- Email: [email protected]
Contribution of NO, ATP-sensitive K+ channels and prostaglandins to adenosine receptor agonists-induced relaxation of the rat tail artery
Pharmacological Reports
Copyright © 2009 by Institute of Pharmacology Polish Academy of Sciences
2009, 61, 330334 ISSN 1734-1140
Short communication
Contribution of NO, ATP-sensitive K+ channels and prostaglandins to adenosine receptor agonists-induced relaxation of the rat tail artery Katarzyna Kêdzior, Renata Szczepañska, Ivan Kociæ Department of Pharmacology, Medical University of Gdañsk, Dêbowa 23, PL 80-204 Gdañsk, Poland Correspondence: Ivan Kociæ, e-mail:
[email protected]
Abstract: The mechanism of relaxation in the rat tail artery induced by the adenosine A1 receptor-selective agonist N6-cyclohexyladenosine (CHA, 10 nM–300 μM) and the adenosine A1/A2a receptor agonist 5’-N-ethylcarboxamidoadenosine (NECA, 10 nM–300 μM) has been characterized. To do this, we used a1-receptor agonist phenylephrine to evoke contraction (10 μM), and inhibitors of nitric oxide synthase (L-NAME, 10 μM), ATP-sensitive K+ channels (glibenclamide, 10 μM) and prostaglandin synthesis (indomethacin, 10 μM). CHA and NECA induced relaxation of rat-tail artery by 80% and 70% in a concentration-dependent manner, respectively. The relaxation effect of NECA was completely abolished in the presence of L-NAME, while glibenclamide and indomethacin prevented CHA-induced relaxation of the rat tail artery by approximately 25% and 40%, respectively. Our results indicate that nonspecific effects such nitric oxide and prostaglandins release or the activation of potassium channels significantly contributed to the effects of CHA and NECA. Key words: adenosine receptor agonists, smooth muscle, nitric oxide, prostaglandins, potassium channels
Introduction The role of adenosine in the regulation of smooth muscle contractility has been extensively studied [2]. Special attention has been paid to the role of specific adenosine receptor subtypes A1, A2a and A2b. in the context of noradrenaline release at the synaptic and postsynaptic levels, calcium changes and the contraction of venous and/or arterial smooth muscles [3, 10]. The majority of studies to date have used selective adenosine receptor agonists to determine the potential contribution of particular receptor subtypes in the regulation of different physiological processes [5, 11, 17]. However, little attention was paid to the nonspecific effects of adenosine-receptors ligands [6].
330
Pharmacological Reports, 2009, 61, 330334
Therefore, in this study, we employed inhibitors of nitric oxide synthase, cyclooxygenase enzyme and ATP-sensitive K+ channels to determine whether these proteins were involved in the action of N6-cyclohexyladenosine (CHA) and 5’-N-ethylcarboxamidoadenosine (NECA) substances, often used to stimulate the A1 and A2 receptors.
Materials and Methods Animals and housing
Three-month old male Wistar rats, weighing 190–260 g were used. Animals were maintained at 24°C in a 12-h
Adenosine receptor agonists and smooth muscle relaxation Katarzyna Kêdzior et al.
light-dark cycle with free access to food and water for seven days prior to experimentation. The experimental protocol was approved by the Local Ethic Committee of Medical University of Gdañsk, Poland. Tissue preparation
After pentobarbital (60 mg/kg b.w. ip in pre-heparinized animals) administration, the tail artery was excised and cleaned, according to previously described methods [15]. The proximal region was cut into 3-mm segments that were mounted on two wires placed through the lumen. The mounted arterial segment was then submerged in a tissue bath filled with oxygenated Krebs’ solution at 37°C, pH = 7.4. The composition of the Krebs’ solution was (mM): NaCl, 6.9; KCl, 0.35; CaCl2, 0.28; MgSO4, 0.29; glucose, 2.0; KH2PO4, 0.16; NaHCO3, 2.1; and dextrose, 2.0. Tissue segments were equilibrated for 75 min, stretched using a preload of 0.5 g of weight and the flow was established at 1.6 ml/min with a perfusion pressure of 7.99 kPa. The solution was constantly aerated by carbogen and circulated using the peristaltic pump PP1 (Zalimp, Poland)
L-NAME and indomethacin on resting tension and on phenylephrine-induced contraction of the rat tail artery were examined in a separate series of experiments. Drugs used
All substances were supplied by Sigma (St. Louis, MO, USA) and dissolved in distilled water. Data analysis
The results were expressed as the per cent of contraction amplitude induced by phenylephrine. The means ± SEM are reported and n values indicate the number of animals in each sample group. To quantify pharmacological drug potencies in the presence of different compounds, IC50 values (effective concentration of CHA or NECA to produce 50% of the maximal effect of relaxation or inhibition of contraction induced by phenylephrine) were determined. The relaxation or inhibition effects were compared among groups using one-way analysis of variance (ANOVA) and Newman- Keuls post-hoc test; p < 0.05 was considered significant.
Measurement of vasoconstricting and vasorelaxing responses
Tissue contraction was recorded using Statham P23P6 force transducers (Statham Laboratories, USA) and an Electromanometer EK4 (Farum, Poland) was used to transfer data to the on line recorder TZ 4200 (Laboratorium Pristroje, Praha, Czech Republic). To study the contraction response in the rat tail artery, control concentration-response curves were constructed by adding phenylephrine (10 μM), and depending on the experimental setting, adenosine receptor agonists CHA or NECA were added in increasing concentrations ranging from 0.01 μM to 300 μM. Following the addition of the greatest concentration of adenosine receptor agonists, tissues were washed three times with Krebs’ solution. After the tissues had reached a steadystate resting level, one of the following compounds was added: 10 μM glibenclamide or 10 μM L-NAME, or 10 μM indomethacin. After equilibration for 30–45 min, tissue contraction was induced using phenylephrine and another concentration-response curve to CHA and NECA was measured. Only one pair of compounds was tested with any given arterial segment. Additionally, the effects of glibenclamide,
Results and Discussion Generally, it is difficult to draw reliable conclusions about the physiological or pathological roles of receptors based solely on pharmacological receptor stimulation or inhibition. According to the IUPHAR classification, there are four subtypes of adenosine receptors, A1, A2a, A2b and A3 [5], and many ligands have different affinities for different receptor subtypes. In addition to issues associated with receptor subtype selectivity, most agents have additional non-specific off-target effects making the interpretation of results extremely complicated. CHA and NECA have traditionally been used as stimulatory agents for adenosine receptors. Therefore, we examined their non-specific effects on phenylephrine-induced contraction of rat tail artery. To evaluate the participation of nitric oxide (NO), prostaglandins (PG) and ATP-sensitive K+ channels (KATP) in the CHA and NECA-induced relaxation of the rat tail artery smooth muscle, we used L-NAME (inhibitor of nitric oxide synthase),
Pharmacological Reports, 2009, 61, 330334
331
indomethacin (inhibitor of synthesis of PG) and glibenclamide (blocker of KATP). Among these substances, only L-NAME had a direct effect on phenylephrine-induced contraction of rat tail artery, resulting in a 40% increase in the amplitude of contraction (Fig. 1). In the absence of other drugs, phenylephrine (10 μM) induced tail artery contraction, which was diminished through the addition of increasing concentrations of CHA and NECA (Fig. 2). CHA was a more potent inhibitor, with significantly stronger relaxation effects at concentrations from 3 to 100 μM and a higher pD2 (–log IC50) (Tab. 1). In the presence of 10 μM L-NAME, NECA, which is non-
Tab. 1. Some of parameters showing efficacy and potency of NECA
and CHA to relax precontracted by phenylephrine rat tail artery in the presence of different blockers
Examined compounds NECA CHA
IC50 (μM)
–logIC50 ± SEM
Number of experiments
21.00
4.68 ± 0.15
6
#
6
**
4
**
6
3.50
CHA + indomethacin CHA + glibenclamide
5.46 ± 0.12
7000 210
2.15 ± 0.15 3.68 ± 0.14
p < 0.05: significant difference between CHA and NECA relaxation
effects; * p < 0.05, ** p < 0.01: significant differences in comparison with effects of CHA alone
Ph 10 μM L-NAME Ind. Glib.
% of phenylephrine Emax
160 140 120 100 80 60 40 20
Fig. 1. The effect of L-NAME (inhibitor of nitric oxide synthase), indomethacin (inhibitor of synthesis of PG) and glibenclamide (blocker of K)62) on arterial tension in the presence of 10 µM of phenylephrine. All substances were added separately after achieving steady-state contraction with phenylephrine at a concentration of 10 µM. The amplitude of contraction was measured after a 20-minute incubation. * p < 0.05 significant difference as compared to the control amplitude induced by phenylephrine alone; N = 46 experiments with every substance. Ph phenylephrine; Ind. Indomethacin; Glib. glibenclamide
0
90 80 70 60 50
Fig. 2. The effects of L-NAME, glibenclamide and indomethacin (all at 10 µM) on the maximal relaxation of phenylephrine-precontracted rat tail artery, induced by N6-cyclohexyadenosine (CHA) and 5-N-ethylcarboxamidoadenosine (NECA). * p < 0.05, ** p < 0.01: significant differences relative to the effects of CHA and NECA alone
% of relaxation
40 30 20 10 0 –10 –20 –30 –40 –50
332
NECA
CHA
NECA + L-NAME NECA + GLB
CHA+ GLB CHA + IND
Pharmacological Reports, 2009, 61, 330334
Adenosine receptor agonists and smooth muscle relaxation Katarzyna Kêdzior et al.
specific agonist of A1 and A2a receptors [5], did not exhibit any relaxation effects on the phenylephrinecontracted tail artery. Moreover, maximal contraction amplitude was increased above the baseline level by about 40% (the same effect as with L-NAME alone). Interestingly, glibenclamide, an inhibitor of ATPsensitive K+ channels, significantly decreased the maximal relaxation effect of NECA (Fig. 2). On the other hand, the relaxing effect of CHA on the rat tail artery was strongly diminished by 10 μM indomethacin, a potent cyclooxygenase-1 (COX-1) inhibitor, reducing the maximal relaxation effect by approximately 40% (Fig. 2). Glibenclamide also decreased CHA-induced relaxation, but to a lesser extent than indomethacin. Our results indicate that NECA owes its relaxation effect to nitric oxide. Notably, inhibition of NOS also increased the phenylephrine-induced contractile efficacy, which indicates that nitric oxide participates in the regulation of rat tail artery tonus at rest (Fig. 1). A slight reduction in the maximal relaxation effect of NECA by glibenclamide could be explained by the direct activation of ATP-sensitive K+ channels at a concentration of 100 μM, which is in accordance with previously published data suggesting that the stimulation of A1 receptors can activate KATP channels [14]. However, it is most likely that a certain level of nitric oxide induces the activation of the of ATP-sensitive K+ channels, since we have demonstrated that nitric oxide is responsible for the full relaxing action of NECA. On the other hand, it seems that CHA induces relaxation of the rat tail artery by partial stimulation of the adenosine receptors, as indomethacin decreased the CHA-induced relaxation effect by approximately 40% (suggesting the participation of prostaglandins in relaxation response) and glibenclamide by 20%. This study confirms previous observations that nitric oxide contributes to the vasodilatory effect of adenosine receptor agonists [4, 7, 8, 13, 16]. Also, the results related to indomethacin and CHA interaction are consistent with data that demonstrated a negative effect of indomethacin on CHA-induced negative inotropic action in guinea pig papillary muscle [9]. However, under the present experimental conditions, the non-specific A1/A2a-receptor agonist, NECA, caused relaxation of the rat tail artery exclusively by a nitric oxide-dependent mechanism. This observation is in accordance with other studies that have demonstrated endothelial-dependence for NECA-induced relaxation of porcine coronary artery [1]. These data
suggest that the previously reported effects of NECA thought to be mediated by the stimulation of adenosine A2 receptors (as hypothermia and hypotension in rabbits) are likely due to nitric oxide release [12]. In summary, the present study provides evidence that CHA and NECA possess non-specific mechanism of actions, such as the activation of NOS, COX-1 and ATP-sensitive K+ channels. These effects should be considered when the examined ligands of adenosine receptors are used in different experimental settings.
References: 1. Abebe W, Makujina SR, Mustafa SJ: Adenosine receptor mediated relaxation of porcine coronary artery in presence and absence of endothelium. Am J Physiol, 1994, 266 (5Pt2), H2018–H2025. 2. Conti A, Monopoli A, Gamba M, Borea PA, Ongini E: Effects of selective A1 and A2 adenosine receptor agonists on cardiovascular tissues. Naunyn-Schmiedebergs Arch Pharmacol, 1993, 348, 108–112. 3. Diniz C, Fresco P, Leal S, Gonçalves J: Adenosine receptors involved in modulation of noradrenaline release in isolated rat tail artery. Eur J Pharmacol, 2004, 504, 17–25. 4. Elliott J, Brady FE: Characterization of vasodilatory adenosine receptors in equine digital veins. J Vet Pharmacol Ther, 1998, 21, 74–81. 5. Fredholm BB, Ijzerman AP, Jacobson KA, Klotz KN, Linden J: International Union of Pharmacology XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev, 2001, 53, 527–52. 6. Go³embiowska K, Dziubina A, Kowalska M, Kamiñska K: Paradoxical effects of adenosine receptor ligands on hydroxyl radical generation by L-DOPA in the rat striatum. Pharmacol Rep, 2008, 60, 319–330. 7. Herrera GM, Resta TC, Candelaria JJ, Walker BR: Maintained vasodilatory response to cromakalim after inhibition of nitric oxide synthesis. J Cardiovasc Pharmacol, 1998, 31, 921–929. 8. Hon WM, Khoo HE Ngoi SS, Moochhala S: Effects of adenosine receptor agonists on nitric oxide release in mouse during endotoxemia. Biochem Pharmacol, 1995, 50, 45–47. 9. Kociæ I: Indomethacin antagonizes the effects of adenosine receptor agonists on the contractility and effective refractory period of guinea pig papillary muscle. Pharm Res, 1996, 34, 143–147. 10. Martin PL: Relative agonist potencies of C2-substituted analogues of adenosine: evidence for adenosine A2B receptors in the guinea pig aorta. Eur J Pharmacol, 1992, 216, 235–242. 11. Martin PL, May JM: Identification and functional characterization of A1 and A2 adenosine receptors in the rat vas deferens: a comparison with A1 receptors in guinea pig left atrium and A2 receptors in guinea pig aorta. J Pharmacol Exp Ther, 1994, 269, 1228–1235. Pharmacological Reports, 2009, 61, 330334
333
12. Matuszek M, G¹ga³o I: The effect of N6-cyclohexyladenosine and 5’-N-ethylcarboxamidoadenosine on body temperature in normothermic rabbits. Gen Pharmacol Vasc Syst, 1996, 27, 467–469. 13. Moritoki H, Matsugi T, Takase H, Ueda HA: Evidence for the involvement of cyclic GMP in adenosine-induced, agedependent vasodilation. Br J Pharmacol, 1990, 100, 569–575. 14. Nakhostine N, Lamontagne D: Adenosine contributes to hypoxia-induced vasodilation through ATP-sensitive K+ channel activation. Am J Physiol, 1993, 265, H1289– H1293. 15. Nicholas TE: A perfused tail artery preparation from the rat. J Pharm Pharmacol, 1969, 21, 826–832.
334
Pharmacological Reports, 2009, 61, 330334
16. 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 (5Pt2), H2001–H2006. 17. Stoggall SM, Shaw JS: The coexistence of adenosine A1 and A2 receptors in guinea pig aorta. Eur J Pharmacol, 1990, 190, 329–335.
Received: June 2, 208; in revised form: February 13, 2009.
Copyright © 2009 by Institute of Pharmacology Polish Academy of Sciences
2009, 61, 330334 ISSN 1734-1140
Short communication
Contribution of NO, ATP-sensitive K+ channels and prostaglandins to adenosine receptor agonists-induced relaxation of the rat tail artery Katarzyna Kêdzior, Renata Szczepañska, Ivan Kociæ Department of Pharmacology, Medical University of Gdañsk, Dêbowa 23, PL 80-204 Gdañsk, Poland Correspondence: Ivan Kociæ, e-mail:
[email protected]
Abstract: The mechanism of relaxation in the rat tail artery induced by the adenosine A1 receptor-selective agonist N6-cyclohexyladenosine (CHA, 10 nM–300 μM) and the adenosine A1/A2a receptor agonist 5’-N-ethylcarboxamidoadenosine (NECA, 10 nM–300 μM) has been characterized. To do this, we used a1-receptor agonist phenylephrine to evoke contraction (10 μM), and inhibitors of nitric oxide synthase (L-NAME, 10 μM), ATP-sensitive K+ channels (glibenclamide, 10 μM) and prostaglandin synthesis (indomethacin, 10 μM). CHA and NECA induced relaxation of rat-tail artery by 80% and 70% in a concentration-dependent manner, respectively. The relaxation effect of NECA was completely abolished in the presence of L-NAME, while glibenclamide and indomethacin prevented CHA-induced relaxation of the rat tail artery by approximately 25% and 40%, respectively. Our results indicate that nonspecific effects such nitric oxide and prostaglandins release or the activation of potassium channels significantly contributed to the effects of CHA and NECA. Key words: adenosine receptor agonists, smooth muscle, nitric oxide, prostaglandins, potassium channels
Introduction The role of adenosine in the regulation of smooth muscle contractility has been extensively studied [2]. Special attention has been paid to the role of specific adenosine receptor subtypes A1, A2a and A2b. in the context of noradrenaline release at the synaptic and postsynaptic levels, calcium changes and the contraction of venous and/or arterial smooth muscles [3, 10]. The majority of studies to date have used selective adenosine receptor agonists to determine the potential contribution of particular receptor subtypes in the regulation of different physiological processes [5, 11, 17]. However, little attention was paid to the nonspecific effects of adenosine-receptors ligands [6].
330
Pharmacological Reports, 2009, 61, 330334
Therefore, in this study, we employed inhibitors of nitric oxide synthase, cyclooxygenase enzyme and ATP-sensitive K+ channels to determine whether these proteins were involved in the action of N6-cyclohexyladenosine (CHA) and 5’-N-ethylcarboxamidoadenosine (NECA) substances, often used to stimulate the A1 and A2 receptors.
Materials and Methods Animals and housing
Three-month old male Wistar rats, weighing 190–260 g were used. Animals were maintained at 24°C in a 12-h
Adenosine receptor agonists and smooth muscle relaxation Katarzyna Kêdzior et al.
light-dark cycle with free access to food and water for seven days prior to experimentation. The experimental protocol was approved by the Local Ethic Committee of Medical University of Gdañsk, Poland. Tissue preparation
After pentobarbital (60 mg/kg b.w. ip in pre-heparinized animals) administration, the tail artery was excised and cleaned, according to previously described methods [15]. The proximal region was cut into 3-mm segments that were mounted on two wires placed through the lumen. The mounted arterial segment was then submerged in a tissue bath filled with oxygenated Krebs’ solution at 37°C, pH = 7.4. The composition of the Krebs’ solution was (mM): NaCl, 6.9; KCl, 0.35; CaCl2, 0.28; MgSO4, 0.29; glucose, 2.0; KH2PO4, 0.16; NaHCO3, 2.1; and dextrose, 2.0. Tissue segments were equilibrated for 75 min, stretched using a preload of 0.5 g of weight and the flow was established at 1.6 ml/min with a perfusion pressure of 7.99 kPa. The solution was constantly aerated by carbogen and circulated using the peristaltic pump PP1 (Zalimp, Poland)
L-NAME and indomethacin on resting tension and on phenylephrine-induced contraction of the rat tail artery were examined in a separate series of experiments. Drugs used
All substances were supplied by Sigma (St. Louis, MO, USA) and dissolved in distilled water. Data analysis
The results were expressed as the per cent of contraction amplitude induced by phenylephrine. The means ± SEM are reported and n values indicate the number of animals in each sample group. To quantify pharmacological drug potencies in the presence of different compounds, IC50 values (effective concentration of CHA or NECA to produce 50% of the maximal effect of relaxation or inhibition of contraction induced by phenylephrine) were determined. The relaxation or inhibition effects were compared among groups using one-way analysis of variance (ANOVA) and Newman- Keuls post-hoc test; p < 0.05 was considered significant.
Measurement of vasoconstricting and vasorelaxing responses
Tissue contraction was recorded using Statham P23P6 force transducers (Statham Laboratories, USA) and an Electromanometer EK4 (Farum, Poland) was used to transfer data to the on line recorder TZ 4200 (Laboratorium Pristroje, Praha, Czech Republic). To study the contraction response in the rat tail artery, control concentration-response curves were constructed by adding phenylephrine (10 μM), and depending on the experimental setting, adenosine receptor agonists CHA or NECA were added in increasing concentrations ranging from 0.01 μM to 300 μM. Following the addition of the greatest concentration of adenosine receptor agonists, tissues were washed three times with Krebs’ solution. After the tissues had reached a steadystate resting level, one of the following compounds was added: 10 μM glibenclamide or 10 μM L-NAME, or 10 μM indomethacin. After equilibration for 30–45 min, tissue contraction was induced using phenylephrine and another concentration-response curve to CHA and NECA was measured. Only one pair of compounds was tested with any given arterial segment. Additionally, the effects of glibenclamide,
Results and Discussion Generally, it is difficult to draw reliable conclusions about the physiological or pathological roles of receptors based solely on pharmacological receptor stimulation or inhibition. According to the IUPHAR classification, there are four subtypes of adenosine receptors, A1, A2a, A2b and A3 [5], and many ligands have different affinities for different receptor subtypes. In addition to issues associated with receptor subtype selectivity, most agents have additional non-specific off-target effects making the interpretation of results extremely complicated. CHA and NECA have traditionally been used as stimulatory agents for adenosine receptors. Therefore, we examined their non-specific effects on phenylephrine-induced contraction of rat tail artery. To evaluate the participation of nitric oxide (NO), prostaglandins (PG) and ATP-sensitive K+ channels (KATP) in the CHA and NECA-induced relaxation of the rat tail artery smooth muscle, we used L-NAME (inhibitor of nitric oxide synthase),
Pharmacological Reports, 2009, 61, 330334
331
indomethacin (inhibitor of synthesis of PG) and glibenclamide (blocker of KATP). Among these substances, only L-NAME had a direct effect on phenylephrine-induced contraction of rat tail artery, resulting in a 40% increase in the amplitude of contraction (Fig. 1). In the absence of other drugs, phenylephrine (10 μM) induced tail artery contraction, which was diminished through the addition of increasing concentrations of CHA and NECA (Fig. 2). CHA was a more potent inhibitor, with significantly stronger relaxation effects at concentrations from 3 to 100 μM and a higher pD2 (–log IC50) (Tab. 1). In the presence of 10 μM L-NAME, NECA, which is non-
Tab. 1. Some of parameters showing efficacy and potency of NECA
and CHA to relax precontracted by phenylephrine rat tail artery in the presence of different blockers
Examined compounds NECA CHA
IC50 (μM)
–logIC50 ± SEM
Number of experiments
21.00
4.68 ± 0.15
6
#
6
**
4
**
6
3.50
CHA + indomethacin CHA + glibenclamide
5.46 ± 0.12
7000 210
2.15 ± 0.15 3.68 ± 0.14
p < 0.05: significant difference between CHA and NECA relaxation
effects; * p < 0.05, ** p < 0.01: significant differences in comparison with effects of CHA alone
Ph 10 μM L-NAME Ind. Glib.
% of phenylephrine Emax
160 140 120 100 80 60 40 20
Fig. 1. The effect of L-NAME (inhibitor of nitric oxide synthase), indomethacin (inhibitor of synthesis of PG) and glibenclamide (blocker of K)62) on arterial tension in the presence of 10 µM of phenylephrine. All substances were added separately after achieving steady-state contraction with phenylephrine at a concentration of 10 µM. The amplitude of contraction was measured after a 20-minute incubation. * p < 0.05 significant difference as compared to the control amplitude induced by phenylephrine alone; N = 46 experiments with every substance. Ph phenylephrine; Ind. Indomethacin; Glib. glibenclamide
0
90 80 70 60 50
Fig. 2. The effects of L-NAME, glibenclamide and indomethacin (all at 10 µM) on the maximal relaxation of phenylephrine-precontracted rat tail artery, induced by N6-cyclohexyadenosine (CHA) and 5-N-ethylcarboxamidoadenosine (NECA). * p < 0.05, ** p < 0.01: significant differences relative to the effects of CHA and NECA alone
% of relaxation
40 30 20 10 0 –10 –20 –30 –40 –50
332
NECA
CHA
NECA + L-NAME NECA + GLB
CHA+ GLB CHA + IND
Pharmacological Reports, 2009, 61, 330334
Adenosine receptor agonists and smooth muscle relaxation Katarzyna Kêdzior et al.
specific agonist of A1 and A2a receptors [5], did not exhibit any relaxation effects on the phenylephrinecontracted tail artery. Moreover, maximal contraction amplitude was increased above the baseline level by about 40% (the same effect as with L-NAME alone). Interestingly, glibenclamide, an inhibitor of ATPsensitive K+ channels, significantly decreased the maximal relaxation effect of NECA (Fig. 2). On the other hand, the relaxing effect of CHA on the rat tail artery was strongly diminished by 10 μM indomethacin, a potent cyclooxygenase-1 (COX-1) inhibitor, reducing the maximal relaxation effect by approximately 40% (Fig. 2). Glibenclamide also decreased CHA-induced relaxation, but to a lesser extent than indomethacin. Our results indicate that NECA owes its relaxation effect to nitric oxide. Notably, inhibition of NOS also increased the phenylephrine-induced contractile efficacy, which indicates that nitric oxide participates in the regulation of rat tail artery tonus at rest (Fig. 1). A slight reduction in the maximal relaxation effect of NECA by glibenclamide could be explained by the direct activation of ATP-sensitive K+ channels at a concentration of 100 μM, which is in accordance with previously published data suggesting that the stimulation of A1 receptors can activate KATP channels [14]. However, it is most likely that a certain level of nitric oxide induces the activation of the of ATP-sensitive K+ channels, since we have demonstrated that nitric oxide is responsible for the full relaxing action of NECA. On the other hand, it seems that CHA induces relaxation of the rat tail artery by partial stimulation of the adenosine receptors, as indomethacin decreased the CHA-induced relaxation effect by approximately 40% (suggesting the participation of prostaglandins in relaxation response) and glibenclamide by 20%. This study confirms previous observations that nitric oxide contributes to the vasodilatory effect of adenosine receptor agonists [4, 7, 8, 13, 16]. Also, the results related to indomethacin and CHA interaction are consistent with data that demonstrated a negative effect of indomethacin on CHA-induced negative inotropic action in guinea pig papillary muscle [9]. However, under the present experimental conditions, the non-specific A1/A2a-receptor agonist, NECA, caused relaxation of the rat tail artery exclusively by a nitric oxide-dependent mechanism. This observation is in accordance with other studies that have demonstrated endothelial-dependence for NECA-induced relaxation of porcine coronary artery [1]. These data
suggest that the previously reported effects of NECA thought to be mediated by the stimulation of adenosine A2 receptors (as hypothermia and hypotension in rabbits) are likely due to nitric oxide release [12]. In summary, the present study provides evidence that CHA and NECA possess non-specific mechanism of actions, such as the activation of NOS, COX-1 and ATP-sensitive K+ channels. These effects should be considered when the examined ligands of adenosine receptors are used in different experimental settings.
References: 1. Abebe W, Makujina SR, Mustafa SJ: Adenosine receptor mediated relaxation of porcine coronary artery in presence and absence of endothelium. Am J Physiol, 1994, 266 (5Pt2), H2018–H2025. 2. Conti A, Monopoli A, Gamba M, Borea PA, Ongini E: Effects of selective A1 and A2 adenosine receptor agonists on cardiovascular tissues. Naunyn-Schmiedebergs Arch Pharmacol, 1993, 348, 108–112. 3. Diniz C, Fresco P, Leal S, Gonçalves J: Adenosine receptors involved in modulation of noradrenaline release in isolated rat tail artery. Eur J Pharmacol, 2004, 504, 17–25. 4. Elliott J, Brady FE: Characterization of vasodilatory adenosine receptors in equine digital veins. J Vet Pharmacol Ther, 1998, 21, 74–81. 5. Fredholm BB, Ijzerman AP, Jacobson KA, Klotz KN, Linden J: International Union of Pharmacology XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev, 2001, 53, 527–52. 6. Go³embiowska K, Dziubina A, Kowalska M, Kamiñska K: Paradoxical effects of adenosine receptor ligands on hydroxyl radical generation by L-DOPA in the rat striatum. Pharmacol Rep, 2008, 60, 319–330. 7. Herrera GM, Resta TC, Candelaria JJ, Walker BR: Maintained vasodilatory response to cromakalim after inhibition of nitric oxide synthesis. J Cardiovasc Pharmacol, 1998, 31, 921–929. 8. Hon WM, Khoo HE Ngoi SS, Moochhala S: Effects of adenosine receptor agonists on nitric oxide release in mouse during endotoxemia. Biochem Pharmacol, 1995, 50, 45–47. 9. Kociæ I: Indomethacin antagonizes the effects of adenosine receptor agonists on the contractility and effective refractory period of guinea pig papillary muscle. Pharm Res, 1996, 34, 143–147. 10. Martin PL: Relative agonist potencies of C2-substituted analogues of adenosine: evidence for adenosine A2B receptors in the guinea pig aorta. Eur J Pharmacol, 1992, 216, 235–242. 11. Martin PL, May JM: Identification and functional characterization of A1 and A2 adenosine receptors in the rat vas deferens: a comparison with A1 receptors in guinea pig left atrium and A2 receptors in guinea pig aorta. J Pharmacol Exp Ther, 1994, 269, 1228–1235. Pharmacological Reports, 2009, 61, 330334
333
12. Matuszek M, G¹ga³o I: The effect of N6-cyclohexyladenosine and 5’-N-ethylcarboxamidoadenosine on body temperature in normothermic rabbits. Gen Pharmacol Vasc Syst, 1996, 27, 467–469. 13. Moritoki H, Matsugi T, Takase H, Ueda HA: Evidence for the involvement of cyclic GMP in adenosine-induced, agedependent vasodilation. Br J Pharmacol, 1990, 100, 569–575. 14. Nakhostine N, Lamontagne D: Adenosine contributes to hypoxia-induced vasodilation through ATP-sensitive K+ channel activation. Am J Physiol, 1993, 265, H1289– H1293. 15. Nicholas TE: A perfused tail artery preparation from the rat. J Pharm Pharmacol, 1969, 21, 826–832.
334
Pharmacological Reports, 2009, 61, 330334
16. 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 (5Pt2), H2001–H2006. 17. Stoggall SM, Shaw JS: The coexistence of adenosine A1 and A2 receptors in guinea pig aorta. Eur J Pharmacol, 1990, 190, 329–335.
Received: June 2, 208; in revised form: February 13, 2009.