased A2AR expression which was accompanied by increased histamine secretion. CONCLUSION: These results suggest a role for decreased A(2A)R expression in enhanced adenosine responsiveness as observed in asthma.     

A study of the purinoceptors mediating contraction in the rat colon.

1. The effects of a number of purine analogues were examined on the rat isolated colon muscularis mucosae. Adenosine, adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), adenosine 5'-triphosphate (ATP), 2-methylthioATP (MeSATP), adenosine 5'-(2-fluorodiphosphate) (ADP beta F), adenosine 5'-(beta, gamma-methylene)triphosphonate (AMPPCP) and adenosine 5'-(alpha, beta-methylene)triphosphonate (AMPCPP) each contracted the muscularis mucosae in the concentration range 1-100 microM. 2. MeSATP was the most potent purine agonist, with a threshold concentration for contraction of 0.05 microM and an EC50 of approximately 0.3 microM, and AMPCPP was less potent than ATP. The enantiomer of AMPPCP, L-AMPPCP, was inactive at concentrations up to 100 microM. 3. The adenosine receptor antagonist 8-(p-sulphophenyl)theophylline (8-SPT, 50 microM) produced approximately 50 fold shifts of the dose-response curves to adenosine, AMP and AMPPCP, whereas those to ATP, MeSATP and substance P (SP) were unaffected. Intermediate shifts were observed for the dose-response curves to ADP, ADP beta F and AMPCPP. With a lower concentration of 8-SPT (10 microM) a dose ratio of approximately 11 was observed for the inhibition of the effects of both adenosine and AMPPCP. 4. ATP was rapidly degraded by the tissue to ADP, AMP and adenosine, ADP beta F was more slowly degraded to AMP and adenosine, and no significant degradation of AMPPCP was detected during 20 min incubation. 5. The results are consistent with the existence in the rat colon muscularis mucosae of a mixed population of purine receptors of P2Y and P1 types. The colon thus contains the first documented incidence of a P2Y-receptor mediating contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence against adenosine analogues being agonists at the growth hormone secretagogue receptor

Adenosine and adenosine analogues have been reported to act as agonists or partial agonists at the growth hormone secretagogue receptor 1a (GHSR1a). We have re-examined this question. A concentration-dependent increase in intracellular calcium concentration ([Ca(2+)](i)) was observed in GHSR1a transfected HEK 293-EBNA cells stimulated with adenosine (EC50: 0.2 microM) or 2-chloroadenosine (EC50: 1.1 microM) but also in untransfected HEK 293-EBNA cells stimulated with 2-chloroadenosine (EC50: 0.67 microM) or 5'-N-ethylcarboxamidoadenosine (NECA) (EC50: 0.045 microM). These findings support endogenous expression of adenosine receptors, presumably A(2B) receptors in HEK 293-EBNA cells. In GHSR1a transfected CHO cells, lacking adenosine receptors, the GHSR1a agonist hGhrelin (EC50: 2.4 nM) increased [Ca(2+)](i), but no effects of adenosine, 2-chloroadenosine or NECA were detected. An inverse agonist of GHSR1a, [d-Arg-1, d-Phe-5, d-Trp-7, 9, Leu-11] substance P, reduced hGhrelin effects but adenosine, 2-chloroadenosine or 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) did not. NECA increased the [Ca(2+)](i) in co-transfected (GHSR1a and A(2B) receptor) CHO cells (EC50: 0.053 microM), but no additive or synergistic effects on [Ca(2+)](i) or cAMP formation were observed after stimulation with NECA in the absence or in the presence of hGhrelin. In binding studies on GHSR1a transfected CHO cell membranes, [(125)I]-hGhrelin binding could be displaced by the GHSR1a agonist MK-0677 (IC50: 0.34 nM), hGhrelin (IC50: 1.5 nM), and the substance P analogue (IC50: 0.64 microM) but not by adenosine or 2-chloroadenosine. We conclude that adenosine and analogues do not act as agonists or partial agonists at the GHSR1a and that cross-talk between the GHSR1a and A(2B) receptors is limited.     

Effects of adenine nucleosides and nucleotides on neuromuscular transmission to the prostatic stroma of the rat

1. The aim of this study was to investigate the effects of adenine nucleosides and nucleotides on contractility of the smooth muscle of rat prostate gland. 2. Nerve terminals within rat isolated prostatic tissues were electrically field stimulated (60 V, 0.5 ms, 10 Hz, 20 pulses every 60 s). Adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) and adenosine had no effect on baseline smooth muscle tone but concentration-dependently inhibited electrically-evoked contractile responses. The relative order of potency was ATP congruent with AMP congruent with adenosine>ADP. 3. The inhibition by ATP and adenosine of field stimulation-induced contractions in the rat prostate was antagonized by 8-phenyltheophylline (10 microM), but not by suramin (100 microM) and only slightly by reactive blue 2 (5 microM). 4. The adenosine metabolizing enzyme adenosine deaminase (0.1 unit ml(-1)) inhibited the inhibitory effects of ATP and adenosine. The P2 purinoceptor agonist 2-methylthio ATP (10 nM - 0.1 mM), had no effect on field stimulation-induced contractions of the rat prostate. 5. ATP and adenosine did not modify the contractile responses of the rat prostate to exogenously added noradrenaline (10 microM). 6. Inhibitory concentration-response curves to a number of adenosine analogues with differing stabilities and selectivities for the different adenosine receptors yielded a relative rank order of agonist potency of: N(6)-cyclopentyladenosine (CPA)>N(6)-cyclohexyladenosine (CHA) congruent with (-)-N(6)-(2-phenylisopropyl)-adenosine (R-PIA) congruent with 5'-(N-ethylcarboxamido)-adenosine (NECA)>(+)-N(6)-(2-phenylisopropyl)-adenosine (S-PIA)>2-p-[2-carboxyethyl]phenethyl-amino-5'-N-ethylcarboxamido-ade nosine (CGS 21680). 7. These results indicate that adenine nucleoside and nucleotide induced inhibition of electrically-evoked contractions in the rat prostate occurs through activation of adenosine but not ATP receptors. The relative order of potency of adenosine analogues is consistent with activation of receptors of the A(1)-adenosine receptor subtype. These receptors appear to be prejunctional.     

Effects of adenosine, adenosine triphosphate and structural analogues on glucagon secretion from the perfused pancreas of rat in vitro

The effects of adenosine, adenosine triphosphate (ATP) and structural analogues have been studied on glucagon secretion from the isolated perfused pancreas of the rat in the presence of glucose (2.8 mM). Adenosine induced a transient increase of glucagon secretion. This effect was concentration-dependent in the range of 0.165 to 165 microM. ATP also induced an increase, but the effect was no greater at 165 microM than at 16.5 microM. 2-Chloroadenosine, an analogue more resistant to metabolism or uptake systems than adenosine, was more effective. Among the three structural analogues of ATP or ADP studied, beta, gamma-methylene ATP which can be hydrolyzed into AMP and adenosine had an effect similar to adenosine or ATP at the same concentrations (1.65 and 16.5 microM); in contrast alpha, beta-methylene ATP and alpha, beta-methylene ADP (resistant to hydrolysis into AMP and adenosine) were ineffective. Theophylline (50 microM) a specific blocker of the adenosine receptor, suppressed the glucagon peak induced by adenosine, 2-chloroadenosine, ATP and beta, gamma-methylene ATP (1.65 microM). An inhibitor of 5' nucleotidase, alpha, beta-methylene ADP (16.5 microM), reduced the glucagon increase induced by ATP and did not affect the response to adenosine (1.65 microM). These results support the hypothesis of adenosine receptors (P1-purinoceptors) on the pancreatic glucagon secretory cells and indicate that ATP acts after hydrolysis to adenosine.     

Dual effects of protein kinase-C on receptor-stimulated cAMP accumulation in a human T-cell leukemia line

In the human T-cell leukemia line Jurkat, cAMP accumulation stimulated by the adenosine receptor agonist 5'-N-ethylcarboxamido adenosine (NECA) was enhanced by tumour-promoting phorbol esters whereas the prostaglandin receptor-stimulated accumulation of cAMP was antagonized. Phorbol esters did not alter the adenosine or prostaglandin receptor-stimulated accumulation of cAMP in cells in which the phospholipid/Ca2+-dependent protein kinase (protein kinase-C) was down-regulated. cAMP stimulation induced by cholera toxin (CT) was enhanced by phorbol esters by 100-300%. The cAMP production induced by forskolin was never enhanced by more than 50% by 4 beta-phorbol-12,13-dibutyrate (PDBu) and there was no stimulation at all after down-regulation of the adenosine receptor by treatment with NECA. Phorbol ester enhanced the NECA-stimulated accumulation of cAMP, even in the presence of concentrations of forskolin that increased the cAMP accumulation several-fold. From these data we conclude that protein kinase-C can interact with receptors coupled to adenylate cyclase in a stimulatory as well as an inhibitory manner. Moreover, protein kinase-C appears to interact with signal transduction at two levels, one highly receptor-specific and one distal to the receptor.     

Pharmacological characterization of adenosine receptors in PGT-beta mouse pineal gland tumour cells

1. The adenosine receptor in mouse pinealocytes was identified and characterized using pharmacological and physiological approaches. 2. Expression of the two adenosine receptor subtypes A2B and A3 was detected in mouse pineal glands and PGT-beta cells by polymerase chain reaction and nucleotide sequencing. 3. Adenosine and 5'-N-ethylcarboxamidoadenosine (NECA) evoked cyclic AMP generation but the A2)-selective agonist 2-(4-(2-carboxyethyl)phenylethylamino)adenosine-5'-N-ethylcarboxamideadenosine (CGS 21680) and the A1-specific agonists R-N(6)-(2-phenylisopropyl)adenosine (R-PIA) and N(6)-cyclopentyladenosine (CPA) had little effect on intracellular cyclic AMP levels. The A2B receptor selective antagonists alloxazine and enprofylline completely blocked NECA-mediated cyclic AMP accumulation. 4. Treatment of cells with the A3-selective agonist N(6)-(3-iodobenzyl)-5'-(N-methylcarbamoyl)adenosine (IB-MECA) inhibited the elevation of the cyclic AMP level induced by NECA or isoproterenol in a concentration-dependent manner with maximal inhibition of 40 - 50%. These responses were blocked by the specific A3 adenosine receptor antagonist MRS 1191. Pretreatment of the cells with pertussis toxin attenuated the IB-MECA-induced responses, suggesting that this effect occurred via the pertussis toxin-sensitive inhibitory G proteins. 5. IB-MECA also caused a concentration-dependent elevation in [Ca(2+)]i and IP3 content. Both the responses induced by IB-MECA were attenuated by treatment with U73122 or phorbol 12-myristate 13-acetate. 6. These data suggest the presence of both A2B and A3 adenosine receptors in mouse pineal tumour cells and that the A2B receptor is positively coupled to adenylyl cyclase whereas the A3 receptor is negatively coupled to adenylyl cyclase and also coupled to phospholipase C.     

Adenosine receptors on human airway epithelia and their relationship to chloride secretion.

1. We have characterized an adenosine receptor subtype present in human airway epithelial cells by measuring the changes in the intracellular levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and the rate of transepithelial Cl- secretion. 2. Primary cultures of human nasal epithelium obtained from excised surgical airway epithelial tissues and the cell lines BEAS39 and CF/T43 derived from human airway epithelium were grown on plastic dishes and labelled with [3H]-adenine for measurement of intracellular cyclic AMP accumulation. Primary cultures were loaded with the calcium indicator fura-2 to measure [Ca2+]i and studied as polarized, ion transporting epithelia on collagen matrix supports for measurement of Cl- secretion. 3. Adenosine analogues stimulated cyclic AMP accumulation with a rank order of potency characteristic of an A2-receptor: 5-N-ethyl-carboxamidoadenosine (NECA) greater than adenosine greater than R-phenylisopropyladenosine (R-PIA), 6-N-cyclopentyladenosine (CPA) greater than S-PIA. NECA increased cyclic AMP accumulation in normal and cystic fibrosis (CF) primary cells as well as in the CF/T43 and BEAS39 cell lines with K0.5 values ranging from 0.3 to 3 microM. Preincubation with NECA resulted in the homologous desensitization of airway epithelial cells. The effect of NECA was specifically inhibited by the adenosine receptor antagonist, aminophylline, in a competitive manner. 4. The A1-adenosine receptor agonists CPA and R-PIA did not inhibit isoprenaline-stimulated cyclic AMP accumulation in CF/T43 cells, and potentiating effects of the adenosine analogues were observed on forskolin-stimulated cyclic AMP accumulation. Adenosine analogues did not cause significant changes in intracellular Ca2+ ([Ca2+]i) in airway epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cibacron blue stimulation of surfactant secretion in rat type II pneumocytes.

1. The effect of cibacron blue, a selective P2y-purinoceptor antagonist in some systems, on the stimulatory effect of adenosine 5'-triphosphate (ATP) on [3H]-phosphatidylcholine secretion was examined in primary cultures of rat type II pneumocytes prelabelled by overnight culture with [3H]-choline. 2. Cibacron blue alone stimulated phosphatidylcholine secretion in a concentration-dependent manner in the range 10(-4)-10(-3) M. At a concentration of 10(-4) or lower, cibacron blue had no effect on ATP-induced phosphatidylcholine secretion but at 10(-4)-10(-3) M it increased the effect of ATP. Enhancement of the ATP effect was apparent whether cibacron blue was added before or together with ATP. Cibacron blue also increased ATP-induced secretion in the presence of the P1-purinoceptor antagonist, xanthine amine congener (10(-5) M). 3. The stimulatory effect of cibacron blue on phosphatidylcholine secretion was additive to those of 5' (N-ethylcarboxyamido) adenosine (NECA) and terbutaline but less than additive to that of ATP. 4. Cibacron blue alone had no effect on formation of cyclic AMP or inositol phosphate and when added simultaneously with ATP it did not affect the ATP-induced increase in these second messengers. Preincubation of the cells with cibacron blue before addition of ATP, however, resulted in antagonism of the ATP-induced increase in cyclic AMP and inositol phosphates. Preincubation with ATP had the same effect. The stimulatory effects of NECA and terbutaline on cyclic AMP formation were enhanced by preincubation with cibacron blue. 5. Thus, ATP-induced phosphatidylcholine secretion in type II cells is not diminished by the P2y-antagonist, cibacron blue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional characterization of the adenosine receptor contributing to glycogenolysis and gluconeogenesis in rat hepatocytes

The adenosine receptor subtype mediating glucose production by glycogenolysis and gluconeogenesis was studied in primary cultured rat hepatocytes. Adenosine and adenosine agonists caused cyclic AMP accumulation in rat hepatocytes. The order of potency was 5'-N-ethylcarboxamidoadenosine (NECA)>R(-)-N(6)-(2-phenylisopropyl)adenosine (RPIA)>adenosine>2-[p-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680). Furthermore, adenosine agonists stimulated glycogenolysis and gluconeogenesis. The order of potency was NECA>RPIA>CGS21680. The rank order of potency is typical for adenosine A(2B) receptors. Glycogenolysis stimulated by NECA was fully inhibited by nonselective adenosine antagonists, 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS15943). However, the adenosine A(2A) receptor-selective antagonist, 8-(3-chlorostyryl)caffeine (CSC), and the adenosine A(1) receptor-selective antagonist, (+)-(R)-[(E)-3-(2-phenylpyrazolo[1,5-alpha]pyridin-3-yl)acryloyl]-2-piperidine ethanol (FK453), had a low inhibitory potency. A strong correlation was found between the inhibitory effect of adenosine antagonists on NECA-induced glucose production and that on intracellular cyclic AMP generation in rat hepatocytes. Our results suggest that adenosine stimulates cyclic AMP formation and regulates glycogenolysis and gluconeogenesis, most likely through the adenosine A(2B) receptor subtype in rat hepatocytes.