Protein kinase C subtypes and retinal ischemic preconditioning.
The purpose of our study was to determine the specific subtypes of protein kinase C involved in the neuroprotection afforded by retinal ischemic preconditioning (IPC), their relationship to the opening of mitochondrial KATP (mKATP) channels, and their role in apoptosis after preconditioning and ischemia. Rats were subjected to retinal ischemia after IPC, or retinas were rendered ischemic after pharmacological opening of mKATP channels. Using immunohistochemistry and image analysis, we determined cellular localization of PKC subtypes. We blocked PKC-delta and -epsilon to study the effect on protection with IPC or with IPC-mimicking by the opening of mKATP channels. PKC subtypes were inhibited pharmacologically or with interfering RNA. Electroretinography assessed functional recovery after ischemia. IPC was effectively mimicked by injection of diazoxide to open the mKATP channel. IPC and/or its mimicking were attenuated by the PKC-delta inhibitor rottlerin and by interfering RNA targeting PKC-delta or -epsilon. Using TUNEL staining and Western blotting for caspase-3 and fodrin breakdown we assessed apoptosis. The injection of interfering RNA to PKC-delta and -epsilon before preconditioning significantly enhanced TUNEL staining as well as the cleavage of caspase-3 and fodrin after ischemia. In summary, our experiments have shown that both PKC-delta and -epsilon subtypes are involved in the cellular signaling that results in neuroprotection from IPC and that both are downstream of the opening of mKATP channels.
Dreixler
John C
JC
Department of Anesthesia and Critical Care, University of Chicago, Chicago, IL 60637, USA.
Shaikh
Afzhal R
AR
Shenoy
Shanti K
SK
Shen
Yang
Y
Roth
Steven
S
D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D001578:Benzopyrans / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D003981:Diazoxide / Q000494:pharmacology; D004596:Electroretinography; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D019194:Ischemic Preconditioning; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D015221:Potassium Channels / Q000502:physiology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D034741:RNA, Small Interfering / Q000493:pharmacokinetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000201:enzymology / Q000517:prevention & control*; D012164:Retinal Diseases / Q000201:enzymology / Q000473:pathology / Q000517:prevention & control*; D012171:Retinal Vessels / Q000201:enzymology* / Q000473:pathology; D014665:Vasodilator Agents / Q000494:pharmacology
D000098:Acetophenones; D001578:Benzopyrans; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D015221:Potassium Channels; D034741:RNA, Small Interfering; D014665:Vasodilator Agents; C117780:mitochondrial K(ATP) channel; C085746:rottlerin; D011493:Protein Kinase C; D003981:Diazoxide