Annals of the New York Academy of SciencesVolume 603, Issue 1 p. 366-378 A Comparison of Ectonucleotidase Activities on Vascular Endothelial and Smooth Muscle Cellsa LINDA L. SLAKEY, LINDA L. SLAKEY Department of Biochemistry University of Massachusetts Amherst, Massachusetts 01003Search for more papers by this authorELLEN L. GORDON, ELLEN L. GORDON Department of Biochemistry University of Massachusetts Amherst, Massachusetts 01003 Department of Neurological Surgery, Harborview Medical Center, Seattle, Washington 98104.Search for more papers by this authorJEREMY D. PEARSON, JEREMY D. PEARSON Medical Research Council Clinical Research Centre Harrow, Middlesex HA1 3UJ, EnglandSearch for more papers by this author LINDA L. SLAKEY, LINDA L. SLAKEY Department of Biochemistry University of Massachusetts Amherst, Massachusetts 01003Search for more papers by this authorELLEN L. GORDON, ELLEN L. GORDON Department of Biochemistry University of Massachusetts Amherst, Massachusetts 01003 Department of Neurological Surgery, Harborview Medical Center, Seattle, Washington 98104.Search for more papers by this authorJEREMY D. PEARSON, JEREMY D. PEARSON Medical Research Council Clinical Research Centre Harrow, Middlesex HA1 3UJ, EnglandSearch for more papers by this author First published: December 1990 https://doi.org/10.1111/j.1749-6632.1990.tb37686.xCitations: 24 a This work was supported by Grants HL 31854 and HL 38130 from the National Institutes of Health. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume603, Issue1Biological Actions of Extracellular ATPDecember 1990Pages 366-378 RelatedInformation

The extracellular reaction sequence ATP----ADP----AMP----adenosine participates in regulating the time course of cellular response during crisis or signaling events, such as thrombus formation or neurotransmission. We have investigated the whole time course of hydrolysis of ATP to adenosine by recirculating adenine nucleotide substrates over smooth muscle cells attached to polystyrene beads. Kinetic parameters were estimated for each reaction by fitting observed time courses to models of the pathway. In spite of the inhibition of 5'-nucleotidase by ADP, adenosine was produced very rapidly by smooth muscle cells. Comparisons of the apparent Km values of ADPase and 5'-nucleotidase (determined from experiments in which each substrate was used as the initial substrate with Km values observed when each substrate was supplied from the upstream reaction) suggest that the local concentrations of substrate supplied from the preceding reactions are very much higher than those in the bulk phase. This enhancement of efficiency overcomes the effect of the feed-forward inhibition to give rise to very rapid adenosine production from ADP or ATP. These observations are in marked contrast to our previous findings with endothelial cells (Gordon, E. L., Pearson, J. D., and Slakey, L. L. (1986) J. Biol. Chem. 261, 15496-15504), on which feed-forward inhibition causes a profound lag in adenosine production from adenine nucleotides and on which there are no apparent surface effects on substrate delivery.

Diadenosine triphosphate is present in platelet-dense granules and released quantitatively on platelet aggregation. We have found that intact porcine aortic endothelial cells can efficiently hydrolyze extracellular diadenosine triphosphate. The products of diadenosine triphosphate hydrolysis are adenosine monophosphate and adenosine diphosphate. Adenosine diphosphate is a potent stimulus of platelet aggregation. Since platelet-dense granules contain high concentrations of adenosine triphosphate and adenosine diphosphate, we examined endothelial cell hydrolysis of a mixture of diadenosine triphosphate and adenosine triphosphate. We find that the presence of adenosine triphosphate severely inhibits the hydrolysis of diadenosine triphosphate. Thus, although endothelial cells can rapidly clear extracellular diadenosine triphosphate, during platelet aggregation the hydrolysis of diadenosine triphosphate may be slow due to the presence of high concentrations of other adenine nucleotides. This phenomenon may be important physiologically if, as current evidence implies, diadenosine triphosphate is involved in the maintenance of hemostasis.

version of ATP to ADP, AMP, and adenosine by ectonucleotidases.There data In the accompanying paper, data are presented for the time course Of Consuggest that inhibition Of 5"nuclaMidare by ATP and/or ADP Causes an inverse dependence Of the rate of adenosine appearance on the initial concentration Of ATP.simulation of the progress Of this reaction sequence and fitting Of the simulated curves t o experimental data were undertaken (1) to understand better the potential of the pathway for regulating the time course of appearance and disappearance Of extracellular nucleotides and adenosine; ( 2 ) to estimate kinetic constants for the nucleotidases; and ( 3 ) to assess which of several models best explain the Observed time courses.W e report in this Appendix the rate equations used to model the pathways, and the procedures Used for aimulation and data fitting.

A procedure for measuring cyclic AMP in samples containing interfering substances, especially high concentrations of other adenine nucleotides, is described. Samples are purified by sequential chromatography on phenyl boronate followed by Dowex - 1. The final elution is with 0.1 normal hydrochloric acid; the samples are immediately ready for acetylation and radioimmunoassay. Cyclic AMP can be detected at 0.5 nanomolar without need to concentrate the sample at any step. The method has been successfully applied to samples containing 100 micromolar ATP, ADP, or AMP in tissue culture medium.

The time course of the extracellular reaction sequence ATP----ADP----AMP----adenosine has been examined during recirculation of substrate solutions over cultured pig aortic endothelial cells attached to polystyrene beads. This permits the study of reactions at volume to cell surface ratios approaching those of small blood vessels. When endothelial cells were presented with an initial bolus of ATP, high concentrations of the intermediates ADP and AMP developed before significant conversion of AMP to adenosine occurred. Further, the higher the initial ATP concentration, the slower the conversion of AMP to adenosine. Kinetic constants for each reaction were estimated by fitting simulated reaction curves to observed time courses. Apparent Km values estimated in this way agreed well with those reported for initial velocity measurements (ATPase = 300 microM; ADPase = 240 microM; and 5'-nucleotidase = 26 microM). The ratio of maximum velocities was ATPase:ADPase:AMPase = 6:1.5:1, with absolute values varying among cell batches. The data could only be fitted if the model incorporated inhibition of 5'-nucleotidase by ATP or ADP, and satisfactory fitting was achieved with a Ki value for ADP of 5 microM. These kinetic properties maximize the time separation of the intermediate pools. In vivo, at sites of platelet degranulation, they would create a time gap proportional to the size of the initial release between release of ADP (a proaggregatory milieu) and the appearance of adenosine (an anti-aggregatory milieu).

The working hypothesis for the experiments presented here is that the reaction sequence: ATP → ADP → AMP Adenosine occuring extracellularly, is a key regulatory sequence. Adenosine and its nucleotides occur extracellularly during many crisis or signalling events. They are agonists for a wide variety of physiologic responses. The antagonistic and competitive interactions among adenosine and its nucleotides, and the kinetic properties of the ectonucleotidases which carry out the sequential hydrolysis of the phosphate groups, provide for great subtlety in regulating the rate and extent of tissue response to the appearance of nucleotides in the pericellular space.