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PubMed1996

Influence of aging on rolipram-sensitive phosphodiesterase activity and [3H]rolipram binding in the rat brain.

To clarify the quantitative and qualitative changes in type IV phosphodiesterase (PDE IV) with aging, phosphodiesterase (PDE) activity and [3H]rolipram binding in the cytosolic fraction from the brains of young and aged rats were examined. In all areas of the aged (100-week-old) rat brain except for hippocampus, the PDE activity was decreased by about half that in the young (10-week-old) animals. However, inhibition % by 100 microM rolipram and by 100 microM isobutylmethylxanthine (IBMX) was not significantly different between the aged and young rats. On the other hand, [3H]rolipram binding and rolipram-sensitive PDE activity did not change with aging in the hippocampus, although both were decreased in other brain regions. These results suggest that PDE IV does not decrease with aging and maintains its cAMP degrading activity in the hippocampus. It may be involved in the dysfunction of the hippocampus with aging.

Aging↗

PubMed1990

Pharmacokinetics of (+)-rolipram and (-)-rolipram in healthy volunteers.

Plasma levels of S-(+)-rolipram and R-(-)-rolipram in six healthy male volunteers were measured by radioimmunoassay after intravenous injection of 0.1 mg and oral administration of 1.0 mg of the pure enantiomers. Following i.v. treatment, plasma levels of both isomers declined in three phases, with half-lives of 0.2 h, 0.6-0.9 h and 6-8 h. Total clearance was 6 ml.min-1.kg-1. Oral administration of 1.0 mg gave a peak concentration of 16 ng.ml-1 after 0.5 h. Bioavailability of (+)-rolipram was 77% and of the (-) enantiomer it was 74%. There was no significant difference in Cmax, half-life, total clearance or bioavailability between the two enantiomers.

Administration, Oral↗

PubMed1993

Stereospecificity of rolipram actions on eosinophil cyclic AMP-specific phosphodiesterase.

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.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1997

Rolipram and its optical isomers, phosphodiesterase 4 inhibitors, attenuated the scopolamine-induced impairments of learning and memory in rats.

We investigated the effects of (+/-)-rolipram, a phosphodiesterase (PDE) 4 inhibitor, and its isomers on scopolamine-induced deficits of learning and memory in rats using an 8-arm radial maze task and a passive avoidance task. 1) In the 8-arm radial maze task, (+/-)-rolipram (0.02-0.2 mg/kg, p.o.), (-)-rolipram (0.01-0.02 and 0.2-0.5 mg/kg, p.o.) and (+)-rolipram (20-50 mg/kg, p.o.) attenuated the scopolamine-induced deficits of spatial cognition. As for the minimum effective dose of each drug, (-)-rolipram was 2 and 2000 times as potent as (+/-)-rolipram and (+)-rolipram, respectively. (-)-Rolipram produced a biphasic dose-response and (+/-)-rolipram produced a broad dose-response. 2) (+/-)-Rolipram and its isomers also attenuated the scopolamine-induced deficits in the passive avoidance response. Also for the minimum effective dose, (-)-rolipram (0.01-0.02 mg/kg) was 2 and 200 times as potent as (+/-)-rolipram (0.02-0.1 mg/kg) and (+)-rolipram (2mg/kg). 3) The behaviorally effective doses of (+/-)-rolipram and its isomers also enhanced the oxotremorine-induced tremors in mice. Comparing these racemic isomers, (-)- and (+/-)-rolipram have more potent effects than (+)-rolipram on scopolamine-induced deficits in the 8-arm radial maze task and passive avoidance task. Especially (+/-)-rolipram has a wide dose range in these behavioral study. These results suggest that the ameliorating effects of rolipram might result from the indirect potentiation of various transmitters including cholinergic and noradrenergic systems by an increase in cAMP with the inhibition of PDE4.

Animals↗

PubMed1997

Suppression of TNF-alpha expression, inhibition of Th1 activity, and amelioration of collagen-induced arthritis by rolipram.

Rolipram is a type IV phosphodiesterase inhibitor that suppresses inflammation and TNF-alpha production. As anti-TNF-alpha therapy is effective in rheumatoid arthritis, we investigated the effect of rolipram on collagen-induced arthritis (CIA), a murine model of rheumatoid arthritis. Rolipram was administered after the onset of clinical arthritis at doses of 0.5, 3, 5, or 10 mg/kg twice daily, with a dose-dependent therapeutic effect on clinical severity and joint erosion. Immunohistochemical analysis of joints of rolipram-treated mice revealed 67% reduction in TNF-alpha-expressing cells compared with control arthritic mice. In vitro studies using bone marrow-derived macrophages confirmed that rolipram directly suppressed TNF-alpha and IL-12 production following stimulation with IFN-gamma and LPS. The effect of rolipram on T cell activity was studied by measuring Th1/Th2 cytokine production by collagen-stimulated draining lymph node cells from arthritic mice treated in vivo with rolipram. Rolipram reduced IFN-gamma production and increased IL-10, indicating that rolipram down-regulated the ongoing Th1 response to type II collagen. Finally, the effect on CIA of combination therapy was studied using rolipram plus either anti-TNF-alpha or anti-CD4 mAbs. Rolipram plus anti-TNF-alpha was not therapeutically additive, whereas rolipram plus anti-CD4 mAb was clearly additive. This result indicates that the therapeutic effects of rolipram overlap with TNF-alpha blockade, but are complementary to anti-CD4 treatment. It is therefore proposed that a major mechanism of action of rolipram in CIA is suppression of TNF-alpha activity. These findings suggest that type IV phosphodiesterase inhibitors may be effective in pathologic conditions, such as RA, with overexpression of TNF-alpha.

Animals↗

PubMed1988

Influence of rolipram on the cyclic 3',5'-adenosine monophosphate response to histamine and adenosine in slices of guinea-pig cerebral cortex.

The effect of the phosphodiesterase (PDE) inhibitor rolipram on the cyclic AMP responses to adenosine, histamine and combinations of these two agonists, was examined in [3H]adenine-labelled slices of guinea-pig cerebral cortex. Constant levels of [3H]-cyclic AMP were achieved within 10 min of agonist addition, both in the presence and absence of rolipram (0.1 mM). Histamine (1 mM) produced an 8-fold increase in [3H]-cyclic AMP (compared with basal) which was increased 7-fold by rolipram. The responses to adenosine (0.1 mM) and adenosine and histamine in combination were larger than that to histamine alone (46-fold or more compared with basal) but the potentiation by rolipram was much smaller (2.5-fold or less). With both agonists the effect of rolipram was dose-dependent, the steady state [3H]-cyclic AMP levels increasing 1-2-fold for a 10-fold increase in rolipram concentration. Removal of the histamine or adenosine stimulus once steady state had been reached resulted in a rapid fall in [3H]-cyclic AMP levels with a half time of less than 5 min. Rolipram (0.1 mM) did not significantly alter the initial rates of fall in [3H]-cyclic AMP levels but increased the time taken for them to return to basal levels. The findings of higher steady state levels of cyclic AMP in the presence of rolipram, together with an almost unaltered rate of cyclic AMP turnover, are consistent with an interaction of rolipram with PDE which is overcome by an increase in cyclic AMP concentration. However, the relatively smaller effects of rolipram on the higher steady levels of cyclic AMP produced by adenosine and the rather shallow dose-dependence of the PDE inhibitor on the responses to both agonists are inconsistent with a simple competitive inhibition of total PDE activity in responding cells. The results can be explained, however, by the involvement of different forms of PDE, with the rolipram-sensitive, calcium-independent form dominating at low cyclic AMP levels and the rolipram-insensitive, calcium-dependent form becoming more important when cyclic AMP levels are higher.

2-Chloroadenosine↗

PubMed1992

Coexpression of human cAMP-specific phosphodiesterase activity and high affinity rolipram binding in yeast.

Studies by various investigators have demonstrated that the low Km, cAMP-specific phosphodiesterase (PDE IV) is selectively inhibited by a group of compounds typified by rolipram and Ro 20-1724. In addition to inhibiting the catalytic activity of PDE IV, rolipram binds to a high affinity binding site present in brain homogenates. Although it has been assumed that the high affinity rolipram-binding site is PDE IV, no direct evidence has been produced to support this assumption. The present studies were undertaken to determine whether the rolipram-binding site is coexpressed with PDE IV catalytic activity in Saccharomyces cerevisiae genetically engineered to express human recombinant monocytic PDE IV (hPDE IV). Expressing hPDE IV cDNA in yeast resulted in a 20-fold increase in PDE activity that was evident within 1 h of induction and reached a maximum by 3-6 h. The recombinant protein represented hPDE IV as judged by its immunoreactivity, molecular mass (approximately 88 kDa), kinetic characteristics (cAMP Km = 3.1 microM; cGMP Km greater than 100 microM), sensitivity to rolipram (Ki = 0.06 microM), and insensitivity to siguazodan (PDE III inhibitor) and zaprinast (PDE V inhibitor). Saturable, high affinity [3H] (R)-rolipram-binding sites (Kd = 1.0 nM) were coexpressed with PDE activity, indicating that both binding activity and catalytic activity are properties of the same protein. A limited number of compounds were tested for their ability to inhibit hPDE IV catalytic activity and compete for [3H](R)-rolipram binding. Analysis of the data revealed little correlation (r2 = 0.35) in the structure-activity relationships for hPDE IV inhibition versus competition for [3H] (R)-rolipram binding. In fact, certain compounds (e.g. (R)-rolipram Ro 20-1724) possessed a 10-100-fold selectivity for inhibition of [3H] (R)-rolipram binding over hPDE IV inhibition, whereas others (e.g. dipyridamole, trequinsin) possessed a 10-fold selectivity for PDE inhibition. Thus, although the results of these studies demonstrate that hPDE IV activity and high affinity [3H](R)-rolipram binding are properties of the same protein, they do not provide clear cut evidence linking the binding site with the PDE inhibitory activity of rolipram and related compounds.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1997

Detailed characterization of a purified type 4 phosphodiesterase, HSPDE4B2B: differentiation of high- and low-affinity (R)-rolipram binding.

We have overexpressed in a baculovirus expression system, and purified to > 95% homogeneity, milligram quantities of a human recombinant rolipram-sensitive cAMP phosphodiesterase, HSPDE4B2B (amino acid residues 81-564). The protein expression levels were approximately 8 mg of HSPDE4B2B (81-564) per liter of Sf9 cells. The Km of the purified enzyme for cAMP was 4 microM and the Ki for the Type 4 phosphodiesterase-specific inhibitor (R)-rolipram was 0.6 microM. The specific activity of the purified protein was 40 mumol/min/mg protein. A nonequilibrium filter binding assay revealed a high-affinity (R)-rolipram binding site on the purified enzyme with a Kd of 1.5 nM and a stoichiometry of 0.05-0.3 mol of (R)-rolipram per mol of HSPDE4B2B (81-564). Equilibrium dialysis experiments revealed a single binding constant of 140 nM with a stoichiometry of 0.75 mol of (R)-rolipram per mol of HSPDE4B2B (81-564). Size exclusion chromatography and analytical ultracentrifugation experiments suggest that the protein exists in multiple association states larger than a monomer. Proteolysis experiments revealed a 43-kDa fragment that contained catalytic and rolipram-inhibitable activities, but the fragment showed no high-affinity (R)-rolipram binding. Based on the proteolytic cleavage studies a 43-kDa protein was constructed, expressed, and purified. This protein, HSPDE4B2B (152-528), had Km and Vmax similar to those of the HSPDE4B2B (81-564) protein, but did not exhibit high-affinity (R)-rolipram binding. The protein did show low-affinity (R)-rolipram binding using the equilibrium binding assay. These results show that a low-affinity binding site for (R)-rolipram is solely contained within the catalytic domain of HSPDE4B2B, whereas high-affinity (R)-rolipram binding requires residues within the catalytic domain and residues flanking N- and/or C-terminal to the catalytic region.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1986

Rolipram, a stereospecific inhibitor of calmodulin-independent phosphodiesterase, causes beta-adrenoceptor subsensitivity in rat cerebral cortex.

Prolonged pretreatment of rats with the atypical antidepressant rolipram attenuates noradrenaline (NA) sensitivity of the cerebral cortical cAMP generating system. The development of this down-regulation is time (7 d treatment required) and dose dependent (EC50 = 0.35 mg/kg). Density of beta-adrenoceptor as measured by (-)-3H-dihydroalprenolol [(-)-3H-DHA] binding is also reduced by rolipram pretreatment. The effect of rolipram is absolutely stereospecific for the (-)-enantiomer (ED50 = 0.18 mg/kg). In addition, only with this isomer, a reduction in daily weight gain was found compared to sham treated controls. Presynaptic denervation using intracerebroventricular (i.c.v.) injections of 6-hydroxydopamine (6-OHDA) prior to or during rolipram treatment did not completely block the effect of a rolipram treatment on down-regulation of cerebral cortical beta-adrenoceptors. The data favor a pre- and postsynaptic action of rolipram different from all other antidepressants studied so far in this experimental setting. Rolipram is known as inhibitor of brain phosphodiesterase. Using partially purified calmodulin-independent phosphodiesterase from brain it is shown that exclusively the (-)-enantiomer of rolipram inhibits phosphodiesterase with an IC50 of 1.25 mumol/l whereas the (+)-isomer possesses little potency. Since a marked stereospecificity for the (-)-isomer of rolipram was displayed in all pharmacological parameters tested so far with (+)- and (-)-rolipram, it is suggested that stereospecific and isozyme specific inhibition of cAMP-phosphodiesterase is, at least in part, related to the mechanism of action of the potential antidepressant drug rolipram and possibly of other antidepressants as well.

Adenylyl Cyclases↗

PubMed1997

Human recombinant phosphodiesterase 4B2B binds (R)-rolipram at a single site with two affinities.

The interactions between (R)-rolipram and purified human recombinant low-Km, cAMP-specific phosphodiesterase (HSPDE4B2B) constructs were investigated using biochemical, kinetic, and biophysical approaches. The full-length protein (amino acids 1-564) and an N-terminal truncated protein (amino acids 81-564) exhibited high-affinity (R)-rolipram binding, whereas an N-terminal and C-terminal truncated protein (amino acids 152-528) lacked high-affinity (R)-rolipram binding. The 152-528 and 81-564 proteins had similar Km's and kcat/Km's and differed less than 4-fold compared with the 1-564 protein. (R)-Rolipram inhibition plots were biphasic for the 1-564 and 81-564 proteins and fit to two states, a high-affinity (Ki = 5-10 nM) state and a low-affinity (Ki = 200-400 nM) state, whereas the 152-528 protein fit to a single state (Ki = 350-400 nM). The stoichiometry for high-affinity binding using a filter binding assay was found to be <1 mol of (R)-rolipram per mole of 1-564 or 81-564 protein. Titration microcalorimetric studies revealed both a high-affinity state with a stoichiometry of 0.3 mol of (R)-rolipram per mole of protein and a low-affinity state with a stoichiometry of 0.6 mol of (R)-rolipram per mole of protein for the 81-564 protein. A single low-affinity state with a stoichiometry of 0.9 mol of (R)-rolipram per mole of protein was seen using the 152-528 protein. The data indicate that purified HSPDE4B2B 1-564 and 81-564 proteins contain a single binding site for (R)-rolipram and suggest that the proteins exist in two different states distinguishable by their affinity for (R)-rolipram. Furthermore, the high-affinity binding state of the protein requires amino acid residues at the N-terminus (81-151) of the protein and catalytic domain (152-528), whereas the low-affinity binding state only requires residues in the catalytic domain (152-528). Phosphorylation at residues 487 and 489 of the 81-564 protein does not appear to alter the substrate kinetics or the stoichiometry and binding affinity of (R)-rolipram.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1989

Autoradiographic mapping of a selective cyclic adenosine monophosphate phosphodiesterase in rat brain with the antidepressant [3H]rolipram.

Rolipram is a clinically effective antidepressant with selective cAMP phosphodiesterase (PDE) inhibiting properties. (+/-)-[3H]Rolipram binds with high affinity (Kd = 2.52 +/- 0.47 nM) to sections of rat brain (Hill number = 0.90 +/- 0.05). Binding is stereospecific. Association of (+/-) [3H]rolipram to sections is rapid (47% of specific binding in the first minute, kobs = 0.52 min-1). Dissociation of (+/-)-[3H]rolipram exhibits non first order kinetics (3 component model; t1/2 = 2.5 min, 50 min and 6 h, respectively). A number of PDE inhibitors reduce (+/-)-[3H]rolipram binding to the level of nonspecific binding ((-)-rolipram, IC50 = 0.9 nM; (+/-)-rolipram, IC50 = 1.5 nM; Ro 20-1724, IC50 = 11 nM; ICI 63.197, IC50 = 35 nM; medazepam, IC50 = 240 nM; diazepam, IC50 = 1200 nM; IBMX, IC50 = 3800 nM). In vitro autoradiography reveals high binding site densities in the cerebellum, olfactory bulb, lateral septal nucleus, frontal cortex, subiculum and CA1 of hippocampus. Most of the labeled structures are part of the limbic system. In vivo autoradiography of (+/-)-[3H]rolipram binding shows much more nonspecific binding than in vitro, nevertheless the distribution pattern of (+/-)-[3H]rolipram binding sites is similar. A comparison of the distribution pattern of (+/-)-[3H]rolipram binding sites with that of an antidepressant (monoamine oxidase inhibitor, monoamine uptake inhibitor) reveals no overlap. Limited, though significant correlations exist with the distribution of beta 1-adrenergic, adenosine1 and glutamate/quisqualate receptors as well as protein kinase C, but not with beta 2-adrenergic receptors and forskolin binding sites.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1996

Preventive but not therapeutic application of Rolipram ameliorates experimental autoimmune encephalomyelitis in Lewis rats.

Experimental autoimmune encephalomyelitis (EAE) in Lewis rats, an animal model mimicking some aspects of multiple sclerosis, was treated with the type IV-specific phosphodiesterase inhibitor Rolipram. Actively induced EAE evoked by immunization with myelin basic protein (MBP) in complete Freund's adjuvant was delayed but only slightly ameliorated in its maximal severity by preventive treatment with Rolipram (2 x 3 mg/kg per day) starting on the day of immunization. Therapeutic administration of Rolipram (2 x 5 mg/kg per day) was begun within hours after onset of first clinical signs of EAE but could not modify the further course of the disease. Both doses had significant side effects. Injection of 5 mg Rolipram/kg provoked transient slackening and unsteady gait while chronic application of 6 mg/kg/day strongly accelerated the weight gain in adolescent rats. EAE adoptively transferred by injection of encephalitogenic T line blasts was shortened and significantly suppressed in its severity by application of Rolipram (2 x 5 mg/kg per day) starting on the day of cell transfer. In corresponding lumbar spinal cord sections density of inflammatory infiltration by T cells and macrophages was reduced. Rolipram did not prevent generation of an antigen-specific immune response in vivo. In vitro the drug inconsistently inhibited MBP-induced activation of encephalitogenic T cells. TNF-alpha secretion by encephalitogenic T cells was limited only when T cell proliferation was also affected. In contrast, TNF-alpha production by LPS-activated macrophages was consistently and markedly suppressed by Rolipram. However, since the encephalitogenic T line cells produced at least 100 times more TNF-alpha than the same number of Rolipram-sensitive macrophages, the impact of Rolipram on the total amount of TNF-alpha synthesized in EAE may be limited. Together with our histological findings, the data suggest that relevant immunosuppressive mechanisms of Rolipram may be the inhibition of migration of leukocytes into the central nervous system and to some extent its inhibitory effect on T cell proliferation and macrophage activity. The downregulatory effects of Rolipram may be partially counteracted by its augmenting impact on the production of nitric oxide by macrophages.

Animals↗

PubMed1996

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

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.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1999

Imaging of cAMP-specific phosphodiesterase-IV: comparison of [11C]rolipram and [11C]Ro 20-1724 in rats.

The phosphodiesterase type IV (PDEIV) family of enzymes contributes to the metabolism of cAMP formed by the stimulation of beta-adrenergic, A2-adenosine, and H2-histamine receptors in the brain. Disturbances in cAMP-mediated signaling have been implicated in several neuropsychiatric disorders, and there is evidence that increasing cAMP levels through PDEIV inhibition improves the symptoms of these disorders. In the present study, the selective PDEIV inhibitors rolipram and Ro 20-1724, labeled with C-11, were evaluated in vivo in rats, as potential radioligands for imaging PDEIV enzymes with positron emission tomography (PET). Biodistribution experiments revealed a greater than threefold increased regional brain retention of [11C]rolipram as compared to [11C]Ro 20-1724. [nC]Rolipram uptake was higher in rat brain areas (e.g., cortical regions and olfactory system) showing higher expression of PDEIV enzymes, as determined previously using [3H]rolipram autoradiography or molecular genetic techniques. Binding of [n1C]rolipram and [11C]Ro 20-1724 was specific, since coadministration of high doses of unlabeled rolipram (10 mg/Kg, i.v.) or Ro 20-1724 (30 mg/Kg with [11C]rolipram and 10 mg/Kg with [11C]Ro 20-1724, i.v.) reduced radioactivity uptake in brain regions. Pretreatment with high doses of the PDEI selective inhibitor vinpocetine (10 mg/Kg, i.p., 15 min prior), or the noradrenaline reuptake inhibitor desipramine (10 mg/Kg, i.p., 30 min prior), or coinjection with the adenylyl cyclase activator forskolin (6.5 or 15 mg/Kg, i.v.), did not inhibit [11C]rolipram uptake in brain areas, suggesting binding selectivity for PDEIV enzymes. We conclude that [11C]rolipram, but not [11C]Ro 20-1724, is a promising radioligand for imaging the PDEIV enzymes with PET.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1986

Stereospecific binding of the antidepressant rolipram to brain protein structures.

The characteristics for the binding of the selective cAMP phosphodiesterase inhibitor and antidepressant agent rolipram to brain and peripheral organs were investigated. (+/-)-[3H]Rolipram equilibrium binding and Scatchard analysis revealed saturable, reversible, stereospecific, Mg2+-dependent and heat-sensitive binding with an apparent Hill number of 1. Binding was detected both to membrane-bound and soluble sites, with dissociation constants Kd of 1.2 and 2.4 nM, respectively, and binding site concentrations (Bmax) of 19.3 and 23.6 pmol/g rat forebrain. The (-)-enantiomer of rolipram was ca. 20 times more effective than the (+)-enantiomer in displacing (+/-)-[3H]rolipram from membranes. Rolipram bound to brain tissue of all mammalian species tested including man, while tissue from bird and fish showed less binding. Organs other than brain exhibited only negligible binding. Only specific cAMP phosphodiesterase inhibitors (ICI 63.197, Ro 20-1724) were potent competitors, while rolipram itself was inactive in a variety of receptor binding assays of neuroactive ligands. The kinetics of (-)-[3H]rolipram binding to the particulate fraction revealed a complex association and dissociation behaviour. The nature of the rolipram binding protein(s) is not clear, but the low affinity binding site evident from binding kinetics may represent a rolipram-sensitive phosphodiesterase isoenzyme also common to some peripheral organs, while the high affinity binding site(s) may be related to PDE isoenzymes more confined to the central nervous system.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1994

Characterization of cAMP-dependent inhibition of LPS-induced TNF alpha production by rolipram, a specific phosphodiesterase IV (PDE IV) inhibitor.

Bacterial endotoxins (lipopolysaccharide or LPS) provoke shock and tissue injury by eliciting the release of toxic factors from reticuloendothelial cells. One of the principal endogenous factors involved in this process is tumor necrosis factor alpha (TNF alpha). In this study, inhibitors selective for different classes of phosphodiesterases (PDE), were examined for their effects on LPS-induced TNF alpha production by human monocytes. The selective cAMP-PDE IV inhibitors, rolipram and RO-20-1724 were capable of inhibiting LPS-induced TNF alpha production by human monocytes in a concentration-dependent manner. Rolipram was used to examine further the cellular pharmacology of PDE IV inhibitors on cytokine production. The IC50 for inhibition of LPS-induced TNF alpha production by rolipram was 0.1 microM, whereas production of IL-1 beta or IL-6 was unaffected. Furthermore, rolipram was equally effective in inhibiting TNF alpha production by a number of other stimuli. Inhibition of TNF alpha production by rolipram was associated with an elevation of intracellular cAMP, consistent with a mechanism involving phosphodiesterase inhibition. Rolipram was efficacious in suppressing LPS-induced TNF alpha mRNA expression, and at the protein level was also active when added to cultures post-stimulated with LPS. This indicates that rolipram may act at both the transcriptional and translational levels. Rolipram inhibited TNF alpha production in vivo in a rat endotoxemia model. Collectively, these data suggest that the prototypic inhibitor of PDE IV isozyme, rolipram, can effectively and selectively inhibit LPS-induced TNF alpha production through elevation of intracellular cAMP.

Animals↗

PubMed1995

The specific type IV phosphodiesterase inhibitor rolipram combined with adenosine reduces tumor necrosis factor-alpha-primed neutrophil oxidative activity.

Monocytes and macrophages produce tumor necrosis factor-alpha (TNF alpha) in response to microbial products including endotoxin. TNF alpha is a potent primer of neutrophil (PMN) oxidative activity. Certain xanthine phosphodiesterase (PDE) inhibitors such as pentoxifylline have been shown to inhibit stimulated oxidative activity in PMN. In the present study, the non-xanthine PDE type IV inhibitor rolipram (4-[3'-cyclopentyloxy-4'-methoxyphenyl]-2-pyrrolidone) alone and in combination with adenosine is examined as a potential modulator of TNF alpha-primed PMN oxidative activity. Attainable in vivo concentrations of rolipram and physiological concentrations of adenosine alone and together synergistically decreased rhTNF alpha-primed suspended PMN oxidative activity stimulated by the chemoattractant f-met-leu-phe. The rolipram effect was reversible by washing, and rolipram had a comparable effect if added before or after priming, indicating that its effect was on the primed response rather than on priming per se. In addition, rolipram especially when combined with adenosine, decreased rhTNF alpha-stimulated PMN adherence to a fibrinogen-coated surface, and the oxidative burst of rhTNF alpha-stimulated adherent PMN. The specific adenosine A2a receptor agonists CGS 21680 and WRC-0474 had comparable activity to adenosine in these experiments. Adenosine (or CGS 21680) combined with rolipram synergistically increased f-met-leu-phe-stimulated PMN cAMP content. The effects of both adenosine and rolipram with adenosine could be only partly counteracted by treatment of the PMN with the protein kinase A inhibitor KT 5720, indicating that protein phosphorylation is only partially involved. Rolipram activity was about 1000 x (by molar concentration) greater than pentoxifylline in comparable assays. Thus, rolipram, especially when combined with adenosine, has potent modulating effects on PMN activation and may be useful in decreasing inflammatory tissue damage in patients with sepsis.

Adenosine↗

PubMed1993

Sequential alteration of [3H]rolipram binding in gerbil brain after transient cerebral ischemia.

The time course of rolipram (Ca2+/calmodulin independent cyclic adenosine monophosphate inhibitor) binding sites changes following gerbil transient forebrain ischemia was determined using receptor autoradiography. Gerbils subjected to 10-min ischemia revealed a significant reduction in rolipram binding in most selectively vulnerable regions early in the recirculation (1-5 h). Marked reduction in the rolipram binding was seen in the selectively vulnerable areas 48 h or 7 days after ischemia. Thereafter, the rolipram binding in the hippocampal CA1 and CA3 sectors, which were most vulnerable to ischemia, was severely reduced up to 1 month after recirculation. In contrast, the reduction of the rolipram binding activity in other regions recovered to sham-operated level or showed a slight recovery. Interestingly, the dentate gyrus, which was resistant to ischemia, also exhibited a significant reduction of the rolipram binding activity up to 1 month after ischemia. Eight months after ischemia, the hippocampal CA1 and CA3 sectors showed severe shrinkage and marked reduction in the rolipram binding. Other regions exhibited no significant reduction in the rolipram binding except for a slight reduction in the thalamus. These results demonstrate that transient cerebral ischemia causes severe reduction in rolipram binding sites in selectively vulnerable areas, and this reduction precedes the neuronal cell loss. These findings may reflect the alteration of an intracellular phosphodiesterase activity after ischemia.

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