We have investigated the inhibitory effects of RP 73401 (piclamilast) and rolipram against human monocyte cyclic AMP‐specific phosphodiesterase (PDE4) in relation to their effects on prostaglandin (PG)E2‐induced cyclic AMP accumulation and lipopolysaccharide (LPS)‐induced TNFα production and TNFα mRNA expression. PDE4 was found to be the predominant PDE isoenzyme in the cytosolic fraction of human monocytes. Cyclic GMP‐inhibited PDE (PDE3) was also detected in the cytosolic and particulate fractions. Reverse transcription polymerase chain reaction (RT‐PCR) of human monocyte poly (A+) mRNA revealed amplified products corresponding to PDE4 subtypes A and B of which the former was most highly expressed. A faint band corresponding in size to PDE4D was also observed. RP 73401 was a potent inhibitor of cytosolic PDE4 (IC50: 1.5 ± 0.6 nM, n = 3). (±)‐Rolipram (IC50: 313 ± 6.7 nM, n = 3) was at least 200 fold less potent than RP 73401. R‐(−)−rolipram was approximately 3 fold more potent than S‐(+)‐rolipram against cytosolic PDE4. RP 73401 (IC50: 9.2 ± 2.1 nM, n = 6) was over 50 fold more potent than (±)‐rolipram (IC50: 503 ± 134 nM, n = 6)) in potentiating PGE2‐induced cyclic AMP accumulation. R‐(−)−rolipram (IC50: 289 ± 121 nM, n = 5) was 4.7 fold more potent than its S‐(+)‐enantiomer (IC50: 1356 ± 314 nM, n = 5). A strong and highly‐significant, linear correlation (r = 0.95, P < 0.01, n = 13) was observed between the inhibitory potencies of a range of structurally distinct PDE4 inhibitors against monocyte PDE4 and their ED50 values in enhancing monocyte cyclic AMP accumulation. A poorer, though still significant, linear correlation (r = 0.67, P < 0.01, n = 13) was observed between the potencies of the same compounds in potentiating PGE2‐induced monocyte cyclic AMP accumulation and their abilities to displace [3H]‐rolipram binding to brain membranes. RP 73401 (IC50: 6.9 ± 3.3 nM, n = 5) was 71 fold more potent than (±)‐rolipram (IC50: 490 ± 260 nM, n = 4) in inhibiting LPS‐induced TNFα release from monocytes. R‐(−)−rolipram (IC50: 397 ± 178 nM, n = 3) was 5.2‐fold more potent than its S‐(+)‐ enantiomer (IC50: 2067 ± 659 nM, n = 3). As with cyclic AMP, accumulation a closer, linear correlation existed between the potency of structurally distinct compounds in suppressing TNFα with PDE4 inhibition (r = 0.93, P < 0.01, n = 13) than with displacement of [3H]‐rolipram binding (r = 0.65, P < 0.01, n = 13). RP 73401 (IC50: 2 nM) was 180 fold more potent than rolipram (IC50: 360 nM) in suppressing LPS (10 ng ml−1)‐induced TNFα mRNA. The results demonstrate that RP 73401 is a very potent inhibitor of TNFα release from human monocytes suggesting that it may have therapeutic potential in the many pathological conditions associated with over‐production of this pro‐inflammatory cytokine. Furthermore, PDE inhibitor actions on functional responses are better correlated with inhibition of PDE4 catalytic activity than displacement of [3H]‐rolipram from its high‐affinity binding site, suggesting that the native PDE4 in human monocytes ex...

The stereospecificity of rolipram inhibition of particulate cyclic AMP-specific phosphodiesterase (PDE IV) from guinea-pig eosinophils has been investigated. (-)-Rolipram (IC50 = 0.22 +/- 0.08 microM) was 2.5-fold more potent than (+)-rolipram (IC50 = 0.58 +/- 0.05 microM) in inhibiting membrane-bound PDE IV. Solubilization of PDE IV with deoxycholate (0.5%) and NaCl (100 mM) increased rolipram stereospecificity [IC50 (-)-rolipram = 0.020 +/- 0.002 microM; IC50 (+)-rolipram = 0.33 +/- 0.07 microM]. Partial purification of this solubilized PDE IV by DEAE-trisacryl anion-exchange chromatography reduced the enantiomeric potency difference compared with the pre-chromatographed activity, with (-)-rolipram (IC50 = 0.20 +/- 0.02 microM) being only 2.9-fold more potent than (+)-rolipram (IC50 = 0.57 +/- 0.14 microM). Vanadate-glutathione complex (V-GSH) stimulated membrane-bound PDE IV activity and increased the potency of (-)-rolipram (IC50 = 0.014 +/- 0.006 microM) but not (+)-rolipram (IC50 = 0.32 +/- 0.07 microM). In intact eosinophils, (-)-rolipram (EC50 = 0.19 +/- 0.02 microM) was 10-fold more potent than (+)-rolipram (EC50 = 1.87 +/- 0.09 microM) in enhancing isoprenaline (10 microM)-stimulated cyclic AMP accumulation. Strong correlations were demonstrated for displacement of [3H]rolipram binding to brain membranes by several PDE inhibitors and their inhibition of solubilized PDE IV (r = 0.98, P < 0.001, n = 7) and stimulation of cyclic AMP accumulation in intact cells (r = 0.98, P < 0.001, n = 6). Rolipram was a relatively weak inhibitor of partially purified pig aortic PDE IV and only slight stereospecificity was exhibited [IC50 (-)-rolipram = 1.47 +/- 0.09 microM; IC50 (+)-rolipram = 2.73 +/- 0.38 microM]. The results indicate the presence of a partially concealed stereospecific site (Sr) on eosinophil PDE IV possibly similar to the high-affinity rolipram-binding site in brain through which rolipram can potently inhibit enzyme activity. This site, which apparently is not present on partially purified pig aortic PDE IV, is concealed in freshly prepared eosinophil membranes but is exposed by solubilization or V-GSH treatment and is important in regulating intracellular cyclic AMP accumulation in intact cells.

Treatment of membranes from guinea-pig peritoneal cosinophils with dcoxycholatc and NaCl solubilized ~95% of the particulate cyclic AMPspecific phosphodicsterase (PDE IV). Solubilizcd PDE IV was at least 10 times more potently inhibited by selective PDE IV inhibitors(e.g. roliprant, denbufyllinc) than bound enzyme. Vanadatclglutathionc complex (VGSH) activated mcmbranc-bound PDE IV and also increased potencies of these same inhibitors by at least IO-fold. Neither solubilization nor V/GSH markedly influenced the inhibitory activities of non-xlcctivc inhibitors (e.g. trequinsin, dipyridamole), Inhibitor effects on solubilixcd PDE IV and cyclic AMP accumulirtion in intact cells wcrc strongly corrclatcd. Thcsc results suggest a biologically important site on cosinophil PDE IV which is conccalcd or partially conceulcd in freshly prcparcd membrdncs and is exposed by solubilixation or VIGSH.

1 The possible role of cyclic AMP phosphodiesterase (PDE) in the inhibitory actions of ibudilast on tracheal smooth muscle contractility and eosinophil thromboxane generation was investigated. 2 Ibudilast was a non-selective inhibitor of partially purified cyclic nucleotide PDE isoenzymes from pig aorta and bovine tracheal smooth muscle, exhibiting only moderate potency against bovine tracheal PDE IV (IC0 = 12 ± 4 AM, n = 3). Similar or slightly lower potencies were displayed against PDEs I, II, III and V. In contrast, rolipram exhibited selectivity for PDE IV (3 0.5 AM, n = 3).3 Ibudilast (IC50 = 0.87 ± 0.37 AM, n = 3), like rolipram (IC = 0.20 0.04 AM, n = 3), was a more potent inhibitor of membrane-bound PDE IV from guinea-pig eosinophils than of partially purified PDE IV from bovine tracheal smooth muscle. The potency of ibudilast increased when the eosinophil enzyme was solubilised with deoxycholate and NaCl (IC50 = 0.11 ± 0.05 JAM, n = 3) or exposed to vanadate/glutathione complex (V/GSH) (ICo=0.11 ± 0.02 JM, n = 3). The potency of rolipram was also increased by solubilization (IC50 = 0.012 0.003, n = 3) or V/GSH (IC,0 = 0.012 ± 0.003, n = 3). 4 In intact eosinophils, ibudilast (0.032 jiM-20 JM) potentiated isoprenaline-induced cyclic AMP accumulation in a concentration-dependent manner, being approximately 20 fold less potent than rolipram. Little or no effect on basal cyclic AMP levels was observed with either compound. The cyclic AMP-dependent protein kinase activity ratio was significantly increased following incubation of eosinophils with either ibudilast (20 AM) or rolipram (20 gM) in the absence or presence of isoprenaline. 5Leukotriene B4 (300 nM)-induced thromboxane generation from guinea-pig eosinophils was inhibited by ibudilast (IC50 = 11.3 ± 3.7 AM, n = 5) and rolipram (IC,0 = 0.280 ± 0.067 AM, n = 5) in a concentration-dependent manner. 6 Ibudilast (10 nM-I AM), whilst generally less potent than rolipram (1 nM-AM), produced concentration-dependent relaxation of spasmogen (methacholine, histamine, LTD4)-induced tone in the guineapig isolated tracheal strip. Ibudilast was less potent in reversing the methacholine (IC50 = 1.95 ± 0.40 JM, n = 6)-induced contraction than those of histamine (IC50 = 0.18 ± 0.70 AM, n =6) or leukotriene D4 (LTD4, IC50 = 0.12 ± 0.05 JM, n = 6). Rolipram also exhibited a similar pattern of activity, although the difference in potency against methacholine (IC50 = 0.1 ± 0.01 JAM, n = 6) compared with the other two spasmogens, histamine (IC50 = 0.034 ± 0.017 JM, n = 7) and LTD4 (IC50 = 0.026 ± 0.008 AM, n = 7), was not as great.7 These results demonstrate that ibudilast, like rolipram, has several biological actions on the eosinophil and airways smooth muscle which may be attributed to inhibition of cyclic AMP PDE. These actions may account, at least in part, for the recently reported anti-asthma effects of ibudilast.

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