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J A Beavo

Publications and source records attributed to J A Beavo.

At least 55 records · Page 3Linked to original sources
PubMed1995

Identification of inhibitory and calmodulin-binding domains of the PDE1A1 and PDE1A2 calmodulin-stimulated cyclic nucleotide phosphodiesterases.

Using a bovine 61-kDa (PDE1A2) calmodulin-stimulated phosphodiesterase (CaM-PDE) cDNA and a bovine lung 59-kDa (PDE1A1) CaM-PDE cDNA reported here, we have identified two new regions within the primary structure of these two related isozymes that are important for regulation by Ca2+/CaM. PDE1A1 is identical to the PDE1A2 isozyme except for the amino-terminal 18 residues. In agreement with earlier studies, the CaM concentration required for half-maximal activation (KCaM) of recombinant PDE1A1 (0.3 nM) was approximately 10-fold less than the KCaM for recombinant PDE1A2 (4 nM). A series of deletion mutations of the PDE1A2 cDNA removing nucleotide sequence encoding the first 46-106 aminoterminal residues were constructed and expressed using the baculovirus system. Deletion of the amino acids encompassing a previously identified, putative CaM-binding domain (residues 4-46) produced a polypeptide that was still activated 3-fold by CaM (KCaM approximately 3 nM). However, complete CaM-independent activation occurred when residues 4-98 were deleted. To determine the location of the additional CaM-binding domain(s), the inhibitory potency of seven overlapping, synthetic peptides spanning amino acids 76-149 of PDE1A2 was tested using the CaM-activated enzyme. One peptide spanning amino acids 114-137 of PDE1A2 appeared to be the most potent inhibitor of CaM-stimulated activity. These results reveal the existence of a CaM-binding domain located approximately 90 residues carboxyl-terminal to the putative CaM-binding domains previously identified within the PDE1A1 and PDE1A2 isozymes. Moreover, a discrete segment important for holding these CaM-PDEs in a less active state at low Ca2+ concentrations is located between the two CaM-binding domains.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

An essential aspartic acid at each of two allosteric cGMP-binding sites of a cGMP-specific phosphodiesterase.

The amino acid sequences of all known cGMP-binding phosphodiesterases (PDEs) contain internally homologous repeats (a and b) that are 80-90 residues in length and are arranged in tandem within the putative cGMP-binding domains. In the bovine lung cGMP-binding, cGMP-specific PDE (cGB-PDE or PDE5A), these repeats span residues 228-311 (a) and 410-500 (b). An aspartic acid (residue 289 or 478) that is invariant in repeats a and b of all known cGMP-binding PDEs was changed to alanine by site-directed mutagenesis of cGB-PDE, and wild type (WT) and mutant cGB-PDEs were expressed in COS-7 cells. Purified bovine lung cGB-PDE (native) and WT cGB-PDE displayed identical cGMP-binding kinetics, with approximately 1.8 microM cGMP required for half-maximal saturation. The D289A mutant showed decreased affinity for cGMP (Kd > 10 microM) and the D478A mutant showed increased affinity for cGMP (Kd approximately 0.5 microM) as compared to WT and native cGB-PDE. WT and native cGB-PDE displayed an identical curvilinear profile of cGMP dissociation which was consistent with the presence of distinct slowly dissociating (koff = 0.26 h-1) and rapidly dissociating (koff = 1.00 h-1) sites of cGMP binding. In contrast, the D289A mutant displayed a single koff = 1.24 h-1, which was similar to the calculated koff for the fast site of WT and native cGB-PDE, and the D478A mutant displayed a single koff = 0.29 h-1, which was similar to that calculated for the slow site of WT and native cGB-PDE. These results were consistent with the loss of a slow cGMP-binding site in repeat a of the D289A mutant cGB-PDE, and the loss of a fast site in repeat b of the D478A mutant, suggesting that cGB-PDE possesses two distinct cGMP-binding sites located at repeats a and b, with the invariant aspartic acid being crucial for interaction with cGMP at each site.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1995

Molecular cloning and characterization of a calmodulin-dependent phosphodiesterase enriched in olfactory sensory neurons.

The sensing of an odorant by an animal must be a rapid but transient process, requiring an instant response and also a speedy termination of the signal. Previous biochemical and electrophysiological studies suggest that one or more phosphodiesterases (PDEs) may play an essential role in the rapid termination of the odorant-induced cAMP signal. Here we report the molecular cloning, expression, and characterization of a cDNA from rat olfactory epithelium that encodes a member of the calmodulin-dependent PDE family designated as PDE1C. This enzyme shows high affinity for cAMP and cGMP, having a Km for cAMP much lower than that of any other neuronal Ca2+/calmodulin-dependent PDE. The mRNA encoding this enzyme is highly enriched in olfactory epithelium and is not detected in six other tissues tested. However, RNase protection analyses indicate that other alternative splice variants related to this enzyme are expressed in several other tissues. Within the olfactory epithelium, this enzyme appears to be expressed exclusively in the sensory neurons. The high affinity for cAMP of this Ca2+/calmodulin-dependent PDE and the fact that its mRNA is highly concentrated in olfactory sensory neurons suggest an important role for it in a Ca(2+)-regulated olfactory signal termination.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms.

In the last few years there has been a veritable explosion of knowledge about cyclic nucleotide phosphodiesterases. In particular, the accumulating data showing that there are a large number of different phosphodiesterase isozymes have triggered an equally large increase in interest about these enzymes. At least seven different gene families of cyclic nucleotide phosphodiesterase are currently known to exist in mammalian tissues. Most families contain several distinct genes, and many of these genes are expressed in different tissues as functionally unique alternative splice variants. This article reviews many of the more important aspects about the structure, cellular localization, and regulation of each family of phosphodiesterases. Particular emphasis is placed on new information obtained in the last few years about how differential expression and regulation of individual phosphodiesterase isozymes relate to their function(s) in the body. A substantial discussion of the currently accepted nomenclature is also included. Finally, a brief discussion is included about how the differences among distinct phosphodiesterase isozymes are beginning to be used as the basis for developing therapeutic agents.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Characterization of cyclic nucleotide phosphodiesterases with cyclic GMP analogs: topology of the catalytic domains.

To help define essential interactions of cGMP with the catalytic site, we tested a series of cGMP analogs as competitive inhibitors of each cyclic nucleotide phosphodiesterase (PDE) family known to hydrolyze cGMP (PDE1, PDE2, PDE3, PDE5, and PDE6). IC50 values, relative to cGMP, were used to predict which functional groups of cGMP contribute to binding by the catalytic sites of each isozyme. The results indicate that the N1-nitrogen of cGMP contributes to binding at the catalytic site of all PDEs, probably as a hydrogen donor. All PDEs tested, with the exception of PDE2, also use the 6-oxo group, probably as a hydrogen acceptor. In contrast to other cGMP-binding enzymes, the 2-amino and 2'-hydroxyl groups of cGMP are not major requirements for binding to any PDE. The 8-bromo- and 8-p-chlorophenylthio-substituted analogs inhibit PDE1, PDE2, and PDE6 activity with high relative affinities, suggesting that these PDEs are not sterically hindered with bulky 8-position substitutions and that they do not preferentially bind the anti-conformation of cGMP. PDE3 and PDE5 have reduced apparent affinity for these analogs and therefore either are sterically hindered with these substitutions or bind cGMP in the anti-conformation. Overall, the data show substantial differences in structural requirements for cGMP binding to the catalytic sites of the different PDE families. Comparisons with published data show different structural requirements for binding to the catalytic, compared with noncatalytic, binding domains of PDEs. Even larger differences are seen between the requirements for binding to PDE catalytic sites and those for the cGMP-dependent protein kinase and the cGMP-gated cation channel.

Animals↗
PubMed1995

Characterization of cyclic nucleotide phosphodiesterases with cyclic AMP analogs: topology of the catalytic sites and comparison with other cyclic AMP-binding proteins.

To define essential interactions of cAMP with the catalytic sites of cyclic nucleotide phosphodiesterases (PDEs) and to begin to map the topology of the sites, we have tested a series of cAMP analogs as competitive inhibitors of the PDEs that hydrolyze cAMP with high efficiency (PDE1, PDE2, PDE3, and PDE4). Comparisons of IC50 values, relative to cAMP, were used to predict which functional groups on cAMP interact with each isozyme. Common to all PDEs tested, except for the calcium/calmodulin-dependent PDE (CaM-PDE, PDE1), is an interaction at the N1-position of cAMP and a distinct lack of binding to the 2'-hydroxyl group of the ribose moiety. Only the cGMP-stimulated (PDE2) and cAMP-specific (PDE4) PDEs appear to interact strongly at the N7-position. The cGMP-inhibited PDE (cGI-PDE, PDE3) may interact less strongly with this nitrogen. The PDE4 and PDE3 both interact with cAMP through the 6-amino group, which most likely serves as a hydrogen bond donor. PDE4 and PDE3 appear to be able to bind to the anti-conformer of cAMP, whereas the PDE1 and PDE2 bind the syn-conformer. The CaM-PDE exhibits no appreciable specificity for any of the analogs tested, showing little or no interaction with the 6-amino group or with any of the ring nitrogens. Large differences exist in the nucleotide-binding requirements for the PDE catalytic sites, compared with the regulatory sites of cAMP-dependent protein kinase and the catabolite activator protein.

Animals↗
PubMed1994

Phosphorylation of the 61-kDa calmodulin-stimulated cyclic nucleotide phosphodiesterase at serine 120 reduces its affinity for calmodulin.

Phosphorylation of the 61-kDa isoform of bovine calmodulin (CaM)-stimulated cyclic nucleotide phosphodiesterase (CaM-PDE) by the catalytic subunit of cyclic AMP-dependent protein kinase A (PKA) results in a decrease in the affinity of the enzyme for calmodulin [Sharma, R. K., & Wang, J. H. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 2603-2607]. In the present study, purified 61-kDa CaM-PDE was phosphorylated in the presence of [gamma-32P]ATP and cleaved with a Lys-C endoproteinase. The resultant phosphopeptides were resolved by reverse-phase HPLC and analyzed by electrospray mass spectrometry and Edman sequencing. Serine residues 120 and 138 were identified as the principal sites of phosphorylation. A cDNA encoding the 61-kDa CaM-PDE [Sonnenburg, W. K., Seger, D., & Beavo, J. A. (1993) J. Biol. Chem. 268, 645-652] was used to generate point mutants in which either or both of these serines were replaced with alanine. The mutants were expressed in COS-7 cells, purified, and phosphorylated. Phosphorylation of the mutant Ser 138-->Ala resulted in a decrease in affinity for CaM that was comparable to that seen with the wild-type enzyme. In contrast, phosphorylation of the mutant Ser 120-->Ala had virtually no effect on CaM affinity. We conclude that phosphorylation of serine 120 by PKA is responsible for the reduction in affinity of the 61-kDa CaM-PDE for CaM.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Cyclic GMP and regulation of cyclic nucleotide hydrolysis.

Several of the different PDE isozyme families have the ability in vitro to hydrolyze cGMP. In particular they include the CaM-dependent PDEs, the cGMP-stimulated PDEs, and the cGMP binding, cGMP-specific PDEs. Existing evidence suggests or demonstrates that in different cell types, each of these can be important determinants for the control of cGMP steady-state levels. Each of these enzymes is differentially expressed and regulated; moreover, the amount of the enzyme expressed and the mode of regulation determine to a large extent the rate of rise, maximal level, rate of fall, and duration of the cGMP signal in the cell. In addition to enzymes that function to degrade cGMP at least two also are regulated by cGMP both in vitro and in the intact cell. The cGMP-stimulated PDE has the ability to decrease cAMP levels in response to cGMP and the cGMP-inhibited PDE can increase cAMP levels in response to cGMP. We are just beginning to define how many different isozymes of PDE exist in mammalian tissues, where they are located, and how they are regulated. Selective inhibitors to each are being developed and studies designed to define structural features that determine the mechanisms of action and regulation of the PDEs have been initiated. It is expected that in the next few years more PDEs will be discovered and the functions of the new an existing ones with be more clearly defined.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Differential expression of the 61 kDa and 63 kDa calmodulin-dependent phosphodiesterases in the mouse brain.

Based on their relative abundance and regulation by Ca2+ and by phosphorylation in vitro, it is thought that the Ca2+/calmodulin-dependent phosphodiesterases (CaM-PDEs) are important modulators of cyclic nucleotide function in the brain. Two of the most abundant CaM-PDEs in the brain are the 61 kDa and 63 kDa isozymes. In this study, the regional and cellular expression of mRNA encoding these two different isoforms in mouse brain has been determined by in situ hybridization. The 63 kDa CaM-PDE mRNA has a wide-spread but uneven distribution. Very strong hybridization signals are present in the caudate-putamen, nucleus accumbens, olfactory tubercle, and dentate gyrus of the hippocampus. Somewhat lesser amounts of 63 kDa CaM-PDE mRNA are present in the olfactory bulb and piriform cortex. Weaker but still easily discernible hybridization signals are seen in several layers of the cerebral cortex, CA1 and CA3 regions of the hippocampus, amygdaloid nuclear complex, thalamus, hypothalamus, midbrain, brainstem, cerebellum, and spinal cord. A weak hybridization signal was detected in the globus pallidus of the basal ganglia. In general, the distribution of the 63 kDa CaM-PDE is very similar to that of dopamine receptors, suggesting that it may modulate dopamine function. In contrast, the 61 kDa CaM-PDE mRNA has a more limited and much different distribution, with the highest level of expression in the cerebral cortex and in the pyramidal cells of the hippocampus. A moderate hybridization signal was detected in the medial habenula and amygdaloid nuclear complex. In addition, small subsets of neurons in several other regions showed specific hybridization. Both PDE mRNAs appear to be localized exclusively in neuronal cell bodies. Their distinct distribution suggests important but different physiological roles for these two isozymes in the regional regulation of cyclic nucleotides in the CNS. Since these two isozymes are differentially phosphorylated by cAMP-dependent and Ca2+/CaM-dependent protein kinases, the differential expression also provides a potential mechanism by which these PDEs can differentially regulate cAMP and cGMP in different brain areas. The high expression levels in specific subsets of neurons also suggest that agents increasing Ca2+ in these neurons will increase the rate of cyclic nucleotide degradation.

Animals↗
PubMed1993

Isolation and characterization of a previously undetected human cAMP phosphodiesterase by complementation of cAMP phosphodiesterase-deficient Saccharomyces cerevisiae.

We have established a highly sensitive functional screen for the isolation of cDNAs encoding cAMP phosphodiesterases (PDEs) by complementation of defects in a Saccharomyces cerevisiae strain lacking both endogenous cAMP PDE genes, PDE1 and PDE2. Three groups of cDNAs corresponding to three distinct human genes encoding cAMP-specific PDEs were isolated from a human glioblastoma cDNA library using this functional screen. Two of these genes are closely related to the Drosophila dunce cAMP-specific PDE. The third gene, which we named HCP1, encoded a novel cAMP-specific PDE. HCP1 has an amino acid sequence related to the sequences of the catalytic domains of all cyclic nucleotide PDEs. HCP1 is a high affinity cAMP-specific PDE (Km = 0.2 microM) that does not share other properties of the cAMP-specific PDE family, i.e. extensive sequence homology to the Drosophila dunce cAMP PDE and sensitivity to rolipram and R020-1724. The PDE activity of HCP1 is not sensitive to cGMP or other inhibitors of the cGMP-inhibitable PDEs, such as milrinone. The biochemical and pharmacological properties of HCP1 suggest that it is a member of a previously undiscovered cyclic nucleotide PDE family. Northern blot analysis indicates that high levels of HCP1 mRNA are present in human skeletal muscle.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

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.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

Molecular cloning of a cDNA encoding the "61-kDa" calmodulin-stimulated cyclic nucleotide phosphodiesterase. Tissue-specific expression of structurally related isoforms.

We have isolated a 2287-bp cDNA encoding the 61-kDa calmodulin-stimulated cyclic nucleotide phosphodiesterase (CaM PDE) from a bovine brain library. A large open reading frame within the cDNA encodes a 530-residue polypeptide which is identical to the sequence of the purified protein previously determined by direct amino acid sequencing. Moreover, COS cells transfected with the cDNA express a cAMP and cGMP hydrolytic activity that is stimulated by calcium and calmodulin, confirming that the cDNA represents a mRNA species encoding a CaM PDE isozyme. RNase protection analyses indicate that either 61-kDa CaM PDE mRNA or structurally related transcripts encoding different CaM PDE isoforms are expressed in a tissue-specific manner. Total RNA isolated from brain (cerebral cortex, basal ganglia, hippocampus, cerebellum, and medulla/spinal cord), heart, aorta, liver, kidney outer medulla, kidney papilla, trachea, and lung completely protected a 410-base antisense riboprobe corresponding to sequence encoding a portion of the catalytic domain. Little or no protection was detected using adrenal cortex, adrenal medulla, liver, kidney cortex, spleen, or T-lymphocyte total RNA. Only brain RNA completely protected a 240-base antisense riboprobe corresponding to the 61-kDa CaM PDE amino terminus encompassing a putative calmodulin-binding domain. However, heart, aorta, liver, kidney, trachea, and lung RNA protected 150 bases of this riboprobe suggesting that these tissues express an isoform structurally related to the 61-kDa CaM PDE. Northern analysis of mRNA isolated from brain, heart, aorta, liver, kidney, lung, and trachea revealed that the cDNA hybridizes with a 3.8- and a 4.4-kb (kilobase) mRNA species. Interestingly, Northern blots of bovine cerebral cortex and heart mRNA probed under stringent conditions with antisense transcripts corresponding to either the 5'- or 3'-untranslated sequence of the 61-kDa CaM PDE cDNA hybridized with only the 4.4-kb mRNA from both tissues. Since different, yet structurally similar CaM PDE isoforms are expressed in brain and in heart, this result, in addition to the RNase protection data, is consistent with the idea that the mRNAs encoding these two CaM PDE isoforms are products of an alternately spliced gene.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1993

The role of protein phosphorylation in the regulation of cyclic nucleotide phosphodiesterases.

The cyclic nucleotide phosphodiesterases constitute a complex superfamily of enzymes responsible for catalyzing the hydrolysis of cyclic nucleotides. Regulation of cyclic nucleotide phosphodiesterases is one of the two major mechanisms by which intracellular cyclic nucleotide levels are controlled. In many cases the fluctuations in cyclic nucleotide levels in response to hormones is due to the hormone responsiveness of the phosphodiesterase. Isozymes of the cGMP-inhibited, cAMP-specific, calmodulin-stimulated and cGMP-binding phosphodiesterases have been demonstrated to be substrates for protein kinases. Here we review the evidence that hormonally responsive phosphorylation acts to regulate cyclic nucleotide phosphodiesterases. In particular, the cGMP-inhibited phosphodiesterases, which can be phosphorylated by at least two different protein kinases, are activated as a result of phosphorylation. In contrast, phosphorylation of the calmodulin-stimulated phosphodiesterases, which coincides with a decreased sensitivity to activation by calmodulin, results in decreased phosphodiesterase activity.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

Immunochemical characterization of the distinct monocyte cyclic AMP-phosphodiesterase from patients with atopic dermatitis.

BACKGROUND: Previous findings have suggested that the immunopathology of patients with atopic dermatitis (AD) results from altered cellular responses caused by cyclic nucleotide regulatory abnormalities. One such defect is the increased degradation of the second messenger, cyclic adenosine monophosphate (cAMP), by elevated cAMP-phosphodiesterase (PDE) activity in patients with AD. METHODS: We used two monoclonal antibodies to identify the major PDE isoform in AD blood monocytes. We have also characterized the abnormal PDE activity by means of chromatofocusing and sucrose gradient centrifugation. RESULTS: The chromatofocusing technique allowed the separation of a PDE-containing fraction (isoelectric point = 6.1) from AD monocytes but not from normal cells. This monocyte fraction accounted for most of the elevated leukocyte-PDE activity and was a cytosolic, cAMP-specific, low Michaelis constant, calcium-calmodulin-dependent enzyme, inhibited by the cAMP-PDE inhibitor, Ro 20-1724. The majority of the PDE activity in this chromatofocused fraction was immunoadsorbed by the solid-phase immobilized antibodies against calcium-calmodulin-dependent PDE. CONCLUSIONS: The increased degradation of cAMP by a unique form of PDE may cause defective regulation of intracellular functions of AD monocytes, leading to the characteristic hyperreactive immune and inflammatory events. Characterization of PDE isoenzymes from different leukocyte subpopulations may allow further expansion of cell-directed therapy for inflammatory disease.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1992

Molecular cloning of cDNA encoding a "63"-kDa calmodulin-stimulated phosphodiesterase from bovine brain.

Partially degenerate oligonucleotides based on peptide sequence were used to isolate cDNA to a 63-kDa bovine brain calmodulin-stimulated phosphodiesterase (CaM-PDE) isozyme. A 412-base pair polymerase chain reaction fragment was obtained and used along with the oligonucleotides to isolate several cDNAs each encoding sequence identical to known peptide sequences from the 63-kDa CaM-PDE. The largest cDNA contained a full-length open reading frame (ORF) encoding a 534 amino acid, 61,005-dalton protein. It had 59% amino acid identity to the 61-kDa bovine brain CaM-PDE and included a carboxyl-terminal conserved domain containing the PDE catalytic domain consensus sequences. The NH2-terminal region fits the criteria for a calmodulin-binding domain. When its expression was driven by a cytomegalovirus promoter on a pCDM8 vector in COS-7 cells, the cDNA encoded a catalytically active, calmodulin-stimulated PDE. Northern analysis of RNA from several tissues with a probe containing much of the conserved PDE catalytic domain showed only a single band of 4.0 kilobases. Hybridization was seen in mRNA from several regions of the central nervous system with the greatest signal in basal ganglia. Strong signals also were seen in other tissues including kidney papilla and adrenal medulla. Antisense RNA probes were used in RNase-protection assays to look for evidence of multiple 63-kDa CaM-PDE transcripts. A catalytic domain probe was fully protected by RNA from cerebral cortex, basal ganglia, cerebellum, hippocampus, adrenal medulla, and kidney papilla. However, a probe to the NH2-terminal region was fully protected only by brain and adrenal medullary RNA indicating the likelihood of one or more isozyme(s) divergent in this region in the kidney papilla.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1992

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.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗