Protein kinase C family members as a target for regulation of blood-brain barrier Na,K,2Cl-cotransporter during in vitro stroke conditions and nicotine exposure.
OBJECTIVE
The aim of the study is to identify specific protein kinase C (PKC) isoforms involvement in K(+) transport mediated at altered blood-brain barrier (BBB) response to stroke conditions with prior nicotine exposure, which provides ways to intervene pharmacologically in PKC-mediated molecular pathways that could lead to effective treatment for smoking stroke patients.


METHODS
Changes in PKC isoform levels were studied in the cytosolic and membrane fractions of bovine brain microvessel endothelial cells subjected to stroke conditions as well as nicotine/cotinine exposure. Furthermore, abluminal Na,K,2Cl-cotransporter (NKCC) activity regulated by specific conventional PKC isoform activators and inhibitors was investigated using rubidium ((86)Rb) uptake studies.


RESULTS
Membrane-bound PKCalpha, PKCbetaI, and PKCepsilon levels were increased after 6 h hypoxia/aglycemia, and this was attenuated by 24-h nicotine/cotinine exposure. Interestingly, membrane-bound PKCgamma protein level was decreased after 6 h hypoxia/aglycemia and increased by 24-h nicotine/cotinine exposure. (86)Rb uptake studies showed that basolateral NKCC activity was down-regulated by both a conventional PKC inhibitor and specific inhibitors for PKCalpha, PKCbeta, and PKCvarepsilon and was up-regulated by an activator of conventional PKCs during 6-h hypoxia/aglycemia treatment.


CONCLUSIONS
Specific PKC inhibitors or activators might be designed to individualize stroke therapies and improve health outcome for smokers by rebalancing ion transport into and out of the brain.
Yang
Tianzhi
T
Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 Coulter Drive, Amarillo, 79106, USA.
Roder
Karen E
KE
Bhat
G Jayarama
GJ
Thekkumkara
Thomas J
TJ
Abbruscato
Thomas J
TJ
D000818:Animals; D001812:Blood-Brain Barrier / Q000187:drug effects*; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D015536:Down-Regulation; D000860:Hypoxia / Q000201:enzymology / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D009538:Nicotine / Q000494:pharmacology*; D018722:Nicotinic Agonists / Q000494:pharmacology*; D011188:Potassium / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D012414:Rubidium Radioisotopes; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D020521:Stroke / Q000378:metabolism*; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007527:Isoenzymes; D018722:Nicotinic Agonists; D012414:Rubidium Radioisotopes; D028021:Sodium-Potassium-Chloride Symporters; D009538:Nicotine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D011188:Potassium