Catalytic and kinetic properties of purified high-affinity cyclic AMP phosphodiesterase from dog kidney
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34.81UConn Health Center37.99University of South AlabamaAbstractHigh-affinity cyclic AMP phosphodiesterase purified to homogeneity from dog kidney was studied with respect to its stability, its catalytic and kinetic properties, and its sensitivity to pharmacological agents. The enzyme was shown to rapidly lose activity upon dilution to low protein concentrations in aqueous media, but this activity loss was largely prevented by the presence of bovine serum albumin or ethylene glycol. Similarly, maximum activity required bovine serum albumin to be present during incubation for activity analysis. Enzyme activity requir Mg2+, Mn2+, and Co2+ each supported activity, but highest activity was obtained with Mg2. The temperature optimum ranged from 30 to 45 °C and depended on sub the Ea = 10,600 cal/mol. The pH optimum of the enzyme was broad, with a maximum from pH 8.0 to 9.5. The enzyme exhibits linear Michaelis-Menton kinetics for hydrolysis of cyclic AMP at all substrate concentrations tested and for hydrolysis of cyclic GMP at & 20 μm. The Km for cyclic AMP hydrolysis was 2 μm, and that for cyclic GMP hydrolysis was 312 μm. The Ki values for the competitive inhibition of hydrolysis of each substrate by the other were similar to their Km values suggesting a single active site. Cyclic AMP hydrolysis was weakly inhibited by cyclic GMP, cyclic IMP, adenine, and adenosine, but was not inhibited by the mono-, di, or trinucleotides of adenosine, guanosine, or inosine. Activity was competitively inhibited with Ki values in the micromolar range by drugs representative of methylxanthines, isoquinolines, pyrazolopyridines, imidazolidinones, triazolopyrimidines, pyridylethylenediamines, phenothiazines, and calcium antagonists. The results are discussed with reference to the similarities and differences between high- and low-affinity phosphodiesterase forms.Do you want to read the rest of this article?Request full-text
CitationsCitations40ReferencesReferences45Data were obtained using heterologously expressed mouse (types VII, VIII, and X) and human (types I, II, IV, V, VII, IX, and XI) PDEs as well as endogenous PDEs from dog kidney (type IV), rat brain (type IV), bovine heart (types III and IV), human heart (type II), bovine aorta (types I, III, and V), rabbit aorta (types I, IV, and V), and bovine photoreceptor (type VI). Data compiled from: 1 (Ahluwalia and Rhoads, 1982), 2 (Ahn et al., 1989), 3 (Beavo, 1988), 4 (Bolger et al., 1993), 5 (Bolger et al., 1997), 6 (Coste and Grondin, 1995), 7 (Epstein et al., 1982), 8 (Fawcett et al., 2000), 9 (Fisher et al., 1998), 10 (Gardner et al., 2000), 11 (Harrison et al., 1988), 12 (Hetman et al., 2000), 13 (Holck et al., 1984), 14 (Lorenz and Wells, 1983), 15 (Loughney et al., 1996), 16 (Loughney et al., 1998), 17 (Nemoz et al., 1985), 18 (Podzuweit et al., 1995), 19 (Rosman et al., 1997), 20 (Soderling et al., 1998a), 21 (Soderling et al., 1998b), 22 (Soderling et al., 1999), 23 (Whalin et al., 1991). Blank spaces indicate that data are not available. ABSTRACT: Phosphodiesterases (PDEs) catalyze the hydrolysis of the second messengers cAMP and cGMP. However, little is known about how PDE activity regulates cyclic nucleotide signals in vivo because, outside of specialized cells, there are few methods with the appropriate spatial and temporal resolution to measure cyclic nucleotide concentrations. We have previously demonstrated that adenovirus-expressed, olfactory cyclic nucleotide-gated channels provide real-time sensors for cAMP produced in subcellular compartments of restricted diffusion near the plasma membrane (Rich, T.C., K.A. Fagan, H. Nakata, J. Schaack, D.M.F. Cooper, and J.W. Karpen. 2000. J. Gen. Physiol. 116:147-161). To increase the utility of this method, we have modified the channel, increasing both its cAMP sensitivity and specificity, as well as removing regulation by Ca(2)+-calmodulin. We verified the increased sensitivity of these constructs in excised membrane patches, and in vivo by monitoring cAMP-induced Ca(2)+ influx through the channels in cell populations. The improved cAMP sensors were used to monitor changes in local cAMP concentration induced by adenylyl cyclase activators in the presence and absence of PDE inhibitors. This approach allowed us to identify localized PDE types in both nonexcitable HEK-293 and excitable GH4C1 cells. We have also developed a quantitative framework for estimating the K(I) of PDE inhibitors in vivo. The results indicate that PDE type IV regulates local cAMP levels in HEK-293 cells. In GH4C1 cells, inhibitors specific to PDE types I and IV increased local cAMP levels. The results suggest that in these cells PDE type IV has a high K(m) for cAMP, whereas PDE type I has a low K(m) for cAMP. Furthermore, in GH4C1 cells, basal adenylyl cyclase activity was readily observable after application of PDE type I inhibitors, indicating that there is a constant synthesis and hydrolysis of cAMP in subcellular compartments near the plasma membrane. Modulation of constitutively active adenylyl cyclase and PDE would allow for rapid control of cAMP-regulated processes such as cellular excitability. Full-text · Article · Aug 2001 +1 more author...Data were obtained using heterologously expressed mouse (types VII, VIII, and X) and human (types I, II, IV, V, VII, IX, and XI) PDEs as well as endogenous PDEs from dog kidney (type IV), rat brain (type IV), bovine heart (types III and IV), human heart (type II), bovine aorta (types I, III, and V), rabbit aorta (types I, IV, and V), and bovine photoreceptor (type VI). Data compiled from: 1 (Ahluwalia and Rhoads, 1982), 2 (Ahn et al., 1989), 3 (Beavo, 1988), 4 (Bolger et al., 1993), 5 (Bolger et al., 1997), 6 (Coste and Grondin, 1995), 7 (Epstein et al., 1982), 8 (Fawcett et al., 2000), 9 (Fisher et al., 1998), 10 (Gardner et al., 2000), 11 (Harrison et al., 1988), 12 (Hetman et al., 2000), 13 (Holck et al., 1984), 14 (Lorenz and Wells, 1983), 15 (Loughney et al., 1996), 16 (Loughney et al., 1998), 17 (Nemoz et al., 1985), 18 (Podzuweit et al., 1995), 19 (Rosman et al., 1997), 20 (Soderling et al., 1998a), 21 (Soderling et al., 1998b), 22 (Soderling et al., 1999), 23 (Whalin et al., 1991). Blank spaces indicate that data are not available. ABSTRACT: Phosphodiesterases (PDEs) catalyze the hydrolysis of the second messengers cAMP and cGMP. However, little is known about how PDE activity regulates cyclic nucleotide signals in vivo because, outside of specialized cells, there are few methods with the appropriate spatial and temporal resolution to measure cyclic nucleotide concentrations. We have previously demonstrated that adenovirus-expressed, olfactory cyclic nucleotide–gated channels provide real-time sensors for cAMP produced in subcellular compartments of restricted diffusion near the plasma membrane (Rich, T.C., K.A. Fagan, H. Nakata, J. Schaack, D.M.F. Cooper, and J.W. Karpen. 2000. J. Gen. Physiol. 116:147–161). To increase the utility of this method, we have modified the channel, increasing both its cAMP sensitivity and specificity, as well as removing regulation by Ca?+-calmodulin. We verified the increased sensitivity of these constructs in excised membrane patches, and in vivo by monitoring cAMP-induced Ca?+ influx through the channels in cell populations. The improved cAMP sensors were used to monitor changes in local cAMP concentration induced by adenylyl cyclase activators in the presence and absence of PDE inhibitors. This approach allowed us to identify localized PDE types in both nonexcitable HEK-293 and excitable GH4C1 cells. We have also developed a quantitative framework for estimating the KI of PDE inhibitors in vivo. The results indicate that PDE type IV regulates local cAMP levels in HEK-293 cells. In GH4C1 cells, inhibitors specific to PDE types I and IV increased local cAMP levels. The results suggest that in these cells PDE type IV has a high Km for cAMP, whereas PDE type I has a low Km for cAMP. Furthermore, in GH4C1 cells, basal adenylyl cyclase activity was readily observable after application of PDE type I inhibitors, indicating that there is a constant synthesis and hydrolysis of cAMP in subcellular compartments near the plasma membrane. Modulation of constitutively active adenylyl cyclase and PDE would allow for rapid control of cAMP-regulated processes such as cellular excitability.Article · Jun 2001 The potency of RS-rolipram (IC 50 =270 nM) as an inhibitor of membrane-bound PDE4 in guinea-pig macrophages was similar to its activity against other particulate PDE4s including guinea-pig and human eosinophils (Souness et al., 1991; Dent et al., 1994a) and murine macrophages (Okonogi et al., 1991). However, these results contrast with PDE4s that are apparently soluble, such as those in human cardiac ventricle (Reeves et al., 1987), human monocyte (Elliott & Leonard, 1989) and canine kidney (Epstein et al., 1982), where rolipram is signi(R)cantly less potent (IC 50 *1 to 6 mM). Although an explanation for this di?erence could relate to changes in the phosphorylation state of the enzyme (Kelly et al., 1996), as shown for recombinant HSPDE4D3 (Alvarez et al., 1995; Sette & Conti, 1996), which is a substrate for cyclic AMP-dependent protein kinase, another possibility might relate simply to the subcellular location of the enzyme (Huston et al., 1996). ABSTRACT: 1. The cyclic AMP phosphodiesterases (PDE) in guinea-pig peritoneal macrophages were isolated, partially characterized and their role in regulating the cyclic AMP content in intact cells evaluated. 2. Differential centrifugation of macrophage lysates revealed that approximately 90% of the PDE activity was membrane-bound and exclusively hydrolyzed cyclic AMP. This activity was not removed by KCl (200 mM) but was readily solubilized by the non-ionic detergent, Triton X-100 (1% v/v). Greater than 80% of the hydrolytic activity was suppressed by the PDE4 inhibitors, R-rolipram and nitraquazone with IC50s of 240 and 540 nM, respectively. 3. Anion-exchange chromatography of the total protein extracted from macrophages resolved two major peaks of cyclic AMP PDE activity that were insensitive to cyclic GMP (10 microM), calmodulin (50 units plus 2 mM CaCl2) and a PDE3 inhibitor, SK&F 95654 (10 microM), but were markedly suppressed by RS-rolipram (10 microM). The two peaks of PDE activity were arbitrarily designated CPPDE4alpha and CPPDE4beta with respect to the order from which they were eluted from the column where the prefix, CP, refers to the species, Cavia porcellus. 4. The hydrolysis of cyclic AMP catalyzed by CPPDE4alpha and CPPDE4beta conformed to Michaelis-Menten kinetic behaviour with similar K(m)s (13.4 and 6.4 microM, respectively). 5. Thermal denaturation of membrane-bound PDE4 at 50 degrees C followed bi-exponential kinetics with t1/2 values of 1.5 and 54.7 min for the first and second components, respectively. In contrast, CPPDE4alpha and CPPDE4beta each decayed mono-exponentially with significantly different thermostabilities (t1/2 = 2.77 and 1.15 min, respectively). 6. Gel filtration of CPPDE4beta separated two peaks of rolipram-sensitive PDE activity. The main peak eluted at a volume indicative of a approximately 180 kDa protein but was preceded by a much larger form of the enzyme that had an estimated weight of 750 kDa. Size exclusion chromatography of CPPDE4alpha resolved a broad peak of activity with molecular weights spanning 50 to 200 kDa. 7. Of ten PDE inhibitors examined, none distinguished CPPDE4alpha from CPPDE4beta with respect to their IC50 values or their rank order of potency. RS-rolipram acted as a purely competitive inhibitor of cyclic AMP hydrolysis with K(i)s of 2 microM and 1.5 microM for CPPDE4alpha and CPPDE4beta, respectively. In contrast to the membrane-associated enzyme(s), R-rolipram and nitraquazone were 4 to 19 fold less potent as inhibitors of CPPDE4alpha and CPPDE4beta. 8. In intact macrophages, Ro 20-1724 and RS-rolipram potentiated isoprenaline-induced cyclic AMP accumulation under conditions where a PDE3 inhibitor, SK&F 94120, was essentially inactive. 9. These data demonstrate that the predominant cyclic AMP hydrolyzing activity in guinea-pig macrophages is a PDE4. Moreover, thermostability studies and size exclusion chromatography indicates the possible expression of two intrinsic, membrane-associated isoenzymes which can regulate the cyclic AMP content in intact cells. The finding that soluble and particulate forms of the same enzyme exhibit different sensitivities to rolipram and nitraquazone implies that PDE4 can change conformation. Finally, the identification of multiple molecular weight species of CPPDE4 suggests that this enzyme(s) might form multimeric complexes of variable association states.Article · Jun 1998 I724 inhibited cGMP hydrolysis, while 10 PM denbufylline inhibited cGMP hydrolysis only about 25%. These three drugs have been reported to inhibit selectively the high-affinity CAMPselective form of PDE (Epstein et al., 1982; Wachtel, 1983; Nicholson et al., 1989). The cardiotonic drugs milrinone (10 FM) and Cl 930 (10 PM), selective for cGMP-inhibited PDE (Harrison et al., 1986; Kincaid and Manganiello, 1988), reduced PDE activity about 20% whether in the presence of 100 I.LM EGTA or with 100 PM Ca2+. ABSTRACT: We show that calmodulin-dependent phosphodiesterase (CAM-PDE) is selectively expressed in mature olfactory receptor neurons within the olfactory mucosa. Immunocytochemical staining reveals neuronal immunoreactivity that is most pronounced within cilia, dendritic knobs, and axon bundles. Neither sustentacular cells nor basal cells display immunoreactivity. The extent of loss of neuronal immunoreactivity following bulbectomy resembles loss of the neuronal population. High-affinity CAM-PDE activity in olfactory cilia is fivefold greater than in brain, when assayed at low micromolar cAMP. This activity is depleted in turbinates following bulbectomy. Olfactory mucosal PDE activity is composed of a minimum of two major forms. In the absence of Ca(2+), rolipram-sensitive PDE comprises 65% of total activity. Following stimulation by Ca2+, CAM-PDE activity is elevated sixfold to become the predominant form, thereby increasing total activity 300%, with half-maximal effect at 1 microM Ca2+. We propose that Ca2+ stimulation of CAM-PDE may be necessary for termination of olfactory signals. Full-text · Article · Apr 1992 +1 more author...f [3H]ACh from rat pheochromocytoma PC12 cells was also enhanced by forskolin (Rabe, Schneider & McGee, 1982) and in electrophysiological studies forskolin increased excitability of myenteric neurones (Nemeth et al. 1984Nemeth et al. , 1986 Zafirov et al. 1985b). The competitive inhibitor of phosphodiesterase 3-isobutyl-1-methylxanthine (IBMX) (cf. Epstein, Strada, Sarada & Thompson, 1982) was used with the intention to enhance neuronal levels of endogenous cyclic nucleotides. The secretion of [3H]ACh was enhanced by IBMX (Fig. 3; cf. Sawynok & Jhamandas, 1979; Alberts & Sellstr6m, 1983) except in a previous study ( Alberts & Stjiirne, 1982a). The hyperbolic concentration-response curve of IBMX (Fig. ABSTRACT: 1. Secretion of [3H]acetylcholine was studied in the guinea-pig ileum longitudinal muscle-myenteric plexus preparation. The transmitter stores of the cholinergic nerves were labelled by pre-incubation with [3H]choline. The preparation was mounted in an organ bath and superfused with Tyrode solution containing hemicholinium-3 and eserine. [3H]Acetylcholine secretion was evoked by electrical stimulation (0.5 Hz, 150 shocks). 2. 8-Bromo cyclic AMP, the adenylate cyclase activator forskolin, and the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine enhanced the [3H]acetylcholine secretion in a concentration-dependent manner. The values of 'maximal enhancement' calculated were similar, viz. 200-300% of control. 3. 8-Bromo cyclic GMP reduced the [3H]acetylcholine secretion. 4. The 'maximal enhancement' of 3-isobutyl-1-methylxanthine was not altered by the presence of forskolin (25 mumol/l) suggesting a common mechanism of action, i.e. elevation of endogenous cyclic AMP levels. 5. The muscarinic acetylcholine receptor antagonist atropine enhanced the [3H]acetylcholine secretion with a 'maximal enhancement' of 506% of control. Presence of neither forskolin (25 mumol/l) nor 3-isobutyl-1-methylxanthine (5 mmol/l) altered the 'maximal enhancement' for atropine. 6. In contrast, atropine (1 mumol/l) and 4-aminopyridine (0.5 mmol/l) additively enhanced the [3H]acetylcholine secretion. 7. The results suggest that neuronal cyclic AMP may be involved in muscarinic acetylcholine receptor-mediated control of [3H]acetylcholine secretion in guinea-pig ileum myenteric plexus.Article · Feb 1988 ABSTRACT: Verschiedene Phosphodiesterasehemmer sind zur Zeit in der Erprobung zur Behandlung der pulmonalen Hypertonie. Einige PDE 5-Inhibitoren sind bereits zur Therapie der pulmonalarteriellen Hypertonie zugelassen, jedoch ist bisher weniger über den therapeutischen Nutzen von PDE 3-, PDE 4- und insbesondere dualselektiven PDE 3/4-Inhibitoren bekannt.
Die vorliegende Studie untersucht die chronisch-therapeutischen Effekte von Pumafentrine, einem oral verfügbaren, gemischtselektiven PDE 3/4-Hemmstoff, in einem Modell der Monocrotalin-induzierten pulmonalen Hypertonie der Ratte. Nach einmaliger subkutaner Injektion von Monocrotalin (60 mg/kg) wurde Pumafentrine vom Tag 28 bis 42 nach Monocrotalin-induzierter Lungensch?digung verabreicht (10 mg/kg/d). Die Behandlung mit Pumafentrine konnte teilweise die pulmonale Hypertonie und die Rechtsherzhypertrophie der Ratten rückg?ngig machen. Zus?tzlich wurden die progressive Muskularisierung kleiner pulmonalarterien, die Mediahypertrophie und die Abnahme der Lumenfl?che gr?sstenteils rückg?ngig gemacht. Die chronische Therapie der Ratten mit Pumafentrine potenzierte die endothelabh?ngige Acetylcholin-induzierte Vasodilation und verbesserte die endothelunabh?ngige vasodilatorische Reaktion auf Natrium-Nitroprussid im Modell der isolierten Rattenlunge.
In vivo wurde die Hemmung der Proliferation von glatten Muskelzellen in der Lunge gezeigt. Pumafentrine übte weiterhin einen pro-apoptotischen Effekt auf Gef?sszellen aus. Darüber hinaus erh?hte Pumafentrine dosisabh?ngig die Spiegel von zyklischem Adenosinmonophosphat und hemmte die Proliferation kultivierter pulmonalarterieller glatter Muskelzellen.
Zusammenfassend ist zu sagen, dass Pumafentrine teilweise die Monocrotalin-induzierte pulmonale Hypertonie, den proliferativen Gef?sswandumbau und die Rechtsherzhypertrophie rückg?ngig machen kann.
Dies ist die erste Studie, die einen therapeutischen Effekt eines oral verfügbaren PDE 3/4 Hemmers in einem experimentellen Modell der pulmonalen Hypertonie zeigt. Die Hemmung der Phosphodiesterasen 3/4 k?nnte eine neue Therapieoption zur Behandlung der humanen pulmonalarteriellen Hypertonie darstellen. Phosphodiesterase (PDE) inhibitors are currently under investigation for the therapy of pulmonary hypertension. Several PDE 5-inhibitors are approved for the treatment of pulmonary arterial hypertension, but less is known about the therapeutical impact of PDE 3-, PDE 4- or dual-selective PDE 3/4-inhibitors.
The present study was designed to investigate chronic effects of oral pumafentrine, a mixed selective PDE 3/4-inhibitor, in monocrotaline (MCT)-induced pulmonary hypertension in rats. After a single subcutaneous injection of monocrotaline (60 mg/kg), when chronically administered from day 28 to 42 after monocrotaline-induced lung injury, pumafentrine (10 mg/kg/d) partially reversed pulmonary hypertension and right heart hypertrophy in rats. In addition, small pulmonary arterial muscularisation, medial hypertrophy and decrease in lumen area were largely reversed. Chronic pumafentrine treatment potentiated endothelial-dependent acetylcholine-induced vasodilation and improved the endothelial-independent vasodilatory response to sodium nitroprusside in an isolated rat lung preparation.
Inhibition of smooth muscle cell proliferation under pumafentrine was demonstrated in vivo and in vitro as was a pro-apoptotic effect of pumafentrine on vascular cells. Moreover, pumafentrine dose-dependently increased cyclic adenosine monophosphate levels and inhibited proliferation of cultured pulmonary arterial smooth muscle cells.
In conclusion, oral pumafentrine partially reverses monocrotaline-induced pulmonary hypertension, lung vascular remodelling and right heart hypertrophy in rats.
This study is the first one which shows a therapeutical effect of an orally bioavailable PDE 3/4 inhibitor in experimental pulmonary hypertension. Phosphodiesterase 3/4 inhibition might be a useful therapeutic option to treat human pulmonary arterial hypertension.Article · The Journal of Physiology ArticleMay 1982 · Archives of Biochemistry and Biophysics · Impact Factor: 3.02A high-affinity form of cyclic AMP phosphodiesterase, purified to apparent homogeneity from dog kidney, was labeled with 125I using a solid-state lactoperoxidaseglucose oxidase system and its purity confirmed by acrylamide gel electrophoresis and isoelectric focusing. Sheep anti-cyclic AMP phosphodiesterase immunoglobulin fraction was analyzed for 125I-enzyme binding and covalently bound to... ArticleDecember 1979 · Biochemistry · Impact Factor: 3.02ArticleOctober 1981 · Biochimica et Biophysica Acta · Impact Factor: 4.66The cyclic nucleotide phosphodiesterase (3':5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) systems of many tissues show multiple physical and kinetic forms. In contrast, the soluble rat uterine phosphodiesterase exists as a single enzyme form with non-linear Lineweaver-Burk kinetics for cyclic AMP (app. Km of approx. 3 and 20 microM) and linear kinetics for cyclic GMP (app. Km of... ArticleAugust 1978 · Biochemistry · Impact Factor: 3.02Soluble cyclic nucleotide phosphodiesterase of rat uterus displays distinct structural and regulatory properties. Like phosphodiesterases from many mammalian sources the soluble uterine enzyme system exhibits nonlinear Lineweaver--Burk kinetics with cyclic adenosine 3':5'-monophosphate (cAMP) as substrate (apparent Kms congruent to 3 and 20 micron) and linear kinetics with cyclic guanosine... Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.This publication is from a journal that may support self archiving.Last Updated: 09 Oct 17}