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Stimulation of the adenosine A3 receptor reverses vascular hyporeactivity after hemorrhagic shock in rats

AIM
To investigate whether adenosine A(3) receptors (A(3)AR) stimulation restore vascular reactivity after hemorrhagic shock through a ryanodine receptor (RyR)-mediated and large conductance calcium-activated potassium (BK(Ca)) channel-dependent pathway.


METHODS
Rat hemorrhagic shock model (40 mmHg) and vascular smooth muscle cell (VSMC) hypoxic model were used. The expression of A(3)AR was determined by Western blot and RT-PCR. The effect of A(3)AR stimulation on RyR-mediated Ca(2+) release in VSMCs was analyzed by the Fura-3/AM loading Ca(2+) imaging. The modulation of vascular reactivity to norepinephrine (NE) by A(3)AR stimulation was monitored by an isolated organ tension instrument.


RESULTS
Decrease of A(3)AR expression is consistent with the loss of vasoreactivity to NE in hemorrhagic shock rats. The stimulation of A(3)AR with a selective agonist, IB-MECA, could partly but significantly restore the vasoreactivity in the rats, and this restorative effect could be counteracted by MRS1523, a selective A(3)AR antagonist. In hypoxic VSMCs, RyR activation by caffeine significantly evoked the rise of [Ca(2+)] compared with the control cells, a phenomenon closely associated with the development of vascular hyporeactivity in hemorrhagic shock rats. The stimulation of A(3)AR with IB-MECA significantly blocked this over activation of RyR-mediated Ca(2+) release. RyR activation by caffeine and BK(Ca) channel activation by NS1619 attenuated the restoration of vasoreactivity to NE resulting from A(3)AR stimulation by IB-MECA after hemorrhagic shock; this attenuation effect could be antagonized by a selective BK(Ca) channel blocker.


CONCLUSION
These findings suggest that A(3)AR is involved in the modulation of vasoreactivity after hemorrhagic shock and that stimulation of A(3)AR can restore the decreased vasoreactivity to NE through a RyR-mediated, BK(Ca) channel-dependent signal pathway.

RyR-mediated Ca 2+ release increases significantly [10] , which might contribute to the activation of the BK Ca channel. Our previous studies suggested that over-activation of the BK Ca channel is closely associated with vascular hyporeactivity after hemorrhagic shock in rats [11,12] , and stimulation of A 3 AR could inhibit RyR-mediated Ca 2+ release [13] . Therefore, we hypothesized that A 3 AR might be involved in the modulation of vasoreactivity through a RyR-mediated Ca 2+ release and BK Ca channel dependent signal pathway after hemorrhagic shock. In this study, we examined: (1) whether A 3 AR is involved in the modulation of vascular reactivity after hemorrhagic shock in rats and (2) whether A 3 AR modulates the vascular reactivity to NE after hemorrhagic shock through a RyR-mediated Ca 2+ release-dependent pathway. To the best of our knowledge, this is the first report to demonstrate that A 3 AR is involved in the modulation of vasoreactivity after hemorrhagic shock and that stimulation of A 3 AR could restore the decreased vasoreactivity to NE through a RyR-mediated, BK Ca channel dependent signal pathway.

Materials and methods
This study was approved by the Research Council and Animal Care and Use Committee of the Research Institute of Surgery, Daping Hospital, Third Military Medical University. All experiments conformed to the guidelines of the ethical use of animals, and all possible efforts were made to minimize animal suffering and to reduce the number of animals used.
Animal model SD rats provided by the Animal Center of the Military Surgery Research Institute of the Third Military Medical University in China (SYXK2002-032), weighing 220±20 g, were anesthetized by pentobarbital sodium (40 mg/kg, ip). Their left femoral arteries were cannulated, connected to a pressure recorder for measuring the mean arterial pressure (MAP), and heparinized with sodium heparin (50 U/kg). The rats were then hemorrhaged, and the MAP was maintained at 40 mmHg for 0, 0.5, 1, 2, and 4 h via a femoral artery catheter.

VSMC preparation
VSMCs were obtained by enzymatic digestion of the abdominal aorta from normal Wistar rats as previously described [14,15] . Briefly, after the endothelium was scraped off, the abdominal aorta was digested in D-Hanks solution, containing collagenase I (2 mg/mL) and bovine serum albumin (BSA) (2 mg/mL), at 37 °C for 30 min. The VSMCs were then cultured in DMEM-F12 with 20% calf bovine serum for 5 to 7 d. Before each experiment, the third to fifth passages of VSMCs were serum-starved for 24 h. On the day of experiment, VSMCs were incubated in a hypoxic culture compartment with an O 2 concentration less than 0.2% for 2 h as previously described and then used for the following experiment.

Western blot of A 3 AR
At different time points (0 min-4 h) after hemorrhage, the abdominal cavities of the rats were opened; the superior mesenteric arteries (SMAs) were obtained, and the protein level of A 3 AR in SMA after hemorrhagic shock was determined by Western blot, as previously described [16] . Briefly, the tissues were put into pre-cooled RIPA (pH 7.6; Hepes 50 mmol/L, NaCl 150 mmol/L, EDTA 1 mmol/L, NP-40 1%, β-glycerophosphate 20 mmol/L, Na 3 VO 4 1 mmol/L, NaF 1 mmol/L, Benzamidine 1 mmol/L, para-nitrophenylphosphate 5 mmol/L, DTT 1 mmol/L, and protein kinase inhibitor cocktail tablets), cut into pieces, and homogenized on ice. Then, the supernatants were collected, thawed on ice for 1 h, and centrifuged at 8000 g for 10 min at 4 °C. Afterward, the supernatants were collected from the lysates, and the protein concentrations were determined by the Bradford method. Aliquots of the lysates (120 µg of protein) were boiled for 5 min and electrophoresed on a 10% SDS-polyacrylamide gel. Blots of the gel were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), which were blocked with 10% nonfat dry milk for 4 h and incubated with a primary A 3 AR antibody (1:1200) for 4 h at room temperature. Then the membranes were further incubated with a horseradish peroxidaseconjugated secondary antibody, developed using an enhanced chemiluminescence Western blotting detection kit (Pierce, Rockland, IL, USA), and exposed to X-ray film. The intensity of the immunoreactive bands was quantified and the results were normalized to β-actin levels.
Reverse transcriptase-polymerase chain reaction (RT-PCR) of A 3 AR At different time points (0 min-4 h) after hemorrhagic shock, the abdominal cavities of the rats were opened and the SMAs were obtained. Then the tissues were gently cut into pieces. One mL of Trizol reagent was added, and total RNA was extracted as per the manufacturer's protocol. RT-PCR was performed as previously described [17] . The PCR conditions for A 3

Experimental protocols
Changes in vascular reactivity to NE in different vasculatures after hemorrhagic shock in rats The artery rings (2-3 mm in length) of the abdominal aorta, SMA stem and SMA branch were prepared from the hemorrhagic shock (40 mmHg for 2 h) rats. Each vascular ring was mounted in a 10 mL organ perfusion system filled with modified Krebs-Henseleit (K-H) solution (in mmol/L: NaCl 119, KCl 4.7, CaCl 2 2.2, NaHCO 3 25, KH 2 PO 4 1.2, MgSO 4 ·7H 2 O 0.45, and glucose 11; pH 7.4) or Ca 2+ -free K-H solution (without Ca 2+ or Mg 2+ , but with 1 mmol/L EGTA in the K-H solution), continuously bubbled with 95% O 2 and 5% CO 2 , and maintained at 37 °C. Each mesenteric arterial or abdominal arterial ring was stretched to a passive force (preload) of about 0.6 g or 0.8 g and equilibrated for 2 h, respectively. The contractile response of each artery ring to NE was recorded by a Powerlab polygraph (AD instrument, Castle Hill, Australia) through a force transducer. NE was added cumulatively from 10 -9 to 10 -5 mol/L. The contractile force of each abdominal or SMA stem artery ring was calculated as the change in tension per g tissue (mN/g), and the contractive force of SMA branch was recorded as the change in tension (mN). The NE cumulative dose-response curve and the maximal contraction induced by 10 -5 mol/L NE (E max ) were used to evaluate the vascular reactivity to NE. There were eight observations in each group.

Involvement of A 3 AR in the modulation of vascular reactivity after hemorrhagic shock in rats
The abdominal artery rings (2-3 mm in length) from the hemorrhagic shock (40 mmHg for 2 h) or the sham-operated control rats were prepared and randomly divided into the following 4 groups (n=8/group): sham-operated control, shock, shock+IB-MECA (4×10 -6 mol/L), and shock+IB-MECA+MRS1523 (10 -7 mol/L). Each vascular ring was mounted in a 10 mL organ perfusion system filled with K-H solution with or without Ca 2+ . The incubation time of IB-MECA was 20 min in the presence or absence of MRS1523. The contractile response of each artery ring to NE was recorded as described above.

Modulation of A 3 AR on the RyR-mediated and the IP 3 R-mediated Ca 2+ release in hypoxic VSMCs
To explore the effect of IB-MECA on RyR-mediated Ca 2+ release in the absence of extracellular Ca 2+ , the following experiments were conducted individually: 1) cells were randomly divided into 4 groups (n=6/group): control, hypoxia, control+NE (10 -6 mol/L), and hypoxia+NE (10 -6 mol/L), to examine the NE-induced changes in [Ca 2+ ] in hypoxic cells; 2) cells were randomly divided into 3 groups (n=6 per group): control+caffeine (10 -3 mol/L), hypoxia+caffeine, and hypoxia+caffeine+IB-MECA (4×10 -6 mol/L), to explore the effects of A 3 AR stimulation on the RyR-mediated Ca 2+ release in hypoxic VSMCs; 3) cells were randomly divided into 3 groups (n=6/group): control+adenophostin A (10 -5 mol/L), hypoxia+adenophostin A, and hypoxia+adenophostin A+IB-MECA (4×10 -6 mol/L), to explore the effects of A 3 AR stimulation on the IP 3 R-mediated Ca 2+ release in hypoxic VSMCs; and 4) in some hypoxia VSMCs, cells were pretreated with thapsigargin (10 -8 mol/L) before incubation with caffeine. The incubation time of each agent was 5 min. The single cell [Ca 2+ ] was measured by a fluorescence indicator, Fura-3/AM, with confocal laser scanning microscopy as previously described [18] . Briefly, Fura-3/AM (10 -5 mol/L) was added and incubated for 30 min at 37 °C, and then the Fura-3/AM-loaded cells were placed on the stage of an inverted fluorescence microscope (Olympus, Japan). With the alternative illumination at 340 nm or 380 nm excitation, fluorescence images were obtained using a silicon-intensified-target video camera (C2400-8, Japan) and digitized by an image processor. The cellular [Ca 2+ ] was represented by mean fluorescence intensity: the single cell mean fluorescence intensity of Ca 2+ =(the sum of the single fluorescence intensity of 1, 2, 3, ..., n)/ (the sum of the area of 1, 2, 3, ..., n).

Involvement of RyR-mediated Ca 2+ release in the modulation of vascular hyporeactivity by A 3 AR stimulation after hemorrhagic shock in rats
We further investigated whether stimulation of A 3 AR with IB-MECA regulates the contractile response to NE associated with RyR-evoked Ca 2+ release. One hundred and twelve abdominal artery rings from the rats subjected to either hemorrhagic shock (40 mmHg, 2 h) or sham-operated control treatment were randomized into the following 7 groups (n=8/group): sham-operated control, shock, shock+caffeine (10 -3 mol/L), shock+ryanodine (10 -5 mol/L), shock+IB-MECA (4×10 -6 mol/L), shock+IB-MECA+caffeine, and shock+IB-MECA+caffeine+ryanodine. The contractile response of each artery ring to NE was recorded both in the K-H solution with 2.2 mmol/L Ca 2+ and in the Ca 2+ -free K-H solution, as previously described. The incubation time and order of each group are described in Table 1.

Involvement of the BK Ca pathway in the modulation of vascular hyporeactivity by A 3 AR stimulation after hemorrhagic shock in rats
Last, we examined whether the stimulation of A 3 AR with IB-MECA regulates the contractile response to NE through a RyR-mediated Ca 2+ release, BK Ca channel dependent pathway. Fifty-six abdominal artery rings from rats subjected to either hemorrhagic shock (40 mmHg, 2 h) or sham-operated control treatment were randomized into the following 7 groups (n=8/group): sham-operated control, shock, shock+IB-MECA (4×10 -6 mol/L), shock+IB-MECA+caffeine npg of each artery ring to NE was recorded as previously described. The incubation time and order of each group are given in Table 1:

Statistical analysis
All data are expressed as mean±SD of "n" observations. The effect of A 3 AR stimulation (with IB-MECA) on vascular reactivity to NE in vitro was assessed by a one-factor analysis of variance, followed by post-hoc Tukey tests. A value of P<0.05 was considered statistically significant, and P<0.01 was considered highly significant.

Results
Decreased vascular reactivity to NE after hemorrhagic shock in rats Three types of vasculatures (abdominal aorta, SMA stem, and SMA branch) were chosen for this study. As shown in Figure  1, in the abdominal aorta, the NE cumulative dose-response curve shifted to the right, and 10 -5 mol/L NE induced a decrease in maximal tension (E max ) from 0.88±0.13 mN/g to 0.39±0.14 mN/g (P<0.01) ( Figure 1A). In SMA stem, the vascular reactivity to NE also decreased, characterized by a shift of the NE cumulative dose-response curve to the right and a decrease of the E max from 0.91±0.18 mN/g to 0.50±0.19 mN/g (P<0.01) ( Figure 1B). In the SMA branch, the vascular reactiv-ity to NE also significantly decreased: the NE cumulative doseresponse curve shifted to the right, and the E max decreased from 11.30±2.10 mN/g to 3.94±1.43 mN/g (P<0.01) ( Figure  1C).

Decreased A 3 AR expression after hemorrhagic shock in rats
The results showed that after hemorrhagic shock (from 30 min to 4 h), there were no significant changes in the A 3 AR mRNA level in the hemorrhagic shock rats or in the controls ( Figure  2A), while the A 3 AR protein level was significantly decreased 2-4 h after hemorrhagic shock ( Figure 2B).
Involvement of A 3 AR in the modulation of vasoreactivity to NE after hemorrhagic shock in rats As shown in Figure 3, in the abdominal aorta, stimulation of A 3 AR with IB-MECA (4×10 -6 mol/L) improved the NEinduced contraction responses of the artery compared with the hemorrhagic shock group, resulting in a shift of the NE cumulative dose-response curve to the left and an increase in the E max of 10 -5 mol/L NE from 0.39±0.14 mN/g to 0.71±0.12 mN/g (P<0.05). In the presence of MRS1523 (10 -7 mol/L), the effects of IB-MECA were significantly counteracted: the NE cumulative dose-response curve was shifted to the right compared with that of the IB-MECA only group, and the E max (induced by 10 -5 mol/L NE) decreased from 0.71±0.12 mN/g to 0.28±0.11 mN/g (P<0.05) (Figure 3).    Figure 4D).

Restoration of the vascular reactivity to NE by A 3 AR stimulation through a RyR-, BK Ca dependent signal pathway
The role of RyR-mediated Ca 2+ release in the decrease of vasoreactivity after hemorrhagic shock was first explored. As shown in Figure 5, the activation of RyR-mediated Ca 2+ release by caffeine further down-regulated the decreased vascular reactivity to NE in hemorrhagic shock rats, characterized by a shift in the NE cumulative dose-response curve to the right and a decrease in the E max (induced by 10 -5 mol/L NE) from 0.39±0.14 mN/g to 0.23±0.11 mN/g in normal K-H solution and from 0.20±0.05 mN/g to 0.05±0.07 mN/g in the Ca 2+ -free K-H solution (P<0.05), respectively. In addition, the inhibition of RyR-mediated Ca 2+ release by ryanodine (10 -5 mol/L) at least partly restored vascular reactivity to NE after hemorrhagic shock: the NE cumulative dose-response curve shifted to the left, and the E max (induced by 10 -5 mol/L NE) increased from 0.39±0.14 mN/g to 0.65±0.11 mN/g in normal K-H solution (P<0.05) and from 0.20±0.05 mN/g to 0.33±0.07 mN/g in the Ca 2+ -free K-H solution (P<0.05), respectively ( Figure 5).   Next, we investigated whether the inhibition of RyRmediated Ca 2+ release by A 3 AR stimulation is involved in the improvement of vasoreactivity to NE in hemorrhagic shock rats. As shown in Figure 6, compared with the IB-MECA treated group, pre-treatment with a RyR agonist, caffeine (10 -7 mol/L), could partly reverse the improvement in vasoreactivity to NE by IB-MECA, as characterized by a significant shift of the NE cumulative dose-response curve to the right and a significant decrease in the E max from 0.71±0.12 mN/g to 0.14±0.12 mN/g (P<0.01) in normal K-H solution and from 0.31±0.06 mN/g to 0.10±0.03 mN/g in the Ca 2+ -free K-H solution, respectively. These effects could be antagonized by a RyR antagonist, ryanodine (10 -5 mol/L): the NE cumulative dose-response curve significantly shifted to the left and the E max increased from 0.14±0.10 mN/g to 0.54±0.18 mN/g (P<0.01) in normal K-H solution and from 0.10±0.03 mN/g to 0.26±0.06 mN/g (P<0.05) in the Ca 2+ -free K-H solution, respectively ( Figure 6).
Last, we examined whether the stimulation of A 3 AR restores vasoreactivity through a RyR-mediated Ca 2+ release, BK Ca channel dependent signal pathway after hemorrhagic shock in rats. Our results showed that pretreatment with a BK Ca opener, NS1619, could partly counteract the restoration of vasoreactivity by IB-MECA, as characterized by a significant shift of NE cumulative dose-response curve to the right and a decrease in the E max from 0.71±0.12 mN/g to 0.25±0.09 mN/g (P<0.01). In addition, a selective BK Ca channel blocker, TEA (0.1 mmol/L), significantly antagonized the further decrease of vascular reactivity to NE induced by NS1619, characterized by a shift of the NE cumulative dose-response curve to the left and an increase in the E max from 0.25±0.09 mN/g to 0.68±0.13 mN/g (P<0.05, Figure 7A), without significant influence on the vascular basal tone (data not shown). TEA (0.1 mmol/L) also counteracted the further reduction of vasoreactivity to NE induced by caffeine: the NE cumulative dose-response curve shifted to the left, and the E max increased from 0.14±0.12 mN/g to 0.72±0.12 mN/g (P<0.05, Figure 7B).

Discussion
The stimulation of A 3 AR has a protective effect against neurodegenerative diseases, myocardial injury [19,20] , and the hypoxia-reoxygen-induced damage of vascular reactivity [21] . However, little is known about whether the stimulation of A 3 AR can improve decreased vascular reactivity after hemorrhagic shock. In this study, we first examined the effect of A 3 AR in decreased vascular reactivity to NE after hemorrhagic shock in rats. We found that the expression of A 3 AR was decreased 2-4 h after hemorrhagic shock (40 mmHg), consistent with the loss of vasoreactivity to NE. IB-MECA, a selective A 3 AR agonist, partly but significantly restored the decreased vasoreactivity in hemorrhagic shock rats; this restorative effect could be antagonized by MRS1523 (a selective A 3 AR antagonist). These results suggest that A 3 AR is involved in the modulation of vasoreactivity, and the stimulation of A 3 AR can at least partly restore vascular reactivity to  There are two types of Ca 2+ release channels located on ER that are closely associated with the modulation of vascular tension: RyR (activated by Ca 2+ increase) and IP 3 R (activated by InsP 3 ) [22,23] . Many reports demonstrated that RyR-mediated Ca 2+ release (also called Ca 2+ spark) activates the BK Ca channel in plasmalemma, leads to VSM membrane hyperpolarization, and negatively regulates vascular tension [24] . Zheng et al reported that RyR-evoked Ca 2+ release is increased in hypoxic SMCs [10] , and our previous studies showed that over-activation of the BK Ca channel plays an important role in the occurrence of vascular hyporeactivity after hemorrhagic shock [12,13] , suggesting that RyR-mediated Ca 2+ release might be related to the development of vascular hyporeactivity after hemorrhagic shock. Because the stimulation of A 3 AR could inhibit RyRmediated Ca 2+ release from the ER, we then explored whether A 3 AR stimulation can reverse the decreased vascular hyporeactivity after hemorrhagic shock through a RyR-mediated Ca 2+ release and BK Ca channel dependent signal pathway.
To clarify the role of RyR-mediated Ca 2+ release in the restoration of vasoreactivity by A 3 AR stimulation, we first used hypoxia-insulted VSMCs to examine the modulation of ER Ca 2+ release by A 3 AR stimulation. Consistent with others' reports, our results showed that RyR-mediated Ca 2+ release is over-activated in hypoxic VSMCs, which might be owing to the increased expression of RyR and the increased sensitivity of RyR to Ca 2+ [25,26] . Furthermore, the over-activation of RyRmediated Ca 2+ release is closely associated with the occurrence of vascular hyporeactivity to NE in hemorrhagic shock rats. The stimulation of A 3 AR significantly antagonized the overactivation of RyR-mediated Ca 2+ release in hypoxic VSMCs, and the restoration of the vasoreactivity to NE by A 3 AR stimulation was partly but significantly counteracted by the activation of RyR-mediated Ca 2+ release by caffeine. These results suggest that RyR-mediated Ca 2+ release is indeed associated with the restoration of vasoreactivity by A 3 AR stimulation after hemorrhagic shock.
RyR-mediated Ca 2+ release contributes to the activation of several Ca 2+ -activated potassium (K Ca ) channels in the nearby sarcolemma, induces membrane hyperpolarization, and leads to the reduction of voltage-dependent Ca 2+ channel activity and vascular smooth muscle relaxation [27,28] . Meanwhile, our previous work showed that over-activation of the BK Ca channel plays an important role in the development of vascular hyporeactivity after hemorrhagic shock. Therefore, we conducted more pharmacological experiments to explore the role of the RyR-mediated, BK Ca channel dependent pathway in the modulation of vascular reactivity by A 3 AR stimulation. We used a selective BK Ca channel opener, NS1619, and a BK Ca channel blocker, TEA (0.1 mmol/L), to explore the role of the BK Ca channel in the restoration of vasoreactivity by A 3 AR stimulation. Our results showed that the selective A 3 AR agonist, IB-MECA, improved vascular reactivity to NE via a RyR-mediated Ca 2+ release and BK Ca channel dependent signal cascade, while the BK Ca selective opener, NS1619, and the RyR activator, caffeine, both counteracted the restoration of the vascular reactivity to NE by IB-MECA; this counteracting effect could be antagonized by 0.1 mmol/L TEA. These results suggest that the stimulation of A 3 AR improves the vascular reactivity to NE through a RyR-mediated Ca 2+ release dependent pathway, in which the BK Ca channel is closely involved.
In a nonpressurized vascular ring exposed to a vasoconstrictor, the frequency of RyR-mediated Ca 2+ release would be low, and vasodilation mechanisms via Ca 2+ spark activation would be blunted. The α1 adrenergic receptor agonist NE strongly inhibits the RyR-mediated Ca 2+ release during vasoconstriction under normal conditions, while in hypoxic VSMCs, RyRmediated Ca 2+ release is over-activated [29] , which might contribute to the over-activation of BK Ca channel. Furthermore, our results suggest that the over-activation of RyR-mediated Ca 2+ release is at least partly involved in the occurrence of vascular hyporeactivity to NE after hemorrhagic shock. The restoration of vasoreactivity by A 3 AR stimulation after hemorrhagic shock is at least partly associated with the inhibition of the over-activation of RyR-mediated Ca 2+ release and BK Ca channel. Because RyR-mediated Ca 2+ release regulates BK Ca channel activity [30] and BK Ca channel is involved in the restoration of the vasoreactivity to NE by A 3 AR stimulation through a RyR-mediated Ca 2+ release dependent pathway, it is rational to conclude that the stimulation of A 3 AR restores vasoreactivity through a RyR-mediated, BK Ca channel dependent signal Figure 7. Involvement of RyR-mediated and BK Ca channel dependent pathway in the modulation of vascular reactivity to NE by A 3 AR stimulation after hemorrhagic shock in rats (mean±SD, n=8) In summary, our results show that A 3 AR is closely involved in the modulation of vascular reactivity after hemorrhagic shock in rats, and stimulation of A 3 AR restores the vascular reactivity to NE after hemorrhagic shock through a RyR-mediated Ca 2+ release and BK Ca channel dependent pathway.