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Am J Physiol Heart Circ Physiol 293: H3685-H3691, 2007. First published October 5, 2007; doi:10.1152/ajpheart.00819.2007
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Protective roles of adenosine A1, A2A, and A3 receptors in skeletal muscle ischemia and reperfusion injury

Jingang Zheng,1 Rubio Wang,1 Edward Zambraski,2 Dan Wu,3 Kenneth A. Jacobson,4 and Bruce T. Liang1

1Pat and Jim Calhoun Cardiology Center, University of Connecticut Health Center, Farmington, Connecticut; 2United States Army Research Institute of Environmental Medicine, Natick, Massachusetts; 3Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut; and 4Molecular Recognition Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland

Submitted 15 July 2007 ; accepted in final form 1 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although adenosine exerts cardio-and vasculoprotective effects, the roles and signaling mechanisms of different adenosine receptors in mediating skeletal muscle protection are not well understood. We used a mouse hindlimb ischemia-reperfusion model to delineate the function of three adenosine receptor subtypes. Adenosine A3 receptor-selective agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IBMECA; 0.07 mg/kg ip) reduced skeletal muscle injury with a significant decrease in both Evans blue dye staining (5.4 ± 2.6%, n = 8 mice vs. vehicle-treated 28 ± 6%, n = 7 mice, P < 0.05) and serum creatine kinase level (1,840 ± 910 U/l, n = 13 vs. vehicle-treated 12,600 ± 3,300 U/l, n = 14, P < 0.05), an effect that was selectively blocked by an A3 receptor antagonist 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS-1191; 0.05 mg/kg). The adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 0.05 mg/kg) also exerted a cytoprotective effect, which was selectively blocked by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.2 mg/kg). The adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680; 0.07 mg/kg)-induced decrease in skeletal muscle injury was selectively blocked by the A2A antagonist 2-(2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e] [1,2,4]triazolo[1,5-C]pyrimidin-5-amine (SCH-442416; 0.017 mg/kg). The protection induced by the A3 receptor was abrogated in phospholipase C-β2/β3 null mice, but the protection mediated by the A1 or A2A receptor remained unaffected in these animals. The adenosine A3 receptor is a novel cytoprotective receptor that signals selectively via phospholipase C-β and represents a new target for ameliorating skeletal muscle injury.

wild-type mice; creatine kinase; phosolipase C

ISCHEMIA AND REPERFUSION can cause significant injury of skeletal muscle, which is the most vulnerable tissue in the extremities (5, 15). Trauma, autogenous skeletal muscle transplantation, surgical incision, vascular clamp application during vascular surgery or musculoskeletal reconstructive surgery, and sustained strenuous exertion can also induce skeletal muscle damage with deleterious systemic consequences (4, 5, 9). Protection of skeletal muscle from ischemia and reperfusion injury is therefore an important therapeutic goal. Although various measures, including a tissue-preserving solution and cold immersion, are used to preserve intact organs and skeletal muscle (18, 34, 38), a more effective method or pharmacological agent to protect skeletal muscle from ischemia-reperfusion injury is needed. Ischemic preconditioning can provide potent protection of the heart (27, 39) as well as the skeletal muscles (7, 8) from ischemia-reperfusion injury. As with cytoprotection of the heart, extracellular adenosine is implicated in mediating the protective effect of preconditioning in skeletal muscle (7, 8, 28). Direct infusion of adenosine can mimic the effect of preconditioning in reducing skeletal muscle injury. Adenosine is an important regulatory agent that exerts its cytoprotective effect via activation of four G protein-coupled receptors: A1, A2A, A2B, and A3 subtypes.

N6-(R-phenyl-2-propyl)adenosine (R-PIA), an adenosine A1 receptor agonist of low selectivity, exerted an anti-ischemic effect in a pig latissimus dorsi muscle flap model (28). The adenosine A1 receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) blocked the protection by adenosine in this model. Although these data suggested a role for the adenosine A1 receptor in mediating protection against ischemia-reperfusion injury in skeletal muscle, it is not known whether other adenosine receptor subtypes also protect skeletal muscle. Activation of the adenosine A3 receptor has been shown to protect the myocardium against ischemia and reperfusion injury (2, 24, 26). However, the activation of the adenosine A3 receptor expressed in rodent mast cells stimulates inflammation (30, 37) with a potentially deleterious effect on skeletal muscle. A systematic investigation of the cytoprotective role of adenosine A1, A2A, and A3 receptors in skeletal muscle is needed, along with a genetic approach and a detailed pharmacological characterization of selective agonists and antagonists.

Our objective was to define the function of various adenosine receptor subtypes in skeletal muscle ischemia and reperfusion injury. We used a mouse hindlimb ischemia-reperfusion injury model and demonstrated for the first time a novel anti-ischemic cytoprotective role of the adenosine A3 receptor. A detailed and specific pharmacological characterization of both A1 and A2A receptors was also carried out. We used phospholipase C (PLC)-β2/β3 knockout mice to determine the signaling role of this enzyme in mediating the cytoprotective role of the adenosine receptor subtypes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mouse hindlimb ischemia and reperfusion model. After 2.5- to 3-mo-old wild-type (WT; C57BL6 strain) or PLC-β2/β3 knockout(in C57BL6 background) mice, each weighing ~23–25 g, were sedated with anesthetic (pentobarbital sodium, 50 mg/kg ip), their right or left hindlimbs (used randomly) were elevated briefly to minimize retained blood before being subjected to ischemia as previously described (1). Ischemia was induced by placement of a constrictor band (Latex O-Rings, Miltex Instruments, York, PA) above the greater trochanter with a McGiveney Hemorrhoidal Ligator (7 in. long; Miltex) according to a modification of a previously described method (1, 11). After 90 min of warm ischemia at 37°C, the constrictor was removed to allow reperfusion for 24 h. The mice were continuously maintained on a 37°C warming pad (Physitemp Instruments, Clifton, NJ) during the reperfusion. After the mice were euthanized by anesthetic overdose, the gastrocnemius muscle was quickly frozen, cut into three slices separated by 2 to 3 mm, and embedded in Shandon Cryomatrix (10% polyvinyl alcohol and 4% polyethylene glycol; Anatomical Pathology, Pittsburgh, PA). Each slice was processed as one 10-µm section on a Thermo Electron/Shandon Cryotome (Anatomical Pathology), fixed in ice-cold acetone, air dried, and washed in phosphate-buffered saline (PBS). Each 10-µm section had seven fields. Gastrocnemius was used because of its high proportion of fast-twitch muscle, which is prone to ischemia and reperfusion injury (1, 11).

Quantification of skeletal muscle injury. Evans blue dye (EBD), prepared as a 1% wt/vol solution to yielding 1 mg of EBD/10 g body wt, was given via a separate intraperitoneal injection 2.5 h before the induction of ischemia. EBD stained only injured muscle, and EBD-positive cells were quantified according to a previously described method (14). The percentage of EBD-positive cells in each field was averaged with those from all seven fields within one 10-µm section. The averaged fraction of EBD-positive cells in each 10-µm section was similar among the three sections. Each 10-µm section was also stained with rabbit polyclonal anti-skeletal muscle actin antibodies (ab15265; Abcam, Cambridge, MA) and goat polyclonal anti-rabbit IgG conjugated with fluorescein isothiocyanate. Sections were mounted, and their cross sections were viewed with fluorescent microscopy (EBD-positive cells via a DM580 band-pass filter 510–560 nm with emission of 590 nm; fluorescein isothiocyanate cells via a DM510 filter of 450–490 nm with emission at 520 nm). Each field was counted at x20 magnification, and their images were captured via the two filters for quantification of muscle injury as follows. Images were acquired and stored as .jpeg files with a Macrofire camera (Macrofire 1.0, Optronics, Goleta, CA). The ImageProPlus Program (version 5.0, Media Cybernetics, Silver Spring, MD) was used for the quantitative determination (31). The percentage of EBD-positive areas was calculated by dividing the area of EBD staining by the total muscle cells, a quantity defined as the total area stained by anti-skeletal muscle actin antibodies and was identical to the total area in each field minus the area not occupied by any cell. The actin-stained area included the EBD-positive area, as shown in superimposed EBD- and actin antibody-stained images. Serum creatine kinase (CK) activity was measured with a previously described procedure (13). The fraction of skeletal muscle staining positive for EBD represented a direct determination of the muscle that was injured, and the serum CK level provided a circulating index of the extent of skeletal muscle injury. Both are established and accepted methods to quantify skeletal muscle injury.

Protocol for administration of adenosine receptor agonist and antagonist. Adenosine receptor agonists [0.07 mg/kg for 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IBMECA) and 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoaden osine (CGS-21680), 0.05 mg/kg for 2-chloro-N6-cyclopentyladen osine (CCPA) and R-PIA], antagonists {0.2 mg/kg for DPCPX, 0.05 mg/kg for 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1, 4-(±)-dihydropyridine-3,5-dicarboxylate (MRS-1191), 0.017 mg/kg for 2-(2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo [4,3-e][1,2,4]triazolo[1,5-C]pyrimidin-5-amine (SCH-442416)}, or vehicle (0.1% DMSO in PBS) was administered in a sterile 0.1-ml volume by intraperitoneal injection 2 h before induction rather than at the onset of ischemia. This protocol allowed time for the absorption of adenosine ligands and for their presence in the circulation before the beginning of ischemia. Previous studies demonstrated that intraperitoneal injection of similar doses of adenosine receptor agonists produced potent pharmacological myocardial protection in the mouse (40, 42). Similar intraperitoneal doses of adenosine receptor antagonists (MRS-1191 and DPCPX) were also given, and only MRS-1191 blocked the myocardial protection afforded by the A3 receptor agonist Cl-IBMECA (41). When both antagonist and agonist were administered, the antagonist was given 30 min before the agonist. Unless otherwise indicated, data are shown as means ± SE. One-way ANOVA followed by posttest Newman-Keuls comparison was used to analyze the statistical significance of differences in more than two groups. P < 0.05 was considered statistically significant.

Materials and chemicals. The adenosine receptor ligands DPCPX, MRS-1191, CGS-21680, CCPA, Cl-IBMECA, and R-PIA were obtained from Sigma Chemicals (St. Louis, MO). The adenosine A2A receptor antagonist SCH-442416 was from Tocris Bioscience (Ellisville, MO).

PLC-β2/β3-deficient mice. PLC-β2/β3 null mice were bred as previously described (21). C57BL6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). All animal experiments were conducted under the guidelines on humane use and care of laboratory animals for research and approved by the Institutional Animal Care and Use Committee of the University of Connecticut Health Center.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Role of adenosine A1 receptors in anti-ischemic skeletal muscle protection. Ischemia followed by reperfusion resulted in significant limb skeletal muscle injury in PBS vehicle-treated mice. The extent of injury was quantified by an increase in the EBD staining of the skeletal myocytes (Fig. 1, A and B). The fraction of total skeletal muscle cross sections that stained positive for EBD was 28 ± 6% (n = 7 mice, means ± SE, Fig. 1B). Administration of the relatively nonselective adenosine receptor agonist R-PIA before ischemia and reperfusion caused a significant reduction in the extent of injury (data not shown). To elucidate the cytoprotective role of different adenosine receptor subtypes, we found that a highly A1 receptor-selective agonist, CCPA, induced a large decrease in the extent of muscle injury (Fig. 1C; EBD-positive area in CCPA-treated mice: 10.8 ± 2%, n = 12 mice vs. PBS vehicle-treated mice: 28 ± 6%, n = 7 mice, P < 0.05). The A1 receptor-selective antagonist DPCPX completely abrogated the cytoprotective response to CCPA (Fig. 1C; DPCPX and CCPA treatment: 22 ± 3.3%, n = 9 mice, P < 0.05 vs. CCPA treatment). The adenosine A3 receptor-selective antagonist MRS-1191 did not affect CCPA-mediated protection (Fig. 1C; MRS-1191 and CCPA treatment: 12.2 ± 2.4%, n = 23 mice, P > 0.05 vs. CCPA treatment), indicating that the adenosine A1 receptor protects skeletal muscle from ischemia-reperfusion injury. The contralateral limb not subjected to ischemia-reperfusion showed no EBD staining (data not shown), indicating an absence of muscle injury in the nonischemic limb.


Figure 1
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  		Fig. 1. Cytoprotective action of adenosine in a quantitative model of mouse hindlimb ischemia-reperfusion (I/R) injury. Adult wild-type (WT) mice were injected with various adenosine ligands, they were subjected to I/R injury, and their skeletal muscle injuries were quantified as described in MATERIALS AND METHODS. A: extent of Evans Blue dye (EBD) staining is shown in mice treated with vehicle (0.1% DMSO in phosphate-buffered saline, pH 7.4, n = 7 mice). The contralateral leg not subjected to I/R showed no EBD uptake. B: the same section was stained with rabbit polyclonal anti-skeletal muscle actin antibodies followed by staining with goat anti-rabbit IgG conjugated with fluorescein isothiocyanate. C: 2-chloro-N6-cyclopentyladenosine (CCPA) decreased the percentage of EBD-positive area (n = 12 mice); this reduction was reversed in mice injected with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) before CCPA treatment (n = 9 mice). Effect of 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS-1191) on CCPA-induced decrease in EBD staining is also shown (n = 23 mice). Average EBD staining (means ± SE) of skeletal muscle sections following treatment with vehicle or adenosine ligand were quantified by blinded observers (n = 8 mice for DPCPX alone and 15 mice for MRS-1191 alone). *P < 0.05 for CCPA or MRS-1191 plus CCPA or N6-(R-phenyl-2-propyl)adenosine (R-PIA) vs. any of the following groups: treatment with PBS, DPCPX, MRS-1191, or CCPA plus DPCPX (Newman-Keuls posttest comparison). P > 0.05 for any comparison between CCPA, MRS-1191 plus CCPA, and R-PIA.

 
A novel anti-ischemic protective role of adenosine A3 receptors in skeletal muscle. Our data demonstrated that the A3 receptor agonist Cl-IBMECA induced a significant reduction in EBD-positive cells (Fig. 2A; Cl-IBMECA treatment: 5.4 ± 2.6%, n = 8 mice, P < 0.05 vs. PBS treatment). This reduction was sensitive to antagonism by MRS-1191 (Fig. 2A; MRS-1191 and Cl-IBMECA treatment: 21.5 ± 3.5%, n = 22 mice, P < 0.05 vs. Cl-IBMECA treatment) but not by DPCPX (Fig. 2A; DPCPX and Cl-IBMECA treatment: 4 ± 1.6%, n = 9 mice, P > 0.05 vs. Cl-IBMECA treatment). Neither MRS-1191 (23 ± 4.4%, n = 15 mice) nor DPCPX (19.3 ± 4.4%, n = 8 mice) alone had any effect on the extent of ischemia-reperfusion-induced skeletal muscle injury.


Figure 2
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  		Fig. 2. Adenosine A2A and A3 receptors mediate a potent anti-ischemic protective response distinct from that mediated by the A1 receptor. Adult WT mice were injected with various adenosine ligands, subjected to I/R injury, and their skeletal muscle injuries were quantified as described in Fig. 1. A: effects on EBD staining following treatment with 2-chloro-N6-(3-iodobenzyl) adenosine-5'-N-methyluronamide (Cl-IBMECA) or 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680) in the presence or the absence of DPCPX, 2-(2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-C]pyrimidin-5-amine (SCH-442416) or MRS-1191 were determined at the indicated combinations. Average EBD staining (means ± SE) of skeletal muscle sections following treatment with vehicle or adenosine ligand is shown. P > 0.05 for comparison between any pair of the following groups: Cl-IBMECA (n = 8 mice), DPCPX plus Cl-IBMECA (n = 9), CGS-21680 (n = 9), CGS-21680 plus MRS-1191 (n = 8), or CGS-21680 plus DPCPX (n = 10). *P < 0.05 for Cl-IBMECA, DPCPX plus Cl-IBMECA, CGS-21680, or CGS-21680 plus MRS-1191 vs. any of the following groups: PBS, MRS-1191 (15 mice), CGS-21680 plus SCH-442416 (n = 5 mice), or MRS-1191 plus Cl-IBMECA (n = 22) in a Newman-Keuls posttest comparison. B: adenosine receptor agonists and antagonists were administered as in the experiments described in Fig. 1. Creatine kinase (CK) was used as a marker of muscle injury. A3 receptor agonist Cl-IBMECA caused a reduction in the serum CK, as did the A1 receptor agonist CCPA or the A2A receptor agonist CGS-21680 (one-way ANOVA and posttest Newman-Keuls comparison, *P < 0.05 for Cl-IBMECA, CCPA, or CGS-21680 vs. any of the others). Data are means ± SE of 14 mice (PBS), 13 mice (Cl-IBMECA), 15 mice (MRS-1191 plus Cl-IBMECA), 9 mice (DPCPX plus CCPA), 11 mice (CCPA), 10 mice (CGS-21680), and 8 mice (CGS-21680 plus SCH-442416).

 
Mice pretreated with the adenosine A2A receptor-selective agonist CGS-21680 showed reduced muscle injury compared with PBS vehicle-treated animals (6.6 ± 3.5%, n = 9 mice, P < 0.05 vs. PBS treatment; Fig. 2A). The protective effect of CGS-21680 was attenuated by DPCPX (DPCPX and CGS-21680 treatment: 14.7 ± 2.3%, n = 10 mice, P > 0.05 vs. DPCPX alone). The A3 antagonist MRS-1191 could not inhibit the CGS-21680-induced skeletal muscle protection (MRS-1191 and CGS-21680 treatment: 2.4 ± 1.25%, n = 8 mice, P < 0.05 vs. MRS-1191 alone). The adenosine A2A receptor-selective antagonist SCH-442416 completely abrogated the CGS-21680-induced protection (Fig. 2A). Animals treated with SCH-442416 and CGS-21680 showed significantly larger EBD-positive areas (26 ± 4%, n = 5 mice, means ± SE) than mice treated with CGS-21680 alone (P < 0.05).

We measured CK levels as another method to quantify skeletal muscle injury induced by ischemia and reperfusion. Cl-IBMECA, CGS-21680, and CCPA were able to reduce these levels when each agonist was administered individually before ischemia and reperfusion (Fig. 2B). CK in Cl-IBMECA-treated mice was 1,840 ± 910 U/L, n = 13 mice. In CCPA-treated mice, CK was 2,340 ± 710 U/L, n = 11 mice. CK in CGS-21680-treated mice was 838 ± 243 U/L, n = 10 mice (P < 0.05 for any agonist vs. vehicle-treated mice, which had a serum CK level of 12,600 ± 3,300 U/L, n = 14 mice). The protection against CK release induced by A1, A2A, or A3 receptors was blocked by an antagonist of each adenosine receptor subtype. In mice treated with MRS-1191 and Cl-IBMECA, serum CK was 14,400 ± 2,900 U/L (n = 15 mice, P < 0.05 vs. Cl-IBMECA alone). Serum CK in mice treated with DPCPX plus CCPA was 11,300 ± 2,200 U/L (n = 9 mice, P < 0.05 vs. CCPA alone). CK in mice treated with SCH-442416 plus CGS-21680 was 15,180 ± 4,420 U/L, n = 8 mice. The data obtained on serum CK activity, derived from the same method of agonist and antagonist administration in the same ischemia-reperfusion injury model, complement those obtained through EBD staining.

Specificity of protection induced by each adenosine receptor subtype. The specificity by which each adenosine receptor agonist mediates its protective response was further illustrated by testing the effect of the agonist in PLC-β2/β3 null mice. Cl-IBMECA-induced protection was completely abrogated in PLC-β2/β3 null mice (Cl-IBMECA treatment: 22 ± 3.6%, n = 8 mice, P > 0.05 vs. PBS treatment: 23.4 ± 4%, n = 9 mice; Fig. 3, A–C), whereas protection induced by CCPA was unaffected (CCPA treatment: 8.5 ± 2.5%, n = 10 mice, P < 0.05 vs. PBS treatment). PLC-β2/β3 deficiency alone had no effect on ischemia-reperfusion-induced skeletal muscle injury (PBS vehicle-treated WT mice had 28 ± 6% EBD-positive area, n = 7; PLC-β2/β3 null mice had 23.4 ± 4% EBD-positive area, n = 9, P > 0.05; Figs. 1C and 3C, respectively).


Figure 3
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  		Fig. 3. The adenosine A3 receptor signals through PLC-β2/β3 to cause its anti-ischemic skeletal muscle protective response. Adult PLC-β2/β3 null mice were injected with vehicle (n = 9 mice), Cl-IBMECA (n = 8 mice), CGS-21680 (n = 6 mice), or CCPA (n = 10 mice); they were subjected to I/R, and the extent of skeletal muscle injury was subsequently quantified as in WT mice. In PLC-β2/β3 null mice not subjected to I/R, skeletal muscles did not show any EBD staining. A: a typical EBD staining in a vehicle-injected PLC-β2/β3 null mouse following I/R is shown. The extent of EBD staining was similar to that obtained in vehicle-injected WT mice after I/R (see Fig. 1C, P > 0.05). B: a representative EBD staining in a Cl-IBMECA-treated mouse is shown. C: treatment with CCPA or CGS-21680 reduced EBD staining, but Cl-IBMECA did not. Average EBD staining (means ± SE) of skeletal muscle sections from PLC-β2/β3 null mice following treatment with vehicle or adenosine ligand is shown. *P < 0.05, CCPA, CGS-21680 vs. either PBS or Cl-IBMECA. P > 0.05, PBS vs. Cl-IBMECA; P > 0.05, CCPA vs. CGS-21680.

 
The A2A agonist CGS-21680 was also able to cause cytoprotection in PLC-β2/β3 null mice (CGS-21680 treatment: 4 ± 1.1%, n = 6 PLC-β2/β3 null mice, P < 0.05 vs. PBS treatment: 23.4 ± 4%, n = 9 PLC-β2/β3 null mice; Fig. 3C). Because the A2A receptor is coupled to stimulation of adenylyl cyclase activity and cAMP accumulation, it was not unexpected that the absence of PLC-β2/β3 had no effect on the cytoprotective effect of the A2A receptor. The cytoprotective action of adenosine A2A receptors in skeletal muscle is independent of and separate from the salutary effect mediated by adenosine A1 or A3 receptors in that tissue.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Ischemia and reperfusion of the skeletal muscle can cause significant injury with deleterious consequences. Effective therapies that reduce such injury will have significant benefits in treatment of trauma, autogenous skeletal muscle transplantation, and vascular and musculoskeletal reconstructive surgery. As with anti-ischemic myocardial protection, adenosine and its receptors have been implicated in protecting the skeletal muscle against ischemia and reperfusion injury. The present study demonstrated for the first time that the adenosine A3 receptor can induce potent cytoprotection of the skeletal muscle against ischemia and reperfusion injury. The adenosine A3 receptor, but not the A1 or A2A receptor, signals via PLC-β2/β3 to achieve its skeletal muscle protective effect.

Several lines of evidence clearly delineate the cytoprotective role of adenosine A1, A2A, and A3 receptors. The highly A1 receptor-selective agonist CCPA decreased skeletal muscle ischemia and reperfusion injury. The protective effect was blocked only by the A1 receptor-selective antagonist DPCPX but not by the A3 receptor-selective antagonist MRS-1191. Conversely, the A3 receptor-selective agonist Cl-IBMECA reduced skeletal muscle injury, and this protective effect, although insensitive to blockade by the A1 receptor antagonist DPCPX, was completely abrogated by the A3 receptor antagonist MRS-1191, which was shown to antagonize the adenosine A3 receptor in mice (3, 38, 39). Similarly, the cytoprotective effect of A2A receptor agonist CGS-21680 was selectively blocked by its antagonist SCH-442416 and was insensitive to the A3 antagonist MRS-1191. The A1 antagonist DPCPX, at the current dosage, was able to attenuate the CGS-21680-induced skeletal muscle protection. Several explanations are possible. Given the interaction between A1 and A2A receptors, it is possible that A2A receptor-mediated effect could be potentiated by A1 receptor activation. A positive interaction between adenosine A1 and A2A receptors was recently demonstrated in rat heart (23). Since DPCPX could inhibit the protective effect of CGS-21680 in the current skeletal muscle ischemia-reperfusion injury model, it is possible that A1 receptor activation contributed to the CGS-21680-induced skeletal muscle protection. Recent evidence suggests that protein kinase C (PKC) activation can potentiate the adenosine A2B receptor signaling during reperfusion in the heart (22). Thus another possible explanation is that PKC activation, induced by adenosine A1 receptor, may also increase the responsiveness of the adenosine A2A receptor signaling during reperfusion in skeletal muscle. Overall, the present data provided detailed characterization of antagonists and agonists associated with A1, A2A, and A3 receptors in the current in vivo model of skeletal muscle ischemia-reperfusion injury. The study confirmed their selectivity at each adenosine receptor subtype and indicates that the cytoprotection afforded by each receptor agonist was due to activation of that specific receptor.

PLC-β2/β3 deficiency selectively abrogated the protective effect of A3 receptor agonist Cl-IBMECA and had no effect on CCPA- or CGS-21680-induced protection. It was unlikely that PLC-β2/β3 deficiency affected the bioavailability or pharmacokinetic properties of Cl-IBMECA for the following reason. CCPA and CGS-21680 have similar size and molecular weights as Cl-IBMECA. Both CCPA and CGS-21680 were fully capable of protecting against skeletal muscle injury in the PLC-β2/β3 knockout mice. In WT mice, CGS-21680 induced anti-ischemic skeletal muscle protection in a manner that was insensitive to blockade by MRS-1191 but was completely abolished by the A2A receptor-selective antagonist SCH-442416. The protective effect of CGS-21680 remained unaffected and intact in PLC-β2/β3 null mice. The exact bioavailability or pharmacokinetic property of Cl-IBMECA in WT or PLC-β knockout mice remains to be determined.

Although PLC-β2/β3 is not involved in mediating the protective effect of A1 or A2A receptors in the skeletal muscle, ATP-sensitive K+ channels appear to be an important effector mechanism for the anti-ischemic effect of the A1 receptor (28). The adenosine A2A receptor serves an important nonredundant role in suppressing immune and lymphoid cells and thus in protecting against inflammatory tissue damage (25, 32, 33). Since activation of adenosine A2A receptors on CD4+ T cells mediated potent protection against renal ischemia-reperfusion injury (12), it is possible that the same mechanism is also responsible for its protection in the skeletal muscle in vivo. Activation of the A3 receptor in rodent immune cells such as mast cells is proinflammatory and may be damaging (10, 20). A genetic absence or antagonism of adenosine A3 receptors augmented an increase in coronary flow or hypotension mediated by adenosine or an A2A receptor agonist (36, 43), pointing to a vasoconstrictive role of the vascular A3 receptor. Activated mast cells and neutrophils mediate skeletal muscle ischemia-reperfusion injury (6, 16, 17). The present data could not determine whether the adenosine A3 receptor-mediated protection was due to direct activation of A3 receptors on the skeletal muscle or the result of an anti-inflammatory action of A3 receptors on the immune cells. EBD is a dye that accumulates in injured tissues as a result of an increase in vascular permeability (35) and a disruption of sarcolemmal integrity of the tissue (such as muscle) supplied by the vasculature. It is possible that a decrease in EBD staining was due to a decrease in vascular permeability induced by one or all of the adenosine receptor subtypes studied. Differentiating a protective effect of adenosine receptor subtypes at the levels of vasculature, circulating immune cells, and skeletal muscle is needed. Bone marrow transplant from adenosine receptor knockout mice, or possible creation of vascular or skeletal muscle specific knockout of adenosine receptor subtypes, would provide a more definitive answer to this question.

That the adenosine A3 receptor exerts a potent cytoprotective effect in mouse skeletal muscle is consistent with its cardioprotective action in the mouse heart (19). Although PLC is currently shown to have an important role in mediating the A3 effect in skeletal muscle, a previous study suggested that PLD, but not PLC, mediated the cardioprotective effect of adenosine A3 receptors in chick embryo ventricular myocytes (29). The reasons for this apparent difference are not clear; however, several plausible explanations are offered. First, species and age differences (chick embryos vs. adult mouse) may be important. Second, the coupling of adenosine A3 receptors to PLC vs. PLD may be different in skeletal than in cardiac muscles. Third, our skeletal muscle ischemia-reperfusion injury preparation was an in vivo and intact animal model, whereas the model used by Parsons et al. (29) was an isolated cell culture model. The present gene knockout approach rendered all cells completely deficient in PLC-β2/β3, including skeletal muscle and circulating immune cells capable of mediating anti-and proinflammatory. It is possible that PLC mediated an anti-inflammatory effect of A3 receptors on circulating immune cells. In this scenario, knockout of PLC would eliminate the anti-inflammatory effect of A3 receptors on immune cells and thus abrogated their cytoprotective effect on skeletal muscles.

The combined use of receptor pharmacological tools and a gene ablation approach delineated, for the first time, a distinct anti-ischemic protective role of adenosine A1, A2A, and A3 receptor subtypes in skeletal muscle. Although both adenosine A1 and A2A receptors have shown anti-ischemic protective properties, agonists at either receptor caused pronounced decreases in blood pressure or heart rate. Our data provide convincing evidence that the adenosine A3 receptor is a novel cytoprotective receptor in skeletal muscle. Because the A3 receptor agonist is not associated with cardiac or hemodynamic depression (2), the A3 receptor represents a potential therapeutic target because of its ability to ameliorate skeletal muscle injury.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by a Department of Defense Grant W81XWH-05-1-0060 (to B. T. Liang) and by intramural funds from National Institute of Diabetes and Digestive and Kidney Diseases (to K. A. Jacobson).


   ACKNOWLEDGMENTS
 
The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. We thank Dr. Kevin Campbell for useful comments and advice.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. T. Liang, Pat and Jim Calhoun Cardiology Ctr., MC-3946, Univ. of Connecticut Health Ctr., 263 Farmington Ave., Farmington, CT 06030 (e-mail: bliang{at}uchc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abonia JP, Friend DS, Austen WG Jr, Moore FD Jr, Carroll MC, Chan R, Afnan J, Humbles A, Gerard C, Knight P, Kanaoka Y, Yasuda S, Morokawa N, Austen KF, Stevens RL, Gurish MF. Mast cell protease 5 mediates ischemia-reperfusion injury of mouse skeletal muscle. J Immunol 174: 7285–7291, 2005.[Abstract/Free Full Text]
  2. Auchampach JA, Ge ZD, Wan TC, Moore J, Gross GJ. A3 adenosine receptor agonist IB-MECA reduces myocardial ischemia-reperfusion injury in dogs. Am J Physiol Heart Circ Physiol 285: H607–H613, 2003.[Abstract/Free Full Text]
  3. Avila MY, Stone RA, Civan MM. Knockout of A3 adenosine receptors reduces mouse intraocular pressure. Invest Ophthalmol Vis Sci 43: 3021–3026, 2002.[Abstract/Free Full Text]
  4. Beyersdorf F, Unger A, Wildhirt A, Kretzer U, Deutschlander N, Kruger S, Matheis G, Hanselmann A, Zimmer G, Satter P. Studies of reperfusion injury in skeletal muscle: preserved cellular viability after extended periods of warm ischemia. J Card Surg 32: 664–676, 1991.[Web of Science]
  5. Blaisdell FW. The pathophysiology of skeletal muscle ischemia and perfusion syndrome: a review. Cardiovasc Surg 10: 620–630, 2002.[CrossRef][Web of Science][Medline]
  6. Bortolotto SK, Morrison WA, Han XL, Messina A. Mast cells play a pivotal role in ischemia reperfusion injury to skeletal muscle. Lab Invest 84: 1–9, 2004.[CrossRef]
  7. Bushell AJ, Klenerman L, Taylor S, Davies H, Grierson I, Helliwell TR, Jackson MJ. Ischaemic preconditioning of skeletal muscle. 1. Protection against the structural changes induced by ischemic/reperfusion injury. J Bone Joint Surg. 84: 1184–1188, 2002.[CrossRef]
  8. Bushell AJ, Klenerman L, Davies H, Grierson I, McArdle A, Jackson MJ. Ischaemic preconditioning of skeletal muscle. 2. Investigation of the potential mechanisms involved. J Bone Joint Surg. 84: 1189–1193, 2002.[Abstract/Free Full Text]
  9. Carrol CMA, Carrole SM, Overgoor MLE, Tobin G, Barker JH. Acute ischemic preconditioning of skeletal muscle prior to flap elevation augments muscle-flap survival. Plast Reconstr Surg 100: 58–65, 1997.[Web of Science][Medline]
  10. Cerniway RJ, Yang Z, Jacobson MA, Linden J, Mathern GP. Targeted deletion of A3 adenosine receptors improves tolerance to ischemia-reperfusion injury in mouse myocardium. Am J Physiol Heart Circ Physiol 281: H1751–H1758, 2001.[Abstract/Free Full Text]
  11. Chan RK, Verna N, Afnan J, Zhang M, Ibrahim S, Carroll MC, Moore FD Jr. Attenuation of skeletal muscle reperfusion injury with intravenous 12 amino acid peptides that bind to pathogenic IgM. Surgery 139: 236–243, 2006.[CrossRef][Web of Science][Medline]
  12. Day YJ, Huang L, Ye H, Li L, Linden J, Okusa MD. Renal ischemia-reperfusion injury and adenosine A2A receptor-mediated tissue protection: the role of CD4+ T cells and IFN-g. J Immunol 176: 3108–3114, 2006.[Abstract/Free Full Text]
  13. Duclos F, Straub V, Moore SA, Venzke DP, Hrstka RF, Crosbie RH, Durbeej M, Lebakken CS, Ettinger AJ, van der Meulen Holt J, KH, Lim LE, Sanes JR, Davidson BL, Faulkner JA, Williamson R, Campbell KP. Progressive muscular dystrophy in {alpha}-sarcoglycan-deficient mice. J Cell Biol 142: 1461–1471, 1998.[Abstract/Free Full Text]
  14. Durbeej M, Sawatzki SM, Barresi R, Schmainda KM, Allamand V, Michele DE, Campbell KP. Gene transfer establishes primacy of striated vs. smotth muscle sarcoglycan complex in limb-girdle muscular dystrophy. Proc Natl Acad Sci USA 100: 8910–8915, 2003.[Abstract/Free Full Text]
  15. Ecker P, Schnackerz K. Ischemic tolerance of human skeletal muscle. Ann Plast Surg 26: 77–84, 1991.[CrossRef][Web of Science][Medline]
  16. Fielding RA, Manfredi TJ, Ding W, Fiatarone MA, Evans WJ, Cannon JG. Acute phase response in exercise. III. Neutrophil and IL-1β accumulation in skeletal muscle. Am J Physiol Regul Integr Comp Physiol 265: R166–R172, 1993.[Abstract/Free Full Text]
  17. Formigli L, Lombardo LD, Adembri C, Brunelleschi S, Ferrari E, Novelli GP. Neutrophils and mediators of human skeletal muscle ischemia-reperfusion syndrome. Hum Pathol 23: 627–634, 1992.[CrossRef][Web of Science][Medline]
  18. Gallin JI, Snyderman R. Inflammation: Basic Principles and Clinical Correlations, edited by Gallin JL, Snyderman R, Fearon DT, Haynes BF, and Nathan C. Philadelphia, PA: Lippincott Willliams & Wilkins, 1999, p. 1047–1061.
  19. Ge ZD, Peart JN, Kreckler LM, Wan TC, Jacobson MA, Gross GJ, Auchampach JA. Cl-IBMECA [2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide] reduces ischemia/reperfusion injury in mice by activating the A3 adenosine receptor. J Pharmacol Exp Ther 319: 1200–1210, 2006.[Abstract/Free Full Text]
  20. Harrison Cerniway GJ, RJ, Peart J, Berr SS, Ashton K, Regan S, Mathern PG, Headrick JP. Effects of A3 adenosine receptor activation and gene knockout in ischemic-reperfused mouse heart. Cardiovasc Res 53: 147–155, 2002.[Abstract/Free Full Text]
  21. Jiang H, Kuang Y, Wu Y, Smrcka A, Simon MI, Wu D. Pertussis toxin-sensitive activation of phospholipase C by the C5a and fMet-Leu-Phe receptors. J Biol Chem 271: 13430–13434, 1996.[Abstract/Free Full Text]
  22. Kuno A, Critz SD, Cui L, Solodushko V, Yang XM, Krahn T, Albrecht B, Philipp S, Cohen MV, Downey JM. Protein kinase C protects preconditioned rabbit hearts by increasing sensitivity of adenosine A2b-dependent signaling during early reperfusion. J Mol Cell Cardiol 43: 262–271, 2007.[CrossRef][Web of Science][Medline]
  23. Lasley RD, Kristo G, Keith BJ, Mentzer RM. The A2a/A2b receptor antagonist ZM-241385 blocks the cardioprotective effect of adenosine agonist pretreatment in in vivo rat myocardium. Am J Physiol Heart Circ Physiol 292: H426–H431, 2007.[Abstract/Free Full Text]
  24. Liang BT, Jacobson KA. A physiological role of adenosine A3 receptor: sustained cardioprotection. Proc Natl Acad Sci USA 95: 6995–6999, 1998.[Abstract/Free Full Text]
  25. Lukashev D, Ohta A, Apasov S, Chen JF, Sitkovsky M. Cutting edge: Physiologic attenuation of proinflammatory transcription by the Gs protein-coupled A2A adenosine receptor in vivo. J Immunol 173: 21–24, 2004.[Abstract/Free Full Text]
  26. Maddock HL, Mocanu MM, Yellon DM. Adenosine A3 receptor activation protects the myocardium from reperfusion/reoxygenation injury. Am J Physiol Heart Circ Physiol 283: H1307–H1313, 2002.[Abstract/Free Full Text]
  27. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74: 1124–1136, 1986.[Abstract/Free Full Text]
  28. Pang CY, Neligan P, Zhong A, He W, Xu H, Forrest CR. Effector mechanism of adenosine in acute ischemic preconditioning of skeletal muscle against infarction. Am J Physiol Regul Integr Comp Physiol 273: R887–R895, 1997.[Abstract/Free Full Text]
  29. Parsons M, Young L, Lee JE, Jacobson KA, Liang BT. Distinct cardioprotective effects of adenosine mediated by differential coupling of receptor subtypes to phospholipases C and D. FASEB J 14: 1423–1431, 2000.[Abstract/Free Full Text]
  30. Ramkumar V, Stiles GL, Beaven MA, Ali H. The A3 adenosine receptor is the unique adenosine receptor which facilitates release of allergic mediators in mast cells. J Biol Chem 268: 16887–16890, 1993.[Abstract/Free Full Text]
  31. Shen JB, Cronin C, Sonin D, Joshi BV, Nieto MG, Harrison D, Jacobson KA, Liang BT. P2X purinergic receptor-mediated ionic current in cardiac myocytes of calsequestrin model of cardiomyopathy: implications for the treatment of heart failure. Am J Physiol Heart Circ Physiol 292: H1077–H1084, 2007.[Abstract/Free Full Text]
  32. Sitkovsky MV, Lukashev D, Apasov S, Kojima H, Koshiba M, Caldwell C, Ohta A, Thiel M. Physiological control of immune response and inflammatory tissue damage by hypoxia-inducible factors and adenosine A2A receptors. Annu Rev Immunol 22: 657–682, 2004.[CrossRef][Web of Science][Medline]
  33. Sitkovsky M, Lukashev D. Regulation of immune cells by local-tissue oxygen tension: HIF1{alpha} and adenosine receptors. Nat Rev Immunol 5: 712–721, 2005.[CrossRef][Web of Science][Medline]
  34. Southard JH, Belzer FO. Organ preservation. Annu Rev Med 46: 235–247, 1995.[CrossRef][Web of Science][Medline]
  35. Souza DG, Cara DC, Cassali GD, Coutinho SF, Silveira MR, Andrade SP, Poole SP, Teixeira MM. Effects of the PAF receptor antagonist UK74505 on local and remote reperfusion injuries following ischemia of the superior mesenteric artery in the rat. Br J Pharmacol 131: 1800–1808, 2000.[CrossRef][Web of Science][Medline]
  36. Talukder Morrison MA, RR, Jacobson MA, Jacobson KA, Ledent C, Mustafa SJ. Targeted deletion of adenosine A3 receptors augments adenosine-induced coronary flow in isolated mouse heart. Am J Physiol Heart Circ Physiol 282: H2183–H2189, 2002.[Abstract/Free Full Text]
  37. Tilley SL, Wagoner VA, Salvatore CA, Jacobson MA, Koller BH. Adenosine and inosine increase cutaneous vasopermeability by activating A3 receptors on mast cells. J Clin Invest 105: 361–367, 2000.[Web of Science][Medline]
  38. Tsuchida T, Kato T, Yamaga M, Ikebe K, Oniki Y, Irie H, Takagi K. The effect of perfusion with UW solution on the skeletal muscle and vascular endothelial exocrine function in rat hindlimbs. J Surg Res 110: 266–271, 2003.[CrossRef][Web of Science][Medline]
  39. Yellon DM, Downey JM. Preconditioning the myocardium: from cellular physiology to clinical cardiology. Physiol Rev 83: 1113–1151, 2003.[Abstract/Free Full Text]
  40. Zhao TC, Hines DS, Kukreja RC. Adenosine-induced late preconditioning in mouse hearts: role of p38 MAP kinase and mitochondrial KATP channels. Am J Physiol Heart Circ Physiol 280: H1278–H1285, 2001.[Abstract/Free Full Text]
  41. Zhao TC, Kukreja RC. Late preconditioning elicited by activation of adenosine A3 receptor in heart: role of NF-kB, iNOS and mitochondrial KATP channel. J Mol Cell Cardiol 34: 263–277, 2002.[CrossRef][Web of Science][Medline]
  42. Zhao TC, Kukreja RC. Protein kinase C-{delta} mediates adenosine A3 receptor-induced delayed cardioprotection in mouse. Am J Physiol Heart Circ Physiol 285: H434–H441, 2003.[Abstract/Free Full Text]
  43. Zhao Z, Makaritsis K, Francis CE, Gavras H, Ravid K. A role for the A3 adenosine receptor in determining tissue levels of cAMP and blood pressure: studies in knock-out mice. Biochim Biophys Acta 1500: 280–290, 2000.[Medline]




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