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Pig aortic endothelial-cell cyclic nucleotide phosphodiesterases. Use of phosphodiesterase inhibitors to evaluate their roles in regulating cyclic nucleotide levels in intact cells.

ABSTRACT: Two cyclic nucleotide phosphodiesterase (PDE) activities were identified in pig aortic endothelial cells, a cyclic GMP-stimulated PDE and a cyclic AMP PDE. Cyclic GMP-stimulated PDE had Km values of 367 microM for cyclic AMP and 24 microM for cyclic GMP, and low concentrations (1 microM) of cyclic GMP increased the affinity of the enzyme for cyclic AMP (Km = 13 microM) without changing the Vmax. This isoenzyme was inhibited by trequinsin [IC50 (concn. giving 50% inhibition of substrate hydrolysis) = 0.6 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 0.6 microM for cyclic GMP hydrolysis] and dipyridamole (IC50 = 5 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 3 microM for cyclic GMP hydrolysis). Cyclic AMP PDE exhibited a Km of 2 microM for cyclic AMP and did not hydrolyse cyclic GMP. This activity was inhibited by trequinsin (IC50 = 0.2 microM), dipyridamole (IC50 = 6 microM) and, selectively, by rolipram (IC50 = 3 microM). Inhibitors of cyclic GMP PDE (M&B 22948) and of low Km (Type III) cyclic AMP PDE (SK&F 94120) only weakly inhibited the two endothelial PDEs. Incubation of intact cells with trequinsin and dipyridamole induced large increases in cyclic GMP, which were completely blocked by LY-83583. Rolipram, SK&F 94120 and M&B 22948 did not significantly influence cyclic GMP accumulation. Dipyridamole enhanced the increase in cyclic GMP induced by sodium nitroprusside. Cyclic AMP accumulation was stimulated by dipyridamole and trequinsin with and without forskolin. Rolipram, although without effect alone, increased cyclic AMP in the presence of forskolin, whereas M&B 22948 and SK&F 94120 had no effects on resting or forskolin-stimulated levels. These results suggest that cyclic GMP-stimulated PDE regulates cyclic GMP levels and that both endothelial PDE isoenzymes contribute to the control of cyclic AMP.

SUBMITTER: Souness JE

PROVIDER: S-EPMC1131105 | biostudies-other | 1990 Feb

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. RESULTS: This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs) from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range. Several PDE inhibitors were found to be active against these PDEs in vitro, and to inhibit cell proliferation. CONCLUSION: The genome of L. major contains only PDE genes that are predicted to code for class I PDEs, and none for class II PDEs. This is more similar to what is found in higher eukaryotes than it is to the situation in Dictyostelium or the fungi that concomitantly express class I and class II PDEs. Functional complementation demonstrated that LmjPDEA, LmjPDEB1 and LmjPDEB2 are capable of hydrolyzing cAMP. In vitro studies with recombinant LmjPDEB1 and LmjPDEB2 confirmed this, and they demonstrated that both are completely cAMP-specific. Both enzymes are inhibited by several commercially available PDE inhibitors. The observation that these inhibitors also interfere with cell growth in culture indicates that inhibition of the PDEs is fatal for the cell, suggesting an important role of cAMP signalling for the maintenance of cellular integrity and proliferation.

Project description:The balanced signaling between the two cyclic nucleotides (cNs) cAMP and cGMP plays a critical role in regulating cardiac contractility. Their degradation is controlled by distinctly regulated phosphodiesterase isoenzymes (PDEs), which in turn are also regulated by these cNs. As a result, PDEs facilitate communication between the β-adrenergic and Nitric Oxide (NO)/cGMP/Protein Kinase G (PKG) signaling pathways, which regulate the synthesis of cAMP and cGMP respectively. The phenomena in which the cAMP and cGMP pathways influence the dynamics of each other are collectively referred to as cN cross-talk. However, the cross-talk response and the individual roles of each PDE isoenzyme in shaping this response remain to be fully characterized. We have developed a computational model of the cN cross-talk network that mechanistically integrates the β-adrenergic and NO/cGMP/PKG pathways via regulation of PDEs by both cNs. The individual model components and the integrated network model replicate experimentally observed activation-response relationships and temporal dynamics. The model predicts that, due to compensatory interactions between PDEs, NO stimulation in the presence of sub-maximal β-adrenergic stimulation results in an increase in cytosolic cAMP accumulation and corresponding increases in PKA-I and PKA-II activation; however, the potentiation is small in magnitude compared to that of NO activation of the NO/cGMP/PKG pathway. In a reciprocal manner, β-adrenergic stimulation in the presence of sub-maximal NO stimulation results in modest cGMP elevation and corresponding increase in PKG activation. In addition, we demonstrate that PDE2 hydrolyzes increasing amounts of cAMP with increasing levels of β-adrenergic stimulation, and hydrolyzes increasing amounts of cGMP with decreasing levels of NO stimulation. Finally, we show that PDE2 compensates for inhibition of PDE5 both in terms of cGMP and cAMP dynamics, leading to cGMP elevation and increased PKG activation, while maintaining whole-cell β-adrenergic responses similar to that prior to PDE5 inhibition. By defining and quantifying reactions comprising cN cross-talk, the model characterizes the cross-talk response and reveals the underlying mechanisms of PDEs in this non-linear, tightly-coupled reaction system.

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Pig aortic endothelial-cell cyclic nucleotide phosphodiesterases. Use of phosphodiesterase inhibitors to evaluate their roles in regulating cyclic nucleotide levels in intact cells.

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