Mechanism of extracellular ATP- and adenosine-induced apoptosis of cultured pulmonary artery endothelial cells.

Apoptosis may be important in the exacerbation of endothelial cell injury or limitation of endothelial cell proliferation. We have found that extracellular ATP (exATP) and adenosine cause endothelial apoptosis and that the development of apoptosis is linked to intracellular metabolism of adenosine [Dawicki, D. D., D. Chatterjee, J. Wyche, and S. Rounds. Am. J. Physiol. 273 (Lung Cell Mol. Physiol. 17): L485-L494, 1997]. In the present study, we investigated the mechanism of this effect. We found that exATP, adenosine, and the S-adenosyl-L-homocysteine (SAH) hydrolase inhibitor MDL-28842 caused apoptosis and decreased the ratio of S-adenosyl-L-methionine to SAH compared with untreated control cells. Using release of soluble [3H]thymidine as a measure of DNA fragmentation, we found that the effect of adenosine on soluble DNA release was potentiated by coincubation with homocysteine. These results suggest that the mechanism of exATP- and adenosine-induced endothelial cell apoptosis involves inhibition of SAH hydrolase. exATP-induced apoptosis was enhanced by an inhibitor of adenosine deaminase, whereas exogenous adenosine-induced apoptosis was partially inhibited by an adenosine deaminase inhibitor. These results suggest that adenosine deaminase may also be involved in the mechanism of adenosine-induced endothelial cell apoptosis. Adenosine and MDL-28842 caused intracellular acidosis as assessed with the fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. The cell-permeant base chloroquine prevented adenosine-induced acidosis but not apoptosis. Thus, although intracellular acidosis is associated with adenosine-induced apoptosis, it is not necessary for this effect. We speculate that exATP- and adenosine-induced endothelial cell apoptosis may be due to an inhibition of methyltransferase(s) activity. Purine-induced endothelial cell apoptosis may be important in limiting endothelial cell proliferation after vascular injury.

Differences in nucleotide effects on intracellular pH, Na+/H+ antiport activity, and ATP-binding proteins in endothelial cells.

Bovine (BPAEC) and human (HPAEC) pulmonary artery endothelial cell monolayers were incubated with either ATP, ATP analogues, or UTP, followed by measurement of intracellular pH (pHi) and the rate of recovery from acidosis. ATP increased baseline pHi and the rate of acid recovery in BPAEC. This response was inhibited by the amiloride analogue, methyisobutylamiloride, demonstrating that activation of the Na+/H+ antiport was responsible for the increase in baseline pHi and the recovery from acidosis. This response had the features of both a P2Y and P2U purinergic receptor, based on the responses to a series of ATP analogues and UTP. In contrast, none of the nucleotides had any significant effect on pHi and Na+/H+ antiport activity in HPAEC. This difference in the response to extracellular nucleotides was not due to a difference in ATP metabolism between cell types, since the ectonucleotidase-resistant analogue. ATP gamma S, also had no effect on HPAEC. Analogues of cAMP had no effect on pHi or acid recovery in either cell type. Incubation of BPAEC and HPAEC with the photoaffinity ligand [32P] 8-AzATP indicated that both BPAEC and HPAEC possess an ATP-binding protein of 48 kDa. However, BPAEC exhibited an additional binding protein of 87 kDa. Thus, the contrasting response to extracellular ATP between bovine and human pulmonary artery endothelial cells may be related to differences in the signal transduction pathway leading to antiport activation, including different ATP-binding sites on the cell membrane.

Studies on the mechanism of short-term regulation of pulmonary artery endothelial cell Na/K pump activity.

The Na/K pump is critically important in maintenance of cell homeostasis in the face of injury. Little is known about the regulation of endothelial cell Na/K-pump activity. We previously reported that short-term (30-minute) oxidant-induced endothelial cell perturbation increased Na/K-pump activity in intact monolayers of bovine pulmonary artery endothelial cells (BPAECs). In this study we investigated the mechanism of oxidant-induced increases in endothelial Na/K-pump activity, focusing on short-term modulation of alpha1-pump subunit. By using immunofluorescence microscopy and confocal scanning laser microscopy, we found alpha1 subunit on both apical and basal aspects of BPAECs without polarized distribution. Short-term (30-minute) incubation of PAEC monolayers with H2O2 (1 mmol/L) did not change the relative amounts of alpha1 subunit in membrane fractions, as assessed by immunoblotting. Phosphorylation of the alpha1 subunit also was not affected by H2O2 treatment. Because protein kinases have been reported to alter Na/K-pump activity in several tissues and because H2O2 has been reported to increase PKC activity of endothelial cells, we determined the effects of inhibition and activation of protein kinase C (PKC) on Na/K-pump activity quantitated as ouabain-inhibitable uptake of 86Rb. We also determined the effects of PKC activation and inhibition on H2O2-induced increases in Na/K-pump activity. Inhibitors of PKC increased Na/K-pump activity over a 30-minute period in intact monolayers. Inhibition or depletion of PKC did not prevent H2O2-induced increases in pump activity. These results indicate that PKC is an endogenous regulator of pulmonary artery endothelial cell Na/K-pump activity but that the effects of H2O2 are not mediated by activation of PKC or by changes in the expression or phosphorylation of alpha1 subunit.

Extracellular ATP and adenosine cause apoptosis of pulmonary artery endothelial cells.

ATP acts as an intracellular energy source and an extracellular signaling molecule. We report that extracellular ATP causes apoptosis in pulmonary artery endothelial cells, as assessed by morphological changes and internucleosomal DNA degradation. We investigated the mechanism of this effect using release of tritiated soluble DNA as a marker for apoptosis. We conclude that the metabolite adenosine is responsible for the apoptotic effect of ATP, since nucleotides that can be degraded to adenosine, as well as adenosine itself, cause DNA damage, whereas nonmetabolizable ATP analogs and uridine 5'-triphosphate are inactive. Furthermore, the ecto-5'-nucleotidase inhibitor alpha, beta-methylene-ADP blocks ATP-induced DNA fragmentation. The adenosine receptor agonist 5'-N-ethylcarboxamide adenosine does not cause DNA fragmentation, and adenosine receptor antagonists do not block adenosine-induced apoptosis. However, the nucleoside transport inhibitor dipyridamole prevents extracellular ATP-induced DNA cleavage. These findings indicate that ATP- and adenosine-mediated apoptosis are mediated via intracellular events rather than through cell surface receptor(s). The adenosine metabolites inosine, hypoxanthine, and xanthine do not cause apoptosis. The adenosine analogs 3-deazaadenosine and MDL-28842, which are not metabolized and are S-adenosylhomocysteine hydrolase inhibitors, also cause DNA fragmentation. Therefore, we speculate that extracellular ATP and adenosine cause apoptosis of pulmonary artery endothelial cells by altering methylation reactions that require S-adenosylmethionine as the methyl donor. We speculate that ATP released from cells undergoing cytolysis or degranulation may cause endothelial cell death. Endothelial cell apoptosis may be important in acute vascular injury or in limiting angiogenesis.

Effect of hypercarbia on surface proteins of cultured bovine endothelial cells.

Hypercarbia is a common complication of respiratory failure, and the technique of "permissive hypercapnia" is used to ventilate individuals with increased peak airway pressures on mechanical ventilators, resulting in elevated arterial PCO2. We studied the effects of hypercarbia on cultured bovine aortic and main pulmonary artery endothelial cell surface proteins, assessing cell surface iodination using lactoperoxidase bound to latex microspheres. We found that 4 h of exposure to 10% CO2 increased the display of substances of apparent molecular masses of 27, 47, and 52 kDa. This effect was not mimicked by acidotic media. Western blots of detergent extracts of main pulmonary artery endothelial cell monolayers did not show increased expression of carbonic anhydrase IV (molecular mass = 52 kDa) after incubation under hypercarbic conditions. Hypercarbia did not change the pattern of [35S]methionine incorporation into endothelial cell proteins. We conclude that hypercarbia of 4-h duration changes iodinated endothelial cell surface proteins. We speculate that this effect may be related to changes in secretion or display of apical cell membrane-associated proteins.

Mechanism of ATP-induced leukocyte adherence to cultured pulmonary artery endothelial cells.

Previously we have shown that ATP enhances the adherence of HL-60 cells and human neutrophils to bovine pulmonary artery endothelial cells. The current investigations extend earlier findings by showing that ATP and UTP dose-dependently stimulate human neutrophil adherence to human pulmonary artery endothelial cells. We have also explore the mechanisms of ATP- and UTP-stimulated adherence. We have found that fucose, a component of selectin receptors, inhibits ATP-stimulated HL-60 cell-bovine pulmonary artery endothelial cell adhesion. Additionally, pretreatment of HL-60 cells with neuraminidase abolishes ATP enhancement. However, fucose does not affect ATP- or thrombin-induced adhesion of freshly isolated human neutrophils to human endothelial cells. Antibodies to human P-selection intercellular adhesion molecule (ICAM)-1, and the beta-subunit of CD11/CD18 do not alter ATP-induced adherence of HL-60 cells to bovine endothelial cells. Similarly, antibodies to human P-selectin and ICAM-1 do not inhibit human neutrophil-human pulmonary artery endothelial cell adhesion. The platelet-activating factor receptor antagonists, WEB-2086 and L-659,989, are effective in attenuating ATP- and UTP-stimulated adherence. Preincubation of neutrophils or human pulmonary artery endothelial cells with ATP or UTP also enhances adherence, an effect that is blocked by L-659,989. Thus platelet activating factor, associated with both neutrophils and endothelial cells, mediates ATP- and UTP-induced neutrophil adherence. ATP, released during vascular injury, may exacerbate neutrophil-endothelial cell interaction and thereby contribute to neutrophil-induced injury.

Extracellular nucleotides stimulate leukocyte adherence to cultured pulmonary artery endothelial cells.

Adenosine, ATP, and various nucleotides were examined for their effects on the adherence of leukocytes to bovine pulmonary artery endothelial cells. Extracellular ATP enhanced adherence of HL-60 cells and human neutrophils to endothelial cells in a dose-dependent fashion. Maximal adherence occurred after 15 min coincubation of ATP and HL-60 cells or neutrophils with endothelial cells. ATP stimulation was mediated by direct effects on both HL-60 cells and endothelial cells. The potency profile of various nucleotides was ATP = 2-MeSATP > beta,gamma-CH2ATP, indicative of a P2y receptor. Interestingly, UTP was as potent as ATP in stimulating HL-60 cell adherence, suggesting the presence of a pyrimidine nucleotide receptor. Photoaffinity labeling of endothelial cells with 8-Az-[alpha-32P]ATP showed the presence of two ATP binding proteins of 48 and 87 kDa. ATP and 2-MeSATP inhibited binding by both proteins. Labeling of the 87-kDa protein was inhibited by beta,gamma-CH2ATP, whereas UTP blocked binding by the 48-kDa protein. Thus photoaffinity labeling experiments support the proposal that endothelial cells possess two ATP receptors, one of which is a P2u nucleotide receptor. These findings show that extracellular nucleotides enhance leukocyte adherence to endothelial cells. Nucleotide release into the extracellular space may be one mechanism of exacerbating vascular cell injury relevant to conditions such as adult respiratory distress syndrome and septic shock.

Identification of a protein-tyrosine phosphatase from human platelet membranes by an immobilon-based solid phase assay.

The present investigations show that a 53-kDa platelet-membrane protein is a protein tyrosine phosphatase. Identification involved a novel methodology in which membrane proteins are resolved by polyacrylamide gel electrophoresis and then transblotted to a polyvinylidene difluoride membrane surface-labeled with [32P-Tyr](Glu4.Tyr1)n. Phosphatase activity appears as clear areas in the autoradiograph of the renatured 32P Western blot. Studies using this new solid-phase system indicate that a 53-kDa platelet-membrane protein dephosphorylates [32P-Tyr](Glu4.Tyr1)n. Dephosphorylation is both time- and dose-dependent. The protein-tyrosine phosphatase antagonists orthovanadate and molybdate block dephosphorylation in a concentration-dependent manner. Inhibitors of protein serine/threonine phosphatases and of acid and alkaline phosphatases do not significantly affect enzymatic activity. The enzyme is active toward phosphotyrosyl proteins but not phosphoseryl proteins. Results from the solid-phase phosphatase assays correlate with data from standard liquid-phase studies. A Lineweaver-Burk plot gives an apparent Km of 4.3 microM and an apparent Vmax = 168 nmol P(i) transferred/min/mg for the membrane enzyme towards the 32P-labeled polymer in a liquid system. Platelet lysate was shown by the solid-phase assay to possess a protein-tyrosine phosphatase of approximately equal to 50 kDa. The tyrosine phosphatase activities associated with a placental preparation, pure recombinant protein-tyrosine phosphatase 1B, and YOPR, the product of the YOP51 gene of Yersinia enterocolitica, which contains the C235R mutation, were also visualized by this technique. However, the catalytic activity of domain 1 of the transmembrane leukocyte antigen-related protein-tyrosine phosphatase was not detected using the solid-phase assay.

The role of microtubules in platelet secretory release.

The role of microtubules in platelet aggregation and secretion has been analyzed using platelets permeabilized with digitonin and monoclonal antibodies to alpha (DM1A) and beta (DM1B) subunits of tubulin. Permeabilized platelets were able to undergo aggregation and secretory release. However, threshold doses of agonists capable of eliciting a second wave of aggregation and the platelet release reaction were higher than in control platelets exposed to dimethyl sulfoxide, the solvent for digitonin. Both antibodies to alpha and beta tubulin caused a further increase in the threshold concentration of agonists and inhibited the secretory release of permeabilized platelets, but were ineffective using intact platelets. Neither monoclonal antibody inhibited polymerization or depolymerization of platelet tubulin in vitro. Antibodies to platelet actin and myosin also exhibited an inhibitory activity on platelet aggregation albeit less severe than that observed with the antibodies to alpha and beta tubulin. There was evidence of an interaction between DM1A and DM1B and the antibodies to actin and myosin. The interaction of platelet tubulin and myosin was investigated by two different methods. (1) Coprecipitation of the proteins at low ionic strength at which tubulin by itself did not precipitate and (2) affinity chromatography on columns of immobilized myosin. Tubulin freed of its associated proteins (MAPs) by phosphocellulose chromatography bound to myosin in a molar ratio which approached 2. Platelet actin competed with tubulin for 1 binding site on the myosin molecule. MAPs also reduced the binding stoichiometry of tubulin/myosin. Treatment of microtubule protein with p-chloromercuribenzoate or colchicine did not influence its binding to myosin. DM1A and DM1B inhibited the interaction of tubulin and myosin. This effect could also be demonstrated by reaction of electrophoretic transblots of extracted platelet tubulin with the respective proteins. We interpret these results as evidence for an interference of the two monoclonal antibodies to the tubulin subunits (DM1A and DM1B) with the translocation of microtubule protein from its submembranous site to a more central one during the activation process.

Permeabilization of platelets: an investigation of biochemical, ultrastructural and functional aspects.

The biochemical, ultrastructural and functional aspects of digitonin-permeabilized platelets were investigated. Human platelets were permeabilized by exposure to the steroid glycoside digitonin. A 60 microM concentration of this permeabilizer produced a very substantial release of cytosolic enzymes from the platelets. Release from subcellular granules was relatively low and did not inhibit the response of platelets to a series of agonists. Although digitonin-permeabilized platelets required higher threshold concentrations of the usual stimulants, both primary and secondary aggregation as well as the release of nucleotides and enzymes from their respective granules remained intact. Transmission electron micrographs revealed discontinuities in the plasma membrane of digitonin-treated platelets, but scanning electron microscopy showed no difference between control and permeabilized platelets. No substantial loss of structural or membrane proteins could be detected by one- and two-dimensional gel electrophoresis. The pore size produced by digitonin treatment was sufficient to allow entry of 125I-labeled IgG into the platelet cytosolic space.

Time resolved analysis of tubulin phosphorylation during platelet activation.

Tubulin phosphorylation was analyzed during the different phases of platelet activation. Platelets preloaded with [32P]-phosphate were stimulated with collagen. Tubulin was immunoprecipitated from serial samples obtained during the activation process. The immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and autoradiographs analyzed by laser densitometry. Agonist induced dephosphorylation of platelets occurred after the onset of shape change at the time of initiation of the secretory release. The dephosphorylation was selective affecting specific peptides.

Adenosine metabolism in human whole blood. Effects of nucleoside transport inhibitors and phosphate concentration.

Adenosine (Ado, 10 microM) was metabolized in whole blood within 1 min, primarily to hypoxanthine and ATP. The concentration of Ado, the activities of adenosine deaminase (ADA) and Ado kinase, the Km values for Ado with ADA and Ado kinase, and the substrate inhibition of Ado kinase are factors that govern the Ado metabolism between deamination and phosphorylation. If ADA activity was blocked by 2'-deoxycoformycin (dCF, 5 microM), a tight-binding inhibitor of ADA, most of the Ado (96%) was incorporated into adenine nucleotides, whereas if Ado kinase activity was blocked with 5-iodotubercidin (10 microM), Ado was mainly (95%) metabolized into hypoxanthine. A high phosphate concentration (25 mM) caused marked increases in the formation of IMP. The nucleoside transport inhibitors dilazep (1 microM), dipyridamole (10 microM) and nitrobenzylthioinosine (NBMPR, 1 microM) strongly blocked cellular Ado metabolism. In the presence of nucleoside transport inhibitors, Ado which slowly enters the cell was metabolized principally by Ado kinase rather than ADA. Dilazep, NBMPR and dipyridamole were more effective in blocking Ado uptake and metabolism by erythrocytes suspended in a protein-free medium than by cells suspended in plasma.

Potentiation of the antiplatelet action of adenosine in whole blood by dipyridamole or dilazep and the cAMP phosphodiesterase inhibitor, RA 233.

Adenosine (Ado, 10-50 microM), a potent inhibitor of ADP-induced human platelet aggregation in platelet-rich plasma (PRP), does not inhibit aggregation in whole blood. However, the Ado analogs, 2-fluoroadenosine, 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine (NECA) which are resistant to deamination (2-fluoroadenosine) or deamination and phosphorylation (2-chloroadenosine and NECA), inhibit aggregation in whole blood with IC50 values of 12 microM, 2.3 microM and 0.26 microM, respectively. The inhibitory effect of NECA (200 nM) is potentiated by the platelet cAMP phosphodiesterase (PDE) inhibitor RA 233 (5 microM). Inhibition of the erythrocytic nucleoside transport system by dilazep (1 microM) or dipyridamole (10 microM), or blockade of Ado metabolism by 2'-deoxycoformycin (5 microM) plus 5-iodotubercidin (10 microM), evokes the antiaggregatory action of Ado in whole blood (IC50 congruent to 2 microM). RA 233 (5 microM) potentiates Ado-mediated inhibition about 10-fold when nucleoside transport or Ado metabolism is blocked. Ado (10 microM or 200 nM) is rapidly metabolized within 1 min in whole blood. When nucleoside transport is inhibited by dilazep or dipyridamole, or when Ado metabolism is blocked by 2'-deoxycoformycin and 5-iodotubercidin, 50-60% of the Ado remains in the plasma after 5 min. These results show that the failure of Ado to inhibit platelet aggregation in whole blood results from its rapid uptake and metabolism by erythrocytes. More importantly, these data emphasize the key role of nucleoside transport inhibition in the antiplatelet actions of dipyridamole and dilazep. In addition, superior therapeutic results may be obtained from the combination of blockade of nucleoside transport system with inhibition of platelet cAMP PDE.

Role of adenosine uptake and metabolism by blood cells in the antiplatelet actions of dipyridamole, dilazep and nitrobenzylthioinosine.

Adenosine (Ado, 10 microM) did not inhibit ADP-induced human platelet aggregation in whole blood. However, if the blood was preincubated with dipyridamole (10 microM), a potent inhibitor of the erythrocytic nucleoside transport system (NTS), Ado acted as a strong inhibitor of platelet aggregation. Similarly, Ado inhibited platelet aggregation in whole blood in the presence of other potent NTS inhibitors, dilazep (1 microM) and p-nitrobenzylthioinosine (NBMPR, 1 microM). RA 233 (10 microM), an analog of dipyridamole which is a potent inhibitor of platelet cAMP phosphodiesterase (PDE), did not evoke the Ado effect in whole blood. However, in platelet-rich plasma (PRP), RA 233 potentiated strongly Ado-mediated inhibition, whereas dipyridamole, dilazep and NBMPR were without activity. 5'-Methylthioadenosine (MTA), an Ado receptor antagonist, reversed the inhibition produced by a nucleoside transport system inhibitor plus Ado in whole blood. Dipyridamole (10 microM), dilazep (1 microM) or NBMPR (1 microM) blocked [14C]Ado (10 microM) uptake by blood cells in whole blood, whereas RA 233 (10 microM) was not effective. The combination of 2'-deoxycoformycin (dCF, 5 microM), a tight-binding inhibitor of adenosine deaminase (ADA), plus 5-iodotubercidin (ITu, 10 microM), a potent inhibitor of adenosine kinase (Ado kinase), gave comparable Ado-mediated inhibition of platelet aggregation in whole blood as was obtained when the blood was pretreated with dilazep. These studies suggest that the in vivo antiplatelet actions of drugs such as dipyridamole and dilazep result from their abilities to block erythrocytic Ado uptake and subsequent metabolism, thus elevating the extracellular steady-state concentration of the physiologically occurring, antiplatelet agent, Ado.