CREB activation and ischaemic preconditioning.

PURPOSE: Previous studies from our laboratory showed that activation of p38 MAPK is one of the triggers of ischaemic preconditioning. The signalling events downstream of p38 MAPK and their links to the putative final effectors of preconditioning are not clear. The cAMP responsive element-binding protein (CREB) is also phosphorylated by exposure to short episodes of ischaemia/reperfusion, suggesting a triggering action. The aim of this study was to systematically investigate (1) the signalling pathways leading to CREB phosphorylation during an ischaemic or beta-adrenergic preconditioning protocol (2) changes in CREB phosphorylation during sustained ischaemia and their significance in ischaemia/reperfusion injury. METHODS: The isolated perfused working rat heart was preconditioned by 1 x 5 min global ischaemia or 3 x 5 min global ischaemia and freeze-clamped. Drugs to manipulate CREB activation were added 5 min before onset of ischaemia. Non-preconditioned and preconditioned hearts were subjected to 25 min global or 35 min regional ischaemia, followed by 30 min reperfusion. Infarct sizes were determined using tetrazolium staining. Phosphorylation of CREB was determined by Western blots. RESULTS: Exposure of hearts to 5 min global ischaemia followed by reperfusion, significantly increased CREB phosphorylation This is mediated by, amongst others, release of endogenous catecholamines and adenosine, as indicated by the use of receptor blockers. Events downstream of receptor stimulation were evaluated using inhibitors for PKA (H89), MSK-1 (H89, Ro318220), PKC (bisindolylmaleimide), p38 MAPK (SB203580) and ERK (PD98059). Activation of PKA, PKC, ERK and p38 MAPK is involved in preconditioning-induced CREB phosphorylation. Ischaemia-induced activation of iPLA(2) and cPLA(2) also contribute to CREB phosphorylation as indicated by the use of the inhibitors 4-bromo-enol-lactone (BEL) and AACOF(3,) respectively. Inhibition of CREB phosphorylation by either BEL or AACOF(3) during a preconditioning protocol partially attenuated cardioprotection. CREB phosphorylation was attenuated during sustained global ischaemia of both non-preconditioned and preconditioned hearts. CONCLUSIONS: These data suggest that CREB phosphorylation during an ischaemic preconditioning protocol may contribute to triggering preconditioning, while reduced phosphorylation during sustained ischaemia does not appear to be associated with cardioprotection.