AMP Hydrolysis Research Articles

Electrostatic potential surface analysis of the transition state for AMP nucleosidase and for formycin 5'-phosphate, a transition-state inhibitor.

AMP nucleosidase hydrolyzes the N-glycosidic bond of AMP to yield adenine and ribose 5-phosphate. Kinetic isotope effects have been used to establish an experimentally based transition-state structure for the native enzyme and a Vmax mutant [Mentch, F., Parkin, D. W., & Schramm, V. L. (1987) Biochemistry 26, 921-930; Parkin, D. W., Mentch, F., Banks, G. A., Horenstein, B. A., & Schramm, V. L. (1991) Biochemistry 30, 4586-4594]. The transition states are characterized by weak reaction coordinate bonds to C1' and substantial carbocation character in the ribose ring. The N9-C1' bond to the leaving group is nearly broken and the adenine ring is protonated at the transition state. Formycin 5'-phosphate and other purine nucleoside 5'-phosphate analogues with syn-glycosyl torsion angles bind better than substrate, supporting a syn configuration in the enzyme-substrate complex and presumably in the transition state [Giranda, V. L., Berman, H. M., & Schramm, V. L. (1988) Biochemistry 27, 5813-5818]. Access to a geometric model of the transition state permits the analysis of its molecular electrostatic potential surface as enforced by the enzyme. Comparison of the molecular electrostatic potential surfaces for AMP, formycin 5'-phosphate, and the transition state reveals a striking similarity in the surface charges of formycin 5'-phosphate and the transition state. The enzyme-stabilized transition state for AMP hydrolysis is characterized by new positive electrostatic potential in the adenine ring as a result of protonation by the enzyme. This is closely matched by the protonated pyrazole ring of formycin 5'-phosphate. The molecular electrostatic potential surfaces of formycin 5'-phosphate and the transition state for AMP are similar and are likely to be a factor in the Km/Ki value of > 10(3) for formycin 5'-phosphate as a transition-state inhibitor of AMP nucleosidase.

Phosphodiesterase activity is a novel property of alkaline phosphatase from osseous plate

Phosphodiesterase activity is a novel property of the still-enigmatic alkaline phosphatase from osseous plate. Bis-(p-nitrophenyl) phosphate was hydrolysed at both pH 7.5 and 9.4 with an apparent dissociation constant (K0.5) of 1.9 mM and 3.9 mM respectively. The hydrolysis of p-nitrophenyl-5'-thymidine phosphate followed hyberbolic kinetics with a K0.5 of 500 microM. For p-nitrophenyl phenylphosphonate, site-site interactions [Hill coefficient (h) = 1.3] were observed in the range between 0.2 and 100 microM, and K0.5 was 32.8 mM. The hydrolysis of cyclic AMP by the enzyme followed more complex kinetics, showing site-site interactions (h = 1.7) and K0.5 = 300 microM for high-affinity sites. The low-affinity sites, representing 85% of total activity, also showed site-site interactions (h = 3.8) and a K0.5 of about 22 mM. ATP and cyclic AMP were competitive inhibitors of bis-(p-nitrophenyl) phosphatase activity of the enzyme and Ki values (25 mM and 0.6 mM for cyclic AMP and ATP respectively) very close to those of the K0.5 (22 mM and 0.7 mM for cyclic AMP and ATP respectively), determined by direct assay, indicated that a single catalytic site was responsible for the hydrolysis of both substrates. Non-denaturing PAGE of detergent-solubilized enzyme showed coincident bands on the gel for phosphomonohydrolase and phosphodiesterase activities. Additional evidence for a single catalytic site was the similar pKa values (8.5 and 9.7) found for the two ionizing groups participating in the hydrolysis of bis-(p-nitrophenyl) phosphate and p-nitrophenyl phosphate. The alkaline apparent pH optima, the requirement for bivalent metal ions and the inhibition by methylxanthines, amrinone and amiloride demonstrated that rat osseous-plate alkaline phosphatase was a type I phosphodiesterase. Considering that there is still confusion as to which is the physiological substrate for the enzyme, the present results describing a novel property for this enzyme could be of relevance in understanding the mineralization process.

Relaxation of guinea-pig trachea by cyclic AMP phosphodiesterase inhibitors and their enhancement by sodium nitroprusside.

1. The effects of agents that elevate either cyclic AMP (the phosphodiesterase (PDE) III inhibitor siguazodan, salbutamol) or cyclic GMP (sodium nitroprusside (SNP)) on the relaxant activity of the PDE IV inhibitor, rolipram, were investigated in carbachol (0.1 microM) precontracted guinea-pig tracheal sheets. 2. Rolipram, siguazodan and SNP caused concentration-related reductions in tone of tissues precontracted with 0.1 microM carbachol (EC50 values 12.5; 2.73 and 0.35 microM respectively). Whilst the concentration-response relationship for the PDE III inhibitor, siguazodan, was monophasic that of the PDE IV inhibitor, rolipram, was biphasic. 3. The relaxant activity of rolipram was markedly enhanced in the presence of 10 microM siguazodan (EC50 < 0.01 microM), 0.1 microM salbutamol (EC50 0.03 microM) and 0.3 microM SNP (EC50 0.03 microM). In contrast, the relaxant activity of siguazodan was unaffected by SNP and only modestly enhanced by rolipram (10 microM) and salbutamol (0.1 microM). 4. The relaxant activity of SNP was enhanced by the PDE V inhibitor SK&F 96231 (30 microM: EC50 0.06 microM) and rolipram (30 microM, EC50 0.08 microM) but was unaffected by 30 microM siguazodan. 5. At concentrations up to 10 microM, neither siguazodan nor rolipram elevated tracheal cyclic AMP levels. However, the combination of 10 microM rolipram and siguazodan caused a two fold increase in the cyclic AMP content (from 2.19 to 4.36 pmol cyclic AMP mg-1 protein). SNP (0.1-10 microM) failed to produce a significant increase in tracheal cyclic AMP levels. At 0.1 microM the effect of SNP on tracheal cyclic AMP levels was significantly (P < 0.05) increased in the presence of rolipram but not siguadozan. 6. The results indicate that the relaxant effects of rolipram are markedly enhanced by agents that inhibit PDE III activity or elevate cyclic GMP. They support the hypothesis that SNP potentiates the effects of rolipram via the inhibitory action of cyclic GMP on hydrolysis of cyclic AMP by PDE III. The findings also suggest that whilst PDE III may be more significant in regulating basal smooth muscle tone in the absence of any exogenous stimulus to cyclic AMP accumulation, PDE IV activity may be more tightly coupled to the pool of adenylyl cyclase stimulated by beta2-adrenoceptor agonists.

Rapid turnover of the AMP-adenosine metabolic cycle in the guinea pig heart.

The intracellular flux rate through adenosine kinase (adenosine-->AMP) in the well-oxygenated heart was investigated, and the relation of the AMP-adenosine metabolic cycle (AMP<-->adenosine) to transmethylation (S-adenosylhomocysteine [SAH]-->adenosine) and coronary flow was determined. Adenosine kinase was blocked in isolated guinea pig hearts by infusion of iodotubercidin in the presence of the adenosine deaminase blocker erythro-9-(2-hydroxy-3-nonyl)adenine (5 mumol/L). Iodotubercidin (1 nmol/L to 4 mumol/L) caused graded increases in venous effluent concentrations of adenosine, from 8 +/- 3 to 145 +/- 32 nmol/L (mean +/- SEM, n = 3), and in coronary flow, which increased to maximal levels. Flow increases were completely abolished by adenosine deaminase (5 to 10 U/mL). Interstitial adenosine concentrations, estimated using a mathematical model, increased from 22 nmol/L during control conditions to 420 nmol/L during maximal vasodilation. The possibility that iodotubercidin caused increased venous adenosine by interfering with myocardial energy metabolism was ruled out in separate 31P nuclear magnetic resonance experiments. To estimate total normoxic myocardial production of adenosine (AMP-->adenosine<--SAH), the time course of coronary venous adenosine release was measured during maximal inhibition of adenosine kinase with 30 mumol/L iodotubercidin. Adenosine release increased more than 15-fold over baseline, reaching a new steady-state value of 3.4 +/- 0.3 nmol.min-1 x g-1 (n = 5) after 4 minutes. In parallel experiments, the relative roles of AMP hydrolysis and transmethylation (SAH hydrolysis) were determined, using adenosine dialdehyde (10 mumol/L) to block SAH hydrolase. In these experiments, adenosine release increased to similar levels of 3.4 +/- 0.5 nmol.min-1 x g-1 (n = 6) during inhibition of adenosine deaminase and adenosine kinase. It is concluded that (1) maximal increases in coronary flow are elicited by increases in interstitial adenosine concentration to approximately 400 nmol/L, (2) more than 90% of the adenosine produced in the heart is normally rephosphorylated to AMP without escaping into the venous effluent, (3) AMP hydrolysis is the dominant pathway for cardiac adenosine production under normoxic conditions, and (4) the high rate of adenosine salvage is due to rapid turnover of a metabolic cycle between AMP and adenosine. Rapid cycling may serve to amplify the relative importance of AMP hydrolysis over transmethylation in controlling cytosolic adenosine concentrations.

Influence of dopamine on cyclic nucleotide enzymes in bovine retinal membrane fractions.

Dopamine is a major neurotransmitter and neuromodulator in vertebrate retina. Although its pharmacological and physiological actions are well understood, the biochemical mechanisms of its signal transduction are less clear. Acting via D1 receptors, dopamine was shown to increase cyclic AMP levels in intact retina and to activate adenylate cyclase in retinal homogenates. The action via activation of D2 receptors is controversial: it was reported to decrease cyclic AMP levels in intact retina but inhibition of cyclase could not be demonstrated in retinal homogenates; also it was reported to activate rod outer segment cyclic GMP phosphodiesterase in vitro but did not decrease cyclic GMP levels in aspartate-treated retinas. We made an attempt to fractionate bovine retinal membranes and to investigate the effects of dopamine, via D1 and D2 receptors, on the synthesis and hydrolysis of cyclic AMP and cyclic GMP. Activation of cyclic AMP synthesis was noted in all fractions, but no effects were evident on cyclic nucleotide hydrolysis or cyclic GMP synthesis in any fraction. Also, D2 agonist did not inhibit cyclic AMP synthesis. These observations suggest that D2 receptors may not be directly coupled to cyclic nucleotide metabolizing enzymes in bovine retina.

Hippocampal localization of 5′-nucleotidase as revealed by immunocytochemistry

The distribution of binding sites for an antibody against ecto-5′-nucleotidase was investigated in the mouse hippocampus by light microscopical immunocytochcmistry. The antibody selectively labels a band corresponding to the innervation area of mossy fibre terminals within area CA3. Area CA1 as well as the dendate gyms are negative. In area CA3 only the proximal but not the distal parts of the apical dendrites of pyramidal cells are labelled. Labelling is in the form of large dots around dendrites of pyramidal cells suggesting that mossy fibre terminals are immunopositive. In contrast, an antibody against the ubiquitous synaptic vesicle protein SV2 labels the large mossy fibre terminals as well as fine and punctate structures in the dendritic and somatic regions throughout the hippocampus. Labelled astrocytes can be found in the entire hippocampus and are frequent in the stratum radiatum and stratum oriens of the CA1 region. Immunopositive astrocytic processes can be found in association with capillary walls. Our results suggest that ecto-5′-nucleotidase may play a crucial role in the hydrolysis of AMP to adenosine at the mossy fibre synapses. Thus, at these synapses, 5′-nucleotidases could function both in completing the extracellular hydrolysis of synaptically released ATP as well as in the extracellular formation of adenosine.

The effect of cyclic AMP and cyclic GMP phosphodiesterase inhibitors on the superoxide burst of guinea‐pig peritoneal macrophages

1. The cyclic nucleotide phosphodiesterase (PDE) activity of guinea-pig peritoneal macrophages was partially characterized and the effects of selective and non-selective inhibitors of adenosine 3':5'-cyclic monophosphate (cyclic AMP PDE) and guanosine 3':5'-cyclic monophosphate (cyclic GMP PDE) phosphodiesterases on superoxide generation were investigated using peritoneal macrophages from horse-serum pretreated guinea-pigs. 2. The non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and the PDE I/V selective inhibitor, zaprinast, inhibited spontaneous superoxide generation with IC50s of 30.7 +/- 11.3 microM and 145 +/- 17 microM respectively (n = 6 and 5). The concentration-response curves for the PDE IV selective inhibitors rolipram and Ro20-1724 were biphasic; mean maximum inhibitions were 56.9 +/- 5.9% and 66.8 +/- 10.5% respectively at 300 microM, but in 2 out of 6 (rolipram) and 2 out of 5 (Ro20-1724) experiments inhibition was < 50%. The PDE III inhibitor SK&F 94120 was without effect. Spontaneous superoxide generation was reduced 57 +/- 10% by 1 microM prostaglandin E2 (PGE2) and 62.6 +/- 3.76% by 1 microM salbutamol. 3. The increase in superoxide generation elicited by FMLP (10(-9)-10(-5)M) was unaffected by any of the PDE inhibitors studied. Inhibition of FMLP-stimulated superoxide generation by PGE2 was enhanced in the presence of 10 microM IBMX. 4. Macrophages were found to contain a predominantly membrane bound cyclic AMP PDE (90% of total activity) which was unaffected by cyclic GMP or calcium/calmodulin. The cyclic AMP PDE activity in the cytosolic fraction was enhanced in the presence of calcium/calmodulin. Selective inhibitors of PDE IV inhibited the particulate cyclic AMP PDE activity (IC50s rolipram 1.5 +/- 0.3 microM, Ro 20-17244.1 +/- 0.6 microm) as did the non-selective inhibitor IBMX (IC50 22 +/- 8 microM). The macrophage particulate PDE activity was resistant to inhibition by the PDE III inhibitor SK&F 94836 and the PDE I/V inhibitor, zaprinast. The cytosolic calcium/calmodulin stimulated cyclic AMP hydrolytic activity was inhibited by zaprinast (IC50 - calcium/calmodulin 123 +/- 39 microM; + calcium/calmodulin IC50 17.7 +/- 6.3 microM).5. The results indicate that guinea-pig peritoneal macrophages contain a type IV cyclic AMP PDE which is predominantly membrane associated and a predominantly cytosolic calcium/calmodulin stimulated cyclic AMP PDE. Functional studies suggest that both of these PDE activities contribute to cyclic AMP hydrolysis and regulation of superoxide generation in these cells. Inhibition of spontaneous superoxide generation, but not that stimulated by FMLP, suggests that the activity of PDE inhibitors is subject to functional antagonism but that this can be overcome by enhancing cyclic AMP formation.

Contribution of the ciliary cyclic nucleotide‐gated conductance to olfactory transduction in the salamander.

1. Flash photolysis of caged cyclic nucleotides was used to examine the contribution of the ciliary cyclic nucleotide-gated conductance to olfactory transduction in the tiger salamander. Brief illumination of solitary olfactory receptor cells loaded with 100 microM caged cyclic AMP caused a large inward current (peak amplitude 355 +/- 200 pA; mean +/- S.D. for eleven cells) under whole-cell voltage clamp at -50 mV. 2. The photolysis response was initiated after a latency of 4-12 ms, whereas an odorant response of identical amplitude had a latency of several hundred milliseconds. The amplitudes of both responses exhibited almost identical voltage dependence between -50 and +25 mV, with both reversing near 0 mV. The time courses of the falling phases of odorant and photolysis responses also exhibited similar voltage dependence, both being prolonged at positive voltages. 3. Photolysis of caged cyclic GMP activated a current similar in amplitude and time course to that produced by photolysis of caged cyclic AMP. 4. When the flash was spatially limited to the cilia, the amplitude and duration of the photolysis response increased linearly with the length of the cilia illuminated (for cilia not longer than 30-40 microns) while the latency remained constant at 4-12 ms. The increase in duration was described semi-quantitatively by a model which incorporated diffusion and saturable hydrolysis of cyclic AMP. When the flash was limited to the soma or proximal dendrite, the response latency was proportional to the square of the distance between the illuminated region and the cilia. 5. Dialysis of cells with 500 microM cyclic AMP from a whole-cell electrode under voltage clamp activated a large transient inward current. Simultaneous suction electrode recording showed that this current originated almost entirely from the ciliary membrane. The density of cyclic nucleotide-gated channels was estimated to be 800-fold higher in the cilia than in the soma. 6. Summation of simultaneous odorant and photolysis responses was non-linear, the flash-induced current being enhanced during a small odorant response and attenuated during a large odorant response. Summation of two photolysis responses was similarly non-linear. The data were consistent with odorant stimuli and cyclic AMP both activating a common cyclic nucleotide-gated conductance with a Hill coefficient, n, of 2.0-4.4. For n = 2.5, the basal cyclic AMP concentration was estimated to be less than 20% of the K 1/2, which predicts a basal current of 5.8 pA, less than 2% of the maximum.(ABSTRACT TRUNCATED AT 400 WORDS)

気管支ぜん息におけるリンパ球Ｐｈｏｓｐｈｏｄｉｅｓｔｅｒａｓｅ活性値について

Phosphodiesterase is the only enzyme that catalyzes the hydrolysis of cyclic AMP to 5′AMP, and the relaxation of smooth muscle is the result of an intracellular increase in the level of cyclic AMP. It has been generally assumed that the bronchodilatory properties of theophylline in asthma therapy are due to its cyclic AMP-phosphodiesterase inhibitory capacity. In recent years, a number of studies on the action of theophylline have led to a general hypothesis which suggests that xanthine derivatives act as adenosine receptor antagonists. In the present study, we reported that the lymphocyte cyclic AMP level showed an obvious increase only in asthma patients after the administration of theophylline, while the level was the same in asthma patients and healthy adults before this administration.The difference in phosphodiesterase activity between patients and healthy adults in one of the causes of the difference in the lymphocyte cyclic AMP level.In this study, we examined lymphocyte phosphodiesterase activity and the following results were obtained.1) The lymphocyte phosphodiesterase activity was higher in patients with bronchial asthma than in healthy adults.2) The lymphocyte phosphodiesterase activity level showed an obvious decrease only in patients with asthma.These results suggest that phosphodiesterase activity is related to the mechanism of bronchial asthma, and the hypothesis that theophylline is an inhibitor of phosphodiesterase is confirmed.

Comprehensive model of transport and metabolism of adenosine and S-adenosylhomocysteine in the guinea pig heart.

Regulation of blood flow and mitochondrial respiration in the heart would be clarified by improved knowledge of interstitial concentrations and cellular production rates of adenosine; however, these variables cannot be measured directly. To interpret indexes that are available, a comprehensive mathematical model was developed, based on a large body of experimental data. The model describes most of the important pathways of capillary-tissue transport and cellular metabolism of adenosine in the guinea pig heart. It includes capillary flow, solute transport between tissue regions, nonlinear enzyme kinetics for adenosine kinase and adenosine deaminase, and reversible biunireactant kinetics for S-adenosylhomocysteine hydrolase in cardiomyocytes and endothelial cells, intracellular production of adenosine via AMP hydrolysis and transmethylation, and extracellular production of adenosine. A single set of parameter values for the model was obtained in the first stage of the analysis by taking certain values directly from published sources, other values were subject to specific constraints, and other values were determined by parameter optimization. The effects of flow and endothelial metabolism on the relation between interstitial and venous adenosine concentrations were determined. The relation between myocardial adenosine production rate and S-adenosylhomocysteine accumulation in the presence of excess homocysteine was estimated. In the second stage of the analysis, the model was used to investigate the mechanism of myocardial adenosine production, without changing the parameter values. Cellular adenosine production rates were estimated by fitting measurements of venous adenosine release obtained during altered energetic conditions in experiments by different investigators. The original results showed a dissociation between measurements of cytosolic AMP concentrations and venous adenosine release. It is concluded that 1) it is essential to account for the effect of flow on interstitial and venous adenosine concentrations, since decreased flow may produce effects outwardly resembling inhibition of the enzyme 5'-nucleotidase, 2) adenosine concentrations in epicardial transudate are not in equilibrium with interstitial fluid, and 3) the rate of cellular adenosine production increases monotonically with free cytosolic concentrations of AMP during a variety of alterations in energy balance of the guinea pig heart.

A novel human phosphotransferase highly specific for adenosine

A novel nucleoside phosphotransferase, referred to as adenosine phosphotransferase (Ado Ptase), was partially purified 1230-fold from human placenta. This enzyme differed from other known nucleoside phosphotransferases in its substrate specificity. Using AMP as the phosphate donor, it readily phosphorylated Ado. Changes in the sugar moiety were tolerated. dAdo and ddAdo were phosphate acceptors and dAMP was a donor. No other nucleotide or nucleoside common in nature displayed appreciable activity as donor or acceptor substrate, respectively. In the absence of nucleoside, the enzyme catalyzed the hydrolysis of AMP, typical of other nucleoside phosphotransferases. However, in the presence of Ado, little, if any, hydrolysis occurred. Ado Ptase had an absolute requirement for a metal cation, with Mg 2+ and, to a lesser extent, Mn 2+ fulfilling this requisite. The apparent K m for Ado was 0.2 m m. However, the donor AMP displayed cooperativity in both transfer and hydrolytic reactions. This cooperativity was eliminated by nucleotides, 2,3-diphosphoglycerate, and inorganic phosphate. ADP and 2,3-diphosphoglycerate were especially potent. In the presence of these effectors, the apparent K m for AMP was 3.0 m m in the transfer reaction and 4.0 m m in the hydrolytic reaction. Kinetic data suggest that there are two nucleotide binding sites on Ado Ptase, one for the donor, the other for an effector. AMP appeared to bind to both sites. Although this novel enzyme might play a role in the anabolism of nucleoside analogues, the normal physiological role of this nucleoside phosphotransferase is not understood.

Regulation of extracellular adenosine production by ectonucleotidases of adult rat ventricular myocytes.

We have investigated the kinetic properties of the extracellular reaction sequence ATP----ADP----AMP----adenosine catalyzed by ectonucleotidases at the surface of adult rat cardiac myocytes. Analysis of progress of reaction curves indicates that depletion of substrate at cell surfaces dominates the regulation of the rate of hydrolysis of ATP or of ADP when it is the initial substrate. Preferential delivery of intermediate products to be substrates at cell surfaces makes a significant contribution to the regulation of adenosine production from ATP or ADP. Preferential delivery has more impact on the delivery of ADP from adenosinetriphosphatase (ATPase) to adenosinediphosphatase (ADPase) than on delivery of AMP from ADPase to 5'-nucleotidase. At high initial ATP concentrations, feed-forward inhibition of AMP hydrolysis also modulates the rate of adenosine production. Taken together, the properties of the ectonucleotidases on the myocyte provide a milieu at the cell surface that tends to be poor in nucleotides, especially ATP and ADP (P2 purinoceptor agonists), and rich in adenosine (a P1 purinoceptor agonist) during periods of supply of extracellular nucleotides.

5′‐Nucleotidase from the electric ray electric lobe

A cDNA encoding a 5'-nucleotidase was identified by screening a lambda gt10 cDNA library from the electric lobe of Discopyge ommata using a cDNA probe containing the complete open reading frame coding for the rat liver enzyme. Nucleotide sequence analysis defines an open reading frame of 577 amino acids, corresponding to a calculated molecular mass of 63,833 Da. The N-terminus of the mature protein, as determined by direct protein sequencing, is preceded by 29 amino acid residues comprising a signal peptide. The C-terminus contains a stretch of hydrophobic amino acids, considered to be cleaved on post-translational modification and exchanged for glycosylphosphatidylinositol as a membrane anchor. The predicted protein contains four potential N-linked glycosylation sites. Electric ray 5'-nucleotidase shares 61% amino acid identity with the enzymes from rat liver and human placenta, and about 23% with bacterial proteins possessing 5'-nucleotidase activity and also additional enzyme activities like UDP-glucose hydrolase. Polyclonal antibodies raised against 5'-nucleotidase from mammalian sources or the electric ray electric organ reveal mutual cross-reactivity. Interestingly, there are 5-7 domains highly conserved in procaryotes and vertebrates in enzymes exhibiting 5'-nucleotidase, 3'-nucleotidase or phosphodiesterase activity. 5'-nucleotidase isolated from Torpedo electric organ hydrolyzes UDP-glucose at 8% of the rate of AMP hydrolysis. The possible phylogenetic origin of vertebrate 5'-nucleotidase from multifunctional nucleotide hydrolases is discussed.

Segment-specific effects of the heat-stable enterotoxin of E. coli on electrolyte transport in the rat coton

The heat-stable enterotoxin of E. coli (STa) induced an increase in short-circuit current (Isc) in the rat colon. The maximal increase in Isc was about three times larger in the proximal than the distal colon. The action of STa was mimicked by 8-Br-cyclic GMP. Unidirectional flux measurements revealed that STa decreased Na + and Cl − absorption in the distal colon, while it decreased Na + absorption and activated Cl − secretion in the proximal colon. In the distal, but not in the proximal colon, indomethacin inhibited the action of STa and of 8-Br-cyclic GMP. Inhibition by indomethacin could be overcome by addition of prostaglandin E 2 or forskolin, but not by addition of a non-hydrolysable analogue of cyclic AMP, suggesting an action of STa on cyclic AMP hydrolysis. Amrinone and trequinsin, two inhibitors of cyclic GMP-inhibited phosphodiesterases, mimicked the action of STa on Isc and inhibited the response to a subsequent administration of the toxin indicating the modulation of a cyclic GMP-inhibited phosphodiesterasc by STa in the distal colon. The results give evidence for different intracellular action sites of STa in the two parts of the rat colon.

Inhibition of eosinophil cyclic nucleotide PDE activity and opsonised zymosan-stimulated respiratory burst by 'type IV'-selective PDE inhibitors.

1. The cyclic nucleotide phosphodiesterase (PDE) of guinea-pig eosinophils was partially characterized and the effects of selective inhibitors of PDE isoenzymes upon opsonized zymosan (OZ)-stimulated respiratory burst were studied. 2. PDE activity in eosinophil lysates appeared to be membrane-associated, displayed substrate specificity for adenosine 3':5' cyclic monophosphate (cyclic AMP) versus guanosine 3':5' cyclic monophosphate (cyclic GMP) and was insensitive to cyclic GMP or Ca2+ and calmodulin. 3. The non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine caused a concentration-dependent inhibition of both OZ-stimulated hydrogen peroxide (H2O2) generation and cyclic AMP hydrolysis. The type IV-selective PDE inhibitors, rolipram and denbufylline, also inhibited H2O2 generation and cyclic AMP hydrolysis in a concentration-dependent manner whilst SK&F 94120 and Org 9935 (type III-selective) and zaprinast (type Ia or V-selective) were ineffective. 4. Dibutyryl cyclic AMP, a cell-permeable, non-hydrolysable analogue of cyclic AMP, caused a concentration-dependent inhibition of H2O2 generation stimulated by OZ. Dibutyryl cyclic GMP was ineffective. 5. It is concluded that eosinophil respiratory burst activity induced by OZ can be regulated by intracellular cyclic AMP and that the levels of cyclic AMP are controlled exclusively by a rolipram- and denbufylline-sensitive PDE isoenzyme that resembles a type IV species.

The stepwise hydrolysis of adenine nucleotides by ectoenzymes of rat renal brush-border membranes

Evidence is presented for the existence of ectoenzymes in rat renal cortical brush-border membrane vesicles that produce adenosine as a final product using either ATP, ADP or AMP as substrate. The enzymes are insensitive to levamisole, ouabain, oligomycin and N-ethylmaleimide, and have absolute requirement for divalent cations with following order of activation Mg 2+ < Ca 2+ < Mn 2+ < Ba 2+ < Zn 2+. At least two separate enzymes can be distinguished. One is capable of hydrolyzing ATP, other nucleoside triphosphates and ADP, but not AMP. The enzyme is insensitive to concanavalin A. The other enzyme hydrolyzes AMP and is strongly inhibited by this lectin. Mg 2+-stimulated ATP hydrolysis displays saturation kinetics which is not of the simple Michaelis-Menten type, but is biphasic with a high-affinity ( K′ m = 0.16 mM) and low-affinity site ( K′ m = 9.0 mM, respectively. The low-affinity site hydrolyzes ATP, ITP and GTP to a similar extent, whereas CTP and UTP with about 40% lower rate. The high-affinity site splits ATP much better than other nucleoside triphosphates. Hydrolysis of ADP follows simple Michaelis-Menten saturation kinetic with apparent K m = 0.38 ± 0.06 mM. Inhibition, activation and substrate specificity studies indicate that nucleoside triphosphatase and nucleoside diphosphatase may reside on the same protein. Kinetics of the AMP hydrolysis is hyperbolic with apparent K m = 76 ± 9 μM. The cascade of ectonucleotidases in the brush-border membrane of the proximal tubule may catalyze the degradation of filtered nucleotides into adenosine and phosphate, the compounds which are thereafter probably reabsorbed by separate transport systems.

Pharmacology of positive inotropic phosphodiesterase III inhibitors

The drugs, new and old, useful in the treatment of acute cardiac failure, are reviewed in the light of its pathophysiological mechanisms and of the biochemical aspects of myocardial contraction. Two major classes of drugs are considered, those that stimulate cell membrane adenylcyclase, i.e. beta-agonists (dopamine, dobutamine and dopexamine) and alpha-agonists (glucagon, forskolin, calcium agonists), and those that inhibit the cellular phosphodiesterases, i.e. bipyridine derivatives (amrinone and milrinone) and imidazolone derivatives (fenoximone and piroximone). Virtually, all the inotropic agents act by increasing the entry of calcium into the cell by increasing the intracellular AMPc concentration. Dopamine has a dose-related triphasic activity. At low doses, stimulation of renal dopaminergic receptors increases renal blood flow, glomerular filtration rate and sodium clearance. At moderate doses, dopamine stimulates, for the most part, cardiac beta-adrenergic receptors. Higher doses stimulate alpha-1-adrenergic receptors, with an increase in systemic arterial and venous pressures. Dobutamine exerts a potent positive inotropic action, with little effect on vascular tone and less tachycardia than with other catecholamines, resulting in only a slight increase in myocardial oxygen consumption. The dopamine analogue, dopexamine, increases renal blood flow, myocardial contractility and produces peripheral vasodilation. The haemodynamic effects of phosphodiesterase inhibitors are similar to those of dobutamine, except that these drugs are vasodilators, their positive inotropic properties are weak and their haemodynamic effects persist for at least 8 h after a single dose in heart failure patients. Beta-adrenergic agonists should be considered as phosphodiesterase inhibitors should probably be used as second-line drugs, when beta-agonists become ineffective, or for patients with chronic congestive heart failure, especially so as it would seem that only oral preparations of phosphodiesterase inhibitors are effective in long-term treatment.

Identification and selective inhibition of four distinct soluble forms of cyclic nucleotide phosphodiesterase activity from kidney

Homogenization of rat kidney under isotonic conditions and in the presence of protease inhibitors showed that some 92% of the cyclic AMP phosphodiesterase activity and some 83% of the cyclic GMP phosphodiesterase activity was released into the soluble fraction. Analysis of soluble phosphodiesterase activity by FPLC on a Mono-Q column resolved four distinct fractions expressing cyclic nucleotide phosphodiesterase activity. Lineweaver-Burk plots for the hydrolysis of both cyclic GMP and cyclic AMP yielded linear results. The first two peaks (KPDE-MQ-II, KPDE-MQ-III) showed higher activities towards cyclic GMP than cyclic AMP with the ratio of their V max values for the hydrolysis of cyclic AMP/cyclic GMP being 0.66 and 0.16, respectively. For the second two peaks (KPDE-MQ-IV, KPDE-MQ-V) the V max ratios for the hydrolysis of cyclic AMP/cyclic GMP were 6.4 and 16.7, respectively. All enzymes exhibited similar low K m values for both cyclic AMP and cyclic GMP but had very different V max values. KPDE-MQ-II was activated by Ca 2+/calmodulin. The cyclic AMP phosphodiesterase activity of KPDE-MQ-III was augmented by the presence of low concentrations of cyclic GMP. Thermal denaturation studies showed that the phosphodiesterase activity of each fraction decayed as a single exponential indicating that each phosphodiesterase fraction contained but a single phosphodiesterase activity. The inhibitors IBMX, zaprinast, milrinone, amrinone, buquineran, carbazeran, ICI 118233, ICI 63197 exerted selective effects on the activities of these enzymes. We compared the action of these compounds on cyclic GMP phosphodiesterases from bovine retina. Over the concentration ranges used, the bovine retinal enzyme was only inhibited by IBMX, zaprinast and carbazeran. The cytosolic isoenzymes of cyclic AMP phosphodiesterases play a much more important role in metabolizing cyclic AMP in kidney compared with liver, where the activity of membrane-bound isoenzymes predominate.

Phospholipid metabolism modulates cyclic nucleotide phosphodiesterase activity in rat heart microsomes

Cyclic nucleotide phosphodiesterase activity in rat heart microsomes is attributable to several isoenzymatic forms: a cyclic AMP-specific, a cyclic GMP-specific, and a cyclic GMP-stimulated enzyme. Incubation of microsomes with an exogenous phospholipase C (C. welchii) induced a marked stimulation (+126%) of cyclic AMP phosphodiesterase and a moderate stimulation (+49%) of cyclic GMP-phosphodiesterase in the membrane-bound fraction. Besides, a notable fraction of activity was solubilized by the treatment. A parallel decrease in the activating effect of cyclic GMP on the hydrolysis of cyclic AMP was observed in the membranes (down to 18% of the control effect). It resulted from a marked stimulation of the basal activity, while the activated level was unaffected. The treatment by an exogenous phospholipase D induced more moderate modifications. The addition to microsomes of oleyl,acetyl-glycerol, but not of long chain-diacylglycerols, partly reproduced the phospholipase C effect. Phosphatidate also induced variations in phosphodiesterase activity, and could thus participate in the phospholipase effects. These results suggest that endogenous phospholipases, the activity of which is modulated by hormonal stimuli, might influence phosphodiesterase activity in cardiac membranes by producing phospholipid metabolites, with potential consequences on heart contractility.

Purification and characterization of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from the cytosol of human platelets

A cyclic GMP-stimulated cyclic nucleotide phosphodiesterase was purified to near homogeneity from the 150,000 g supernatant fraction of human platelets by a combination of DEAE-cellulose chromatography and cyclic GMP affinity chromatography. Overall purification was about 7400-fold with a 10% to 15% recovery of activity. On NaDodSO4-containing polyacrylamide gels, the purified enzyme migrates as a single band M r = 105,000. Phosphodiesterase activity co-migrates with the protein band on native polyacrylamide gels. Both Mg 2+ and Mn 2+ support the activity of this phosphodiesterase. The enzyme hydrolyzes both cyclic AMP and cyclic GMP with similar maximal rates. The hydrolysis of both nucleotides exhibits positive homotropic cooperativity with S 0.5 values of 50 ± 12 μM for cyclic AMP and 35 ± 15 μM for cyclic GMP and Hill coefficients of 1.2 to 1.5 for both nucleotides. Low levels of cyclic GMP stimulate the rate of cyclic AMP hydrolysis from 3- to 10-fold. The activity of this phosphodiesterase is not stimulated by the calcium binding protein, calmodulin. The cyclic GMP stimulation of cyclic AMP hydrolysis by this phosphodiesterase may provide a possible regulatory link between the metabolism of these two nucleotides in platelets.