Elsevier

European Journal of Pharmacology

Volume 805, 15 June 2017, Pages 125-130
European Journal of Pharmacology

Full length article
Oxidative stress inactivates ecto-5'-nucleotidase by inhibiting protein kinase C in rat hearts in vivo

https://doi.org/10.1016/j.ejphar.2017.02.019Get rights and content

Abstract

Examined in the present study, allopurinol are xanthine oxidase inhibitors for use in rat hearts in vivo dialysis technology and ventricular myocardial intersitial adenosine production can increase. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rat hearts and the tissue in the vicinity of the dialysis was perfused with Tyrode's solution containing adenosine 5'-monophosphate (AMP) through the dialysis probe at a rate of 1.0 ml/min to assess the activity of ecto-5'-nucleotidase. Allopurinol (10 μM) significantly increased the level of adenosine in rat heart dialysate (n=6, p<0.05), which was inhibited by chelerythrine, 10 μM, an antagonist of protein kinase C (PKC). Another free radical scavenger, coenzyme Q10 (CoQ10, 100 μM) or ascorbic acid (Vitamin C; 100 μM) also increased adenosine production. In addition, allopurinol enhanced the diacylglycerol (DAG; 50 μM)-induced also increases in adenosine production by 71.5±12.0% (n=6, P<0.05), to a level significantly (P<0.05) greater than the increase caused by DAG alone (33.0±10.6%). In the presence of allopurinol (10 μM), a marked elevation of AMP-primed dialysate adenosine in ischemia/reperfused rat hearts was observed. Free radical generation may suppress adenosine production via activation of PKC. The results suggest that oxidative stress may cause inactivation of nucleotidase, adenosine production in rat heart.

Introduction

Adenosine, a degradation substance of adenosine triphosphate (ATP), may act as an endogenous substance in physiological conditions of the heart (Kolwicz et al., 2013; Johnson and Ogbi, 2011). Adenosine derived from ischemic myocardium is produced by degradation of adenine nucleotides, via enzymatic dephosphorylation of AMP by 5'-nucleotidase (EC 3.1.3.5) (Bauerle et al., 2011, Ipata and Balestri, 2013). Conversely, adenosine release is decreased when excess oxygen is supplied by overperfusion (Erlichman et al., 2010). It is widely believed that reactive oxygen free radicals occur during of ischemic myocardium (Obata et al., 1994, Johnstone and Hool, 2014). Some investigators (Sato et al., 1997, Obata, 2002) reported that enhanced activation of protein kinase C (PKC) increased 5'-nucleotidase activity, leading to an increased release of adenosine, in isolated rat cardiomyocytes, and the activation of 5'-nucleotidase was shown to be mediated by the activation of PKC.

Intriguingly, oxygen-derived free radical have been reported to reduce ecto-5'-nucleotidase and adenosine production during ischemia (Kitakaze et al., 1992). Only ecto-5'-nucleotidase has been found to be deactivated by superoxide anion (O2-) (Kitakaze et al., 1992, Kitakaze et al., 1993). Allopurinol, a xanthine oxidase inhibitor (Azarmi et al., 2014), may exert protection against O2- mediated damage to ecto-5'-nucleotidase and adenosine release. The present study was undertaken to clarify further the mechanism of O2- on the production of interstitial adenosine in rat myocardium. To achieve goal, we measured the concentration of interstitial adenosine in in vivo hearts, with the use of a flexibly mounted microdialysis technique that we developed (Obata et al., 1994). Major pathway of adenosine formation are dephosphorylation of adenosine-5'-monophosphate (AMP) by 5'-nucleotidase (Kitakaze et al., 1993) and the hydrolysis of S-adenosylhomocysteine (SAH) by SAH hydrolase (Shu et al., 2006); the hydrolysis of AMP by ecto-5'-nucleotidase, the main pathway for adenosine production under ischemic conditions (Kitakaze et al., 1993, Hart et al., 2008). We measured the concentration of dialysate adenosine under continuous supply of AMP (the substrate for 5'-nucleotidase) through the microdialysis probe. With the use of this system, we have reported that the level of AMP-primed dialysate adenosine reflects the activity of ecto-5'-nucleotidase in the particular site of interstitial space of the myocardium (Sato et al., 1997, Obata et al., 2001). The results of this study, oxidative stres suppresses the production of interstitial adenosine via activation of ecto-5'-nucleotidase.

Section snippets

Materials

Allopurinol, diacylglycerol (DAG), coenzyme Q10 (CoQ10) and ascorbic acid (Vitamin C) (Sigma Chemical Co. St. Louis, MO, USA) and adenosine 5'-monophosphate (AMP, Wako Pure Chemical Co., Osaka, Japan) were prepared immediately before the start of experiments by directly dissolving an appropriate amount of each agent in the Tyrode solution or Ringer's solution to acquire the desired final concentrations, as given in the text. α,β−Methyleneadenosine-5'-diphosphate (α,β-meADP) and chelerythrine

In vivo estimation of 5'-nucleotidase activity

After injection with a standard mixture of 5 picomole each inosine and adenosine, two peaks were obtained (Fig. 1). The authentic standards of adenosine and inosine had an identical retention. In the absence of AMP, the concentrations of adenosine in the dialysate within the first 15 min after implantation of the microdialysis probe was 5 μM. There was the a gradual decline in adenosine concentration so that it reached a virtually steady-state level of 0.51±0.09 μM (n =14) at 45–60 min. When the

Discussion

In the this study, to test a possible link between the O2- and ecto-5'-nucleotidase, the effect of allopurinol, a xanthine oxidase inhibitor, were examined, with use of flexibly mounted microdialysis technique (Obata et al., 1994). While this technology is stable, long-term measurements of interstitial adenosine that is impossible.. We previously reported that the sequential change of dialysate adenosine measured under a constant supply of AMP reflects the activity of endogenous

Acknowledgement

This study was supported in part by Grants-in-Aid for Exploratory Research (No. 08877011) and for Scientific Research (No. 08457014) from the Ministry of Education, Science, Sports and Culture of Japan to M.A.

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