Characterization of phosphodiesterase 4 in guinea-pig macrophages: Multiple activities, association states and sensitivity to selective inhibitors

1 The cyclic AMP phosphodiesterases (PDE) in guinea-pig peritoneal macrophages were isolated, partially characterized and their role in regulating the cyclic AMP content in intact cells evaluated. 2 Differential centrifugation of macrophage lysates revealed that similar to 90% of the PDE activity was membrane-bound and exclusively hydrolyzed cyclic AMP. This activity was not removed by KCl (200 mM) but was readily solubilized by the non-ionic detergent, Triton X-100 (1% v/v). Greater than 80% of the hydrolytic. activity was suppressed by the PDE4 inhibitors, R-rolipram and nitraquazone with IC(50)s of 240 and 540 nM, respectively. 3 Anion-exchange chromatography of the total protein extracted from macrophages resolved two major peaks of cyclic AMP PDE activity that were insensitive to cyclic GMP (10 mu M), calmodulin (50 units plus 2 mM CaCl2) and a PDE3 inhibitor, SK&F 95654 (10 mu M), but were markedly suppressed by RS-rolipram (10 mu M). The two peaks of PDE activity were arbitrarily designated CPPDE4 alpha and CPPDE4 beta with respect to the order from which they were eluted from the column where the prefix. CP, refers to the species, Cavia porcellus. 4 The hydrolysis of cyclic AMP catalyzed by CPPDE4 alpha and CPPDE4 beta conformed to Michaelis-Menten kinetic behaviour with similar K(m)s (13.4 and 6.4 mu M, respectively). 5 Thermal denaturation of membrane-bound PDE4 at 50 degrees C followed bi-exponential kinetics with t(1/2) values of 1.5 and 54.7 min for the first and second components, respectively. In contrast, CPPDE4 alpha and CPPDE4 beta each decayed mono-exponentially with significantly different thermostabilities (t(1/2) = 2.77 and 1.15 min, respectively). 6 Gel filtration of CPPDE4 beta separated two peaks of rolipram-sensitive PDE activity. The main peak eluted at a volume indicative of a similar to 180 kDa protein but was preceded by a much larger form of the enzyme that had an estimated weight of 750 kDa. Size exclusion chromatography of CPPDE4 alpha resolved a broad peak of activity with molecular weights spanning 50 to 200 kDa. 7 Of ten PDE inhibitors examined, none distinguished CPPDE4 alpha from CPPDE4 beta with respect to their IC50 values or their rank older of potency. RS-rolipram acted as a purely competitive inhibitor of cyclic AMP hydrolysis with K(i)s of 2 mu M and 1.5 mu M for CPPDE4 alpha and CPPDE4 beta, respectively. In contrast to the membrane-associated enzyme(s), R-rolipram and nitraquazone were 4 to 19 fold less potent as inhibitors of CPPDE4 alpha and CPPDE4 beta. 8 In intact macrophages, Ro 20-1724 and RS-rolipram potentiated isoprenaline-induced cyclic AMP accumulation under conditions where a PDE3 inhibitor, SK&F 94120, was essentially inactive. 9 These data demonstrate that the predominant cyclic AMP hydrolyzing activity in guinea-pig macrophages is a PDE4. Moreover, thermostability studies and size exclusion chromatography indicates the possible expression of two intrinsic, membrane-associated isoenzymes which can regulate the cyclic AMP content in intact cells. The finding that soluble and particulate forms of the same enzyme exhibit different sensitivities to rolipram and nitraquazone implies that PDE4 can change conformation. Finally, the identification of multiple molecular weight species of CPPDE4 suggests that this enzyme(s) might form multimeric complexes of variable association states.