The release of prostacyclin (PGI2) and von Willebrand factor (vWF) from human umbilical vein endothelial cells (HUVEC) was examined to determine if aprotinin had any effects on these endothelial cell reactions. These end-points were chosen to indicate if this serine protease inhibitor caused alterations in the control of haemostatic function by endothelium, in the light of the improvement in haemostasis seen in patients given aprotinin therapy at the time of open heart surgery. Stimuli used to promote secretion of prostacyclin and vWF were human alpha-thrombin, histamine, protamine sulphate, poly-L-lysine and phorbol myristate acetate. Aprotinin (30 microMs) had no significant effect on the basal or stimulated release of PGI2 or vWF from HUVEC.

The effects of bradykinin and ATP on L-arginine transport and nitric oxide (NO) production were studied in porcine aortic endothelial cells cultured and perfused on microcarriers and deprived of L-arginine for 24 h. Stimulation of cells with bradykinin (100 nM) or ATP (100 microM) resulted in a rapid increase in L-arginine uptake and NO release. In the presence of nitro-L-arginine (100 microM), an inhibitor of NO synthase, the stimulatory effect of bradykinin on L-arginine uptake was partially inhibited while NO release was completely abolished. Nitro-L-arginine alone was not an inhibitor of basal L-arginine transport, suggesting that its inhibitory action was not directly on the L-arginine transporter but a result of the inhibition of NO generation. These data indicate that during agonist-stimulated NO production there is a concomitant increase in the transport of L-arginine into endothelial cells providing a mechanism for the continual generation of NO.

The ability of sera from patients with SLE to stimulate endothelial cell prostacyclin production was studied using a standardized assay system for testing the effects of serum on cultured human endothelial cell monolayers. The effects of 20 normal and 32 SLE sera on endothelial prostacyclin production were measured. No differences between the rates of prostacyclin production were seen between the two groups, either basally or when prostacyclin release was stimulated with thrombin or bradykinin. In the SLE samples there was no correlation between anticardiolipin IgG or IgM titres and their ability to stimulate basal or agonist-induced prostacyclin release. These results suggest that the elevated risk of thrombosis in SLE patients is not associated with inhibition of endothelial cell prostacyclin synthesis.

Biologically active concentrations of potently vasoactive and platelet-active adenine nucleotides are generated in plasma by a variety of pathophysiological mechanisms. Although there is evidence that ATP and ADP are inactivated by endothelial ectonucleotidases, there has been little attempt to study the metabolic routes of their catabolism in blood or to assess the contribution of this process to their clearance in vivo. Therefore, we have studied the rates and patterns of catabolism of ATP, ADP, and AMP in whole blood, plasma, and isolated blood cells. Rates of degradation of each nucleotide in cell-free plasma ranged from 0.07-0.32 nmol/min/ml with 1 microM substrates to 1.1-3.6 nmol/min/ml with 100 microM substrates. The pattern of catabolism indicated that sequential dephosphorylation from ATP----ADP----AMP----adenosine occurs. In whole blood, the pattern was similar although ATP and ADP (but not AMP) breakdown was more rapid. This was due to leukocyte ectonucleotidase activity. The use of selective inhibitors demonstrated that catabolism was not due to nonspecific phosphatase activity and that plasma 5'-nucleotidase is distinct from ATPase or ADPase. In leukocytes, ATPase and ADPase activities were distinguishable, and each contributed substantially to the rates of catabolism in whole blood. Leukocyte 5'-nucleotidase did not measurably contribute to AMP dephosphorylation in blood. By comparison, ecto-ATPase and ecto-ADPase activities on cultured human umbilical vein endothelial cells were similar to those on leukocytes while endothelial 5'-nucleotidase per 10(6) cells was equivalent to the soluble activity in 1 ml of blood or plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

We have used the rat isolated, perfused heart to study the metabolism of adenine nucleotides on a single passage through the coronary circulation. Low doses (3-30 nmol) of ATP, ADP, or AMP injected as a bolus were extensively catabolized by ectoenzymes. Increasing doses of each nucleotide demonstrated saturability of catabolism that occurred at significantly lower doses of AMP than of ADP or ATP. The patterns of catabolites formed in each case were consistent with the major pathway of metabolism being sequential dephosphorylation of ATP----ADP----AMP----adenosine, although from experiments in which [3H]ATP was co-injected with unlabeled ADP, it appears that some direct conversion of ATP----AMP can occur. Furthermore, particularly in the presence of excess unlabeled ATP, [3H]ADP was phosphorylated to [3H]ATP, indicating that ectoenzymes capable of interconverting nucleotides are present. By evaluating recovery and metabolism in serial samples collected rapidly after bolus injection, we were able to use the integrated form of the Michaelis-Menten equation as developed by Bronikowski et al. (Math. Biosci. 61: 237-266, 1982) to derive Michaelis constant (Km) and maximum velocity times capillary plasma volume (Amax) values for adenosinetriphosphatase, adenosine diphosphatase, and 5'-nucleotidase (450, 300, and 93 microM; and 5.3, 5.9, and 1.7 mumol/min, respectively). This analysis also indicated that there is a high degree of heterogeneity of path lengths within the coronary circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine is a well-recognised vasoactive mediator that is believed to play an important role in reactive hyperaemia. More recently, ATP has also been recognised as a powerful vasoactive agent, with receptor-mediated actions independent of those of adenosine. It is likely that ATP released into the circulation during trauma, platelet aggregation, or possibly as a consequence of hypoxia, significantly modulates vascular tone.

The adenosine diphosphatase (ADPase) activity of rat lung has been investigated. Subcellular fractionation of lung tissue homogenates by sucrose density gradient centrifugation has shown the ADPase activity to be associated with the plasma membrane. ADPase was solubilised from the membranes and fractionated by ammonium sulphate precipitation to separate a specific, low-Km ADPase from non-specific alkaline phosphatase activity. The solubilised ADPase has a Km of 50 microM at pH 7.5 and appears to be distinct from ATPase.

The effects of ischaemia on adenylate energy charge of tissues from fetal rats and fetal guinea pigs were measured. Adult rat and guinea-pig tissues, as well as human placentae, were also studied. The largest differences observed were between the fetuses from different pregnant animals (P = 4.74 X 10(-15). Reductions in energy charge in placentae were slower than in other defined fetal tissues, especially brain. In the rat, an immature species at birth, greater 'stability' was observed in placentae of 14 days of gestation than near term at 20 days of gestation. As contrast, in the guinea pig, a mature species at birth, there was no difference in 'stability' in placenta or other fetal tissues between about 40 days of gestation and near term, about 60 days of gestation. In addition to these tissue and maturity effects in the fetus, it has been confirmed that fetal tissues are more 'resistant' than adult tissues to failures of energy supply. Concentrations of adenosine, uridine, guanosine and cytidine nucleotides in placenta show similar patterns in rats and guinea pigs. Fetal liver contains more uridine nucleotides and brain more cytidine nucleotides. It is suggested that the placenta retains an early fetal ability to maintain itself during ischaemia; this might be advantageous during parturition. Possible endocrine and other mechanisms 'damping' fetoplacental metabolism are linked with a discussion of the large maternal effect.

Respiratory parameters were studied in mitochondria from human placenta. Respiratory control and ADP/O ratios were low in this preparation. The adenine nucleotide content of placental mitochondria was found to be only one quarter of that found for adult uterine muscle tissue mitochondria prepared in the same way. Loading placental mitochondria with adenine nucleotides by incubation in the presence of ATP produced increased respiratory control ratios but no improvement in ADP/O ratios. Our evidence is consistent with the developmental changes shown to occur in rat liver, in which an increased concentration of adenine nucleotides is responsible for changes in respiratory parameters.

We compared the properties of the ectonucleotidases (nucleoside triphosphatase, EC 3.6.1.15; nucleoside diphosphatase, EC 3.6.1.6; 5′-nucleotidase, EC 3.1.3.5) in intact pig aortic smooth-muscle cells in culture with the properties that we previously investigated for ectonucleotidases of aortic endothelial cells [Cusack, Pearson & Gordon (1983) Biochem. J. 214, 975-981]. In experiments with nucleotide phosphorothioate diastereoisomers, stereoselective catabolism of adenosine 5′-[β-thio]triphosphate, but not of adenosine 5′-[α-thio]triphosphate, by the triphosphatase and stereoselective catabolism of adenosine 5′-[α-thio]diphosphate by the diphosphatase were found, as occurs in endothelial cells. In contrast with endothelial ecto-5′-nucleotidase, the smooth-muscle-cell enzyme catabolized adenosine 5′-monophosphorothioate (AMPS) to adenosine: the affinity of the enzyme for AMPS was greater than for AMP, and Vmax for AMPS was about one-sixth that for AMP. In both cell types AMPS was an apparently competitive inhibitor of AMP catabolism by 5′-nucleotidase. The relative rates of catabolism of nucleotide enantiomers in which the natural D-ribofuranosyl moiety is replaced by an L-ribofuranosyl moiety were similar to those in endothelial cells. No ectopyrophosphatase activity was detected in smooth-muscle cells, in contrast with endothelial cells, where modest activity is present.

1. After decreasing muscle ATP by a 2 min period of intense exercise, we have studied purine metabolism by using high-pressure liquid chromatography. 2. A major increase in hypoxanthine concentration in plasma and urine was found with increases in xanthine and, in plasma, inosine. Erythrocyte hypoxanthine rose with the level in plasma, but there was no corresponding rise in IMP, the first intracellular metabolite of hypoxanthine. No rises in uridine or urate were found in plasma. 3. Plasma adenosine did not rise and fall significantly after exercise, but a small rise and fall in adenine nucleotide concentrations in plasma was found. 4. Running, swimming and games, which tended to be at the weekend, were associated with a rise in hypoxanthine and xanthine excretion; exercise was probably the cause of the higher excretion during the day than at night. Such activities do not produce changes in concentrations of ATP in muscle, although turnover must rise. 5. The results are consistent with widespread purine exchange between tissues and a ‘circulating hypoxanthine pool’.

ATP, ADP and AMP in concentrations at least 1 mumol/l have been found by high pressure liquid chromatography (HPLC) in plasma from peripheral venous blood. Total adenine nucleotide concentrations of about 15-20 mumol/l can be found in some conventional clinical samples of blood using EDTA as an anticoagulant. EDTA prevented adenine nucleotide conversion to inosine in plasma. In order to estimate concentrations in vivo, the contribution derived from the cell breakage inherent even in careful venous blood sampling has been estimated by extrapolation to zero 'haemoglobin' concentration in plasma and minimum values in samples of small volume. Available results appear to be consistent with the release of small amounts of ATP in or near the peripheral circulation at the time of venepuncture. In CSF, ATP, ADP and AMP concentrations were less than 0.05 mumol/l suggesting that membrane activity in the central nervous system is not associated with non-specific leakage. The high Km variant of lymphocyte ecto-5'-nucleotidase was not associated with a significantly higher concentration of its substrate AMP in plasma. However, this enzyme may function on the lymphocyte in the thymus and spleen.

In vitro dual circuit perfusion of the placenta with well-oxygenated medium results in the continuous and stable consumption of oxygen and glucose over a 2-h perfusion period. This is reflected in a stable production of lactate and an energy charge which is higher at the end of the perfusion period than that seen in fresh placental tissue immediately after vaginal delivery. Anoxic perfusion causes an increase in glucose consumption which is more than twofold higher than that seen in the oxygenated perfusion, resulting finally in placental uptake of glucose not only from the maternal but also from the fetal circulation. Lactate production is increased during the anoxic perfusion, while the final tissue energy charge value lies between the values observed for fresh tissue and for the oxygenated perfusion. The shift to anaerobic metabolism shown by placental tissue in anoxic conditions enables continued functioning of the tissue over the 2-h perfusion period but it appears that under anoxic conditions the tissue may incur an energy debt not observed in oxygenated perfusions.

In growing tissues there is an increased turnover of tRNA and consequent increases in tRNA breakdown. The methylated bases and pseudouridine characteristic of tRNA are not ‘salvaged’ unlike the majority of the constituent bases of tRNA. There is therefore an increased concentration of methylated purine nucleosides and pseudouridine in extracellular fluids in many cancer patients (Mrochek et al., 1974). We have shown such an increase in human pregnancy and in children.

In order to study the effects of raising the hypoxanthine concentration in plasma on its metabolism and renal handling, the effects of intense exercise have been investigated in a patient with xanthine oxidase deficiency. Despite the 90-fold increased concentration of hypoxanthine in plasma above resting levels in normal individuals, the intracellular concentration of the initial product of hypoxanthine in cells, IMP, was unaffected. Evolution may have stabilized intracellular nucleotide concentrations against the large fluctuations in plasma hypoxanthine which occur during exercise. The renal handling of hypoxanthine is consistent with ‘filtration’. In contrast, xanthine clearances may exceed those for creatinine and urinary concentrations do not correlate with those for creatinine; ‘secretion’ may be involved. Xanthine excretion may reflect guanine breakdown. A retrospective survey of urate concentrations in blood from 47 420 patients followed by further selected investigations detected 2 women with persistent marked hypouricaemia and high urinary urate clearances, ‘Dalmatian’ hypouricaemia. High pressure liquid chromatographic analysis of plasma extracts can distinguish xanthine oxidase deficiency from other causes of hypouricaemia.

Summary. The ratio of the urinary concentrations of the ATP metabolite, hypoxanthine, to that of creatinine was determined in normal newborn infants. An increase in this ratio reflects high hypoxanthine excretion and thus ATP breakdown. The ratio can be determined on random urine samples, thus simplifying sampling. Urinary changes are persistent; abnormalities are detectable on the second day of life after intrapartum hypoxia. Preliminary results suggest that this ratio on a sample during the second day of life could‘diagnose’intrapartum hypoxia and might therefore quantitatively assess those obstetric‘risk factors’believed to operate through hypoxia.

The association between a reduction in cellular ATP and reduced cell survival has been established for the erythrocyte by many groups (see Seegmiller 1980) and more recently for the kidney (Buhl, 1976a, b). The ATP concentrations in the erythrocyte have guided a search for better condictions of erythrocyte storage for blood transfusion. The factors altering intracellular ATP concentration are therefore of practical interest. A large number of factors reduce ATP concentration but it is difficult to raise it. One condition associated with raised ATP concentration in erythrocytes is renal failure (Mansell and Grimes, 1979).

Nucleotides, nucleosides and purine bases in trichloroacetic acid extracts of freeze clamped samples of human placenta have been measured by high-pressure liquid chromatography. The concentrations of the nucleotides concerned with energy transduction, ATP, ADP and AMP, and especially the energy charge, are stable over periods of ischaemia of 30 min. Concentrations of 14 nucleotides, including UDPAG, GDP Man, UDP and CTP, have now been defined. In addition, the concentrations of hypoxanthine, xanthine, uridine, adenine and inosine are indicated. Concentrations of the vasodilator adenosine are similar to the apparent Michaelis constants of its main metabolizing enzymes adenosine kinase and adenosine deaminase. The availability of ‘normal’ values of adenine nucleotide concentrations in human placenta should permit the detection of ‘placental insufficiency’ of energy supply, if this condition exists.