alpha B crystallin translocation and phosphorylation: Signal transduction pathways and preconditioning in the isolated rat heart

In this program of studies we have characterized in detail the translocatio n (assessed by Triton-insolubility) and phosphorylation (using serine-45 or -59 phosphospecific antibodies) of alphaB crystallin during myocardial isc hemia [both with or without ischemic preconditioning (IPC)]. Pharmacologica l activators and inhibitors allowed us to characterize the signaling pathwa ys involved in alphaB crystallin phosphorylation during ischemia. Ischemic preconditioning alone caused 30% of the heart's alphaB crystallin pool to t ranslocate, providing a significant translocation 'head-start' in protected tissue. This enhanced translocation is coupled with increased (3-fold) alp haB crystallin phosphorylation at both serine residues. The possible role o f aB crystallin in the protection afforded by ischemic preconditioning is s upported by the signal transduction data: which showed preconditioning-indu ced aB crystallin phosphorylation,can be blocked by tyrosine kinase inhibit ion (using genistein) and by p38 MAP kinase or PKC inhibition (using SB2035 80 or bisindolylmaleimide, respectively). The activation of both p38 MAP ki nase and PKC are recognized requirements for the induction of preconditioni ng and their inhibition is known to block protection. Western immunoblottin g analysis after isoelectric focusing electrophoresis, confirmed the observ ations made with the phosphospecific antibodies: but also showed that 27 +/ - 4% of total cardiac crystallin was phosphorylated after 30 min of ischemi a. alphaB crystallin exists as large polymeric aggregates in cardiac tissue under basal conditions (approximate to 1MDa as determined by gel filtratio n chromatography). We induced phosphorylation of alphaB crystallin during a erobic perfusion by the administration of phenylephrine. However this treat ment did not alter the molecular aggregate size of alphaB crystallin. It ap pears that alphaB crystallin molecular aggregate size is not simply regulat ed by phosphorylation. alphaB crystallin may have a role to play in the myo cardial protection induced by ischemic preconditioning, as both translocati on and phosphorylation are both accelerated and enhanced by ischemic precon ditioning. (C) 2001 Academic Press.