Regulation of adrenal steroidogenesis by the high-affinity phosphodiesterase 8 family.

The main function of cyclic AMP phosphodiesterases (PDEs) is to degrade cAMP, a ubiquitous second messenger. Therefore, PDEs can function as prime regulators of cAMP/PKA-dependent processes such as steroidogenesis. Until recently, the roles of the PDE8 family have been largely unexplored, presumably due to the lack of a selective inhibitor. This review focuses on recent reports about the regulatory roles of the PDE8 family in adrenal steroidogenesis, as well as the inhibitory properties and specificity of a new PDE8-selective inhibitor, PF-04957325. We also describe a method of measuring urinary corticosterone levels in vivo as a minimally invasive way of monitoring the stress level in a mouse.

Regulation and function of cyclic nucleotides.

Recent work has greatly expanded our knowledge of the structure, regulation and diversity of enzymes involved in the synthesis and degradation of cyclic nucleotides. This review focuses on recent work that provides insight into the structure and function of the cyclases and phosphodiesterases that regulate cyclic nucleotide metabolism. Particular emphasis is given to the roles played by multiple isoforms of each enzyme system.

Regulation of cAMP and cGMP signaling: new phosphodiesterases and new functions.

The past eighteen months have provided much progress in the cyclic nucleotide phosphodiesterase (PDE) field. Six new phosphodiesterase genes have been discovered and characterized. In addition, several new highly specific PDE inhibitors have been developed and approved for clinical use. Finally, new strategies have been employed to determine PDE function in model systems including the use of antisense oligonucleotide and disruption techniques.

Immunofluorescent localization of cyclic nucleotide-dependent protein kinases on the mitotic apparatus of cultured cells.

Cyclic nucleotides and cyclic nucleotide-dependent protein kinases have been implicated in the regulation of cell motility and division, processes that depend on the cell cytoskeleton. To determine whether cyclic nucleotides or their kinases are physically associated with the cytoskeleton during cell division, fluorescently labeled antibodies directed against cyclic AMP, cyclic GMP, and the cyclic nucleotide-dpendent protein kinases were used to localize these molecules in mitotic PtK1 cells. Both the cyclic GMP-dependent protein kinase and the type II regulatory subunit of the cyclic AMP-dependent protein kinase were localized on the mitotic spindle. Throughout mitosis, their distribution closely resembled that of tubulin. Antibodies to cyclic AMP, cyclic GMP, and the type I regulatory and catalytic subunits of the cyclic AMP-dependent protein kinase did not label the mitotic apparatus. The association between specific components of the cyclic neucleotide system and the mitotic spindle suggests that cyclic nucleotide-dependent phosphorylation of spindle proteins, such as those of microtubules, may play a fundamental role in the regulation of spindle assembly and chromosome motion.

CD3- and CD28-dependent induction of PDE7 required for T cell activation.

Costimulation of both the CD3 and CD28 receptors is essential for T cell activation. Induction of adenosine 3',5'-monophosphate (cAMP)-specific phosphodiesterase-7 (PDE7) was found to be a consequence of such costimulation. Increased PDE7 in T cells correlated with decreased cAMP, increased interleukin-2 expression, and increased proliferation. Selectively reducing PDE7 expression with a PDE7 antisense oligonucleotide inhibited T cell proliferation; inhibition was reversed by blocking the cAMP signaling pathways that operate through cAMP-dependent protein kinase (PKA). Thus, PDE7 induction and consequent suppression of PKA activity is required for T cell activation, and inhibition of PDE7 could be an approach to treating T cell-dependent disorders.

Phosphorylation and inhibition of olfactory adenylyl cyclase by CaM kinase II in Neurons: a mechanism for attenuation of olfactory signals.

Acute desensitization of olfactory signaling is a critical property of the olfactory system that allows animals to detect and respond to odorants. Correspondingly, an important feature of odorant-stimulated cAMP increases is their transient nature, a phenomenon that may be attributable to the unique regulatory properties of the olfactory adenylyl cyclase (AC3). AC3 is stimulated by receptor activation and inhibited by Ca2+ through Ca2+/calmodulin kinase II (CaMKII) phosphorylation at Ser-1076. Since odorant-stimulated cAMP increases are accompanied by elevated intracellular Ca2+, CaMKII inhibition of AC3 may contribute to termination of olfactory signaling. To test this hypothesis, we generated a polyclonal antibody specific for AC3 phosphorylated at Ser-1076. A brief exposure of mouse olfactory cilia or primary olfactory neurons to odorants stimulated phosphorylation of AC3 at Ser-1076. This phosphorylation was blocked by inhibitors of CaMKII, which also ablated cAMP decreases associated with odorant-stimulated cAMP transients. These data define a novel mechanism for termination of olfactory signaling that may be important in olfactory responses.

Leptin inhibits insulin secretion by activation of phosphodiesterase 3B.

The molecular signaling events by which leptin exerts its functions in vivo are not well delineated. Here, we show a novel leptin signaling mechanism that requires phosphoinositide 3-kinase (PI 3-kinase)-dependent activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and subsequent suppression of cAMP levels. In pancreatic beta cells, leptin causes the activation of PDE3B, which leads to marked inhibition of glucagon-like peptide-1-stimulated insulin secretion. The effect of leptin is abolished when insulin secretion is induced with cAMP analogues that cannot be hydrolyzed by PDE3B. Selective inhibitors of PDE3B and PI 3-kinase completely prevent the leptin effect on insulin secretion and cAMP accumulation. The results demonstrate that one of the physiological effects of leptin, suppression of insulin secretion, is mediated through activation of PDE3B and suggest PDE3B as a mediator of leptin action in other tissues.

Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1C) is induced in human arterial smooth muscle cells of the synthetic, proliferative phenotype.

The diversity among cyclic nucleotide phosphodiesterases provides multiple mechanisms for regulation of cAMP and cGMP in the cardiovascular system. Here we report that a calmodulin-stimulated phosphodiesterase (PDE1C) is highly expressed in proliferating human arterial smooth muscle cells (SMCs) in primary culture, but not in the quiescent SMCs of intact human aorta. High levels of PDE1C were found in primary cultures of SMCs derived from explants of human newborn and adult aortas, and in SMCs cultured from severe atherosclerotic lesions. PDE1C was the major cAMP hydrolytic activity in these SMCs. PDE expression patterns in primary SMC cultures from monkey and rat aortas were different from those from human cells. In monkey, high expression of PDE1B was found, whereas PDE1C was not detected. In rat SMCs, PDE1A was the only detectable calmodulin-stimulated PDE. These findings suggest that many of the commonly used animal species may not provide good models for studying the roles of PDEs in proliferation of human SMCs. More importantly, the observation that PDE1C is induced only in proliferating SMCs suggests that it may be both an indicator of proliferation and a possible target for treatment of atherosclerosis or restenosis after angioplasty, conditions in which proliferation of arterial SMCs is negatively modulated by cyclic nucleotides.

Downregulation of fasting-induced cAMP response element-mediated gene induction by leptin in neuropeptide Y neurons of the arcuate nucleus.

States of increased metabolic demand such as fasting modulate hypothalamic neuropeptide gene expression and decrease circulating leptin levels. This study tested the hypotheses that fasting stimulates gene induction mediated by cAMP response element (CRE)-dependent increases in gene transcription and that fasting-induced decreases in leptin can regulate this CRE-mediated gene induction. Using C57BL/6J mice transgenic for a CRE-lacZ construct, an immunocytochemical study showed that fasting activated reporter gene expression in the hypothalamic arcuate nucleus (Arc) in a small subset of neurons and increased phosphorylation of CRE binding protein. The increase of beta-galactosidase expression caused by fasting was inhibited by a protein kinase A inhibitor, Rp-8-Br-cAMPS, when the compound was microinjected into the medial basal hypothalamus, and enhanced by intraperitoneal injection of selective phosphodiesterase inhibitors. In situ hybridization studies showed that neuropeptide Y (NPY) mRNA levels increased in the Arc during fasting, whereas proopiomelanocortin (POMC) mRNA levels decreased. Double labeling of mRNA and beta-galactosidase immunoreactivity in the fasted brain indicated that the subpopulation of the neurons expressing beta-galactosidase all produced NPY but not POMC. To study the possible involvement of decreased circulating leptin during starvation on CRE-mediated gene induction, leptin was administered intraperitoneally to fasted mice. Leptin significantly attenuated both beta-galactosidase expression and NPY gene expression stimulated by fasting, suggesting that leptin inhibits fasting-stimulated NPY gene expression at least in part through downregulation of CRE-mediated gene induction in the Arc. Leptin-induced modification of CRE-mediated gene induction in the Arc may play an essential role in the central regulation of feeding behavior and energy expenditure.

Upregulation of phosphodiesterase 1A1 expression is associated with the development of nitrate tolerance.

BACKGROUND: The efficacy of nitroglycerin (NTG) as a vasodilator is limited by tolerance, which develops shortly after treatment begins. In vascular smooth muscle cells (VSMCs), NTG is denitrated to form nitric oxide (NO), which activates guanylyl cyclase and generates cGMP. cGMP plays a key role in nitrate-induced vasodilation by reducing intracellular Ca(2+) concentration. Therefore, one possible mechanism for development of nitrate tolerance would be increased activity of the cGMP phosphodiesterase (PDE), which decreases cGMP levels. METHODS AND RESULTS: To test this hypothesis, rats were made tolerant by continuous infusion of NTG for 3 days (10 microgram kg(-1). min(-1) SC) with an osmotic pump. Analysis of PDE activities showed an increased function of Ca(2+)/calmodulin (CaM)-stimulated PDE (PDE1A1), which preferentially hydrolyzes cGMP after NTG treatment. Western blot analysis for the Ca(2+)/CaM-stimulated PDE revealed that PDE1A1 was increased 2.3-fold in NTG-tolerant rat aortas. Increased PDE1A1 was due to mRNA upregulation as measured by relative quantitative reverse transcription-polymerase chain reaction. The PDE1-specific inhibitor vinpocetine partially restored the sensitivity of the tolerant vasculature to subsequent NTG exposure. In cultured rat aortic VSMCs, angiotensin II (Ang II) increased PDE1A1 activity, and vinpocetine blocked the effect of Ang II on decrease in cGMP accumulation. CONCLUSIONS: Induction of PDE1A1 in nitrate-tolerant vessels may be one mechanism by which NO/cGMP-mediated vasodilation is desensitized and Ca(2+)-mediated vasoconstriction is supersensitized. Inhibiting PDE1A1 expression and/or activity could be a novel therapeutic approach to limit nitrate tolerance.

Primary sequence of cyclic nucleotide phosphodiesterase isozymes and the design of selective inhibitors.

Primary sequence information has been reported for more than 15 different mammalian cyclic nucleotide phosphodiesterases. Moreover, recent observations suggest that many of these isozymes are selectively expressed in a limited number of cell types. The fact that nearly all these different phosphodiesterases have unique primary sequences in their catalytic or regulatory domains and that they are often selectively expressed implies that it may be possible to modulate individual isozymes using specific drugs. Joe Beavo and David Reifsnyder summarize much of the evidence that has led to our current understanding of multiple isozymes of phosphodiesterase, with emphasis on aspects that may be relevant to drug design. They also discuss why many previous attempts to isolate isozyme-selective inhibitors may have failed.

Identification and characterisation of a human calmodulin-stimulated phosphodiesterase PDE1B1.

A cDNA encoding a calmodulin-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE) was isolated from a human brain cDNA library. The cDNA, designated HSPDE1B1, encoded a protein of 536 amino acids that shared 96% sequence identity with the bovine "63 kDa" calmodulin-stimulated PDE. The recombinant protein had cyclic nucleotide phosphodiesterase activity that was stimulated approximately 2-fold by Ca2+/calmodulin and preferred cGMP as substrate. In addition, the enzymatic activity of HSPDE1B1 was inhibited by phosphodiesterase inhibitors with potencies similar to that displayed toward the bovine PDE1 enzymes: IBMX approximately equal to 8-methoxymethyl-IBMX > vinpocetine approximately equal to zaprinast > cilostamide > rolipram. HSPDE1B1 mRNA was found predominantly in the brain. Lower mRNA levels were found in heart and skeletal muscle. In situ hybridisation of brain revealed expression of HSPDE1B1 predominately in neuronal cells of the cerebellum, hippocampus and caudate. The HSPDE1B1 gene was mapped to human chromosome 12. A partial genomic sequence of HSPDE1B1 was isolated and shown to contain two splice junctions that are conserved in the rat PDE4 and the Drosophila dunce genes.

Immunocytochemical localization of cyclic guanosine monophosphate-dependent protein kinase in endocrine tissues.

Antisera have been produced against purified soluble cyclic guanosine monophosphate (cGMP) dependent-protein kinase (ATP: protein phosphotransferase EC 2.7.1.37) isolated from bovine lung. No cross-reactivity was observed between the antisera and structurally related components of cAMP-dependent protein kinases (cAMP kinase), as judged by the immunodiffusion and immunoprecipitation techniques. Immunocytochemical specificity was determined by absorption of antisera with pure antigen. The distribution of cGMP kinase has been examined in several rat tissues, using an indirect immunofluorescence technique, and compared with the immunocytochemical distribution of cGMP. In skeletal muscle, cGMP kinase was localized primarily to A bands on the muscle fiber and along the Z line in I band regions. Densitometric determinations of immunoperoxidase staining indicated that absorbance over A band areas was greater than absorbance over the I band regions. In small intestine, cGMP kinase is distributed primarily along the villus brush border membrane. In testis, cGMP kinase is observed in several cell types adjacent to the seminiferous tubular wall, including Sertoli cells and spermatogonia, as well as in association with meiotic chromosomes of pachytene spermatocytes. In the cortex of the adrenal glands from dexamethasone-suppressed rats, chronic ACTH treatment induced an increase in cGMP kinase fluorescence in nuclei. In each of the tissues examined, a striking correlation was observed between the distribution of cGMP kinase and cGMP, supporting the hypothesis that cGMP-mediated actions occur via cGMP kinases.

Hormonal effects on the immunocytochemical location of 3',5'-cyclic adenosine monophosphate-dependent protein kinase in rat tissues.

Homogeneous preparations of type I and type II regulatory subunits (RI and RII, respectively) of cAMP-dependent protein kinase (cAMP kinase) were utilized as antigens to obtain isozyme specific antisera. Injections of pure catalytic subunit (C) from the type I isozyme resulted in antisera that reacted with C subunit obtained from either isozyme type. Cross-reactivity of the antisera raised against isolated subunits of the kinase was assessed by immunodiffusion analysis and by measuring the cAMP binding and phosphotransferase activities of the subunits after immunoprecipitation. These antisera were used to localize subunits of type I and type II cAMP kinases in rat skeletal muscle, liver, and adrenal by using indirect immunofluorescence and immunoperoxidase techniques. Specificity of the immunofluorescence was shown by absorption of the antisera with pure homologous antigens. In skeletal muscle, both R and C subunits of the type I and type II cAMP kinases were localized in the area of the sarcoplasmic reticulum and in periodic crossbands. Specific fluorescence for these components was observed in both isotropic and anisotropic band regions of the sarcomere. Densitometric determinations of immunoperoxidase staining revealed a larger amount of RI, RII, and C subunits in the isotropic band than in the anisotropic band regions. In liver, C, RI, and RII subunits were distributed both in cytoplasmic and nuclear areas and along plasma membranes of hepatocytes; however, there were qualitative differences observed among these various subcellular sites. With each antiserum, fluorescence was blocked by prior absorption with homologous antigen. After treatment of rats with glucagon, dramatic changes in the relative distribution patterns of C and RII were noted in the nucleus. In the adrenal gland, RI, RII, and C subunits were localized in both cytoplasmic and nuclear areas, and an apparent redistribution of these subunits occurred after treatment of (dexamethasone-suppressed) rats with ACTH. The application of this immunocytochemical approach provides a tool for examining and monitoring the subcellular distribution of these components of cAMP kinase in biological systems.

Isolation and characterization of human cDNAs encoding a cGMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase.

Human cyclic GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE2A3) cDNAs were cloned from hippocampus and fetal brain cDNA libraries. A 4.2-kb composite DNA sequence constructed from overlapping cDNA clones encodes a 941 amino acid protein with a predicted molecular mass of 105,715 Da. Extracts prepared from yeast expressing the human PDE2A3 hydrolyzed both cyclic AMP (cAMP) and cyclic GMP (cGMP). This activity was inhibited by EHNA, a selective PDE2 inhibitor, and was stimulated three-fold by cGMP. Human PDE2A is expressed in brain and to a lesser extent in heart, placenta, lung, skeletal muscle, kidney and pancreas. The human PDE2A3 differs from the bovine PDE2A1 and rat PDE2A2 proteins at the amino terminus but its amino-terminal sequence is identical to the bovine PDE2A3 sequence. The different amino termini probably arise from alternative exon splicing of the PDE2A mRNA.

Isolation and characterization of cDNAs encoding PDE5A, a human cGMP-binding, cGMP-specific 3',5'-cyclic nucleotide phosphodiesterase.

Human cGMP-binding, cGMP-specific 3',5'-cyclic nucleotide phosphodiesterase (PDE5A) cDNAs were isolated. A 3.1-kb composite DNA sequence assembled from overlapping cDNAs encodes an 875-amino-acid protein with a predicted molecular mass of 100012 Da (PDE5A1). Extracts prepared from yeast expressing human PDE5A1 hydrolyzed cGMP. This activity was inhibited by the selective PDE5 inhibitors zaprinast and DMPPO. PDE5A mRNA is expressed in aortic smooth muscle cells, heart, placenta, skeletal muscle and pancreas and, to a much lesser extent, in brain, liver and lung. A 5'-splice variant, PDE5A2, encodes an 833-amino-acid protein with eight unique amino acids at the amino terminus. PDE5A maps to chromosome 4q 25-27.

Affinities of bovine photoreceptor cGMP phosphodiesterases for rod and cone inhibitory subunits.

Rods and cones have analogous phototransduction components and cycles, but differ from each other in their physiological response to light. Differences between the affinities of rod and cone phosphodiesterase (PDE) catalytic subunits for their respective inhibitory subunits could potentially contribute to these physiological differences. To test this idea, we expressed both the 13 kDa PDE subunit, unique to a subset of bovine retinal cones [(1990) J. Biol. Chem. 265, 11259-11264], and the rod PDE 11 kDa inhibitory subunit in E. coli, purified them, and compared their abilities to inhibit rod and cone PDE catalytic subunits. Rod PDE has similar Ki values (approximately 80 pM) for both the rod and cone recombinant inhibitory subunits. Activated cone PDE has Ki values of 200 pM for the cone 13 kDa subunit and 600 pM for rod PDE gamma.

Purification of calmodulin-stimulated cyclic nucleotide phosphodiesterase by monoclonal antibody affinity chromatography.

Immobilized ACC-1 and ACAP-1 antibodies are effective tools for the purification of active calmodulin-dependent phosphodiesterases. ACC-1 antibody binds all bovine and rat brain isozymes in a Ca2+-dependent manner and has been used for their purification. Since ACC-1 binds both bovine brain isozymes (61- and 63-kDa forms) and ACAP-1 recognizes only the 61-kDa isozyme, ACAP-1 can be used to separate and purify the two brain isozymes. The procedures described here for phosphodiesterase isolation from brain are rapid and require few enzymatic assays, resulting in preparations of good purity, specific activity, and yield (Tables II, III). The procedures for brain tissue can be easily adapted for use with larger amounts of tissue. The cross-reactivity of ACP-1 for rat brain phosphodiesterase suggests that this antibody may recognize isozymes from other mammalian tissues.

Analysis of the functional role of cGMP-dependent protein kinase in intact human platelets using a specific activator 8-para-chlorophenylthio-cGMP.

8-(p-Chlorophenylthio)-cGMP (8-pCPT-cGMP) and 8-bromo-cGMP were compared with respect to their chemical and biological properties in order to evaluate their potential as selective activators of cGMP-dependent protein kinase (cGMP-PK; EC 2.7.1.37) in intact human platelets. 8-pCPT-cGMP, 8-Br-cGMP and cGMP were shown to be potent and selective activators of purified bovine lung cGMP-PK and of cGMP-PK present in human platelet membranes when compared with the activation of cAMP-dependent protein kinase (cAMP-PK; EC 2.7.1.37). 8-pCPT-cGMP was not hydrolysed by the purified cGMP-stimulated phosphodiesterase (cGS-PDE), cGMP-inhibited phosphodiesterase (cGI-PDE) and Ca(2+)-calmodulin-dependent phosphodiesterase (CaM-PDE), whereas cGMP and, to a lesser extent, 8-Br-cGMP were hydrolysed by all three types of 3',5' cyclic nucleotide phosphodiesterases (EC 3.1.4.17) examined. Also, 8-pCPT-cGMP was not hydrolysed by a human platelet homogenate which contains a high level of the cGMP-specific cGMP-binding phosphodiesterase (cGB-PDE). Additionally, 8-pCPT-cGMP did not activate the cGS-PDE or inhibit the cGI-PDE, whereas half-maximal inhibition of cGI-PDE occurred at 8 microM 8-Br-cGMP. The apparent lipophilicity of 8-pCPT-cGMP was higher than that of 8-Br-cGMP. Extracellular application of 8-pCPT-cGMP to intact human platelets reproduced the pattern of protein phosphorylation induced by sodium nitroprusside (SNP), a cGMP-elevating inhibitor of platelet activation. Quantitatively, 8-pCPT-cGMP was more effective than 8-Br-cGMP in inducing phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein, a major substrate of cGMP-PK in intact platelets. As observed with SNP, pretreatment of human platelets with 8-pCPT-cGMP prevented the aggregation induced by thrombin. The results suggest that 8-pCPT-cGMP is a very potent and selective activator of cGMP-PK in cell extracts and in intact human platelets and, in this respect, is superior to 8-Br-cGMP and other cGMP analogs used for intact cell studies. The data also suggest that inhibition of platelet activation in intact human platelets by nitrovasodilators is mediated by cGMP-PK.

Cyclic AMP-dependent and cyclic AMP-independent actions of a novel cardiotonic agent, OPC-8212.

Possible cAMP-dependent and cAMP-independent mechanisms of action for the cardiac effects of OPC-8212, a novel piperazinyl-quinolinone derivative, were evaluated. OPC-8212 was tested for in vitro potency as an inhibitor of soluble bovine cardiac phosphodiesterases using a rapid isolation and assay method involving monoclonal antibodies that distinguish among isozymes. The drug was selective for a low-Km, cGMP-inhibited phosphodiesterase (CGI-PDE) with an IC50 (half-maximal inhibition concentration) of 7.4 mumol/l when measured at a substrate level of 0.35 mumol/l cAMP. Under the conditions used, sulfolane, the solvent for OPC-8212, did not affect CGI-PDE activity. In electrophysiological measurements, OPC-8212 prolonged the action potential duration in canine Purkinje strand preparations up to 148% (APD90) at 10 mumol/l. Concomitantly, OPC-8212 produced a 100% increase in developed force. Both prolongation of the action potential duration and the positive inotropic effect were readily reversed after exposure to tetrodotoxin, 3 mumol/l. Using Na-selective microelectrodes, intracellular Na+ ion activity increased 225% upon exposure to 10 mumol/l OPC-8212. OPC-8212 represents a novel type of positive inotropic agent, possessing both cAMP-dependent (selective PDE isozyme inhibition) and cAMP-independent (activation of intracellular Na+) mechanism of action.