Adenosine receptors mediating cardiac depression

Several adenosine analogs were evaluated for their effects on rate and contractility in guinea pig isolated atria. Among adenosine agonists, (?)-N-(1-methyl-2-phenylethyl) adenosine (?-phenylisopropyladenosine; ?-PIA) and N-cyclohexyladenosine (CHA), decreased rate and force at nanomolar concentrations, whereas 2-chloroadenosine, N, N-dimethyladenosine (N⁶-dimetyl-adenosine) and (+)-N-(1-methyl-2-phenylethyl) adenosine (d-phenyl-isopropyladenosine; d-PIA) were less potent cardiac depressants. The degree and order of potency of these agonists suggest that the cardiac depressant effects of adenosine are mediated via A₁-receptors. The cardiac depressant effects of CHA and ?-PIA were antagonized by theophylline and 1,3-diethyl-8-phenylxanthine (DPX).     

The effects of adenosine agonists on human neutrophil function

Adenosine is a potent physiologic substance with a variety of biologic activities. Many of the effects of adenosine appear to be mediated by two populations of cell-surface adenosine receptors (A1 and A2). We have examined the effects of several adenosine receptor agonists on human neutrophils stimulated with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). The results indicate that both superoxide anion generation and degranulation (as assessed by lysozyme release) are inhibited. Inhibition correlated most strongly with A2 receptor affinity for both parameters and was reversible by the adenosine receptor antagonist 8-phenyltheophylline. Because toxic oxygen metabolites and degradative enzymes are implicated in a variety of inflammatory disorders, adenosine agonists may be useful probes to help expand our knowledge of the role of these mediators in human disease.     

Pain-relieving prospects for adenosine receptors and ectonucleotidases

Adenosine receptor agonists have potent antinociceptive effects in diverse preclinical models of chronic pain. By contrast, the efficacy of adenosine and adenosine receptor agonists in treating pain in humans is unclear. Two ectonucleotidases that generate adenosine in nociceptive neurons were recently identified. When injected spinally, these enzymes have long-lasting adenosine A(1) receptor-dependent antinociceptive effects in inflammatory and neuropathic pain models. Furthermore, recent findings indicate that spinal adenosine A(2A) receptor activation can enduringly inhibit neuropathic pain symptoms. Collectively, these studies suggest the possibility of treating chronic pain in humans by targeting specific adenosine receptor subtypes in anatomically defined regions with agonists or with ectonucleotidases that generate adenosine.     

Inhibition of human tumour cell proliferation by analogues of adenosine

The effects of adenosine and several structural analogues of adenosine upon thymidine incorporation into human tumour cells and rat cervical lymphocytes were investigated. The analogue NECA, which has equal specificity for the A₁ and A₂ receptor, had the most inhibitory effect on lymphocyte proliferation while the A₁ agonists had limited effects, suggesting that these cells possess principally A₂ adenosine receptors. In the case of human tumour cells, however, the most inhibitory effect on proliferation was obtained with the A₁-specific analogues. The general order of inhibitory effects of adenosine analogues on thymidine incorporation in human tumour cells was: S-ENBA>CPA=R-PIA>S-PIA>NECA. These findings suggest that in the cells presently studied the A₁ adenosine receptor predominates. Removal of exogenous adenosine by growth in the presence of adenosine deaminase inhibited thymidine incorporation. The effect of adenosine removal lends further support to the proposal that adenosine has some, as yet unidentified, regulatory role in the control of human tumour cell proliferation. © 1997 John Wiley & Sons, Ltd.     

PD 116,948, a highly selective A1 adenosine receptor antagonist

(R)-N-(1-Methyl-2-phenylethyl) adenosine (R-PIA), an adenosine receptor agonist has both negative chronotropic activity and coronary vasodilator activity. These actions of R-PIA are proposed to be mediated by subtypes (A1 and A2) of adenosine receptors. PD 116,948 is a xanthine derivative which is a highly selective A1 adenosine receptor ligand. In this study PD 116,948 selectively antagonized the negative chronotropic activity of R-PIA in the isolated rat heart. These results are consistent with, and add further support to the hypothesis that adenosine receptor agonists mediate their negative chronotropic activity via A1 receptors and their vasodilator activity via A2 receptors.     

Adenosine, a cytoprotective autocoid: effects of adenosine on neutrophil plasma membrane viscosity and chemoattractant receptor display

At inflammatory sites neutrophils are stimulated to produce a variety of toxic agents, yet rarely harm the endothelium across which they migrate. We have recently found that endothelium releases adenosine which, acting via receptors on the surface of human neutrophils, inhibits generation of toxic metabolites by stimulated neutrophils but, paradoxically, promotes chemotaxis. Agents which diminish plasma membrane viscosity affect neutrophil function similarly, possibly by modulating chemoattractant receptor number or affinity. We therefore determined whether adenosine receptor agonists modulate neutrophil function by decreasing membrane viscosity and/or changing the affinity of chemoattractant (N-fMet-Leu-Phe, FMLP) receptors. Surprisingly, 5'-(N-ethylcarboxamido)adenosine (NECA, 10 microM), the most potent agonist at neutrophil adenosine receptors, increased plasma membrane viscosity, as measured by fluorescence anisotropy of the plasma membrane specific probe 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene (TMA-DPH), in unstimulated neutrophils from a mean microviscosity of 1.67 +/- 0.02 (S.E.) to 1.80 +/- 0.02 (p less than 0.001) while inosine (10 microM), a poor adenosine receptor agonist, had no effect (1.73 +/- 0.04, p = n.s. vs. control, p less than 0.01 vs. NECA). Adenosine receptor agonists increased plasma membrane viscosity in neutrophils with the same order of potency previously seen for inhibition of superoxide anion generation and enhancement of chemotaxis (NECA greater than adenosine = N6-phenylisopropyladenosine). The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline reversed the effect of NECA on plasma membrane viscosity. Unlike other agents which modulate plasma membrane viscosity, NECA (10 microM) did not significantly change the number or affinity of [3H]FMLP binding sites on neutrophils. In contrast to the hypothesis of Yuli et al. these results indicate that occupancy of adenosine receptors on neutrophils increases plasma membrane viscosity without affecting chemoattractant receptor display.     

Adenosine receptor-adenylate cyclase system in the trout testis: involvement in the regulation of germ cell proliferation

To ascertain the presence of adenosine receptors in the trout testis, cells isolated from testes at different spermatogenetic stages were cultured in the presence or absence of adenosine, adenosine receptor agonists, or antagonists and of cAMP analogs, for up to 20 min, or 20 hr, or 4.5 days. Cyclic AMP production was then assayed or 3H-thymidine incorporation was measured. Cellular content of cAMP was enhanced by adenosine, by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), and by 2-p(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), an adenosine A2A receptor-selective agonist. The increase in cAMP induced by the adenylate cyclase activator L-858051 was inhibited by the adenosine A1)receptor-selective agonists R-N6-(2-phenylisopropyl)adenosine (R-PIA) and N6-cyclopentyladenosine (CPA). These effects were antagonized by the two adenosine A2)receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 8-(3-chlorostyryl)caffeine (CSC), and by the adenosine A1)receptor-selective antagonist 8-cyclopentyl-1,3dipropylxanthine (CPX), respectively. Increase in the cAMP content induced by adenosine was inhibited by the cell permeable adenylate cyclase inhibitor 2',5'-dideoxyadenosine. These data suggest that A(1) and A(2) adenosine receptors which respectively inhibit and stimulate adenylate cyclase activity are present on trout testicular cells (unidentified), while the presence of A3 adenosine receptor subtype was not apparent. 3H-thymidine incorporation decreased in the presence of the adenylate cyclase activator L-858051 and of the cAMP analogs 8-CPT cAMP and Sp-5,6-DCI-cBiMPS, regardless of the presence or absence of the phosphodiesterase inhibitor RO 20-1724. This suggests that an increase in testicular cAMP may act as a negative growth regulator for the mitotic germ cells. In agreement with these data, the activation of A2 stimulatory receptors inhibited short-term (20 hr) DNA synthesis. However, the activation of A1 inhibitory receptors had the same effect. This suggests that events, cAMP-dependent or independent, induced by the activation of testicular adenosine receptors, may participate in the regulation of trout male germ cell proliferation.     

The effect of adenosine receptor agonists on cytokine release by human mononuclear cells depends on the specific Toll-like receptor subtype used for stimulation

In the present study, we determined whether the immunomodulatory effect of adenosine receptor stimulation depends on the Toll-like Receptor (TLR) used for stimulation of cytokine release. Therefore, human mononuclear cells were stimulated by different TLR agonists in the absence and presence of A1 (CPA), A2a (CGS21680), and A3 (Cl-IB-MECA) adenosine receptor agonists. Effects of these agonists on Il-6, Il-10, IFN-gamma, TNF-alpha, and Il-1beta production were expressed as percentage inhibition/stimulation after TLR stimulation. CGS21680 inhibited TLR4-mediated TNF-alpha release and potentiated TLR3- and TLR5-mediated IL-6 release. Cl-IB-MECA inhibited TLR4-agonist-induced IFN-gamma release. Interestingly, CPA en Cl-IB-MECA tended to inhibit cytokine release only after TLR4 stimulation. In more detail, CPA potentiated TLR5-mediated IL-6 production, TLR3-mediated IFN-gamma production and TLR3-mediated Il-1beta-production compared to TLR4-mediated stimulation. Cl-IB-MECA potentiated TLR5-mediated IL-6 and Il-1beta formation as compared to TLR4-mediated stimulation. Finally, CGS21680 potentiated TLR5-mediated IL-6 production compared to TLR1-2 stimulation, and potentiated TLR3- and TLR5-mediated IL-10 production compared to TLR1-2-mediated stimulation. In conclusion, the effect of adenosine agonists on cytokine production depends on the specific TLR agonist used for stimulation. These findings suggest that well-known anti-inflammatory effects of adenosine agonists on LPS-induced inflammation cannot be extrapolated to situations in which stimulation of other TLR subtypes is involved.     

Effects of stable adenosine receptor agonists on bone marrow hematopoietic cells as inferred from the cytotoxic action of 5-fluorouracil

The aim of the study was to investigate the effects of stable adenosine receptor agonists on bone marrow hematopoiesis by utilizing the model of hematopoietic damage induced by 5-fluorouracil (5-FU), a cycle-specific cytotoxic agent. Effects of a non-selective agonist NECA activating all the known adenosine receptors (A1, A2A, A2B, A3) and of the selective agonists for A1 (CPA), A2A (CGS 21680), and A3 (IB-MECA) adenosine receptors were investigated. Experiments were performed with B10CBAF1 mice under in vivo conditions. Adenosine receptor agonists were given in single injections before 5-FU administration and the effects were determined 4 days later. The numbers of femoral marrow nucleated cells and hematopoietic progenitor cells (CFC-GM and BFU-E) were taken as indices of the effects. The non-selective agonist NECA given at a dose of 200 nmol/kg induced biphasic time-dependent effects, i.e. protection and sensitization, when given 10 h and 22 h before 5-FU administration, respectively. The use of isomolar doses of selective receptor agonists indicated that the protective effects of NECA were induced by activation of A2A and A2B receptors, while the sensitizing action of NECA was mediated via A3 receptors. In addition, it was observed that A1 receptors induced protection when activated by administration of CPA 22 h before 5-FU. These findings are discussed with respect to the action of adenosine receptor agonists on the cell cycle state and on the cell cycle-independent cellular protective mechanisms.     

An adenosine analog (IB-MECA) inhibits anchorage-dependent cell growth of various human breast cancer cell lines

A3 adenosine receptor agonists have been reported to influence cell death and survival. Here we report the effects of an A3 adenosine receptor agonist, IB-MECA, on the cell growth of human breast cancer cell lines, MCF-7 (estrogen receptor positive) and MDA-MB468 (estrogen receptor negative). Therefore, this study was aimed to investigate the expression and possible action of A3 receptor in the human breast cancer cell lines. IB-MECA, at 1-100 microM, resulted in a significant cell growth inhibition (P < 0.05) which reached the maximum at 48 h, in the cell lines. In both cell lines, agonist-induced effects were antagonized by pretreatment with a selective A3 adenosine receptor antagonist, MRS1220. Using RT-PCR method, further confirmation was provided by the presence of mRNA of A3 receptor in the cells. In addition, IB-MECA was able to inhibit forskolin-stimulated cAMP levels, which indicate the functional form of A3 receptor on the cell surface of these breast cancer cell lines. These results suggest that the inhibitory effect of IB-MECA on the growth of human breast cancer cell lines is mediated through activation of A3 adenosine receptor.     

Neutrophil adherence to endothelium is enhanced via adenosine A1 receptors and inhibited via adenosine A2 receptors.

We have recently demonstrated that human neutrophils (PMN) possess two different classes of adenosine receptors (A1 and A2) that, when occupied, promote chemotaxis and inhibit the generation of reactive oxygen species (e.g., O2- and H2O2), respectively. We have previously demonstrated that adenosine protects endothelial cells (EC) from injury by stimulated neutrophils (PMN) both by diminishing generation of H2O2 and inhibiting adherence of PMN to EC. We therefore determined whether occupancy of A1 or A2 adenosine receptors regulated adherence of PMN to EC. At concentrations similar to those required to inhibit release of O2- by ligation of A2 receptors, both adenosine (IC50 = 56 nM) and 5'N-ethylcarboxamidoadenosine (NECA, IC50 = 8 nM), the most potent A2 agonist, inhibited adherence to EC by stimulated PMN (FMLP, 0.1 microM). In direct contrast, the specific A1 agonists N6-phenylisopropyladenosine and N6-cyclopentyladenosine (CPA) promoted PMN adherence to EC at concentrations of 1-100 nM. To further investigate the mechanisms by which adenosine receptor agonists affected the adherence of stimulated PMN we examined the effect of NECA (A2) and CPA (A1) on the adherence of PMN to fibrinogen (a ligand for the beta 2 integrin CD11b/CD18) and to gelatin. In a dose-dependent manner (IC50 = 2 nM), NECA inhibited the adherence of FMLP-treated PMN to fibrinogen- but not gelatin-coated plates. In contrast, CPA (A1) promoted adherence of stimulated PMN to gelatin-(EC50 = 13 pM) but not fibrinogen-coated plates. Theophylline (10 microM), an adenosine receptor antagonist, reversed the inhibition by NECA (0.3 microM) of stimulated neutrophil adherence to fibrinogen. These observations not only confirm the presence of A1 and A2 receptors on PMN but also suggest two opposing roles for adenosine in inflammation. Occupancy of A1 receptors promotes neutrophil adherence to endothelium and chemotaxis (a proinflammatory role) whereas occupancy of A2 receptors inhibits adherence and generation of toxic oxygen metabolites (an antiinflammatory role).     

Adenosine; a physiologic modulator of superoxide anion generation by human neutrophils. Adenosine acts via an A2 receptor on human neutrophils

Adenosine specifically inhibits superoxide anion generation by N-formyl-methionyl-leucyl-phenylalanine-stimulated neutrophils without affecting either degranulation or "aggregation." We present data that also supports the hypothesis that adenosine engages a specific cell surface receptor to mediate inhibition of stimulated neutrophils. Theophylline (10 and 100 mu M), a competitive antagonist at adenosine receptors, reversed the effects of adenosine (0.1 mu M) on superoxide anion generation by stimulated neutrophils. The adenosine analogue 5'N-ethylcarboxamidoadenosine (NECA) was a more potent inhibitor of superoxide anion generation than either N6-phenylisopropyladenosine (PIA) or adenosine, an order of potency consistent with that previously demonstrated for adenosine A2 receptors. 2-Chloroadenosine inhibited superoxide anion generation at concentrations similar to NECA. [3H]-NECA and [3H]-2-chloroadenosine bound to a single receptor on intact neutrophils. The characteristics of the receptors for [3H]-NECA and [3H]-2-chloroadenosine were similar (Kd = 0.22 and 0.23 mu M, respectively; number of binding sites = 9.31 and 11.1 X 10(3) sites/cell, respectively). NECA, 2-chloroadenosine, adenosine, and PIA inhibited binding of [3H]-NECA with a rank order similar to that for inhibition of superoxide anion generation (NECA = 2-chloroadenosine greater than adenosine greater than PIA). There was 50% inhibition of superoxide anion generation by NECA at approximately 20% receptor occupancy. Adenosine, derived from damaged tissues, may serve as a specific, endogenous modulator of superoxide anion generation by activated neutrophils through interaction at this newly described receptor on human neutrophils.     

Adenosine inhibits macrophage colony-stimulating factor-dependent proliferation of macrophages through the induction of p27kip-1 expression.

Adenosine is produced during inflammation and modulates different functional activities in macrophages. In murine bone marrow-derived macrophages, adenosine inhibits M-CSF-dependent proliferation with an IC50 of 45 microM. Only specific agonists that can activate A2B adenosine receptors such as 5'-N-ethylcarboxamidoadenosine, but not those active on A1 (N6-(R)-phenylisopropyladenosine), A2A ([p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamido adenosine), or A3 (N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide) receptors, induce the generation of cAMP and modulate macrophage proliferation. This suggests that adenosine regulates macrophage proliferation by interacting with the A2B receptor and subsequently inducing the production of cAMP. In fact, both 8-Br-cAMP (IC50 85 microM) and forskolin (IC50 7 microM) inhibit macrophage proliferation. Moreover, the inhibition of adenylyl cyclase and protein kinase A blocks the inhibitory effect of adenosine and its analogues on macrophage proliferation. Adenosine causes an arrest of macrophages at the G1 phase of the cell cycle without altering the activation of the extracellular-regulated protein kinase pathway. The treatment of macrophages with adenosine induces the expression of p27kip-1, a G1 cyclin-dependent kinase inhibitor, in a protein kinase A-dependent way. Moreover, the involvement of p27kip-1 in the adenosine inhibition of macrophage proliferation was confirmed using macrophages from mice with a disrupted p27kip-1 gene. These results demonstrate that adenosine inhibits macrophage proliferation through a mechanism that involves binding to A2B adenosine receptor, the generation of cAMP, and the induction of p27kip-1 expression.     

Gs protein-coupled adenosine receptor signaling and lytic function of activated NK cells

The effect of adenosine and its analogues on the cytotoxic activity of IL-2-activated NK cells was investigated. Adenosine is an endogenous ligand for four different adenosine receptor (AdoR) subtypes (AdoRA1, AdoRA2A, AdoRA2B, and AdoRA3). Increased concentrations of adenosine were found in ascites of MethA sarcoma or in culture medium of 3LL Lewis lung carcinoma growing under hypoxic conditions. We hypothesize that intratumor adenosine impairs the ability of lymphokine-activated killer (LAK) cells to kill tumor cells. The effect of AdoR engagement on LAK cells cytotoxic activity was analyzed using AdoR agonists and antagonists as well as LAK cells generated from AdoR knockout mice. Adenosine and its analogues efficiently inhibited the cytotoxic activity of LAK cells. CGS21680 (AdoRA2A agonist) and 5-N-ethylcarboxamide adenosine (NECA) (AdoRA2A/ADoRA2B agonist) inhibited LAK cell cytotoxicity in parallel with their ability to increase cAMP production. The inhibitory effects of stable adenosine analog 2-chloroadenosine (CADO) and AdoRA2 agonists were blocked by AdoRA2 antagonist ZM 241385. Adenosine and its analogues impair LAK cell function by interfering with both perforin-mediated and Fas ligand-mediated killing pathways. Studies with LAK cells generated from AdoRA1-/- and AdoRA3-/- mice ruled out any involvement of these AdoRs in the inhibitory effects of adenosine. LAK cells with genetically disrupted AdoRA2A were resistant to the inhibitory effects of adenosine, CADO and NECA. However, with extremely high concentrations of CADO or NECA, mild inhibition of LAK cytotoxicity was observed that was probably mediated via AdoRA2B signaling. Thus, by using pharmacological and genetic blockage of AdoRs, our results clearly indicate the prime importance of cAMP elevating AdoR2A in the inhibitory effect of adenosine on LAK cell cytotoxicity. The elevated intratumor levels of adenosine might inhibit the antitumor effects of activated NK cells.     

Inflammatory cytokines regulate function and expression of adenosine A(2A) receptors in human monocytic THP-1 cells

Adenosine, acting at its receptors, particularly A(2A) receptors, is a potent endogenous anti-inflammatory agent that modulates the functions and differentiation of inflammatory and immune cells. Because the inflammatory milieu abounds in proinflammatory cytokines, we investigated the effects of Th1-inflammatory cytokines on function and expression of adenosine A(2A) receptors in the human monocytic cell line THP-1. We found that, consistent with previous reports, adenosine and 2-[p-(2-carnonylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS-21680), a selective A(2A) receptor agonist, suppress IL-12 production but increase IL-10 production in LPS-activated THP-1 cells. These effects were blocked by the A(2A) receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385). More importantly, the suppressive effect of adenosine and CGS-21680 on IL-12 production was significantly enhanced in cells pretreated with either IL-1 (10 U/ml) or TNF-alpha (100 U/ml) but markedly attenuated in cells pretreated with IFN-gamma (100 U/ml). Similarly, IL-1 and TNF-alpha treatment potentiated the stimulatory effect of adenosine and CGS-21680 on IL-10 production, whereas IFN-gamma treatment almost completely abolished this effect. CGS-21680 stimulated an increase in intracellular cAMP in a time- and dose-dependent manner in IL-1- and TNF-alpha-treated cells but not in control or IFN-gamma-treated cells. Both IL-1 and TNF-alpha increased A(2A) receptor mRNA and protein. In parallel with its effect on A(2A) receptor function, IFN-gamma down-regulated A(2A) receptor message and protein. Because adenosine mediates many of the antiinflammatory effects of drugs such as methotrexate, these observations suggest that local changes in the cytokine milieu may influence the therapeutic response to those drugs by altering the expression and function of adenosine receptors on inflammatory cells.     

Pannexin 1 Channels Link Chemoattractant Receptor Signaling to Local Excitation and Global Inhibition Responses at the Front and Back of Polarized Neutrophils

Neutrophil chemotaxis requires excitatory signals at the front and inhibitory signals at the back of cells, which regulate cell migration in a chemotactic gradient field. We have previously shown that ATP release via pannexin 1 (PANX1) channels and autocrine stimulation of P2Y₂ receptors contribute to the excitatory signals at the front. Here we show that PANX1 also contributes to the inhibitory signals at the back, namely by providing the ligand for A_2A adenosine receptors. In resting neutrophils, we found that A_2A receptors are uniformly distributed across the cell surface. In polarized cells, A_2A receptors redistributed to the back where their stimulation triggered intracellular cAMP accumulation and protein kinase A (PKA) activation, which blocked chemoattractant receptor signaling. Inhibition of PANX1 blocked A_2A receptor stimulation and cAMP accumulation in response to formyl peptide receptor stimulation. Treatments that blocked endogenous A_2A receptor signaling impaired the polarization and migration of neutrophils in a chemotactic gradient field and resulted in enhanced ERK and p38 MAPK signaling in response to formyl peptide receptor stimulation. These findings suggest that chemoattractant receptors require PANX1 to trigger excitatory and inhibitory signals that synergize to fine-tune chemotactic responses at the front and back of neutrophils. PANX1 channels thus link local excitatory signals to the global inhibitory signals that orchestrate chemotaxis of neutrophils in gradient fields.     

Adenosine, a cytoprotective autocoid: effects of adenosine on neutrophil plasma membrane viscosity and chemoattractant receptor display

At inflammatory sites neutrophils are stimulated to produce a variety of toxic agents, yet rarely harm the endothelium across which they migrate. We have recently found that endothelium releases adenosine which, acting via receptors on the surface of human neutrophils, inhibits generation of toxic metabolites by stimulated neutrophils but, paradoxically, promotes chemotaxis. Agents which diminish plasma membrane viscosity affect neutrophil function similarly, possibly by modulating chemoattractant receptor number or affinity. We therefore determined whether adenosine receptor agonists modulate neutrophil function by decreasing membrane viscosity and/or chaning the affinity of chemoattractant (N-fMet-Leu-Phe, FMLP) receptors. Surprisingly, 5′-(N-ethylcar☐amido)adenosine (NECA, 10 μM), the most potent agonist at neutrophil adenosine receptors, increased plasma membrane viscosity, as measured by fluorescence anisotropy of the plasma membrane specific probe 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene (TMA-DPH), in unstimulated neutrophils from a mean microviscosity of 1.67 ± 0.02 (S.E.) to 1.80 ± 0.02 (p < 0.001) while inosine (10 μM), a poor adenosine receptor agonist, had no effect (1.73 ± 0.04, p =n.s. vs. control, p < 0.01 vs. NECA). Adenosine receptor agonists increased plasma membrane viscosity in neutrophils with the same order of potency previously seen for inhibition of superoxide anion generation and enhancement of chemotaxis (NECA > adenosine = N⁶-phenylisopropyladenosine). The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline reversed the effect of NECA on plasma membrane viscosity. Unlike other agents which modulate plasma membrane viscosity, NECA (10 μM) did not significantly change the number or affinity of [³H]FMLP binding sites on neutrophils. In contrast to the hypothesis of Yuli et al. these results indicate that occupancy of adenosine receptors on neutrophils increases plasma membrane viscosity without affecting chemoattractant receptor display.     

Inhibitory actions of adenosine on follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells

To determine the effects of adenosine on follicle-stimulating hormone (FSH)-induced differentiation, granulosa cells isolated from the ovaries of diethylstilbestrol-treated immature rats were cultured with increasing concentrations of the nucleoside and modulators of adenosine action. Although adenosine had no effect on basal granulosa cell function during 48 h of culture, concentrations of the nucleoside from 10 microM to 1 mM progressively inhibited FSH-induced responses, including progesterone production and expression of FSH and luteinizing hormone (LH) receptors. Adenosine had biphasic effects on FSH-stimulated cAMP accumulation, causing inhibition of cAMP production at 10 to 100 microM and stimulation at higher concentrations. The enhancement of cAMP production by 1 mM adenosine occurred during the first 24 h of culture, while both 100 microM and 1 mM adenosine reduced FSH-stimulated cAMP production from 24 to 48 h. The inhibitory effects of adenosine were prevented by adenosine deaminase and dipyridamole, an inhibitor of adenosine transport, and were antagonized by 1-methyl-3-isobutylxanthine. The inhibition of cAMP and progesterone production by adenosine was partially overcome when cells were washed and reincubated with forskolin, but not with FSH. Adenine, guanosine, and inosine at concentrations of 100 microM did not modify FSH-induced cAMP formation or LH receptor induction. These results indicate that adenosine exerts predominantly inhibitory actions on hormone-induced granulosa cell differentiation, as manifested by prominent reductions in steroidogenesis and gonadotropin receptor expression.     

Lysophosphatidylcholine modulates neutrophil oxidant production through elevation of cyclic AMP

Lysophosphatidylcholine (LPC) is an oxidized phospholipid present in micromolar concentrations in blood and inflamed tissues. The effects of LPC on neutrophil functions remain incompletely understood, because conflicting reports exist for its stimulatory and inhibitory roles. We report in this study that LPC inhibits superoxide generation in fMLP- and PMA-stimulated neutrophils without affecting fMLP-induced Ca(2+) mobilization and cell viability. This effect was observed with LPC dissolved in ethanol, but not with LPC stock solutions prepared in water or in BSA-containing aqueous solution with sonication. Under the same experimental conditions, platelet-activating factor primed neutrophils for superoxide generation. The inhibitory effect of LPC was observed within 30 s after its application and was maximal at LPC concentrations between 0.1 and 1 muM. Inhibition of superoxide generation was accompanied by a 2.5-fold increase in the intracellular cAMP concentration. In addition, LPC reduced fMLP-stimulated phosphorylation of ERK and Akt and membrane translocation of p67(phox) and p47(phox). The protein kinase A inhibitors H-89 and adenosine 3'5'-cyclic monophosphorothioate Rp-isomer (Rp-cAMP) partially restored superoxide production in LPC-treated neutrophils, indicating involvement of protein kinase A in LPC-mediated inhibition. Using an ex vivo mouse lung perfusion model that measures lung weight change and capillary filtration coefficient, we found that LPC prevented lung vascular injury mediated by fMLP-activated neutrophils. Taken together, these results suggest that LPC-induced elevation of intracellular cAMP is partially responsible for its inhibition of neutrophil NADPH oxidase activation. A similar mechanism of inhibition may be used for the control of neutrophil-mediated tissue injury.