Introduction Our previous studies demonstrated that activation of the prostaglandin D 2 (PGD 2 ) selective prostanoid receptor (DP receptor) could inhibit histamine release from anti-IgE stimulated rat peritoneal mast cells (RPMC) [1]. Since the DP receptor is known to be positively coupled to the G s pro- tein, which subsequently leads to the activation of adenylate cyclase [2], we further investigated the effects of PGD 2 on the level of intracellular cyclic AMP in RPMC . Materials and methods Mast cells from ovalbumin-sensitized male Sprague Dawley rats were purified by centrifugation through Percoll density gradients [1]. Puri- fied mast cells were pre-incubated with ibuprofen (10 mM, Cayman Chemical) for 20 min at 37 °C to eliminate interference by endo- genously produced prostanoids. 100 ml of ibuprofen treated cells (10 5 ) were added to tubes containing 50 ml of PGD 2 (Cayman Chemical) and/or anti-IgE (ICN Biomedicals) for different periods of incubation which were then stopped by the addition of 10 ml of 70 % perchloric acid. Samples were immediately snap-frozen in liquid nitrogen and were stored at –70 ° C until assay. Cyclic AMP in the samples were extracted by adding 100 ml of a 1 : 1 mixture of tri-n-octylamine and 1,1,2-trichlorotrifluoroethane to 150 ml of samples. 100 ml of the top layer, which was free from organic solvents, was then transferred to fresh micro-centrifuge tubes for cyclic AMP quantification using the cyclic AMP Enzyme Immunosorbent assay (EIA) kit from Cayman Chemical. The increase in cyclic AMP was normalized against the basal cyclic AMP level obtained from cells incubated in buffer alone and was expressed as the percentage of increase in cyclic AMP. Histamine re- lease from the same pools of cells incubated for 5 min in the same com- binations of anti-IgE and/or PGD 2 was also determined spectrofluoro- metrically for each experiment [1]. Unless specified, all materials were purchased from Sigma Chemicals. Results and discussion When incubated with RPMC alone, both PGD 2 (10 μM) and the immunological stimulus, anti-IgE (1/250 dilution), in- duced a transient and monophasic elevation of intracellular cyclic AMP levels by around 200% of basal levels within the Inflamm. res. 50, Supplement 2 (2001) S 61– S 62 1023-3830/01/02S61-02 $ 1.50+0.20/0 © Birkhäuser Verlag, Basel, 2001 Inflammation Research Modulation of intracellular cyclic AMP in immunologically activated rat peritoneal mast cells by prostaglandin D 2 H.Y. A. Lau and C. L. Chan Department of Pharmacology, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, SAR, China, Fax: 852 2603 5139, e-mail: hyalau@cuhk.edu.hk Correspondence to: H. Y. A. Lau first 30 s (Table 1). Both elevations gradually returned to basal levels after 120 s. 35.8 % ± 1.4 % and – 1.5 % ± 0.5 % of total cellular histamine were released by anti-IgE and PGD 2 , respectively, at the end of the 5 min incubation period (values were corrected for the spontaneous histamine release of 7.3 % ± 0.2 % in buffer alone). The observation with anti-IgE confirmed previous reports which described similar kinetics of cyclic AMP elevation induced by mast cell activators which mediated their effects by cross-linking the high af- finity IgE receptors [3]. Although mast cells were obviously activated by anti-IgE, whether this immunologically-induced cyclic AMP elevation plays an enhancing or inhibitory role in the activation of mast cell exocytosis remains controversial [4, 5]. The elevation of cyclic AMP induced by PGD 2 con- firmed that PGD 2 alone can activate adenylate cyclase and was comparable to that previously reported [6]. In contrast to anti-IgE, the elevated cyclic AMP induced by PGD 2 alone did not affect the basal level of histamine release. When RPMC was activated by anti-IgE (1/250) in the presence of PGD 2 Table. 1. Effects of anti-IgE (1/250 dilution), PGD 2 (10 μM) and anti- IgE (1/250) with PGD 2 (10 μM) on cyclic AMP changes in rat peritone- al mast cells. Results are increase in cyclic AMP expressed as a percen- tage of the basal cyclic AMP level [increase in cyclic AMP (%)]. All data are mean ± SEM for 6 experiments. The mean basal cyclic AMP level = 1.9 ± 0.1 pmol/10 6 cells. Time Increase in cyclic AMP (%) (s) Anti-IgE (1/250) PGD 2 (10 μM) Anti-IgE (1/250) + PGD 2 (10 μM) 0 13.8 ± 15.7 37.3 ± 36.3 – 29.0 ± 14.7 5 29.7 ± 9.5 131.5 ± 52.8 395.4 ± 361.4 8 130.0 ± 35.4 151.5 ± 42.6 1210.5 ± 621.1 10 138.9 ± 23.7 94.3 ± 28.3 2545.0 ± 1739.7 15 195.6 ± 28.1 108.3 ± 77.1 3480.3 ± 1113.9 20 170.0 ± 49.5 176.7 ± 93.9 3811.3 ± 1029.9 30 243.3 ± 80.4 144.4 ± 91.8 4227.1 ± 1540.3 60 136.2 ± 45.4 103.1 ± 67.5 5431.7 ± 1914.1 120 34.6 ± 17.6 70.5 ± 41.8 3423.7 ± 1464.0 180 37.5 ± 12.3 36.2 ± 22.8 1426.6 ± 669.0 240 49.4 ± 11.0 41.2 ± 24.2 771.1 ± 486.4 300 26.5 ± 22.2 4.3 ± 10.2 558.2 ± 301.5