Molecular Pharmacology, Vol 14, 848-855, Copyright © 1978 by the American Society for Pharmacology and Experimental Therapeutics

Rat Mast Cell High Affinity Cyclic Nucleotide Phosphodiesterases: Separation and Inhibitory Effects of Two Antiallergic Agents

¹ Research and Development Laboratories, AB Draco, Fack, S-221 01 Lund, Sweden

Rat mast cells, harvested from the thoracic and peritoneal cavities and purified to at least 90% by density gradient centrifugation in 38% bovine serum albumin (BSA) were examined for cyclic nucleotide phosphodiesterase activities. The soluble fraction (200,000 x g) of disrupted mast cells was found to contain approximately 80% of the total activity at a substrate concentration of 0.13 µM or 6.0 µM cyclic AMP or 0.12 µM cyclic GMP. It could be separated by DEAE-Sephadex chromatography into two major fractions (I_MC and III_MC) and one minor fraction (II_MC). Fraction I_MC contained phosphodiesterase activities with high apparent affinity for both cyclic GMP and cyclic AMP (app. K_m values in the order of 0.1 to 0.2 µM), cyclic GMP being the preferred substrate. Fraction III_MC hydrolyzed primarily cyclic AMP; the app. K_m value was 0.4 µM. The particulate fraction (200,000 x g) of rat mast cells, which was found to contain approximately 20% of the total activity at the indicated substrate concentrations, hydrolyzed cyclic AMP and cyclic GMP with similar apparent K_m values (0.5 µM) and maximum velocities. The phosphodiesterase activity pattern of supernatants from nonpurified thoracic and peritoneal cells was different from that of mast cells, indicating that contaminating cells are not the source of the two major phosphodiesterase fractions in mast cell supernatants. The antiallergic agents disodiumcromoglycate (DSCG) and 2-o-propoxyphenyl-8-azapurin-6-one (M&B 22,948) inhibited the activity of the separated mast cell cyclic AMP phosphodiesterase activities. The lowest I₅₀ values observed were 250 µM and 10 µM, respectively, i.e., concentrations that exceed those sufficient for inhibition of antigen-induced histamine release from mast cells.