Superoxide Generation Links Protein Kinase C Activation to Impaired ATP-Sensitive K + Channel Function After Brain Injury

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Abstract
Background and Purpose —Endothelin-1, in concentrations similar to that present in cerebrospinal fluid after fluid percussion brain injury (FPI), increases superoxide anion (O 2 − ) production. Endothelin-1 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 NBT reduction from 1±1 to 37±5 pmol/mm 2 . Staurosporine (10 −7 mol/L), a PKC antagonist, blocked the NBT reduction after phorbol 12,13-dibutyrate and blunted the NBT 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 NBT 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.