Modulation of vascular ATP diphosphohydrolase by fatty acids.

Endothelial cells (EC) possess several protective thromboregulatory mechanisms that may be perturbed by cell activation or injury. Vascular ATP-diphosphohydrolase (ATPDase) has been demonstrated on both aortic EC and smooth muscle cells and may play a key regulatory role in hemostasis and platelet reactivity by converting extracellular ATP and ADP to AMP. We have examined the role of exogenous saturated or unsaturated fatty acids in the modulation of EC associated ATPDase activity in vitro. EC growth was not dramatically influenced by supplementation with fatty acids whereas viability was enhanced by oleic, butyrate and eicosapentaenoic acid. EC cultures supplemented with saturated or a monounsaturated (oleic acid) fatty acid(s) had markedly increased ATPDase activity, whereas those exposed to polyunsaturated fatty acids showed substantive decreases. Exogenous oleic acid could also protect against the significant loss of ATPDase activity, following exposure to reactive oxygen intermediates in vitro. We conclude that endothelial ATPDase activity may be regulated by exogenous fatty acids and that underlying mechanisms include alterations in the nature of the phospholipid composition of EC membranes that influence responses to oxidative stress reactions.

Purification of pancreas type-I ATP diphosphohydrolase and identification by affinity labelling with the 5'-p-fluorosulphonylbenzoyladenosine ATP analogue.

The enzyme recently identified as type-I ATP diphosphohydrolase (ATPDase; EC 3.6.1.5) has been purified from the zymogen granule membrane of pig pancreas. After solubilization with Triton X-100 and chromatographies on ion-exchange and Affi-Gel Blue columns an approximate 3500-fold purification was obtained. The enzyme preparation with a specific activity of 45 units/mg of protein was much further purified by PAGE under non-denaturing conditions. The active band localized on the gel contained two proteins after SDS/PAGE and silver staining, corresponding to apparent molecular masses of 56 and 54 kDa. The identity of the ATPDase was confirmed by an affinity labelling technique with 5'-p-fluorosulphonylbenzoyladenosine (FSBA) as an ATP analogue. The latter was detected by a Western blot technique. A strong reaction was observed with the band corresponding to 54 kDa. N-terminal sequence analysis revealed that the 56 kDa protein has significant similarities (50-72%) with lipases, whereas the 54 kDa enzyme has no significant similarity with any known proteins. N-glycosidase F treatment confirmed the glycoprotein nature of the enzyme and suggested that the enzyme bears several N-glycosylation sites. Comparisons of molecular masses and biochemical properties show that this ATPDase is different from other reported mammalian ATPDases.

Thrombin-receptor agonist peptides, in contrast to thrombin itself, are not full agonists for activation and signal transduction in human platelets in the absence of platelet-derived secondary mediators.

Synthetic thrombin receptor peptides (TRPs), comprising the first 6-14 amino acids of the new N-terminus tethered ligand of the thrombin receptor that is generated by thrombin's proteolytic activity, were reported to activate platelets equally with thrombin itself and are considered to be full agonists [Vu et al. (1991) Cell 64, 1057-1068]. Using aspirin plus ADP-scavengers or the ADP-receptor antagonist adenosine 5'-[alpha-thio]triphosphate to prevent the secondary effects of the potent agonists that are normally released from stimulated platelets (i.e. ADP and thromboxane A2), we assessed the direct actions of thrombin and TRPs (i.e. TRP42-47 and TRP42-55). Compared with thrombin, under these conditions, TRPs: (1) failed to aggregate platelets completely; (2) produced less activation of glycoprotein (GP)IIb-IIIa; (3) did not cause association of GPIIb and pp60c-src with the cytoskeleton; and (4) caused less alpha-granule secretion, phosphorylation of cytoplasmic phospholipase A2, arachidonic acid release and phosphatidyl inositol (PtdOH) production. Furthermore, TRPs induced transient increases in protein phosphorylation mediated by protein kinase C and protein tyrosine phosphorylation, whereas these same responses to thrombin were greater and more sustained. Hirudin added after thrombin accelerated protein dephosphorylation, thereby mimicking the rate of spontaneous dephosphorylation seen after stimulation by TRPs. Platelets totally desensitized to very high concentrations of TRPs, by prior exposure to maximally effective concentrations of the peptides, remained responsive to alpha- and gamma-thrombins. Thrombin-stimulated PtdOH production in permeabilized platelets desensitized to TRPs was abolished by guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), as in normal platelets. These results are discussed in terms of the allosteric Ternary Complex Model for G-protein linked receptors [Samama et al. (1993) J. Biol. Chem. 268, 4625-4636]. We conclude that: (1) TRPs are partial agonists for the thrombin receptor and produce incomplete receptor desensitization in keeping with their lower intrinsic activity; (2) thrombin's effects in platelets, even in TRP-desensitized platelets, are entirely mediated through the recently cloned G-protein linked receptor, and (3) thrombin's ability to produce sustained signals, compared with TRPs, may require the continued progressive proteolytic activation of naive thrombin receptors.

Demonstration of a novel type of ATP-diphosphohydrolase (EC 3.6.1.5) in the bovine lung.

A novel type of ATP-diphosphohydrolase (ATPDase) is demonstrated in bovine lung. The enzyme has an optimum pH of 7.5 and catalyzes the hydrolysis of the beta- and gamma-phosphate residues from diphospho- and triphosphonucleosides. It requires Ca2+ or Mg2+ and is insensitive to ouabain, an inhibitor of Na+/K(+)-ATPase, P1,P5-di(adenosine 5')-pentaphosphate, an inhibitor of adenylate kinase, and tetramisole, an inhibitor of alkaline phosphatase. In contrast, sodium azide (10 mM), a known inhibitor of ATPDases and mitochondrial ATPases, as well as mercuric chloride (10 microM) and gossypol (2,2'-bis[8-formyl-1,6,7-trihydroxy-5-isopropyl-3-methylnaphthalene]) (35 microM) are powerful inhibitors of this enzyme. The same inhibition profile is obtained with ATP or ADP as substrate, thereby supporting the concept of a common catalytic site for these substrates. This is further confirmed by enzyme localization after polyacrylamide gel electrophoresis under nondenaturing conditions and by kinetic properties, namely pH dependence profiles, heat inactivation, and 60Co irradiation-inactivation curves. The native molecular mass of the enzyme calculated from 60Co gamma-irradiation-inactivation curves is estimated at 70 +/- 3 kDa, whereas Km,app and Vmax,app of the ATPDase are evaluated at 7 +/- 2 microM and 1.1 +/- 0.3 mumol of Pi/min/mg protein, respectively. A comparison of the kinetic properties of this ATPDase with those of pig pancreas (Type I) and bovine aorta (Type II) lead us to believe that this enzyme is an hitherto undescribed type of ATPDase. By reference to the previously described ATPDase, we propose to identify this enzyme as ATPDase Type III (EC 3.6.1.5).

Kinetic properties of type-II ATP diphosphohydrolase from the tunica media of the bovine aorta.

The kinetic properties of type-II ATP diphosphohydrolase are described in this work. The enzyme preparation from the inner layer of the bovine aorta, mostly composed of smooth muscle cells, shows an optimum at pH 7.5. It catalyzes the hydrolysis of tri- and diphosphonucleosides and it requires either Ca2+ or Mg2+ for activity. It is insensitive to ouabain (3 mM), an inhibitor of Na+/K(+)-ATPase, to tetramisole (5 mM), an inhibitor of alkaline phosphatase, and to Ap5A (100 microM), an inhibitor of adenylate kinase. In contrast, sodium azide (10 mM), a known inhibitor for ATPDases and mitochondrial ATPase, is an effective inhibitor. Mercuric chloride (10 microM) and 5'-p-fluorosulfonylbenzoyl adenosine are also powerful inhibitors, both with ATP and ADP as substrates. The inhibition patterns are similar for ATP and DP, thereby, supporting the concept of a common catalytic site for these substrates. Apparent Km and Vmax, obtained with ATP as the substrate, were evaluated at 23 +/- 3 microM and 1.09 mumol Pi/min per mg protein, respectively. The kinetic properties of this enzyme and its localization as an ectoenzyme on bovine aorta smooth muscle cells suggest that it may play a major role in regulating the relative concentrations of extracellular nucleotides in blood vessels.

Characterization of ATP-diphosphohydrolase activities in the intima and media of the bovine aorta: evidence for a regulatory role in platelet activation in vitro.

The inner layer of the aorta contains the enzyme ATP diphosphohydrolase (ATPDase: EC 3.6.1.5) which catalyzes the sequential phosphorolysis of ATP----ADP----AMP. Two zones of the inner layer, the intima and media, were separated and both were shown to contain ATPDase activity of similar specific activity (0.08 and 0.10 U/mg protein, respectively). However, the media exhibited about 100-times more enzyme activity than the intima. Both preparations were virtually identical with respect to pH optima (7.5), migration patterns after electrophoresis under non-denaturing conditions, relative rates of ATP and ADP hydrolysis and potency to inhibit ADP-induced platelet aggregation in both human platelet-rich plasma and whole blood. The IC50 values for ADP (2 microM)-induced aggregation were 6.8 and 12.9 mU/ml in platelet-rich plasma and whole blood, respectively. Addition of ATPDase to platelets pre-aggregated with ADP resulted in a dose-dependent disaggregation in platelet-rich plasma (IC50 4.9 mU/ml), but not in whole blood. When both ATPDase (5.6-58.7 mU/ml) and ATP (0.5-10 microM) were added to platelet-rich plasma, there was an immediate dose-dependent aggregation of platelets followed by a slowly developing disaggregation. These data show that ATPDase is present in both the intima and media layers of bovine aorta and suggest a dual role for this enzyme in platelet activation. By converting ATP released from damaged cells into ADP, the enzyme could facilitate platelet aggregation at the site of vascular injury, whereas the subsequent conversion of ADP to AMP could inhibit or reverse platelet aggregation. The consequence of these activities would be to control the growth of a platelet thrombus.

Identification and localization of ATP-diphosphohydrolase (apyrase) in bovine aorta: relevance to vascular tone and platelet aggregation.

In this work, we confirm the existence of an ATP-diphosphohydrolase (apyrase) in bovine aorta and we show that its properties are different from the previously described pancreas ATP-diphosphohydrolase. Hence the aorta enzyme should be considered as a novel type of apyrase. The demonstration is based on pH dependency profiles, heat denaturation curves, 60Co irradiation-inactivation curves and enzyme localization after polyacrylamide gel electrophoresis under non-denaturing conditions. In addition, the irradiation-inactivation curves clearly showed that for both pancreas and aorta enzymes preparations, the same catalytic site is responsible for the hydrolysis of ATP and ADP. The molecular masses of enzymes calculated with this method are 132 +/- 19 kDa (mean +/- S.D.) and 189 +/- 30 kDa (mean +/- S.D.) for the pancreas and aorta enzymes, respectively. Preliminary observations on isolated bovine brain capillaries revealed a high level of enzyme activity strongly suggesting that an ATP-diphosphohydrolase is associated with endothelial cells. The presence of the enzyme on this type of cells was confirmed with pulmonary endothelial cells in culture. Considering the high proportions of smooth muscle cells relative to endothelial cells and the high level of enzyme activity in the aorta preparation, an ATP-diphosphohydrolase activity is definitely present in smooth muscle cells. The ATP-diphosphohydrolase activities described above could regulate the relative concentrations of purine nucleotides both in the plasma and within the vascular wall and hence could play a role both in platelet aggregation and in the control of vascular tone.

Endothelin-1 induces prostacyclin release from bovine aortic endothelial cells.

The effects of endothelin-1 (ET-1) on the release of prostacyclin from cultured bovine aortic endothelial cells were studied. ET-1 induced a time- and dose-dependent release of 6-keto PGF1 alpha, the stable metabolite of prostacyclin, with an apparent EC50 value of 3.0 +/- 0.9 nM (n = 6). ET-1 up to a concentration of 500 nM did not affect cellular integrity. Preincubation of the cells for 30 min with 10 microM indomethacin inhibited ET-1 (100 nM) - induced prostacyclin release by 90%. These findings indicate that ET-1 can directly stimulate prostacyclin release from endothelial cells probably through a receptor mediated mechanism.