Suppression of anti-CD3-induced interleukin-4 and interleukin-5 release from splenocytes of Mesocestoides corti-infected BALB/c mice by phosphodiesterase 4 inhibitors.

We investigated the suppressive effects of rolipram, RP 73401 (piclamilast), and other structurally diverse inhibitors of adenosine 3'5'-cyclic monophosphate (cAMP)-specific phosphodiesterase (PDE4) on anti-CD3-stimulated interleukin (IL)-4 and IL-5 generation by splenocytes from BALB/c mice infected with Mesocestoides (M) corti. RP 73401 (IC40: 0.011 +/- 0.004 microM) was a very potent inhibitor of anti-CD3-induced IL-4 release, being approximately 40-fold more potent than (+/-)-rolipram (IC40: 0.43 +/- 0.09 microM). A maximal inhibition of 60-70% of the response was achieved at the top concentrations of RP 73401 (1 microM) and rolipram (100 microM). These PDE inhibitors also suppressed IL-5 generation over the same concentration ranges, but the maximal suppression achieved was only 30-40%. R-(-)-rolipram (IC40: 0.39 +/- 0.09 microM) was approximately 6-fold more potent than S-(+)- rolipram (IC40: 2.6 +/- 0.95 microM) in inhibiting IL-4 release. A close correlation (r2 = 0.82) was observed between suppression of IL-4 release by PDE inhibitors and inhibition of CTLL cell PDE4, a form against which R-(-)-rolipram displayed relatively weak inhibitory potency. A poorer correlation (r2 = 0.26) was observed between suppression of IL-4 release and affinities of cAMP PDE inhibitors for the high-affinity rolipram binding site in mouse brain membranes. The cGMP-inhibited PDE (PDE3) inhibitor, siguazodan, had little or no effect (IC40 > 100 microM) on anti-CD3-stimulated release of either IL-4 or IL-5 and did not significantly enhance the inhibitory action of RP 73401 on the release of either of these cytokines. Finally, RP 73401 (IC50: 0.41 +/- 0.19 nM) inhibited anti-CD3-stimulated DNA synthesis in splenocyte preparations from M. corti-infected mice and siguazodan (10 microM) had no effect on this response, either alone or in combination with the PDE4 inhibitor. The results show that PDE4 inhibitors suppress the release of Th2 cytokines from anti-CD3-stimulated murine spenocytes and that this effect is correlated with inhibition of a low-affinity PDE4 form.

Evidence that cyclic AMP phosphodiesterase inhibitors suppress interleukin-2 release from murine splenocytes by interacting with a 'low-affinity' phosphodiesterase 4 conformer.

1. We have investigated the suppressive effects of rolipram, RP 73401 (piclamilast) and other structurally diverse inhibitors of cyclic AMP-specific phosphodiesterase 4 (PDE4) on interleukin (IL)-2 generation from Balb/c mouse splenocytes exposed to the superantigen, Staphylococcocal enterotoxin-A (Staph. A). The purpose was to determine whether their potencies are more closely correlated with inhibition of PDE4 from CTLL cells, against which rolipram displays weak potency (low-affinity PDE4), or displacement of [3H]-(+/-)-rolipram from its high-affinity binding site (HARBS) in mouse brain cytosol. 2. RP 73401 (IC50 0.46 +/- 0.07 nM, n = 4) was a very potent inhibitor of Staph. A-induced IL-2 release from Balb/c mouse splenocytes, being > 1100 fold more potent than (+/-)-rolipram (IC50 540 +/- 67 nM, n = 3). 3. A close correlation (r = 0.95) was observed between suppression of IL-2 release by PDE inhibitors and inhibition of PDE4. In contrast, little correlation (r = 0.39) was observed between suppression of IL-2 release and their affinities for the high-affinity rolipram binding site (HARBS). 4. RP 73401 only inhibited partially (30-40%) Staph. A-induced incorporation of [3H]-thymidine into splenocyte DNA. The PDE3 inhibitor, siguazodan (10 microM), had little or no effect on IL-2 release or DNA synthesis. This concentration of siguazodan did not enhance the inhibitory action of RP 73401 on IL-2 release but potentiated its effect on DNA synthesis, increasing potency and efficacy. 5. Staph. A-induced DNA synthesis was only partially inhibited by anti-IL-2 neutralizing antibody, whereas dexamethazone (100 nM) and cyclosporine A (100 nM) completely blocked the response. 6. RP 73401 (IC50 6.3 +/- 1.9 nM, n = 4) was 140 fold more potent than rolipram (IC50 900 +/- 300 nM, n = 3) in inhibiting Staph. A-induced [3H]-thymidine incorporation into splenocyte DNA. 7. The results implicate a low-affinity form of PDE4 in the suppression of Staph. A-induced IL-2 release from murine splenocytes by PDE inhibitors. The data also indicate that mitogenic factors other than IL-2, whose elaboration or responses to which are regulated by PDE3 as well as PDE4, contribute to the superantigen-induced DNA synthesis.

Characterisation of cytosolic phospholipase A2 as mediator of the enhanced arachidonic acid release from dimethyl sulphoxide differentiated U937 cells.

Studies were performed to characterise the phospholipase A2 (PLA2) responsible for the greatly increased capacity to release arachidonic acid (AA) of dimethyl sulphoxide (DMSO) differentiated U937 monocytic cells compared to undifferentiated cells (18-fold increase in response to Ca2+ ionophore A23187). Cytosolic PLA2 (cPLA2) activity could be measured in homogenates of differentiated cells, and the highly selective cPLA2 inhibitor arachidonic acid trifluoromethyl ketone reduced A23187 induced [3H]AA release from pre-labelled cells by at least 80%, with an IC50 (12.7 +/- 1.4 microM) not significantly different from that for inhibiting authentic cPLA2 (9.3 +/- 2.0 microM). On the other hand, type II PLA2 activity was not detected in cell homogenates, and [3H]AA release was not inhibited by heparin (1 mg/mL), which binds secreted type II PLA2 and reduces its ability to degrade membrane phospholipids. Stimulation of intact cells with A23187 plus phorbol myristate acetate (PMA) under conditions that released [3H]AA did not increase cPLA2 activity of the cell homogenate, and there was little difference between DMSO differentiated and undifferentiated cells in cPLA2 protein content, cPLA2 specific activity of homogenates, or distribution of cPLA2 between membrane and cytosol in the resting cell. Following stimulation with A23187 plus PMA, no increase in [33P] labelling of cPLA2 immunoprecipitates was seen in cells pre-labelled with [33P] orthophosphate, nor a change in electrophoretic mobility of cPLA2. It was concluded that cPLA2 releases the bulk of AA from stimulated, DMSO differentiated U937 cells. The failure to observe increased cPLA2 specific activity following cellular stimulation could be explained by increased [3H]AA release requiring the activation of only a small proportion of the cell pool of cPLA2 or, alternatively, by increased release reflecting greater Ca(2+)-dependent association of cPLA2 with membrane substrate rather than increased specific activity per se. There was no evidence that any such increased membrane association resulted from cPLA2 phosphorylation. The relative inability of undifferentiated cells to release AA was not due to the absence of cPLA2 or an altered distribution between membrane and cytosol, but suggested the presence of a repressor mechanism that prevents elevated Ca2+ from functionally activating the enzyme intracellularly.

Protein tyrosine kinase but not protein kinase C inhibition blocks receptor induced alveolar macrophage activation.

The selective enzyme inhibitors genistein and Ro 31-8220 were used to assess the importance of protein tyrosine kinase (PTK) and protein kinase C (PKC), respectively, in N-formyl-methionyl-leucyl-phenylalanine (FMLP) induced generation of superoxide anion and thromboxane B(2) (TXB(2)) in guinea-pig alveolar macrophages (AM). Genistein (3-100 muM) dose dependently inhibited FMLP (3 nM) induced superoxide generation in non-primed AM and TXB(2) release in non-primed or in lipopolysaccharide (LPS) (10 ng/ml) primed AM to a level > 80% but had litle effect up to 100 muM on phorbol myristate acetate (PMA) (10 nM) induced superoxide release. Ro 31-8220 inhibited PMA induced superoxide generation (IC(50) 0.21 +/- 0.10 muM) but had no effect on or potentiated (at 3 and 10 muM) FMLP responses in non-primed AM. In contrast, when present during LPS priming as well as during FMLP challenge Ro 31-8220 (10 muM) inhibited primed TXB(2) release by > 80%. The results indicate that PTK activation is required for the generation of these inflammatory mediators by FMLP in AM. PKC activation appears to be required for LPS priming but not for transducing the FMLP signal; rather, PKC activation may modulate the signal by a negative feedback mechanism.

Synthesis and biological activity of trans(+-)-N-methyl-2-(3-pyridyl)-2-tetrahydrothiopyrancarbothioamide 1-oxide (RP 49356) and analogues: a new class of potassium channel opener.

The synthesis and biological activity of trans-(+-)-N-methyl-2-(3-pyridyl)-2-tetrahydrothiopyrancarbothioamid+ ++ e 1-oxide (8a, RP 49356) and analogues is reported. These compounds constitute a new structural class of K(+)-channel opener. The effects of changes in pyridyl group, thioamide, and thiane ring on in vitro K(+)-channel opening reactivity are discussed. A 3-pyridyl or 3-quinolyl group, a small N-alkyl thioamide function, and a thiane oxide ring, in which the sulfoxide is in a trans relationship to the thioamide, are preferred for activity. Selected compounds were tested intravenously in the normotensive anaesthetized rat for hypotensive effects, and the activities reflect their in vitro K(+)-channel opening activity. This led to further evaluation of compound 8a and the selection of the (-)-enantiomer 8b (RP 52891) for development as an antihypertensive and antianginal agent.

Calcium regulation of phospholipase A2 is independent of calmodulin.

There are conflicting data in the literature as to whether or not the Ca2+ activation of phospholipase A2 is mediated by the calcium binding protein calmodulin. In the present study the membrane-bound phospholipase A2 enzymes in rat and human platelets were shown to be absolutely Ca2+ dependent but were not stimulated by the addition of calmodulin. A partially purified phospholipase A2 from rat platelet membrane, which contained little endogenous calmodulin, also was not stimulated by calmodulin addition. Both isolated and membrane-bound phospholipase A2 were inhibited by the non-specific calmodulin antagonist trifluoperazine but the inhibition was not overcome by adding calmodulin. There was thus no evidence from these studies that phospholipase A2 is calmodulin regulated.

Synthesis and antifertility activity of omega-chain phenyl- and 16-phenoxy-analogues of (+/-)-11-deoxyprostaglandin F1 alpha.

Some omega-chain phenyl- and 16-phenoxy- analogues of (+/-)-11-deoxyprostaglandin F1 alpha have been synthesized and evaluated for anti-fertility activity in the hamster. 11-Deoxy-16-phenoxy-17,18,19,20-tetranor-PGF1 alpha was the most active member of the series with an ED50 equal to that of PGF2 alpha. 11-Deoxy-17-phenyl-18,19,20-trinor-PGF1 alpha, which was one third as active as PGF2 alpha, was more potent than the corresponding 16- and 18-phenyl compounds. Aryl ring substitution was found to lower activity, except that with the 16-phenyl compound, p-bromo and m-trifluoromethyl substitution increased the potency. The antifertility activity of the phenoxy compounds, which were poor substrates for 15-hydroxyprostaglandin dehydrogenase, was shown to correlate well with the binding affinity for the bovine corpus luteum PGF2 alpha receptor. Some quantitative structure-activity data supporting this finding are presented.