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J A Beavo

Publications and source records attributed to J A Beavo.

At least 37 records · Page 2Linked to original sources
PubMed1998

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.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1998

Cloning and characterization of a cAMP-specific cyclic nucleotide phosphodiesterase.

Cyclic nucleotide phosphodiesterases (PDEs) regulate intracellular levels of cAMP and cGMP by hydrolyzing them to their corresponding 5' monophosphates. We report here the cloning and characterization of a novel cAMP-specific PDE from mouse testis. This unique phosphodiesterase contains a catalytic domain that overall shares <40% sequence identity to the catalytic domain of all other known PDEs. Based on this limited homology, this new PDE clearly represents a previously unknown PDE gene family designated as PDE8. The cDNA for PDE8 is 3,678 nucleotides in length and is predicted to encode an 823 amino acid enzyme. The cDNA includes a full ORF as it contains an in-frame stop codon before the start methionine. PDE8 is specific for the hydrolysis of cAMP and has a Km of 0.15 microM. Most common PDE inhibitors are ineffective antagonists of PDE8, including the nonspecific PDE inhibitor 3-isobutyl-1-methylxanthine. Dipyridamole, however, an inhibitor that is generally considered to be relatively specific for the cGMP selective PDEs, does inhibit PDE8 with an IC50 of 4.5 microM. Tissue distribution studies of 22 different mouse tissues indicates that PDE8 has highest expression in testis, followed by eye, liver, skeletal muscle, heart, 7-day embryo, kidney, ovary, and brain in decreasing order. In situ hybridizations in testis, the tissue of highest expression, shows that PDE8 is expressed in the seminiferous epithelium in a stage-specific manner. Highest levels of expression are seen in stages 7-12, with little or no expression in stages 1-6.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Identification and characterization of a novel family of cyclic nucleotide phosphodiesterases.

We report the cloning, expression, and characterization of a new family of cyclic nucleotide phosphodiesterase (PDE) that has unique kinetic and inhibitor specificities. A clone corresponding to the C terminus of this PDE was initially identified by a bioinformatic approach and used to isolate a cDNA that is likely full-length. This novel PDE, designated as MMPDE9A1, shows highest mRNA expression in kidney with lower levels in liver, lung, and brain. The mRNA size by Northern blot analysis is approximately 2.0 kilobases, and the cDNA encoding PDE9A1 is 1929 base pairs in length. The largest open reading frame predicts a protein of 534 amino acids with a molecular mass of 62,000 Da. When expressed in COS-7 cells, PDE9A1 activity was not inhibited well by either the nonselective inhibitor 3-isobutyl-1-methyl-xanthine or the new selective PDE5 inhibitor, sildenafil, but it is inhibited by the PDE1/5 inhibitor (+)-cis-5,6a, 7,8,9 hyl] phenylmethyl]-5-methyl-cylopent[4,5]imidao[2, 1-b]purin-49(3H)one (SCH51866) with an IC50 of 1.55 microM. This new phosphodiesterase is highly specific for cGMP. Its Km of approximately 0.07 microM for cGMP is the lowest yet reported for a PDE, being at least 40-170 times lower than that of PDE5 and PDE6, respectively.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1998

Characterization of human and mouse rod cGMP phosphodiesterase delta subunit (PDE6D) and chromosomal localization of the human gene.

The mammalian multisubunit photoreceptor cGMP phosphodiesterase PDE alpha beta gamma 2 (PDE6 family) is a peripherally membrane-associated enzyme. A novel subunit, termed PDE delta (HGMW-approved symbol, PDE6D; MW 17 kDa), is able to detach PDE partially from bovine rod outer segment membranes under physiological conditions. Cloning of human and mouse PDE delta cDNAs revealed that PDE delta is a nearly perfectly conserved polypeptide of 150 amino acids that shows partial sequence homology to photoreceptor RG4 of unknown function. Multiple-species Southern blot analysis demonstrates that the PDE delta gene has been well conserved during evolution and is detectable at high stringency in invertebrates. The human and mouse genes are contained in less than 8 kb of genomic DNA and consist of four exons and three introns (0.7-4 kb in human, 0.7-2.2 kb in mouse). The PDE delta gene structure is identical to that of the C27H5.1 gene identified in the eyeless nematode Caenorhabditis elegans. The human PDE delta gene (locus designation PDE6D) was localized to the long arm of chromosome 2 (2q35-q36) by fluorescence in situ hybridization. By synteny, the mouse PDE delta gene is predicted to reside on chromosome 1.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1998

Identification, quantitation, and cellular localization of PDE1 calmodulin-stimulated cyclic nucleotide phosphodiesterases.

The calmodulin-stimulated cyclic nucleotide phosphodiesterases (PDE1s) constitute a large gene family and are found in a wide variety of tissues and cells. Because of the functional diversity of PDE1 genes and the observation that these isozymes often make up a major component of the total cyclic nucleotide hydrolytic activity in certain cell types, PDE1s are of growing interest as targets for therapeutic intervention. Here we describe a series of methodologies to identify, quantitate, and determine the cellular expression of PDE1 isozymes. We describe first the resolution of different PDEs using high-performance anion-exchange chromatography and then a Western blotting methodology for identifying or authenticating PDE1 activities. Next we present an immunoprecipitation method that can be used for quantitating specific PDE1 isoforms and describe the use of RNase protection analysis for further identification of PDE1 subtypes. Finally, we provide a simple, immunocytochemical method for determining the cellular expression of PDE1 isozymes. Combined, the above methodologies should allow an investigator to identify, quantitate, and determine the cellular localization of PDE1 isozymes in any tissue with little ambiguity.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

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.

Adenylyl Cyclase Inhibitors↗
PubMed1997

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.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

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.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1997

Attenuation of insulin secretion by insulin-like growth factor 1 is mediated through activation of phosphodiesterase 3B.

Both insulin and insulin-like growth factor 1 (IGF-1) are known to reduce glucose-dependent insulin secretion from the beta cells of pancreatic islets. In this paper we show that the mechanism by which IGF-1 mediates this effect is in large part through activation of a specific cyclic nucleotide phosphodiesterase, phosphodiesterase 3B (PDE3B). More specifically, in both isolated pancreatic islets and insulin-secreting HIT-T15 cells, IGF-1 inhibits insulin secretion that has been increased by glucose and glucagonlike peptide 1 (GLP-1). Moreover, IGF-1 decreases cAMP levels in parallel to the reduction of insulin secretion. Insulin secretion stimulated by cAMP analogs that activate protein kinase A and also are substrates for PDE3B is also inhibited by IGF-1. However, IGF-1 does not inhibit insulin secretion stimulated by nonhydrolyzable cAMP analogs. In addition, selective inhibitors of PDE3B completely block the ability of IGF-1 to inhibit insulin secretion. Finally, PDE3B activity measured in vitro after immunoprecipitation from cells treated with IGF-1 is higher than the activity from control cells. Taken together with the fact that pancreatic beta cells express little or no insulin receptor but large amounts of IGF-1 receptor, these data strongly suggest a new regulatory feedback loop model for the control of insulin secretion. In this model, increased insulin secretion in vivo will stimulate IGF-1 synthesis by the liver, and the secreted IGF-1 in turn feedback inhibits insulin secretion from the beta cells through an IGF-1 receptor-mediated pathway. This pathway is likely to be particularly important when levels of both glucose and secretagogues such as GLP-1 are elevated.

Animals↗
PubMed1997

A subset of olfactory neurons that selectively express cGMP-stimulated phosphodiesterase (PDE2) and guanylyl cyclase-D define a unique olfactory signal transduction pathway.

Odorant information is encoded by a series of intracellular signal transduction events thought to be mediated primarily by the second messenger cAMP. We have found a subset of olfactory neurons that express the cGMP-stimulated phosphodiesterase (PDE2) and guanylyl cyclase-D (GC-D), suggesting that cGMP in these neurons also can have an important regulatory function in olfactory signaling. PDE2 and GC-D are both expressed in olfactory cilia where odorant signaling is initiated; however, only PDE2 is expressed in axons. In contrast to most other olfactory neurons, these neurons appear to project to a distinct group of glomeruli in the olfactory bulb that are similar to the subset that have been termed "necklace glomeruli." Furthermore, this subset of neurons are unique in that they do not contain several of the previously identified components of olfactory signal transduction cascades involving cAMP and calcium, including a calcium/calmodulin-dependent PDE (PDE1C2), adenylyl cyclase III, and cAMP-specific PDE (PDE4A). Interestingly, these latter three proteins are expressed in the same neurons; however, their subcellular distribution is distinct. PDE1C2 and adenylyl cyclase III are expressed almost exclusively in the olfactory cilia whereas PDE4A is present only in the cell bodies and axons. These data strongly suggest that selective compartmentalization of different PDEs and cyclases is an important feature for the regulation of signal transduction in olfactory neurons and likely in other neurons as well. In addition, the data implies that an olfactory signal transduction pathway specifically modulated by cGMP is present in some neurons of the olfactory neuroepithelium.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1997

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.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Identification and tissue-specific expression of PDE7 phosphodiesterase splice variants.

Type 7 cyclic nucleotide phosphodiesterases (PDE7s) are a newly described family of enzymes having high affinity and specificity for cAMP. However, little is known about their structure, function, or regulation. We have isolated a mouse skeletal muscle cDNA representing a new alternative splice variant (PDE7A2) of the PDE7 gene. The ORF encodes a 456-amino acid protein having a predicted molecular weight of 52.4 kDa. The 5' end of the mouse PDE7A2 is divergent from the 5' end of the human PDE7A1 sequence and is more hydrophobic. A comparison of the 5' ends of the two cDNA clones with human genomic sequence indicates that they represent alternate splice products rather than species variation. RNase protection analysis of several mouse tissues indicates that PDE7 is expressed widely with highest levels in skeletal muscle. HPLC fractionation and Western blot analysis of two human lymphocyte T-cell lines shows that an unknown PDE activity described by Ichimura and Kase [Ichimura, M. & Kase, H. (1993) Biochem. Biophys. Res. Commun. 193, 985-990] is most likely to be PDE7A1. A single immunoreactive band of approximately 55 kDa, which comigrates with PDE7A1, is seen in fractions of the HPLC profile containing this activity suggesting that the original human PDE7A1 clone contains a full-length ORF, and is not truncated at the 5' end as was originally postulated. In a human lymphocyte B-cell line and also in mouse skeletal muscle, a large amount of PDE7 mRNA but little PDE7 protein or activity is expressed suggesting that the translation or stability of PDE7 protein may be highly regulated in these tissues.

Alternative Splicing↗
PubMed1996

The calmodulin-dependent phosphodiesterase gene PDE1C encodes several functionally different splice variants in a tissue-specific manner.

We report here the identification of cDNAs for three new mouse PDE1C splice variants and the characterization of their kinetics, regulation by Ca2+, sensitivities to inhibitors, and tissue/cellular expression patterns. Sequence analysis indicated that these three cDNAs (PDE1C1, PDE1C4, and PDE1C5), together with our previously reported PDE1C2 and PDE1C3, are alternative splice products of the PDE1C gene. The results from RNase protection analysis and in situ hybridization indicated that the expression of the different PDE1C splice variants is differentially regulated in a tissue/cell-specific manner. Particularly, high levels of PDE1C mRNAs were found in the olfactory epithelium, testis, and several regions of mouse brain such as cerebellar granule cells. All of these splice variants have similar kinetic properties, showing high affinities and approximately the same relative Vmax values for both cAMP and cGMP. However, they responded to Ca2+ stimulation differently. In addition, they show different sensitivities to the calmodulin-dependent phosphodiesterase inhibitors, KS505a and SCH51866. Substrate competition experiments suggested the presence of only one catalytic site on these PDE1C isozymes for both cAMP and cGMP. In summary, these findings suggest that the PDE1C gene undergoes tissue-specific alternative splicing that generates structurally and functionally diverse gene products.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Solubilization of membrane-bound rod phosphodiesterase by the rod phosphodiesterase recombinant delta subunit.

Retinal rod and cone phosphodiesterases are oligomeric enzymes that consist of a dimeric catalytic core (alpha'2 in cones and alphabeta in rods) with inhibitory subunits (gamma) that regulate their activity. In addition, a 17-kDa protein referred to as the delta subunit co-purifies with the rod soluble phosphodiesterase and the cone phosphodiesterase. We report here partial protein sequencing of the rod delta subunit and isolation of a cDNA clone encoding it. The predicted amino acid sequence is unrelated to any other known protein. Of eight bovine tissue mRNA preparations examined by Northern analysis, the strongest delta subunit-specific signal was present in the retina. A less intense signal was seen in the brain and adrenal mRNA. In bovine retinal sections, rod delta subunit anti-peptide antibodies label rod but not cone outer segments. delta subunit, added back to washed outer segment membranes, solubilizes a large fraction of the membrane-bound phosphodiesterase, indicating that this subunit binds to the classical membrane associated phosphodiesterase. The subunit forms a tight complex with native, but not trypsin-released phosphodiesterase, suggesting that the isoprenylated carboxyl termini of the catalytic subunits may be involved in binding of the delta subunit to the phosphodiesterase holoenzyme.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1996

Isolation and characterization of cDNAs corresponding to two human calcium, calmodulin-regulated, 3',5'-cyclic nucleotide phosphodiesterases.

cDNAs corresponding to two human calcium, calmodulin (CaM)-regulated 3',5'-cyclic nucleotide phosphodiesterases (PDEs) were isolated. One, Hcam1 (PDE1A3), corresponds to the bovine 61-kDa CaM PDE (PDE1A2). The second, Hcam3 (PDE1C), represents a novel phosphodiesterase gene. Hcam1 encodes a 535-amino acid protein that differs most notably from the bovine 61-kDa CaM PDE by the presence of a 14-amino acid insertion and a divergent carboxyl terminus. RNase protection studies indicated that Hcam1 is represented in human RNA from several tissues, including brain, kidney, testes, and heart. Two carboxyl-terminal splice variants for Hcam3 were isolated. One, Hcam3b (PDE1C1), encodes a protein 634 amino acids (72 kDa) in length. The other, Hcam3a (PDE1C3), diverges from Hcam3b 4 amino acids from the carboxyl terminus of Hcam3b, and extends an additional 79 amino acids. All the cDNAs isolated for Hcam3a are incomplete; they do not include the 5'-end of the open reading frame. Northern analysis revealed that both splice variants were expressed in several tissues, including brain and heart, and that there may be additional splice variants. Amino-truncated recombinant proteins were expressed in yeast and characterized biochemically. Hcam3a has a high affinity for both cAMP and cGMP and thus has distinctly different kinetic parameters from Hcam1, which has a higher affinity for cGMP than for cAMP. Both PDE1C enzymes were inhibited by isobutylmethylxanthine, 8-methoxymethyl isobutylmethylxanthine, zaprinast, and vinpocetine.

3',5'-Cyclic-AMP Phosphodiesterases↗