AMP Hydrolyzing Activity Research Articles

Endothelium-independent vasorelaxant effect of a Berberis orthobotrys root extract via inhibition of phosphodiesterases in the porcine coronary artery

BackgroundBerberis orthobotrys Bien ex Aitch. (Berberidaceae) is a plant indigenous of Pakistan that is locally used for the treatment of hypertension. HypothesisThis study evaluated the vasoactive properties of a Berberis orthobotrys root extract and its fractions, and investigated the role of the endothelium and the underlying mechanism. Study designAn aqueous methanolic extract of Berberis orthobotrys roots was prepared and submitted to a multi-step liquid-liquid fractionation with solvents of increasing polarity. Vascular reactivity of the different fractions was assessed using porcine coronary artery rings either with or without endothelium, and in the presence or absence of specific pharmacological tools. The ability of Berberis orthobotrys extracts to affect phosphodiesterase (PDE) activity was evaluated using a radioenzymatic method and purified phosphodiesterases. ResultsThe aqueous methanol extract induced similar relaxations in coronary artery rings with and without endothelium, and, amongst the three derived preparations, the butanol fraction (BFBO) was slightly but significantly more effective than the ethyl acetate fraction and the aqueous residue in rings without endothelium. Analysis of the butanol fraction (BFBO) by LC-ELSD-MS indicated the presence of four major isoquinoline alkaloids including berberine. BFBO significantly potentiated the relaxations induced by cyclic GMP- and cyclic AMP-dependent relaxing agonists, and inhibited contractions to KCl, CaCl2, and U46619 in endothelium denuded rings. In contrast, BFBO did not affect relaxations to endothelium-dependent vasodilators. BFBO concentration-dependently inhibited the cyclic GMP-hydrolyzing activity of basal PDE1, calmodulin-activated PDE1 and PDE5, and of cyclic AMP-hydrolyzing activity of PDE3 and PDE4 with IC50 values ranging from 40 to 130µg/ml. ConclusionThe butanol fraction of the aqueous methanol extract of Berberis orthobotrys roots induced pronounced endothelium-independent relaxations and inhibited contractile responses by acting directly at the vascular smooth muscle in the coronary artery. Moreover, BFBO potentiated relaxations induced by both cyclic GMP- and cyclic AMP-dependent vasodilators most likely due to its ability to inhibit several vascular PDEs, and in particular PDE4 and PDE5.

Ontogenetic profile of ecto-5′-nucleotidase in rat brain synaptic plasma membranes

Ecto-5′-nucleotidase (CD73; EC 3.1.3.5, e-5NT) is regarded as the key enzyme in the extracellular formation of adenosine, which acts as a neuromodulator and important trophic and homeostatic factor in the brain. In the present study, we have investigated e-5NT activity, kinetic properties concerning AMP hydrolysis and the enzyme protein abundance in the purified synaptic plasma membrane (SPM) preparations isolated from whole female rat brain at different ages. We observed pronounced increase in AMP hydrolyzing activity in SPM during maturation, with greatest increment between juvenile (15-day-old) and pre-pubertal (30-day-old) rats. Immunodetection of e-5NT protein in the SPM displayed the reverse pattern of expression, with the maximum relative abundance at juvenile and minimum relative abundance in the adult stage. Negative correlation between the enzyme activity and the enzyme protein abundance in the SPM indicates that e-5NT has additional roles in the synaptic compartment during postnatal brain development, other than those related to AMP hydrolysis. Determination of kinetic parameters, K m and V max, suggested that the increase in the enzyme activity with maturation was entirely due to the increase in the enzyme catalytic efficiency ( V max/ K m). Finally, double immunofluorescence staining against e-5NT and presynaptic membrane marker syntaxin provided first direct evidence for the existence of this ecto-enzyme in the presynaptic compartment. The results of the study suggest that e-5NT may be a part of general scheme of brain development and synapse maturation and provide rationale for the previously reported inconsistencies between enzyme immunohistochemical and biochemical studies concerning localization of e-5NT in the brain.

Identification of the Nucleotidase Responsible for the AMP Hydrolysing Hyperactivity Associated with Neurological and Developmental Disorders

Nucleoside monophosphate phosphohydrolases comprise a family of enzymes dephosphorylating nucleotides both in intracellular and extracellular compartments. Members of this family exhibit different sequence, location, substrate specificity and regulation. Besides the ectosolic 5'-nucleotidase, several cytosolic and one mitochondrial enzymes have been described. Nevertheless, researchers refer any AMP-dephosphorylating activity to as 5'-nucleotidase, lacking a more accurate identification. Increase of AMP hydrolysing activity has been associated with neurological and developmental disorders. The identification of the specific enzyme involved in these pathologies would be fundamental for the comprehension of the linkage between the enzyme activity alteration and brain functions. We demonstrate that the described neurological symptoms are associated with increased ectosolic 5'-nucleotidase activity on the basis of radiochemical assays and immunoblotting analysis. Furthermore, present data evidence that the assay conditions normally applied for the determination of cytosolic 5'-nucleotidases activity in crude extracts are affected by the presence of solubilised ectosolic nucleotidase.

Effect of CI-930 [3-(2 H)-pyridazinone-4,5-dihydro-6-[4-(1 H-imidazolyl) phenyl]-5-methyl-monohydrochloride] and rolipram on human coronary artery smooth muscle cell proliferation

Experiments were conducted to determine how selective inhibitors of certain cyclic nucleotide phosphodiesterase (PDE) families, namely CI-930 (PDE3 inhibitor; 3-(2 H)-pyridazinone-4,5-dihydro-6-[4-(1 H-imidazolyl) phenyl]-5-methyl monohydro chloride) and rolipram (PDE4 inhibitor), may affect human coronary artery smooth muscle cell (HCASMC) proliferation. CI-930- and rolipram-inhibitable PDEs accounted for most of the cyclic AMP hydrolyzing activity in HCASMC. Twenty micromolar CI-930 and 20 μM rolipram used individually attenuated proliferation of HCASMC from some, but not all donors, as measured by flow cytometry. The simultaneous addition of 10 μM CI-930 plus 10 μM rolipram caused greater attenuation. This attenuation represented a reduction of the number of cells entering the S phase of the cell cycle and not merely a delay in cell cycle traverse. No statistically significant elevation of cyclic AMP was detected following the addition of either PDE inhibitor individually, but the combination produced significant elevations. It is concluded that CI-930- and rolipram-inhibitable PDE isozymes are expressed in HCASMC and that selective inhibitors of these isozymes can attenuate HCASMC proliferation. The data suggest that selective PDE inhibitors may prevent restenosis in patients following percutaneous transluminal coronary angioplasty because of their effect on HCASMC proliferation, and they may also be useful in retarding the progression of atherosclerosis in individuals at risk. PDE3 and PDE4 inhibitors in combination are more effective than the inhibitors used individually.

Characterization of phosphodiesterase 4 in guinea‐pig macrophages: multiple activities, association states and sensitivity to selective inhibitors

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.

In vitro differentiation of human monocytes to macrophages: change of PDE profile and its relationship to suppression of tumour necrosis factor-alpha release by PDE inhibitors.

1. During in vitro culture in 10% human AB serum, human peripheral blood monocytes acquire a macrophage-like phenotype. The underlying differentiation was characterized by increased activities of the macrophage marker enzymes unspecific esterase (NaF-insensitive form) and acid phosphatase, as well as by a down-regulation in surface CD14 expression. 2. In parallel, a dramatic change in the phosphodiesterase (PDE) profile became evident within a few days that strongly resembled that previously described for human alveolar macrophages. Whereas PDE1 and PDE3 activities were augmented, PDE4 activity, which represented the major cyclic AMP-hydrolysing activity of peripheral blood monocytes, rapidly declined. 3. Monocytes and monocyte-derived macrophages responded to lipopolysaccharide (LPS) with the release of tumour necrosis factor-alpha (TNF). In line with the change in CD14 expression, the EC50 value of LPS for induction of TNF release increased from approximately 0.1 ng ml-1 in peripheral blood monocytes to about 2 ng ml-1 in macrophages. 4. Both populations of cells were equally susceptible towards inhibition of TNF release by cyclic AMP elevating agents such as dibutyryl cyclic AMP, prostaglandin E2 (PGE2) or forskolin, which all led to a complete abrogation of TNF production in a concentration-dependent manner and which were more efficient than the glucocorticoid dexamethasone. 5. In monocytes, PDE4 selective inhibitors (rolipram, RP73401) suppressed TNF formation by 80%, whereas motapizone, a PDE3 selective compound, exerted a comparatively weak effect (10-15% inhibition). Combined use of PDE3 plus PDE4 inhibitors resulted in an additive effect and fully abrogated LPS-induced TNF release as did the mixed PDE3/4 inhibitor tolafentrine. 6. In monocyte-derived macrophages, neither PDE3- nor PDE4-selective drugs markedly affected TNF generation when used alone (< 15% inhibition), whereas in combination, they led to a maximal inhibition of TNF formation by about 40-50%. However, in the presence of PGE2 (10 nM), motapizone and rolipram or RP73401 were equally effective and blocked TNF release by 40%. Tolafentrine or motapizone in the presence of either PDE4 inhibitor, completely abrogated TNF formation in the presence of PGE2. Thus, an additional cyclic AMP trigger is necessary for PDE inhibitors to become effective in macrophages. 7. Finally, the putative regulatory role for PDE1 in the regulation of TNF production in macrophages was investigated. Zaprinast, at a concentration showing 80% inhibition of PDE1 activity (100 micromol l-1), did not influence TNF release. At higher concentrations (1 mmol l-1), zaprinast became effective, but this inhibition of TNF release can be attributed to a significant inhibitory action of this drug on PDE3 and PDE4 isoenzymes. 8. In summary, the in vitro differentiation of human peripheral blood monocytes to macrophages is characterized by a profound change in the PDE isoenzyme pattern. The change in the PDE4 to PDE3 ratio is functionally reflected by an altered susceptibility towards selective PDE inhibitors under appropriate stimulating conditions.

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.

Regulation of rat heart cytosol 5'-nucleotidase by adenylate energy charge.

A 5'-nucleotidase (EC 3.1.3.5) was highly purified from the soluble fraction of rat heart. The preparation appeared homogeneous by the criterion of polyacrylamide-gel electrophoresis. The enzyme was activated by ATP and ADP, and inhibited by Pi. When AMP was used as substrate, the velocity/substrate-concentration plot was sigmoidal. ATP or ADP changed the plot to hyperbolic and decreased S0.5. Pi increased both the sigmoidicity of the plot and S0.5. When IMP was used as substrate, the velocity/substrate plot was hyperbolic. ATP or ADP decreased Km and increased V. Pi changed the plot to sigmoidal and increased S0.5. Within the range of adenylate energy charge observed in surviving mammalian cells (0.7-0.9), the rate of AMP-hydrolysing activity catalysed by the 5'-nucleotidase increased sharply with decreasing energy charge. The highest activity was observed at an energy-charge value of about 0.6. The response was also observed in the presence of Pi. No change in IMP-hydrolysing activity was observed in the physiological range of adenylate energy charge, but in the presence of Pi the activity gradually increased with increasing energy charge. These results suggest the possibility that this enzyme participates in production of adenosine, a vasodilator, during hypoxia and in removal of IMP, which accumulates during the hypoxia, in the heart.

Properties of an ecto-5'-nucleotidase of the renal brush border.

A decrease of glomerular filtration rate can be observed during accelerated catabolism of ATP in kidney. It has been proposed that this effect is due to the increase in the renal production of adenosine from ATP. The last reaction in the pathway concerned is the conversion of 5'-AMP to adenosine. We found that brush border membranes purified from homogenates of the rat renal cortex carry out this reaction. The enzyme involved in the hydrolysis has the characteristic properties of ecto-5'-nucleotidases: It is inhibited by ATP, ADP, and by alpha, beta-methyleneadenosine-5'-diphosphate, and it is not stimulated by magnesium. All catalytic sites are accessible from the outside of the vesicles. The Km of the enzyme for 5'-AMP is 5.77 microM. The enrichment of the 5'-AMP-hydrolyzing activity in the brush border fraction as compared to the homogenate is 9.2 +/- 1.5 times. Histochemical staining of kidney sections reveals hydrolysis of 5'-AMP only at the brush border of the proximal tubule. We conclude that the brush border of the proximal tubule of the rat kidney possesses an ecto-5'-nucleotidase which has the same properties as the ecto-5'-nucleotidases in other tissues.

Age-related changes in 5′-nucleotidase and alkaline phosphatase activities in mouse thymocytes

Enzymatic activities of thymocytes isolated from Swiss albino mice were studied at various ages from immediately post weaning until 100 weeks of age, approaching the life expectancy of these animals. Between 5 and 10 weeks of age, the activities of lactate dehydrogenase and alkaline phosphatase decreased to a level that was maintained throughout the remainder of the aging profile. Neutral β-glycerophosphatase (pH 7.5) activity in a thymus membrane preparation was similar in all age groups. The activity of membrane-bound 5′-nucleotidase, that is, AMP-hydrolyzing activity inhibited by 100 μM α,β-methyleneadenosine 5′-diphosphate, progressively increased as a function of age, indicating thymocyte population changes occurring very late in life. In thymocytes of the oldest mice examined (100 weeks of age), 5′-nucleotidase specific activity was approximately ten-fold greater than the activity found in 5-week-old mice. Thus, membrane-bound 5′-nucleotidase activity in thymocytes increased markedly as a function of age in Swiss albino mice; yet several other enzymatic activities, including alkaline phosphatase, remained relatively unchanged in mature mice.

Absence of 5′-nucleotidase in porcine polymorphonuclear leucocyte membranes

A fraction enriched in plasma membranes from porcine polymorphonuclear leucocytes, isolated by sucrose density centrifugation was shown to possess considerable AMP hydrolysing activity (150 nmol/min per mg protein). However all of this activity could be inhibited using excess p- nitrophenyl phosphate in the incubation medium. Furthermore the hydrolysis of AMP by the membrane was unaffected by the 5′-nucleotidase inhibitor α,β-methyleneadenosine diphosphate and by the lectin concanavalin A, another potent inhibitor of 5′-nucleotidase. An antibody against mouse liver 5′-nucleotidase also did not inhibit the activity. These results suggest that the hydrolysis of AMP by porcine polymorph membranes is not accomplished by a specific 5′-nucleotidase and the necessity for distinguishing between true 5′-nucleotidase and non-specific phosphatase activity is discussed.

Regulation of cytosol 5′-nucleotidase by adenylate energy charge

In the physiological range of the adenylate energy charge in liver (0.7–0.9), the rate of AMP-hydrolysis catalyzed by rat liver cytosol 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) increased sharply with decreasing energy charge. In addition, a decrease in the concentration of P i caused marked acceleration of the AMP-hydrolysing activity over the physiological range of adenylate energy charge. These responses seem to serve to protect the cells against a metabolic stress which could result from sudden utilization of ATP by removal of AMP. The AMP-hydrolysing activity of this enzyme decreased sharply as the size of the adenine nucleotide pool decreased in the physiological range. This effect may be a self-limiting response to prevent excess depletion of the pool. IMP-hydrolysing activity of this enzyme increased with increasing adenylate energy charge. But no marked response to its variation within the physiological range was observed. On the basis of the data obtained in this study, the IMP-hydrolysing activity of the cytosol 5′-nucleotidase in rat liver cells seems to be comparable to that of AMP deaminase reaction, but the AMP-hydrolysing activity was estimated to be less than 10% of AMP deaminase reaction at energy charge value of about 0.7. This strongly suggests that the AMP → IMP → inosine pathway is more significant than the AMP → adenosine → inosine pathway in rat liver.

Inhibitory effect of 8-substituted adenosine derivatives on Ca ++ and modulator protein-dependent phosphodiesterase activity

8-Substituted adenosine and cyclic AMP derivatives exhibited some negative Cotton effects in circular Dichroism at B 2u band in pH 7.5 solution, suggesting that these derivatives take syn conformation. The adenosine derivatives, as well as cyclic AMP derivatives, competitively inhibited the cyclic AMP hydrolyzing activity in Ca ++ and modulator protein-dependent phosphodiesterase preparation from hog brain cortex. The inhibitory potential of an adenosine derivative was lower than that of the cyclic AMP derivative having the same substituent by the lack of the phosphate moiety for which affinity was 0.5 kcal / mol. These results may suggest that the cyclic AMP hydrolyzing site on the enzyme requires the syn conformation of purine riboside.