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Am J Physiol Heart Circ Physiol 282: H1933-H1943, 2002. First published January 17, 2002; doi:10.1152/ajpheart.00771.2001
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Vol. 282, Issue 5, H1933-H1943, May 2002

Enhanced IPC by activation of pertussis toxin-sensitive and -insensitive G protein-coupled purinoceptors

Hideki Ninomiya, Hajime Otani, Kejie Lu, Takamichi Uchiyama, Masakuni Kido, and Hiroji Imamura

Department of Thoracic and Cardiovascular Surgery, Kansai Medical University, Moriguchi, Osaka 570-8507, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Extracellular ATP plays an important role in ischemic preconditioning (IPC) through the activation of P2y purinoceptors. This study examined whether ATP-stimulated P2y purinoceptors are coupled to pertussis toxin (PTX)-insensitive G protein and whether activation of this pathway enhances myocardial protection afforded by IPC. The rat was treated with PTX for 48 h, and the heart was then isolated and buffer perfused. The heart underwent IPC by three cycles of 5-min ischemia and 5-min reperfusion before 25 min of global ischemia. Isovolumic left ventricular function was measured, and functional recovery at 30 min after reperfusion was taken as an end point of myocardial protection. PTX pretreatment partially inhibited functional protection by IPC. Treatment with 100 µM 8-(p-sulfophenyl) theophylline (SPT) during IPC had no further effect on PTX-induced inhibition of functional protection by IPC, whereas suramin (300 µM) or reactive blue (RB) (10 µM) completely abolished myocardial protection in the preconditioned heart pretreated with PTX. Supplementation with adenosine (30 µM), ATP (30 µM), or UTP (50 µM) significantly enhanced IPC-induced functional protection, although preconditioning with these nucleotides without IPC had no protective effect. Adenosine-enhanced IPC was inhibited by pretreatment with PTX and SPT but not by suramin or RB, whereas ATP-enhanced IPC was inhibited by suramin or RB in combination with PTX pretreatment. On the other hand, UTP-enhanced IPC was not affected by PTX pretreatment but was inhibited by suramin or RB. Adenosine supplemented IPC without PTX pretreatment and ATP supplemented IPC with PTX pretreatment were not affected by nitric oxide synthase inhibitor Nomega -nitro-L-arginine methyl ester (100 µM). Although the protein kinase C inhibitor Ro318425 (0.3 µM) or tyrosine kinase inhibitor genistein (50 µM) had no significant effect on the functional protection afforded by adenosine-supplemented IPC without PTX pretreatment and ATP-supplemented IPC with PTX pretreatment, the combination of Ro318425 and genistein attenuated functional protection afforded by both the purinoceptor agonist-supplemented IPC. These results suggest the crucial involvement of PTX-sensitive and -insensitive G protein coupled purinoceptors in enhanced IPC by supplementation with adenosine, ATP, and UTP.

preconditioning; P2 purinoceptors


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACTIVATION OF G-PROTEIN-COUPLED receptors plays a pivotal role in eliciting ischemic preconditioning (IPC). G protein-coupled receptors are either pertussis toxin (PTX)-sensitive or -insensitive. Former receptors are coupled with Gi/o, which consists of heterotrimeric G protein Galpha beta gamma , whereas later receptors are coupled with Gq/11 family, which consists of the heterotrimetric G protein Galpha q. These receptors fulfill both distinctive and overlapping functions in mediating cellular responses. Activation of Gi/o-coupled receptors induces dissociation of Galpha from Gbeta gamma , which activates phospholipase C and a resultant generation of the second messengers D-myo-inositol 1,4,5-trisphosphate and diacylglycerol, leading to activation of protein kinase C (PKC). PKC activation is an essential step for mediating IPC in most studies (4, 42). On the other hand, signaling pathways linked with activation of Galpha q also converge at the level of phospholipase C (38), although the phospholipase C isoform coupled with Galpha q activation may be different (13, 52).

Involvement of Gi/o-coupled receptors in IPC has been well documented by several investigators, although the relative contribution of Gi/o-coupled receptors to IPC remains a matter of debate. Gi/o proteins have been implicated in cardioprotective effects of IPC in the isolated rat heart (15, 37) and in the intact rat, rabbit, and dog hearts (31, 41, 44). Conflicting results have been reported by Liu and Downey (23), who have demonstrated that preconditioning against infarction does not involve a PTX-sensitive G protein in the intact rat heart. Such a discrepant observation strongly suggests the involvement of both PTX-sensitive and -insensitive G proteins in IPC.

Our previous study (34) has demonstrated that adenosine receptors and P2y purinoceptors play a complementary role in mediating IPC. Adenosine A1 receptors, a major adenosine receptor subtype involved in IPC, are exclusively coupled with PTX-sensitive Gi/o proteins (43), whereas P2y purinoceptors, which are preferentially stimulated by the physiological agonists UTP or ATP are known to be coupled with both Gi/o and PTX-insensitive Galpha q proteins (16, 32) or predominantly Galpha q protein (29, 46), depending on species and cell types. It is therefore anticipated that IPC is mediated by PTX-sensitive adenosine receptors and PTX-insensitive P2y purinoceptors in the rat heart.

Extracellular adenosine has been thought to be derived predominantly from extracellular ATP in the preconditioned heart through hydrolysis by the ectonucleotidase system (18, 19). If this is the case, a significant amount of ATP, the parent molecule of adenosine, should be released into the interstitial space during IPC and sequentially dephosphorylated to adenosine via the ecto-nucleotidase system, which is located on endothelial cells in the microvascular beds (6). Our previous study (34) has demonstrated that IPC significantly increased interstitial fluid (ISF) ATP to a concentration comparable to or even greater than ISF adenosine in some hearts. Further analysis of ISF concentrations of ATP and adenosine revealed a reciprocal correlation between ISF concentrations of ATP and adenosine. Functional studies using antagonists against adenosine receptors and P2y purinoceptors demonstrated that adenosine receptors and P2y purinoceptors play a complementary role in mediating IPC. The present study has tested the additional hypothesis that P2y purinoceptor-mediated preconditioning is coupled to PTX-insensitive G proteins, whereas that mediated by adenosine receptors is coupled to PTX-sensitive G proteins. To achieve this goal, we have examined relative contribution of PTX-sensitive adenosine receptors and -insensitive P2y purinoceptors to myocardial protection afforded by IPC. We then tested whether preconditioning with adenosine and P2y agonists, ATP and UTP mimic IPC or enhance myocardial protection conferred by IPC in a PTX-sensitive and -insensitive manner. Finally, we have investigated the role of nitric oxide (NO) and the signal-transduction pathways involved in PTX-sensitive and -insensitive G protein-coupled purinoceptor-mediated IPC. The results suggest crucial involvement of PTX-insensitive G protein-coupled P2y purinoceptors in conventional as well as in enhanced IPC by supplementation with ATP and UTP in the rat heart. The results also suggest no obligatory role of endogenous NO but additive or synergistic interaction of PKC with tyrosine kinases in PTX-insensitive G protein-coupled P2y purinoceptor-mediated IPC.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Perfusion technique. Male Sprague-Dawley rats weighing 250-300 g were used in the present study. These animals received humane care according to the animal welfare regulations of the Kansai Medical University. The isolated and buffer-perfused rat heart model was prepared as described previously (34).

A latex ballon was interted into the left ventricle through the left atrium to measure isovolumic left ventricular (LV) function. After the baseline measurement, the heart was treated with 1 µM acetylcholine for 5 min to confirm blockade of Gi/o by PTX (23). The heart was then perfused with normal Krebs-Henseleit buffer for 10 min to allow recovery of baseline functions before entering regular protocols. The balloon was filled with saline to produce LV end-diastolic pressure (LVEDP) of 5-10 mmHg at the baseline, and the balloon volume was kept constant throughout the experiment. Coronary flow (CF) was measured by timed collection of the coronary effluent. The hearts producing LV developed pressure of <80 mmHg or a heart rate of <240 beats/min at the baseline were excluded from the study. IPC was introduced by three repeated times of 5-min ischemia and 5-min reperfusion. The recovery of CF, rate pressure products (RPP), and LVEDP obtained 30 min after reperfusion was taken as an end point of myocardial protection because recovery of RPP and LVEDP was correlated well with cardiomyocyte necrosis in this model (26).

Drugs. PTX was purchased from List Biological Laboratories (Campbell, CA). Each 50-µg vial was reconstituted with 0.5 ml of sterile distilled water, mixed with 1.5 ml of phosphate-buffered saline, and administered as a dose of 25 µg/kg ip 48 h before the study. Adenosine receptor antagonist 8-(p-sulfophenyl) theophylline (SPT; 100 µM), and the structurally distinct P2y purinoceptor antagonists suramin (300 µM) or reactive blue (RB; 10 µM) were included in the perfusion buffer 10 min before and during IPC, followed by 10 min of washout perfusion. The NO synthase inhibitor Nomega -nitro-L-arginine methyl ester (L-NAME; 100 µM), the PKC inhibitor Ro318425 (0.3 µM), and the tyrosine kinase inhibitor genistein (50 µM) were included in the perfusion buffer for 40 min in the time-matched perfusion groups or 10 min before and during IPC, followed by 10 min of washout perfusion in the IPC groups. Ro318425 and genistein were dissolved in dimethyl sulfoxide with final concentrations <0.05%. Acetylcholine, SPT, ATP, UTP, and L-NAME were obtained from Sigma (Tokyo, Japan). Suramin and RB were purchased from Alexis (San Diego, CA) and Research Biochemical International (Natick, MA), respectively. Ro318425 and genistein were obtained from Calbiochem (La Jolla, CA) and Wako (Osaka, Japan), respectively.

Statistics. All numerical data are expressed as means ± SE. Statistical analysis was performed with one-way ANOVA and Scheffé's multiple-comparison test. A value of P < 0.05 was considered to be statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PTX reverses acetylcholine-induced bradycardia. PTX blockade of Gi/o proteins was confirmed by elimination of bradycardia induced by acetylcholine (23). The basal contractile function was not significantly different between PTX-pretreated and nontreated heart under Langendorff's perfusion. After the baseline measurements, the rat heart was administered 1 µM acetylcholine for 5 min. In consecutive 113 PTX-nontreated animals, acetylcholine caused a decrease of heart rate from 279 ± 2 (ranging from 240 to 360 beats/min) to 153 ± 3 (ranging from 86 to 232 beats/min), whereas in consecutive 101 PTX-pretreated animals, the muscarinic response was 283 ± 2 (ranging from 240 to 355 beats/min) at the baseline and 242 ± 3 (ranging from 159 to 301 beats/min) after acetylcholine. The minimal heart rate of the mean - 2SD after the acetylcholine test was 182 beats/min in PTX-pretreated hearts. Therefore, PTX-pretreated hearts showing heart rate <182 beats/min after the acetylcholine test were excluded from the study.

PTX partially, but PTX plus suramin or RB completely, inhibits IPC-induced functional protection. Pretreatment with PTX had no significant effect on IPC-induced depression of RPP (Fig. 1A), although administration of suramin or RB tended to mitigate IPC-induced depression of RPP in the heart pretreated with PTX. IPC had no significant effect on LVEDP in all groups of hearts (Fig. 1B). PTX with or without SPT partially and with suramin or RB completely inhibited IPC-induced increase in CF (Fig. 1C). Although pretreatment with PTX had no significant effect on the recovery of RPP, LVEDP, and CF compared with the control heart, IPC-induced improvement of LV function was partially but significantly inhibited by pretreatment with PTX. SPT had no further effect on the PTX-induced inhibition of functional improvement afforded by IPC, whereas the functional improvement was completely abrogated when suramin or RB was administered during IPC in the PTX-pretreated heart.


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  		Fig. 1.   Effect of pertussis toxin (PTX) on functional protection conferred by ischemic preconditioning (IPC). A: rate pressure product (RPP); B: left ventricular end-diastolic pressure (LVEDP); C: coronary flow. PTX (25 µg/kg) was administered intraperitoneally 48 h before the study. 8-(p-sulfophenyl) theophylline (SPT) (100 µM), suramin (Su) (300 µM), or reactive blue (RB) (10 µM) was administered 10 min before and during IPC (hatched boxes). Solid boxes represent ischemia. , Control (n = 7); open circle , PTX (n = 6); black-triangle, IPC (n = 7); triangle , PTX + IPC (n = 6); , PTX + SPT + IPC (n = 6); , PTX + Su + IPC (n = 6); black-lozenge , PTX + RB+ IPC (n = 6). Each symbol represents means ± SE. * P < 0.05, ** P < 0.01 compared with control; dagger  P < 0.05, dagger dagger P < 0.01 compared with IPC.

ATP exerts positive inotropic effect by pretreatment with PTX. To characterize the physiological responses produced by PTX-sensitive adenosine receptors and PTX-insensitive P2y purinoceptors in the isolated rat heart, ATP, UTP, and adenosine were added to the perfusate buffer. ATP and UTP were employed to activate P2y purinoceptors because P2y purinoceptors are both adenine and uridine trisphosphate specific (33), although conflicting results still exist as to the cardiovascular effect of P2y purinoceptor subtypes, i.e., P2y2 or P2y4 purinoceptors, which are activated by both ATP and UTP.

The addition of adenosine produced a negative inotropic effect (NIE) in a dose-dependent manner (Fig. 2A). Pretreatment with PTX significantly inhibited the adenosine-induced NIE, but the addition of suramin had no effect on PTX inhibition of adenosine-induced NIE. ATP also produced NIE comparable to adenosine in a dose-dependent manner (Fig. 2). However, ATP produced a positive inotropic effect (PIE) in the heart pretreated with PTX. The PIE exerted by ATP in hearts pretreated with PTX was abolished by suramin or RB. In contrast with ATP and adenosine, treatment with UTP produced only PIE in a concentration-dependent manner. The PIE was not affected by pretreatment with PTX but was abolished by suramin or RB.


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  		Fig. 2.   Effect of PTX, Su, and RB on adenosine-, ATP-, and UTP-induced changes in RPP (A) and coronary flow (B). PTX (25 µg/kg) was administered intraperitoneally 48 h before study. Adenosine, ATP, and UTP at concentrations ranging between 10 and 100 µM were administered in a cumulative manner. Su (300 µM) or RB (10 µM) was administered 10 min before addition of the purinoceptor agonists and was present throughout the dose-response study. Each symbol represents means ± SE of 6 experiments.

Adenosine increased CF in a dose-dependent manner (Fig. 2B). The increase in CF induced by adenosine was abrogated by pretreatment with PTX, but the addition of suramin or RB had no effect on PTX-inhibition of adenosine-induced increase in CF. ATP increased CF with a magnitude similar to that observed with adenosine at the same concentration. CF was increased with an increasing concentration of ATP, giving rise to 60% increase in CF with 100 µM ATP. The ATP-induced increase in CF was significantly attenuated by pretreatment with PTX and was completely abrogated by suramin or RB in the heart pretreated with PTX. CF was also increased by UTP in a concentration-dependent manner, but the magnitude of CF increase was much less than that observed with ATP and adenosine. The increase in CF induced by UTP was inhibited by pretreatment with PTX and was abolished by subsequent addition of suramin or RB.

Enhanced IPC supplemented with adenosine, ATP, or UTP. To further clarify the role of PTX-sensitive adenosine receptors and PTX-insensitive P2y purinoceptors in IPC-induced functional protection, adenosine, ATP, and UTP were used as tools for pharmacological preconditioning. We chose 30 µM adenosine and ATP because this concentration of adenosine and ATP increased ISF ATP and adenosine much higher than those induced by IPC but yielded only a modest NIE, which is acceptable for clinical setting of myocardial protection. UTP at a concentration of 50 µM was chosen because this concentration of UTP produced PIE comparable to 30 µM ATP in the PTX-pretreated heart and was thought to be equipotent with 30 µM ATP in stimulating P2y purinoceptors. Adenosine, ATP, and UTP were administered during IPC or a time-matched preconditioning period, followed by 10-min washout perfusion before 25 min of ischemia.

Preconditioning with adenosine and ATP depressed RPP but increased CF, whereas preconditioning with UTP increased RPP and CF (Fig. 3). However, these hemodynamic changes were returned to the baseline after 10-min washout perfusion. On the sustained ischemia, LVEDP rose in these hearts with a time course and a magnitude similar to the control heart. Consequently, preconditioning with adenosine, ATP, and UTP had no salutary effect on the recovery of RPP, LVEDP, and CF. We then investigated whether supplementation with adenosine, ATP, and UTP with IPC could enhance functional protection afforded by IPC, providing that cardioprotection mediated by IPC requires not only G protein-coupled receptor stimulation but also unknown triggers induced by a brief period of ischemia and reperfusion. The appreciable changes in hemodynamics during the P2y-supplemented IPC were that UTP significantly attenuated IPC-induced depression of RPP and all the purinergic agonists significantly augmented an IPC-induced increase in CF. These purinergic agonists-supplemented IPC significantly shortened the time to onset of ischemic contracture induced by IPC. However, IPC supplemented with adenosine, ATP, or UTP conferred superior functional protection over IPC, as indicated by enhanced recovery of RPP and CF and lowered LVEDP.


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  		Fig. 3.   Effect of preconditioning with adenosine, ATP, and UTP and IPC supplemented with adenosine, ATP, and UTP on functional recovery. Adenosine (30 µM), ATP (30 µM), and UTP (50 µM) were administered for 40 min (gray box) and excluded for 10 min before 25 min of ischemia. These purinergic agonists were also included 10 min before and during IPC (hatched boxes). Solid boxes represent ischemia. A: RPP; B: LVEDP; C: coronary flow. ×, IPC (n = 7); open circle , adenosine (n = 7); , adenosine + IPC (n = 7); triangle , ATP (n = 7); black-triangle, ATP + IPC (n = 7); , UTP (n = 7); , UTP + IPC (n = 7). Each symbol represents means ± SE. * P < 0.05, ** P < 0.01 compared with IPC.

Role of PTX-sensitive and -insensitive G protein in enhanced IPC by supplementation with adenosine, ATP, and UTP. We then examined PTX-sensitive and -insensitive G protein involvement in enhanced IPC by supplementation with adenosine, ATP, and UTP (Fig. 4). RPP, LVEDP, and CF were evaluated 30 min after reperfusion. Enhanced functional protection afforded by adenosine-supplemented IPC was abolished by pretreatment with PTX and SPT but not by suramin or RB. Additional slight inhibition of adenosine-enhanced functional protection was caused by suramin or RB after pretreatment with PTX. In contrast, enhanced functional protection afforded by ATP-supplemented IPC was not inhibited by PTX, SPT, suramin, or RB alone but was abrogated by PTX plus suramin or RB. On the other hand, UTP-enhanced IPC was not affected by PTX pretreatment nor SPT but was inhibited by suramin or RB.


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  		Fig. 4.   Effect of PTX, SPT, Su, and RB on enhanced functional protection conferred by adenosine-, ATP-, and UTP-supplemented IPC. PTX (25 µg/kg) was administered intraperitoneally 48 h before the study. Adenosine (Ad) (30 µM), ATP (30 µM), or UTP (50 µM) was administered 10 min before and during IPC. SPT (100 µM), Su (300 µM), or RB (10 µM) was also administered 10 min before and during IPC. Each bar graph represents means ± SE of 6 or 7 experiments. * P < 0.01 vs. Ad+PC; dagger  P < 0.01 vs. ATP+PC; #P < 0.01 vs. UTP+PC. A: RPP; B: LVEDP; C: coronary flow.

Role of NO in PTX-sensitive and -insensitive G protein-coupled purinoceptor-mediated IPC. Because NO may contribute to the early as well as to the late IPC (2, 3, 25), we have investigated the role of NO in PTX-sensitive and -insensitive G protein-coupled purinoceptor-mediated IPC. Robust activation of PTX-sensitive or -insensitive G protein-coupled purinoceptors was performed by supplementation with adenosine or ATP during IPC in the absence or presence of pretreatment with PTX, respectively. Treatment with L-NAME before and during IPC, followed by 10 min of washout perfusion, had no significant effect on functional protection afforded by adenosine-supplemented IPC (Fig. 5). Time-matched treatment with L-NAME without IPC did not modify functional recovery compared with the control heart. In addition, functional protection afforded by ATP-supplemented IPC after pretreatment with PTX also was not significantly affected by treatment with L-NAME.


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  		Fig. 5.   Effect of Nomega -nitro-L-arginine methyl ester (L-NAME) on functional protection conferred by adenosine-supplemented IPC without PTX pretreatment and ATP-supplemented IPC with PTX pretreatment. L-NAME (100 µM) was administered 10 min before and during IPC. Time-matched L-NAME treatment was performed for 40 min, followed by 10-min washout perfusion. Each bar graph represents means ± SE of 6 or 7 experiments. A: RPP; B: LVEDP; C: coronary flow.

Role of PKC and tyrosine kinases in PTX-sensitive and -insensitive G protein-coupled purinoceptor-mediated IPC. We have also investigated signal transduction pathways involved in IPC mediated by PTX-sensitive and -insensitive G protein-coupled purinoceptors with the use of PKC inhibitor Ro318425 and tyrosine kinase inhibitor genistein. Treatment with Ro318425 and genistein alone or in combination for 40 min, followed by 10 min of washout perfusion before 30 min of ischemia, had no significant effect on the functional recovery 30 min after reperfusion (Fig. 6). Functional protection afforded by adenosine-supplemented IPC was not significantly affected by treatment with Ro318425 or genistein alone. Similarly, functional protection afforded by ATP-supplemented IPC after pretreatment with PTX was not significantly inhibited by treatment with Ro318425 or genistein alone. On the contrary, functional protection afforded by adenosine-supplemented IPC without PTX pretreatment or ATP-supplemented IPC with PTX pretreatment was significantly inhibited by combined treatment with Ro318425 and genistein, although this treatment modality was more potent in inhibiting adenosine-supplemented IPC without PTX pretreatment than ATP-supplemented IPC with PTX pretreatment.


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  		Fig. 6.   Effect of Ro318425 (Ro) and genistein (Ge) on functional protection conferred by adenosine-supplemented IPC without PTX pretreatment and ATP-supplemented IPC with PTX pretreatment. Ro318425 and genistein were administered 10 min before and during IPC. Time-matched Ro318425 and genistein treatments were performed for 40 min, followed by 10-min washout perfusion. Each bar graph represents means ± SE of 6 or 7 experiments. * P < 0.05; ** P < 0.01 vs. Ad + IPC; dagger  P < 0.05 vs. PTX + ATP + IPC. A: RPP; B: LVEDP; C: coronary flow.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We (34) have previously shown that extracellular adenosine and ATP play a complementary role in IPC via activation of adenosine receptors and P2y purinoceptors, respectively. Several lines of evidence obtained in the present study suggest further that IPC mediated by adenosine is coupled with PTX-sensitive G protein whereas that mediated by ATP is coupled primarily with PTX-insensitive G protein. Functional protection afforded by IPC was partially inhibited by pretreatment with PTX whereas it was completely abrogated by P2y antagonist suramin or RB in combination with PTX pretreatment, indicating that PTX-insensitive component of functional protection is derived from P2y purinoceptors. SPT exerted no additional inhibition of IPC-induced functional protection over pretreatment with PTX, suggesting that adenosine receptors are exclusively coupled with PTX-sensitive G protein. Supplementation with exogenous adenosine, ATP, and UTP during IPC enhanced functional protection afforded by IPC. Functional protection mediated by supplementation with adenosine was abrogated by pretreatment with PTX or STP, indicating that exogenous adenosine potentiated functional protection via activation of PTX-sensitive G protein-coupled adenosine receptors. In contrast, ATP enhanced IPC was not affected by PTX nor SPT but was abrogated by PTX plus suramin or RB, indicating that exogenous ATP potentiated functional protection via activation of both PTX-sensitive G protein-coupled adenosine receptors and PTX-insensitive G protein-coupled P2y purinoceptors. The inability of suramin or RB to abolish ATP-enhanced functional protection may be because there is a sufficient amount of ISF adenosine hydrolyzed from exogenous ATP that could strongly activate PTX-sensitive adenosine receptors. On the other hand, UTP-enhanced IPC was not affected by PTX pretreatment nor SPT but was abolished by suramin or RB, indicating that exogenous UTP potentiated functional protection exclusively via activation of PTX-insensitive G protein-coupled P2y purinoceptors.

PTX-sensitive G protein involvement in IPC remains a controversial issue. Liu and Downey (23) reported that pretreatment with PTX failed to block the protective effects of IPC in the rat heart, whereas the same group of investigators demonstrated the blockade of infarct size-limiting effect of IPC by PTX in the rabbit heart (44). The species difference in response to PTX in preconditioned hearts is consistent with the fact that adenosine receptors do not play a significant role in IPC in the rat heart (22), whereas they do play an important role in IPC in the rabbit heart (24). These observations support the notion that PTX-insensitive signaling pathways are operated in IPC in the rat heart. However, it has been reported (41) even in the rat heart that PTX completely blocked IPC-induced infarct size limitation. These conflicting observations may be related to the difference in activity of the ecto-nucleotidase system, which determines ISF adenosine and ATP as proposed in the previous study (34).

We have employed ATP and UTP to stimulate P2y purinoceptors. It is known that ATP and UTP stimulate common and distinct P2y purinoceptor subtypes. Studies that use expression cloning (33) indicate that ATP stimulates most potently P2y2 and weakly P2y4 purinoceptors, whereas UTP stimulates P2y2 and P2y4 purinoceptors equally. Thus P2y2 purinoceptors are likely a common subtype potently stimulated by ATP and UTP. In the present study, ATP and UTP provoked an increase in CF in a dose-dependent manner, although the potency was much greater with ATP. When the coronary vasodilatation response by ATP was tested in the heart pretreated with PTX, the increase in CF was blunted. Because the adenosine-induced increase in CF was abrogated by pretreatment with PTX, a greater increase in CF observed with ATP compared with UTP was thought to be due to the formation of adenosine. It should also be noted that UTP or its degradation products may provoke coronary vasodilatation through certain PTX-sensitive P2y purinoceptor subtypes because PTX pretreatment blunted UTP-induced increase in coronary flow. However, the increase in CF by ATP and UTP is at least partially attributed to the stimulation of PTX-insensitive P2y purinoceptors because suramin or RB eliminated ATP- or UTP-induced increase in CF in the PTX-pretreated heart. Because P2y2 receptor stimulation provokes vasodilatation (53) whereas P2y4 receptor stimulation provokes vasoconstriction (30), it is suggested that P2y2 receptors are the most likely candidate for suramin- or RB-sensitive CF increase by ATP and UTP. Other candidates of P2y purinoceptor subtypes responsible for the PTX-insensitive and the suramin- or RB-sensitive coronary vascular response are the P2y1 receptor and P2y6 receptors, which are potently stimulated by ADP and UDP after degradation of ATP and UTP, respectively. Although P2y6 receptor stimulation produces vasoconstriction (30), P2y1 receptor stimulation could produce vasodilatation (28). Therefore, involvement of P2y1 receptors in coronary vasodilatation response produced by ATP cannot be excluded.

PTX blunted NIE produced by adenosine and converted ATP-induced NIE to PIE. Because UTP also produced PIE in the heart pretreated with PTX and suramin or RB completely abrogated PIE produced by ATP and UTP, it is suggested that this PIE is mediated through PTX-insensitive P2y purinoceptor stimulation. It has been shown (35) that ATP and UTP produces PIE through the signaling pathway independent of phosphoinositide response, which is commonly provoked by all of the known agonists coupled with Gi/o or Gq/11. It is therefore suggested that the mechanism of PIE produced by ATP and UTP in the PTX-pretreated heart is distinct from that mediating coronary vasodilatation.

Although preconditioning with adenosine, ATP, or UTP failed to elicit cardioprotection, supplementation of these purinergic agonists successfully enhanced functional protection afforded by IPC. Similar cardioprotection by combination of adenosine and IPC has been reported by Toyoda and associates (45) who demonstrated that adenosine-enhanced IPC exerted antistunning and anti-infarct effects, whereas bolus infusion of adenosine alone conferred only anti-infarct effect in the sheep heart. The inability of adenosine to induce functional protection is not due to insufficient concentrations of ISF adenosine because addition of 10 µM adenosine increased an ISF concentration of adenosine nearly twofold of that observed with IPC (34). We have employed adenosine with a "preconditioning" but not "pretreatment" modality. Because IPC was capable of mediating myocardial protection even after ISF adenosine had been washed away (34), continuing presence of adenosine into the sustained ischemia appears not to be necessary. The cardioprotective effect of pretreatment with adenosine against ischemic injury is associated with reductions of ATP depletion, acidosis, and accumulation of calcium that may be attributed to slower rate of energy consumption (8). The ATP saving effect of adenosine is consistent with the fact that adenosine slows the onset of ischemic contracture (8, 17, 21). The present study has, however, clearly shown that adenosine preconditioning had no effect on ischemic contracture and provided no functional protection when adenosine was eliminated from the buffer before the sustained ischemia. IPC, in contrast, shortened the time to onset of ischemic contracture and increased the magnitude of contracture but conferred myocardial protection. Lasley and Konyn (20) and Cave (5) documented similar unsuccessful cardioprotection by adenosine used as a preconditioning modality. Thus the mechanism of cardioprotection mediated by pretreatment with adenosine and that mediated by IPC may be fundamentally different. This assumption may not necessarily contradict the hypothesis that adenosine is involved in IPC. Rather, it is suggested that the cardioprotective effect of IPC is elicited by adenosine in concert with other yet-unidentified triggers produced by a brief ischemia and reperfusion.

Our present study suggests that both PTX-sensitive and -insensitive G protein-coupled P2y purinoceptors can stimulate the common signaling cascade in augmenting IPC. Supplementation with adenosine, ATP, and UTP during IPC conferred similar enhancement of functional protection. Adenosine-supplemented IPC-induced functional protection was abolished by PTX and SPT, and we did not note additional inhibition of functional protection by suramin or RB, suggesting that adenosine-supplemented IPC-induced functional protection is mediated predominantly by PTX-sensitive G protein-coupled adenosine receptors. In contrast, ATP enhanced IPC was not inhibited by PTX, SPT, suramin, nor RB alone but was abrogated by PTX plus suramin or RB. On the other hand, UTP-enhanced IPC was not affected PTX nor SPT but was abolished by suramin or RB. These observations provide a rationale for employing agonists that stimulate either PTX-sensitive or -insensitive G protein-coupled receptors during IPC to enhance cardioprotection.

The fact that ischemic contracture was attenuated by treatment with P1 and P2y purinoceptor antagonists suggests that enhanced ischemic contracture by IPC was a purinoceptor-triggered process. This idea was supported by the fact that IPC-induced acceleration of ischemic contracture was enhanced by supplementation with adenosine, ATP, and UTP and was attenuated by pretreatment with PTX and suramin or RB. It is known that contracture usually occurs at the cessation of anaerobic glycolysis. A recent study (40) suggests that inhibition of ischemia-induced activation of p38 mitogen-activated protein (MAP) kinase by IPC inhibits anaerobic glycolysis and enhances ischemic contracture, but reduces intracellular Ca2+ overload by inhibiting acidosis and subsequent Na+/H+ exchange activation. Thus IPC-induced inactivation of p38 MAP kinase during index ischemia may represent a mechanism of accelerated contracture and paradoxical alleviation of ischemia and reperfusion injury afforded by IPC. This attractive hypothesis remains to be investigated.

We have investigated the role of NO in preconditioning with PTX-sensitive and -insensitive G protein-coupled purinoceptor stimulation. Treatment with L-NAME at a concentration (100 µM) known to abolish NO synthesis in comparable experimental models (9) failed to inhibit functional protection afforded by adenosine-supplemented IPC without PTX pretreatment and ATP-supplemented IPC with PTX pretreatment. These results tend to suggest that endogenous NO is not a necessary component in early IPC mediated by both PTX-sensitive and -insensitive G-protein-coupled purinoceptor stimulation in our experimental model. The contribution of NO in early IPC has been controversial. This is in contrast with a well-established role of NO in late IPC (2, 3). The argument for the involvement of NO in early IPC was supported by the observations that L-NAME attenuated IPC-induced protection against cell death, LV dysfunction, and arrhythmias (7, 25, 49). In contrast, the NO synthase antagonist was unable to abrogate protective effects of IPC against cell death, LV dysfunction and arrhythmias (27, 36, 51). Possible explanation for these divergent observations with respect to the contribution of NO to early and late IPC may be attributed to differences in species and protocols and phases of IPC studied. In some experimental models, NO acts as an essential trigger of preconditioning, whereas in others there are alternative pathways that are able to bypass NO-induced signal transduction to elicit preconditioning for cardioprotection.

We then investigated signal transduction pathways downstream of PTX-sensitive and -insensitive G protein-coupled purinoceptors because our present study suggested that both Gi/o and Gq/11 proteins equally contribute to IPC. Recent studies have demonstrated that PKC and tyrosine kinases play a crucial role in IPC, although relative positions and interactions of these signaling pathways remain elusive. Baines et al. (1) have hypothesized that tyrosine kinases, especially MAP kinases, are downstream of PKC in the rabbit model of IPC, whereas Vahlhaus at al. (47) and Fryer et al. (11) have proposed that PKC and tyrosine kinases exist in parallel pathways to confer cardioprotection in the pig and the rat models of IPC, respectively. Our study demonstrated that combination of PKC inhibitor Ro318425 with a tyrosine kinase inhibitor genistein but not each alone attenuated functional protection afforded by adenosine-supplemented IPC without PTX pretreatment and ATP-supplemented IPC with PTX pretreatment. The results tend to suggest that PKC and tyrosine kinases responsible for mediating both PXT-sensitive and -insensitive IPC exist in a parallel position and that these pathways act in an additive or a synergistic fashion to provoke powerful cardioprotective signaling, although specific PKC and tyrosine kinases involved in IPC remain to be identified. This notion is consistent with PKC signaling complex hypothesis in that receptors for activated C kinase acts as adaptors for not only PKC but also for enzymes of other signaling pathways that exist in parallel to activation of PKC (39). Such a signaling module formation could amplify activities of another kinase. Vondriska et al. (50) have documented that association of Src tyrosine kinases with PKC-epsilon in the conscious rabbit model of NO-induced late preconditioning dramatically increases Src tyrosine kinase activity. The opposite may be true for Src tyrosine kinase-dependent increase in PKC-epsilon activity, because Hattori et al. (14) have demonstrated that inhibition of Src tyrosine kinases by PP1 abrogated PKC-epsilon activity in the rat model of early IPC. Thus simultaneous activation of Src tyrosine kinase and PKC-epsilon can increase activities of another kinase in a synergistic fashion, although these kinases are activated by independent triggers. The PKC-epsilon -Src module is merely a paradigm of signaling complexes. Perhaps many more PKC-tyrosine kinase-signaling complexes are recruited on IPC that is thought to render IPC-induced cardioprotective signaling pathways redundant and resistant to inhibition by conventional doses of PKC or tyrosine kinase inhibitors. IPC mediated by PTX-sensitive and -insensitive G protein-coupled purinoceptors appears to share such signaling complexes downstream of Gi/o and Gq/11 proteins, respectively. However, the fact that combined treatment with Ro318425 and genistein was more potent in inhibiting adenosine-supplemented IPC without PTX pretreatment than ATP-supplemented IPC with PTX pretreatment suggests that there may be additional signaling pathways besides PKC and tyrosine kinases in Gq/11 protein-coupled purinoceptor-mediated IPC. This possibility remains to be investigated.

In conclusion, the results of the present study suggest that PTX-sensitive adenosine receptors and -insensitive P2y purinoceptors are involved in early IPC. These purinoceptor stimulations alone do not successfully trigger cardioprotection, but in concert with yet-unidentified triggers produced on a brief ischemia and reperfusion, they integrate cardioprotective signaling through a mechanism independent of NO but dependent on a cooperative interaction of PKC with tyrosine kinase signal-transduction pathways.


    ACKNOWLEDGEMENTS

This work was supported in part by Research Grant 10671275 from the Ministry of Education, Science, and Culture of Japan.


    FOOTNOTES

Present address of K. Lu: Department of Cardiothoracic Surgery, Capital University of Medical Science, Beijing Friendship Hospital, 95 Yongan Road, Beijing 100050, China.

Address for reprint requests and other correspondence: H. Otani, Dept. of Thoracic and Cardiovascular Surgery, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8507, Japan (E-mail: otanih{at}takii.kmu.ac.jp).

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.

10.1152/ajpheart.00771.2001

Received 30 August 2001; accepted in final form 16 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Baines, CP, Wang L, Cohen MV, and Downey JM. Protein tyrosine kinase is downstream of protein kinase C for ischemic preconditioning's anti-infarct effect in the rabbit heart. J Mol Cell Cardiol 30: 383-392, 1998.

2.   Bolli, R, Bhatti ZA, Tang XL, Qiu Y, Zhang Q, Guo Y, and Jadoon AK. Evidence that late preconditioning against myocardial stunning in coucious rabbits is triggered by the generation of nitric oxide. Circ Res 81: 42-52, 1997.

3.   Bolli, R, Dawn B, Tang XL, Qiu Y, Ping P, Xuan YT, Jones WK, Takano H, Guo Y, and Zhang J. The nitric oxide hypothesis of late preconditioning. Basic Res Cardiol 93: 325-338, 1998.

4.   Brooks, G, and Hearse DJ. Role of protein kinase C in ischemic preconditioning: player or spectator? Circ Res 79: 627-630, 1996.

5.   Cave, AC. Improved functional recovery by ischaemic preconditioning is not mediated by adenosine in the globally ischaemic isolated rat heart. Cardiovasc Res 45: 131-133, 2000.

6.   Coade, SB, and Pearson JD. Metabolism of adenine nucleotides in human blood. Circ Res 65: 531-537, 1989.

7.   Csnoka, C, Szilvassy Z, Fulop F, Pali T, Blasig IE, Tosaki A, Schulz R, and Ferdinandy P. Classic preconditioning decreases the harmful accumulation of nitric oxide during ischemia and reperfusion in rat hearts. Circulation 100: 2260-2266, 1999.

8.   Dickeson, JM, Blank JL, and Hill SJ. Human adenosine A1 receptor and P2y2-purinoceptor-mediated activation of the mitogen-activated protein kinase cascade in transfected CHO cells. Br J Pharmacol 124: 1491-1499, 1998.

9.   Engelman, DT, Watanabe M, Engelman RM, Rousou JA, Flack JE, III, Deaton DW, and Das DK. Constitutive nitric oxide release is impaired after ischemia and reperfusion. J Thorac Cardiovasc Surg 110: 1047-1053, 1995.

10.   Fralix, TA, Murphy E, London RE, and Steenbergen C. Protective effects of adenosine in the perfused rat heart: changes in metabolism and intracellular ion homeostasis. Am J Physiol Cell Physiol 264: C986-C994, 1993.

11.   Fryer, RM, Schultz JEJ, Hsu AK, and Gross GJ. Importance of PKC and tyrosine kinase in single or multiple cycles of preconditioning in rat hearts. Am J Physiol Heart Circ Physiol 276: H1229-H1235, 1999.

12.   Harper, S, Webb TE, Charlton SJ, Ng LL, and Boarder MR. Evidence that P2y4 nucleotide receptors are involved in the regulation of rat aortic smooth muscle cells by UTP and ATP. Br J Pharmacol 124: 703-710, 1998.

13.   Harrison, GJ, Willis RJ, and Headrick JP. Extracellular adenosine levels and cellular energy metabolism in ischemically preconditioned rat heart. Cardiovasc Res 40: 74-87, 1998.

14.   Hattori, R, Otani H, Uchiyama T, Imamura H, Cui J, Maulik N, Cordis JA, Zhu L, and Das DK. Src tyrosine kinase is the trigger but not the mediator of ischemic preconditioning. Am J Physiol Heart Circ Physiol 281: H1066-H1074, 2001.

15.   Heasley, LE, Storey B, Fanger GR, Butterfield L, Zamarripa J, Blumberg D, and Maue RA. GTPase-deficient Galpha 16 and Galpha q induce PC12 cell differentiation and persistent activation of cJun NH2-terminal kinases. Mol Cell Biol 16: 648-656, 1996.

16.   Hu, K, and Nattel S. Mechanism of ischemic preconditioning in rat hearts. Involvement of alpha 1B-adrenoceptors, pertussis toxin-sensitive G proteins, and protein kinase C. Circulation 92: 2259-2265, 1995.

17.   Janier, MF, Vanoverschelde JL, and Bergmann SR. Adenosine protects ischemic and reperfused myocardium by receptor-mediated mechanisms. Am J Physiol Heart Circ Physiol 264: H163-H170, 1993.

18.   Kitakaze, M, Hori M, Takashima S, Sato H, Inue M, and Kamada T. Ischemic preconditioning increases adenosine release and 5'-nucleotidase activity during myocardial ischemia and reperfusion in dogs: implication for myocardial salvage. Circulation 87: 208-215, 1993.

19.   Kitakaze, M, Hori M, Takashima S, Sato H, Shinozaki Y, Chujo M, Mori H, Inoue M, and Kamada T. Infarct size-limiting effect of ischemic preconditioning is blunted by inhibition of 5'-nucleotidase activity and attenuation of adenosine release. Circulation 89: 1237-1246, 1994.

20.   Lasley, RD, and Konyn PJ. Effects of ischemic and adenosine preconditioning on interstitial fluid adenosine and myocardial infarct size. Am J Physiol Heart Circ Physiol 269: H1460-H1466, 1995.

21.   Lasley, RD, Rhee JW, Wylen V, and Mentzer RM, Jr. Adenosine A1 receptor mediated protection of the globally ischemic isolated rat heart. J Mol Cell Cardiol 22: 39-47, 1990.

22.   Li, Y, and Kloner RA. The cardioprotective effects of ischemic "preconditioning" are not mediated by adenosine receptors in rat hearts. Circulation 87: 1642-1648, 1993.

23.   Liu, Y, and Downey J. Preconditioning against infarction in the rat heart does not involve a pertussis toxin sensitive G protein. Cardiovasc Res 27: 608-611, 1993.

24.   Liu, GS, Thornton J, Van Winkle DM, Stanley AWH, Olsson RA, and Downey JM. Protection against infarction afforded by preconditioning is mediated by A1 adenosine receptors in rabbit heart. Circulation 84: 350-356, 1991.

25.   Lochner, A, Marais E, Genade S, and Moolman JA. Nitric oxide: a trigger for classic preconditioning? Am J Physiol Heart Circ Physiol 279: H2752-H2765, 2000.

26.   Lu, K, Otani H, Yamamura Y, Nakao Y, Hattori R, Ninomiya H, Osako M, and Imamura H. Protein kinase C isoform-dependent myocardial protection by ischemic preconditioning and potassium cardioplegia. J Thorac Cardiovasc Surg 121: 137-148, 2001.

27.   Lu, HR, Remeysen P, and Parratt J. Does the anti-arrhythmic effect of ischemic preconditioning in rats involve the L-arginine nitric oxide pathway? J Cardiovasc Pharmacol 25: 524-530, 1995.

28.   Marrelli, SP, Khorovets A, Johnson TD, Childres WF, and Bryan RM. P2 purinoceptor-mediated dilations in the rat middle cerebral artery after ischemia-reperfusion. Am J Physiol Heart Circ Physiol 276: H33-H41, 1999.

29.   Matsubayashi, T, Matsuura H, and Ehara T. On the mechanism of the enhancement of delayed rectifier K+ current by extracellular ATP in guinea-pig ventricular myocytes. Pflügers Arch 437: 635-642, 1999.

30.   Matsumoto, T, Nakane T, and Chiba S. UTP induces vascular responses in the isolated and perfused canine coronary artery via UTP-preferring P2y receptors. Br J Pharmacol 122: 1625-1632, 1997.

31.   Miura, K, Kano S, Nakai T, Satoh K, Hoshi K, and Ichihara K. Inhibitory effects of glibenclamide and pertussis toxin on the attenuation of ischemia-induced myocardial acidosis following ischemic preconditioning in dogs. Jpn Circ J 61: 709-714, 1997.

32.   Murthy, KS, and Makhlouf GM. Coexpression of ligand-gated P2x and G protein-coupled P2y receptors in smooth muscle. Preferential activation of P2y receptors coupled to phospholipase C (PLC)-beta 1 via Galpha q/11 and to PLC-beta 3 via Gbeta gamma i3. J Biol Chem 273: 4695-4704, 1998.

33.   Nicholas, RA, Watt WC, Lazarowski ER, Li Q, and Harden K. Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: identification of a UDP- and UTP-specific receptor. Mol Pharmacol 50: 224-229, 1996.

34.   Ninomiya, H, Otani H, Lu K, Uchiyama T, Nakao Y, Yamamura T, Kido M, Osako M, and Imamura H. Complementary role of extracellular adenosine and ATP in ischemic preconditioning in rat heart. Am J Physiol Heart Circ Physiol 282: H1810-H1820, 2002.

35.   Podrasky, E, Xu D, and Liang BT. A novel phospholipase C- and cAMP-independent positive inotropic mechanism via a P2 purinoceptor. Am J Physiol Heart Circ Physiol 273: H2380-H2387, 1997.

36.   Post, H, Schulz R, Behrends M, Gres P, Umschlag C, and Heusch G. No involvement of endogenous nitric oxide in classical ischemic preconditioning in swine. J Mol Cell Cardiol 32: 725-733, 2000.

37.   Ravingerova, T, Pyne NJ, and Parratt JR. Ischaemic preconditioning in the rat heart: the role of G-proteins and adrenergic stimulation. Mol Cell Biochem 147: 123-128, 1995.

38.   Rocca, GJD, van Biesen T, Daaka Y, Luttrell DK, Luttrell LM, and Lefkowitz RJ. Ras-dependent mitogen-activated protein kinase activation by G protein-coupled receptors. J Biol Chem 272: 19125-19132, 1997.

39.   Schechtman, D, and Mochly-Rosen D. Adaptor proteins in protein kinase C-mediated signal transduction. Oncogene 20: 6339-6347, 2001.

40.   Schneider, S, Chen W, Hou J, Steenbergen C, and Murphy E. Inhibition of p38 MAPK alpha /beta reduces ischemic injury and does not block protective effects of preconditioning. Am J Physiol Heart Circ Physiol 280: H499-H508, 2001.

41.   Schultz, JEJ, Hsu AK, Barbieri JT, Li PL, and Gross GJ. Pertussis toxin abolishes the cardioprotective effect of ischemic preconditioning in intact rat heart. Am J Physiol Heart Circ Physiol 275: H495-H500, 1998.

42.   Simkhovich, BZ, Przyklenk K, and Kloner RA. Role of protein kinase C as a cellular mediator of ischemic preconditioning: a critical review. Cardiovasc Res 40: 9-22, 1998.

43.   Stiles, GL. Adenosine receptors: physiologic regulation and biochemical mechanisms. News Physiol Sci 6: 161-165, 1991.

44.   Thornton, JD, Liu GS, and Downey JM. Pretreatment with pertussis toxin blocks the protective effects of preconditioning: evidence for a G-protein mechanism. J Mol Cell Cardiol 24: 311-320, 1993.

45.   Toyoda, Y, Gregorio VD, Parker RA, Levitsky S, and McCully JD. Anti-stunning and anti-infarct effects of adenosine-enhanced ischemic preconditioning. Circulation 102, Suppl3: III326-III331, 2000.

46.   Tu, MT, Luo SF, Wang CC, Chien CS, Chiu CT, Lin CC, and Yang CM. P2y2 receptor-mediated proliferation of C6 glioma cells via activation of Ras/Raf/MEK/MAPK pathway. Br J Pharmacol 129: 1481-1489, 2000.

47.   Vahlhaus, C, Schulz R, Post H, Rose J, and Heusch G. Prevention of ischemic preconditioning only by combined inhibition of protein kinase C and protein tyrosine kinase in pigs. J Mol Cell Cardiol 30: 197-209, 1998.

48.   Van Wylen, DGL, Willis J, Sodhi J, Weiss RJ, Lasley RD, and Mentzer RM. Cardiac microdialysis to estimate interstitial adenosine and coronary blood flow. Am J Physiol Heart Circ Physiol 258: H1642-H1649, 1990.

49.   Vegh, A, Szekeres L, and Parratt J. Preconditioning of the ischaemic myocardium; involvement of the L-arginine nitric oxide pathway. Br J Pharmacol 107: 648-652, 1992.

50.   Vondriska, TM, Zhang J, Song C, Tang XL, Cao X, Baines CP, Pass JM, Wang S, Bolli R, and Ping P. Protein kinase C epsilon -Src modules direct signal transduction in nitric oxide-induced cardioprotection. Circ Res 88: 1306-1313, 2001.

51.   Weselcouch, EO, Baird AJ, Sleph P, and Grover GJ. Inhibition of nitric oxide synthesis does not affect ischemic preconditioning in isolated perfused rat hearts. Am J Physiol Heart Circ Physiol 268: H242-H249, 1995.

52.   Yang, L, Bafy G, Rhee SG, Manning D, Hansen CA, and Williamson JR. Pertussis toxin-sensitive Gi protein involvement in epidermal growth factor-induced activation of phospholipase C-gamma in rat hepatocytes. J Biol Chem 266: 22451-22458, 1991.

53.   You, J, Johnson TD, Marrelli SP, Mombouli JV, and Bryan RM. P2u receptor-mediated release of endothelium-derived relaxing factor/nitric oxide and endothelium-derived hyperpolarizing factor from cerebrovascular endothelium in rats. Stroke 30: 1125-1133, 1999.

Am J Physiol Heart Circ Physiol 282(5):H1933-H1943
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