Jeremy D. Pearson

A Comparison of Ectonucleotidase Activities on Vascular Endothelial and Smooth Muscle Cellsa

Extracellular adenine nucleotides can be sequentially hydrolyzed to yield adenosine by ectoenzymes found on many cell types. Because the extent of phosphorylation can profoundly modify the physiologic effects of extracellular adenine nucleotides, we thought it highly likely that the regulation of the time course of their hydrolysis would be complex and would be tuned to serve different purposes in different locations. Regulation of the rates of nucleotide hydrolysis, together with an array of cell-specific responses to adenine nucleotides and adenosine, provides a rich network of regulatory elements with which to integrate the time course of cell and tissue response during crisis or signaling. We have investigated the whole time course of hydrolysis of ATP, ADP, and AMP by cultured endothelial cells and smooth muscle cells from pig aorta.*, Cells were grown attached to polystyrene beads. Cell-coated beads were loaded into chromatography columns, and substrates were perfused over them (FIG. 1 ). In single-pass perfusion, this gives an incubation volume-to-cell-surface ratio close to that observed in intact issue. When substrates are recirculated over the cells, the volumeto-surface ratio is comparable to that found at the surface of large blood vessels. We found that endothelial cells and smooth muscle cells differ strikingly in their means of regulation of the rate of adenosine appearance from ATP or ADP.

Adenosine Uptake and Adenine Nucleotide Metabolism by Vascular Endothelium

Recent research has emphasized that extracellular purines (particularly adenosine and A TP) may be important regulators of vessel tone, commonly producing vasodilation, through their binding to receptors currently classified as P1 (adenosine) and P2 (ATP) [1]. The direct control of vascular tone has usually been thought of in terms of the action of mediators at receptors on vascular smooth muscle cells, causing contraction or relaxation. However, within the last two years it has been recognized that endothelial cells are necessary modulators of the vasodilator response to several agents, including ATP [2–4]; before the publication of these results, such a role for endothelium in the control of vascular tone had been largely unconsidered. Indeed, until fairly recently, the concept that endothelial cell functions could contribute in any way to the regulation of blood flow, for example, by the metabolism of circulating vasoactive compounds, was not widely recognized.

Monoamine oxidase activities of porcine vascular endothelial and smooth muscle cells

Abstract Amine uptake by cultured vascular cells was studied under conditions minimizing nonenzymic oxidation. 5-Hydroxytryptamine (5HT) was accumulated only very poorly; detailed kinetic analysis couid not be performed, but there was no evidence for a saturable high affinity process. Comparison of β-phenylethylamine (PEA) and 5HT metabolism in intact cells and lysed cells demonstrated that the rates of entry of the amines into cells usually limited their metabolism especially at low (μM) concentrations. Primary cultures of aortic endothelial cells metabolised 5HT and PEA substantially faster than did subcultured endothelium. Subcultured aortic vascular smooth muscle cells and endothelial cells metabolised PEA and 5HT with comparable specific enzyme activities to those found in aortic medial tissue. Inhibition by clorgyline of PEA, 5HT and benzylamine (BZA) metabolism reveaied, however, that while aortic tissue possessed monoamine oxidase (MAO) types A and B and a comparable amount of a clorgyline resistant amine oxidase(s) (CRAO), cultured vascular cells possessed MAO-A, but little or no CRAO or MAO-B. Cultured venous endothelium, and smooth muscle from several vascular sites, metabolised PEA and 5HT at similar rates to those found in aortic cells. the studies demonstrate that although cultured porcine endothelial and smooth muscle cells from large blood vessels contain MAO, they do not apparently possess the amine transport process present in the lung. Additionally, conditions of culture can affect both the extent of amine metabolism and the pattern of amine oxidase present.

Effects of Extracellular ATP on the Release of Vasoactive Mediators from Endothelium

Endothelium-dependent vasodilatation in response to ATP was first described by De Mey and Vanhoutte. It was subsequently demonstrated that this action, now known to be due to the synthesis and release of endothelium-derived relaxing factor (EDRF, nitric oxide): occurred by stimulation of P,-purinoceptors, that is, those recognizing ATP or ADP but not AMP or aden~sine.~ The use of ATP analogues, such as 2-methylthio-ATP (more potent than ATP) and L-ATP (almost inactive), indicated further that the P2-purinoceptor on endothelium mediating the generation of EDRF is of the P,, ~ubtype ,~ according to the classification of Burnstock and Kennedy. Concurrent experiments with endothelial cells in culture showed that ATP or ADP acting at P,-purinoceptors stimulated the transient release of prostacyclin ( PGI,).6 The receptor was again subtyped as P2, by the use of a variety of analogues. Despite some apparent differences in the relative abilities of certain ATP analogues at stimulating EDRF and PGI, release, the most straightforward conclusion is that both of these responses are triggered by the binding of ATP to a single P,-purinoceptor subtype. Nonetheless, there are significant differences in the time course of each response, its desensitization, and its dependence on extracellular calcium, which indicate that discrete intracellular transduction pathways are likely to be involved in the generation and regulation of EDRF and PGI,.9

Metabolism of serotonin and adenosine

The major role played by endothelial cells in the inactivation of a wide variety of vasoactive and platelet-active compounds is becoming apparent as our knowledge of endothelial biology increases, mainly because of the ability to isolate and culture these cells. Endothelium is currently believed to be responsible for the inactivation of circulating thrombin, bradykinin, several prostaglandins and amines, adenosine and purine nucleotides. In this chapter we shall discuss the metabolism by endothelial cells of three of these molecules: serotonin (5-hydroxytryptamine, 5HT), adenosine and ATP. 5HT is released into the plasma when platelets are activated. It is a powerful vasoconstrictor in most vascular beds, although in some species it is a vasodilator in certain small vessels (1). Virtually all the 5HT in the blood is carried by the platelets in dense storage granules; it is released in common with other granule contents when platelets are stimulated by a variety of agents (e.g. ADP, thrombin, collagen) and is itself a platelet aggregating agent (2).

Endothelial Cell P2 Purinoceptors

Endothelial cells possess P2 purinoceptors of the P2Y subclass. These are coupled to the generation of inositol trisphosphate and hence the mobilization of intracellular calcium stores. P2Y receptor-mediated changes in intracellular ionized calcium have been followed in detail in cell populations and in individual endothelial cells, and consist of a second phase of calcium elevation requiring calcium entry, in addition to the first transient rise which is due to discharge from internal stores. Synthesis of prostacyclin, a potent vasodilator and inhibitor of platelet function, is driven by the first phase of the calcium response. The properties of the second phase of the calcium response, in contrast, are consistent with a causal role for this phase in the synthesis of nitric oxide (endothelium-derived relaxing factor), although this has yet to be established directly.

Lysosomal hydrolases of human vascular cells: response to agonists of endothelial function.

Endothelial injury has been proposed as a feature of a wide variety of vascular diseases, and release of endothelial lysosomal hydrolases could contribute to the pathological changes seen. We have determined the relative activities of 14 glycosidases, two esterases and four peptide hydrolases in human umbilical vein endothelial cells and investigated whether known agonists of endothelial function, or materials known to modulate hydrolase secretion in other phagocytic cells, influenced the activity or secretion of these enzymes by human umbilical vein endothelial cells. Hexosaminidase, beta-galactosidase, beta-glucuronidase and alpha-iduronidase accounted for most of the measured glycosidase activity. Acid phosphatase activity greatly exceeded arylsulphatase activity, and most of the measured peptidase activity was due to acid peptidases. Optimum pH and apparent Km values were determined for the most abundant hydrolases. Exposure of human umbilical vein endothelial cells to bradykinin, thrombin or interleukin-1 resulted in negligible release of either hexosaminidase or lactate dehydrogenase (LDH), in contrast to phorbol myristate acetate, which caused a parallel, dose-dependent release of both enzymes. Treatment of these cells with calcium ionophore A23187, trypsin or platelet-activating factor, caused less than 10% release of either hexosaminidase or LDH. Agents known to modulate lysosomal enzyme secretion by other phagocytic cells failed to induce selective secretion of lysosomal enzymes by human umbilical vein endothelial cells.