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ABSTRACTGlioblastoma (GBM) is the most aggressive primary brain tumor, exhibiting a high rate of recurrence and poor prognosis. Surgery and chemoradiation with temozolomide (TMZ) represent the standard of care, but, in most cases, the tumor develops resistance to further treatment and the patients succumb to disease. Therefore, there is a great need for the development of well-tolerated, effective drugs that specifically target chemoresistant gliomas. NEO214 was generated by covalently conjugating rolipram, a PDE4 (phosphodiesterase 4) inhibitor, to perillyl alcohol, a naturally occurring monoterpene related to limonene. Our previous studies in preclinical models showed that NEO214 harbors anticancer activity, is able to cross the blood-brain barrier (BBB), and is remarkably well tolerated. In the present study, we investigated its mechanism of action and discovered inhibition of macroautophagy/autophagy as a key component of its anticancer effect in glioblastoma cells. We show that NEO214 prevents autophagy-lysosome fusion, thereby blocking autophagic flux and triggering glioma cell death. This process involves activation of MTOR (mechanistic target of rapamycin kinase) activity, which leads to cytoplasmic accumulation of TFEB (transcription factor EB), a critical regulator of genes involved in the autophagy-lysosomal pathway, and consequently reduced expression of autophagy-lysosome genes. When combined with chloroquine and TMZ, the anticancer impact of NEO214 is further potentiated and unfolds against TMZ-resistant cells as well. Taken together, our findings characterize NEO214 as a novel autophagy inhibitor that could become useful for overcoming chemoresistance in glioblastoma.Abbreviations:ATG: autophagy related; BAFA1: bafilomycin A1; BBB: blood brain barrier; CQ: chloroquine; GBM: glioblastoma; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MGMT: O-6-methylguanine-DNA methyltransferase; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; POH: perillyl alcohol; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TMZ: temozolomide

A preprint abstract from biorxiv.org discusses the potential of the PDE4 inhibitor Rolipram in breast cancer treatment. The study found that Rolipram can down-regulate MMP2 and MMP9 levels, which are important markers of metastasis in breast cancer cell lines. The inhibitor was also found to alter the enzymatic activities of these MMPs independently of cAMP-PKA signaling. In vitro experiments using primary cells from breast cancer patients further supported these findings. The study suggests that Rolipram may have off-target effects on proteins involved in metastasis, making it a potential therapeutic option for breast cancer. However, it is important to note that this preprint has not been peer-reviewed. [Extracted from the article]

A preprint abstract from biorxiv.org discusses the potential therapeutic implications of the PDE4 inhibitor Rolipram in breast cancer treatment. The abstract highlights that Rolipram has been found to down-regulate MMP2 and MMP9 levels, which are important markers of metastasis in breast cancer cell lines. The study also explores the inhibitory mechanism of Rolipram on MMP2 and MMP9 through zymographic studies, ligand-based target prediction, and molecular docking. In vitro experiments using primary cells from breast cancer patients further support the ability of Rolipram to control breast cancer by altering the enzymatic activities of MMP2 and MMP9. However, it is important to note that this preprint has not been peer-reviewed. [Extracted from the article]

A preprint abstract from biorxiv.org discusses the potential use of the PDE4 inhibitor rolipram in repressing the hedgehog signaling pathway to regress breast cancer. The study found that rolipram increased levels of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA), which led to the transformation of GLI2FL and GLI3FL transcription factors into repressor forms. This resulted in a reduction in the level of GLI1 transcription factor over time. Rolipram also showed promising results in altering wound healing capability and reducing tumor weight and volume in mice models. The study suggests that rolipram could be an effective inhibitor of the hedgehog signaling pathway in controlling breast cancer progression and metastasis. However, it is important to note that this preprint has not been peer-reviewed. [Extracted from the article]

Cognitive impairment often occurs in Parkinson's disease (PD), but the mechanism of onset remains unknown. Recently, we reported that PD model mice produced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) show facilitation of hippocampal memory extinction, which may be the cause of cognitive impairment in PD. When we examined the cAMP/CREB signaling in the hippocampus, decreased levels of cAMP and phosphorylated CREB were observed in the dentate gyrus (DG) of MPTP-treated mice. Administration of rolipram improved the memory deficits with concomitant recovery of cAMP and phosphorylated CREB levels, suggesting that reduced cAMP/CREB signaling in the DG leads to cognitive impairment in MPTP-treated mice.

Latent toxoplasmosis always has the risk of reactivation leading to significant sequelae. The available medications, for chronic toxoplasmosis, are awfully limited by resistance of Toxoplasmacysts. Therefore, there is a growing necessity for novel therapeutic approaches. Agents increasing cAMP levels and downregulating proinflammatory cytokine could inhibit Toxoplasmaconversion to the bradyzoite stage. This study explores a potential immunomodulatory effect of rolipram, a PDE4 inhibitor, on the course of experimental toxoplasmosis and links this role to deterrence of the resistant chronic phase of the disease.

Objective: Forskolin, an activator of adenylate cyclase and rolipram a selective inhibitor of phosphodiesterase 4, stimulate lipolysis and inhibit body weight increase by increasing cyclic adenosine monophosphate (cAMP) levels. This study was aimed to investigate the effects of them on leptin, resistin and adiponectin levels in diet induced obesity in rats.

Here we show that administration of the phosphodiesterase type 4 (PDE4) inhibitor rolipram into the basolateral complex of the amygdala (BLA) at a specific time interval after training enhances memory consolidation and induces memory persistence for novel object recognition (NOR) in rats. Intra-BLA infusion of rolipram immediately, 1.5 h, or 6 h after training had no effect on retention tested at 1, 7, and 14 d later. However, rolipram infused 3 h post-training promoted memory persistence for up to at least 14 d. The findings suggest that PDE4 inhibition in the BLA can enhance long-term memory formation when induced specifically 3 h after learning.

AbstractBackground/Objective:High cervical spinal cord hemisection interrupts descending respiratory drive from the rostral ventral respiratory group in the medulla to the ipsilateral phrenic motoneurons. Hemisection results in the paralysis of the ipsilateral hemidiaphragm. Chronic administration of rolipram, a specific phosphodiesterase-IV inhibitor, promotes synaptic plasticity and restores phrenic nerve function after a high cervical spinal cord lesion. Here, we test the hypothesis that an acute administration of rolipram will increase spinal and medullary levels of 3′,5′-cyclic adenosine monophosphate (cAMP) and induce phrenic nerve recovery after cervical (C2) spinal cord hemisection.Methods:Male Sprague-Dawley rats were subjected to left C2 hemisection surgery 1 week before experimentation. Bilateral phrenic nerve activity was recorded in anesthetized, vagotomized, and pancuronium paralyzed rats, and rolipram was intravenously applied (2 mg/kg).Results:Intravenous administration of rolipram increased phrenic nerve output in uninjured control and left C2 spinal cord-hemisected rats. In addition, rolipram restored respiratory-related activity to the left phrenic nerve made quiescent by the hemisection. In both uninjured and hemisected rats, rolipram significantly enhanced phrenic inspiratory burst amplitude and burst area compared with predrug values. Also, rolipram concomitantly increased spinal and medullary cAMP.Conclusions:These results suggest that a phosphodiesterase inhibitor capable of elevating cAMP levels can enhance phrenic nerve output and restore respiratory-related phrenic nerve function after high cervical spinal cord injury. Thus, targeting the cAMP signaling cascade can be a useful therapeutic approach in promoting synaptic efficacy and respiratory recovery after cervical spinal cord injury.

Periventricular white matter injury is the leading cause of cerebral palsy in premature infants for which no effective treatments are available. Our previous studies have demonstrated that pharmacological activation of the cAMP response element-binding protein (CREB) signaling pathway, before hypoxic-ischemia protected against neuronal injury in neonatal rats. We examined whether rolipram, a phosphodiesterase type IV inhibitor, treatment after hypoxic-ischemia is protective against white matter injury in neonatal rats. Rats were exposed to hypoxia-ischemia (HI) on P7 and then treated with daily injections of various doses of rolipram (P7–P11). Immunohistochemical staining for myelin basic protein, ED1, glial fibrillary acidic protein, CREB and O1 were examined on P11. We found that the periventricular white matter and deep cortical lesions were exacerbated by rolipram administration after HI injury. The lesions in the rolipram-treated group also showed increased astrogliosis and increased CREB phosphorylation in the activated microglia and astrocytes. Furthermore, the rolipram-posttreated HI group had markedly depleted preoligodendrocytes in the ipsilateral hemisphere, which may be related to decreased preoligodendrocytes proliferation after rolipram treatment per se. These data suggest that rolipram treatment after hypoxic-ischemia is not protective; in contrast, rolipram may exacerbate hypoxic-ischemic white matter injury in neonatal rat brains.

Periventricular white matter injury is the leading cause of cerebral palsy in premature infants for which no effective treatments are available. Our previous studies have demonstrated that pharmacological activation of the cAMP response element-binding protein (CREB) signaling pathway, before hypoxic-ischemia protected against neuronal injury in neonatal rats. We examined whether rolipram, a phosphodiesterase type IV inhibitor, treatment after hypoxic-ischemia is protective against white matter injury in neonatal rats. Rats were exposed to hypoxia-ischemia (HI) on P7 and then treated with daily injections of various doses of rolipram (P7–P11). Immunohistochemical staining for myelin basic protein, ED1, glial fibrillary acidic protein, CREB and O1 were examined on P11. We found that the periventricular white matter and deep cortical lesions were exacerbated by rolipram administration after HI injury. The lesions in the rolipram-treated group also showed increased astrogliosis and increased CREB phosphorylation in the activated microglia and astrocytes. Furthermore, the rolipram-posttreated HI group had markedly depleted preoligodendrocytes in the ipsilateral hemisphere, which may be related to decreased preoligodendrocytes proliferation after rolipram treatment per se. These data suggest that rolipram treatment after hypoxic-ischemia is not protective; in contrast, rolipram may exacerbate hypoxic-ischemic white matter injury in neonatal rat brains.

The aim of this study was to compare the positive inotropic effects of 3 different agents with 3 different mechanisms of actions-levosimendan, rolipram, and dobutamine-on human atrial trabecular muscles. Samples of right atrial appendage (1 cm2, 500-1000 mg) were removed and immersed in preoxygenated and modified Tyrode solution. In oxygenated Tyrode solution, preparations were used to investigate the concentration-effect relationship of levosimendan, dobutamine, and rolipram on percentage developed tension (DT), from 10−9to 10−4M, each concentration for 15 minutes. All 3 agents produced concentration-dependent increments in DT. We found that levosimendan was the most efficacious positive inotropic agent on isolated human atrial trabeculae. Both the sensitivity (pD2) and maximum response (Emax) of human atrial trabeculae to levosimendan (6.711 ± 0.26 and 23.2 ± 2.2 mN, respectively) were significantly greater than those of dobutamine (6.663 ± 0.19 and 17.6 ± 2.8 mN) and rolipram (6.497 ± 0.18 and 15.0 ± 1.0 mN). pD2and Emaxvalues for dobutamine were significantly higher than those for rolipram. It was suggested that because of its potential to enhance cardiac performance without predisposition to calcium-induced arrhythmias, levosimendan might be more useful as a positive inotropic agent in clinical practice.

In this study, we examined whether morphine dependence was inhibited by rolipram, a cyclic AMP selective phosphodiesterase inhibitor in mice, since a role for the cyclic AMP systems in the development of morphine dependence has been reported.Mice, which received morphine (10 mg kg−1s.c.) twice a day for 5 days showed withdrawal syndromes such as jumping, rearing and forepaw tremor following naloxone challenge (5 mg kg−1i.p.) on the 6th day.Such mice exhibited a significant elevation of cyclic AMP levels in the thalamus compared to control mice. However, co‐administration of rolipram (1 mg kg−1i.p.) with morphine for 5 days significantly attenuated the severity of the withdrawal syndrome and the increase in the cyclic AMP levels after the administration of naloxone.In naïve mice, acute morphine treatment (10 mg kg−1s.c.) decreased cyclic AMP levels in the thalamus and cerebral cortex 10 min later. The decrease of cyclic AMP levels induced by acute morphine treatment was blocked by co‐administration of rolipram (1 mg kg−1i.p.). However, acute rolipram did not affect the naloxone‐precipitated morphine withdrawal syndrome.These results suggest that the elevation of the cyclic AMP levels is involved in the development of morphine withdrawal syndrome and that blockade of the morphine‐induced reduction of cyclic AMP levels by chronic rolipram inhibits the development of dependence and the behavioural and biochemical changes induced by naloxone. Furthermore, rolipram may be a useful drug for attenuating the development of morphine dependence.

1 In this study, we examined whether morphine dependence was inhibited by rolipram, a cyclic AMP selective phosphodiesterase inhibitor in mice, since a role for the cyclic AMP systems in the development of morphine dependence has been reported. 2 Mice, which received morphine (10 mg kg−1 s.c.) twice a day for 5 days showed withdrawal syndromes such as jumping, rearing and forepaw tremor following naloxone challenge (5 mg kg−1 i.p.) on the 6th day. 3 Such mice exhibited a significant elevation of cyclic AMP levels in the thalamus compared to control mice. However, co-administration of rolipram (1 mg kg−1 i.p.) with morphine for 5 days significantly attenuated the severity of the withdrawal syndrome and the increase in the cyclic AMP levels after the administration of naloxone. 4 In naïve mice, acute morphine treatment (10 mg kg−1 s.c.) decreased cyclic AMP levels in the thalamus and cerebral cortex 10 min later. The decrease of cyclic AMP levels induced by acute morphine treatment was blocked by co-administration of rolipram (1 mg kg−1 i.p.). However, acute rolipram did not affect the naloxone-precipitated morphine withdrawal syndrome. 5 These results suggest that the elevation of the cyclic AMP levels is involved in the development of morphine withdrawal syndrome and that blockade of the morphine-induced reduction of cyclic AMP levels by chronic rolipram inhibits the development of dependence and the behavioural and biochemical changes induced by naloxone. Furthermore, rolipram may be a useful drug for attenuating the development of morphine dependence. British Journal of Pharmacology (2001) 132, 1111 – 1117

This study investigates the role of adrenal‐derived catecholamines and corticosterone on the inhibition by rolipram, a phosphodiesterase (PDE)‐4 inhibitor, of pulmonary eosinophilia and airway hyperresponsiveness (AHR) in allergic mice.The following experimental groups were studied in mice sensitized and challenged with ovalbumin (OVA): normal, adrenalectomized, propranolol (β‐adrenoceptor antagonist) and metyrapone (corticosterone synthesis inhibitor) treated. These interventions were studied both in the absence and in the presence of rolipram. Eosinophil numbers in the bronchoalveolar lavage (BAL) and AHR to methacholine were measured 24 h after OVA challenge.Treatment of sensitized mice with rolipram (0.3–10 mg kg−1, p.o.), inhibited pulmonary eosinophilia and the AHR to methacholine in OVA‐challenged mice.Adrenalectomy increased the number of eosinophils in the BAL of OVA‐challenged mice but had no effect on AHR to methacholine. Adrenalectomy attenuated both the rolipram‐induced inhibition of BAL eosinophilia and AHR to methacholine in OVA challenged mice. Propranolol (10 mg kg−1, p.o.) had no effect on the inhibition of eosinophilia by rolipram but attenuated the inhibition of AHR to methacholine in OVA challenged mice. On the other hand, metyrapone (10 mg kg−1, p.o.) attenuated the inhibition of eosinophilia by rolipram but had no effect on the inhibition of AHR to methacholine in OVA challenged mice. Metyrapone‐treatment alone increased the number of eosinophils in the BAL of OVA‐challenged mice.These results identify an important role for adrenal‐derived catecholamines and corticosterone on the inhibition of pulmonary eosinophilia and AHR by rolipram in allergic mice.

U937 monocytic cells are shown here to express a range of PDE4, cAMP-specific phosphodiesterase (PDE) isoenzymes: the long isoenzymes, PDE4A4, PDE4D5 and PDE4D3, plus the short isoenzyme, PDE4B2. These isoenzymes provide around 76% of the total cAMP PDE activity of U937 cells. The specific activities of the total PDE4A, PDE4B and PDE4D activities were 0.63±0.09, 8.8±0.2 and 34.4±2.9 pmol/min per mg of protein respectively. The PDE4 selective inhibitor, rolipram, inhibited immunopurified PDE4B and PDE4D activities similarly, with IC50 values of approx. 130 nM and 240 nM respectively. In contrast, rolipram inhibited immunopurified PDE4A activity with a dramatically lower IC50 value of around 3 nM. Rolipram increased phosphorylation of cAMP-response-element-binding protein (CREB) in U937 cells in a dose-dependent fashion, which implied the presence of both high affinity (IC50 value approx. 1 nM) and low affinity (IC50 value approx. 120 nM) components. Rolipram dose-dependently inhibited the interferon-γ (IFN-γ)-stimulated phosphorylation of p38 mitogen-activated protein (MAP) kinase in a simple monotonic fashion with an IC50 value of approx. 290 nM. On this basis, it is suggested that rolipram inhibition of PDE4A4 is involved in regulating CREB phosphorylation but not IFN-γ-stimulated p38 MAP kinase phosphorylation. PDE4A4 was also selectively activated by challenge of U937 cells with either bacterial lipopolysaccharide (LPS) or IFN-γ through a process which was attenuated by both wortmannin and rapamycin. It is proposed that the PDE4A4 isoform is involved in compartmentalized cAMP signalling responses in U937 monocytes.

Brain ischemia stimulates neurogenesis. However, newborn neurons show a progressive decrease in number over time. Under normal conditions, the cAMP-cAMP responsive element binding protein (CREB) pathway regulates the survival of newborn neurons. Constitutive activation of CREB after brain ischemia also stimulates hippocampal neurogenesis. Thus, activation of cAMP-CREB signaling may provide a promising strategy for enhancing the survival of newborn neurons. We examined whether treatment of mice with the phosphodiesterase-4 inhibitor rolipram enhances hippocampal neurogenesis after ischemia.

The cAMP-specific phosphodiesterase (PDE) HSPDE 4A4B(pde46) selectively bound SH3 domains of SRC family tyrosyl kinases. Such an interaction profoundly changed the inhibition of PDE4 activity caused by the PDE4-selective inhibitor rolipram and mimicked the enhanced rolipram inhibition seen for particulate, compared with cytosolic pde46 expressed in COS7 cells. Particulate pde46 co-localized with LYN kinase in COS7 cells. The unique N-terminal and LR2 regions of pde46 contained the sites for SH3 binding. Altered rolipram inhibition was triggered by SH3 domain interaction with the LR2 region. Purified LYN SH3 and human PDE4A LR2 could be co-immunoprecipitated, indicating a direct interaction. Protein kinase A-phosphorylated pde46 remained able to bind LYN SH3. pde46 was found to be associated with SRC kinase in the cytosol of COS1 cells, leading to aberrant kinetics of rolipram inhibition. It is suggested that pde46 may be associated with SRC family tyrosyl kinases in intact cells and that the ensuing SH3 domain interaction with the LR2 region of pde46 alters the conformation of the PDE catalytic unit, as detected by altered rolipram inhibition. Interaction between pde46 and SRC family tyrosyl kinases highlights a potentially novel regulatory system and point of signaling system cross-talk.

Ser-13 and Ser-54 were shown to provide the sole sites for the protein kinase A (PKA)-mediated phosphorylation of the human cAMP-specific phosphodiesterase isoform HSPDE4D3. The ability of PKA to phosphorylate and activate HSPDE4D3 was mimicked by replacing Ser-54 with either of the negatively charged amino acids, aspartate or glutamate, within the consensus motif of RRES54. The PDE4 selective inhibitor rolipram {4-[3-(cyclopentoxy)-4-methoxyphenyl]-2-pyrrolidone} inhibited both PKA-phosphorylated HSPDE4D3 and the Ser-54 → Asp mutant, with an IC50 value that was ∼ 8-fold lower than that seen for the non-PKA-phosphorylated enzyme. Lower IC50 values for inhibition by rolipram were seen for a wide range of non-activated residue 54 mutants, except for those which had side-chains able to serve as hydrogen-bond donors, namely the Ser-54 → Thr, Ser-54 → Tyr and Ser-54 → Cys mutants. The Glu-53 → Ala mutant exhibited an activity comparable with that of the PKA phosphorylated native enzyme and the Ser-54 → Asp mutant but, in contrast to the native enzyme, was insensitive to activation by PKA, despite being more rapidly phosphorylated by this protein kinase. The activated Glu-53 → Ala mutant exhibited a sensitivity to inhibition by rolipram which was unchanged from that of the native enzyme. The double mutant, Arg-51 → Ala/Arg-52 → Ala, showed no change in either enzyme activity or rolipram inhibition from the native enzyme and was incapable of providing a substrate for PKA phosphorylation at Ser-54. No difference in inhibition by dipyridamole was seen for the native enzyme and the Ser-54 → Asp and Ser-54 → Ala mutants. A model is proposed which envisages that phosphorylation by PKA triggers at least two distinct conformational changes in HSPDE4D3; one of these gives rise to enzyme activation and another enhances sensitivity to inhibition by rolipram. Activation of HSPDE4D3 by PKA-mediated phosphorylation is suggested to involve disruption of an ion-pair interaction involving the negatively charged Glu-53. The increase in susceptibility to inhibition by rolipram upon PKA-mediated phosphorylation is suggested to involve the disruption of a hydrogen-bond involving the side-chain hydroxy group of Ser-54.

Summary: The purpose of this study was to determine whether the selective type IV cAMP-phosphodiesterase inhibitor rolipram could reduce the reperfusion injury that occurs during myocardial infarction in the anesthetized dog. This question was tested in pentobarbital-anesthetized dogs subject to 90 min of regional myocardial ischemia and 5 h of reperfusion. Dogs were treated with 1 mg/kg of rolipram (i.v., 15 min before reperfusion) followed by a 1 mg/kg/h infusion over the duration of the 5 h of reperfusion. Rolipram was tested in vitro for efficacy in inhibition of isolated human neutrophil superoxide generation. Rolipram produced significant inhibition of superoxide production over the concentration range of 0.1–100 μM- rolipram when neutrophils were stimulated with a 10 -7Mconcentration of the chemotactic peptide f-Met-Leu-Phe. Rolipram significantly inhibited superoxide generation from human and canine granulocytes in whole blood stimulated by zymosan. Therapeutic concentrations of rolipram in the blood of dogs were achieved during the course of the experiments with a plasma concentration of 0.761 ± 0.095 μg/ml (2.76 ± 0.34 μ- M) at the time of reperfusion, and 0.574 ± 0.098 μg/ml (2.08 ± 0.36 μ M) at the end of the reperfusion period. The relative severity of myocardial ischemia between the two treatment groups was similar as assessed with radiolabeled microsphere measurement of myocardial blood flow. Transmural myocardial blood flows were not significantly different between the two groups after coronary occlusion (control, 0.05 ± 0.01 ml/min/g, n = 6, vs. rolipram, 0.18 ± 0.07 ml/min/g, n = 6; p = 0.48). There was not a significant reduction in the accumulation of neutrophils in the myocardium as assessed by the marker enzyme myeloperoxidase (control, 2.33 ± 1.07 U/g, n = 5, vs. rolipram, 0.54 ± 0.28 U/g, n = 6; p = 0.0548). Myocardial infarct size determined postmortem was not limited by rolipram treatment (control, 31.1 ± 2.6 infarct/ area at risk, n = 6, vs. rolipram, 23.3 ± 7.8 infarct/area at risk, n = 6; p > 0.05). Even though rolipram did not result in statistically significant protection as measured by infarct size, there was a significant attenuation of the “no-reflow” phenomenon with rolipram treatment. Circumflex coronary blood flow declines that were evident in the vehicle-treated group did not occur in the rolipram treatment group. These data indicate that cAMP-phosphodiesterase inhibition with rolipram is not sufficient to provide protection against myocardial reperfusion injury.

A cyclic AMP specific phosphodiesterase (type IV) was purified 450,000‐fold from human peripheral blood mononuclear cells through a sequence of chromatographic steps involving anion exchange, affinity chromatography on a matrix coupled to a derivative of the type IV inhibitor rolipram, and gel filtration. The enzyme showed apparent molecular masses of 70 kDa on gel filtration and 35 kDa on denaturing or native PAGE, indicating a possible dimerization or cleavage under certain conditions. The isoelectric point was 4.6. Kinetic parameters were Km= 2.2μM, Ki= 1.2 μM (rolipram) and νmax= 80 μmol/min per mg protein. The most probable N‐terminal sequence was determined as SLTNTNIPRF, 80% identical to part of the deduced amino acid sequence from cDNA sequences of PDE IVAand PDE IVD.

The effect of the selective type 4 phosphodiesterase (PDE 4) inhibitor rolipram on human eosinophil activation and migration mediated by eotaxin was investigated.Studies were performed with human freshly isolated eosinophils from peripheral blood of healthy donors by a magnetic cell separation (MACS) technique to a purity>99%. To test the effect of rolipram, eosinophils were stimulated with recombinant human eotaxin and the cell surface activation markers CD11b and L‐selectin were analysed by flow cytometry. Furthermore, eotaxin mediated eosinophil migration was measured in a transendothelial chemotaxis assay.Our results indicate that rolipram inhibited eotaxin‐induced CD11b up‐regulation up to 60.6±7.6% at the highest tested dose (10μm), whereas transendothelial chemotaxis was partially inhibited reaching a plateau of approx. 30% at a rolipram concentration of 0.1μm.We conclude that the selective PDE 4 inhibitor rolipram decreases eotaxin mediated eosinophil activation, an observation that may contribute to elucidate the mechanism by which PDE 4 inhibitors reduce antigen‐induced eosinophil infiltration in different animal models of allergic inflammation.

Transfection of COS7 cells with a plasmid encoding the human cyclic AMP-specific PDE4A phosphodiesterase PDE-46 (HSPDE4A4B) led to the expression of a rolipram-inhibited PDE4 activity, which contributed ∼96% of the total COS cell PDE activity. A fusion protein was generated which encompassed residues (788-886) at the extreme C terminus of PDE-46 and was used to generate an antiserum that detected PDE-46 in transfected COS7 cells. Immunoblotting studies identified PDE-46 as a ∼125-kDa species that was associated with both the soluble and particulate fractions. The relative Vmaxof particulate PDE-46 was ∼56% that of cytosolic PDE-46. Particulate PDE-46 was not solubilized using Triton X-100 or high NaCl concentrations. Immunofluorescence analysis by laser scanning confocal microscopy showed that PDE-46 was located at discrete margins of the cell, indicative of association with membrane cortical regions. The human PDE4A species, h6.1 (HSPDE4A4C), which lacks the N-terminal extension of PDE-46, was found as an entirely soluble species when expressed in COS7 cells. h6.1 was shown to have an ∼11-fold higher Vmaxrelative to that of PDE-46. In dose-response studies rolipram inhibited particulate PDE-46 at much lower concentrations (IC50= 0.195 μM) than those needed to inhibit the cytosolic enzyme (IC50= 1.6 μM). The basis of this difference lay in the fact that rolipram served as a simple competitive inhibitor of the cytosol enzyme (Ki= 1.6 μM) but as a partial competitive inhibitor of the particulate enzyme (Ki= 0.037 μM; Ki′ = 2.3 μM). Particulate PDE-46 thus showed a ∼60-fold higher affinity for rolipram than cytosolic PDE-46.

1We have investigated the inhibitory potency of RP 73401, a novel, highly selective and potent inhibitor of cyclic AMP‐specific phosphodiesterase (PDE IV), against partially‐purified PDE isoenzymes from smooth muscle and the particulate PDE IV from guinea‐pig eosinophils. The inhibitory effects of RP 73401 on the generation of superoxide (.O2−), major basic protein (MBP) and eosinophil cationic protein (ECP) from guinea‐pig eosinophils have also been studied.2RP 73401 potently inhibited partially‐purified cyclic AMP‐specific phosphodiesterase (PDE IV) from pig aortic smooth muscle (IC50= 1.2 nM); it was similarly potent against the particulate PDE IV from guinea‐pig peritoneal eosinophils (IC50= 0.7 nM). It displayed at least a 19000 fold selectivity for PDE IV compared to its potencies against other PDE isoenzymes. Rolipram was approximately 2600 fold less potent than RP 73401 against pig aortic smooth muscle PDE IV (IC50= 3162 nM) and about 250 times less potent against eosinophil PDE IV (IC50= 186 nM).3Solubilization of the eosinophil particulate PDE IV increased the potency of rolipram 10 fold but did not markedly affect the potency of RP 73401. A similar (10 fold) increase in the PDE IV inhibitory potency of rolipram, but not RP 73401, was observed when eosinophil membranes were exposed to vanadate/glutathione complex (V/GSH).4Reverse transcription polymerase chain reaction (RT‐PCR), using primer pairs designed against specific sequences in four distinct rat PDE IV subtype cDNA clones (PDE IVA‐D), showed only mRNA for PDE IVDin guinea‐pig eosinophils. PDE IVDwas also the predominant subtype expressed in pig aortic smooth muscle cells.5RP 73401 (Kiapp= 0.4 nM) was 4 fold more potent than (±)‐rolipram (Kiapp=1.7 nM) in displacing [3H]‐(±)‐rolipram from guinea‐pig brain membranes.6In intact eosinophils, RP 73401 potentiated isoprenaline‐induced cyclic AMP accumulation (EC50= 79 nM). RP 73401 also inhibited leukotriene B4‐induced generation of O2−(IC50= 25 nM), and the release of major basic protein (IC50‐ 115 nM) and eosinophil cationic protein (IC50= 7nM). Rolipram was 3–14 times less potent than RP 73401.7Thus RP 73401 is a very potent and selective PDE IV inhibitor which suppresses eosinophil function suggesting that it may be a useful agent for the treatment of inflammatory diseases such as asthma. The greatly different inhibitory potencies of rolipram against PDE IV from smooth muscle and eosinophils (in contrast to the invariable effects of RP 73401) are unlikely to be attributable to diverse PDE IV subtypes but suggest distinct interactions of the two inhibitors with the enzyme. We have investigated the inhibitory potency of RP 73401, a novel, highly selective and potent inhibitor of cyclic AMP‐specific phosphodiesterase (PDE IV), against partially‐purified PDE isoenzymes from smooth muscle and the particulate PDE IV from guinea‐pig eosinophils. The inhibitory effects of RP 73401 on the generation of superoxide (.O2−), major basic protein (MBP) and eosinophil cationic protein (ECP) from guinea‐pig eosinophils have also been studied. RP 73401 potently inhibited partially‐purified cyclic AMP‐specific phosphodiesterase (PDE IV) from pig aortic smooth muscle (IC50= 1.2 nM); it was similarly potent against the particulate PDE IV from guinea‐pig peritoneal eosinophils (IC50= 0.7 nM). It displayed at least a 19000 fold selectivity for PDE IV compared to its potencies against other PDE isoenzymes. Rolipram was approximately 2600 fold less potent than RP 73401 against pig aortic smooth muscle PDE IV (IC50= 3162 nM) and about 250 times less potent against eosinophil PDE IV (IC50= 186 nM). Solubilization of the eosinophil particulate PDE IV increased the potency of rolipram 10 fold but did not markedly affect the potency of RP 73401. A similar (10 fold) increase in the PDE IV inhibitory potency of rolipram, but not RP 73401, was observed when eosinophil membranes were exposed to vanadate/glutathione complex (V/GSH). Reverse transcription polymerase chain reaction (RT‐PCR), using primer pairs designed against specific sequences in four distinct rat PDE IV subtype cDNA clones (PDE IVA‐D), showed only mRNA for PDE IVDin guinea‐pig eosinophils. PDE IVDwas also the predominant subtype expressed in pig aortic smooth muscle cells. RP 73401 (Kiapp= 0.4 nM) was 4 fold more potent than (±)‐rolipram (Kiapp=1.7 nM) in displacing [3H]‐(±)‐rolipram from guinea‐pig brain membranes. In intact eosinophils, RP 73401 potentiated isoprenaline‐induced cyclic AMP accumulation (EC50= 79 nM). RP 73401 also inhibited leukotriene B4‐induced generation of O2−(IC50= 25 nM), and the release of major basic protein (IC50‐ 115 nM) and eosinophil cationic protein (IC50= 7nM). Rolipram was 3–14 times less potent than RP 73401. Thus RP 73401 is a very potent and selective PDE IV inhibitor which suppresses eosinophil function suggesting that it may be a useful agent for the treatment of inflammatory diseases such as asthma. The greatly different inhibitory potencies of rolipram against PDE IV from smooth muscle and eosinophils (in contrast to the invariable effects of RP 73401) are unlikely to be attributable to diverse PDE IV subtypes but suggest distinct interactions of the two inhibitors with the enzyme.

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.

Analyses were done on a human type-IV cyclic AMP (cAMP) phosphodiesterase (hPDE-IVA-h6.1) expressed in an engineered strain of Saccharomyces cerevisiae. This strain (YMS6) expressed soluble PDE activity, together with an insoluble activity which was not released by re-homogenization, treatment with high-ionic-strength solutions or with the detergent Triton X-100. Pellet and soluble PDE activities were typical of type-IV PDE. They were cAMP-specific, insensitive to the addition of either cGMP (1 microM) or Ca2+/calmodulin, and inhibited by rolipram. Thermostability studies showed both activities to decay as single exponentials, indicating the presence of homogeneous PDE protein species in each fraction. Pellet PDE activity was more thermostable than the soluble enzyme. Mg2+ and Mn2+ dose-dependently increased PDE activity and reversed the inactivating effect of EDTA.h6.1 was engineered to express a C-terminal five-histidine motif (h6.1his5). This allowed purification of the PDE to apparent homogeneity in a simple two-step process involving a rolipram affinity column and a Ni2(+)-chelate column. A single monomeric protein of subunit molecular mass approximately 73 kDa and native molecular mass approximately 74 kDa resulted after a approximately 53000-fold purification. This exhibited a Km for cAMP of 8 microM, a true Vmax. of 0.8 mumol of cAMP hydrolysed/min per mg of PDE protein, a kcat. of 3702 s-1, and a value of the specificity constant kcat/Km of 4.6 x 10(8) M-1.s-1, the last implying a diffusion controlled reaction. Rolipram (Ki 0.4 soluble; 0.7 microM pellet) and 3-isobutyl-1-methylxanthine (Ki 15 soluble; 19 microM pellet) served as simple competitive inhibitors for both soluble and pellet forms of h6.1, respectively.

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.

The involvement of rolipram-sensitive phosphodiesterase (PDE IV) in regulation of cardiac contraction was investigated by studying the effect of selective inhibitors (rolipram, denbufylline, Ro 20-1724) on guinea pig left atria contraction. In contrast to milrinone and SK&F 94120 (inhibitors of the cyclic GMP-inhibited PDE, PDE III), (±)-rolipram and denbufylline (0.1-30 µM) did not produce any positive inotropic effect in normal (2.5 m M) or elevated (3-3.2 m M) external CaCl2concentration. In these conditions, Ro 20-1724 produced only a slight but significant increase of contraction over control levels. In the presence of forskolin (an adenylate cyclase activator) or SK&F 94120 (a PDE III inhibitor), which produced an increase of the response to electrical stimulation of ˜10, (±)-rolipram, denbufylline, and Ro 20-1724 all exerted concentration-dependent positive inotropic effects (mean EC50values were 20, 25, and 125 n M, respectively, in the presence of forskolin). Rolipram exhibited stereospecificity the (-)-enantiomer was 10 times more potent than the ()-enantiomer. Neither preincubation of the atria with atenolol nor pretreatment of the guinea pigs with reserpine significantly modified the effect of PDE IV inhibitors obtained in the presence of forskolin. These data show that in the presence of cyclic AMP-dependent positive inotropic agents, PDE IV inhibitors exert a positive inotropic effect which probably does not involve enhanced catecholamine release from sympathetic nerve endings. This suggests that PDE IV may play a role in regulation of cardiac contraction in physiologic conditions in which the sympathetic outflow produces a stimulation of adenylate cyclase in cardiac cells

The effect of a new cardiotonic agent Y-20487 6-(3,6-dihydro-2-oxo-2H-1,3,4-thiadiazin-5-yl)-3,4- dihydro-2(1H)-quinolinone on cyclic AMP levels of rat ventricular cardiomyocytes and the contractile force of papillary muscles was investigated in comparison with selective cyclic AMP phosphodiesterase (PDE) inhibitors, milrinone (PDE-III selective), rolipram and Ro 20-1724 (PDE-IV selective), and a nonselective inhibitor 3-isobutyl-I-methylxanthine (IBMX). Rolipram and Ro 20-1724 did not elicit cyclic AMP accumulation and positive inotropy, but they potentiated the isoproterenol (ISO)-induced cyclic AMP accumulation more effectively than IBMX. Rolipram was more effective than Ro 20-1724 in enhancing ISO-induced cyclic AMP accumulation but was less effective in enhancing the ISO-induced positive inotropic effect, indicating that these agents produce a differential action on cyclic AMP metabolism and inotropy. Milrinone and Y-20487 elicited cyclic AMP accumulation and positive inotropy by themselves. Whereas milrinone scarcely affected the ISO-induced effects, Y-20487 shifted the concentration-response curve for the positive inotropic effect of ISO to the left to the same extent that IBMX did. Y-20487, however, was much less effective than IBMX in enhancing the ISO-induced cyclic AMP accumulation. The present results indicate that in rat ventricular myocardium, PDE-IV may play a crucial role in breakdown of cyclic AMP generated by (3-adrenoceptor stimulation, whereas other types of PDE isoenzymes, including PDE-III, may be responsible for the cyclic AMP accumulation and direct positive inotropic effect induced by PDE inhibitors. Y-20487 may exert a characteristic action different from other agents, probably through inhibition of PDE-III and PDE-IV isoenzymes

Transfection of COS7 cells with a plasmid encoding the human cyclic AMP-specific PDE4A phosphodiesterase PDE-46 (HSPDE4A4B) led to the expression of a rolipram-inhibited PDE4 activity, which contributed approximately 96% of the total COS cell PDE activity. A fusion protein was generated which encompassed residues (788-886) at the extreme C terminus of PDE-46 and was used to generate an antiserum that detected PDE-46 in transfected COS7 cells. Immunoblotting studies identified PDE-46 as a approximately 125-kDa species that was associated with both the soluble and particulate fractions. The relative Vmax of particulate PDE-46 was approximately 56% that of cytosolic PDE-46. Particulate PDE-46 was not solubilized using Triton X-100 or high NaCl concentrations. Immunofluorescence analysis by laser scanning confocal microscopy showed that PDE-46 was located at discrete margins of the cell, indicative of association with membrane cortical regions. The human PDE4A species, h6.1 (HSPDE4A4C), which lacks the N-terminal extension of PDE-46, was found as an entirely soluble species when expressed in COS7 cells. h6.1 was shown to have an approximately 11-fold higher Vmax relative to that of PDE-46. In dose-response studies rolipram inhibited particulate PDE-46 at much lower concentrations (IC50 = 0. 195 microM) than those needed to inhibit the cytosolic enzyme (IC50 = 1.6 microM). The basis of this difference lay in the fact that rolipram served as a simple competitive inhibitor of the cytosol enzyme (Ki = 1.6 microM) but as a partial competitive inhibitor of the particulate enzyme (Ki = 0.037 microM; Ki' = 2.3 microM). Particulate PDE-46 thus showed a approximately 60-fold higher affinity for rolipram than cytosolic PDE-46.

A cyclic AMP specific phosphodiesterase (type IV) was purified 450,000-fold from human peripheral blood mononuclear cells through a sequence of chromatographic steps involving anion exchange, affinity chromatography on a matrix coupled to a derivative of the type IV inhibitor rolipram, and gel filtration. The enzyme showed apparent molecular masses of 70 kDa on gel filtration and 35 kDa on denaturing or native PAGE, indicating a possible dimerization or cleavage under certain conditions. The isoelectric point was 4.6. Kinetic parameters were Km= 2.2μM, Ki= 1.2 μM (rolipram) and ν max= 80 μmol/min per mg protein. The most probable N-terminal sequence was determined as SLTNTNIPRF, 80% identical to part of the deduced amino acid sequence from cDNA sequences of PDE IV Aand PDE IV D.

In multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) the cytokines tumour necrosis factor-α (TNF), lymphotoxin-α (LT), and interferon-gamma (IFN-γ) are of central pathogenetic importance. A therapy capable of stopping neurological deterioration in MS patients is not yet available. Here, we report that rolipram, a selective type IV phosphodiesterase inhibitor, stereospecifically suppresses the production of TNF/LT and less strongly also IFN-γ in human and rat auto-reactive T cells. Moreover, we show that rolipram is an effective treatment for EAE. Rolipram has extensively been studied in humans for the treatment of depression, but has not yet been marketed. The data presented here identify rolipram as potential therapy for multiple sclerosis and provoke the immediate initiation of clinical trials.

Intraperitoneal administration of the phosphodiesterase type 4 (PDE4) inhibitor rolipram (1–30 mg/kg) caused a dose-dependent increase in the circulating levels of both corticosterone and adrenaline in male Balb/c mice. These increases were maximal 0.5–1 h after administration of rolipram and had declined to control levels by 4 h. Rolipram (10 mg/kg i.p.) substantially inhibited the production of tumour necrosis factor α (TNF-α) ex vivo in blood from normal mice but was ineffective in adrenalectomized mice, suggesting that the inhibitory effect of rolipram is dependent on intact adrenal function. The corticosterone antagonist, RU 486, and the β-adrenoceptor antagonist, popranolol, both partially reversed the inhibitory activity of rolipram while the combination of RU 486 and popranolol abrogated the effect of the rolipram to the same degree as adrenalectomy. These data suggest the release of both corticosterone and adrenaline contribute to the ability of rolipram to inhibit TNF-α production in mouse blood ex vivo.

Full-length cDNA for the rat brain rolipram-sensitive cyclic AMP phosphodiesterase (PDE), RD1 was introduced into the expression vector pSVL. COS cells transfected with the recombinant vector pSVL-RD1 exhibited a 30-55% increase in homogenate PDE activity, which was abolished by rolipram (10 microM). Removal of the first 67 nucleotides of the RD1 cDNA yielded a truncated enzyme called Met26-RD1 which lacked the N-terminal first 25 amino acids. Whereas approx. 75% of RD1 activity was membrane-associated, Met26-RD1 activity was found exclusively in the cytosol fraction. Expression of RD1 nearly doubled membrane-associated PDE activity, while expression of Met26-RD1 increased cytosolic activity by approx. 30%. Membrane RD1 activity was found to be primarily associated with the plasma membrane, was not released by either high concentrations of NaCl or by a ‘hypotonic shock’ treatment, but was solubilized with low concentrations of Triton X-100. Phase separation of membrane components with Triton X-114 showed partition of RD1 into both the aqueous and detergent-rich phases, whereas Met26-RD1 partitioned exclusively into the aqueous phase. Both RD1 and Met26-RD1 specifically hydrolysed cyclic AMP; were unaffected by either Ca2+/calmodulin or by low cyclic GMP concentrations; exhibited linear Lineweaver-Burke plots with similar Km values for cyclic AMP (4 microM); both were potently and similarly inhibited by rolipram (Ki approx. 0.5 microM) and were similarly inhibited by cilostamide and 3-isobutyl-1-methylxanthine. Thermal inactivation, at 50 degrees C, showed that while the cytosolic-located fraction of RD1 (t0.5 approx. 3 min) and Met26-RD1 (t0.5 approx 3 min) were similarly thermolabile, membrane-bound RD1 was considerably more thermostable (t0.5 approx. 11 min). Treatment of both cytosolic RD1 and Met26-RD1 with Triton X-100 did not affect their thermostability, but solubilization of membrane RD1 activity with Triton X-100 markedly decreased its thermostability (t0.5 approx. 5 min). The N-terminal domain of RD1 appears not to influence either the substrate specificity or inhibitor sensitivity of this enzyme, but it does contain information which can allow RD1 to become plasma membrane-associated and thereby adopt a conformation which has enhanced thermostability.

The effects of rolipram, a selective cAMP phosphodiesterase inhibitor, on locomotor activity, rearing, and stereotyped behavior (sniffing, repetitive head movements) induced by methamphetamine (MAP) over 1 hour were investigated in rats. Coadministration of rolipram (4 mg/kg IP) significantly attenuated the responses of locomotor activity, rearing and repetitive head movements to MAP (2, 4 or 8 mg/kg IP). Rolipram (0.5, 1, 2, or 4 mg/kg IP) dose-dependently inhibited locomotor hyperactivity and rearing induced by 4 mg/kg of MAP. The rearing was completely inhibited by 4 mg/kg of rolipram, whereas the maximal inhibition of the locomotor hyperactivity was about 50%. However, rolipram did not alter MAP-induced sniffing and repetitive head movements. These results indicate that there is heterogeneity in the response of MAP-induced behavior to rolipram, suggesting that MAP-induced behavioral alteration may be partly regulated by cAMP levels in the brain.Neuropsychopharmacology (1995) 13 33–39.

1We have investigated the effects of the phosphodiesterase (PDE) type IV inhibitor rolipram and of the glucocorticoid methylprednisolone on the induction of tumour necrosis factor alpha (TNF‐α) mRNA and protein in brains of rats after peripheral administration of lipopolysaccharide (LPS).2After intravenous administration of LPS, a similar time‐dependent induction of both TNF‐α mRNA and protein was observed in rat brain. Peak mRNA and protein levels were found 7 h after administration of LPS.3In situhybridization experiments with a specific antisense TNF‐α riboprobe suggested that the cells responsible for TNF‐α production in the brain were microglia.4Intraperitoneal administration of methylprednisolone inhibited the induction of TNF‐α protein in a dose‐dependent manner. A maximal inhibition of TNF‐α protein production by 42.9±10.2% was observed at a dose regimen consisting of two injections of each 30 mg kg−1methylprednisolone.5Intraperitoneal administration of rolipram also inhibited the induction of TNF‐α protein in a dose‐dependent manner. The maximal inhibition of TNF‐α protein production was 96.1±12.2% and was observed at a dose regimen of three separate injections of each 3 mg kg−1rolipram.6In situhybridization experiments showed that the level of TNF‐α mRNA induced in rat brain by LPS challenge was reduced by intraperitoneal administration of methylprednisolone (2×15 mg kg−1) and of rolipram (3×3 mg kg−1).7We suggest that peripheral administration of LPS induces a time‐dependent expression of TNF‐α in rat brain, presumably in microglial cells, and that methylprednisolone and rolipram inhibit LPS‐induced expression of TNF‐α in these cells via a decrease of TNF‐α mRNA stability and/or TNF‐α gene transcription.

We have investigated the effects of the selective phosphodiesterase (PDE) type 4 inhibitor, rolipram (0.01–1 μM) on cytosolic Ca2+fluxes in FMLP‐activated human neutrophils, as well as on superoxide production by, and release of elastase from, these cells.Cytosolic Ca2+fluxes were measured by use of fura‐2 spectrofluorimetry in combination with a radiometric procedure that enables distinction between net efflux and influx of the cation. Superoxide production and elastase release were measured by lucigenin‐enhanced chemiluminescence and a colorimetric procedure, respectively.Pretreatment of neutrophils with rolipram did not affect the FMLP‐activated release of Ca2+from intracellular stores, but was associated with dose‐related acceleration of the rate of decline in fura‐2 fluorescence and with decreased efflux, as well as store‐operated influx of 45Ca2+, indicative of enhancement of resequestration of the cation by the endo‐membrane Ca2+‐ATPase.Inhibition of superoxide production and elastase release was observed at concentrations of rolipram which accelerated the clearance of Ca2+from the cytosol of FMLP‐activated neutrophils.These effects of rolipram on FMLP‐activated Ca2+fluxes, superoxide generation and elastase release were mimicked by pretreatment of neutrophils with dibutyryl cyclic AMP (0.5–4 mM), while theophylline (10–150 μM), a non‐specific PDE inhibitor, as well as the β2‐agonist, salbutamol, were less effective.We conclude that rolipram deactivates FMLP‐stimulated human neutrophils by enhancement of cyclic AMP‐dependent resequestration of cytosolic Ca2+.

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.

We have isolated cDNA clones representing cyclic AMP (cAMP)-specific phosphodiesterases (PDEases) from a human monocyte cDNA library. One cDNA clone (hPDE-1) defines a large open reading frame of ca. 2.1 kilobases, predicting a 686-amino-acid, ca. 77-kilodalton protein which contains significant homology to both rat brain and Drosophila cAMP PDEases, especially within an internal conserved domain of ca. 270 residues. Amino acid sequence divergence exists at the NH2 terminus and also within a 40- to 100-residue domain near the COOH-terminal end. hPDE-1 hybridizes to a major 4.8-kilobase mRNA transcript from both human monocytes and placenta. The coding region of hPDE-1 was engineered for expression in COS-1 cells, resulting in the overproduction of cAMP PDEase activity. The hPDE-1 recombinant gene product was identified as a low-Km cAMP phosphodiesterase on the basis of several biochemical properties including selective inhibition by the antidepressant drug rolipram. Known inhibitors of other PDEases (cGMP-specific PDEase, cGMP-inhibited PDEase) had little or no effect on the hPDE-1 recombinant gene product. Human genomic Southern blot analysis suggests that this enzyme is likely to be encoded by a single gene. The presence of the enzyme in monocytes may be important for cell function in inflammation. Rolipram sensitivity, coupled with homology to the Drosophila cAMP PDEase, which is required for learning and memory in flies, suggests an additional function for this enzyme in neurobiochemistry.

Biochemical and immunofluorescence analyses revealed that phosphodiesterase variants encoded by thePDE4Dgene are targeted to discrete subcellular structures. In quiescent FRTL-5 thyroid cells, the rolipram-sensitive phosphodiesterase (PDE) activity (cAMP-PDE) was recovered both in the soluble and particulate fractions of the homogenate. Although an immunoreactive 93-kDa PDE (PDE4D3) variant was recovered in both compartments, a 105-kDa variant with the properties of PDE4D4 was recovered mostly in the particulate fraction. The PDE4D3 form was readily solubilized with nonionic detergents. Conversely, the PDE4D4 form required buffers containing ionic detergents for extraction, suggesting that different mechanisms target these variants to insoluble structures. A 15-min stimulation with thyroid-stimulating hormone (TSH) led to an activation of the cAMP-PDE in both compartments and was correlated with a shift in electrophoretic mobility of the PDE4D3 polypeptide. Long term incubation with TSH caused an increase of the PDE activity in the soluble fraction and the appearance of a 68-kDa immunoreactive polypeptide with the properties of PDE4D2. Immunofluorescence analysis showed, in addition to diffuse staining, a signal localized on regions adjacent to the plasma membrane on cytoskeletal structures and in a perinuclear region of quiescent cells. Long term incubation with TSH caused an increase in the immunofluorescence signal in the soluble compartment. These data demonstrate that three PDE4D splicing variants are targeted to discrete subcellular compartments and that hormones cause the activation of these isoforms in a temporally and spatially dependent manner.

Biochemical and immunofluorescence analyses revealed that phosphodiesterase variants encoded by the PDE4D gene are targeted to discrete subcellular structures. In quiescent FRTL-5 thyroid cells, the rolipram-sensitive phosphodiesterase (PDE) activity (cAMP-PDE) was recovered both in the soluble and particulate fractions of the homogenate. Although an immunoreactive 93-kDa PDE (PDE4D3) variant was recovered in both compartments, a 105-kDa variant with the properties of PDE4D4 was recovered mostly in the particulate fraction. The PDE4D3 form was readily solubilized with nonionic detergents. Conversely, the PDE4D4 form required buffers containing ionic detergents for extraction, suggesting that different mechanisms target these variants to insoluble structures. A 15-min stimulation with thyroid-stimulating hormone (TSH) led to an activation of the cAMP-PDE in both compartments and was correlated with a shift in electrophoretic mobility of the PDE4D3 polypeptide. Long term incubation with TSH caused an increase of the PDE activity in the soluble fraction and the appearance of a 68-kDa immunoreactive polypeptide with the properties of PDE4D2. Immunofluorescence analysis showed, in addition to diffuse staining, a signal localized on regions adjacent to the plasma membrane on cytoskeletal structures and in a perinuclear region of quiescent cells. Long term incubation with TSH caused an increase in the immunofluorescence signal in the soluble compartment. These data demonstrate that three PDE4D splicing variants are targeted to discrete subcellular compartments and that hormones cause the activation of these isoforms in a temporally and spatially dependent manner.

Our previous work demonstrated that bacterial lipopolysaccharide (LPS), administered by aerosol, induced tumour necrosis factor (TNF‐α) synthesis leading to the infiltration of neutrophils into mice lungs. The treatment of animals with prostaglandin E2or dibutyryl cyclic AMP impaired both processes. In this study, the target cell for LPS and the modulation by cyclic AMP of TNF‐α production and neutrophil recruitment were investigated.One hour after inhalation of 2 ml of 0.3 mg ml−1LPS, TNF‐α levels measured by an ELISA method increased in the bronchoalveolar lavage fluid (BALF) of BALB/c mice, reaching a maximal level 3 h after inhalation. The immunocytochemistry assay demonstrated that 1 h after inhalation, 21.2% of alveolar macrophages collected in the BALF were immunopositive for TNF‐α.When mice were pretreated, i.p., with 20 mg kg−1rolipram, a selective inhibitor of phosphodiesterase type 4, TNF‐α levels in the BALF were significantly reduced and only 7.3% of alveolar macrophages were immunopositive for TNF‐α.Alveolar macrophages from rolipram‐treated mice collected 30 min after inhalation of LPS had a significant increase in the intracellular concentrations of cyclic AMP. This was accompanied by a marked reduction of TNF‐α levels in the BALF that were associated with a suppression of TNF‐α mRNA expression.Systemic treatment with 20 mg kg−1rolipram almost completely inhibited the LPS‐induced neutrophil recruitment, whereas it did not significantly reduce the recruitment induced by rmTNF‐α.Our results indicate that alveolar macrophages may be the target cells for both the induction and control of the lung inflammatory response to LPS. They also suggest that systemic treatment with cyclic AMP‐elevating agents may be useful to control local inflammation resulting from inhalation of bacterial endotoxin.