Enhanced expression and activity of xanthine oxidoreductase in the failing heart.

The molecular basis for heart failure is unknown, but oxidative stress is associated with the pathogenesis of the disease. We tested the hypothesis that the activity of xanthine oxidoreductase (XOR), a free-radical generating enzyme, increases in hypertrophied and failing heart. We studied XOR in two rat models: (1) The monocrotaline-induced right ventricular hypertrophy and failure model; (2) coronary artery ligation induced heart failure, with left ventricular failure and compensatory right ventricular hypertrophy at different stages at 3 and 8 weeks post-infarction, respectively. XOR activity was measured at 30 degrees C and the reaction products were analysed by HPLC. In both models XOR activity in hypertrophic and control ventricles was similar. In the monocrotaline model, the hearts showed enhanced XOR activity in the failing right ventricle (65+/-5 mU/g w/w), as compared to that in the unaffected left ventricle (47+/-3 mU/g P<0.05, n=6-7). In the coronary ligation model, XOR activities did not differ at 3 and 8 weeks. In the infarcted left ventricle, XOR activity increased from 29.4+/-1.4 mU/g (n=6) in sham-operated rats, to 48+/-3 and 80+/-6 mU/g (n=8 P<0.05 v sham) in the viable and infarcted parts of failing rat hearts, respectively. With affinity-purified polyclonal antibody, XOR was localized in CD68+ inflammatory cells of which the number increased more in the failing than in sham-operated hearts. Our results show that the expression of functional XOR is elevated in failing but not in hypertrophic ventricles, suggesting its potential role in the transition from cardiac hypertrophy into failure.

The role of adenosine in preconditioning.

Preconditioning is a powerful form of (myocardial) protection that follows brief sublethal ischemia. G-protein-coupled receptors constitute the trigger for entrance to the preconditioned state. In conjunction with other receptors, various membrane adenosine receptors play an important role in the transduction of extracellular signals, leading to protection by preconditioning, lasting 1-3 hr. Adenosine A(1)- and A(3)-receptors mediate inhibition of adenylate cyclase via a guanine nucleotide binding inhibitory protein (G(i/o)). A(2)-receptors couple to a comparable stimulatory protein (G(s)). Adenosine receptors are especially abundant in the central nervous system; in lesser numbers, they are found in many tissues, including the heart. A(1)-receptors are located on cardiomyocytes and vascular smooth muscle cells, A(2)-receptors on endothelial and vascular smooth muscle cells, and A(3)-receptors on ventricular myocytes. Ischemic preconditioning by endogenous adenosine takes place through A(1)- and A(3)-receptors. A(2A/B)-receptor activation results in vasodilation. The relevance of cellular mediators, such as 5'-nucleotidase, to generate adenosine for preconditioning is controversial. In contrast, the role of protein kinase C (PKC) is clearly established. Signals from different receptors converge at PKC, reaching a threshold activation of the kinase necessary to induce protection. Tyrosine and mitogen-activated protein kinases may play a role in addition to PKC. The exact products downstream responsible for the memory of preconditioning are elusive. A prime candidate for the end-effector of preconditioning is the K(ATP) channel. Preconditioning with adenosine-receptor agonists offers the possibility for treatment of coronary artery disease, but research in this field is still in its infancy.

Purine in bronchoalveolar lavage fluid as a marker of ventilation-induced lung injury.

OBJECTIVE: To investigate in a rat model of ventilation-induced lung injury whether metabolic changes in the lung are reflected by an increased purine concentration (adenosine, inosine, hypoxanthine, xanthine, and urate; an index of adenosine-triphosphate breakdown) of the bronchoalveolar lavage fluid and whether purine can, thus, indirectly serve as a marker of ventilation-induced lung injury. DESIGN: Prospective, randomized, controlled trial. SETTING: Research laboratory. SUBJECTS: Forty-two male Sprague-Dawley rats. INTERVENTIONS: Five groups of Sprague-Dawley rats were subjected to 6 mins of mechanical ventilation. One group was ventilated at a peak inspiratory pressure of 7 cm H2O and a positive end-expiratory pressure of 0 cm H2O. A second group was ventilated at a peak inspiratory pressure of 45 cm H2O and a positive end-expiratory pressure of 10 cm H2O. Three groups of Sprague-Dawley rats were ventilated at a peak inspiratory pressure of 45 cm H2O without positive end-expiratory pressure. Before mechanical ventilation, two of these groups received intratracheal administration of saline or exogenous surfactant at a dose of 100 mg/kg and one group received no intratracheal administration. A sixth group served as the nonventilated controls. MEASUREMENTS AND MAIN RESULTS: Bronchoalveolar lavage fluid was collected in which both purine concentration (microM; mean +/- SD) and protein concentration (mg/mL; mean +/- SD) were determined. Statistical differences were analyzed using the one-way analysis of variance (ANOVA) with a Student-Newman-Keul's post hoc test. Purine and protein concentrations were different between groups (ANOVA p value for purine and protein, <.0001). Both purine and protein concentrations in bronchoalveolar lavage fluid were increased in Group 45/0 (3.2 +/- 1.9 and 4.2 +/- 1.6, respectively) compared with Group 7/0 (0.4 +/- 0.1 [p < .05] and 0.4 +/- 0.2 [p < .001]) and controls (0.2 +/- 0.2 [p < .01] and 0.2 +/- 0.1 [p < .001]) and in Group 45/Na (5.8 +/- 2.5 and 4.2 +/- 0.5) compared with Group 7/0 (purine and protein, p < .001) and the controls (purine and protein, p < .001). Positive end-expiratory pressure prevented an increase in purine and protein concentrations in bronchoalveolar lavage fluid (0.4 +/- 0.3 and 0.4 +/- 0.2, respectively) compared with Group 45/0 (purine, p < .01; protein, p < .001) and Group 45/Na (purine and protein, p < .001). Surfactant instillation preceding lung overinflation reduced purine and protein concentration in bronchoalveolar lavage fluid (2.1 +/- 1.6 and 2.7 +/- 1.0) compared with Group 45/Na (purine, p < .001; protein (p < .01). Surfactant instillation reduced protein concentration compared with Group 45/0 (p < .01). CONCLUSIONS: This study shows that metabolic changes in the lung as a result of ventilation-induced lung injury are reflected by an increased level of purine in the bronchoalveolar lavage fluid and that purine may, thus, serve as an early marker for ventilation-induced lung injury. Moreover, the study shows that both exogenous surfactant and positive end-expiratory pressure reduce protein infiltration and that positive end-expiratory pressure decreases the purine level in bronchoalveolar lavage fluid after lung overinflation.

Ischemic nucleotide breakdown increases during cardiac development due to drop in adenosine anabolism/catabolism ratio.

Our earlier work on reperfusion showed that adult rat hearts released almost twice as much purine nucleosides and oxypurines as newborn hearts did [Am J Physiol 254 (1988) H1091]. A change in the ratio anabolism/catabolism of adenosine could be responsible for this effect. We therefore measured the activity of adenosine kinase, adenosine deaminase, nucleoside phosphorylase and xanthine oxidoreductase in homogenates of hearts and myocytes from neonatal and adult rats. In hearts the activity of adenosine deaminase and nucleoside phosphorylase (10-20 U/g protein) changed relatively little. However, adenosine kinase activity decreased from 1.3 to 0.6 U/g (P less than 0.025), and xanthine oxidoreductase activity increased from 0.02 to 0.85 U/g (P less than 0.005). Thus the ratio in activity of these rate-limiting enzymes for anabolism and catabolism dropped from 68 to 0.68 during cardiac development. In contrast, the ratio in myocytes remained unchanged (about 23). The large difference in adenosine anabolism/catabolism ratio, observed in heart homogenates, could explain why ATP breakdown due to hypoxia is lower in neonatal than in adult heart. Because this change is absent in myocytes, we speculate that mainly endothelial activities of adenosine kinase and xanthine oxidoreductase are responsible for this shift in purine metabolism during development.

Release of purine nucleosides and oxypurines from the isolated perfused rat heart.

In the ischemic heart, high-energy phosphates are rapidly broken down. We studied the release of AMP catabolites from the isolated perfused rat heart which was temporarily made ischemic or anoxic. We measured the concentration of purine nucleosides and oxypurines with a novel high-pressure liquid chromatographic technique. The postischemic working heart released adenosine, inosine, hypoxanthine, and also substantial amounts of xanthine. The latter could indicate that xanthine oxidase is present in rat heart. Further evidence for the myocardial occurrence of this enzyme was obtained from experiments with hearts perfused retrogradely with allopurinol, an inhibitor of xanthine oxidase. This drug greatly enhanced the release of hypoxanthine, both during normoxic and anoxic perfusions. We conclude that xanthine oxidase could play an essential role in the myocardial breakdown of AMP catabolites.

Nifedipine reduces adenine nucleotide breakdown in ischemic rat heart.

An ATP-sparing effect has been demonstrated for a number of calcium antagonists. Nifedipine probably has a similar action, but data supporting this view are limited. Therefore we decided to study the effect of nifedipine on high-energy phosphate (and carbohydrate) metabolism in the ischemic rat heart. Langendorff preparations were made ischemic for less than 15 min. The reduction in coronary flow was 60 or 70%. Apex displacement during ischemia, a measure of contractility, was comparable for nifedipine-treated and untreated hearts. Ischemia caused a considerable release of the AMP catabolites adenosine, inosine and (hypo)xanthine, and of lactate. Nifedipine (10-100 micrograms/l) prevented this in a dose-dependent way. The highest dose reduced the release of purines and lactate by 90% (P less than 0.01) and 60% (P less than 0.001), respectively. The drug acted in a similar way during reperfusion. Due to ischemia, the adenylate energy charge (ATP + 0.5 ADP)/(ATP + ADP + AMP), decreased 15% (P less than 0.001); nifedipine at a concentration of 100 micrograms/l prevented this decrease (P less than 0.05). We conclude that nifedipine exerts a beneficial effect on myocardial adenine nucleotide metabolism during ischemia and reperfusion.

Dipyridamole affects myocardial adenosine kinase activity.

Adenosine kinase (EN 2.7.1.20) from rat and dog heart was purified until it was devoid of adenosine deaminase activity. A stimulation of adenosine kinase activity by dipyridamole was observed when the enzyme was assayed under optimal conditions. At low substrate concentrations adenosine kinase was inhibited by the drug. It increased the Km for adenosine sevenfold. The effects of dipyridamole were Mg2+-dependent. The adenosine-sparing action of dipyridamole at low substrate concentrations is in keeping with the vasodilatory action of the drug.