RESUMEN
Isoforms in the PDE1 family of cyclic nucleotide phosphodiesterases were recently found to comprise a significant portion of the cGMP-inhibited cAMP hydrolytic activity in human hearts. We examined the expression of PDE1 isoforms in human myocardium, characterized their catalytic activity, and quantified their contribution to cAMP hydrolytic and cGMP hydrolytic activity in subcellular fractions of this tissue. Western blotting with isoform-selective anti-PDE1 monoclonal antibodies showed PDE1C1 to be the principal isoform expressed in human myocardium. Immunohistochemical analysis showed that PDE1C1 is distributed along the Z-lines and M-lines of cardiac myocytes in a striated pattern that differs from that of the other major dual-specificity cyclic nucleotide phosphodiesterase in human myocardium, PDE3A. Most of the PDE1C1 activity was recovered in soluble fractions of human myocardium. It binds both cAMP and cGMP with K(m) values of approximately 1 microm and hydrolyzes both substrates with similar catalytic rates. PDE1C1 activity in subcellular fractions was quantified using a new PDE1-selective inhibitor, IC295. At substrate concentrations of 0.1 microm, PDE1C1 constitutes the great majority of cAMP hydrolytic and cGMP hydrolytic activity in soluble fractions and the majority of cGMP hydrolytic activity in microsomal fractions, whereas PDE3 constitutes the majority of cAMP hydrolytic activity in microsomal fractions. These results indicate that PDE1C1 is expressed at high levels in human cardiac myocytes with an intracellular distribution distinct from that of PDE3A and that it may have a role in the integration of cGMP-, cAMP- and Ca(2+)-mediated signaling in these cells.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Miocitos Cardíacos/enzimología , Animales , Línea Celular , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/genética , Regulación Enzimológica de la Expresión Génica , Humanos , Hidrólisis , Isoenzimas/metabolismo , Cinética , Spodoptera , Fracciones Subcelulares/enzimologíaRESUMEN
This study assessed the effects of selective inhibitors of 3',5'-cyclic nucleotide phosphodiesterases (PDEs) on adipocyte lipolysis. IC224, a selective inhibitor of type 1 phosphodiesterase (PDE1), suppressed lipolysis in murine 3T3-L1 adipocytes (69.6 +/- 5.4% of vehicle control) but had no effect in human adipocytes. IC933, a selective inhibitor of PDE2, had no effect on lipolysis in either cultured murine 3T3-L1 adipocytes or human adipocytes. Inhibition of PDE3 with cilostamide moderately stimulated lipolysis in murine 3T3-L1 and rat adipocytes (397 +/- 25% and 235 +/- 26% of control, respectively) and markedly stimulated lipolysis in human adipocytes (932 +/- 7.6% of control). Inhibition of PDE4 with rolipram moderately stimulated lipolysis in murine 3T3-L1 adipocytes (291 +/- 13% of control) and weakly stimulated lipolysis in rat adipocytes (149 +/- 7.0% of control) but had no effect on lipolysis in human adipocytes. Cultured adipocytes also responded differently to a combination of PDE3 and PDE4 inhibitors. Simultaneous exposure to cilostamide and rolipram had a synergistic effect on lipolysis in murine 3T3-L1 and rat adipocytes but not in human adipocytes. Hence, the relative importance of PDE3 and PDE4 in regulating lipolysis differed in cultured murine, rat, and human adipocytes.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/fisiología , Adipocitos/metabolismo , Lipólisis/fisiología , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Células 3T3-L1/efectos de los fármacos , Adipocitos/fisiología , Animales , Células Cultivadas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Relación Dosis-Respuesta a Droga , Exonucleasas/antagonistas & inhibidores , Exonucleasas/fisiología , Humanos , Lipólisis/efectos de los fármacos , Ratones , Fosfodiesterasa I/antagonistas & inhibidores , Fosfodiesterasa I/fisiología , Inhibidores de Fosfodiesterasa/farmacología , RatasRESUMEN
PURPOSE: cAMP phosphodiesterase (PDE) 4 is a family of enzymes the inhibition of which induces chronic lymphocytic leukemia (CLL) apoptosis. However, leukemic cells from a subset of CLL patients are relatively resistant to treatment with the PDE4 inhibitor rolipram, particularly when this drug is used in the absence of an adenylate cyclase stimulus such as forskolin. Elevated cAMP levels induce compensatory up-regulation of several cyclic nucleotide PDE families in other model systems. We here examine the hypothesis that CLL cells that survive treatment with rolipram do so as a result of residual PDE activity that is not inhibited by this drug. EXPERIMENTAL DESIGN: We examined by Western analysis the effect of rolipram treatment on CLL expression of PDE3B, PDE4A, PDE4B, PDE4D, and PDE7A. We also examined the ability of rolipram (PDE4 inhibitor) or cilostamide (PDE3 inhibitor), alone or together, to induce apoptosis or elevate cyclic AMP in leukemic cells from patients with CLL. RESULTS: Rolipram increased levels of PDE4B and, to a variable extent, PDE4D. When combined with forskolin, rolipram also increased levels of a second family of PDEs, PDE3B. Addition of the specific PDE3 inhibitor, cilostamide, modestly augmented rolipram-induced apoptosis in five of seven "rolipram-resistant" CLL samples. CONCLUSIONS: Although this work confirms that PDE4 appears to be the most important PDE target for induction of apoptosis in CLL, combination therapy with PDE3 and PDE4 inhibitors or use of dual-selective drugs may be of benefit in a subset of relatively PDE4-inhibitor resistant CLL patients.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Apoptosis , Leucemia Linfocítica Crónica de Células B/patología , Inhibidores de Fosfodiesterasa/farmacología , 3',5'-AMP Cíclico Fosfodiesterasas/biosíntesis , Northern Blotting , Western Blotting , Línea Celular , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7 , Humanos , Isoenzimas/biosíntesis , Isoformas de Proteínas , Quinolonas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rolipram/farmacología , Factores de Tiempo , Regulación hacia ArribaRESUMEN
PDE7A is a recently described 3',5'-cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase (PDE) whose expression has been detected in T-cells. As treatment with the methylxanthine theophylline, a nonspecific PDE inhibitor, induces apoptosis in leukemic cells from patients with the B-lineage malignancy chronic lymphocytic leukemia (CLL), we sought to determine if PDE7A was a target of theophylline therapy in such cells. Western analysis revealed expression of PDE7A in normal human splenic B-cells, primary CLL cells, and in a CLL-derived cell line (WSU-CLL). Among the six cAMP PDEs (PDE1B, PDE3B, PDE4A, PDE4B, PDE4D, and PDE7) examined in WSU-CLL, only PDE7A levels were augmented by treatment with methylxanthines. The activity of PDE7A isolated from the WSU-CLL cell line by immunoprecipitation was inhibited by theophylline and IBMX with IC50 values of 343.5 and 8.6 microM, respectively. WSU-CLL PDE7A was also up-regulated by a novel specific inhibitor (IC242), which inhibits PDE7A from WSU-CLL cells with an IC50 value of 0.84 microM. IC242-mediated up-regulation of PDE7A was blocked by the protein kinase A (PKA) inhibitor H-89.