Effects of diabetes and vascular occlusionon adenosine-induced relaxant response of ratcommon carotid artery

(1)

Effects of diabetes and vascular occlusion on adenosine-induced relaxant response of rat common carotid artery

Miroslav Radenkoviæ¹, Marko Stojanoviæ¹, Radmila Jankoviæ², Mirko Topaloviæ¹, Milica Stojiljkoviæ¹

1Department of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Belgrade, PO Box 38, 11129 Belgrade, Serbia

2Institute of Pathology, Faculty of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade, Serbia Correspondence: Miroslav Radenkoviæ, e-mail: miroslavr@beotel.net

Abstract:

Background: The aim of this study was to investigate effect of adenosine on isolated rat common carotid artery (CA) submitted to occlusion in non-diabetic or diabetic animals, and to determine whether endothelium denudation or potassium conductance block af- fects adenosine action.

Methods: Experiments were conducted on Wistar rat CA with or without endothelium. Diabetes was induced by alloxan. Occlusion of CA was performed in randomly selected non-diabetic or diabetic animals anesthetized with urethane. Thus, experiments were performed in four groups of rats: non-operated (control) animals without or with diabetes and operated animals submitted to the occlusion of CA without or with diabetes. Concentration-response curves for adenosine were obtained in a cumulative fashion on precontracted arteries.

Results: Adenosine produced concentration-dependent and endothelium-independent relaxation of CA with comparable maximal effects in all groups. Analysis of pEC₅₀values showed that responsiveness of CA decreased in following order: [diabetes (–) / occlu- sion (–)] = [diabetes (–) / occlusion (+)] > [diabetes (+) / occlusion (–)] > [diabetes (+) / occlusion (+)]. In the presence of high K⁺ maximal relaxant response of CA from non-operated rats without diabetes was reduced. The recorded inhibition was even stronger in animals subjected to CA occlusion. Conversely, in non-operated diabetic animals obtained reduction of adenosine effect was less pronounced in regard to non-diabetic rats.

Conclusions: Adenosine produced equi-effective endothelium-independent relaxation of CA in all groups. Pharmacological po- tency of adenosine was reduced in diabetic animals solely, but even more in diabetic rats submitted to CA occlusion. The enhanced potassium transmembrane flow has certain protective role on adenosine-induced action in occluded CA from non-diabetic rats. Con- versely, diabetes solely inhibited adenosine-evoked cascade connected to increased potassium conductance.

Key words:

adenosine, common carotid artery, occlusion, diabetes, endothelium, potassium channels

(2)

Introduction

Adenosine is an ubiquitous purine nucleoside and it is a product of complete dephosphorylation of adenine nucleotides catalyzed by the membrane or intracellular 5’-nucleotidases. This nucleoside has a wide dis- tribution in almost every tissue and it is an important signal molecule engaged in transduction mechanisms regarding the modulation of many physiological processes [32]. Accordingly, it can be assumed that the main role of adenosine is to maintain homeostasis, and it is involved in key biochemical processes [35].

In physiological conditions, the concentration of adenosine in various body fluids and tissues is low.

However, in different pathological conditions under- lined by tissue ischemia or anoxia, the concentration of adenosine rapidly increases, which is a protective tissue response [24].

Adenosine actions are related to the activation of adenosine receptors that belong to a well defined group of G protein-coupled receptors [39]. Among adenosine receptors, four types have already been dis- tinguished: A₁, A_2A, A_2B, and A₃ [5]. So far, it has been established that vasodilatations in response to adenosine are mainly due to the direct activation of adenosine receptors located on vascular smooth muscle cells [4, 18, 29]. However, in other blood vessels adenosine induces indirect action after activation of adenosine receptors located on endothelial cells and secondary release of different endothelial relaxing factors [2, 12, 38]. After the activation of specific adenosine receptor, the further signaling pathway would typically involve an opening of specific potassium channels or activation of Na⁺/K⁺-ATPase on the vascular smooth muscle cell [29]. Namely, increased potassium channel conductance hyperpolarizes smooth muscle and thereby reduces smooth muscle contrac- tion via reduced calcium entry through the voltage- dependent calcium channels [13]. This all underlines the importance of increased potassium conductance as a notable part in adenosine-induced vascular effects.

It is known that the carotid artery supplies cerebral blood flow from the systemic circulation, suggesting that this artery participates as an important regulator of cerebral circulation [20]. Likewise, there is a notable degree of evidence that adenosine contributes to the overall cerebral blood flow during physiological and different pathological conditions [26]. Accord- ingly, it has been for example shown that transient intracarotid infusion of adenosine increases cerebral

blood flow and decreases cerebrovascular resistance without affecting intracranial pressure or evoking ad- verse systemic hemodynamic side effects, which led to an assumption that intracarotid adenosine might be useful for evaluation of intraarterial vasodilator ther- apy and for controlled manipulation of cerebrovascular resistance [16]. It is also known that adenosine can be released from carotid bodies in response to hypoxia, thus contributing to the carotid body chemore- ceptor sensitivity [6]. Still, the underlying mechanism of the vasodilatory effect of adenosine in carotid arteries from different species is under investigation.

Moreover, limited data are available considering possible changes in transduction mechanism of adenosine action in this blood vessel regarding the vascular occlusion or diabetes, especially regarding up-to-date knowledge that vascular responsiveness and the functional features of different blood vessels are altered in diabetes mellitus. This is also in accordance to the fact that relation between diabetic micro- and macro- angiopathy and endothelial dysfunction is complex and it is still a subject of extensive research [41].

Taking into account previous considerations, this study was undertaken in order to investigate an effect of adenosine on isolated rat common carotid artery submitted to occlusion in non-diabetic or diabetic animals, and to determine the effects of endothelial denudation or potassium conductance block on adenosine action in investigated blood vessel.

Materials and Methods

The standards from the European Convention for the Protection of Vertebrate Animals Used for Experi- mental and other Scientific Purposes, as well as guidelines from the Good Laboratory Practice were fully applied in regard to procedures with used animals [3]. The methodology described in this article was in accordance with our previous investigations [29, 30, 33, 34] and was approved by the Ethical Commission for the Welfare Protection of Experi- mental Animals (Medical Faculty, University of Bel- grade, 2010 – 1500/05).

Our experiments involved male Wistar rats that were two months old and 220–250 g of weight at the beginning of the study. The experimental design included two groups of rats – the first group without

Adenosine action on carotid artery in diabetes

Miroslav Radenkoviæ et al.

(3)

diabetes, and the second group with alloxan-induced diabetes. Each group was further divided into three clusters: non-operated, sham-operated and operated rats. Experimental diabetes was induced in randomly selected animals with single intraperitoneal injection of 150 mg/kg alloxan dissolved in normal saline [21, 40]. Blood sample for glucose estimation was col- lected every seven days from rat tail vein [7]. Only the rats with glucose higher than 11 mM after four weeks were included in the study [27]. Thus, the animals with short-lasting diabetes of 4 weeks and their age-matched controls were used for further experiments. In the group of operated rats, the left and the right common carotid artery were occluded for 45 min by cords [33]. Those animals were previously subjected to anesthesia conducted with single intraperitoneal application of 1.25 g/kg urethane [22]. After the induction of anesthesia, the rats were placed on a heated table and fitted with a tracheal cannula to fa- cilitate spontaneous breathing. Animals were allowed to stabilize for a period of 90 min before carotid artery occlusion. The same protocol was conducted in the group of sham-operated rats, except of the carotid artery occlusion procedure.

Common carotid artery was carefully isolated from rat, dissected from surrounding fat and connective tissue, cut into circular segments (which were 4 mm long) and immediately placed in Krebs-Ringer bicarbonate solution (composition in mM: NaCl 118.3; KCl 4.7;

CaCl₂ 2.5; MgSO₄ 1.2; KH₂PO₄ 1.2; NaHCO₃ 25.0;

Ca-EDTA 0.026; glucose 11.1). The endothelium was

removed from some rings by gently rubbing the inti- mal surface with stainless-steel wire. Vascular rings were mounted between two stainless-steel triangles in an organ bath containing Krebs-Ringer bicarbonate solution (37°C, pH 7.4) and aerated with 95% O₂and 5% CO₂. One of the triangles was attached to a displacement unit allowing a fine adjustment of tension, and further connected to a force-displacement trans- ducer (Hugo Sachs Elektronik F30 Type 372, Frei- burg, Germany). Isometric tension was continuously recorded on a Rikadenki R-62 multi-pen electronic re- corder (Rikadenki Kogyo Co., Ltd, Tokyo, Japan).

At the beginning of each experiment, endothelium functional integrity was examined by precontraction of isolated carotid artery with submaximal concentration (EC₅₀–EC₇₀) of serotonin, followed by the addition of acetylcholine (1 µM). This procedure was re- peated three times in 20 min intervals (Fig. 1). Re- laxation > 80% of serotonin precontraction was indicative for the functionally intact endothelium. The morphological integrity of vascular endothelium was additionally confirmed at the end of randomly selected experiments by preparing histological preparations of rat carotid artery with standard hematoxylin and eosin stain. Concentration-response curves for adenosine were obtained in a cumulative fashion on serotonin-precontracted arteries. Since separate experiments in preparations of carotid artery demonstrated that the first and the second concentration- response curve (determined 45 min apart) for adenosine were not significantly different (Fig. 1), a multi-

demonstrated that the first and the second concentration-response curve (determined 45 min apart) for adenosine were not significantly different.

ACh = acetylcholine; 5-HT = serotonin

(4)

ple curve design protocol was applied for the experiments with high K⁺. Oppositely, in experiments with denuded arteries only one concentration-response curve was obtained on rings with or without endothelium, but always from the same rat.

The effect induced by each concentration of adenosine was expressed as a relaxation percentage of serotonin-produced precontraction. The results were expressed as the means ± SEM and n refers to the number of experiments. The concentration of adenosine producing 50% of its own maximum response (EC₅₀) was determined by using a non-linear least square fitting procedure of the individual experimental data, and was presented as pEC₅₀ (pEC₅₀ = –log EC₅₀). All calculations were done by using the com- puter program Graph Pad Prism (Graph Pad Software Inc., San Diego, USA). Statistical significance of differences between two means was determined with the Student’s t-test. A value of p < 0.05 was considered to be statistically significant.

The following chemicals were used: adenosine, alloxan, urethane (Sigma-Aldrich, St Louis, MO, USA);

acetylcholine iodide (Serva, Heidelberg, Germany);

serotonin (ICN, Irvine, CA, USA). All agents were

dissolved in distilled water and diluted to the desired concentration with the buffer. During experimental procedure all agents were added directly to the organ bath in a volume of 150 µl and the concentrations given are the calculated final concentrations in the bath solution.

Results

Adenosine (0.01–100 µM) produced comparable concentration-dependent relaxation of carotid arteries obtained from non-operated (max. % relaxation

= 97.0 ± 5.8; pEC₅₀= 5.94 ± 0.22) and sham-operated (max. % relaxation = 99.4 ± 2.3; pEC₅₀= 5.13 ± 0.25) rats without induced diabetes. Equivalent result was recorded after endothelial denudation (non-operated rats: max. % relaxation = 108.4 ± 3.8; pEC₅₀ = 5.64

± 0.26 and sham-operated rats: max. % relaxation = 98.5 ± 2.0; pEC₅₀= 5.58 ± 0.24). In addition, similar findings were obtained in diabetic rats (data not shown). Taking into account that we did not detect any significant difference between adenosine-induced

Fig. 2. Adenosine-induced relaxation in isolated rat common carotid artery after the endothelial denudation. Vas- cular rings have been obtained from non-diabetic (A, B) and diabetic (C, D) animals. The vascular occlusion was additionally performed on randomly selected non-diabetic (B) or diabetic (D) rats. Each point represents the mean ± SEM (n = 4–7). Vascular relaxations induced by adenosine are expressed as percentages of the precontraction induced by serotonin

(5)

action in non-operated and sham-operated rats, and also in order to avoid further unnecessary use of experimental animals in accordance to “The 3Rs” prin- ciple [9, 25, 28], from this point the control group consisted only of non-operated rats.

The adenosine-induced maximal relaxant response of rings with or without endothelium was similar in all investigated groups, indicating an equi-effective action of adenosine irrespective of diabetes or vascular occlusion (Fig. 2, Tab. 1, p > 0.05). Likewise, the analysis of the median effective concentrations (pEC₅₀) showed that the response of intact or denuded vessels to adenosine was comparable within each group, thus confirming an endothelium-independent relaxation (Fig. 2, Tab. 1, p > 0.05). In accordance, the histologi-

cal evaluation did not show any evidence of morphological differences in preparations obtained from non-operated animals without (Fig. 3A,B) or with (Fig. 3C,D) diabetes, which suggests the preservation of vascular endothelial and smooth muscle cells after short-term diabetes. Similar finding was observed in other investigated groups (pictures not shown).

The calculated median effective concentrations gradually decreased in-between investigated groups in the following order: [diabetes (–) / occlusion (–)] = [diabetes (–) / occlusion (+)] > [diabetes (+) / occlu- sion (–)] > [diabetes (+) / occlusion (+)], which actu- ally corresponds to the progressive decrease in adenosine pharmacological potency. Although it was found that diabetes solely reduced the responsiveness of ca-

Diabetes (–) Diabetes (+)

Occlusion (–) Occlusion (+) Occlusion (–) Occlusion (+)

Max. % relaxation

pEC50 Max. % relaxation

pEC50

Endothelium (+) 97.0 ± 5.8 5.94 ± 0.22 99.7 ± 2.1 5.77 ± 0.27 94.7 ± 3.2 5.13 ± 0.23 100.1 ± 5.9 4.59 ± 0.34 Endothelium (–) 108.4 ± 3.8 5.64 ± 0.26 95.2 ± 3.2 5.45 ± 0.25 95.3 ± 1.8 4.91 ± 0.27 104.1 ± 4.1 4.85 ± 0.29

A B

C D

E ( + ) E ( - )

Fig. 3. Histological preparations of in- tact and denuded rat common carotid arteries obtained from non-diabetic (A, B) and diabetic (C, D) animals that were not subjected to the vascular occlusion (hematoxylin and eosin stain, magnification = 400´; E = endothelium)

(6)

rotid artery to adenosine, the most prominent difference was noted between non-operated rats without diabetes and the group of diabetic animals additionally subjected to the carotid artery occlusion (Tab. 1, p < 0.05). This was also accompanied with the match- ing rightward shift of cumulative concentration-response curves for adenosine on intact or endothelium denuded rings, which is separately presented in Fig- ure 4A,B.

Although our statistical evaluation indicated that adenosine induced endothelium-independent relaxation within each group, it was also of interest to exam- ine if short term diabetes or transient vascular occlusion independently influenced an endothelial input in the effect of investigated purine nucleoside. Thus, in endothelium-free preparations from non-operated diabetic animals, the concentration-response curve for adenosine was notably shifted to the right if compared to endothelium-intact rings from non-operated and diabetes-free rats (Fig. 4C, Tab. 1, p < 0.01). In agree- ment with previous statistical analysis considering diabetes, the similar, yet less pronounced trend was detected in regard to the separate influence of tran-

sient vascular occlusion on the involvement of endothelial cells in adenosine relaxant action (Fig. 4D, Tab. 1, p < 0.05).

In the presence of high K⁺(100 mM), maximal relaxant response of carotid arteries obtained from both non-operated and operated rats without diabetes was significantly reduced (Fig. 5A,B, Tab. 2, p < 0.01).

Still, the recorded inhibition of adenosine-induced relaxation in diabetes-free rats was more evident in animals subjected to the occlusion than in non- operated rats (max % relaxation in the presence of high K⁺: 15.8 ± 9.5 vs. 31.2 ± 11.3, respectively, Tab. 2, p < 0.05). On the other hand, in non-operated diabetic animals the obtained reduction of adenosine- induced effect was also apparent (p < 0.05), yet if compared to the corresponding group without diabetes this inhibition was notably less pronounced (Fig.

5C; Tab. 2; p < 0.01). Interestingly, in the group of diabetic animals subjected to the carotid artery occlusion, adenosine-induced relaxation in the presence of high K⁺was reduced at the same extent as in arteries obtained from non-operated rats without diabetes (Fig. 5D, Tab. 2).

Fig. 4. The effect of concomitant pres- ence of diabetes and vascular occlusion on action induced by adenosine on endothelium-intact (A) or endothe- lium-denuded vascular rings (B), and the comparison of individual input of diabetes (C) or vascular occlusion (D) to the contribution of intact endothelium in adenosine-induced vascular relaxation. Each point represents the mean ± SEM (n = 4–7). Vascular relaxations induced by adenosine are expressed as percentages of the precontraction induced by serotonin

(7)

Discussion

In the present study, adenosine produced concentration-dependent relaxation with comparable maximal relaxant response in all investigated groups. There- fore, we can assume that short-term diabetes and/or transient vascular occlusion did not affect overall pharmacological efficacy of investigated purine nucleoside. This is also in accordance with the per-

formed histological evaluation suggesting the morphological preservation of endothelial and smooth muscle cells in physiological or investigated pathological conditions.

In our previous investigations on isolated rat aorta, inferior mesenteric or femoral artery we have reported that adenosine induced concentration-dependent relaxation, which was unaffected by endothelial denudation [11, 29, 31]. Accordingly, in the present study, it also appears that intact endothelium does not have

(A, B) and diabetic (C, D) animals. The vascular occlusion was additionally performed on randomly selected non- diabetic (B) or diabetic (D) rats. Each point represents the mean ± SEM (n = 5–7). Vascular relaxations induced by adenosine are expressed as percentages of the precontraction induced by serotonin. * p < 0.05 and ** p < 0.01 compared to the control relaxant effect

Tab. 2. Adenosine-induced relaxation in isolated rat common carotid artery in the presence of hyperpolarizing solution (KCl = 100 mM).

The concentration of adenosine eliciting 50% of its own maximum response (EC₅₀) is presented as pEC₅₀(pEC₅₀= –log EC₅₀), whereas maximal obtained relaxation is expressed as a percentage of the precontraction produced by serotonin. Each result represents the mean ± SEM (n = 5–7; E = endothelium)

Diabetes (–) Diabetes (+)

Occlusion (–) Occlusion (+) Occlusion (–) Occlusion (+)

Max. % relaxation

pEC50 Max. % relaxation

pEC50

Control, E (+) 92.1 ± 10.1 5.86 ± 0.27 97.7 ± 2.0 5.82 ± 0.25 98.4 ± 2.9 5.37 ± 0.25 102.8 ± 2.8 4.77 ± 0.31 KCl, 100 mM 31.2 ± 11.3 4.78 ± 0.11 15.8 ± 9.5 4.33 ± 0.16 70.9 ± 12.5 5.14 ± 0.34 32.1 ± 11.9 4.79 ± 0.36

(8)

significant role in the carotid artery response to adenosine in any of investigated groups of rats. This for the most part excludes the established impact of diabetes-induced functional changes on endothelial cells that were consequently related to the altered pharmacological action of various endogenous va- sorelaxant agonists in experiments on rats [1, 8, 37, 42, 43]. From our findings it can be also suggested that the carotid artery relaxation was most probably initiated by activation of specific adenosine binding sites located solely on smooth muscle cells irrespective of diabetes or vascular occlusion.

Although the evaluation of relaxant responses to the highest adenosine concentration did not demon- strate significant difference in-between investigated groups, an additional analysis of the calculated median effective concentrations was performed. The finding that the vascular occlusion in the group of non-diabetic animals did not change adenosine- induced relaxation on rings with or without endothelium would exclude any significant alterations regarding the pharmacological potency of adenosine connected to the applied transient procedure on the carotid artery. This is also indicative for the assumption that vascular occlusion most probably did not affect transduction mechanism of adenosine-evoked effect on the level of vascular smooth muscle cells.

On the other hand, the presence of diabetes solely led to a notable reduction in the blood vessel responsiveness to adenosine if compared to non-diabetic animals, which clearly indicates probable changes at the level of adenosine binding sites on smooth muscle cells or further intracellular signalling mechanisms.

Still, the most pronounced increase in the median effective concentration was obtained in the group of diabetic rats concomitantly submitted to the carotid artery occlusion. This was accompanied with corresponding rightward shift of the cumulative concentration-response curves for adenosine. Therefore, it appears that in pathological setting of experimental diabetes, the short-term ischemia of investigated blood vessel additionally contributed to the decreased vascular responsiveness to adenosine. In other words, taking into account that the median effective concentration as a pharmacological term corresponds to the potency of investigated drug [23], it can be also proposed that the potency of adenosine action was significantly inhibited by functional changes at the level of smooth muscle cells induced concurrently by diabetes and vascular occlusion.

As previously mentioned, the analysis of endothelium input in carotid artery response was performed within each group, thus suggesting an endothelium- independent mechanism. However, if one would com- pare concentration-response curve and corresponding median effective concentration obtained in endothelium-intact rings from non-operated and diabetic-free rats with one acquired in endothelium-denuded preparations from diabetic rats, it could be proposed the presence of endothelium-dependent relaxation in dia- betic versus healthy animals. The same assumption could be put forward for non-diabetic animals submitted to vascular occlusion opposed to non-diabetic and non-operated animals. Still, since we consistently obtained endothelium-independent relaxation within each group of investigated animals, further experiments are necessary to confirm, at this point speculative hypothe- sis, that the short-term diabetes or transient occlusion can actually stimulate an endothelium-dependent, thus most probably protective mechanism in adenosine- evoked relaxations of isolated rat carotid artery.

In the second part of our study, it was of interest to investigate if the transmembrane potassium ion flow contributes to the adenosine-induced action in investigated blood vessel. This question was substantiated by earlier reports that in different animal or human blood vessels adenosine-evoked vascular responses were associated with an opening of different types of potassium ion channels. This signalling pathway regarding the vasodilatation induced by adenosine or adenosine analogues have been, for example, described in mouse coronary vessels, porcine coronary and retinal arterioles, rat tail and renal arteries or human small coronary arteries and arterioles [10, 12, 14, 15, 17, 19, 36]. This pattern of adenosine action was also detected in our study. Namely, in all investigated groups the adenosine-induced relaxation has been significantly inhibited in the presence of high K⁺, thus suggesting that the transduction mechanism of carotid artery response to adenosine was strongly associated with an opening of potassium ion channels located on smooth muscle cells. Still, the obtained results were not consistent in all investigated groups. Namely, in the group of non-diabetic rats an inhibition obtained in the presence of high K⁺was more prominent in the presence of concomitant vascular occlusion. This suggests that the short-term ischemia of investigated blood vessel additionally stimulated signalling mechanism connected to the transmembrane potassium flow. In fact, it is plausible to imply that this can be an

(9)

adenosine-induced relaxation by high K⁺was significantly less pronounced in diabetic animals not subjected to the vascular occlusion, if compared to the corresponding non-diabetic group of rats. This clearly suggests that diabetes-related pathological process significantly inhibited apparently very important transduction cascade of adenosine action connected to transmembrane potassium flow on smooth muscle cells. Finally, in the group of diabetic animals submitted to the vascular occlusion the obtained result did not differ from the one recorded in the group of non- diabetic and non-operated animals. Still, there was a similar trend of less pronounced reduction of adenosine-evoked relaxation in preparations obtained after carotid artery occlusion in diabetic animals, compared to those without induced diabetes. How- ever, this final result requires further clarification.

Conclusions

Adenosine produced an equi-effective concentration- dependent and endothelium-independent relaxation of the carotid artery irrespective of vascular occlusion or the presence of diabetes. The obtained results indicate that the pharmacological potency of adenosine has been reduced at the highest level by the simultaneous presence of diabetes and vascular occlusion. Taking into apart the endothelium-independent mechanism involved in carotid artery response to adenosine, it can be postulated that this process is related to the functional changes on smooth muscle cells. The transmembrane potassium ion flow notably contributes to the adenosine-induced action in investigated blood vessel. In the occluded carotid arteries obtained from non-diabetic rats the input of potassium ion flow was more pronounced compared to the non-occluded vascular rings. On the other hand, it was shown that diabetes solely reduced adenosine-evoked transduction cascade connected to transmembrane potassium flow.

Acknowledgments:

This work was supported by the Ministry of Education and Science – Republic of Serbia with the Grant 175073. The authors would like to thank Mrs. Spomenka Luèiæ for technical assistance.

from diabetic rats. Life Sci, 2008, 82, 279–289.

2.Abebe W, Mustafa SJ: Effect of low density lipoprotein on adenosine receptor-mediated coronary vasorelaxation in vitro. J Pharmacol Exp Ther, 1997, 282, 851–857.

3.Beernaert H, Vanherle AM, Bertrand S: Critical aspects in implementing the OECD monograph No. 14 “The application of the principles of GLP to in vitro studies”.

Ann Ist Super Sanita, 2008, 44, 348–356.

4.Cheng DY, DeWitt BJ, Suzuki F, Neely CF, Kadowitz PJ: Adenosine A1 and A2 receptors mediate tone- dependent responses in feline pulmonary vascular bed.

Am J Physiol, 1996, 270, H200–H207.

5.Chroœciñska-Krawczyk M, Jargie³³o-Baszak M, Wa³ek M, Tylus B, Czuczwar SJ. Caffeine and the anticonvul- sant potency of antiepileptic drugs: experimental and clinical data. Pharmacol Rep, 2011, 63, 12–18.

6.Conde SV, Monteiro EC: Hypoxia induces adenosine release from the rat carotid body. J Neurochem, 2004, 89, 1148–1156.

7.Diniz SF, Amorim FP, Cavalcante-Neto FF, Bocca AL, Batista AC, Simm GE, Silva TA: Alloxan-induced diabetes delays repair in a rat model of closed tibial fracture.

Braz J Med Biol Res, 2008, 41, 373–379.

8.Fahim M, Hussain T, Mustafa SJ: Relaxation of rat aorta by adenosine in diabetes with and without hypertension:

role of endothelium. Eur J Pharmacol, 2001, 412, 51–59.

9.Festing MF, Altman DG: Guidelines for the design and statistical analysis of experiments using laboratory animals. ILAR J, 2002, 43, 244–58.

10.Flood A, Headrick JP: Functional characterization of coronary vascular adenosine receptors in the mouse.

Br J Pharmacol, 2001, 133, 1063–1072.

11.Grboviæ L, Radenkoviæ M: Analysis of adenosine vascular effect in isolated rat aorta: possible role of Na⁺/K⁺- ATPase. Pharmacol Toxicol, 2003, 92, 265–271.

12.Grboviæ L, Radenkoviæ M, Prostran M, Pešiæ S: Charac- terization of adenosine action in isolated rat renal artery.

Possible role of adenosine A_2Areceptors. Gen Pharma- col, 2001, 35, 29–36.

13.Heaps CL, Bowles DK: Gender-specific K⁺-channel contribution to adenosine-induced relaxation in coronary arterioles. J Appl Physiol, 2002, 92, 550–558.

14.Heaps CL, Tharp DL, Bowles DK: Hypercholestero- lemia abolishes voltage-dependent K⁺channel contribution to adenosine-mediated relaxation in porcine coronary arterioles. Am J Physiol Heart Circ Physiol, 2005, 288, H568–H576.

15.Jeppesen P, Aalkjaer C, Bek T: Adenosine relaxation in small retinal arterioles requires functional Na-K pumps and K_ATPchannels. Curr Eye Res, 2002, 25, 23–28.

16.Joshi S, Hartl R, Wang M, Feng L, Hoh D, Sciacca RR, Mangla S: The acute cerebrovascular effects of intracarotid adenosine in nonhuman primates. Anesth Analg, 2003, 97, 231–237.

17.Kêdzior K, Szczepañska R, Kociæ I: Contribution of NO, ATP-sensitive K⁺channels and prostaglandins to adeno-

(10)

sine receptor agonists-induced relaxation of the rat tail artery. Pharmacol Rep, 2009, 61, 330–334.

18.Keef KD, Pasco JS, Eckman DM: Purinergic relaxation and hyperpolarization in guinea pig and rabbit coronary artery: role of the endothelium. J Pharmacol Exp Ther, 1992, 260, 592–600.

19.Kemp BK, Cocks TM: Adenosine mediates relaxation of human small resistance-like coronary arteries via A_2Bre- ceptors. Br J Pharmacol, 1999, 126, 1796–1800.

20.Kinoshita H, Kimoto Y, Nakahata K, Iranami H, Dojo M, Hatano Y: The role of K⁺channels in vasorelaxation induced by hypoxia and the modulator effects of lidocaine in the rat carotid artery. Anesth Analg, 2003, 97, 333–338.

21.Manjunath S, Kugali SN, Deodurg PM: Effect of clo- nidine on blood glucose levels in euglycemic and alloxan-induced diabetic rats and its interaction with glibenclamide. Indian J Pharmacol, 2009, 41, 218–220.

22.Nakagiri A, Sunamoto M, Takeuchi K, Murakami M:

Evidence for the involvement of NADPH oxidase in ischemia/reperfusion-induced gastric damage via angio- tensin II. J Physiol Pharmacol, 2010, 61, 171–179.

23.Neubig RR, Spedding M, Kenakin T, Christopoulos A:

International Union of Pharmacology Committee on Re- ceptor Nomenclature and Drug Classification. XXXVIII.

Update on terms and symbols in quantitative pharmacology. Pharmacol Rev, 2003, 55, 597–606.

24.Peart JN, Headrick JP: Adenosinergic cardioprotection:

multiple receptors, multiple pathways. Pharmacol Ther, 2007, 114, 208–221.

25.Perry P: The ethics of animal research: a UK perspec- tive. ILAR J, 2007, 48, 42–46.

26.Phillis JW: Adenosine and adenine nucleotides as regula- tors of cerebral blood flow: roles of acidosis, cell swel- ling, and KATP channels. Crit Rev Neurobiol, 2004, 16, 237–270.

27.Preet A, Gupta BL, Yadava PK, Baquer NZ: Efficacy of lower doses of vanadium in restoring altered glucose me- tabolism and antioxidant status in diabetic rat lenses.

J Biosci, 2005,30, 221–230.

28.Radenkoviæ M: The OECD principles of good laboratory practice and the current bioethics. In: Bioethics and Pharmacology: Ethics in Preclinical and Clinical Drug Development. Eds. Todoroviæ Z, Prostran M, Turza K, Transworld Research Network, India, 2012, 43–50.

29.Radenkoviæ M, Grboviæ L, Pešiæ S, Stojiæ D: Isolated rat inferior mesenteric artery response to adenosine: possible participation of Na⁺/K⁺-ATPase and potassium channels. Pharmacol Rep, 2005, 57, 824–832.

30.Radenkoviæ M, Stojanoviæ M, Jankoviæ R, Topaloviæ M, Stojiljkoviæ M: Combined contribution of endothelial relaxing autacoides in the rat femoral artery response to CPCA: an adenosine A₂receptor agonist. Scienti- ficWorldJournal, 2012, 2012, 143818.

doi:10.1100/2012/143818.

31.Radenkoviæ M, Topaloviæ M, Stojanoviæ M: Isolated rat femoral artery response to adenosine: involvement of po-

tassium channels. In: Inflammation, Lipids and Immuno- logic Reactions During Atherogenesis. Eds. Heinle H, Marx N, Lorkowski S, Schaefer J, Deutsche Gesellschaft fuer Arterioskleroseforschung, Tuebingen, 2010, 189–192.

32.Ralevic V, Burnstock G: Receptors for purines and py- rimidines. Pharmacol Rev, 1998, 50, 413–492.

33.Roganoviæ J, Radenkoviæ M, Stojiæ D: ACh- and VIP- induced vasorelaxation in rabbit facial artery after carotid artery occlusion. Arch Oral Biol, 2010, 55, 333–342.

34.Roganoviæ J, Radenkoviæ M, Taniæ N, Taniæ N, Petroviæ N, Stojiæ D: Impairment of acetylcholine-mediated endothelium-dependent relaxation in isolated parotid artery of the alloxan-induced diabetic rabbit. Eur J Oral Sci, 2011,119, 352–360.

35.Samsel M, Dzierzbicka K. Therapeutic potential of adenosine analogues and conjugates. Pharmacol Rep, 2011, 63, 601–617.

36.Sato A, Terata K, Miura H, Toyama K, Loberiza FR Jr, Hatoum OA, Saito T et al.: Mechanism of vasodilation to adenosine in coronary arterioles from patients with heart disease. Am J Physiol Heart Circ Physiol, 2005, 288, H1633–H1640.

37.Shi Y, Ku DD, Man RY, Vanhoutte PM: Augmented endothelium-derived hyperpolarizing factor-mediated relaxations attenuate endothelial dysfunction in femoral and mesenteric, but not in carotid arteries from type I diabetic rats. J Pharmacol Exp Ther, 2006, 318, 276–281.

38.Steinhorn RH, Morin FC 3rd, Van Wylen DG, Gugino SF, Giese EC, Russell JA: Endothelium-dependent relaxations to adenosine in juvenile rabbit pulmonary arteries and veins. Am J Physiol, 1994, 266, H2001–H2006.

39.Tabrizchi R, Bedi S: Pharmacology of adenosine receptors in the vasculature. Pharmacol Ther, 2001, 91, 133–147.

40.Turlapaty PD, Lum G, Altura BM: Vascular responsiveness and serum biochemical parameters in alloxan diabetes mellitus. Am J Physiol, 1980, 239, E412–E421.

41.van den Oever IA, Raterman HG, Nurmohamed MT, Simsek S: Endothelial dysfunction, inflammation, and apoptosis in diabetes mellitus. Mediators Inflamm, 2010, 2010, 792393.

42.Wigg SJ, Tare M, Tonta MA, O’Brien RC, Meredith IT, Parkington HC: Comparison of effects of diabetes mellitus on an EDHF-dependent and an EDHF-independent artery. Am J Physiol Heart Circ Physiol, 2001, 281, H232–H240.

43.Yousif MH, Benter IF, Akhtar S: The role of tyrosine kinase-mediated pathways in diabetes-induced alterations in responsiveness of rat carotid artery. Auton Auta- coid Pharmacol, 2005, 25, 69–78.

Received: July 25, 2012; in the revised form: January 3, 2013;

accepted: January 11, 2013.