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

Abstract

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 Di erential centrifugation of macrophage lysates revealed that *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 mM), calmodulin (50 units plus 2 mM CaCl 2 ) and a PDE3 inhibitor, SK&F 95654 (10 mM), but were markedly suppressed by RS-rolipram (10 mM). The two peaks of PDE activity were arbitrarily designated CPPDE4a and CPPDE4b with respect to the order from which they were eluted from the column where the pre®x, CP, refers to the species, Cavia porcellus. 4 The hydrolysis of cyclic AMP catalyzed by CPPDE4a and CPPDE4b conformed to MichaelisMenten kinetic behaviour with similar K m s (13.4 and 6.4 mM, respectively). 5 Thermal denaturation of membrane-bound PDE4 at 508C followed bi-exponential kinetics with t 1/2 values of 1.5 and 54.7 min for the ®rst and second components, respectively. In contrast, CPPDE4a and CPPDE4b each decayed mono-exponentially with signi®cantly di erent thermostabilities (t 1/2 =2.77 and 1.15 min, respectively). 6 Gel ®ltration of CPPDE4b separated two peaks of rolipram-sensitive PDE activity. The main peak eluted at a volume indicative of a *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 CPPDE4a resolved a broad peak of activity with molecular weights spanning 50 to 200 kDa. 7 Of ten PDE inhibitors examined, none distinguished CPPDE4a from CPPDE4b with respect to their IC 50 values or their rank order of potency. RS-rolipram acted as a purely competitive inhibitor of cyclic AMP hydrolysis with K i s of 2 mM and 1.5 mM for CPPDE4a and CPPDE4b, respectively. In contrast to the membrane-associated enzyme(s), R-rolipram and nitraquazone were 4 to 19 fold less potent as inhibitors of CPPDE4a and CPPDE4b. 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 ®nding that soluble and particulate forms of the same enzyme exhibit di erent sensitivities to rolipram and nitraq...

Modulation of eotaxin formation and eosinophil migration by selective inhibitors of phosphodiesterase type 4 isoenzyme

“…This implies that like rolipram, salbutamol is also able to prevent eotaxin production in vivo , which prompted us to investigate a potential synergism between rolipram and salbutamol as regards eosinophil influx blockade. It is noteworthy that the β2‐agonist isoprenaline worked in a synergistic manner with rolipram in guinea‐pig macrophages to elevate the cyclic AMP content (Kelly et al ., 1998). In another study, low concentrations of β2‐agonists, including salbutamol, increased the relaxant effect of PDE type 4 inhibitors on antigen‐induced contraction of guinea‐pig isolated trachea (Planquois et al ., 1996).…”

IL‐12 as a therapeutic target for pharmacological modulation in immune‐mediated and inflammatory diseases: regulation of T helper 1/T helper 2 responses

“…The predominant form of cyclic AMP PDE in monocytes/macrophages is the PDE IV isotype, but lower amounts of PDE III and PDE I can also be found in these cells ( Verghese et al ., 1995 ; Souness et al ., 1996 ; Gantner et al ., 1997 ; Kelly et al ., 1998 ). Selective inhibition of these enzymes suppresses IL‐12 production in various in vivo models.…”

Tracheal Relaxant Effect of Triazine Derivatives: Correlation with Phosphodiesterase 4 Inhibitory Activity

“…The PDE 4 family has a marked preference for cAMP as a substrate. Inhibition of PDE 4 activity results in increased intracellular levels of the nucleotide, which is often associated with dampening effects on airway smooth muscle tone and on activation and mediator release from in¯ammatory cells (Nicholson & Shahid 1994;Souness & Giembycz 1994;Santamaria et al 1997;Houslay et al 1998;Kelly et al 1998). Thus, it is generally accepted that inhibitors of this isoenzyme may have useful bronchodilator and anti-in¯ammatory properties for treating both the symptoms and the underlying causes of asthma.…”