P Tacchi

A2 Adenosine Receptors in Neutrophils from Health Volunteers and Patients with Rheumatic Disease

Inflammation is the primary response to tissue injury or microbial invasion and is characterized by the local accumulation of neutrophils. While neutrophils are essential for limiting the spread of infection, stimulated neutrophyls are capable of damaging injured tissues while en route to sites of infection or inflammation. It has been recently discovered that release of adenosine is one mechanism by which normal cells may protect themselves from activated neutrophils (Cronstein et al., 1986). Extracellular adenosine and its analogues diminish generation of toxic oxygen product (e. g. O2− or H2O2) by activated neutrophils, yet promote neutropil chemiotaxis (Cronstein et al., 1985; Roberts et al., 1985; Rose et al., 1988). There are two types of cell surface adenosine receptors, A1 and A2 which are differentiated by their affinity for adenosine, the order of potency of adenosine analogues and opposing effects on cellular cAMP metabolism (van Calker et al., 1979). Recently it has been possible to label adenosine receptors by adenosine analogues such as 3H-N6-cyclohexyl adenosine (CHA) for A1 adenosine receptors and 3H-5’N-ethylcarboxamido adenosine (NECA) for A2 receptors in cells from synovial fluid from rheumatic patients (Martini et al., 1989). In the present report we described the presence of A2 adenosine receptors in neutrophils from health volunteers and from rheumatic patients. Initially, the sites were labeled by 3H-NECA but the binding properties of this ligand did not agree with the pharmacology of A2 receptors and indicated the presence of A2 -like binding protein. Then, the characterization of A2 adenosine binding sites in human neutrophils was carried out using 3H- (2 [p- (2- carboxyethyl)-phen ethylamido]-5’ -N- ethylcarboxamido adenosine (CGS 21680), a novel high affinity and selectivity ligand for striatal A2 receptors ( Jarvis et al., 1989).

Characterization of adenosine receptors in Mullus surmuletus

Abstract The intent of the present study was to investigate adenosine receptor sites in brain membranes of the saltwater teleost fish, Mullus surmuletus, using the A1 receptor selective agonist, [3H]CHA, and A2a receptor selective agonist [3H]CGS 21680. The A1 selective agonist, [3H]CHA, bound saturably, reversibly and with high affinity to a single-class of binding sites (Kd 1.47 nM; Bmax 100–190 fmol/mg protein, dependent on fish length). The A2a selective agonist, [3H]CGS 21680, also bound saturably, reversibly and with relative high affinity to a single-class of binding sites (Kd 44.2 nM; Bmax 150–300 fmol/mg protein dependent on fish length). In equilibrium competition experiments, adenosine analogous, NECA, CGS 21680, CHA, CPA, S-PIA, R-PIA, CPCA, DPMA, and xanthine antagonists, DPCPX, XAC, and THEO all displaced [3H]CHA and [3H]CGS 21680 specifically bound to brain membranes from Mullus surmuletus. Specific binding of both [3H]CHA and [3H]CGS 21680 was inhibited by GDPβS. For [3H]CHA the IC50 value was 2.5 ± 0.1 μM, while for [3H]CGS 21680 the IC50 value was 7.7 ± 0.3 μM. Our results indicate that the high affinity binding sites for [3H]CHA have some pharmacological characteristics of mammalian A1 adenosine receptors, while the binding sites for [3H]CGS 21680 appear to be virtually identical to the binding sites for [3H]CHA.

A2 Adenosine Receptors in Neutrophils from Healthy Volunteers and Patients with Rheumatoid Arthritis

At physiological concentrations, adenosine can modulate a variety of biological activities by engaging specific cells surface receptors, termed A1 and A2, with different affinity for adenosine and adenosine analogues 1. Engagement of A2 adenosine receptors induces an increase in cAMP levels in several cells types, in contrast stimulation of A1 receptors causes opposite effects 2. It has been shown that adenosine and its analogues inhibit O2 generation 3, 4, phagocytosis and adherence by occupancy of specific adenosine A2 receptors, while the occupancy of A1 adenosine receptors enhance chemiotaxis 5, phagocytosis and adherence 6. In general, activation of adenosine receptors on leukocytes reduces immune and inflammatory responses7. Therefore, it may be suggested that release of adenosine is one mechanism by which normal cells protect themselves from activated neutrophils. Since it is possible that a decreased adenosine receptor functions are implicated in diseases like rheumatic pathologies characterised by an excess of inflammation. In the present report we described characteristics of adenosine binding sites on human neutrophils from healthy volunteers and rheumatic patients afflicted with rheumatoid arthritis by using [3 H]N ethylcarboxamidoadenosine (NECA) and [3 H] 2-p-(2-carboxyethyl) phenethylamino 5’ N-ethylcarboxamidoadenosine (CGS 21680) as ligands.

3H-NECA BINDING TO POLYMORPHONUCLEAR MEMBRANE : EFFECT OF SERA FROM PATIENTS WITH HODGKIN'S DISEASE

Several studies have shown that sera from patients with Hodgkins disease contain factors capable of inhibiting polymorphonuclear functions, among them chemotaxis. In the present study, we investigated whether these sera, which were able to inhibit PMN chemotaxis in the agarose test, were also able to affect the 3H-NECA binding to PMN membrane obtained from healthy donors. Control experiments were carried out using PMN incubated with a pool of sera from healthy volunteers. No significant difference was found in the maximum number of binding sites; on the contrary, the equilibrium dissociation constant was significantly increased in the membrane preparation of PMN incubated with pathological serum.