Reperfusion kinase phosphorylation is essential but not sufficient in the mediation of pharmacological preconditioning: Characterisation in the bi-phasic profile of early and late protection.
OBJECTIVE
Pharmacological preconditioning (PPC) triggers early (ePPC) and delayed protection (dPPC), occurring within 1 h or after 24 h following the preconditioning stimulus, respectively, through recruitment of protein kinase signalling. Angiotensin II (ATII) is a recognised trigger of PPC, recruiting kinases and transcription factors known to be involved in both phases of protection. Our objectives were to determine whether ATII is capable of triggering dPPC and whether recruitment of pro-survival kinases, Akt and extracellular signal-regulated kinase (ERK), following the injurious ischaemic insult is essential for the mediation of PPC.


METHODS
In a mouse Langendorff model of ischaemia/reperfusion injury, we undertook to determine whether ATII triggers both ePPC and dPPC. Western blot analysis was used to determine kinase phosphorylation at reperfusion, and kinase inhibitors wortmannin and PD98059 were used to ascertain the significance of kinase regulation.


RESULTS
We demonstrated that ATII triggered PPC with attenuation of infarction at 1 and 24 h (19+/-4% and 25+/-4% versus control, 35+/-4% of risk zone, p < 0.05), consistent with the ePPC and dPPC time-course. This bi-phasic protection was associated with significant post-ischaemic phosphorylation of both Akt and ERK within the first 5 min of reperfusion. Akt and ERK phosphorylation was increased following ePPC by 4.5+/-0.5 and 1.9+/-0.6 fold, respectively (p < 0.001), and dPPC by 24+/-2.0 and 2.1+/-0.1 fold, respectively (p < 0.001). Both wortmannin and PD98059 administered during reperfusion ameliorated the phosphorylation of Akt and ERK and abrogated the resistance to infarction resulting from both ePPC and dPPC (33+/-3% and 35+/-4%, respectively, versus controls 33+/-4% and 33+/-5%, p = NS). There was no evidence of augmented phosphorylation of either p38 kinase or JNK at either time point.


CONCLUSIONS
We demonstrate that PPC results in a clearly delineated time-course of bi-phasic protection against injurious ischemic injury that is correlated with reperfusion kinase phosphorylation of both Akt and ERK. These data indicate a novel mechanism of early and particularly delayed preconditioning.
Bell
Robert M
RM
Cardiac Physiology, King's College London, Cardiovascular Division, The Rayne Institute, St. Thomas' Hospital, London, SE1 7EH, UK.
Clark
James E
JE
Hearse
David J
DJ
Shattock
Michael J
MJ
D000730:Androstadienes / Q000494:pharmacology; D000804:Angiotensin II / Q000378:metabolism / Q000627:therapeutic use*; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D019157:Ischemic Preconditioning, Myocardial / Q000379:methods*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D023421:Models, Animal; D009203:Myocardial Infarction / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology; D015428:Myocardial Reperfusion Injury / Q000201:enzymology / Q000517:prevention & control*; D009206:Myocardium / Q000201:enzymology / Q000473:pathology; D051058:Oncogene Protein v-akt / Q000378:metabolism; D010477:Perfusion; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013997:Time Factors; D000077191:Wortmannin
D000730:Androstadienes; D005419:Flavonoids; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D000804:Angiotensin II; D051058:Oncogene Protein v-akt; D048049:Extracellular Signal-Regulated MAP Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D000077191:Wortmannin