RESUMEN
The pain associated with lancing can be a significant barrier to self-monitoring of blood glucose (SMBG). The OneTouch® Delica™ lancing device contains features to reduce lancing pain, including improved lancet control and stability, reduced vibration, and a thinner, 33-gauge lancet. This 2-visit, randomized controlled trial assessed perceived pain of lancing with the OneTouch® Delica™ compared with 4 other common lancing devices: OneTouch® Comfort™, ACCU-CHEK® Softclix, ACCU-CHEK® Multiclix, and Ascensia® Microlet™2. Two hundred patients with type 1 or type 2 diabetes mellitus were assigned to the OneTouch® Delica™ and also randomized to 1 of the 4 comparator devices (n=50 per device pair). At visit 1, we determined the minimum depth settings required to produce≥1 µL of fingertip blood for each patient with each device. At visit 2, patients lanced their fingertips with the devices at the predetermined depths and used a 150-mm visual analog scale (VAS) to rate lancing pain relative to their "usual pain" associated with SMBG. The VAS ranged from "much less painful" (0 mm) to "much more painful" (150 mm), with the midpoint (75 mm) labeled as "usual pain." Fingertip pain scores from patients using OneTouch® Delica™ were significantly lower than those obtained using OneTouch® Comfort™, ACCU-CHEK® Multiclix, and Ascensia® Microlet™2. Pain scores for OneTouch® Delica™ and ACCU-CHEK® Softclix were not significantly different. In conclusion, OneTouch® Delica™ was either less painful or no different than the comparator devices when used for fingertip lancing. Innovative lancing devices that cause less pain may improve compliance and persistence with prescribed SMBG.
Asunto(s)
Automonitorización de la Glucosa Sanguínea/instrumentación , Adolescente , Adulto , Anciano , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 2/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Dolor/etiología , Dimensión del Dolor , Adulto JovenRESUMEN
cAMP-specific PDE (phosphodiesterase) 4 isoforms underpin compartmentalized cAMP signalling in mammalian cells through targeting to specific signalling complexes. Their importance is apparent as PDE4 selective inhibitors exert profound anti-inflammatory effects and act as cognitive enhancers. The p38 MAPK (mitogen-activated protein kinase) signalling cascade is a key signal transduction pathway involved in the control of cellular immune, inflammatory and stress responses. In the present study, we show that PDE4A5 is phosphorylated at Ser147, within the regulatory UCR1 (ultraconserved region 1) domain conserved among PDE4 long isoforms, by MK2 (MAPK-activated protein kinase 2, also called MAPKAPK2). Phosphorylation by MK2, although not altering PDE4A5 activity, markedly attenuates PDE4A5 activation through phosphorylation by protein kinase A. This modification confers the amplification of intracellular cAMP accumulation in response to adenylate cyclase activation by attenuating a major desensitization system to cAMP. Such reprogramming of cAMP accumulation is recapitulated in wild-type primary macrophages, but not MK2/3-null macrophages. Phosphorylation by MK2 also triggers a conformational change in PDE4A5 that attenuates PDE4A5 interaction with proteins whose binding involves UCR2, such as DISC1 (disrupted in schizophrenia 1) and AIP (aryl hydrocarbon receptor-interacting protein), but not the UCR2-independent interacting scaffold protein ß-arrestin. Long PDE4 isoforms thus provide a novel node for cross-talk between the cAMP and p38 MAPK signalling systems at the level of MK2.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Especificidad por SustratoRESUMEN
Clearance of fibrin through proteolytic degradation is a critical step of matrix remodeling that contributes to tissue repair in a variety of pathological conditions, such as stroke, atherosclerosis, and pulmonary disease. However, the molecular mechanisms that regulate fibrin deposition are not known. Here, we report that the p75 neurotrophin receptor (p75(NTR)), a TNF receptor superfamily member up-regulated after tissue injury, blocks fibrinolysis by down-regulating the serine protease, tissue plasminogen activator (tPA), and up-regulating plasminogen activator inhibitor-1 (PAI-1). We have discovered a new mechanism in which phosphodiesterase PDE4A4/5 interacts with p75(NTR) to enhance cAMP degradation. The p75(NTR)-dependent down-regulation of cAMP results in a decrease in extracellular proteolytic activity. This mechanism is supported in vivo in p75(NTR)-deficient mice, which show increased proteolysis after sciatic nerve injury and lung fibrosis. Our results reveal a novel pathogenic mechanism by which p75(NTR) regulates degradation of cAMP and perpetuates scar formation after injury.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Fibrosis , Receptor de Factor de Crecimiento Nervioso/fisiología , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Animales , Cicatriz/etiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Fibrinólisis , Regulación de la Expresión Génica , Ratones , Ratones Noqueados , Inhibidor 1 de Activador Plasminogénico/genética , Nervio Ciático/lesiones , Heridas y LesionesRESUMEN
PDE4A11 is a novel cAMP-specific phosphodiesterase that is conserved in humans, mouse, rat, pig, and bat. Exon-1(4A11) encodes its unique, 81 amino acid N-terminal region. Reverse-transcriptase polymerase chain reaction performed across the splice junction, plus identification of expressed sequence tags, identifies PDE4A11 as a long isoform possessing UCR1 and UCR2 regulatory domains. Transcript analysis shows that PDE4A11 is widely expressed compared with PDE4A10 and PDE4A4B long isoforms. Truncation analysis identifies a putative promoter in a 250-base pair region located immediately upstream of the start site in Exon-1(4A11). Recombinant PDE4A11, expressed in COS-7 cells, is a 126-kDa protein localized predominantly around the nucleus and in membrane ruffles. PDE4A11 exhibits a K(m) for cAMP hydrolysis of 4 microM, with relative V(max) similar to that of PDE4A10 and PDE4A4B. PDE4A11 is dose-dependently inhibited by rolipram, 4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone (Ro 20-1724), cilomilast, roflumilast, and denbufylline, with IC(50) values of 0.7, 0.9, 0.03, 0.004, and 0.3 microM, respectively. Soluble and particulate PDE4A11 exhibit distinct rates of thermal inactivation (55 degrees C; T((0.5)) = 2.5 and 4.4 min, respectively). Elevating cAMP levels in COS-7 cells activates PDE4A11 concomitant with its phosphorylation at Ser119 by protein kinase A (PKA). PDE4A11 differs from PDE4A4 in sensitivity to cleavage by caspase-3, interaction with LYN SH3 domain, redistribution upon long-term rolipram challenge, and sensitivity to certain PDE4 inhibitors. PDE4A11, PDE4A10, and PDE4A4 all can interact with betaarrestin. PDE4A11 is a novel, widely expressed long isoform that is activated by PKA phosphorylation and shows a distinct intracellular localization, indicating that it may contribute to compartmentalized cAMP signaling in cells in which it is expressed.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/biosíntesis , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Regulación Enzimológica de la Expresión Génica/fisiología , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Chlorocebus aethiops , AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Líquido Intracelular/enzimología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/biosíntesis , Isoenzimas/genética , Ratones , Datos de Secuencia Molecular , Inhibidores de Fosfodiesterasa/farmacología , Transducción de SeñalRESUMEN
The cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4A5 interacted with the immunophilin XAP2 in a yeast two-hybrid assay. The interaction was confirmed in biochemical pull-down analyses. The interaction was specific, in that PDE4A5 did not interact with the closely related immunophilins AIPL1, FKBP51, or FKBP52. XAP2 also did not interact with other PDE4A isoforms or typical isoforms from the three other PDE4 subfamilies. Functionally, XAP2 reversibly inhibited the enzymatic activity of PDE4A5, increased the sensitivity of PDE4A5 to inhibition by the prototypical PDE4 inhibitor 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (rolipram) and attenuated the ability of cAMP-dependent protein kinase to phosphorylate PDE4A5 in intact cells. XAP2 maximally inhibited PDE4A5 by approximately 60%, with an IC50 of 120 nm, and reduced the IC50 for rolipram from 390 nm to 70-90 nm. Co-expression of XAP2 and PDE4A5 in COS7 cells showed that they could be co-immunoprecipitated and also reduced both the enzymatic activity of PDE4A5 and its IC50 for rolipram. Native XAP2 and PDE4A5 could be co-immunoprecipitated from the brain. The isolated COOH-terminal half of XAP2 (amino acids 170-330), containing its tetratricopeptide repeat domain, but not the isolated NH2-terminal half (amino acids 1-169), containing the immunophilin homology region, similarly reduced PDE4A5 activity and its IC50 for rolipram. Mutation of Arg271 to alanine, in the XAP2 tetratricopeptide repeat region, attenuated its ability to both interact with PDE4A5 in two-hybrid assays and to inhibit PDE4A5 activity. Either the deletion of a specific portion of the unique amino-terminal region or specific mutations in the regulatory UCR2 domain of PDE4A5 attenuated its ability be inhibited by XAP2. We suggest that XAP2 functionally interacts with PDE4A5 in cells.