Adenosine receptors on rabbit alveolar macrophages: binding characteristics and effects on cellular function
Hasday, J.D.; Sitrin, R.G.
Journal of Laboratory and Clinical Medicine 110(3): 264-273
1987
ISSN/ISBN: 0022-2143 PMID: 3039032 Document Number: 299749
Adenosine and its synthetic analogues are known to affect many leukocyte functions, in some cases by binding to specific cell surface receptors coupled to adenylate cyclase. In this study, adenosine receptors were demonstrated on normal rabbit alveolar macrophages by examining specific binding of tritiated 5-N-ethylcarboxamide adenosine (NECA) to intact cells. Scatchard analysis suggested a single class of approximately 33,000 binding sites per cell and an estimated Kd of 0.46 .mu.mol/L. Competitive inhibition of tritiated NECA binding was demonstrated for 2-chloroadenosine (2-CA; Ki = 3.68 .mu.mol/L) and L-phenylisopropyl adenosine (L-PIA; Ki > 100 .mu.mol/L), a rank order of binding affinities indicative of an A2 receptor. Theophylline and isobutyl methylxanthine had Kis of 368 and 27.6 .mu.mol/L, respectively. For functional correlation, NECA was found to be 10-fold more potent than L-PIA in stimulating an increase in intracellular cyclic adenosine monophosphate. In addition, macrophages were cultured for 24 hours with NECA, 2-CA, or L-PIA to determine whether these analogues modulated expression of either cell-associated procoagulant activity or elaboration of plasminogen activator. Procoagulant activity was suppressed by as much as 62% (P < 0.05); the rank order of potency and blockade of the effect with theophylline suggest that suppression of procoagulant activity occurred primarily by stimulation of A2 receptors. By contrast, these analogues stimulated production and release of plasminogen activator by 30% (P < 0.05), but this effect had none of the features of an A2-mediated mechanism. Macrophages were cotreated with nitrobenzylthioinosine (10 .mu.mol/L) and adenosine deaminase (2 U/ml) to allow adenosine accumulation exclusively within the cell. This resulted in both suppression of procoagulant activity and stimulation of plasminogen activator release, suggesting that adenosine may act at intracellular sites as well as at A2 receptors in the plasma membrane.