Characterization of particulate cyclic nucleotide phosphodiesterases from bovine brain: purification of a distinct cGMP-stimulated isoenzyme

In the absence of detergent, approximately 80-85% of the total cGMP-stimulated phosphodiesterase (PDE) activity in bovine brain was associated with washed particulate fractions; approximately 85-90% of the calmodulin-sensitive PDE was soluble. Particulate cGMP-stimulated PDE was higher in cerebral cortical gray matter than in other regions. Homogenization of the brain particulate fraction in 1% Lubrol increased cGMP-stimulated activity approximately 100% and calmodulin-stimulated approximately 400-500%. Although 1% Lubrol readily solubilized these PDE activities, approximately 75% of the cAMP PDE activity (0.5 microM [3H]cAMP) that was not affected by cGMP was not solubilized. This cAMP PDE activity was very sensitive to inhibition by Rolipram but not cilostamide. Thus, three different PDE types, i.e., cGMP stimulated, calmodulin sensitive, and Rolipram inhibited, are associated in different ways with crude bovine brain particulate fractions. After solubilization and purification by chromatography on cGMP-agarose, heparin-agarose, and Superose 6, the brain particulate cGMP-stimulated PDE cross-reacted with antibody raised against a cGMP-stimulated PDE purified from calf liver supernatant. The brain enzyme exhibited a slightly greater subunit Mr than did soluble forms from calf liver or bovine brain, as evidenced by protein staining or immunoblotting after polyacrylamide gel electrophoresis under denaturing conditions. Incubation of brain particulate and liver soluble cGMP-stimulated PDEs with V8 protease produced several peptides of similar size, as well as at least two distinct fragments of approximately 27 kDa from the brain and approximately 23 kDa from the liver enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of bovine brain calmodulin-dependent cGMP phosphodiesterase by peptide and non-peptide angiotensin receptor ligands.

Inhibition of a purified 60 KDa bovine brain calmodulin-dependent cGMP phosphodiesterase (PDE) was investigated for a number of peptides and non-peptides which are known to bind to angiotensin (ANG) receptors. The peptide antagonists sarilesin and sarmesin had KI = 120 and greater than 200 microM respectively, and the peptide agonists ANG II and ANG III had KI = greater than 200 and 45 microM respectively. Non-peptide ANG receptor antagonists related to DuP 753 exhibited KI values in the same range. For both peptide and non-peptide antagonists, inhibitory activities in the PDE assay reflected the order of antagonist potencies at ANG receptors in the rat isolated uterus assay and binding affinities at ANG receptors in rat uterine membranes, suggesting that molecular recognition factors are similar for both ANG receptors and cGMP PDE. The vasodilatory and blood pressure lowering effects of compounds related to DuP 753 may be due in part to inhibition of cGMP PDE. The differential effects of ANG II and ANG III at target tissues may relate in part to the marked differences in cGMP PDE inhibition associated with these two peptides hormones.     

Identification of the retinal cyclic GMP phosphodiesterase inhibitory gamma-subunit interaction sites on the catalytic alpha-subunit.

Retinal rod outer segment phosphodiesterase (PDE) consists of two similar catalytic subunits (alpha and beta) and two identical inhibitory subunits (gamma 2). A trypsin-activated soluble PDE exhibiting the ability to be reinhibited by PDE gamma was shown by peptide antisera to retain both N and C termini. Synthetic peptides corresponding to residues 16-30, 78-90, 389-403, and 535-563 of PDE alpha used in a PDE activity assay with trypsin-activated PDE partially prevented inhibition by exogenous PDE gamma; however, only competitions by peptides 16-30 and 78-90 (corresponding to PDE alpha 16-30 and 78-90) were concentration-dependent below 100 nmol of peptide. Binding studies using radio-immunoassays and PDE alpha peptides confirmed that peptides 16-30 and 78-90 (corresponding to PDE alpha 16-30 and 78-90, respectively) were able to bind PDE gamma. Additionally, peptides corresponding to the PDE alpha region 453-534 bound PDE gamma in the binding assay. This suggests that several regions on PDE alpha interact with the PDE gamma inhibitor. While some regions may be involved in binding to PDE gamma, other sites may be involved in PDE gamma inhibition of catalytic activity. Our results suggest that the major regions of PDE alpha that interact with PDE gamma reside within the N terminus (16-30 and 78-90), with weaker interaction regions within or near the hypothesized catalytic domain (453-563). Sequence analysis of three retinal phosphodiesterases (rod outer segment alpha, beta, and cone outer segment alpha') revealed the highest region of dissimilarity in the N and C termini.     

Endogenous Peptides That Inhibit Brain Cyclic AMP Phosphodiesterase

Peptide extracts of rat brain powerfully inhibited the cyclic AMP phosphodiesterase activity of rat brain homogenate. Similar extracts of ox brain showed comparable although less potent activity. Preliminary investigation of the physicochemical properties of brain extracts indicated that the rat brain extract contained an active peptide of low molecular weight (about 1400), whereas ox brain contained two such peptides (about 1400 and 900). These studies indicate that endogenous oligopeptides that inhibit cyclic AMP phosphodiesterase are present in brain. Experiments on several pure peptides known to be present in brain. Experiments on several pure peptides known to be present in the CNS showed that the majority were inactive against brain phosphodiesterase, but ACTH(1-24), somatostatin, substance P and Lys8-vasopressin, in descending order of potency, were active. To help distinguish the peptides found in rat and ox brain extracts from known peptides, preliminary analyses of amino acid composition were performed. These suggested that the peptides found in brain extracts were distinct from known peptides having the ability to inhibit cyclic AMP phosphodiesterase.     

Regional Distribution of Calmodulin Activity in Rat Brain

Calmodulin activity in 68 discrete areas of rat brain, obtained by micropunch technique, was assessed by its capacity to activate a calmodulin-sensitive form of phosphodiesterase. In general, the activity of calmodulin was higher in the telencephalon, limbic system, and hypothalamus than in the mesencephalon, pons, cerebellum, and medulla. However, there were substantial differences in calmodulin activity in discrete nuclei of each region. The regional distribution of calmodulin activity in rat brain does not appear to correlate with that of any of the known putative neurotransmitters or peptides.     

Immunohistochemical localization of phosphodiesterase 10A in multiple mammalian species.

A monoclonal antibody directed against the amino terminal of rat phosphodiesterase 10A (PDE10A) was used to localize PDE10A in multiple central nervous system (CNS) and peripheral tissues from mouse, rat, dog, cynomolgus macaque, and human. PDE10A immunoreactivity is strongly expressed in the CNS of these species with limited expression in peripheral tissues. Within the brain, strong immunoreactivity is present in both neuronal cell bodies and neuropil of the striatum, in striatonigral and striatopallidal white matter tracks, and in the substantia nigra and globus pallidus. Outside the brain, PDE10A immunoreactivity is less intense, and distribution is limited to few tissues such as the testis, epididymal sperm, and enteric ganglia. These data demonstrate that PDE10A is an evolutionarily conserved phosphodiesterase highly expressed in the brain but with restricted distribution in the periphery in multiple mammalian species.     

Characterization of phosphodiesterase catalytic sites by means of cyclic nucleotide derivatives

Cyclic nucleotide derivatives have been used as a tool to characterize distinct catalytic sites on phosphodiesterase enzyme forms: the cGMP-stimulated enzyme from rat liver and the calmodulin-sensitive enzyme from rat or bovine brain. Under appropriate assay conditions, the analogues showed linear competitive inhibition with respect to cAMP (adenosine 3',5'-monophosphate) as substrate. The inhibition sequence of the fully activated cGMP-stimulated phosphodiesterase was identical to the inhibition sequence of the desensitized enzyme, i.e. the enzyme which has lost its ability to be stimulated by cGMP. The inhibition pattern could, therefore, not be attributed to competition with cGMP at an allosteric-activating site. Also, the inhibition sequence of the calmodulin-sensitive phosphodiesterase was maintained whether activity was basal or fully stimulated by calmodulin. When cAMP and cGMP, with identical chemical ligands substituted at the same position, were compared as inhibitors of the calmodulin-sensitive phosphodiesterase, the cGMP analogues were always the more potent suggesting that, for that enzyme, the catalytic site was sensitive to a guanine-type cyclic nucleotide structure. Comparing the two phosphodiesterases, it was possible to establish both similar and specific inhibitor potencies of cyclic nucleotide derivatives. In particular, the two enzymes exhibited large differences in analogue specificity modified at C-6, 6-chloropurine 3',5'-monophosphate or purine 3',5'-monophosphate.     

A protein inhibitor of calmodulin-regulated cyclic nucleotide phosphodiesterase in amphibian ovaries

The cytosol fraction of an extract of Xenopus laevis ovaries contains a protein inhibitor that can specifically block the activation of calmodulin-sensitive cyclic nucleotide phosphodiesterase (PDE I) found in that tissue. This inhibitor was purified by DEAE-cellulose chromatography, gel filtration on Sephacryl S-200, and affinity chromatography on calmodulin-Sepharose. It has a molecular weight of approximately 90,000, and is heat-labile and susceptible to inactivation by chymotrypsin. The inhibitor blocks calmodulin activation of cyclic nucleotide phosphodiesterases from amphibian ovary and bovine brain and of the myosin light chain kinase from rabbit smooth muscle, but does not affect the activity of a calmodulin-insensitive cyclic nucleotide phosphodiesterase. The inhibitor not only affects the activation of Xenopus PDE I and of the bovine brain phosphodiesterase by calmodulin, but also inhibits the stimulation of these enzymes by lysophosphatidylcholine. The inhibitor also acts on PDE I activated by partial tryptic proteolysis, but the enzyme fully activated by trypsin is only slightly susceptible to inhibition by this protein. The inhibition of PDE I activation caused by this ovarian factor can be reversed by adding excess amounts of calmodulin or lysophosphatidylcholine. The presence of this inhibitor provides a possible explanation for the previously observed inactivity of PDE I in vivo.     

Immunological distinction between calmodulin-sensitive and calmodulin-insensitive adenylate cyclases

Previous studies using calmodulin-Sepharose affinity chromatography have suggested that bovine brain may contain a mixture of calmodulin-sensitive and -insensitive adenylate cyclase activities (Wescott, K. R., La Porte, D. C., and Storm, D. R. (1979) Proc. Natl. Acad. Sci. U.S.A. 82, 3086-3090). In this study, mice were immunized with a purified preparation of the calmodulin-sensitive adenylate cyclase from bovine brain, and a polyclonal antiserum was obtained which was specific to the calmodulin-sensitive form of the enzyme. The antiserum was not inhibitory and precipitated enzyme activity from a homogeneous preparation of the calmodulin-sensitive adenylate cyclase catalytic subunit. Furthermore, the antiserum did not interact with calmodulin-insensitive adenylate cyclase which was resolved from the calmodulin-sensitive form of the enzyme by calmodulin-Sepharose affinity chromatography. Since the only polypeptide specifically precipitated by the antiserum had an Mr of 135,000, which was identical to the Mr of the catalytic subunit of the enzyme, it is concluded that the antiserum interacted directly and specifically with the catalytic subunit of the calmodulin-sensitive isozyme of adenylate cyclase. Detergent-solubilized membranes from several rat tissues were examined for the presence of calmodulin-sensitive adenylate cyclase using anti-calmodulin-sensitive adenylate cyclase antiserum. Approximately 40-60% of the total adenylate cyclase activity of rat brain and kidney were immunoprecipitated by the antiserum, whereas liver and testes contained no detectable calmodulin-sensitive adenylate cyclase. Approximately 15% of the total adenylate cyclase activity in rat heart and lung was the calmodulin-sensitive form. These data indicate that the calmodulin-sensitive and insensitive adenylate cyclases from bovine brain are immunologically distinct and support the proposal that there may be two or more distinct adenylate cyclase isozymes in brain.     

Phosphorylation and activation of calmodulin-sensitive cyclic nucleotide phosphodiesterase by a brain Ca2+, calmodulin-dependent protein kinase

A Ca2+, calmodulin-dependent protein kinase from rat brain with a MW of 640,000 phosphorylated calmodulin-sensitive phosphodiesterase from the brain cytosol. The Km of the enzyme for the phosphodiesterase was 5.0 microM and the Vmax was 212 nmol/mg/min. The amount of phosphate incorporated into the phosphodiesterase was 0.7 mol/mol subunit. Phosphorylation of the phosphodiesterase enhanced the enzyme activity by about 20% for hydrolysis of a higher concentration of cyclic AMP.     

Human PDE4A8, a novel brain-expressed PDE4 cAMP-specific phosphodiesterase that has undergone rapid evolutionary change

We have isolated cDNAs encoding PDE4A8 (phosphodiesterase 4 isoform A8), a new human cAMP-specific PDE4 isoform encoded by the PDE4A gene. PDE4A8 has a novel N-terminal region of 85 amino acids that differs from those of the related 'long' PDE4A4, PDE4A10 and PDE4A11 isoforms. The human PDE4A8 N-terminal region has diverged substantially from the corresponding isoforms in the rat and other mammals, consistent with rapid evolutionary change in this region of the protein. When expressed in COS-7 cells, PDE4A8 localized predominantly in the cytosol, but approx. 20% of the enzyme was associated with membrane fractions. Cytosolic PDE4A8 was exquisitely sensitive to inhibition by the prototypical PDE4 inhibitor rolipram (IC(50) of 11+/-1 nM compared with 1600 nM for PDE4A4), but was less sensitive to inhibition by cilomilast (IC(50) of 101+/-7 nM compared with 61 nM for PDE4A4). PDE4A8 mRNA was found to be expressed predominantly in skeletal muscle and brain, a pattern that differs from the tissue expression of other human PDE4 isoforms and also from that of rat PDE4A8. Immunohistochemical analysis showed that PDE4A8 could be detected in discrete regions of human brain, including the cerebellum, spinal cord and cerebral cortex. The unique tissue distribution of PDE4A8, combined with the evolutionary divergence of its N-terminus, suggest that this isoform may have a specific function in regulating cAMP levels in human skeletal muscle and brain.     

The neural type II regulatory subunit of cAMP-dependent protein kinase is present and regulated by hormones in the rat ovary

In this study purified isoforms of rat ovarian regulatory subunit of type II cAMP-dependent protein kinase (R-II) were compared with R-II purified from rat brain. A special neural form of R-II has been previously described in bovine brain. Analysis by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved three isoforms of rat ovarian R-II (R-II54, Mr = 54,000; R-II52, Mr = 52,000; and R-II51, Mr = 51,000) compared to two R-II isoforms in rat brain (R-II54 and R-II52). Polychromatic silver-stained peptide maps of purified R-II subunits indicated that peptides generated from both rat ovarian R-II52 and R-II51 were similar (if not identical) to the peptides of the neural form, R-II52, purified from rat brain. These peptides differed markedly from those generated from R-II54 of either rat ovary, brain, or heart. Ovarian R-II52/51 photoaffinity labeled with 8-N3-[32P]cAMP and analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis was shown to consist of three (rather than two) isoelectric variants, which were similar to three variants resolved from rat brain R-II and clearly distinct from that of rat heart R-II54. An antibody which recognized both the R-II54 and R-II52/51 isoforms of rat ovarian extracts also recognized both forms of rat brain R-II (R-II54 and R-II52) and similar forms in extracts of rat adrenal and parotid glands. These results strongly suggest that the R-II52 isoform previously designated as a neural specific form of R-II is present in high concentrations in a nonneural tissue, the rat ovary.     

Noradrenergic Activity Differentially Regulates the Expression of Rolipram-Sensitive, High-Affinity Cyclic AMP Phosphodiesterase (PDE4) in Rat Brain

Abstract: In a previous study, it was observed that the activity of rolipram-sensitive, low- K _m, cyclic AMP phosphodiesterase (PDE4) was decreased in vivo with diminished noradrenergic stimulation. The results of the present experiments indicated that the reduction in the activity may be associated with down-regulation of PDE4 protein. Immunoblot analysis using PDE4-specific, subfamily-nonspecific antibody (K116) revealed four major bands of PDE4 in rat cerebral cortex; those with apparent molecular masses of 109 and 102 kDa are variants of PDE4A. Diminished noradrenergic activity, produced by intracerebroventricular infusion of 6-hydroxydopamine (6-OHDA) or chronic subcutaneous infusion of propranolol, decreased the intensities of the protein bands for the 109- and 102-kDa PDE4A variants in rat cerebral cortex but not of the 98- or 91-kDa PDE4 forms. 6-OHDA-induced noradrenergic lesioning also decreased the content of 102-kDa PDE4A in hippocampus as labeled by PDE4A-specific antibody (C-PDE4A). Enhanced noradrenergic stimulation up-regulated PDE4 in cerebral cortex. This was indicated by the finding that repeated treatment with desipramine increased the intensity of the protein band for the 102-kDa PDE4 but not for the other variants of PDE4. These results suggest that PDE4 subtypes are differentially regulated at the level of expression, as evidenced by an apparent change in the amount of PDE4 protein, following changes in noradrenergic activity. These observations are consistent with the notion that PDE4s, especially the PDE4A variants with molecular masses of 109 and 102 kDa, play an important role in maintaining the homeostasis of the noradrenergic signal transduction system in the brain and may be involved in the mediation of antidepressant activity.     

Important role of phosphodiesterase 3B for the stimulatory action of cAMP on pancreatic beta-cell exocytosis and release of insulin

Cyclic AMP potentiates glucose-stimulated insulin release and mediates the stimulatory effects of hormones such as glucagon-like peptide 1 (GLP-1) on pancreatic beta-cells. By inhibition of cAMP-degrading phosphodiesterase (PDE) and, in particular, selective inhibition of PDE3 activity, stimulatory effects on insulin secretion have been observed. Molecular and functional information on beta-cell PDE3 is, however, scarce. To provide such information, we have studied the specific effects of the PDE3B isoform by adenovirus-mediated overexpression. In rat islets and rat insulinoma cells, approximate 10-fold overexpression of PDE3B was accompanied by a 6-8-fold increase in membrane-associated PDE3B activity. The cAMP concentration was significantly lowered in transduced cells (INS-1(832/13)), and insulin secretion in response to stimulation with high glucose (11.1 mm) was reduced by 40% (islets) and 50% (INS-1). Further, the ability of GLP-1 (100 nm) to augment glucose-stimulated insulin secretion was inhibited by approximately 30% (islets) and 70% (INS-1). Accordingly, when stimulating with cAMP, a substantial decrease (65%) in exocytotic capacity was demonstrated in patch-clamped single beta-cells. In untransduced insulinoma cells, application of the PDE3-selective inhibitor OPC3911 (10 microm) was shown to increase glucose-stimulated insulin release as well as cAMP-enhanced exocytosis. The findings suggest a significant role of PDE3B as an important regulator of insulin secretory processes.     

Identification of angiotensin-(1-7) in rat brain. Evidence for differential processing of angiotensin peptides

Tissue and plasma forms of angiotensin (Ang) peptides were characterized by reverse-phase high performance liquid chromatography and three specific radioimmunoassays. This method allowed resolution of 10 Ang peptides and revealed distinctive distributions for the three principal Ang peptides in the brain, adrenal gland, and plasma. In extracts from the rat hypothalamus, approximately equimolar amounts of Ang-(1-7), Ang-II, and Ang-I were detected (1.10, 1.18, and 1.45 pmol/g of tissue, respectively). A similar profile was observed in the medulla oblongata and amygdala, although the content of these three peptides was 40-70% less than that seen in the hypothalamus. In the adrenal gland, the predominant peptide was Ang-II (1.07 pmol/g); levels of Ang-(1-7) (0.19 pmol/g) and Ang-I (0.14 pmol/g) were approximately 20% that of Ang-II. In plasma, the major angiotensin was Ang-I (0.13 pmol/ml), with lower levels of Ang-(1-7) and Ang-II (0.01-0.02 pmol/ml). This study is the first demonstration of the endogenous presence of Ang-(1-7) in central and peripheral tissues of the rat. Moreover, the data suggest tissue-specific processing of angiotensins, with Ang-(1-7) being a predominant Ang peptide in the central nervous system. In light of the recent biological properties described for this peptide, Ang-(1-7) may represent an active member of Ang peptides in the brain.     

Concanavalin A stimulates the Rolipram-sensitive isoforms of cyclic nucleotide phosphodiesterase in rat thymic lymphocytes

Pretreatment of rat thymic lymphocytes with Concanavalin A induced a very early (30 min) and substantial increase (+90%) of the soluble cAMP phosphodiesterase activity. The crude cytosolic phosphodiesterase activity of rat thymocytes could reproducively be resolved by Mono-Q ion exchange high performance liquid chromatography into four separate phosphodiesterase peaks: a cGMP-stimulated, two cAMP-specific Rolipram-sensitive and a cGMP-inhibited cardiotrope-sensitive peaks. Concanavalin A stimulated very specifically the activity of the two predominant cAMP-specific Rolipram sensitive peaks whereas it only slightly modified the cGMP-stimulated and the cGMP-inhibited forms. The present results strongly suggest that the Rolipram-sensitive cAMP PDE activity may play a key role in the control and regulation of mitogen-induced thymocyte proliferation.     

Cyclic nucleotide phosphodiesterase of the olfactory membrane in rats: its distribution and possible connection with the estradiol receptor

In the rat olfactory tissue the existence of soluble and membrane-bound forms of cyclic nucleotide phosphodiesterase (PDE) with quite similar kinetic parameters was demonstrated (KM = 220 and 200 microM, VMaX = 10 and 7 nmoles cAMP/mg protein per minute, respectively). 17 beta-estradiol (10(-7)-10(-5) M) decreased the soluble PDE activity by 25%, whereas non-hydrolysable GTP analogue (Gpp (NHp)) abolished their effect. On the other hand, this analogue in a dose-dependent manner inhibited the specific binding of 3H-estradiol to cytosolic receptors. The data indicate possible functional interrelations between the cytosolic estradiol receptors, GTP-binding proteins and PDE in the olfactory tissue which is a target organ for steroid hormones.     

Identification of regions of arrestin that bind to rhodopsin

Arrestin facilitates phototransduction inactivation through binding to photoactivated and phosphorylated rhodopsin (RP). However, the specific portions of arrestin that bind to RP are not known. In this study, two different approaches were used to determine the regions of arrestin that bind to rhodopsin: panning of phage-displayed arrestin fragments against RP and cGMP phosphodiesterase (PDE) activity inhibition using synthetic arrestin peptides spanning the entire arrestin protein. Phage display indicated the predominant region of binding was contained within amino acids 90-140. A portion of this region (residues 95-140) expressed as a fusion protein with glutathione S-transferase is capable of binding to rhodopsin regardless of the activation or phosphorylation state of the receptor. Within this region, the synthetic peptide of residues 109-130 was shown to completely inhibit the binding of arrestin to rhodopsin with an IC50 of 1.1 mM. The relatively high IC50 of this competition suggests that this portion of the molecule may be only one of several regions of binding between arrestin and RP. A survey of synthetic arrestin peptides in the PDE assay indicated that the two most effective inhibitors of PDE activity were peptides of residues 111-130 and 101-120. These results indicate that at least one of the principal regions of binding between arrestin and RP is contained within the region of residues 109-130.     

Patterns of cyclic AMP phosphodiesterases during rat testis development

Summary Cyclic AMP phosphodiesterase (PDE; E.C.: 3.1.4.17) plays a crucial role in the regulation of intracellular cAMP levels arising from the hormonal activation of membrane-bound adenylcyclase in the target cell. In the present study, we revealed a complex sequence of appearance and disappearance of individual molecular electrophoretic forms of PDE during the development of the rat testis. Kinetic analysis of cAMP hydrolysis in crude testis homogenates of developmental stages where multiple PDE isozymes are expressed revealed complex kinetic behavior of PDE. After separation of individual isozymes by elution from starch gel blocks, several enzymatic forms still act with complex kinetics, indicating negatively cooperative behavior. The stage specificity of the kinetic properties of PDE appears to be related to the hormonally regulated events leading to the initiation of male puberty.