No abstract

1 Chronic hypoxic treatment of rats (to induce pulmonary hypertension, PHT) for 14 days increased cGMP-inhibited cAMP speci®c phosphodiesterase (PDE3) and cGMP binding cGMP speci®c phosphodiesterase (PDE5) activities in pulmonary arteries. The objective of this study was to establish the molecular basis for these changes in both animal and cell models of PHT. In this regard, RT ± PCR and quantitative Western blotting analysis was applied to rat pulmonary artery homogenates and human pulmonary`artery' smooth muscle cell (HPASMC) lysates. 2 PDE3A/B gene transcript levels were increased in the main, ®rst, intrapulmonary and resistance pulmonary arteries by chronic hypoxia. mRNA transcript and protein levels of PDE5A2 in the main and ®rst branch pulmonary arteries were also increased by chronic hypoxia, with no eect on PDE5A1/A2 in the intra-pulmonary and resistance vessels. 3 The expression of PDE3A was increased in HPASMCs maintained under chronic hypoxic conditions for 14 days. This may be mediated via a protein kinase A-dependent mechanism, as treatment of cells with Br-cAMP (100 mM) mimicked chronic hypoxia in increasing PDE3A expression, while the PKA inhibitor, H8 peptide (50 mM) abolished the hypoxic-dependent increase in PDE3A transcript. 4 We also found that the treatment of HPASMCs with the inhibitor of kB degradation TosylLeucyl-Chloro-Ketone (TLCK, 50 mM) reduced PDE5 transcript levels, suggesting a role for this transcription factor in the regulation of PDE5 gene expression. 5 Our results show that increased expression of PDE3 and PDE5 might explain some changes in vascular reactivity of pulmonary vessels from rats with PHT. We also report that NF-kB might regulate basal PDE5 expression.

Cyclic nucleotide phosphodiesterases (PDEs) comprise a family of enzymes (PDE1-PDE11) which hydrolyse cyclic AMP and cyclic GMP to their biologically inactive 5′ derivatives. Cyclic AMP is an important physiological amplifier of glucose-induced insulin secretion. As PDEs are the only known mechanism for inactivating cyclic nucleotides, it is important to characterise the PDEs present in the pancreatic islet beta cells. Several studies have shown pancreatic islets or beta cells to contain PDE1C, PDE3B and PDE4, with some evidence for PDE10A. Most evidence suggests that PDE3B is the most important in relation to the regulation of insulin release, although PDE1C could have a role. PDE3-selective inhibitors augment glucose-induced insulin secretion. In contrast, activation of beta-cell PDE3B could mediate the inhibitory effect of IGF-1 and leptin on insulin secretion. In vivo, although PDE3 inhibitors augment glucose-induced insulin secretion, concomitant inhibition of PDE3B in liver and adipose tissue induce insulin resistance and PDE3 inhibitors do not induce hypoglycaemia. The development of PDE3 inhibitors as anti-diabetic agents would require differentiation between PDE3B in the beta cell and that in hepatocytes and adipocytes. Through their effects in regulating beta-cell cyclic nucleotide concentrations, PDEs could modulate beta-cell growth, differentiation and survival; some work has shown that selective inhibition of PDE4 prevents diabetes in NOD mice and that selective PDE3 inhibition blocks cytokine-induced nitric oxide production in islet cells. Further work is required to understand the mechanism of regulation and role of the various PDEs in islet-cell function and to validate them as targets for drugs to treat and prevent diabetes. [Diabetologia (2003[Diabetologia ( ) 46:1179[Diabetologia ( -1189

Cyclic 3'5'AMP (cAMP) is an important physiological amplifier of glucose-induced insulin secretion by the pancreatic islet beta-cell, where it is formed by the activity of adenylyl cyclases, which are stimulated by glucose, through elevation in intracellular calcium concentrations, and by the incretin hormones (GLP-1 and GIP). cAMP is rapidly degraded in the pancreatic islet beta-cell by various cyclic nucleotide phosphodiesterase (PDE) enzymes. Many steps involved in glucose-induced insulin secretion are modulated by cAMP, which is also important in regulating pancreatic islet beta-cell differentiation, growth and survival. This chapter discusses the formation, destruction and actions of cAMP in the islets with particular emphasis on the beta-cell.

cGMP signaling regulates epithelial fluid transport by Drosophila Malpighian (renal) tubules. In order to directly evaluate the importance of cGMP-degrading phosphodiesterases (PDEs) in epithelial transport, bovine PDE5 (a bona fide cGMP-PDE), was ectopically expressed in vivo. Transgenic UAS-PDE5 Drosophila were generated, and PDE5 expression was driven in specified tubule cells in vivo by cell-specific GAL4 drivers. Targeted expression was verified by PCR and Western blotting. Immunolocalization of PDE5 in tubule confirmed specificity of expression and demonstrated localization to the apical plasma membrane. GAL4/UAS-PDE5 tubules exhibit increased cG-PDE activity and reduced basal cGMP levels compared with control lines. We show that wild-type and control tubules are sensitive to the PDE5-specific inhibitor sildenafil and that GAL4/UAS-PDE5 tubules display enhanced sensitivity to sildenafil, compared with controls. cGMP content in GAL4/UAS-PDE5 tubules is restored to control levels by treatment with sildenafil. Thus bovine PDE5 retains cGMP-degrading activity and inhibitor sensitivity when expressed in Drosophila. Expression of PDE5 in tubule principal cells results in an epithelial phenotype, reducing rates of basal and cGMP-/Cardioaccelatory peptide 2b (CAP 2b )-stimulated fluid transport. Furthermore, inhibition of PDE5 activity by sildenafil restores basal and cGMP-stimulated fluid transport rates to control levels. However, corticotrophin releasing factorlike-stimulated transport, which is activated by cAMP signaling, was unaffected, confirming that only cGMPstimulated signaling events in tubule are compromised by overexpression of PDE5. Successful ectopic expression of a vertebrate cG-PDE in Drosophila has shown that cG-PDE has a critical role in tubule function in vivo and that cG-PDE function is conserved across evolution. The transgene also provides a generic tool for the analysis of cGMP signaling in Drosophila.

1 We examined various type-selective phosphodiesterase (PDE) inhibitors on glucose-induced insulin secretion from rat isolated islets, on islet PDE activity and on islet cyclic AMP accumulation in order to assess the relationship between type-selective PDE inhibition and modification of insulin release. 2 The non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX, I0 -i0 -M), as well as the type III selective PDE inhibitors SK&F 94836 (10-_-i0-3 M), Org 9935 (10-7-l0-4 M), SK&F 94120 (l0-_-i0-4 M) and ICI 118233 (10-6_ i0-4 M) each caused concentration-dependent augmentation (up to 40% increase) of insulin release in the presence of a stimulatory glucose concentration (10 mM), but not in the presence of 3 mM glucose.3 Neither the type IV PDE inhibitor rolipram (10-4 M) nor the type I and type V PDE inhibitor, zaprinast (10-4-10o-M) modified glucose-induced insulin release when incubated with islets, although a higher concentration of rolipram (10-3 M) inhibited secretion by 55%. However, when islets were preincubated with these drugs followed by incubation in their continued presence, zaprinast (10-6_ 10-4 M) produced a concentration-dependent inhibition (up to 45% at 10-4 M). Under these conditions, rolipram inhibited insulin secretion at a lower concentration (10-4 M) than when simply incubated with islets. 4 A combination of SK&F 94836 (10-5 M) and forskolin (5 x 10i-M) significantly augmented glucoseinduced insulin secretion (30% increase), although neither drug alone, in these concentrations, produced any significant effect. 6 Homogenates of rat islets showed a low Km (1.7 gM) and high Km (13 gM) cyclic AMP PDE in the supernatant fractions (from 48,000 g centrifugation), whereas the particulate fraction showed only a low Km (1.4 gM) cyclic AMP PDE activity. 7 The PDE activity of both supernatant and pellet fractions were consistently inhibited by SK&F 94836 or Org 9935, the concentrations required to reduce particulate PDE activity by 50% being 5.5 and 0.05 pM respectively. 8 Rolipram (i05-i0-4 M) did not consistently inhibit PDE activity in homogenates of rat islets and zaprinast (10-4 M) consistently inhibited activity by 30% in the supernatant fraction, but not consistently in the pellet. 9 These data are consistent with the presence of a type III PDE in rat islets of Langerhans.

Levels of the G-protein alpha-subunits alpha-Gi-2, alpha-Gi-3 and the 42 kDa, form of alpha-Gs were markedly decreased in hepatocyte membranes from streptozotocin-diabetic animals as compared with normals. In contrast, no detectable changes in alpha-Gi subunits were seen in liver plasma membranes of streptozotocin-diabetic animals, although levels of the 45 kDa form of Gs were increased. G-protein beta subunits in plasma membranes were unaffected by diabetes induction. Analysis of whole-liver RNA indicated that the induction of diabetes had little effect on transcript levels of Gi-3, caused an increase in Gs transcripts and decreased transcript number for Gi-2, albeit to a much lesser extent than was observed upon analysis of hepatocyte RNA. In both hepatocyte and liver plasma membranes, immunoblot analysis showed that levels of the catalytic unit of adenylate cyclase were increased upon induction of diabetes. Under basal conditions, alpha-Gi-2 from hepatocytes of diabetic animals was found to be both phosphorylated to a greater extent than alpha-Gi-2 isolated from hepatocytes of normal animals, and furthermore was resistant to any further phosphorylation upon challenge of hepatocytes with angiotensin, vasopressin or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. Treatment of isolated plasma membranes from normal, but not diabetic, animals with purified protein kinase C caused the phosphorylation of alpha-Gi-2. Treatment of membranes from diabetic animals with alkaline phosphatase caused the dephosphorylation of alpha-Gi-2 and rendered it susceptible to subsequent phosphorylation with protein kinase C. Low concentrations of the non-hydrolysable GTP analogue guanylyl 5'-imidodiphosphate inhibited adenylate cyclase activity in both hepatocyte and liver plasma membranes from normal, but not diabetic, animals.

The signaling pathways by which sphingosine 1-phosphate (S1P) potently stimulates endothelial cell migration and angiogenesis are not yet fully defined. We, therefore, investigated the role of protein kinase C (PKC) isoforms, phospholipase D (PLD), and Rac in S1P-induced migration of human pulmonary artery endothelial cells (HPAECs). S1P-induced migration was sensitive to S1P 1 small interfering RNA (siRNA) and pertussis toxin, demonstrating coupling of S1P 1 to G i . Overexpression of dominant negative (dn) PKC-⑀ or -, but not PKC-␣ or -␦, blocked S1P-induced migration. Although S1P activated both PLD1 and PLD2, S1P-induced migration was attenuated by knocking down PLD2 or expressing dnPLD2 but not PLD1. Blocking PKC-⑀, but not PKC-, activity attenuated S1P-mediated PLD stimulation, demonstrating that PKC-⑀, but not PKC-, was upstream of PLD. Transfection of HPAECs with dnRac1 or Rac1 siRNA attenuated S1P-induced migration. Furthermore, transfection with PLD2 siRNA, infection of HPAECs with dnPKC-, or treatment with myristoylated PKC-peptide inhibitor abrogated S1P-induced Rac1 activation. These results establish that S1P signals through S1P 1 and G i to activate PKC-⑀ and, subsequently, a PLD2-PKC--Rac1 cascade. Activation of this pathway is necessary to stimulate the migration of lung endothelial cells, a key component of the angiogenic process.Sphingosine 1-phosphate (S1P) 3 is a naturally occurring bioactive sphingolipid that elicits multiple cellular responses such as differentiation, proliferation, survival, and angiogenesis (1-5). S1P acts as an intracellular second messenger. Extracellular S1P also activates intracellular signaling pathways through ligation to a family of G-protein-coupled S1P receptors, S1P 1-5 (previously known as endothelial differentiation gene receptors) (6). The S1P-Rs are differentially expressed in different cell types and are coupled to G i , G q , or G 12/13 (7-9). Coupling of S1P to S1P 1 via G i activates Rac and Rho (2, 10) and stimulates cell proliferation (4), cortical actin formation (11), assembly of adherens junction, and angiogenesis (2). Binding of S1P to S1P 3 induces signaling through G q or G 13 to activate Rho (2, 10, 12), promotes the formation of stress fibers and adherens junctions (2), stimulates phospholipase D (PLD) (13), and activates phospholipase C/intracellular Ca 2ϩ /protein kinase C (PKC) pathways (7). Ligation of S1P to S1P 1 also initiates cross-talk with other receptors, especially growth factor receptors including those for epidermal growth factor (EGF), platelet-derived growth factor, and vascular endothelial growth factor (14). The functional platelet-derived growth factor (PDGF)-␤/S1P 1 signaling complex was postulated to be involved in regulating migration of mouse embryonic fibroblasts in response to PDGF (15). Furthermore, S1P binding to S1P 2 inhibits cell migration via G q or G 13 (9, 12, 16) and activates adenylate cyclase (17) and mitogen-activated protein kinases (MAPKs) (18). There are few studies related to S1P signaling via S1P 4 and...