PDE7A is expressed in human B-lymphocytes and is up-regulated by elevation of intracellular cAMP

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 μM, 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 μM. IC242-mediated up-regulation of PDE7A was blocked by the protein kinase A (PKA) inhibitor H-89.     

Nitric oxide induces phosphodiesterase 4B expression in rat pulmonary artery smooth muscle cells

Phosphodiesterases (PDE) metabolize cyclic nucleotides limiting the effects of vasodilators such as prostacyclin and nitric oxide (NO). In this study, DNA microarray techniques were used to assess the impact of NO on expression of PDE genes in rat pulmonary arterial smooth muscle cells (rPASMC). Incubation of rPASMC with S-nitroso-l-glutathione (GSNO) increased expression of a PDE isoform that specifically metabolizes cAMP (PDE4B) in a dose- and time-dependent manner. GSNO increased PDE4B protein levels, and rolipram-inhibitable PDE activity was 2.3 +/- 1.0-fold greater in GSNO-treated rPASMC than in untreated cells. The soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one, and the cAMP-dependent protein kinase inhibitor, H89, prevented induction of PDE4B gene expression by GSNO, but the protein kinase G (PKG) inhibitors, Rp-8-pCPT-cGMPs and KT-5823, did not. Incubation of rPASMC with IL-1beta and tumor necrosis factor-alpha induced PDE4B gene expression, an effect that was inhibited by l-N(6)-(1-iminoethyl)lysine, an antagonist of NO synthase 2 (NOS2). The GSNO-induced increase in PDE4B mRNA levels was blocked by actinomycin D but augmented by cycloheximide. Infection of rPASMC with an adenovirus specifying a dominant negative cAMP response element binding protein (CREB) mutant inhibited the GSNO-induced increase of PDE4B gene expression. These results suggest that exposure of rPASMC to NO induces expression of PDE4B via a mechanism that requires cGMP synthesis by sGC but not PKG. The GSNO-induced increase of PDE4B gene expression is CREB dependent. These findings demonstrate that NO increases expression of a cAMP-specific PDE and provide evidence for a novel "cross talk" mechanism between cGMP and cAMP signaling pathways.     

Nonredundant function of phosphodiesterases 4D and 4B in neutrophil recruitment to the site of inflammation

Neutrophils have been implicated in the pathogenesis of many inflammatory lung diseases, including chronic obstructive pulmonary disease and asthma. With this study, we investigated how disruption of cAMP signaling impacts the function of neutrophil recruitment to the lung. Four genes code for type 4 phosphodiesterases (PDE4s), enzymes critical for regulation of cAMP levels and cell signaling. Ablation of two of these genes, PDE4B and PDE4D, but not PDE4A, has profound effects on neutrophil function. In a paradigm of mouse lung injury induced by endotoxin inhalation, the number of neutrophils recovered in the bronchoalveolar lavage was markedly decreased in PDE4D(-/-) and PDE4B(-/-) mice 4 and 24 h after exposure to LPS. Acute PDE4 inhibition with rolipram had additional inhibitory effects on neutrophil migration in PDE4B(-/-) and, to a lesser extent, PDE4D(-/-) mice. This decreased neutrophil recruitment occurred without major changes in chemokine accumulation in bronchoalveolar lavage, suggesting a dysfunction intrinsic to neutrophils. This hypothesis was confirmed by investigating the expression of adhesion molecules on the surface of neutrophils and chemotaxis in vitro. CD18 expression was decreased after ablation of both PDE4B and PDE4D, whereas CD11 expression was not significantly affected. Chemotaxis in response to KC and macrophage inflammatory protein-2 was markedly reduced in PDE4B(-/-) and PDE4D(-/-) neutrophils. The effect of PDE4 ablation on chemotaxis was comparable, but not additive, to the effects of acute PDE4 inhibition with rolipram. These data demonstrate that PDE4B and PDE4D play complementary, but not redundant, roles in the control of neutrophil function.     

Concerted regulation of cGMP and cAMP phosphodiesterases in early cardiac hypertrophy induced by angiotensin II

Left ventricular hypertrophy leads to heart failure and represents a high risk leading to premature death. Cyclic nucleotides (cAMP and cGMP) play a major role in heart contractility and cyclic nucleotide phosphodiesterases (PDEs) are involved in different stages of advanced cardiac diseases. We have investigated their contributions in the very initial stages of left ventricular hypertrophy development. Wistar male rats were treated over two weeks by chronic infusion of angiotensin II using osmotic mini-pumps. Left cardiac ventricles were used as total homogenates for analysis. PDE1 to PDE5 specific activities and protein and mRNA expressions were explored.Rats developed arterial hypertension associated with a slight cardiac hypertrophy (+24%). cAMP-PDE4 activity was specifically increased while cGMP-PDE activities were broadly increased (+130% for PDE1; +76% for PDE2; +113% for PDE5) and associated with increased expressions for PDE1A, PDE1C and PDE5A. The cGMP-PDE1 activation by Ca(2+)/CaM was reduced. BNP expression was increased by 3.5-fold, while NOX2 expression was reduced by 66% and AMP kinase activation was increased by 64%. In early cardiac hypertrophy induced by angiotensin II, all specific PDE activities in left cardiac ventricles were increased, favoring an increase in cGMP hydrolysis by PDE1, PDE2 and PDE5. Increased cAMP hydrolysis was related to PDE4. We observed the establishment of two cardioprotective mechanisms and we suggest that these mechanisms could lead to increase intracellular cGMP: i) increased expression of BNP could increase "particulate" cGMP pool; ii) increased activation of AMPK, subsequent to increase in PDE4 activity and 5'AMP generation, could elevate "soluble" cGMP pool by enhancing NO bioavailability through NOX2 down-regulation. More studies are needed to support these assumptions. Nevertheless, our results suggest a potential link between PDE4 and AMPK/NOX2 and they point out that cGMP-PDEs, especially PDE1 and PDE2, may be interesting therapeutic targets in preventing cardiac hypertrophy.     

Heat-induced salivary gland hypertrophy: change during chronic heat exposure (34 degrees C). (author's transl)

1 A slight rise in ambient temperature (34 degrees C) increased saliva secretion in the rat and this was accompanied by licking behavior, thus increasing heat loss by evaporation. 2 The rise in ambient temperature was accompanied by a hypertrophy of the submaxillary glands (Fig.1, 2), arising from activation of seromucous acini (Fig. 4, 5). 3 The hypertrophy reached a maximum after 2 to 4 days of heat exposure (Fig. 2); it was followed by a slow decrease. Normal values were obtained only after at least 3 weeks in the climatic chamber (Fig. 2, 3), which agreed with behavioral observations. Increased secretion of saliva may help to increase heat loss and maintain survival in a hot environment. 4 The absence of morphological changes in the sublingual g and and the evolution of the granular circonvoluted tubules (Fig. 6) contrast greatly with the hypertrophy of the submaxillary acinus. It is possible that heat-induced saliva secretion may be controlled by both sympathetic and parasympathetic systems.     

Phosphodiesterase isoform‐specific expression induced by traumatic brain injury

Traumatic brain injury (TBI) results in significant inflammation which contributes to the evolving pathology. Previously, we have demonstrated that cyclic AMP (cAMP), a molecule involved in inflammation, is down‐regulated after TBI. To determine the mechanism by which cAMP is down‐regulated after TBI, we determined whether TBI induces changes in phosphodiesterase (PDE) expression. Adult male Sprague Dawley rats received moderate parasagittal fluid‐percussion brain injury (FPI) or sham injury, and the ipsilateral, parietal cortex was analyzed by western blotting. In the ipsilateral parietal cortex, expression of PDE1A, PDE4B2, and PDE4D2, significantly increased from 30 min to 24 h post‐injury. PDE10A significantly increased at 6 and 24 h after TBI. Phosphorylation of PDE4A significantly increased from 6 h to 7 days post‐injury. In contrast, PDE1B, PD4A5, and PDE4A8 significantly decreased after TBI. No changes were observed with PDE1C, PDE3A, PDE4B1/3, PDE4B4, PDE4D3, PDE4D4, PDE8A, or PDE8B. Co‐localization studies showed that PDE1A, PDE4B2, and phospho‐PDE4A were neuronally expressed, whereas PDE4D2 was expressed in neither neurons nor glia. These findings suggest that therapies to reduce inflammation after TBI could be facilitated with targeted therapies, in particular for PDE1A, PDE4B2, PDE4D2, or PDE10A.     

Effect in vivo of beta-adrenergic stimulation, angiotensin II, dibutyryl cyclic AMP, and theophylline on tonin concentration in rat saliva and submaxillary gland.

Tonin (an enzyme present in rat submaxillary gland and saliva) has previously been shown to be able, unlike renin and reninlike substances, to release angiotensin II either directly by acting on an appropriate substrate or from angiotensin I. The administration of a beta-adrenergic drug, isoproterenol, produces a rise of tonin concentration in saliva without affecting its concentration in the submaxillary gland. Prior administration of a beta blocker, propranolol, partially prevents this effect. The administration of theophylline increases the tonin concentration in both saliva and the submaxillary gland, whereas dibutyryl cyclic AMP increases tonin concentration in the former. These results suggest that beta-adrenergic stimulation enhances both tonin release into the saliva and tonin synthesis in the submaxillary gland, and that these effects might be mediated by cyclic AMP. Infusion of angiotensin II blocked the stimulatory effect of isoproterenol on salivary tonin. 1Sar-8Ile-angiotensin II is both a weak antagonist of angiotensin II in this respect and a strong agonist in terms of blocking the effect of isoproterenol another role mirrored in other physiological mechanisms of derivatives of angiotensin II.     

Microheterogeneity of molecular forms of arginase in mammalian tissues

Two isoforms of arginase, A1 and A2, were found in rat liver, submaxillary gland and kidney as well as beef kidney. In beef liver, however, A2 was the only detectable form. Two additional forms, A3 and A4, found only in rat kidney were probably artifactitious. A1 and A2 exhibited chromatographic and immunological microheterogeneity. While A1 in rat liver and submaxillary gland was excluded by DEAE-cellulose (pH 8.3) and retained on CM-cellulose (pH 7.5), that (A'1) in beef and rat kidneys was excluded by both ion-exchangers. A2 in all tissues was retained on DEAE-cellulose, but not on CM-cellulose. Both A1 and A2 in rat liver and beef kidney, A1 from rat submaxillary gland and A2 from beef liver were precipitated by antibodies to rat and beef liver arginases. None of the forms in rat kidney (A1, A2, A3 and A4) showed any cross-reactivity to either antibody. Rat submaxillary gland A2 was precipitated by anti-rat liver arginase, but activated by anti-beef liver arginase. While the major molecular forms were A1 in rat liver and submaxillary gland and A2 in beef liver and rat kidney, the two forms occurred in equal proportions in beef kidney. It appears that different isoforms might function as components of the urea cycle in the liver of different mammals and of the arginine catabolic pathway in different extrahepatic tissues.