Elisabeth Keijzer

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.

Intravenous adenosine protects the myocardium primarily by activation of a neurogenic pathway.

1 Endogenous adenosine is a trigger for ischemic myocardial preconditioning (IPC). Although intravascular administration of adenosine has been used to further unravel the mechanism of protection by IPC, it is questionable whether adenosine and IPC employ the same signaling pathways to exert cardioprotection. 2 We therefore investigated whether the active metabolic barrier of the endothelium prevents an increase in myocardial interstitial adenosine concentrations by intravenous adenosine, using microdialysis, and also the role of NO and activation of a neurogenic pathway in the cardioprotection by adenosine. 3 In pentobarbital‐anesthetized rats, area at risk and infarct size (IS) were determined 120 min after a 60‐min coronary artery occlusion (CAO), using trypan blue and nitro‐blue‐tetrazolium staining, respectively. 4 IPC with a single 15‐min CAO and a 15‐min adenosine infusion (ADO, 200 μg min−1 i.v.) limited IS to the same extent (IS=41±6% and IS=40±4%, respectively) compared to control rats (IS=63±3%, both P<0.05). However, IPC increased myocardial interstitial adenosine levels seven‐fold from 4.3±0.7 to 27.1±10.0 μM (P<0.05), while ADO had no effect on interstitial adenosine (4.1±1.2 μM), or any of the other purines. 5 The NO synthase inhibitor Nω‐nitro‐L‐arginine (LNNA), which did not affect IS (IS=62±3%), attenuated the protection by ADO (IS=56±3%; P<0.05 vs ADO, P=NS vs LNNA). The ganglion blocker hexamethonium, which had also no effect on IS (IS=66±3%), blunted the protection by ADO (IS=55±4%; P<0.05 vs ADO and vs hexamethonium). 6 These observations demonstrate that cardioprotection by ADO is dependent on NO, and is primarily mediated by activation of a neurogenic pathway.

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.

Further purification of adenosine kinase from rat heart using affinity and ion-exchange chromatography

Abstract Adenosine kinase (EC 2.7.1.20) in a cytoplasmic fraction of rat heart was subjected to 5′-AMP-Sepharose 4B chromatography. The enzyme showed affinity for the column in contrast to adenosine deaminase, and was eluted with adenosine plus MgATP. Fractions containing adenosine kinase were put on a column of DEAE-Sephacel and eluted with a gradient. The enzyme was purified up to 3000-fold (yield 10%). The specific activity exceeded 8000 units per gram of protein and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed only one band. We conclude that the method presented is a simple, quick, and elegant way of purifying myocardial adenosine kinase to virtual homogeneity.

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.

Combined Use of Radioenzymatic Assay and High Pressure Liquid Chromatography for the Detection of Myocardial Xanthine Oxidase/Dehydrogenase

One of the current interests in xanthine oxidase (XO; EC 1.2.3.2; electron acceptor is O2, ref. 1) is its possible role in the initiation of atherosclerosis (refs. 2,3). To study the effects of bovine milk XO in rat heart, more knowledge is needed of the XO activity in this tissue, since data with respect to specific activity vary (refs.4–6). Several methods are available to measure XO activity (refs. 7,8). At the moment XO is thought to be present in mammalian tissues mainly as xanthine dehydrogenase (XD; EC 1.2.1.37; physiologic electron acceptor NAD+, ref. 1). The present paper describes the detection of XO and XD in rat heart by radioenzymatic assay in which the oxypurines are separated by high pressure liquid chromatography (HPLC).

Lower Xanthine Oxidoreductase Activity in Isolated Perfused Hearts if Xanthine Replaces Hypoxanthine as Substrate

In normal cardiac tissue xanthine oxidoreductase is present in the dehydrogenase form. During ischemia xanthine dehydrogenase is converted to xanthine oxidase, which generates free radicals during reperfusion. Both forms catalyze the breakdown of hypoxanthine to xanthine and xanthine to urate. Its activity can be measured with hypoxanthine or xanthine as substrate. Most widely xanthine is used as substrate because only one product is formed and therefore the enzyme activity can easily be determined. With hypoxanthine as substrate the difficult two-step reaction can raise problems if one wants to calculate the activity. Since either substrate is used in studies of xanthine oxidoreductase, this can explain the controversial data reported for various species.1,2,3

Purification of myocardial adenosine kinase using affinity and ion-exchange chromatography.

Myocardial adenosine kinase (AK; EC 2.7.1.20) presumably plays a key role in the maintenance of adequate adenine nucleotide levels in the heart cell1–3. In order to study this enzyme in detail, we purified rat-heart AK to apparent homogeneity after a previous report on partial purification from this source4. The method presented here includes elution of AK from a 5′-AMP-Sepharose 4B column with a buffer containing adenosine. The endogenous adenosine in the fractions altered the specific activity of the radioactive substrate in the AK assay. This could be corrected for by means of HPLC adenosine measurements.

The Ca-Antagonist Nifedipine Reduces Purine Nucleoside and Oxypurine Release from Ischemic Heart

Nifedipine* is a blocker of slow Ca-channels1,2, which is presently used for the treatment of angina pectoris and hypertension. Nifedipine -like other Ca-antagonists- is thought to have an ATP-sparing effect, but the data available are conflicting3–5. We studied the effect of nifedipine on nucleotide metabolism in the isolated (ischemic) rat heart. We used release of adenosine (catabolites) as a sensitive indicator for adenine nucleotide breakdown6. Nifedipine decreased the release of adenosine, inosine and (hypo)-xanthine during ischemia and reperfusion.