NTPDase and 5′-nucleotidase activities in rats with alloxan-induced diabetes

https://doi.org/10.1016/j.diabres.2003.11.016Get rights and content

Abstract

Diabetes is associated with a hypercoagulable state. In this study, we investigated the potential effects of alloxan-induced diabetes on the activities of the enzymes NTPDase (E.C. 3.6.1.5, apyrase, ATP diphosphohydrolase, ecto/CD39) and 5′-nucleotidase (E.C. 3.1.3.5, CD73) that can control the levels of ADP and adenosine, two substances that regulates platelet aggregation. In the alloxan-treated rats, NTPDase activity was significantly increased by 88 and 35% with ATP as substrate and by 156 and 58% with ADP as substrate in platelets and synaptosomes, respectively (P<0.05). AMP hydrolysis was increased by 142% (platelets) and 70% (synaptosomes) in diabetic rats compared to control. These results demonstrate that alloxan-induced diabetes interferes with ATP, ADP, and AMP hydrolysis in platelets and synaptosomes. Taken together, these results may indicate that in diabetic rats both NTPDase and 5′-nuleotidase from the central nervous system (CNS) and platelets respond similarly with increased activity. Thus, we speculate that platelets could be used as a potential peripheral marker of central alterations in NTPDase and 5′-nucleotidase activities in diabetes.

Introduction

Several factors can contribute to the accelerated atherosclerosis observed in diabetic patients, including hypertension, dyslipidemia, endothelial dysfunction, increased propensity for thrombosis, and impaired fibrinolysis, and increased platelet aggregation [1], [2], [3]. Of particular importance, platelets are essential for hemostasis and thromboregulation, and changes in platelet functioning occur early in the diabetic state and contribute to the development of microvascular disease in this pathological situation [1], [2], [3].

In addition, diabetes mellitus in humans and in animal models is associated with neurochemical, neurophysiological, and behavioral modifications and vascular disturbances in the brain impairing its functional and structural integrity [4], [5]. In fact, diabetes in animal models can cause an increase in markers of oxidative stress in the brain [6], [7].

An enormous quantity of experimental evidence has demonstrated that ectonucleotidases such as NTPDase (E.C. 3.6.1.5) and 5′-nucleotidase (E.C. 3.1.3.5) participate in the control of extracellular ATP levels in the synaptic cleft and in the control of purinergic neuromodulation and neurotransmission [8], [9]. NTPDase (ecto-apyrase, ecto/CD39) hydrolyzes the extracellular nucleosides tri- and di-phosphates in the presence of Ca2+ or Mg2+ and has been well characterized in the central nervous system (CNS), in platelets and in other tissue [8], [9], [10], [11], [12]. A role for NTPDase in neurotransmission has been suggested and alterations in this enzyme activity appear to be associated with different brain processes like learning and memory acquisition [13], [14].

Most importantly, platelet NTPDase play a fundamental role in the regulation of platelet aggregation and consequently in thrombus formation. In fact, NTPDase is now accepted as a potent antithrombotic agent [15], [16], [17], [18]. In addition, ecto 5′-nucleotidase catalyzes the hydrolysis of AMP, playing an important role in adenosine production. Adenosine is an important molecule in the CNS [19], a modulator of vascular tone and a well known inhibitor of platelet aggregation [20], [21].

Recent data from our laboratory have indicated a significant increase in NTPDase and 5′-nucleotidase activities in human platelets from diabetic type 2, hypertensive and diabetic type 2/hypertensive patients, which have been interpreted as an adaptive response trying to counteract the pathophysiology of these pathologies [22].

In the current study, we examined potential alterations in NTPDase and 5′-nucleotidase in platelets and synaptosomes from rats made diabetic by alloxan administration in an attempt to study the role of these enzymes in these pathological states in a more controlled model of diabetes.

Section snippets

Animals

Adult male Wistar rats (200–250 g) obtained from the Central Animal House, Federal University of Santa Maria, Santa Maria, RS, Brazil, were housed 3–4 per cage. Animals were maintained on a 12 h:12 h light/dark cycle in an air-conditioned constant temperature 22±1° C colony room, with free access to water and commercial protein chow (SUPRA, Brazil). All animal procedures were approved by the Institutional Commission of the Federal University of Santa Maria and were in agreement with the

Results

Plasma cholesterol and triglycerides were determined only at the end of treatment (45 days). Blood glucose was verified three times after the alloxan induction using an accu-chek system (15, 30, and 45 days after the beginning of treatment). Only animals that maintained fasting glycemia higher than 200 mg/dl were considered diabetic and selected for enzymatic assays. Data concerning body weight, glucose, cholesterol, and triglyceride levels of control and diabetic rats are presented in Table 1.

Discussion

NTPDase and 5′-nucleotidase activities in platelets and synaptosomes play a fundamental role in ATP, ADP, and AMP hydrolysis in these different cellular compartments. In every situation studied, experimental diabetes induced an activation of hydrolytic activities, indicating that alloxan treatment alters nucleotide hydrolysis. These modifications in nucleotide hydrolysis may cause distinct cellular responses. In platelets, a fast ADP hydrolysis can interfere with platelet aggregation and

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