AMP Hydrolysis Research Articles

Characterization of the basic amphiphilic alpha-helix calmodulin-binding domain of a 61.5 kDa tobacco calmodulin-binding protein.

A 19-amino acid residue peptide, Gly-Trp-Leu-Lys-Ile-Lys-Ala-Ala-Met-Arg-Trp-Gly-Phe-Phe-Val-Arg-Lys-Lys- Ala, corresponding to the basic amphiphilic alpha-helix (BAA) motif at the C-terminus of a recombinant tobacco calmodulin-binding protein, TCB60, was synthesized. The interaction of the synthetic binding domain with calmodulin (CaM) was analyzed by gel mobility shift assays, phosphodiesterase competition assays, and fluorescence, circular dichroism, and nuclear magnetic resonance spectroscopy. Mobility shift assays showed an apparent 2 kDa increase in CaM Mr in presence of synthetic peptide and CaCl2 in 4 M urea polyacrylamide gel electrophoresis. HPLC measurements of hydrolysis of cyclic AMP by CaM-dependent phosphodiesterase indicated the synthetic peptide competitively inhibits (Ki = 15-20 nM) stimulation of phosphodiesterase activity by CaM. Upon binding CaM, the fluorescence emission maximum of the synthetic peptide, which contained two tryptophanyl residues, shifted toward blue and increased in intensity. The circular dichroism spectra indicated the ellipticity of CaM increased at 208 and 222 nm upon complex formation with the synthetic peptide. 1H NMR studies showed that the peptide interacts with the aromatic residues in domains I and III of CaM. Taken together, these data provide direct evidence that the structurally conserved basic amphiphilic alpha-helix CaM-binding domain of the recombinant tobacco CaM-binding protein interacts with CaM at physiologically significant nanomolar concentrations and the microenvironments of both CaM and the synthetic binding domain are modified upon complex formation.

Immunohistochemical colocalization of the α-subunit of neutrophil NADPH oxidase and ecto-5′-nucleotidase in kidney and liver

In kidney and liver, fibroblasts and fibroblast-like cells, respectively, are sources of erythropoietin (Epo) formation, and these cells also bear a number of other similarities. Renal Epo expression is localized in peritubular type 1 fibroblasts of the cortical labyrinth, and in the liver, apart from parenchymal cells, transcription is found in Ito cells. Both the renal peritubular cells and Ito cells contain ecto-5'-nucleotidase (5'NT). It had been suggested that 5'NT is involved in the oxygen sensing mechanism via a hydrolysis of AMP to adenosine, which in turn may stimulate EPO synthesis. However, the molecular mechanism of the cellular response to hypoxia is currently not well understood. Based on the notion that a heme protein probably acts as the oxygen sensor, it has recently been proposed that a b-type cytochrome as part of the neutrophil NADPH oxidase may influence intracellular superoxide levels depending on local oxygen tension. Superoxide levels were otherwise shown to determine the EPO production in hepatoma cell lines. By double immunofluorescence labeling the alpha-subunit of cytochrome b558 (alpha-SU) and 5'NT were simultaneously localized in rat kidney and liver, and in the kidney Epo mRNA and alpha-SU were double-labeled. Positive signal for alpha-SU was found in the majority of renal peritubular fibroblasts in the cortex and outer medulla, and in Ito cells. In both organs, the cells that coexpress 5'NT and Epo mRNA also contain an immunoreactivity for alpha-SU. In these cells, cytochrome b558 as part of an NADPH oxidase may be involved in a presumptive oxygen sensing mechanism using H2O2 as a possible second messenger for EPO gene regulation.

Extracellular metabolism of nucleotides in the nervous system.

1. A variety of surface-located enzymes are involved in the metabolism of extracellular nucleotides. The biochemical properties of some of these are briefly discussed. 2. The molecular identity of ecto-diadenosine polyphosphate hydrolase has not yet been revealed. On neural cells the enzyme catalyses the hydrolysis of ApnA to Apn-1 and AMP. 3. The molecular structure of ATP-diphosphohydrolase has recently been identified. The enzyme occurs in essentially all tissues where it catalyses the extracellular hydrolysis of ATP and ADP with the formation of AMP. 4. Ecto-5'-nucleotidase is a GPI-anchored glycoprotein and catalyses the formation of AMP to adenosine. In the adult brain, and as revealed by immunocytochemistry, the enzyme is mainly associated with astrocytes. It is associated with developing nerve cells and cultured neural cells. In vitro its inhibition or suppression of its synthesis result in the inhibition of neurite formation and long-time survival of neural cells. Continued extracellular hydrolysis of AMP and formation of adenosine thus appear to be essential for neural differentiation and survival.

Identification of cyclic AMP phosphodiesterases 3, 4 and 7 in human CD4+ and CD8+ T-lymphocytes: role in regulating proliferation and the biosynthesis of interleukin-2.

1. The cyclic AMP phosphodiesterases (PDE) expressed by CD4+ and CD8+ T-lymphocytes purified from the peripheral blood of normal adult subjects were identified and characterized, and their role in modulating proliferation and the biosynthesis of interleukin (IL)-2 and interferon (IFN)-gamma evaluated. 2. In lysates prepared from both subsets, SK&F 95654 (PDE3 inhibitor) and rolipram (PDE4 inhibitor) suppressed cyclic AMP hydrolysis indicating the presence of PDE3 and PDE4 isoenzymes in these cells. Differential centrifugation and subsequent inhibitor and kinetic studies revealed that the particulate fraction contained, predominantly, a PDE3 isoenzyme. In contrast, the soluble fraction contained a PDE4 (approximately 65% of total activity) and, in addition, a novel enzyme that had the kinetic characteristics of the recently identified PDE7. 3. Reverse transcription-polymerase chain reaction (RT-PCR) studies with primer pairs designed to recognise unique sequences in the human PDE4 and PDE7 genes amplified cDNA fragments that corresponded to the predicted sizes of HSPDE4A, HSPDE4B, HSPDE54D and HSPDE7. No message was detected for HSPDE4C after 35 cycles of amplification. 4. Functionally, rolipram inhibited phytohaemagglutinin- (PHA) and anti-CD3-induced proliferation of CD4+ and CD8+ T-lymphocytes, and the elaboration of IL-2, which was associated with a three to four fold increase in cyclic AMP mass. In all experiments, however, rolipram was approximately 60 fold more potent at suppressing IL-2 synthesis than at inhibiting mitogenesis. In contrast, SK&F 95654 failed to suppress proliferation and cytokine generation, and did not elevate the cyclic AMP content in T-cells. Although inactive alone, SK&F 95654 potentiated the ability of rolipram to suppress PHA- and anti-CD3-induced T-cell proliferation, and PHA-induced IL-2 release. 5. When a combination of phorbol myristate acetate (PMA) and ionomycin were used as a co-mitogen, rolipram did not affect proliferation but, paradoxically, suppressed IL-2 release indicating that cyclic AMP can inhibit mitogenesis by acting at, or proximal to, the level of inositol phospholipid hydrolysis. 6. Collectively, these data suggest that PDE3 and PDE4 isoenzymes regulate the cyclic AMP content, IL-2 biosynthesis and proliferation in human CD4+ and CD8+ T-lymphocytes. However, the ability of rolipram to suppress markedly mitogen-induced IL-2 generation without affecting T-cell proliferation suggests that growth and division of T-lymphocytes may be governed by mediators in addition to IL-2. Finally, T-cells have the potential to express PDE7, although elucidating the functional role of this enzyme must await the development of selective inhibitors.

Diminished noradrenergic stimulation reduces the activity of rolipram-sensitive, high-affinity cyclic AMP phosphodiesterase in rat cerebral cortex.

The present study examined the in vivo regulation of rolipram-sensitive, high-affinity cyclic AMP phosphodiesterase (PDE4) in rat cerebral cortex. The hydrolysis of cyclic AMP, formed by stimulation of beta-adrenergic receptors, was measured in cerebral cortical slices. Hydrolysis of cyclic AMP formed under these conditions was inhibited by the PDE4-selective inhibitor rolipram but not by selective inhibitors of other PDE families. Intraventricular infusion of 6-hydroxydopamine (6-OHDA; 200 micrograms) decreased the rate constant of cyclic AMP hydrolysis and increased the cyclic AMP half-life 17 days, but not 1 or 7 days, following the treatment. A reduction in norepinephrine (NE) content occurred first; the NE level was reduced to 42, 24, and 6% of control at 1, 7, and 17 days after 6-OHDA infusion, respectively. This was followed by the development of supersensitivity of beta-adrenergic receptor-linked adenylyl cyclase, which occurred 7 days after the infusion. The reduction in PDE4 activity occurred last. When a higher dose of 6-OHDA (300 micrograms) was used, the reduction in the rate constant of cyclic AMP hydrolysis occurred by 7 days; at this time NE content was depleted to 6% of control. Similar to 6-OHDA treatment, continuous blockade of beta-adrenergic receptors, produced by chronic propranolol infusion, decreased the rate constant of cyclic AMP hydrolysis. Therefore, the current results indicate that diminished stimulation of beta-adrenergic receptors, either by loss of noradrenergic innervation or by receptor blockade, reduces the activity of PDE4. This suggests that PDE4 regulation may contribute in the homeostasis of the noradrenergic receptor-effector system in the brain.

Developmental Differences in Distribution of Cyclic Nucleotide Phosphodiesterase Isoforms in Cardiomyocytes and the Ventricular Tissue from Newborn and Adult Rats

We characterized cyclic nucleotide phosphodiesterase (PDE) activities in neonatal rat cardiomyocytes and in whole ventricle from newborn and adult rats, to determine whether cyclic nucleotide hydrolytic activities might be influenced by the developmental stage of the animal or by cell isolation and culture procedures. Using anion-exchange high-performance liquid chromatography (HPLC), we obtained four soluble PDE activities from adult rat ventricle. Peak 1, a cyclic GMP-specific PDE was stimulated by Ca2+/calmodulin; peak 2 hydrolyzed cyclic AMP and cyclic GMP, the addition of cyclic GMP stimulating the hydrolysis of cyclic AMP. Peaks 3 and 4 selectively hydrolyzed cyclic AMP, peak 3 being very sensitive to inhibition by rolipram and peak 4 consisting of two different enzyme activities, one inhibited by cyclic GMP (peak 4b) and the other inhibited by rolipram (peak 4a). In cardiomyocytes and whole ventricle from newborn rats, the response of the two cyclic GMP-sensitive PDE isoforms (cyclic GMP-stimulated and cyclic GMP-inhibited PDE) to the effector cyclic GMP was markedly reduced as compared with that of the corresponding isoforms (peaks 2 and 4b) present in the heart ventricle of adult rats. The other peaks were analogous to peaks 1, 3, and 4a of the whole ventricle. The profile of PDE activities in nonmyocardial cells did not differ from that of myocytes. Therefore, the lack of cyclic GMP effects was not due to culture conditions. Differences in PDE activities observed between cardiomyocytes from newborn rats and ventricle from adult rats might be age dependent.

The dephosphorylation of AMP and IMP by a soluble low Km 5′nucleotidase from human seminal plasma: Some regulatory aspects

In this study, a soluble low K m 5′nucleotidase, dephosphorylating IMP with a V max/ K m ratio 10-times higher than that of AMP, has been purified from human seminal plasma. The effect of inorganic phosphate (P i) and adenylate energy charge variations on the activity of this enzyme has also been investigated. In the physiological range, with IMP as substrate, the activity of the enzyme does not change whereas the hydrolysis of AMP increases with decreasing energy charge values. In the presence of both the substrates, phosphate exerts an inhibitory effect on the enzyme activity with a similar concentration dependence pattern. The results show that AMP-hydrolysing activity responds to variations of energy charge by increasing the AMP degradation thus protecting the value of energy charge at the expense of a decrease in the total adenylate pool. In contrast, the dephosphorylation of IMP is not regulated by changes in energy charge. This data suggests that the degradation of IMP and AMP, although carried out by the same enzyme, is controlled by different regulatory mechanisms.

Inhibition of extracellular ATP degradation in endothelial cells

The plasma membrane ATPase on the human umbilical vein cndothelial cell line (ECV304) was demonstrated to be an ecto-enzyme. Hydrolysis of ATP was measured by monitoring the appearance of inorganic phosphorus. Hydrolysis of extracellular ATP was insensitive to oligomycin, vanadate, ouabain and N-ethylmaleimide, compounds that inhibit the intracellular ion pumping ATPases. β-Glycerophosphate (1–10 mM) or p-nitrophenyl phosphate (1–10 mM) did not inhibit hydrolysis of ATP, ruling out the involvement of non-specific phosphatases. Enzyme activity in buffer that had previously been incubated with cells was < 7%, showing that the enzyme activity measured did not result from release of intracellular enzymes. Consistent with this, the cell preparations used were estimated to be > 95 % intact as judged by release of cytosolic enzyme lactate dehydrogenase. The enzyme activity was Ca 2+/Mg 2+ dependent. Gramicidin S (20 μM), suramin (100–300 μM), chlorpromazine (250 μM), trifluoperazine (50–250 μM), and thioridazine (100 μM) inhibited the hydrolysis of ATP (3 mM) by 45–80%. The percentage inhibition produced by these substances was not altered in the presence of a concentration of α,β-methylene ADP (10 μM) which inhibited hydrolysis of AMP (3 mM) by 90%, suggesting that these compounds inhibit ecto-ATPase and/or ecto-ADPase. Measurements of absolute amounts of ATP released from various tissues, including the heart, have been hindered because ATP is rapidly and sequentially hydrolysed to adenosine. Identification of compounds that inhibit ATP degradation would prove to be useful to overcome this problem and would lead to the development of invaluable pharmacological tools in many other aspects of purine research.

Kinetics of extracellular ATP hydrolysis by microvascular endothelial cells from rat heart.

We have characterized the ectonucleotidases that catalyse the reaction sequence ATP-->ADP-->AMP-->adenosine on microvascular endothelial cells cultured from the rat heart. Computer simulation and data fitting of progress of reaction curves showed that depletion of substrate at the cell surface dominates the regulation of the rate of hydrolysis of ATP when it is presented to the cells. Preferential delivery of AMP by ADPase to 5'-nucleotidase makes a significant contribution to the regulation of adenosine production from ATP or ADP. By contrast, we found no evidence for the preferential delivery of ADP from ATPase to ADPase. Feed-forward inhibition of AMP hydrolysis by ADP and/or ATP also modulated the rate of adenosine production. The properties of the ectonucleotidases on rat heart microvascular cells are such that adenosine is produced at a steady rate over a wide range of ATP concentrations.

5′‐Nucleotidase Activates and an Inhibitory Antibody Prevents Neuritic Differentiation of PC12 Cells

Ecto-5'-nucleotidase catalyses the hydrolysis of AMP at the surface of a variety of cells whereas it is absent from others. In addition to its catalytic activity, a function in neural development and also its interaction with extracellular matrix proteins has been reported. In order to further elucidate the biological function of ecto-5'-nucleotidase we have investigated the effect of 5'-nucleotidase on nerve growth factor-induced differentiation of PC12 cells. Furthermore, we compared the effect of an inhibitory versus a non-inhibitory monospecific antibody against the enzyme on neuritic differentiation and survival of PC12 cells that constitutively express the enzyme. When coverslips are coated with the soluble form of ecto-5'-nucleotidase in addition to collagen, there is a considerable increase in nerve growth factor-induced neurite length during the first 24 h of culture. Addition of an antibody to a culture medium that inhibits 5'-nucleotidase activity to 33% of control values dramatically reduces the number of neurites per cell within 3 days of culture. The cells round up, cluster and eventually die. On the contrary, another antibody that had no significant effect on enzyme activity affected neither nerve growth factor-induced neurite formation nor survival of PC12 cells. Addition of adenosine (200 nM, 10 or 20 microM) to the culture medium did not influence PC12 cell differentiation. The effects induced by the inhibitory antibody could be only partially prevented by simultaneous application of adenosine. Our results suggest that 5'-nucleotidase is essential for nerve growth factor-induced neurite outgrowth and survival of PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonenzymatic hydrolysis of adenosinetriphosphate (ATP) at high temperatures and high pressures

Marine organisms living in the deep sea near to hot wells show a fascinating tolerance to extremely high temperatures and pressures. Under the given conditions the synthesis and stability of biomolecules seem to be limiting facts for the basis of life. We studied the influence of high pressures and high temperatures on the hydrolysis of ATP, a universal component for the storage of energy in all known organisms and therefore an extremely interesting and important molecule. The hydrolysis of ATP, ADP and AMP was studied in unbuffered solutions at temperatures between 353 and 369 K at pH values between 3.4 and 10.0. The pressure dependence was determined to p max = 220 MPa also at pH 5. All data can be explained by a proton catalyzed mechanism that removes in consecutive steps the final phosphate group. In none of the experiments could pyrophosphate be detected. The influence of phosphate and magnesium ions on the hydrolysis is discussed.

Adenosine triphosphate catabolism in bovine spermatozoa

Adenosine triphosphate metabolism in caudal epididymis bovine spermatozoa was studied. Measurements by HPLC at appropriate time intervals of the spermatozoa content of ATP and its derivatives were carried out under different experimental conditions. In the presence of 2- D-glucose, cellular ATP was transformed almost quantitatively into ADP and AMP at a rate of 2.3 nmol/min per 10 8 cells. At the same time, ADP and AMP accumulated at a rate of 1.52 and 0.58 nmol/min per 10 8 cells, respectively. In the first 4 min, about 50% of total ATP was degraded, the AEC of the cells dropped to non-physiological values while the content of other nucleosides did not vary significantly. Inorganic P i content also remained unchanged. Under non-induced conditions up to 240 min, no variations of the adenylic content and of the EC value was observed. Under induced and non-induced conditions, IMP and adenosine were not detected within the spermatozoa. The lack of IMP might be ascribed either to the absence of AMP deaminase, whose activity has never been found in the spermatozoa or to the intracellular environment which down regulates the activity of the enzyme. In order to explain low levels and absence of variations of adenosine, several enzymic investigations were carried out. Adenosine kinase activity was not determined, therefore the transformation of adenosine into AMP had to be excluded. Nevertheless, enzymic activities potentially able to dephosphorylate the formed AMP are present in the spermatozoa. Our findings are indicative of the existence in the spermatozoa of acid and alkaline phosphatase and of 5′-nucleotidase membrane-derived. The effects of some intracellular conditions on both AMP and PNPP hydrolysing activities were examined. This paper indicates that the intracellular pH and the content of the adenylic nucleotides and P i of the cell exert a negative control of the rate of hydrolysis of AMP, which might explain the lack of production and variations in the spermatozoa adenosine level.

Cyclic nucleotide phosphodiesterase isozymes in rat mesangial cells

We characterized cyclic nucleotide phosphodiesterases isolated from rat mesangial cells and asserted their roles in regulating cellular cyclic nucleotide levels. Three peaks of phosphodiesterase activity were eluted by a linear sodium acetate gradient from a Q Sepharose column loaded with the mesangial cell extract. The first peak activity was stimulated by Ca 2+-calmodulin and inhibited by calmodulin-stimulated phosphodiesterase inhibitors but not by a selective cGMP specific phosphodiesterase V inhibitor. The second, minor activity peak was stimulated by cyclic GMP and inhibited by EHNA [erythro-9-(2-hydroxy-3-nonyl)-adenine], a selective inhibitor of cyclic GMP-stimulated phosphodiesterase II. The last peak activity was not inhibited by cyclic GMP but selectively inhibited by rolipram [4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidene] or Ro 20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone], inhibitors of cyclic AMP specific, cyclic GMP insensitive phosphodiesterase IV. Based on their order of chromatographic elution, kinetic properties and sensitivity to allosteric agents and inhibitors, the peak 1, 2 and 3 correspond to phosphodiesterase I, II and IV. The basal cyclic GMP level was raised more effectively by selective inhibitor of phosphodiesterase I than phosphodiesterase II. In contrast, the atrial natriuretic factor-induced cyclic GMP elevation was potentiated more effectively by selective inhibitors of phosphodiesterase II than phosphodiesterase I. The forskolin-induced cyclic AMP increase was greatly potentiated by selective phosphodiesterase IV inhibitors but not by other phosphodiesterase inhibitors. These data suggest that phosphodiesterase I and II are responsible for cyclic GMP hydrolysis whereas phosphodiesterase IV is mainly responsible for cyclic AMP hydrolysis. The relative importance of phosphodiesterase I and II varied with the intracellular cyclic GMP concentration.

Poly(A) Tail Length Control Is Caused by Termination of Processive Synthesis

Poly(A) polymerase synthesizes poly(A) tails rapidly and processively only when the substrate RNA is bound simultaneously by two stimulatory proteins, the cleavage and polyadenylation specificity factor (CPSF) and poly(A)-binding protein II (PAB II). A burst of synthesis terminates after the addition of about 250 nucleotides, a length corresponding to that of newly synthesized poly(A) tails in vivo. Further elongation is slow. Length control can be reproduced with premade poly(A) tails of different lengths and is insensitive to large changes in the elongation rate. Thus, the control mechanism truly measures the length of the poly(A) tail. The stimulatory action of PAB II is similar on long and short tails. Coating of poly(A) with one PAB II molecule for approximately 30 nucleotides is required, such that the number of PAB II molecules in the polyadenylation complex is a direct measure of poly(A) tail length. CPSF also stimulates poly(A) polymerase on long and short tails. Long tails differ from short ones only in that they do not permit the simultaneous stimulation of poly(A) polymerase by CPSF and PAB II. Consequently, elongation of long tails is distributive. Thus, length control is brought about by an interruption of the interactions responsible for rapid and processive elongation of short tails. The 3'-end of the poly(A) tail is not sequestered in the protein-RNA complex when the correct length has been reached. Neither ATP hydrolysis nor turnover of the polymerized AMP is involved in length control.

Differential effects of phosphodiesterase inhibitors on accumulation of cyclic AMP in isolated ventricular cardiomyocytes

The intracellular actions of phosphodiesterase (PDE) inhibitors on the accumulation of cyclic nucleotides were studied in isolated ventricular cardiomyocytes from adult Sprague-Dawley rats. Elevated levels of cyclic AMP, due to the effects of selective PDE inhibitors, were detected only when the levels of cyclic nucleotide were enhanced with forskolin (10 μM). The time course for the elevation of cyclic AMP levels was similar for all the PDE inhibitors tested, following the pattern of an initial rise in the first 2–4 min, proceeded by a steady state at 67 ± 6% of the maximum stimulation. HN-10200 (2-[3-methoxy-5-methylsulfinyl-2-thienyl]-1 H-imidazo-[4,5- c]-pyridine hydrochloride), a new imidazopyridine derivative, had a similar concentrationdashdependent profile to the structurally related compound, sulmazole (AR-L 115 BS, 2-[2-methoxy-4-methylsulfinyl)phenyl]-1 H-imidazo-[4,5- b]-pyridine). Both the nondashselective inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the selective PDE IV inhibitor, Ro 20-1724 (4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone), potentiated the forskolindashstimulated levels of cyclic AMP with a much greater efficacy than sulmazole or Hndash10200. The concentrations of forskolin required by IBMX, sulmazole and Hndash10200 (10 −3 M) to increase levels of cyclic AMP by 4 pmol/mg protein were 3.2 × 10 −6 M, 1.32 × 10 −5 M and 1.46 × 10 −5 M, respectively. Enoximone failed to cause an increase in the levels of cyclic AMP, even when stimulated with maximal concentrations of forskolin. Furthermore, in the presence of forskolin, enoximone attenuated the response of Ro 20-1724 and IBMX in a concentrationdashdependent manner. Enoximone, similarly to Hndash10200, sulmazole, Ro 20-1724 and IBMX did not produce any significant effect on levels of cyclic GMP under elevated conditions in the presence of sodium nitroprusside. The combined action of Ro 20-1724, with either Hndash10200, sulmazole, or IBMX (10 −4 M), on intracellular levels of cyclic AMP, was not greater than the response to Ro 20-1724 alone. These data demonstrate the differential actions of PDE III and PDE IV inhibitors in rat ventricular cardiomyocytes. It is suggested that enoximone has a high selectivity for the PDE III isoenzyme so that hydrolysis of cyclic AMP by the PDE IV isoenzyme is not inhibited, in accordance with the lack of increase in cyclic AMP by enoximone in rat cardiomyocytes. Hndash10200 and sulmazole, producing small increases in intracellular levels of cyclic AMP, are less selective PDE III inhibitors than enoximone. In contrast, the nondashselective PDE inhibitor, IBMX, and the PDE IV inhibitor, Ro 20-1724, produced large increases in intracellular levels of cyclic AMP and it is likely, therefore, that Ro 20-1724 has effects on both the PDE III and PDE IV isoforms in rat cardiomyocytes.

Further analysis of the mechanisms underlying the tracheal relaxant action of SCA40.

1. SCA40 (1nM-10 microM), isoprenaline (1-300 nM) and levcromakalim (100 nM-10 microM) each produced concentration-dependent suppression of the spontaneous tone of guinea-pig isolated trachea. Propranolol (1 microM) markedly (approximately 150 fold) antagonized isoprenaline but did not antagonize SCA40. The tracheal relaxant action of SCA40 was unaffected by suramin (100 microM) or 8-(p)-sulphophenyltheophylline (8-SPT; 140 microM). 2. An isosmolar, K(+)-rich (80 mM) Krebs solution increased tracheal tone, antagonized SCA40 (approximately 60 fold), antagonized isoprenaline (approximately 20 fold) and very profoundly depressed the log concentration-effect curve for levcromakalim. Nifedipine (1 microM) did not itself modify the relaxant actions of SCA40, isoprenaline or levcromakalim. However, nifedipine prevented the rise in tissue tone and the antagonism of SCA40 and isoprenaline induced by the K(+)-rich medium. In contrast, nifedipine did not prevent the equivalent antagonism of levcromakalim. 3. Charybdotoxin (100 nM) increased tracheal tone, antagonized SCA40 (approximately 4 fold) and antagonized isoprenaline (approximately 3 fold). Nifedipine (1 microM) prevented the rise in tissue tone and the antagonism of SCA40 and isoprenaline induced by charybdotoxin. 4. Quinine (30 microM) caused little or no change in tissue tone and did not modify the relaxant action of isoprenaline. However, quinine antagonized SCA40 (approximately 2 fold). Nifedipine (1 microM) prevented the antagonism of SCA40 induced by quinine. 5. Tested on spontaneously-beating guinea-pig isolated atria SCA40 (1 nM-10 microM) increased the rate of beating in a concentration-dependent manner. Over the concentration-range 1 microM-10 microM, SCA40 also caused an increase in the force of atrial contraction. 6. Intracellular electrophysiological recording from guinea-pig isolated trachealis showed that the relaxant effects of SCA40 (1 micro M) were often accompanied by the suppression of spontaneous electrical slow waves but no change in resting membrane potential. When the concentration of SCA40 was raised to 10 micro M, its relaxant activity was accompanied both by slow wave suppression and by plasmalemmal hyperpolarization.7. SCA40 (10 nM- 100 micro M) more potently inhibited the activity of cyclic AMP phosphodiesterase (PDE)than that of cyclic GMP PDE derived from homogenates of guinea-pig trachealis. Theophylline(1 micro M- 1O mM) also inhibited these enzymes but was less potent than SCA40 in each case and did not exhibit selectivity for inhibition of cyclic AMP hydrolysis.8. Tested against the activity of the isoenzymes of cyclic nucleotide PDE derived from human blood cells and lung tissue, SCA40 proved highly potent against the type III isoenzyme. It was markedly less potent against the type IV and type V isoenzymes and even less potent against the isoenzymes types I and II.9. It is concluded that the tracheal relaxant action of SCA40 (1 nM- 1 micro M) does not involve the activation of beta-adrenoceptors or P1 or P2 purinoceptors. Furthermore, this action is unlikely to depend upon the opening of BKca channels with consequent cellular hyperpolarization and voltage-dependent inhibition of Ca2+ influx. The tracheal relaxant action of SCA40 (up to 1 micro M) is more likely to depend upon its selective inhibition of the type III isoenzyme of cyclic nucleotide PDE. At concentrations above 1 micro M, SCA40 exerts more general inhibition of the isoenzymes of cyclic nucleotide PDE and may then promote the opening of BKca channels.

Milrinone, a cyclic AMP-phosphodiesterase inhibitor, has differential effects on regional myocardial work and oxygen consumption in experimental left ventricular hypertrophy.

The aim was to test the hypothesis that local myocardial work and O2 consumption would respond differentially to milrinone, a selective cyclic AMP-phosphodiesterase inhibitor, in left ventricular hypertrophy due to differences in myocardial cyclic AMP-phosphodiesterase activity. The effect of milrinone on regional segment work and regional O2 consumption was measured in 12 open chest anaesthetised dogs with left ventricular hypertrophy induced by valvular aortic stenosis and in 10 age matched control dogs. Regional myocardial work was calculated as the integrated product of instantaneous force development (miniature transducer) and segment shortening (sonomicrometer). Regional O2 consumption was calculated from coronary blood flow (radiolabelled microspheres) and O2 saturations in small regional vessels (microspectrophotometry). Low Km phosphodiesterase activity was assayed by measuring the hydrolysis of radiolabelled cyclic AMP. Milrinone increased left ventricular dP/dtmax by approximately 60-70% in both control [2808(SEM 314) to 4584(660) mm Hg.s-1] and left ventricular hypertrophy [3279(258) to 5589(470) mm-Hg.s-1]. Regional work increased significantly in control [612(88) to 955(101) g.mm.min-1], while the increase was not significant in left ventricular hypertrophy [859(139) to 974(172) g.mm.min-1]. Regional O2 consumption increased significantly with milrinone in left ventricular hypertrophy [8.1(1.2) to 13.1(2.4) ml O2.min-1.100 g-1], but the increase was not significant in control [6.9(1.2) to 7.4(1.0) ml O2.min-1.100 g-1]. Myocardial stiffness during ejection was increased by milrinone to a significantly greater extent in animals with left ventricular hypertrophy. These effects were not related to differences in cyclic AMP-phosphodiesterase activity between control hearts and hearts with left ventricular hypertrophy [393(45) v 402(36) pmol.mg protein-1.1]. Differences between the hypertrophied and normal canine myocardium in response to milrinone are either due to altered levels of cyclic AMP production in left ventricular hypertrophy, to effects of milrinone that are unrelated to cyclic AMP-phosphodiesterase inhibition, or to other differences in hypertrophied hearts. The greater stiffness of the myocardium in left ventricular hypertrophy may require a greater energy expenditure to increase the amount of work it performs.

The identification of apparently novel cyclic AMP and cyclic GMP phosphodiesterase activities in guinea‐pig tracheal smooth muscle

Phosphodiesterase (PDE) activities that were capable of hydrolysing cyclic AMP (Km = 6.8 +/- 2 microM) and cyclic GMP (Km = 6.7 +/- 1.6 microM) were isolated from tracheal smooth muscle. These enzyme(s) activities were insensitive to stimulation by calcium/calmodulin and to inhibition by cyclic GMP, rolipram (type IV inhibitor) and siguazodan (type III inhibitor). Zaprinast was a relatively poor inhibitor of both cyclic AMP and cyclic GMP hydrolysis (IC50 = 46 +/- 9 microM and 45 +/- 14 microM respectively). These results suggest that tracheal smooth muscle may contain an apparently novel PDE. However, KCl (30 mM) which facilitates calcium entry in cells, depressed bradykinin-stimulated intracellular cyclic AMP formation, suggesting that the type I PDE may be functionally present. We suggest that considerable caution be exercised in identifying apparently novel PDE isoforms.

Electrostatic potential surfaces of the transition state for AMP deaminase and for (R)-coformycin, a transition state inhibitor.

The transition state for the hydrolysis of AMP by AMP deaminase has been characterized by heavy atom kinetic isotope effects (Merkler, D.J., Kline, P.C., Weiss, P., and Schramm, V.L. (1993) Biochemistry 32, 12993-13001). The experimentally established transition state includes a bond order of 0.8 to the attacking water nucleophile, a full bond order to the exocyclic 6-amino group, rehybridization of C-6 of the purine ring to sp3 and protonation of N-1 by Glu633. The transition state is one the path to formation of an unstable tetrahedral intermediate in which the exocyclic amine undergoes rapid protonation followed by its departure. In this mechanism, the highest energetic barrier on the reaction coordinate is the attack of the zinc-activated water. In a further test of this transition state structure, the electrostatic potential surface for the purine ring of the transition state has been determined by molecular orbital calculations and compared to that of the base of (R)-coformycin 5'-monophosphate, a slow onset, tight binding inhibitor of AMP deaminase that binds with an overall dissociation constant of 10(-11) M. The electrostatic potential surfaces of the aglycones of the transition state and (R)-coformycin are compared to the adenine ring of the substrate and to an alternative transition state structure in which the transition state is late, with fully bonded hydroxyl and fully protonated exocyclic amine. The results indicate a near-match of the electrostatic potential surfaces for the early transition state and (R)-coformycin. The electrostatic nature of the late transition state with a protonated amine leaving group differs both from the transition state determine by kinetic isotope effects and from that of (R)-coformycin analogues. The results provide evidence that the nature of the enzyme-stabilized transition state for adenine deamination involves an early transition state with a partially bonded hydroxyl group. The observed tight binding inhibition by (R)-coformycin analogues as transition state inhibitors results from the similarity of the partial charges on the inhibitors to that of the enzymatic transition state stabilized by AMP deaminase.

Atrial natriuretic peptide reduces cyclic AMP by activating cyclic GMP-stimulated phosphodiesterase in vascular endothelial cells.

Bovine aortic endothelial cells contain cyclic GMP-stimulated phosphodiesterase (PDE) regulating intracellular cyclic AMP and cyclic GMP levels. To investigate the roles of this PDE isoform for cyclic AMP hydrolysis in intact endothelial cells (EC), we used an adenine prelabeling method to determine cyclic AMP accumulation in response to agents that might produce effects that increase cyclic GMP levels. Atrial natriuretic peptide (ANP), which dramatically increased cyclic GMP accumulation, reduced cyclic AMP in cultured EC from bovine aorta with or without addition of L-isoproterenol (L-ISO), whereas sodium nitroprusside (SNP) and 8-bromo cyclic GMP had no effect. The reduction in cyclic AMP by ANP was dose dependent (> or = 1.0 nM) and rapid (significant reduction was induced in < or = 15 s) and was abolished when EC were preincubated with 3-isobutyl-1-methylxanthine (IBMX) and HL-725, both nonselective PDE inhibitors. ANP had no effects on adenylate cyclase activity, nor any direct effects on the activities of partially purified cyclic GMP-stimulated PDE isoform or cyclic AMP-specific isoform. However, cyclic AMP hydrolyzing activities of EC were enhanced when EC were pretreated with 0.1 microM ANP. ANP activates cyclic GMP-stimulated PDE and induces reduction of cyclic AMP accumulation in intact EC, which may modify cyclic GMP-dependent endothelial function involved in ANP.