Stereospecificity of rolipram actions on eosinophil cyclic AMP-specific phosphodiesterase

Abstract

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.

Inhibitor Binding to Type 4 Phosphodiesterase (PDE4) Assessed Using [3H]Piclamilast and [3H]Rolipram

“…The potency order of PDE4 inhibitors tested in this study was in agreement with the potency orders of these inhibitors reported previously for inhibition of enzyme activity (Schneider et al, 1986;Souness et al, 1997Souness et al, , 1999Saldou et al, 1998 3 H]rolipram binding by non-PDE4 inhibitors was less potent and showed no significant differences between the two radioligands. Consistent with their low binding affinities, these drugs also have been shown to be very poor inhibitors of PDE4 enzymatic activity (Schneider et al, 1986;Souness and Scott, 1993;Makhay et al, 2001).…”

“…They reported that the peripheral organs tested showed either no detectable [ 3 H]rolipram binding or a very low specific binding capacity. Although the nature of the HARBS is not yet clear, it has been suggested to be one of the two distinct conformations of PDE4 (Torphy et al, 1992;Souness and Scott, 1993 ICI 63,197, and Ro 20-1724 were better described by a two-site model, while the competition curves for piclamilast, cilomilast, roflumilast, and CDP 840 were adequately described by a one-site model. K i values are shown in Table 3.…”

“…Binding to the HARBS, but not the LARBS, depends on the presence of the N-terminal region. Although their exact natures are still unclear, it appears the HARBS and the LARBS are more accurately described as two distinct binding affinity states, rather than separate sites (Schneider et al, 1986;Torphy et al, 1992;Souness and Scott, 1993;Jacobitz et al, 1996).…”

Enantiodivergent Synthesis of (R)- and (S)-Rolipram

“…Although 1 has been often used as the racemate in biological experiments, the biological activity of the enantiomers may be widely divergent [9][10][11][12]. Therefore we considered it desirable to use this compound routinely as a single enantiomer for both in vitro and in vivo studies.…”

Synthesis and Structure-Activity Relationship of N-Arylrolipram Derivatives as Inhibitors of PDE4 Isozymes.

“…While it has been shown that the eudismic ratio can vary depending on the source of the PDE enzyme, there is general agreement that (R)-(Ϫ)-rolipram is the more potent of the two enantiomers. 22,33) As representative examples, Fig. 3 shows a comparison of the IC 50 's for PDE4D inhibition of the (R)-and (S)-enantiomers of compounds (1) (rolipram) and (11).…”