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Phosphodiesterase 4 in macrophages: relationship between cAMP accumulation, suppression of cAMP hydrolysis and inhibition of [3H]R-(-)-rolipram binding by selective inhibitors.

ABSTRACT: A perplexing phenomenon identified in several tissues is the lack of correlation between inhibition of phosphodiesterase 4 (PDE4) and certain functional responses such as smooth muscle relaxation, gastric acid secretion and cAMP accumulation. Interpretation of these data is complicated further by the finding that function correlates with the ability of PDE4 inhibitors to displace [3H]rolipram [4-(3-cyclopentenyloxy-4-methoxyphenyl)-2-pyrrolidone] from a high-affinity site in rat brain that is apparently distinct from the catalytic centre of the enzyme. We have investigated this discrepancy by using guinea pig macrophages as a source of PDE4 and have confirmed that the ability of a limited range of structurally dissimilar PDE inhibitors (Org 20241, nitraquazone and the enantiomers of rolipram and benafentrine) to increase cAMP content did not correlate with their potency as inhibitors of partly purified PDE4, whereas a significant linear and rank order correlation was found when cAMP accumulation was related to the displacement of [3H]R-(-)-rolipram from a specific site identified in macrophage lysates. An explanation for these data emerged from the finding that the IC50 values and rank order of potency of these compounds for inhibition of partly purified PDE4 and the native (membrane-bound) form of the same enzyme were distinct. Similarly, no correlation was found when membrane-bound PDE4 was compared with the same enzyme that had been solubilized with Triton X-100. These unexpected results were attributable to a selective decrease in the potency of those inhibitors [nitraquazone, R-(-)- and S-(+)-rolipram] that interacted preferentially with the rolipram binding site. Indeed, if membrane-bound PDE4 was used as the enzyme preparation, excellent linear and rank order correlations between inhibition of cAMP hydrolysis, displacement of [3H]R-(-)-rolipram and cAMP accumulation were found, which improved further in the presence of the vanadyl (Vo)/2. GSH complex. Moreover, using Vo/2.GSH-treated membranes, the IC50 values of nitraquazone and the enantiomers of rolipram for the inhibition of PDE4 approached their affinity for the rolipram binding site. Collectively, these data suggest that the rolipram binding site and the catalytic domain on CPPDE4 might represent part of the same entity. In addition, these results support the concept that PDE4 can exist in different conformational states [Barnett, Manning, Cieslinski, Burman, Christensen and Torphy (1995) J. Pharmcol. Exp. Ther. 273, 674-679] and provide evidence that the cAMP content in macrophages is regulated primarily by a conformer of PDE4 for which rolipram has nanomolar affinity.

SUBMITTER: Kelly JJ

PROVIDER: S-EPMC1217639 | biostudies-other | 1996 Sep

REPOSITORIES: biostudies-other

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Project description:Inhibitors of cAMP-phosphodiesterase 4 (PDE4) exert a number of promising therapeutic benefits, but adverse effects, in particular emesis and nausea, have curbed their clinical utility. Here, we show that PAN-selective inhibition of PDE4, but not inhibition of PDE3, causes a time- and dose-dependent accumulation of chow in the stomachs of mice fed ad libitum without changing the animals' food intake or the weight of their intestines, suggesting that PDE4 inhibition impairs gastric emptying. Indeed, PDE4 inhibition induced gastric retention in an acute model of gastric motility that traces the passage of a food bolus through the stomach over a 30 minutes time period. In humans, abnormal gastric retention of food is known as gastroparesis, a syndrome predominated by nausea (>90% of cases) and vomiting (>80% of cases). We thus explored the abnormal gastric retention induced by PDE4 inhibition in mice under the premise that it may represent a useful correlate of emesis and nausea. Delayed gastric emptying was produced by structurally distinct PAN-PDE4 inhibitors including Rolipram, Piclamilast, Roflumilast, and RS25344, suggesting that it is a class effect. PDE4 inhibitors induced gastric retention at similar or below doses commonly used to induce therapeutic benefits (e.g., 0.04 mg/kg Rolipram), thus mirroring the narrow therapeutic window of PDE4 inhibitors in humans. YM976, a PAN-PDE4 inhibitor that does not efficiently cross the blood-brain barrier, induced gastroparesis only at significantly higher doses (≥1 mg/kg). This suggests that PDE4 inhibition may act in part through effects on the autonomic nervous system regulation of gastric emptying and that PDE4 inhibitors that are not brain-penetrant may have an improved safety profile. The PDE4 family comprises four subtypes, PDE4A, B, C, and D. Selective ablation of any of these subtypes in mice did not induce gastroparesis per se, nor did it protect from PAN-PDE4 inhibitor-induced gastroparesis, indicating that gastric retention may result from the concurrent inhibition of multiple PDE4s. Thus, potentially, any of the four PDE4 subtypes may be targeted individually for therapeutic benefits without inducing nausea or emesis. Acute gastric retention induced by PDE4 inhibition is alleviated by treatment with the widely used prokinetic Metoclopramide, suggesting a potential of this drug to alleviate the side effects of PDE4 inhibitors. Finally, given that the cause of gastroparesis remains largely idiopathic, our findings open the possibility that a physiologic or pathophysiologic downregulation of PDE4 activity/expression may be causative in a subset of patients.

Project description: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.

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Phosphodiesterase 4 in macrophages: relationship between cAMP accumulation, suppression of cAMP hydrolysis and inhibition of [3H]R-(-)-rolipram binding by selective inhibitors.

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