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I Feoktistov

Publications and source records attributed to I Feoktistov.

16 recordsLinked to original sources
PubMed2001

Inhibition of human mast cell activation with the novel selective adenosine A(2B) receptor antagonist 3-isobutyl-8-pyrrolidinoxanthine (IPDX)(2).

The antiasthmatic drug enprofylline was the first known selective, though not potent, A(2B) antagonist. On the basis of structure-activity relationships (SARs) of xanthine derivatives, we designed a novel selective adenosine A(2B) receptor antagonist, 3-isobutyl-8-pyrrolidinoxanthine (IPDX), with potency greater than that of enprofylline. IPDX displaced [3H]ZM241385 ([3H]4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a]-[1,3,5]triazin-5-ylamino]ethyl)phenol) from human A(2B) adenosine receptors with a K(i) value of 470 +/- 2 nM and inhibited A(2B)-dependent cyclic AMP (cAMP) accumulation in human erythroleukemia (HEL) cells with a K(B) value of 625 +/- 71 nM. We found that IPDX was more selective than enprofylline toward human A(2B) receptors. It was 38-, 55-, and 82-fold more selective for human A(2B) than for human A(1) (K(i) value of 24 +/- 8 microM), human A(2A) (K(B) value of 36 +/- 8 microM), and human A(3) (K(i) value of 53 +/- 10 microM) adenosine receptors, respectively. IPDX inhibited NECA (5'-N-ethylcarboxamidoadenosine)-induced interleukin-8 secretion in human mast cells (HMC-1) with a potency close to that determined for A(2B)-mediated cAMP accumulation in HEL cells, thus confirming the role of A(2B) adenosine receptors in mediating human mast cell activation. Since adenosine triggers bronchoconstriction in asthmatic patients through human mast cell activation, IPDX may become a basis for the development of new antiasthmatic drugs with improved properties compared with those of enprofylline. Our data demonstrate that IPDX can be used as a tool to differentiate between A(2B) and other adenosine receptor-mediated responses.

Adenosine↗
PubMed2001

Proliferation, migration, and ERK activation in human retinal endothelial cells through A(2B) adenosine receptor stimulation.

PURPOSE: The nucleoside adenosine has been implicated in angiogenesis. A previous study demonstrated that activation of the A(2B) adenosine receptor (AdoR) increases cAMP accumulation, cell proliferation, and VEGF expression in human retinal endothelial cells (HRECs). In the present study, the role of this receptor was further characterized by examination of the effects of the selective A(2B) AdoR antagonists 3-N-propylxanthine (enprofylline) and 3-isobutyl-8-pyrrolidinoxanthine (IPDX) on AdoR-mediated HREC proliferation, capillary tube formation, and signal-transduction pathways. METHODS: HRECs were exposed to the adenosine analogue 5'-N-ethylcarboxamido-adenosine (NECA) in the absence or presence of AdoR antagonists. Migration was measured using Boyden chambers. Proliferation was assessed by counting cells. Western analysis was used to assess extracellular signal-related kinase (ERK) and cAMP response element-binding protein (CREB) in cell lysates. The effect of AdoR activation on tube formation was studied using cells grown on a synthetic basement membrane matrix. RESULTS: NECA induced proliferation in a concentration-dependent manner that was inhibited by enprofylline and IPDX. NECA stimulated chemotaxis in a concentration-dependent manner that was also blocked by both A(2B) AdoR antagonists. NECA activated ERK and CREB in HRECs. Both A(2B) AdoR antagonists diminished activation of ERK by NECA exposure. ERK activation was also blocked by the ERK-mitogen-activated protein kinase (MAPK) inhibitor PD98059, but not by the protein kinase A (PKA) inhibitor H-89. CREB activation was blocked by H-89, but not by PD98059, suggesting that ERK activation is independent of PKA. NECA enhanced tube formation on the matrix, whereas both A(2B) AdoR antagonists attenuated this effect. CONCLUSIONS: The selective A(2B) AdoR antagonists, enprofylline and IPDX, inhibited NECA-stimulated proliferation, ERK activation, cell migration, and capillary tube formation. A(2B) AdoR inhibition may offer a way to inhibit retinal angiogenesis and provide a novel therapeutic approach to treatment of diseases associated with aberrant neovascularization, such as diabetic retinopathy and retinopathy of prematurity.

Adenosine-5'-(N-ethylcarboxamide)↗
PubMed2000

Cyclic AMP and protein kinase A stimulate Cdc42: role of A(2) adenosine receptors in human mast cells.

The functional activity of Cdc42 is known to be regulated by proteins that control its GDP/GTP-bound state. However, there is still limited information on how Cdc42 is controlled by G-protein-coupled receptors. Adenosine receptors belong to the G-protein-coupled receptor family of cell surface receptors. Human HMC-1 mast cells express the high-affinity A(2A) and the low-affinity A(2B) subtypes of adenosine receptors known to increase intracellular cAMP levels. We found that both subtypes of A(2) adenosine receptors activate Cdc42 in HMC-1 cells. Furthermore, stimulation of adenylate cyclase with forskolin, or loading of HMC-1 with the cell-permeable cAMP analog 8-Br-cAMP, activated Cdc42. Stimulation of Cdc42 by cAMP was also observed in CHO-K1 and COS-7 cells. Protein kinase A (PKA)-mediated phosphorylation is likely involved in cAMP-dependent Cdc42 activation, because transient expression of the PKA catalytic subunit in COS-7 cells activated Cdc42. Inhibition of protein phosphatases 1 and 2A with calyculin A potentiated the effects of 5'-N-ethylcarboxamidoadenosine and 8-Br-cAMP, whereas the selective PKA inhibitor H-89 reversed the activation of Cdc42. We demonstrated that Cdc42 is a poor substrate for PKA phosphorylation in vitro and in intact cells. Our data suggest that PKA does not phosphorylate Cdc42 directly. Instead, the proteins that modulate the GDP/GTP-bound state of Cdc42 may be the primary targets of PKA phosphorylation.

Adenosine↗
PubMed2000

Adenosine A(2A) and A(2B) receptors in cultured human and porcine coronary artery endothelial cells.

We investigated the role of the cAMP link to the signal transduction mechanism coupled with adenosine A(2A) and A(2B) receptors in cultured human coronary artery endothelial cells (HCAEC) and porcine coronary artery endothelial cells (PCAEC). 2-[4-[2-¿2-[(4-aminophenyl)methylcarbonylamino]ethylaminocarbon yl¿eth yl]phenyl]ethylamino-5'- ethylcarboxamidoadenosine ((125)I-PAPA-APEC) (PAPA-APEC) was used to demonstrate the specific binding in PCAEC membranes. The specific binding was saturable and reversible with a maximal number of binding sites (B(max)) of 240 fmol/mg protein, and scatchard analysis revealed a single class of binding site with an equilibrium dissociation constant (K(d)) of 1. 17 +/- 0.035 nM. In competition experiments, adenosine receptor agonists showed the following order of potency (based on IC(50)): 5'-(N-ethylcarboxamido)adenosine (NECA) >/= CGS-21680 > 2-chloroadenosine. This order appears to be consistent with the A(2) adenosine receptor classification. We also studied the effects of adenosine agonists on the accumulation of cAMP as an indirect approach to show the presence of functional A(2) receptors. Similarly, the same adenosine agonists (10(-7)-10(-4) M) elicited the production of cAMP in intact endothelial cells in a dose-dependent manner, exhibiting consistently with the A(2) adenosine receptor classification. A selective A(2A) adenosine receptor antagonist (ZM-241385, 10(-8) M) significantly inhibited the effect of CGS-21680 on cAMP but only partly inhibited the effect of NECA, suggesting the presence of both A(2A) and A(2B) receptors. Western blot analysis further showed the immunoreactivity of A(2A) and A(2B) receptor at 45 and 36 kDa, respectively, in both HCAEC and PCAEC. Direct evidence for the presence of A(2A) and A(2B) receptors in cultured HCAEC and PCAEC by reverse transcription-polymerase chain reaction (RT-PCR), revealed expected PCR product sizes (205 and 173 bp) for A(2A) and A(2B) receptors in HCAEC and PCAEC, respectively. The data show that adenylate cyclase-coupled adenosine A(2A) and A(2B) receptors are present in coronary endothelial cells.

2-Chloroadenosine↗
PubMed1999

Adenosine receptor activation induces vascular endothelial growth factor in human retinal endothelial cells.

Adenosine, released in increased amounts by hypoxic tissues, is thought to be an angiogenic factor that links altered cellular metabolism caused by oxygen deprivation to compensatory angiogenesis. Adenosine interacts with 4 subtypes of G protein-coupled receptors, termed A(1), A(2A), A(2B), and A(3). We investigated whether adenosine causes proliferation of human retinal endothelial cells (HRECs) and synthesis of vascular endothelial growth factor (VEGF) and, if so, which adenosine receptor subtype mediates these effects. The nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), in a concentration-dependent manner, increased both VEGF mRNA and protein expression by HRECs, as well as proliferation. This proliferative effect of NECA was inhibited by the addition of anti-human VEGF antibody. NECA also increased insulin-like growth factor-I and basic fibroblast growth factor mRNA expression in a time-dependent manner and cAMP accumulation in these cells. In contrast, neither the A(1) agonist N(6)-cyclopentyladenosine nor the A(2A) agonist 2-p-(2-carboxyethyl) phenethylamino-NECA caused any of the above effects of NECA. The effects of NECA were not significantly attenuated by either the A(2A) antagonist SCH58261 or the A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine. However, the nonselective adenosine receptor antagonist xanthine amine congener completely inhibited the effects of NECA. Addition of antisense oligonucleotide complementary to A(2B) adenosine receptor mRNA inhibited VEGF protein production by HRECs after NECA stimulation. Thus, the A(2B) adenosine receptor subtype appears to mediate the actions of adenosine to increase growth factor production, cAMP content, and cell proliferation of HRECs. Adenosine activates the A(2B) adenosine receptor in HRECs, which may lead to neovascularization by a mechanism involving increased angiogenic growth factor expression.

Adenosine-5'-(N-ethylcarboxamide)↗
PubMed1999

Prostaglandin E-prostanoid-3 receptor activation of cyclic AMP response element-mediated gene transcription.

The prostaglandin E-prostanoid (EP)3 receptor signals primarily through the inhibitory G protein Gi, thereby decreasing intracellular cAMP levels. To study the signal transduction properties of the rabbit EP3 receptor, five splice variants were expressed in HEK293tsA201 cells: 72A, 74A, 77A, 80A and the novel splice variant NT, which lacks the C-terminal sequence. The ability of the EP3 receptor splice variants to modulate expression of a beta-galactosidase reporter gene under the control of a promoter containing cAMP response elements (CRE) was assessed. Each splice variant induced sulprostone-mediated increase in beta-galactosidase enzymatic activity with EC50 ranging from 0.8 nM for the NT splice variant to 3.1 nM for the 77A splice variant. Substitution of either Asp338 with Ala, or Arg329 with Ala or Glu in the 77A splice variant resulted in a loss of receptor-evoked increases in beta-galactosidase activity, whereas substitution of Lys300 with alanine had no effect on signal transduction. These phenotypes correlate with the inhibition of cAMP generation by direct cAMP measurement. Signal transduction was insensitive to pretreatment of cells with pertussis toxin, suggesting that a nonGi/Go pathway is activated by the EP3 receptor. Direct measurement of second messenger levels confirmed that there was no increase in cAMP levels mediated by the 77A splice variant, however, there was a modest increase in intracellular Ca2+. Partial blockade of the reporter activity with kinase inhibitors demonstrates that CRE activation is mediated in part by a Ca2+-dependent kinase pathway. These data suggest that the EP3 receptor signals through a novel cAMP response element binding protein/CRE pathway.

Alternative Splicing↗
PubMed1999

Role of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinase kinase in adenosine A2B receptor-mediated interleukin-8 production in human mast cells.

The endogenous nucleoside adenosine is thought to play a role in the pathophysiology of asthma by stimulating mast cells. We previously showed that the human mast cell line HMC-1 expresses A2A and A2B receptors, and that both receptors activate adenylate cyclase via Gs-protein but that only A2B receptors are also coupled to phospholipase C via Gq proteins. Stimulation of A2B but not A2A receptors induced production of interleukin-8 (IL-8) from HMC-1 cells. The mechanism by which adenosine promotes IL-8 synthesis has not been defined. In this study, we tested the hypothesis that mitogen-activated protein kinase (MAPK) signaling pathways are involved in this process. Stimulation of HMC-1 with the stable adenosine analog NECA (5'-N-ethylcarboxamidoadenosine) activated p21(ras) and both p42 and p44 isoforms of extracellular signal-regulated kinase (ERK). NECA (10 microM) induced a 1.9 +/- 0. 06-fold increase in ERK activity, whereas 10 microM of the selective A2A agonist CGS 21680 (4-((N-ethyl-5'-carbamoyladenos-2-yl)-aminoethyl)-phenylpropionic acid) had no effect. NECA, in parallel with the activation of ERK, also stimulated the p46 isoform of c-Jun N-terminal kinase (MEK) and p38 MAPK. Furthermore, the selective MAPK/ERK kinase 1 inhibitor PD 98059 (2'-amino-3'-methoxyflavone), and p38 MAPK inhibitors SB 202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole) and SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H- imidaz ole) blocked A2B receptor-mediated production of IL-8. These results indicate that extracellular adenosine can regulate ERK, c-Jun N-terminal kinase, and p38 MAPK signaling cascades and that activation of ERK and p38 MAPK pathways are essential steps in adenosine A2B receptor-dependent stimulation of IL-8 production in HMC-1.

Adenosine↗
PubMed1998

Pharmacological characterization of adenosine A2B receptors: studies in human mast cells co-expressing A2A and A2B adenosine receptor subtypes.

Characterization of A2B receptors is hampered by the lack of selective pharmacological probes and often relies on their relative affinity to agonists that are selective at other receptor types. This approach is limited because the affinity of A2B receptors for putative A3 agonists has not been determined. Using the human erythroleukemia cell line HEL as a cellular model for A2B-mediated adenylate cyclase activation, we found the following potencies (pD2) for the non-selective agonist 5'-N-ethylcarboxamidoadenosine (NECA) (5.65 +/- 0.04), the putative A3 agonists N6-benzyl-NECA (4.17 +/- 0.06) and N6-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (IB-MECA) (3.7 +/- 0.02), and the A2A agonist 4-[(N-ethyl-5'-carbamoyladenos-2-yl)-aminoethyl]-phenylpropionic acid (CGS21680) (2.8 +/- 0.1). Because of the lack of a selective agonist, characterization of A2B receptor function is difficult in cells co-expressing A2A receptors. In the human mast cell line HMC-1, NECA induced cAMP accumulation with a concentration-response relationship best fitted to a two-sited model (pD2 7.69 +/- 0.42 and 5.92 +/- 0.21 for high- and low-affinity sites), suggesting the presence of both A2A and A2B receptors in these cells. We demonstrated that A2B receptors can be selectively activated with NECA in the presence of the selective A2A antagonist 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c ]pyrimidine (SCH 58261). Under these conditions, the concentration-response relationship of NECA for cyclic AMP accumulation was now best fitted to a one-site model (pD2 5.68 +/- 0.03, Hill slope 0.93 +/- 0.06, 95% confidence intervals 0.8 to 1.06) corresponding to selective activation of A2B receptors. Using the approaches developed in this study, we determined that A2B, and not A2A or A3, receptors account for all the calcium mobilization induced by NECA in HMC-1 cells.

Adenosine↗
PubMed1998

Adenosine A2B receptors: a novel therapeutic target in asthma?

Adenosine is an endogenous nucleoside that modulates many physiological processes. Its actions are mediated by interaction with specific cell membrane receptors. Four subtypes of adenosine receptor have been cloned: A1, A2A, A2B and A3. Significant advancement has been made in our understanding of the molecular pharmacology and physiological relevance of adenosine receptors but our knowledge of A2B receptors lags behind that of other receptor types. Only recently have potentially important functions been discovered for the A2B receptors, prompting a renewed interest in this receptor type. A2B receptors have been implicated in the regulation of vascular smooth muscle tone, cell growth, intestinal function and neurosecretion. In this review, Igor Feoktistov, Riccardo Polosa, Stephen Holgate and Italo Biaggioni focus on the role of A2B receptors in mast cell activation and the potential relevance of this action on asthma.

Anti-Asthmatic Agents↗
PubMed1997

Prostanoid receptor with a novel pharmacological profile in human erythroleukemia cells.

The purpose of this study was to characterize the prostanoid receptors coupled to intracellular calcium in human erythroleukemia (HEL) cells, a cell line with platelet/megakaryocytic characteristics. Both prostaglandin E1 (PGE1) and iloprost increased cyclic AMP (cAMP) in HEL cells, but modulated [Ca2+]i by different mechanisms. Iloprost (10(-9) to 10(-6) M) had no effect on basal [Ca2+]i, but greatly potentiated the increase in [Ca2+]i produced by thrombin. This effect was mimicked by cholera toxin and other Gs-coupled receptors, and involved calcium influx since iloprost had no effect on [Ca2+]i in cells incubated in Ca2+-free buffer. Furthermore, iloprost did not increase the generation of baseline or thrombin-induced inositol phosphates at these concentrations. In contrast, PGE1 (10(-7) to 10(-5) M), but not iloprost, increased basal [Ca2+]i through a pertussis toxin-sensitive mechanism that involved stimulation of inositol phosphate generation and mobilization of intracellular calcium. The order of potencies of other prostaglandins that increased [Ca2+]i was not consistent with known IP, EP, DP, FP, or TP receptors. 11-Deoxy-16,16-dimethyl PGE2 was the most potent of the analogs tested (EC50 = 28 nM). In summary, at least two prostaglandin receptors are functionally coupled to intracellular calcium in HEL cells: a putative IP receptor coupled to Gs proteins that increases cAMP and enhances calcium influx, and a novel prostanoid receptor that evokes calcium mobilization through stimulation of phospholipase C by a pertussis toxin-sensitive pathway.

16,16-Dimethylprostaglandin E2↗
PubMed1996

Role of adenosine in asthma.

Several lines of evidence support the hypothesis that adenosine contributes to asthma. Inhaled adenosine provokes bronchoconstriction in asthmatics, but not in nonasthmatics. This process appears to be mediated by mast cell activation, because it can be blocked by antihistamines and inhibitors of mast cell activation. Inhaled adenosine evokes release of mast cell mediators in bronchoalveolar lavage fluid, including histamine, prostaglandin D2, and tryptase, a specific mast cell marker. Also, adenosine potentiates the immunological activation of mast cells in vitro, including rat peritoneal mast cells, mouse bone marrow-derived mast cells, human lung mast cells, and the human mast cell line HMC-1. The receptor subtype that mediates this activation differs between mast cell type, but preliminary evidence suggests that human lung mast cells express A2B receptors. An argument against the contribution of adenosine in asthma has been the "enprofylline paradox." This xanthine (3-(n-propylyl)xanthine) is as effective an antiasthmatic as theophylline (1,3-dimethyl xanthine) but was initially thought not to be an adenosine receptor antagonist. More recent evidence has confirmed that enprofylline blocks A2B receptors with a Ki (7 microM) similar to that of theophylline (13 microM) and well within its therapeutic plasma levels (5-25 microM). This finding, we believe, resolves the enprofylline paradox and supports the hypothesis that adenosine, through A2B receptor activation, contributes to asthma. Preliminary evidence suggests that A2B receptors are indeed present in human lung mast cells. A2B receptors, therefore, may be a potential target for the development of antiasthmatic drugs.

Adenosine↗
PubMed1995

Adenosine A2b receptors evoke interleukin-8 secretion in human mast cells. An enprofylline-sensitive mechanism with implications for asthma.

Adenosine potentiates mast cell activation, but the receptor type and molecular mechanisms involved have not been defined. We, therefore, investigated the effects of adenosine on the human mast cell line HMC-1. Both the A2a selective agonist CGS21680 and the A2a/A2b nonselective agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased cAMP, but NECA was fourfold more efficacious and had a Hill coefficient of 0.55, suggesting the presence of both A2a and A2b receptors. NECA 10 microM evoked IL-8 release from HMC-1, but CGS21680 10 microM had no effect. In separate studies we found that enprofylline, an antiasthmatic previously thought to lack adenosine antagonistic properties, is as effective as theophylline as an antagonist of A2b receptors at concentrations achieved clinically. Both theophylline and enprofylline 300 micro completely blocked the release of IL-8 by NECA. NECA, but not CGS21680, increases inositol phosphate formation and intracellular calcium mobilization through a cholera and pertussis toxin-insensitive mechanism. In conclusion, both A2a and A2b receptors are present in HMC-1 cells and are coupled to adenylate cyclase. In addition, A2b receptors are coupled to phospholipase C and evoke IL-8 release. This effect is blocked by theophylline and enprofylline, raising the possibility that this mechanism contributes to their antiasthmatic effects.

Adenosine↗
PubMed1994

Positive modulation of intracellular Ca2+ levels by adenosine A2b receptors, prostacyclin, and prostaglandin E1 via a cholera toxin-sensitive mechanism in human erythroleukemia cells.

Human erythroleukemia (HEL) cells express megakaryocyte/platelet membrane markers and thus have been used as a model for studying platelet membrane receptors and their coupling to cell signaling pathways. Our previous studies, however, indicated that platelets and HEL cells possess different subtypes of adenosine A2 receptors. Furthermore, we now report that, whereas adenosine inhibits intracellular Ca2+ increases in platelets, it potentiates the rise in intracellular Ca2+ produced by thrombin, prostaglandin E1, thapsigargin, and the calcium ionophore A23187 in HEL cells. Stable adenosine analogs potentiated intracellular Ca2+ increases with a rank order of potencies of 5'-N-ethylcarboxamidoadenosine (NECA) > (R)-(-)-N6-(2-phenylisopropyl)adenosine (R-PIA) >> CGS 21680, suggesting that this effect is mediated by A2b receptors. EC50 values for NECA and R-PIA were 0.8 and 42 microM, respectively. NECA (100 microM) potentiated by 2-3-fold the increase in intracellular Ca2+ produced by 0.3 unit/ml thrombin. This effect was mimicked by cholera toxin and was shared by other Gs-coupled receptors, such as those activated by the prostacyclin analog iloprost and prostaglandin E1, indicating the involvement of Gs proteins. Adenosine analogs also increased intracellular cAMP with the same rank order of potencies. The membrane-permeable analog 8-bromo-cAMP, however, had no effect on intracellular Ca2+ levels, indicating that the potentiation of intracellular Ca2+ increases and the activation of adenylate cyclase are parallel but independent events. The increase in intracellular Ca2+ produced by adenosine is due not to an increase in phosphoinositide hydrolysis but, rather, to an increase in calcium influx, and it is lost if cells are studied in the absence of extracellular Ca2+. We conclude, therefore, that adenosine A2b receptors in HEL cells are coupled to Gs proteins and their activation leads to stimulation of adenylate cyclase and, independently, to potentiation of the rise in intracellular Ca2+. We speculate that A2b receptors in HEL cells activate a calcium channel through a cholera toxin-sensitive mechanism that requires an initial increase in intracellular Ca2+.

Alprostadil↗
PubMed1993

Characterization of adenosine receptors in human erythroleukemia cells and platelets: further evidence for heterogeneity of adenosine A2 receptor subtypes.

Adenosine receptors are present in platelets, and their activation results in accumulation of cAMP and inhibition of aggregation. The study of platelet adenosine receptors, however, is limited by the impossibility of maintaining these cells in vitro. Human erythroleukemia (HEL) cells express megakaryocytic/platelet markers and have been used as a model to study platelet receptors. Therefore, we sought to determine whether adenosine receptors were present in HEL cells. Adenosine agonists produced an accumulation of cAMP in HEL cells, implying the presence of A2 receptors. Xanthine and nonxanthine adenosine receptor antagonists blocked this effect in a simple competitive manner (Schild analysis). Therefore, both platelets and HEL cells possess A2 adenosine receptors. There were, however, significant differences between them. Adenosine agonists were, in general, less potent in HEL cells, compared with platelets. In particular, the adenosine analog CGS 21680, one of the most potent agonists in platelets, was virtually inactive in HEL cells. The orders of potencies for agonists (and their EC50 values for cAMP production) were 5'-N-ethylcarboxamidoadenosine (0.19 microM) = CGS 21680 (0.18 microM) > (R)-(-)-N6-(2-phenylisopropyl)adenosine (0.5 microM) in platelets and 5'-N-ethylcarboxamidoadenosine (2.4 microM) > (R)-(-)-N6-(2-phenylisopropyl)adenosine (160 microM) >> CGS 21680 (1600 microM) in HEL cells. In contrast to the decreased potency of agonists in HEL cells, the antagonist 1,3-dipropyl-8-p-sulfophenylxanthine was more potent in HEL cells, compared with platelets. Based on the striking differences in the rank orders of potencies of agonists and antagonists, we propose that HEL cells and platelets have different subtypes of adenosine A2 receptors. We found CGS 21680 particularly helpful in distinguishing between these receptor subtypes.

Adenosine↗
PubMed1990

Adenosine inhibits the rise in intracellular calcium and platelet aggregation produced by thrombin: evidence that both effects are coupled to adenylate cyclase.

Platelet aggregation and secretion are associated with a rise in intracellular calcium concentration ([Ca2+]i). Adenosine has been postulated as an endogenous inhibitor of platelet aggregation. The antiaggregatory effects of adenosine are related to activation of adenylate cyclase. We studied the effect of adenosine on the rise in [Ca2+]i and platelet aggregation produced by thrombin. Human platelets were obtained from dextrose/citrate-treated plasma. [Ca2+]i was determined by fluorescence-dye techniques (fura-2). Adenosine inhibited the slope of the first phase of aggregation and the rise in [Ca2+]i produced by thrombin, in a dose-dependent manner. The dose that produced 50% inhibition of both aggregation and the rise in [Ca2+]i was approximately 500 nM. The effects of adenosine on [Ca2+]i were shared by its stable analogs, 5'-N-ethylcarboxamidoadenosine being approximately 10-fold more potent than (-)N6-phenylisopropyladenosine, suggesting that these effects were mediated through adenosine A2 receptors. Furthermore, caffeine antagonized the inhibitory effects of adenosine on platelet aggregation and [Ca2+]i. The effects of adenosine on [Ca2+]i appear to be mediated through a rise in intracellular cAMP, because they were prevented by the adenylate cyclase inhibitor 2',5'-dideoxyadenosine (1 mM) and were potentiated by phosphodiesterase inhibition with papaverine (1 microM). Adenosine also inhibits the rise in [Ca2+]i produced by thrombin in a calcium-free medium, suggesting that adenosine inhibits both calcium influx and the release of calcium from intracellular stores.

Adenosine↗