Trauma to the central nervous system subsequent to cerebrovascular hemorrhage or head injury clearly results in a compromised blood-brain barrier. As a result, nerve cells are directly exposed to blood-borne factors. Among these factors which are produced at the site of tissue injury are thromblin and bradykinin. Incubation of cultured nerve cells (murine neuroblastoma clone N1E-115) with low-nanomolar concentrations of thrombin or bradykinin results in a rapid and marked increase in cyclic guanosine 3 feet, 5 feet monophosphate (cyclic GMP) formation. This clone of neuroblastoma cells has been extensively studied both biochemically and electrophysiologically and has been shown to possess many of the properties of normal differentiated neurons. Thus, this neuronal cell population represents a model system which is ideally suited to investigate the effects of thrombin and bradykinin on nerve cells. The long-term objectives of this proposal are to address the question of the specific neuronal effects of endogenous compounds (such as thrombin and bradykinin) produced at the site of tissue injury. Understanding the neuronal response to these endogenous agents is potentially of great importance to the eventual pathophysiological outcome of cerebral hemorrhage or other CNS trauma. To this end, studies will be conducted to assess the biochemical, morphological and electrophysiological consequences of exposing murine neuroblastoma cells to thrombin and bradykinin. Methods used to accomplish the specific aims of this proposal include: 1) a precursor labeling assay for cyclic GMP formation intact cells: 2) intracellular microelectrode studies of voltage or ionic current changes; 3) morphological examination by microscopy and analysis of changes in the cell surface protein distribution produced by thrombin; 4) analysis by chromatographic techniques (TLC, HPLC) of changes in the membrane phospholipid profile of intact cells exposed to these agents; 5) analysis by chromatography of arachidonic acid metabolism changes in these cells; 6) characterization of the thrombin (and bradykinin) receptors in intact cells by radioligand binding studies; and 7) investigate the ability of antagonists of phospholipase A2 (mepacrine), lipoxygenase (NDGA) and cyclooxygenase (indomethacin) to alter the changes in cyclic GMP formation, phospholipid turnover and arachidonic acid metabolism which are produced by thrombin and/or bradykinin.
