Superoxide generation links protein kinase C activation to impaired ATP-sensitive K+ channel function after brain injury

Background and Purpose-Endothelin-1, in concentrations similar to that present in cerebrospinal fluid after fluid percussion brain injury (FPI), increases superoxide anion (P-2(-)) production. Endothelin-l also contributes to altered cerebral hemodynamics after FPI through impairment of ATP-sensitive K+ (K-ATP) channel function through protein kinase C (PKC) activation. Generation of O-2(-) additionally occurs after FPI. Nitric oxide and cGMP elicit pial artery dilation through K-ATP channel activation. The present study was designed to determine whether PKC activation generates O-2(-), which, in turn, could link such activation to impaired K-ATP channel function after FPI. Methods-Injury of moderate severity (1.9 to 2.1 atm) was produced by the lateral FPI technique in anesthetized newborn pigs equipped with a closed cranial window. Superoxide dismutase-inhibitable nitroblue tetrazolium (NBT) reduction was determined as an index of O-2(-) generation. Results-Phorbol 12,13-dibutyrate (10(-6) mol/L), a PKC activator, increased superoxide dismutase-inhibitable NET reduction from 1+/-1 to 37+/-5 pmol/mm(2). Staurosporine (10(-7) mol/L), a PKC antagonist, blocked the NET reduction after phorbol 12,13-dibutyrate and blunted the NET reduction observed after FPI (1+/-1 to 15+/-2 versus 1+/-1 to 5+/-1 pmol/mm(2) after FPI in the absence versus presence of staurosporine). Exposure of the cerebral cortex to a xanthine oxidase O-2(-)-generating system increased NET reduction in a manner similar to FPI and blunted pial artery dilation to the K-ATP channel agonists cromakalim and calcitonin gene-related peptide, the nitric oxide releasers sodium nitroprusside and S-nitroso-N-acetylpenicillamine, and the cGMP analogue 8-bromo-cGMP (10+/-1% and 21+/-1% versus 4+/-1% and 9+/-1% for 10(-8) and 10(-6) mol/L cromakalim before and after activated oxygen-generating system exposure). Conclusions-These data show that PKC activation increases O-2(-) production and contributes to such production observed after FPI. These data also show that an activated system that generates an amount of O-2(-) similar to that observed with FPI blunted pial artery dilation to K-ATP channel agonists and nitric oxide/cGMP. These data suggest, therefore, that O-2(-) generation links PKC activation to impaired K-ATP channel function after FPI.