Preconditioning of Bovine Endothelial Cells
The Protective Effect Is Mediated by an Adenosine A2 Receptor Through a Protein Kinase C Signaling Pathway
Abstract We tested the hypothesis that anoxic preconditioning could protect coronary endothelial cells against anoxic and reoxygenation injury and that this preconditioning effect could be mediated by an adenosine A2 receptor via the protein kinase C (PKC) pathway. Cells were preconditioned with 10-minute anoxia and 10-minute reoxygenation and were then subjected to anoxia for 60 minutes, followed by 120 minutes of reoxygenation. In some groups, the preconditioning effect was prevented by 8-sulfophenyltheophylline (SPT [50 μmol/L], a nonselective adenosine receptor antagonist) or calphostin C (100 nmol/L, a PKC inhibitor). In other groups, 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS-21680 [20 nmol/L], an adenosine A2 receptor agonist), R-(−)-N6-(2-phenylisopropyl)-adenosine (R-PIA [50 nmol/L], an adenosine A1 receptor agonist), or 4β-phorbol 12-myristate 13-acetate (PMA [100 nmol/L], a PKC activator) was given as a pretreatment to mimic the preconditioning effect. Endothelial cells were also pretreated with 100 nmol/L calphostin C to confirm whether inhibition of PKC can block the effects of adenosine A2 receptor activation by CGS-21680 on anoxia and reoxygenation injury. Preconditioning reduced LDH release, increased adenosine release, promoted translocation of PKC from cytosol to membrane, increased cell viability, and preserved ATP content and cell morphology. Pretreatment with either CGS-21680 or PMA resulted in protection similar to that seen with anoxic preconditioning. The protection was totally abolished by SPT or calphostin C. The results suggest that (1) preconditioning protects coronary endothelial cells against anoxia and reoxygenation injury, (2) the protection is probably mediated by activation of adenosine A2 receptors through the PKC pathway, and (3) the preservation of endothelial cells may be one of the mechanisms of myocardial preconditioning.
Ischemic preconditioning has been extensively studied in whole hearts. Recently, preconditioning has been characterized in isolated myocytes,1 but it is not fully known whether a similar phenomenon exists in coronary endothelial cells and whether it shares the same mechanism as in myocytes. Vascular endothelial cells play an integral role in cardiovascular homeostasis, and their participation in myocardial injury during reperfusion and acute inflammation has been well established. Because deleterious consequences of sustained ischemia are not restricted to the myocytes, the beneficial effects of preconditioning of the heart should not be limited to myocytes. However, recent studies have provided conflicting results regarding whether the protective effects of preconditioning can extend to the coronary vasculature.2 3 4 Therefore, whether ischemic preconditioning protects coronary microvascular endothelial cells still remains unclear. Endothelial cells form an excellent model for the study of the basic mechanisms of preconditioning, since these cells can be cultured as a homogeneous population with little or no contamination by other cell types and they are maintained in a defined environment that can be readily manipulated.
Adenosine A1 receptors located on cardiac myocytes mediate the negative chronotropic, dromotropic, and antiadrenergic effects of adenosine.5 Adenosine A2 receptors, located predominantly on endothelial cells, mediate coronary blood flow,6 and stimulation of adenosine A2 receptors coupled to Gs proteins attenuates not only free radical generation by activated leukocytes but also the aggregation of platelets.7 It is generally accepted that adenosine released during ischemia improves contractility and metabolic function by improving coronary perfusion, preventing free radical generation, and attenuating platelet aggregation. Most of the evidence supporting the involvement of an adenosine A1 receptor in preconditioning comes from studies in intact hearts or cardiac myocytes. It is unknown, however, whether activation of the A2 adenosine receptor elicits preconditioning in endothelial cells.
How adenosine receptor activation enhances myocardial resistance against ischemic injury is poorly understood. Several recent studies have revealed the possible linkage between adenosine receptors and the activation of PKC during ischemic preconditioning.8 9 10 The rationale in these studies was that a short period of ischemia causes PKC activation and that the resultant preconditioning was blocked by PKC inhibitors. However, in those studies, confirmation of PKC activity in the preconditioning effect primarily relied on pharmacological probes. The direct measurement of PKC activity is important to further elucidate the relation between adenosine receptors and PKC activity in the cellular mechanisms of preconditioning.
The major purposes of the present study were to determine (1) whether coronary endothelial cells could be preconditioned against anoxia and reoxygenation injury, (2) whether the preconditioning could be mediated by an adenosine A2 receptor, and (3) whether the preconditioning could be mediated through a PKC signaling pathway.
Materials and Methods
SPT, R-PIA, and CGS-21680 were from Research Biochemical International. Calphostin C and PMA were from Sigma Chemical Co. PKC substrate peptide Ser25 was from GIBCO BRL.
Endothelial Cell Cultures
A bovine coronary microvascular endothelial cell line was developed by cloning techniques. This strain (B-88) was obtained from Gensia Pharmaceutical through the late Dr A. Rymaszewski at the University of Cincinnati. Strain B-88 was grown in medium 199 with 10% calf serum, 2 mmol/L glutamine, 100 μg/mL streptomycin, and 100 U/mL penicillin at 37°C in a humidified atmosphere of 95% air/5% CO2. Experiments were performed on cells at passages 6 to 8. These cells had been shown to stain positively for factor VIII–related antigen, to exhibit angiotensin I–converting enzyme activity, to take up diacetylated low-density lipoproteins, and to be free of mycoplasma and virus. Strain B-88 maintained normal morphology, a constant cellular saturation density, and uniform size. Endothelial cells in culture retain their ability to proliferate actively and, once confluent, can adopt the morphological configuration and phenotypic expression similar to those in situ. Nevertheless, we cannot exclude the possibility that the endothelial cell line somewhat differs from endothelial cells in situ, because the artificial conditions in which the cells are kept may alter their behavior.
Anoxia and Reoxygenation
Endothelial cells in medium 199 were transferred into the anoxic chamber (Forma 1025 anaerobic system) through the gas interchange cycle. Once inside, cells were washed three times with anoxic Tyrode’s solution containing (mmol/L) NaCl 125, KCl 2.6, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.0, and HEPES 25, pH 7.4, bubbled with 100% N2 at 37°C for 2 hours. It took 2 minutes to wash each dish, and this time was not included in the “anoxic incubation period” calculation. The cells were then subjected to anoxic incubation at 37°C with 3 mL anoxic Tyrode’s solution per dish. The O2 content of the air inside the chamber and that of the Tyrode’s solution was <0.1% during the entire period of experiment, as measured by the Cavitron/LexO2 CON-KTM DC-60 total O2 content analyzer. This is at the limit of instrument resolution (0.1%). At the end of the anoxic incubation, the cells were transferred to a CO2 water-jacketed incubator adjusted to a humidified atmosphere of 95% O2/5% CO2 for reoxygenation.
Cells were randomly assigned to nine groups (Fig 1⇓). All experiments were performed in six replicates (n=6 per group).
Group 1: Normal Control
Endothelial cells were not subjected to anoxia but were incubated in the aerobic Tyrode’s solution throughout the experiments.
Group 2: Anoxic Control
After a 20-minute preincubation with aerobic Tyrode’s solution, cells were subjected to 60 minutes of anoxic incubation followed by 120 minutes of reoxygenation.
Group 3: Preconditioning
Preconditioning was induced with 10 minutes of anoxic incubation and 10 minutes of reoxygenation. Then, cells were subjected to 60 minutes of anoxic incubation followed by 120 minutes of reoxygenation.
Group 4: Preconditioning and an Adenosine Receptor Antagonist
The protocol was similar to that of group 3, except that a nonselective adenosine receptor antagonist (50 μmol/L SPT) was added to Tyrode’s solution during the preconditioning period.
Group 5: Preconditioning With an Adenosine A2 Receptor Agonist
After a 10-minute pretreatment with aerobic Tyrode’s solution containing a specific adenosine A2 receptor agonist (20 nmol/L CGS-21680), endothelial cells were exposed to 60 minutes of anoxic incubation followed by 120 minutes of reoxygenation.
Group 6: Preconditioning and a PKC Inhibitor
The protocol was similar to that of group 3, except that a PKC inhibitor (100 nmol/L calphostin C) was added to Tyrode’s solution during the preconditioning period.
Group 7: Preconditioning With a PKC Activator
After a 10-minute pretreatment with aerobic Tyrode’s solution containing a PKC activator (100 nmol/L PMA), endothelial cells were exposed to 60 minutes of anoxic incubation followed by 120 minutes of reoxygenation.
Group 8: Preconditioning With an Adenosine A1 Receptor Agonist
After a 10-minute pretreatment with aerobic Tyrode’s solution containing a specific adenosine A1 receptor agonist (50 nmol/L R-PIA), endothelial cells were exposed to 60 minutes of anoxia followed by 120 minutes of reoxygenation.
Group 9: Preconditioning With an Adenosine A2 Receptor Agonist and Effect of a PKC Inhibitor
After a 10-minute pretreatment with aerobic Tyrode’s solution containing a specific adenosine A2 receptor agonist (20 nmol/L CGS-21680) and a PKC inhibitor (100 nmol/L calphostin C), endothelial cells were exposed to 60 minutes of anoxia followed by 120 minutes of reoxygenation.
Samples were collected at the end of reoxygenation for analysis of LDH and adenosine release. Cells were collected at individual time points during the experiments for PKC, ATP, and creatine phosphate, and some were processed for cell viability and morphology.
Measurement of Protein Content of Endothelial Cells
Protein content was measured by the method discussed previously.11 The method we used is based on the reaction of brilliant blue G with protein in an acid-alcohol medium to form a blue-colored protein dye complex (Sigma). The color measured at 595 nm is proportional to the protein concentration. Cells were mixed with 2 mL of 6% cold perchloric acid. The cell suspension was heated at 70°C for 20 minutes and then centrifuged at an acceleration of 1000g for 20 minutes. The pellet was dissolved in 2 mL of 0.1N NaOH solution and was cooled in ice for 20 minutes, after which it was centrifuged again at 1000g for 20 minutes. For standards, 300 μg/mL bovine serum albumin fraction V, dissolved in 0.1N NaOH, was used.
Measurement of LDH
One milliliter of sample was collected for determination of LDH. A spectrophotometric enzyme assay was performed with a Sigma assay kit. Measurement of enzyme activity was based on the oxidation of lactate and the rate of increase in absorbance at 340 nm. The activity of LDH was expressed as units per milligram protein.
Measurement of Adenosine
Adenosine concentration was assessed by HPLC as described in our earlier studies.12 13 One milliliter of sample was added with 0.5 mL of 20 μmol/L methyladenosine, which was an internal standard. Extraction was performed with ice-cold 6% trichloroacetic acid. After centrifugation, the supernatant was evaporated under N2 in a water bath at 45°C. The dried sample was dissolved in 0.5 mL distilled water, and 20 μL of the solution was injected into the reverse-phase HPLC unit (model 110B, Beckman Instruments Inc). The mobile phase was 90% of 4 mmol/L KH2PO4 buffer and 10% methanol at a flow rate of 1.0 mL/min on a Hibar RT column (10 μm, 25 cm×4 mm, Lichrosorb-RP-18). The detector coupled with an IBM-PC computer was set at a wavelength of 254 nm. Adenosine concentration was determined by comparison of internal standard and expressed as nanomoles per milligram protein.
Analysis of PKC Activity
PKC activity was measured with a modified method as described previously.14 15 Phosphorylation of the Ser residue in the PKC substrate peptide (Liu-Arg-Arg-Tyr-Ser-Leu-Gly) catalyzed by the catalytic subunit of protein kinase at pH 7.5 causes a spectral change at 430 nm, which permits monitoring of the reaction. The cells were homogenized with 2.5 vol of homogenizing buffer (mmol/L: Tris 50, EDTA 4, and 2-mercaptoethanol 15, pH 7.5) at 4°C. The homogenate was centrifuged at 1500g for 10 minutes. The supernatant was collected and centrifuged at 100 000g for 1 hour. The supernatant was used to measure cytosol PKC activity. The membrane pellet was resuspended in 2 vol of homogenizing buffer containing 0.3% Triton X-100 and mixed gently for 1 hour on ice, followed by centrifugation at 100 000g for 1 hour. The supernatant was used to measure the membrane PKC activity. One milliliter of supernatant was treated with 0.5 mL reaction medium containing 50 mmol/L Tris, 10 mmol/L MgCl2, 0.15 mol/L KCl, 0.2 mmol/L dithiothreitol, 0.2 mg/mL bovine serum albumin, 2.0 mmol/L ATP, and 100 μmol/L substrate peptide Ser25. After incubation for 10 minutes at 30°C, the absorbance of reaction of PKC with substrate peptide Ser25 was measured at 430 nm with a Beckman DV-40 spectrophotometer. The activity of PKC was expressed as nanomoles per milligram protein.
Evaluation of Cell Viability
A small aliquot of cells was incubated in 0.1% trypan blue for a few minutes, and the cells were viewed under a light microscope. Dead cells were permeable to trypan blue and thus became colored, whereas viable cells did not take up the dye. By counting 100 cells (dyed and nondyed), the percentage of viable cells was calculated.
Measurement of ATP and Creatine Phosphate
ATP and creatine phosphate were determined as previously described.13 Cells were ground and homogenized with 6% trichloroacetic acid. The supernatant was collected and centrifuged at 25 000g for 10 minutes. ATP and creatine phosphate were analyzed by absorbance at 340 nm. The results were expressed as micromoles per milligram protein.
All assays were performed without previous knowledge of treatments. Statistical analysis was based on the guidelines described by Wallenstein et al.16 All data were expressed as mean±SEM. A one-way ANOVA was first carried out to test for any differences between the mean values within the same study. When a significant F value was obtained, comparisons between individual means of groups were performed by the Student-Newman-Keuls test. A difference of P<.05 was considered significant.
LDH release measured at 120 minutes of reoxygenation after 1 hour of anoxia is illustrated in Fig 2⇓. LDH release was 0.425±0.031 U/mg protein in the anoxic control group and was markedly reduced by anoxic preconditioning or pretreatment with CGS-21680 or PMA (0.216±0.018, 0.252±0.021, and 0.228±0.019 U/mg protein, respectively; P<.05 versus anoxic control). Treatment with SPT or calphostin C completely blocked the protective effect of anoxic preconditioning on LDH release (0.410±0.029 and 0.395±0.025 U/mg protein, respectively; P>.05 versus anoxic control). R-PIA pretreatment did not reduce LDH release (0.412±0.031 U/mg protein) compared with the anoxic control. Pretreatment with calphostin C abolished the beneficial effect of CGS-21680 on LDH release (0.405±0.027 U/mg protein, P<.05 versus CGS-21680 group). The results indicate that stimulation of adenosine A2 receptor led to the attenuation of cellular enzyme release via PKC activation.
Adenosine concentration measured at 120 minutes of reoxygenation after 1 hour of anoxia is illustrated in Fig 3⇓. Adenosine concentration was 1.25±0.19 nmol/mg protein in the anoxic control group and was significantly increased by anoxic preconditioning (2.69±0.18 nmol/mg protein, P<.05 versus anoxic control). Treatment with SPT or calphostin C did not influence the adenosine production induced by anoxic preconditioning (2.61±0.22 and 2.57±0.16 nmol/mg protein, respectively; P<.05 versus anoxic control). In addition, the adenosine production was not affected by pretreatment with CGS-21680, PMA, R-PIA, or CGS-21680 plus calphostin C treatment. These results indicate that adenosine release was significantly higher after preconditioning than after other treatments.
Subcellular distribution of PKC was investigated to determine whether PKC becomes activated during anoxic preconditioning. The membrane PKC activity in the anoxic control group remained unchanged before, during, and after a longer period of anoxia (Fig 4⇓). The membrane PKC activity was markedly augmented by anoxic preconditioning or pretreatment with CGS-21680 or PMA (P<.05 versus anoxic control). On the contrary, treatment with SPT or with calphostin C completely abolished the membrane PKC activation expression induced by anoxic preconditioning. R-PIA failed to stimulate membrane PKC activity. Pretreatment with calphostin C, however, completely blocked the membrane PKC activation expression induced by CGS-21680. Fig 5⇓ demonstrates the time course of cytosol PKC activity changes throughout the experiment. The cytosol PKC activity in the control group remained steady before, during, and after a longer period of anoxia (Fig 5⇓). Upon preconditioning or pretreatment, cytosol PKC activity was markedly reduced by anoxic preconditioning or pretreatment with CGS-21680 or PMA (P<.05 versus anoxic control). Treatment with SPT or calphostin C completely abolished the translocation of cytosol PKC during anoxic preconditioning. R-PIA did not influence cytosol PKC activity. Pretreatment with calphostin C, however, completely blocked the translocation of cytosol PKC induced by CGS-21680. These data suggest that 10 minutes of anoxia was sufficient to activate and mobilize PKC from the cytosol to the membrane, which was mediated through A2 adenosine receptors.
When endothelial cells were exposed to 1 hour of anoxia followed by 2 hours of reoxygenation, the percentage of viable cells progressively decreased from 95±5% to 18±5% in the anoxic control group (Fig 6⇓). When endothelial cells were preconditioned or pretreated with CGS-21680 or PMA, cell viability was significantly increased, and the percentage of viable cells increased from 18±5% to 62±4% in the preconditioned group, 53±4% in the CGS-21680 group, and 57±5% in the PMA group (P<.05 versus anoxic control). On the contrary, the beneficial effect of preconditioning on the survival rate of endothelial cells was completely blocked by SPT (17±2%) or calphostin C (20±3%). Pretreatment with R-PIA did not prevent cell death caused by anoxia (19±4%). The increase of viable endothelial cells induced by CGS-21680 pretreatment was lost by the treatment with calphostin C (22±5%, P<.05 versus CGS-21680 group). These results indicate that activation of an adenosine A2 receptor and PKC significantly increased cell viability.
ATP and Creatine Phosphate
The cellular contents of ATP and creatine phosphate at 120 minutes of reoxygenation after 1 hour of anoxia are summarized in the Table⇓. When endothelial cells were exposed to anoxia and reoxygenation, ATP and creatine phosphate contents were reduced to 0.4±0.2 and 0.9±0.3 μmol/mg protein, respectively, in the anoxic control compared with the normoxic control (7.4±0.8 and 16.3±0.7 μmol/mg protein, respectively). Preconditioned cells had significantly higher ATP and creatine phosphate contents (3.2±0.9 and 7.3±1.6 μmol/mg protein, respectively; P<.05 versus anoxic control). Similarly, cells pretreated with CGS-21680 or PMA had significantly higher ATP and creatine phosphate contents (3.1±0.7 and 6.9±1.0 μmol/mg protein, respectively, for CSG-21680 and 4.1±0.7 and 6.1±1.2 μmol/mg protein, respectively, for PMA; P<.05 versus anoxic control). Treatment with SPT and calphostin C completely resulted in depletion of ATP and creatine phosphate. R-PIA pretreatment did not preserve ATP and creatine phosphate contents. Calphostin C pretreatment abolished the preservation of ATP and creatine phosphate induced by CGS-21680. The data suggest that activation of an adenosine A2 receptor and PKC led to preservation of cellular high-energy phosphates.
Preconditioning and pretreatment with CGS-21680 or PMA preserved the typical cobblestone pattern of endothelial cells (Fig 7⇓). Phase microscopy examination revealed that most of these cells were homogeneous in appearance, closely attached to each other without overlapping, and exhibited a well-defined nucleus surrounded by moderately dense cytoplasm. On the contrary, treatment with SPT or calphostin C resulted in loss of their typical cobblestone appearance. Pretreatment with R-PIA failed to prevent the morphological changes of cells resulting from anoxia. The CGS-21680–mediated preservation of cell morphology was lost after calphostin C pretreatment. Phase microscopy revealed that these cells exhibited a rounded, triangular, or polygonal appearance, partially or completely retracted from each other, and exhibited a poorly defined nucleus surrounded by edematous cytoplasm.
Since the phenomenon of ischemic preconditioning was first reported, numerous studies have been performed to elucidate the mechanism of preconditioning. Although the beneficial effects of preconditioning have been well characterized at the level of myocytes, it is not fully known whether a similar effect extends to coronary vascular cells (especially endothelial cells) and whether it shares the same mechanism as in myocytes. The major aims of the present study were to determine whether anoxic preconditioning could protect endothelial cells against anoxia and reoxygenation injury and whether this preconditioning effect could be mediated by an adenosine A2 receptor via a PKC signaling pathway. The results reported here clearly demonstrate that a short episode of anoxia and reoxygenation can precondition endothelial cells against subsequent sustained anoxia and reoxygenation injury, that this preconditioning effect is mediated by adenosine A2 receptors through the PKC pathway, and that preservation of endothelial cells may be one of the mechanisms of myocardial preconditioning.
Preconditioning Can Protect Endothelial Cells Against Anoxia/Reoxygenation Injury
Brief episodes of anoxia can induce a similar protection from anoxic injury in intact hearts. A recent study has shown that 5 minutes of hypoxic perfusion of hearts was as effective in inducing protection as 5 minutes of zero- or low-flow ischemia.17 In the present study, 10 minutes of anoxia with 10 minutes of reoxygenation preconditioned endothelial cells against subsequent sustained anoxia and reoxygenation injury. This protection accounted for the attenuation of cellular enzyme release, augmentation of adenosine release, increased membrane PKC activity, increased cell viability, increased high-energy phosphate contents, and well-preserved cell morphology.
Vascular endothelial cells release vasoactive substances,18 control vascular permeability,19 interact with blood cells,20 and play an integral role in cardiovascular homeostasis.21 Endothelial cell dysfunction is a manifestation of myocardial ischemia and reperfusion injury. Therefore, the protective effects of preconditioning should involve coronary endothelial cells as well. Through measurement of coronary arteriolar diameters in canine hearts using intravital microscopy, DeFily and Chilian2 have shown that 60 minutes of ischemia and reperfusion impaired the endothelium-dependent regulation of coronary arteriolar reactivity and that ischemic preconditioning induced by 10 minutes of ischemia and reperfusion preceding longer ischemia reduced the endothelial dysfunction of coronary arterioles. Richard et al3 studied the effect of preconditioning on coronary endothelial dysfunction induced by ischemia and reperfusion in coronary segments. They found that ischemia followed by reperfusion markedly impaired coronary relaxation function. In addition to salvaging myocardial cells, preconditioning also protects coronary endothelial cells against ischemia/reperfusion injury. On the contrary, studies from Bauer et al4 provided evidence that ischemic preconditioning does not attenuate vascular endothelium dysfunction after subsequent sustained occlusion and reperfusion. In spite of these conflicting reports, the present study provides further direct evidence that preconditioning can protect endothelial cells against anoxic injury at the cellular level and that preservation of endothelial cells may be one of the mechanisms by which preconditioning reduces tissue necrosis in myocardial ischemia/reperfusion injury.
Preconditioning Is Mediated by Adenosine A2 Receptors in Endothelial Cells
There has been ample evidence that adenosine A1 receptors located on myocytes mediate the preconditioning effect against ischemia and reperfusion injury. The mechanism by which preconditioning prevents anoxic injury in coronary endothelial cells is probably somewhat different from that in myocytes. In the present study, therefore, we tested the hypothesis that preconditioning against anoxic injury was mediated by an adenosine A2 receptor in the coronary endothelial cells. The hypothesis was formulated on the basis of the predominant presence of adenosine A2 receptors on endothelial cells.22 Indeed, pretreatment with a specific adenosine A2 receptor agonist (CGS-21680) markedly reduced intracellular enzyme release, increased membrane PKC activity, preserved high-energy phosphate content, augmented cell viability, and preserved cell structure. In contrast, a specific A1 adenosine receptor agonist (R-PIA) failed to precondition endothelial cells against anoxic injury. The failure of the A1 adenosine receptor in the preconditioning of endothelial cells could be due to the fact that adenosine A2 receptors are located predominantly on endothelial cells and that there are few or no adenosine A1 receptors located on endothelial cells.6 Some studies have shown the role of adenosine A2 receptors in the reduction of ischemia/reperfusion injury. Norton et al23 compared the efficacy of various doses of adenosine with a selective A1 agonist (cyclopentyladenosine) and a selective A2 receptor agonist (CGS-21680) in the rabbit heart. A significant reduction in infarct size was noted with all three doses of adenosine, intermediate and low doses of cyclopentyladenosine, and high and intermediate doses of CGS-21680. The two adenosine receptor agonists afforded a similar degree of protection. Lasley et al24 showed in an isolated heart model that the protection of the ischemic myocardium with adenosine was mediated by interaction with adenosine A1 receptors and not with A2 receptors. The reasons for these discrepancies could be (1) species variations (rat, rabbit, and bovine), (2) different experimental models (isolated cells and intact hearts), and (3) different protocols. Taken together, the results from the present study strongly suggest that adenosine triggers the preconditioning effect and protects endothelial cells against anoxia/reoxygenation injury, probably through adenosine A2 receptors on coronary endothelial cells.
Adenosine A2 Receptor–Triggered Protection via a PKC Pathway
The following question arises: How does adenosine receptor activation enhance cell resistance against ischemic injury? Many second messenger systems have been postulated to couple with adenosine receptors, including cAMP, cGMP, phospholipase C, PKC, the ATP-sensitive potassium channel, and the calcium channel.6 It is unknown which second messengers, if any, are coupled with adenosine receptors activated by preconditioning. Several recent studies have revealed the possible linkage between adenosine receptors and the activation of PKC in ischemic preconditioning. A recent study in isolated rat myocytes has shown that both 5′-nucleotidase activity and adenosine release due to activation of PKC are cardioprotective against hypoxic and reoxygenation injury.9 More recently, Ytrehus et al25 have reported that PKC activation is required for the preconditioning of the ischemic rabbit heart and that the protective effect is mimicked by PKC activators, suggesting that PKC is an important step in the mechanism of preconditioning. In the present study, a short period of anoxia/reoxygenation or pretreatment with CGS-21680 or PMA caused PKC activation. SPT and calphostin C completely abolished PKC activity triggered by anoxic preconditioning. Pretreatment with calphostin C blocked the activation of PKC by CGS-21680. Collectively, all of these data provide strong evidence that stimulation of adenosine A2 receptor on endothelial cells can trigger the protective effect of preconditioning via PKC. The antagonistic potency of calphostin C on PMA-induced protection and that of SPT on the CGS-21680–induced effect in the endothelial cells were not determined in the present study. It has been previously reported that calphostin C blocked the PMA-induced effect and that SPT can abolish CGS-21680–induced protection. The intense neovascularization induced by PMA was totally suppressed by coadministration of calphostin C.26 Similarly, Abebe et al27 have provided evidence that CGS-21680, an A2 agonist, can effectively relax endothelium-intact coronary artery rings and that SPT significantly attenuated the relaxant responses induced by CGS-21680, suggesting that the effect of an A2 agonist can be blocked by a nonspecific adenosine receptor antagonist. This further suggests the presence of A1 receptors on endothelial cells along with the predominant A2 receptors.
Strasser et al28 demonstrated that short periods of ischemia (10 minutes) led to a rapid translocation of PKC from the cytosol to the plasma membranes, indicating PKC activation. Physiological and pharmacological activation of PKC is known to induce such redistribution of the enzyme in the heart.29 The present study is in agreement with Strasser et al,28 who reported that 10 minutes of anoxia itself induced translocation of PKC from cytosol to membrane and was the first step in a cascade of events leading to reduction in anoxic injury. The PKC remained activated during reoxygenation after 10 minutes of anoxia.
The mechanisms of the coupling between adenosine receptors and PKC on endothelial cells remain to be elucidated. Recently, it has been observed that adenosine receptors can couple phospholipase C by a pertussis toxin G protein.30 31 Stimulation of receptor-linked phospholipase C causes hydrolysis of membrane phosphoinositides, resulting in the generation of at least two intracellular second messengers, inositol tris-phosphate, which mobilizes intracellular calcium, and diacylglycerol, which activates PKC.32 None of these studies is, however, related to endothelial cells. Limited data are now available to confirm any coupling between A2 adenosine receptors and PKC during ischemic preconditioning of endothelial cells. However, we have recently observed that the activation of PKC triggered by preconditioning can be blocked by a phospholipase C inhibitor, suggesting a coupling between phospholipase C and PKC during the preconditioning of endothelial cells.33
How PKC plays a role in the preconditioning effect is still a mystery. Recently, Brooks et al34 have reported that preconditioning of isolated myocytes apparently upregulates the PKC pathway so that protein phosphorylation can occur early in the second ischemic period of preconditioning. Therefore, we can reasonably assume that PKC activation might phosphorylate yet unknown cellular proteins that are essential for the protective effect of preconditioning. These unknown proteins could be membrane ATP-sensitive potassium channels,35 5′-nucleotidase,9 antioxidant enzymes,36 or stress proteins.37
In conclusion, a short episode of anoxia and reoxygenation can precondition endothelial cells against subsequent sustained anoxic injury. Activation of an adenosine A2 receptor may mediate its protection through the PKC pathway. PKC activation during preconditioning may occur mainly in the cell membrane, where it participates in various signaling pathways, leading to the attenuation of anoxic injury. The protection of endothelial cells may be one of the mechanisms of ischemic preconditioning in hearts.
Selected Abbreviations and Acronyms
|HPLC||=||high-performance liquid chromatography|
|PKC||=||protein kinase C|
|PMA||=||4β-phorbol 12-myristate 13-acetate|
This study was supported by National Institutes of Health research grant HL-23597.
Presented in part at the 67th Scientific Sessions of the American Heart Association in Dallas, Texas, November 14-17, 1994.
- Received March 15, 1995.
- Accepted September 15, 1995.
- © 1996 American Heart Association, Inc.
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