Figure 4

ANO was refractory to deamination by adenosine deaminase. Stability of 10 μM ANO or adenosine in RMPM 1640 medium supplemented with 10% FBS was assessed in the presence or absence of EHNA (10 μM) for up to 24 h at 37°C. Concentrations of ANO, adenosine or inosine at selected time points were quantified by HPLC (A). Reactivity of 10 μM ANO or adenosine with 6.7 U/L of adenosine deaminase enzyme was assessed for up to 60 min at 37°C in 53.3 mM potassium phosphate buffer containing 0.003% bovine serum albumin. Data are expressed as percentages of residual amounts of ANO or adenosine at each point in time (B). Anti-inflammatory effects of adenosine in the presence or absence of EHNA (10 μM) on TNF-α and IL-6 production by LPS (2 μg/mL)-stimulated peritoneal macrophages were compared with those of ANO. Levels of TNF-α and IL-6 in the culture supernatants were determined by ELISA after incubation for 24 h at 37°C (C and D). Results are representative of three separate experiments with similar results (A and B). In (C) and (D), values represent the means ± S.D. of triplicate cultures and are representative of two separate experiments with similar results. Inosine (←Adenosine) denotes amounts of inosine converted from 10 μM adenosine by adenosine deaminase. *p < 0.05; **p < 0.01, significantly different when compared with control culture.