Hormone-sensitive cyclic GMP-inhibited cyclic AMP phosphodiesterase in rat adipocytes. Regulation of insulin- and cAMP-dependent activation by phosphorylation.

In 32PO4-labeled adipocytes, isoproterenol (ISO) or physiologically relevant concentrations of insulin rapidly increased phosphorylation of a particulate 135-kDa protein which has been identified as a cGMP-inhibited "low Km" cAMP phosphodiesterase (CGI-PDE) by several criteria, including selective immunoprecipitation with anti-CGI-PDE IgG (Degerman, E., Smith, C.J., Tornqvist, H., Vasta, V., Belfrage, P., and Manganiello, V.C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 533-537). The time courses and concentration dependences for phosphorylation of CGI-PDE by ISO and insulin correlated with CGI-PDE activation in the presence of these agents; effects of ISO were somewhat more rapid than those of insulin. Adenosine deaminase, which metabolizes the adenylate cyclase inhibitor adenosine, also rapidly induced phosphorylation and activation of CGI-PDE. Phenylisopropyladenosine (an adenosine deaminase-resistant adenosine analog) prevented or reversed both adenosine deaminase-stimulated phosphorylation and activation of CGI-PDE (IC50 approximately 0.2 nM). Incubation of adipocytes with 0.1 nM insulin in the presence of ISO rapidly produced 30-200% greater activation and phosphorylation of CGI-PDE than the expected added effects of insulin and ISO individually; both effects preceded the insulin-induced decreases in protein kinase A activity and inhibition of lipolysis. These and other results indicate that CGI-PDE can be phosphorylated at distinct sites and activated by cAMP-dependent and insulin-dependent serine kinase(s), that the activation state of CGI-PDE reflects its relative phosphorylation state, and that synergistic phosphorylation/activation of CGI-PDE may be important in the antilipolytic action of insulin

Receptor-Mediated Gonadotropin Action in Ovary

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65826/1/j.1432-1033.1979.tb12873.x.pd

Culture Enriched Molecular Profiling of the Cystic Fibrosis Airway Microbiome

The microbiome of the respiratory tract, including the nasopharyngeal and oropharyngeal microbiota, is a dynamic community of microorganisms that is highly diverse. The cystic fibrosis (CF) airway microbiome refers to the polymicrobial communities present in the lower airways of CF patients. It is comprised of chronic opportunistic pathogens (such as Pseudomonas aeruginosa) and a variety of organisms derived mostly from the normal microbiota of the upper respiratory tract. The complexity of these communities has been inferred primarily from culture independent molecular profiling. As with most microbial communities it is generally assumed that most of the organisms present are not readily cultured. Our culture collection generated using more extensive cultivation approaches, reveals a more complex microbial community than that obtained by conventional CF culture methods. To directly evaluate the cultivability of the airway microbiome, we examined six samples in depth using culture-enriched molecular profiling which combines culture-based methods with the molecular profiling methods of terminal restriction fragment length polymorphisms and 16S rRNA gene sequencing. We demonstrate that combining culture-dependent and culture-independent approaches enhances the sensitivity of either approach alone. Our techniques were able to cultivate 43 of the 48 families detected by deep sequencing; the five families recovered solely by culture-independent approaches were all present at very low abundance (<0.002% total reads). 46% of the molecular signatures detected by culture from the six patients were only identified in an anaerobic environment, suggesting that a large proportion of the cultured airway community is composed of obligate anaerobes. Most significantly, using 20 growth conditions per specimen, half of which included anaerobic cultivation and extended incubation times we demonstrate that the majority of bacteria present can be cultured

Cyclic nucleotide phosphodiesterases (PDEs): diverse regulators of cyclic nucleotide signals and inviting molecular targets for novel therapeutic agents

Introduction: Cyclic adenosine 3’5’-monophosphate (cAMP) and cyclic guanosine 3’5’-monophoshpate (cGMP) are critical intracellular second-messengers involved in the transduction of a wide variety of extracellular stimuli, including peptide hormones, growth factors, cytokines, neurotransmitters and light. These messengers modulate many fundamental biological processes, including growth, differentiation, apoptosis, glycogenolysis, lipolysis, immune/inflammatory responses, etc. By catalyzing hydrolysis of cAMP and cGMP, cyclic nucleotide phosphodiesterases (PDEs) are important determinants in regulating the intracellular concentrations and, consequently, the biological actions of these second-messengers (Fig. 1). The advent of molecular genetics has revealed the extraordinary complexity and diversity of the mammalian PDE superfamily, which contains at least 10 highly regulated and structurally-related gene families (PDEs 1-10).1-8 As depicted in Figure 1, some PDEs are highly specific for hydrolysis of cAMP (PDEs 4,7,8), some are cGMP-specific (PDEs 5,6,9), and some exhibit mixed specificity (PDEs 1,2,3,10). Most gene families are comprised of more than one isogene (indicated by A-D in Table 1). At least 19 genes encoding more than 30 isoforms have been identified. PDE families differ with respect to their primary structures, sensitivity to specific inhibitors, tissue distribution, subcellular localization, and mechanisms of regulation (Table 1).2-6 Within individual families, different mRNAs are generated from the same gene by use of different transcription initiation sites or by alternative mRNA splicing. These variant PDE isoforms are often tissue-specific and selectively expressed in various tissues and cell types.2-6 The importance of cyclic nucleotide signaling in cell regulation and the molecular diversity of PDEs has presented targets for selective interventions and development of family-specific PDE inhibitors as therapeutic agents. This brief review will discuss some general characteristics of PDEs and then focus on the cellular biology and diverse functions of different PDE isoforms and their potential as therapeutic targets

Cyclic Nucleotide Phosphodiesterase 3 Signaling Complexes

The superfamily of cyclic nucleotide phosphodiesterases is comprised of 11 gene families. By hydrolyzing cAMP and cGMP, PDEs are major determinants in the regulation of intracellular concentrations of cyclic nucleotides and cyclic nucleotide-dependent signaling pathways. Two PDE3 subfamilies, PDE3A and PDE3B, have been described. PDE3A and PDE3B hydrolyze cAMP and cGMP with high affinity in a mutually competitive manner and are regulators of a number of important cAMP- and cGMP-mediated processes. PDE3B is relatively more highly expressed in cells of importance for the regulation of energy homeostasis, including adipocytes, hepatocytes, and pancreatic beta-cells, whereas PDE3A is more highly expressed in heart, platelets, vascular smooth muscle cells, and oocytes. Major advances have been made in understanding the different physiological impacts and biochemical basis for recruitment and subcellular localizations of different PDEs and PDE-containing macromolecular signaling complexes or signalosomes. In these discrete compartments, PDEs control cyclic nucleotide levels and regulate specific physiological processes as components of individual signalosomes which are tethered at specific locations and which contain PDEs together with cyclic nucleotide-dependent protein kinases (PKA and PKG), adenylyl cyclases, Epacs (guanine nucleotide exchange proteins activated by cAMP), phosphoprotein phosphatases, A-Kinase anchoring proteins (AKAPs), and pathway-specific regulators and effectors. This article highlights the identification of different PDE3A- and PDE3B-containing signalosomes in specialized subcellular compartments, which can increase the specificity and efficiency of intracellular signaling and be involved in the regulation of different cAMP-mediated metabolic processes