Brain protein kinase C assay using MARCKS substrate reveals no translocation due to profound insulin-induced hypoglycemia.
Hypoglycemia sufficient to produce EEG isoelectricity or coma leads to neuronal death by an excitotoxic mechanism due to elevated extracellular levels of glutamate, aspartate and increased intracellular calcium. Since an elevated intracellular calcium concentration is known to translocate protein kinase C (PKC) from the cytosol to the membrane (a process thought to represent the in vivo activation of the enzyme), the objective of this investigation was to determine if calcium-dependent isoforms of PKC were translocated in specific brain regions of rats subjected to 40 min of insulin-induced hypoglycemic coma. The caudate nucleus and hippocampus (regions damaged by hypoglycemia showing extensive neuronal necrosis), and cerebellum (an undamaged, control region) of hypoglycemic rats were microdissected. Soluble and detergent (Triton X-100)-solubilized particulate fractions were partially purified by DEAE-Sephacel chromatography. PKC activity in both fractions was then measured using a novel assay based on the calcium- and lipid (phosphatidylserine and diolein)-dependent phosphorylation of the specific substrate myristoylated alanine rich C kinase substrate (MARCKS). The percentage distribution of PKC in the soluble and particulate (membrane-bound) fractions of all the brain regions from hypoglycemic rats was not significantly different from that in the control brains, indicating that 40 min of hypoglycemia does not result in PKC translocation as measured in subcellular fractions from brain tissue.
Anderson
L G
LG
MRC Signal Transduction Group, University of Calgary, Alta., Canada.
Zhao
D
D
Dell
K R
KR
Severson
D L
DL
Auer
R N
RN
D000818:Animals; D001345:Autoradiography; D001692:Biological Transport; D001921:Brain / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007003:Hypoglycemia / Q000139:chemically induced* / Q000201:enzymology*; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C