RESUMEN
Annexin A1 (AnxA1) is a glucocorticoid-regulated protein known for its anti-inflammatory and pro-resolving effects. We have shown previously that the cAMP-enhancing compounds rolipram (ROL; a PDE4 inhibitor) and Bt2cAMP (a cAMP mimetic) drive caspase-dependent resolution of neutrophilic inflammation. In this follow-up study, we investigated whether AnxA1 could be involved in the pro-resolving properties of these compounds using a model of LPS-induced inflammation in BALB/c mice. The treatment with ROL or Bt2cAMP at the peak of inflammation shortened resolution intervals, improved resolution indices, and increased AnxA1 expression. In vitro studies showed that ROL and Bt2cAMP induced AnxA1 expression and phosphorylation, and this effect was prevented by PKA inhibitors, suggesting the involvement of PKA in ROL-induced AnxA1 expression. Akin to these in vitro findings, H89 prevented ROL- and Bt2cAMP-induced resolution of inflammation, and it was associated with decreased levels of intact AnxA1. Moreover, two different strategies to block the AnxA1 pathway (by using N-t-Boc-Met-Leu-Phe, a nonselective AnxA1 receptor antagonist, or by using an anti-AnxA1 neutralizing antiserum) prevented ROL- and Bt2cAMP-induced resolution and neutrophil apoptosis. Likewise, the ability of ROL or Bt2cAMP to induce neutrophil apoptosis was impaired in AnxA-knock-out mice. Finally, in in vitro settings, ROL and Bt2cAMP overrode the survival-inducing effect of LPS in human neutrophils in an AnxA1-dependent manner. Our results show that AnxA1 is at least one of the endogenous determinants mediating the pro-resolving properties of cAMP-elevating agents and cAMP-mimetic drugs.
Asunto(s)
Anexina A1/agonistas , Bucladesina/uso terapéutico , AMP Cíclico/agonistas , Infiltración Neutrófila/efectos de los fármacos , Inhibidores de Fosfodiesterasa 4/uso terapéutico , Pleuresia/tratamiento farmacológico , Rolipram/uso terapéutico , Animales , Anexina A1/antagonistas & inhibidores , Anexina A1/genética , Anexina A1/metabolismo , Apoptosis/efectos de los fármacos , Bucladesina/antagonistas & inhibidores , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Lipopolisacáridos/toxicidad , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Inhibidores de Fosfodiesterasa 4/química , Fosforilación/efectos de los fármacos , Pleuresia/inmunología , Pleuresia/metabolismo , Pleuresia/patología , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Células RAW 264.7 , Rolipram/antagonistas & inhibidoresRESUMEN
Chronic challenge of cyclic AMP phosphodiesterase-4A4 (PDE4A4) with certain PDE4 selective inhibitors causes it to reversibly form intracellular aggregates that are not membrane-encapsulated. These aggregates are neither stress granules (SGs) nor processing bodies (PBs) as they contain neither PABP-1 nor Dcp1a, respectively. However, the PDE4 inhibitor rolipram decreases arsenite-induced SGs and increases the amount of PBs, while arsenite challenge ablates rolipram-induced PDE4A4 aggregates. PDE4A4 aggregates are neither autophagic vesicles (autophagosomes) nor aggresomes, although microtubule disruptors ablate PDE4A4 aggregate formation. PDE4A4 constitutively co-immunoprecipitates with p62 protein (sequestosome1, SQSTM1), which locates to both PDE4A4 aggregates and autophagosomes in cells constitutively challenged with rolipram. The mTor inhibitor, rapamycin, activates autophagy, prevents PDE4A4 from forming intracellular aggregates and triggers the loss of bound p62 from PDE4A4. siRNA-mediated knockdown of p62 attenuates PDE4A4 aggregate formation. The p62-binding protein, light chain 3 (LC3), is not found in PDE4A4 aggregates. Blockade of proteasome activity and activation of autophagy with MG132 both increases the level of ubiquitinated proteins found associated with PDE4A4 and inhibits PDE4A4 aggregate formation. Activation of autophagy with either thapsigargin or ionomycin inhibits PDE4A4 aggregate formation. Inhibition of autophagy with either wortmannin or LY294002 activates PDE4A4 aggregate formation. The protein kinase C inhibitors, RO 320432 and GO 6983, and the ERK inhibitors UO 126 and PD 98059 all activated PDE4A4 aggregate formation, whilst roscovitine, thalidomide and the tyrosine kinase inhibitors, genistein and AG17, all inhibited this process. We suggest that the fate of p62-containing protein aggregates need not necessarily be terminal, through delivery to autophagic vesicles and aggresomes. Instead, we propose a novel regulatory mechanism where a sub-population of p62-containing protein aggregates would form in a rapid, reversible manner so as to sequester specific cargo away from their normal, functionally important site(s) within the cell. Thus an appropriate conformational change in the target protein would confer reversible recruitment into a sub-population of p62-containing protein aggregates and so provide a regulatory function by removing these cargo proteins from their functionally important site(s) in a cell.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/análisis , Autofagia , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/análisis , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Inhibidores de Cisteína Proteinasa/farmacología , Gránulos Citoplasmáticos/enzimología , Vesículas Citoplasmáticas/enzimología , Células HeLa , Humanos , Leupeptinas/farmacología , Inhibidores de Proteasoma , Rolipram/antagonistas & inhibidores , Rolipram/farmacología , Proteína Sequestosoma-1 , UbiquitinaciónRESUMEN
The adenosine A2A receptor agonist CGS21680 (50, 100 and 200 microg/ml), the phosphodiserease type IV (PDE IV) inhibitor Rolipram (50, 100 and 200 microg/ml) and, ATP-sensitive K+ channels activator Cromakalim (30 and 40 microg/ml), when added separately, inhibit oxygen free radicals production from isolated human polymorphoneuclear leukocytes (PMNLs), stimulated with phorbol myristate acetate (PMA), in a dose dependent manner. When both CGS21680 and Rolipram were combined, in vitro, the inhibitory effect on PMNLs free radicals production was synergistic. On the other hand, when both the ATP-sensitive K+ channels opener (KATP) Cromakalim and the type IV PDE inhibitor Rolipram were combined, produced negative synergism (the inhibitory effect of both drugs disappeared). Furthermore, CGS21680, Rolipram, Cromakalim and Forskolin produced no significant inhibitory effect on PMNLs aggregation when added separately. But when various combinations of the above drugs were used, produced significant inhibition of aggregation. Only CGS21680 exhibited a scavenging effect on free radicals production. From the above results, combination of adenosine A2A agonists and type IV PDE inhibitors could serve as potentially novel anti-inflammatory drugs. Furthermore, ATP-sensitive K+ channels activators should be considered for further investigation as anti-inflammatory drug.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Agonistas del Receptor de Adenosina A2 , Adenosina/análogos & derivados , Sinergismo Farmacológico , Radicales Libres/metabolismo , Neutrófilos/citología , Canales de Potasio de Rectificación Interna/farmacología , Adenosina/farmacología , Agregación Celular/efectos de los fármacos , Agregación Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cromakalim/antagonistas & inhibidores , Cromakalim/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Relación Dosis-Respuesta a Droga , Radicales Libres/antagonistas & inhibidores , Humanos , Mediciones Luminiscentes/métodos , Luminol/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Fenetilaminas/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Especies Reactivas de Oxígeno/metabolismo , Receptor de Adenosina A2A/efectos de los fármacos , Estallido Respiratorio/fisiología , Rolipram/antagonistas & inhibidores , Rolipram/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
1. The mechanisms and receptors involved in the vasoactive intestinal peptide (VIP)- and pituitary adenylate cyclase-activating polypeptide (PACAP)-induced relaxations of the pig intravesical ureter were investigated. 2. VIP, PACAP 38 and PACAP 27 concentration-dependently relaxed U46619-contracted ureteral strips with a similar potency. [Ala(11,22,28)]-VIP, a VPAC(1) agonist, showed inconsistent relaxations. 3. The neuronal voltage-gated Ca(2+) channel inhibitor, omega-conotoxin GVIA (omega-CgTX, 1 microm), reduced the VIP relaxations. Urothelium removal or blockade of capsaicin-sensitive primary afferents, nitric oxide (NO) synthase and guanylate cyclase with capsaicin (10 microm), N(G)-nitro-l-arginine (l-NOARG, 100 microm) and 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 microm), respectively, did not change the VIP relaxations. However, the PACAP 38 relaxations were reduced by omega-CgTX, capsaicin, l-NOARG and ODQ. 4. The VIP and VIP/PACAP receptor antagonists, [Lys(1), Pro(2,5), Arg(3,4), Tyr(6)]-VIP (1 microm) and PACAP (6-38) (0.4 microm), inhibited VIP and VIP and PACAP 38, respectively, relaxations. 5. The nonselective and large-conductance Ca(2)-activated K(+) channel blockers, tetraethylammonium (3 mm) and charybdotoxin (0.1 microm), respectively, and neuropeptide Y (0.1 microm) did not modify the VIP relaxations. The small-conductance Ca(2)-activated K(+) channel blocker apamin (1 microm) did not change the PACAP 27 relaxations. 6. The cAMP-dependent protein kinase A (PKA) blocker, 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate (Rp-8-CPT-cAMPS, 100 microm), reduced VIP relaxations. The phosphodiesterase 4 inhibitor rolipram and the adenylate cyclase activator forskolin relaxed ureteral preparations. The rolipram relaxations were reduced by Rp-8-CPT-cAMPS. Forskolin (30 nm) evoked a potentiation of VIP relaxations. 7. These results suggest that VIP and PACAP relax the pig ureter through smooth muscle receptors, probably of the VPAC(2) subtype, linked to a cAMP-PKA pathway. Neuronal VPAC receptors localized at motor nerves and PAC(1) receptors placed at sensory nerves and coupled to NO release, seem also to be involved in the VIP and PACAP 38 relaxations.
Asunto(s)
AMP Cíclico/análogos & derivados , Neuropéptidos/farmacología , Fragmentos de Péptidos/farmacología , Receptores de Péptido Intestinal Vasoactivo/fisiología , Células Receptoras Sensoriales/fisiología , Uréter/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Apamina/farmacología , Capsaicina/farmacología , Caribdotoxina/administración & dosificación , Colforsina/farmacología , AMP Cíclico/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Guanilato Ciclasa/farmacología , Masculino , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , NG-Nitroarginina Metil Éster/farmacología , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/fisiología , Neuropéptido Y/fisiología , Neuropéptidos/antagonistas & inhibidores , Óxido Nítrico Sintasa/farmacología , Oxadiazoles/farmacología , Fragmentos de Péptidos/antagonistas & inhibidores , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Canales de Potasio Calcio-Activados/fisiología , Quinoxalinas/farmacología , Receptores de Péptido Relacionado con el Gen de Calcitonina , Receptores de Péptidos/antagonistas & inhibidores , Receptores de Péptidos/efectos de los fármacos , Rolipram/antagonistas & inhibidores , Rolipram/farmacología , Porcinos , Tetraetilamonio/administración & dosificación , Uréter/citología , Uréter/lesiones , Péptido Intestinal Vasoactivo/antagonistas & inhibidores , omega-Conotoxina GVIA/farmacologíaRESUMEN
Piclamilast is a type 4 phosphodiesterase (PDE4) inhibitor with equal affinity for the high-affinity rolipram binding site (HARBS) and low-affinity rolipram binding site (LARBS). The binding of [(3)H]piclamilast to preparations of rat brain and peripheral tissue was investigated and compared with that of [(3)H]rolipram. [(3)H]piclamilast binding was high-affinity, saturable, reversible, and partially Mg(2+)-dependent. Binding was detected both to membrane and soluble fractions, with K(d) values of 3.1 and 4.5 nM, respectively. The B(max) values for [(3)H]piclamilast were about 1.5-fold greater than that of [(3)H]rolipram binding, suggesting that [(3)H]piclamilast, but not [(3)H]rolipram, binds to LARBS as well as the HARBS. The HARBS was present in all the brain regions examined, but not in peripheral tissues. All PDE4 inhibitors tested were potent competitors for [(3)H]piclamilast binding; the competition curves for rolipram, desmethylpiclamilast, ICI 63,197, and Ro 20-1724 were better described by a two-site model, while the competition curves for piclamilast, cilomilast, roflumilast, and CDP 840 were adequately described by a one-site model. Inhibitors of other PDE families were much less potent. The inhibition of [(3)H]piclamilast was further tested in the presence of 1 microM rolipram to isolate the LARBS. Under this condition, the competition curves for all the inhibitors were adequately described by a one-site model, with K(i) values close to that for the LARBS. The results indicated that [(3)H]piclamilast is a useful tool to directly study inhibitor interaction with the HARBS and the LARBS in rat brain.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Benzamidas/metabolismo , Inhibidores de Fosfodiesterasa/metabolismo , Piridinas/metabolismo , Rolipram/metabolismo , Animales , Benzamidas/antagonistas & inhibidores , Sitios de Unión/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Corteza Cerebral/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Citosol/metabolismo , Técnicas In Vitro , Magnesio/metabolismo , Masculino , Membranas/metabolismo , Piridinas/antagonistas & inhibidores , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Rolipram/antagonistas & inhibidoresRESUMEN
Stimulation of N-methyl-D-aspartate (NMDA) receptors on neurons activates both cAMP and cGMP signaling pathways. Experiments were carried out to determine which phosphodiesterase (PDE) families are involved in the hydrolysis of the cyclic nucleotides formed via this mechanism, using primary neuronal cultures prepared from rat cerebral cortex and hippocampus. The nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) potentiated the ability of NMDA to increase cAMP and cGMP. However, among the family-selective inhibitors, only the PDE4 inhibitor rolipram enhanced the ability of NMDA to increase cAMP in the neurons. In contrast, only the PDE2 inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) enhanced the ability of NMDA to increase cGMP. Neither adenosine nor an adenosine deaminase inhibitor mimicked the effect of EHNA; this suggests that EHNA's inhibition of PDE2, not its effects on adenosine metabolism, mediates its effects on NMDA-stimulated cGMP concentrations. The PDE inhibitor-augmented effects of NMDA on cAMP and cGMP formation were antagonized by 5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate (MK-801), verifying NMDA receptor mediation. In contrast, only NMDA-mediated cGMP formation was affected by altering either nitric oxide signaling or guanylyl cyclase; this suggests that NMDA-induced changes in cAMP are not secondary to altered cGMP concentrations. Overall, the present findings indicate that cAMP and cGMP formed in neurons as a result of NMDA receptor stimulation are hydrolyzed by PDE4 and PDE2, respectively. Selective inhibitors of the two PDE families will differentially affect the functional consequences of activation of these two signaling pathways by NMDA receptor stimulation.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Adenina/análogos & derivados , Corteza Cerebral/enzimología , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Hipocampo/enzimología , Neuronas/enzimología , Hidrolasas Diéster Fosfóricas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Adenina/antagonistas & inhibidores , Adenina/farmacología , Adenosina/farmacología , Animales , Células Cultivadas , Corteza Cerebral/citología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Maleato de Dizocilpina/farmacología , Inhibidores Enzimáticos/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Guanilato Ciclasa/antagonistas & inhibidores , Hipocampo/citología , N-Metilaspartato/antagonistas & inhibidores , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Inhibidores de Fosfodiesterasa/farmacología , Embarazo , Ratas , Ratas Sprague-Dawley , Rolipram/antagonistas & inhibidores , Rolipram/farmacologíaRESUMEN
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 (beta-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.