Calcium Preconditioning Elicits Strong Protection Against Ischemic Injury via Protein Kinase C Signaling Pathway
We tested the hypothesis that elevation of [Ca2+]i during Ca2+ preconditioning (CPC) is a strong activator of protein kinase C (PKC) and confers unique protection against ischemic injury. CPC consisted of three cycles of Ca2+ depletion (1 minute each) and Ca2+ repletion (5 minutes each). Langendorff-perfused rat hearts were subjected to 40 minutes of global ischemia followed by 30 minutes of reperfusion. Significant functional recovery and decreased lactate dehydrogenase release were observed in CPC hearts compared with ischemic control hearts. In addition, ATP contents were significantly higher and cell structure was better preserved in CPC hearts than in ischemic control hearts. Administration of chelerythrine, a specific PKC inhibitor, completely abolished the CPC-induced cardioprotection. In other groups, in which Ca2+ influx during CPC was inhibited with verapamil, amiloride, and low Na+ perfusion, cardioprotection was significantly reduced. The prominent increase in the membrane PKC activity after CPC was in agreement with immunolocalization of PKC-α and PKC-δ in the cell membrane of CPC hearts. These results demonstrate that (1) a transient increase in [Ca2+]i is a prominent feature of CPC and is a strong stimulus for the activation of PKC, (2) the elevation of [Ca2+]i likely occurs via an L-type Ca2+ channel and Na+-Ca2+ exchanger, and (3) PKC plays a crucial role in the subcellular mechanisms of protection by CPC.
We have recently demonstrated1 that CPC (repetitive Ca2+ depletion and repletion for short duration) conferred cardioprotection against the lethal injury induced by the Ca2+ paradox. The Ca2+ paradox2 3 4 is characterized by irreversible cell damage resulting from reperfusion of the heart with a Ca2+-containing fluid after a longer period of Ca2+-free perfusion. In our previous study,1 CPC reduced LDH release and cellular damage and preserved tissue ATP content in the rat hearts subjected to the Ca2+ paradox. The study suggested that the stress to the heart by CPC triggered cellular events that led to the protection against Ca2+-overload injury. We therefore hypothesized that CPC should likewise provide significant protection against myocardial ischemic injury.
Brief intermittent periods of ischemia reduce myocardial injury induced by subsequent prolonged ischemia. This phenomenon is known as ischemic preconditioning.5 The principal mechanisms underlying IPC are currently under intensive investigation. However, recently it has been proposed that the activation of PKC, which is known to be an important player in numerous intracellular signal-transduction pathways,6 7 plays a central role in the protection of IPC in rats8 9 and rabbits.10 11 Since sudden augmentation of contractility observed during CPC suggests a transient increase in [Ca2+]i, it is possible that CPC effectively activates PKC, particularly Ca2+-dependent PKC isoforms. It is therefore likely that PKC also plays an important role in the cellular mechanism of CPC-induced protection.
The purpose of the present study was to test our hypotheses that (1) CPC triggers several subcellular Ca2+-dependent pathways, leading to protection against ischemic injury in the isolated rat heart; (2) stimulation, augmentation, and translocation of PKC activity are evoked by CPC; (3) PKC inhibition abolishes the beneficial effect of CPC on ischemic injury; and (4) the blockade of transient increase in [Ca2+]i during CPC does not activate PKC and attenuates the protective effect of CPC.
Materials and Methods
Male Sprague-Dawley rats weighing 220 to 250 g were anesthetized by intraperitoneal injection of 30 mg/kg pentobarbital. After intraperitoneal injection of 500 U/kg heparin sodium, hearts were removed and retrogradely perfused through the aorta in a noncirculating Langendorff apparatus with KH buffer as previously described.1 12 KH buffer consisted of (mmol/L): NaCl 118, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, CaCl2 2.5, NaHCO3 25, and glucose 11. The buffer was saturated with 95% O2-5% CO2 (pH 7.4, 37°C) for 50 minutes. Hearts were perfused at a constant pressure of 80 mm Hg. Left ventricular pressure was measured by the method described by Grupp and Grupp.13 Briefly, a catheter was inserted through a pulmonary vein into the left atrium and advanced into the left ventricle, forced through the apex. The proximal end of the catheter was flanged to prevent it from slipping out of the ventricle. The distal end was connected through a larger-diameter catheter to a pressure transducer (Gould P23Db). The first derivative of left ventricular pressure (±dP/dt) was obtained from a differentiator (model 7P20, Grass Instrument Co). Heart rate, left ventricular pressure, and dP/dt were monitored on a Grass 7D polygraph. The hearts were maintained at 37°C during the ischemic period by immersion in a water-jacketed beaker containing perfusate. The coronary effluent was collected in a beaker, and the flow was determined volumetrically at the end of equilibration, during CPC and reperfusion.
After equilibration, hearts were randomly divided into 10 groups using 2 protocols.
Protocol 1: CPC and Inhibition of PKC Activation
These experiments tested the hypothesis that CPC triggered the activation of PKC, which was responsible for protection against myocardial ischemic injury. To block the activation of PKC during CPC, chelerythrine chloride, a specific PKCI,1414 was used.
Group 1: Normal control
Hearts (n=5) were perfused for 90 minutes with KH buffer as a normal control for different experimental groups.
Group 2: Ischemic control
Hearts (n=6) were perfused with KH buffer for 20 minutes and then subjected to global ischemia for 40 minutes, followed by reperfusion for 30 minutes (I/R).
Group 3: CPC
Hearts (n=8) were perfused for three cycles of 1 minute each with Ca2+-free KH buffer. Each cycle was followed by 5 minutes of Ca2+-containing KH buffer, and then the hearts were subjected to the I/R sequence as in group 2.
Group 4: CPC and PKCI
The procedure was similar to that for group 3, except 100 μmol/L chelerythrine chloride, a specific inhibitor of PKC, was infused through the aorta at the rate of 0.2 mL/min during CPC (n=6). The dose of chelerythrine was chosen on the basis of a previous study by Speechly-Dick et al.8 Chelerythrine was dissolved directly in the buffer.
Group 5: PKCI
The procedure was similar to that for group 2, except 100 μmol/L chelerythrine was infused at a rate of 0.2 mL/min for 20 minutes before I/R (n=5).
Protocol 2: Blockade of Ca2+ Influx During CPC
These experiments tested the hypothesis that a transient increase in [Ca2+]i that occurred during CPC activated PKC, which was responsible for the cardioprotection. The Ca2+ entry was blocked by (1) verapamil, an L-type Ca2+ channel blocker; (2) amiloride, a Na+-H+ exchanger inhibitor; and (3) low Na+ perfusion in a Ca2+-free phase during CPC.
Group 6: CPC and Ca2+ blocker
This group was similar to group 3, except that increasing doses of verapamil (0.2, 0.5, and 2 μmol/L), a Ca2+ channel blocker, were infused at a rate of 0.2 mL/min from 3 minutes before the first cycle of CPC to the end of the third cycle of CPC (n=3 to 6 for each subgroup). After a 5-minute washout by the normal perfusate, I/R was instituted.
Group 7: CPC and amiloride
Amiloride effectively inhibits the intracellular Na+ accumulation and Ca2+ uptake through the inhibition of Na+-H+ exchange linked to Na+-Ca2+ exchange in ischemic isolated rat heart.15 16 In this procedure, hearts were perfused with KH buffer containing 1 mmol/L amiloride during CPC (n=6). After a 5-minute washout period, hearts were subjected to I/R.
Group 8: Low Na+ perfusion during CPC
Ca2+ influx during CPC was reduced by lowering the [Na+] in the perfusate to 60 mmol/L. It has been shown that lowering [Na+]o reduces Na+ influx and subsequently decreases Ca2+ entry modulated by Na+-Ca2+ exchange.17 18 In this procedure, hearts were perfused with Ca2+-free KH buffer containing low NaCl (60 mmol/L) during the Ca2+-depletion phase of the CPC protocol (n=6). To maintain the equimolarity of the perfusate, LiCl was added to the low-NaCl/KH buffer.
Group 9: Verapamil
The procedure was similar to group 2, except 2 μmol/L verapamil was infused at a rate of 0.2 mL/min for 15 minutes followed by 5 minutes of washout. After that, hearts were subjected to I/R (n=5).
Group 10: Amiloride
Hearts (n=5) were perfused with KH buffer containing 1 mmol/L amiloride for 15 minutes. After a 5-minute washout period, I/R was instituted.
Measurement of LDH
LDH, an indicator of myocardial tissue injury, was determined in coronary effluent. This enzyme was assayed by a coupled-enzyme spectrophotometric technique using a Sigma assay kit. Measurement of enzyme activity was based on oxidation of lactate and the rate of increase in absorbance at 340 nm. The accumulated amount of LDH released during 30 minutes of reperfusion was obtained by integrating the area underneath the individual time-course curve for 30 minutes of reperfusion.
Measurement of Tissue ATP
The heart was immediately frozen in liquid nitrogen and freeze-dried for 24 hours. Fifty to 100 mg of freeze-dried tissue was crushed in a precooled glass tube, and ATP was extracted with 5 mL of cold 6% trichloroacetic acid. The extract was analyzed for ATP by a spectrophotometric method.1
Measurement of PKC Activity
At the end of CPC and pretreatment, hearts were excised, immediately frozen in liquid nitrogen, and stored at −70°C until use. Twenty to 30 mg of the freeze-dried heart tissue was used for analysis. Subsequent extraction was performed at 4°C. The left ventricles were dissected and minced. The minced tissue was homogenized with a Brinkmann Polytron homogenizer in 1.5 mL of buffer containing (mmol/L) Tris 50, EDTA 4, and mercaptoethanol 15. The homogenate was subjected to differential centrifugation at 1500g for 10 minutes. The supernatant was centrifuged at 100 000g for 1 hour and served as the cytosolic fraction. The pellet was resuspended in 1.5 mL of homogenizing buffer containing 0.3% Triton X-100, 50 mmol/L Tris, 4 mmol/L EDTA, and 15 mmol/L mercaptoethanol and incubated for 1 hour on ice. The homogenate was centrifuged at 100 000g for 1 hour, and the supernatant contained the membrane fraction. PKC activity was measured after incubation for 10 minutes at 30°C in a reaction mixture containing 5 mmol/L Tris, 10 mmol/L MgCl2, 0.15 mol/L KCl, 0.2 mmol/L dithiothreitol, 0.2 mg/mL BSA, 2.0 mmol/L ATP, and 100 μmol/L PKC substrate peptide [Ser25](19-31).19 The phosphorylation of the peptide as PKC activity was analyzed by absorbance at 430 nm in a Gilford spectrophotometer.20 21 The activity of PKC was expressed as nmol/mg dry weight.
Tissue from the midventricular wall was cut into 1.0-mm pieces, fixed in 2.5% buffered glutaraldehyde, postfixed in 1% buffered osmium tetroxide, and processed for transmission electron microscopy.1 A semiquantitative estimate of cell damage was carried out on 1-μm-thick sections. Three randomly chosen blocks from each heart were examined for quantification of cell damage without prior knowledge of the treatment. Approximately 500 cells were analyzed in each heart, and cells were classified according to presence and degree of damage, as follows: (1) normal (compact myofibers with uniform staining of nucleoplasm, well-defined rows of mitochondria between the myofibrils, and no separation of opposing intercalated disks); (2) mild damage (same as above, except some vacuoles were present adjacent to the mitochondria); and (3) severe damage (reduced staining of cytoplasmic organelles, clumped chromatin material, wavy myofibers, and granularity of cytoplasm; cells with contraction band necrosis were added to this category).
Subcellular localization and translocation studies of PKC isoforms in hearts after CPC were performed by immunofluorescence staining and compared with those of control hearts. In these experiments, hearts were perfused as described above. Specimens were obtained at the end of CPC. Control specimens were obtained after 20 minutes of equilibration. Tissue of left ventricle was excised from the isolated heart and cut into five pieces. These pieces were embedded in OCT compound (Miles Laboratories) in an aluminum foil tube, rapidly frozen in liquid nitrogen, and stored at −70°C. Then the immunofluorescence microscopic study,9 using rabbit polyclonal isoform-specific anti-PKC antibodies (anti-PKC-α, -β1, -β2, -γ, -δ, -ε, -ζ, and -η from Research and Diagnostic Antibodies), was performed by Dr Anirban Banerjee (University of Colorado Health Science Center). Sections were viewed and photographed with a microscope equipped with fluorescence optics (Axiskop with MC-100 camera, Zeiss).
Data were expressed as mean±SEM. Group comparisons were done by analysis of variance with multiple comparisons or t test when appropriate. A value of P<.05 was considered significant.
Ischemic Control Hearts
Ischemic control hearts showed a significant decrease in functional recovery on reperfusion (Fig 1⇓). The recovery of +dP/dt, heart rate, and coronary flow in ischemic control hearts was 28.7±7.2%, 58.1±7.2%, and 51.1±7.7%, respectively. A multifold increase in LDH release was observed after reperfusion, and ATP levels were reduced to 6.6±1.8 μmol/g dry weight compared with the normal control value of 20.3±0.9 μmol/g dry weight (Fig 2⇓).
The ultrastructure of the normal control hearts was well maintained (see the Table⇓ and Fig 3A⇓). Glycogen was abundant, and nuclear chromatin material was uniformly dispersed. Mitochondria were intact. Severe cell damage was observed in the ischemic control hearts (Fig 3B⇓). Glycogen was depleted, nuclear chromatin was markedly clumped, myofibrils were partly disrupted, mitochondria were severely swollen, and cristae were disrupted. The semiquantitative estimate of cell injury showed that only 6.9±3.4% of cells were normal; 11.4±3.8% and 81.3±6.6% of cells were mildly and severely damaged, respectively (Table⇓).
CPC and Ischemia
After the first cycle of CPC, the hearts transiently exhibited a 30% increase in ±dP/dt and showed enhanced functional recovery after I/R (±dP/dt, 90.5±1.2%; heart rate, 81.9±3.1%; coronary flow, 77.9±5.3%), in contrast with ischemic control hearts (Fig 1⇑). In CPC hearts, LDH release was significantly decreased (7.1±0.4 U/g) and ATP contents were markedly preserved (14.3±0.3 μmol/g dry weight) compared with ischemic control hearts (Fig 2⇑).
The cellular structure was extremely well preserved in the CPC hearts: 60.9±3.9% of cells were normal; 21.2±3.5% and 15.9±1.4% were mildly and severely damaged, respectively. The degree of cell damage was much less than that in the ischemic control hearts (Table⇑). At the electron microscopic level (Fig 3C⇑), the normal structural features of myocytes were well preserved. Glycogen deposits were frequently observed, myofibrils were intact, and mitochondria were elongated but occasionally slightly swollen. Cells showing severe damage were not different from the ischemic control hearts.
Role of PKC in CPC
To determine whether the beneficial effects of CPC were mediated by PKC, we administered chelerythrine, a specific PKCI, during CPC. Treatment with chelerythrine during CPC abolished the beneficial effects of CPC (Figs 1 and 2⇑⇑). The cell damage (Fig 3D⇑ and Table) was similar to the ischemic control group.
Blockade of Ca2+ Entry During CPC
To determine whether a transient increase in [Ca2+]i during CPC triggers the second-messenger signaling pathways, leading to cardioprotection against ischemia, we attempted to reduce Ca2+ entry during CPC (protocol 2). In group 6, low doses of verapamil (0.2 and 0.5 μmol/L) did not influence the cardioprotection of CPC (Fig 4⇓). The functional recovery was similar to that in CPC hearts. However, the treatment with a moderate dose of verapamil (2 μmol/L), which was associated with decrease in +dP/dt during CPC (Fig 4⇓), attenuated the functional recovery (Fig 4⇓). In hearts treated with 2 μmol/L verapamil, LDH release was significantly increased, to 16.3±0.8 U/g, and ATP content was reduced, to 8.8±0.4 μmol/g dry weight compared with values in CPC hearts (Fig 5⇓). In these hearts, 24.3±1.6% of cells were normal and 19.5±2.5% and 56.8±0.4% were mildly and severely damaged, respectively (Table⇑). Thus, there was less protection compared with CPC hearts. At the ultrastructural level (Fig 3E⇑), nuclear chromatin material was clumped, mitochondria were swollen and partly disrupted, glycogen was depleted, and myofibrils were partly transformed into contraction bands. These data suggested that verapamil reduced the Ca2+ entry in a dose-dependent fashion via an L-type Ca2+ channel during CPC, resulting in less activation of Ca2+-dependent second-messenger signals.
In groups 7 and 8, both amiloride and perfusion with low Na+ also blunted the CPC-induced increase of +dP/dt (Fig 4A⇑) and resulted in less cardioprotection (Figs 3F, 4, and 5⇑⇑⇑, and Table). Morphological changes in CPC hearts with low Na+ perfusion are the same as in the amiloride-treated CPC hearts and are not shown in Fig 3⇑. To completely block Ca2+ entry through both the L-type Ca2+ channel and Na+-Ca2+ exchange, we attempted the combined pretreatment of verapamil and amiloride during CPC in a couple of experiments. The use of both agents resulted in severe ventricular dysfunction before sustained ischemia (data not provided). Pretreatment with verapamil alone or amiloride alone without CPC did not provide significant protection against the subsequent prolonged I/R (Figs 4 and 5⇑⇑), which was consistent with the previous findings that verapamil22 23 or amiloride16 24 showed a pronounced cardioprotective effect if they were continuously administered before sustained ischemia and through I/R.
It was noteworthy that neither verapamil nor lowering of [Na+]i completely abolished the effect of CPC. Thus, it is clear that both the L-type Ca2+ channel and Na+-Ca2+ exchange contributed to the CPC-induced increase in [Ca2+]i.
Distribution of PKC After CPC
We assessed the distribution of PKC after CPC. Translocation of PKC from the cytoplasm to the cell membrane has been recognized as an index of PKC activation. A prominent increase (8.6±0.8 nmol/mg dry weight) in PKC activity of the membrane fraction was observed in CPC hearts compared with control hearts (1.8±0.3 nmol/mg dry weight; Fig 6⇓, right). On the other hand, chelerythrine completely blocked the CPC-induced increase in PKC activity of the membrane fraction. Treatment with 2 μmol/L verapamil, 1 mmol/L amiloride, and low Na+ perfusion significantly reduced PKC activity of the membrane fraction in CPC hearts (Fig 6⇓, right). There were no significant differences among the groups in PKC activity of the cytosolic fraction (Fig 6⇓, left).
Immunofluorescence microscopic study using isoform-specific anti-PKC antibodies strengthened our hypothesis on the Ca2+-mediated activation of PKC during CPC. Fig 7⇓ demonstrates that both PKC-α and PKC-δ were translocated to the plasma membrane of several hearts after CPC. The cell membranes were deeply stained with the isoform-specific antibodies. The controls showed diffuse staining or lack of staining. The visualization of the other PKC isoforms was not as clear as that of the α and δ isoforms.
The major new findings of the present study are: (1) CPC initiates several subcellular pathways, leading to strong cardioprotection against ischemia, (2) a transient elevation of [Ca2+]i during CPC triggers the activation of Ca2+-dependent PKC, and (3) the CPC-induced protection is mediated by PKC activation. It is generally believed that a major Ca2+ influx causes pathological changes in I/R injury.15 16 25 26 27 28 29 Sustained increase in [Ca2+]i after prolonged ischemia has been postulated to activate a proteolytic process by Ca2+-dependent proteases, which cause irreversible plasma membrane damage.30 31 However, it is intriguing to note that a transient and mild increase in [Ca2+]i that occurs during CPC confers cardioprotection against ischemia (this study) or Ca2+-overload injury.1
Ca2+-Dependent Activation of PKC in CPC
Recently, it has been reported by several studies that PKC activation is essential for the protection induced by IPC.8 9 10 11 In this study, we proposed that a brief Ca2+ loading during CPC could effectively activate PKC, especially Ca2+-dependent isoforms of PKC (conventional PKC). CPC is characterized by transient stress induced by repetitive Ca2+ depletion and repletion of short duration. During the Ca2+ repletion phase of CPC, the contractility of the hearts was augmented by 30% (Fig 1⇑). Although we did not directly measure [Ca2+]i during CPC, this phenomenon strongly suggested a rise in [Ca2+]i, which was ascribed to the increased Ca2+ influx from the extracellular space through the Na+-Ca2+ exchanger and voltage-dependent Ca2+ channel and to Ca2+-induced Ca2+ release from the SR.31 The participation of the L-type Ca2+ channel and Na+-Ca2+ exchanger in Ca2+ mobilization during CPC was supported by our data (Figs 4, 5, and 6⇑⇑⇑). In addition to direct [Ca2+]i-dependent activation of conventional PKC,32 33 the increase in [Ca2+]i may activate PKC via the phosphatidylinositol cycle, through the activation of G protein34 35 linked to Ca2+-dependent phospholipase C.34 36 As a consequence of phospholipase C activation, diacylglycerol is produced from phosphatidylcholine, and a subsequent sustained increase in the amount of diacylglycerol maintains the activation of PKC.7
In the present study, the measurement of PKC activity (Fig 6⇑) confirmed the activation and translocation of PKC by CPC. Moreover, the immunofluorescent microscopy (Fig 7⇑) strongly supported the biochemical findings that CPC caused the translocation of a Ca2+-independent isoform of PKC (novel PKC), PKC-δ, as well as a conventional PKC, PKC-α. Banerjee's group9 suggested that PKC-δ activation after brief ischemia and α1-adrenergic stimulation was involved in preconditioning. It remains to be determined whether different PKC isoforms independently act in preconditioning and what roles the individual isoforms play in preconditioning. However, it is possible that CPC shows its strong cardioprotection through the activation of both Ca2+-dependent and Ca2+-independent PKC isoforms.
Possible Mechanisms of Cardioprotection by PKC
Although no precise mechanism of cardioprotection by PKC is known at present, some subcellular mechanisms for PKC-dependent modulation and preservation of contractile function37 may be suggested, as below. It has been reported that activated PKC directly phosphorylates contractile proteins such as troponin I38 39 and myosin light chain 2 .40 41 Phosphorylation of troponin I may cause a negative inotropic response mediated by reduced myofibrillar actomyosin Mg2+ ATPase activity,39 while phosphorylation of myosin light chain 2 leads to stimulation of myofibrillar actomyosin Mg2+ ATPase activity followed by exertion of a positive inotropism. The cooperation of PKC with myosin light chain kinase increases the sensitivity of contractile protein to Ca2+.40 The augmentation of myofilament sensitivity to Ca2+ may be involved with intracellular alkalization induced by the activation of PKC.42 PKC stimulates Ca2+ uptake from the cytosol by the SR through activation of SR Ca2+-Mg2+ ATPase as a result of the phosphorylation of phospholamban.43 Activated PKC inhibits the L-type Ca2+ channel44 45 46 and decreases the Ca2+ influx. Taken together, these findings lead us to propose that PKC attenuates the deleterious increase in [Ca2+]i that occurs during prolonged ischemia and after reperfusion by enhancing the Ca2+ uptake by the SR and depressing Ca2+ influx through inhibiting the L-type Ca2+ channel and modifying Na+-Ca2+ exchange47 and that PKC also preserves myocardial function by augmenting myofibrillar sensitivity to Ca2+ and modulating contractile proteins. These plausible mechanisms have been schematically represented in Fig 8⇓.
[Ca 2+]i Increase During Preconditioning Is a Potential Common Factor of IPC and CPC
It is likely that the increment of [Ca2+]i during the preconditioning period triggers the protection initiated by IPC. Generally, the protective effect of IPC is induced by 5 minutes or less of ischemia followed by 5 to 10 minutes of reperfusion.48 Several investigators27 49 50 have reported the rapid increase in [Ca2+]i during brief ischemia (≈15 minutes). Kihara et al27 observed the gradual increment of [Ca2+]i during 3 minutes of global ischemia in the isolated ferret heart. Mohabir et al50 have shown the biphasic increase in [Ca2+]i during 15 minutes of global ischemia in the isolated rabbit heart. Thus, brief ischemia in IPC could evoke an increase in [Ca2+]i, which may be an important common event in both IPC and CPC. This hypothesis is supported by our data that blockade of Ca2+ entry by verapamil, amiloride, or low Na+ perfusion during CPC attenuated the CPC-induced protection against ischemia.
Recent studies9 51 52 have indicated that both α1-adrenergic stimulation and transient ischemia precondition the isolated rat heart through activation of PKC. The stimulation of the α1-adrenoceptor increases cytosolic free Ca2+53 and shows a [Ca2+]i-dependent enhancement of PKC activity54 in isolated cardiac myocytes. Keeping in view the role of [Ca2+]i during preconditioning, we speculate that α1-adrenergic-mediated preconditioning is also affected by the [Ca2+]i-dependent PKC pathway.
Other Mechanisms of CPC
The other mechanism of CPC is the increase of adenosine release during CPC, which triggers various signaling pathways, leading to protection against ischemic damage. In our previous study, we demonstrated that the protection by CPC against the Ca2+ paradox results from Gi protein-modulated adenosine A1 receptor stimulation.1 Adenosine release was increased 11-fold by the end of CPC, the adenosine A1 receptor agonist (R)-phenylisopropyl adenosine mimicked CPC, and the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline blocked the beneficial effects of CPC in that study.1 It is well known that increased adenosine formation and release yield beneficial effects by activating the adenosine A1 receptor or through antiadrenergic effects.55 Increased responsiveness of Gi protein56 could promote this mechanism. Furthermore, it has recently been reported that PKC activated by α-adrenergic stimulation increases ecto-5′-nucleotidase and adenosine release.52 Second, the activation of A1 receptors coupled to the ATP-sensitive K+ channel through G protein may cause the opening of the ATP-sensitive K+ channel.57 This may shorten the action-potential duration, followed by a decrease in Ca2+ influx, and lead to a reduction in cellular energy consumption and preservation of ATP and myocardial function (Fig 8⇑).
In summary, our results indicate that CPC offers strong protection against ischemic injury through the activation of PKC and that the transient increase in [Ca2+]i during CPC is a trigger for the induction of CPC. CPC provides an important experimental model to study the role of Ca2+ in initiating multiple intracellular signaling pathways leading to attenuation of ischemic injury.
Selected Abbreviations and Acronyms
|Gi protein||=||inhibitory G protein|
|PKC||=||protein kinase C|
This study was supported in part by grants HL-23597 and HL-44358 from the National Institutes of Health, Heart, Lung, and Blood Institute. We thank Dr Anirban Banerjee (Department of Surgery, University of Colorado Health Science Center) for immunocytochemistry and Gulnar Durrani for her technical assistance.
Correspondence to Muhammad Ashraf, PhD, Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, 231 Bethesda Ave, Cincinnati, OH 45267-0529.
- Received November 8, 1995.
- Accepted March 29, 1996.
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