Earl F. Ellis - US grants

Previous experiments indicate that following experimental concussive brain injury oxygen free radicals produced during cyclooxygenase metabolism of arachidonic acid cause endothelial lesions, irreversible dilation and reduced responsiveness to hypocapnia in cerebral arterioles. The exact chemical factor(s) which stimulate arachidonic acid metabolism following injury are uncertain. We wish to test the hypothesis that endogenous brain bradykinin plays an important role in the generation of these cyclooxygenase dependent abnormalities and in the generation of the transient hypertension and brain edema which follows concussive brain injury. This hypothesis is supported by our knowledge that the brain contains all the components of the kallikrein-kinin system, the known actions of bradykinin and our extensive preliminary investigations showing that endogenous brain bradykinin can produce intense cerebral arteriolar dilation by cyclooxygenase dependent mechanisms. In order to test our hypothesis we will: 1) measure brain bradykinin and kininogen levels after fluid-percussion brain injury, 2) determine whether the kallikrein inhibitor, aprotinin, or a newly developed bradykinin antagonist prevents the arteriolar abnormalities following injury, 3) elucidate whether endogenous brain bradykinin increases local prostaglandin levels, 4) determine whether kallikrein-kinin antagonists prevent formation of prostaglandins following injury, 5) examine the role of bradykinin in the vasogenic brain edema which follows injury, 6) test whether concussive brain injury induces in vivo formation of bradykinin receptors or alters reactivity to other agonists, and 7) determine if bradykinin is important in the CNS generation of the Cushing response which follows concussive injury. To achieve these aims we will utilize in vivo microscopy, RIA, chronic cannulation techniques, 125I-serum albumin and kallikrein-kinin antagonists. Extremely little is known concerning the role of bradykinin in normal or injured neural tissue. These studies will increase our understanding of the CNS kallikrein-kinin system and give us important information concerning the possible role of the pro-inflammatory peptide, bradykinin, in the sequelae of neural trauma. The proposed studies are, therefore, consistent wit our long term goal of elucidating chemical mediators of brain injury.

Previous experiments indicate that following experimental concussive brian injury oxygen free radicals produced during cyclooxygenase metabolism of arachidonic acid cause endothelial lesions, dilation, reduce responsiveness to hypocapnia and abnormal responsiveness to acetylcholine in acid cause endothelial lesions, dilation, reduced responsiveness to hypocapnia and abnormal responsiveness to acetylcholine in cerebral arterioles. We now have evidence that specific agonists, including acetylcholine and bradykinin, may be in part responsible for stimulating arachidonic acid metabolism following injury. We therefore wish to test the following hypotheses (see figure). Hypothesis 1: Following concussive brain injury receptor-mediated mechanisms contribute to an increase metabolism of polyunsaturated fatty acids and the production of oxygen radicals which cause cerebrovascular and brain dysfunction. Hypothesis 2: Pharmacologic inhibition of fatty acid metabolism, free radical production or free radical action will reduce the cerebrovascular dysfunction caused by traumatic brain injury (TBI). Our general aims are to understand 1) factors responsible for initiation and regulation of fatty acid metabolism following injury, 2) the pathways and products of this metabolism, 3) the cerebrovascular consequences of increased fatty acid metabolism and radical production and 4) how pharmacologic intervention can prevent, reduce or reverse the injury process. To accomplish our aims we will utilize microscopy and radioimmunoassay to correlate simultaneous in vivo arteriolar diameter responses and in vivo cyclooxygenase and lipoxygenase synthetic responses. We will also employ gas chromatography/mass spectrometry to identify and measure fatty acids and their metabolites. Little is known about regulation of the changes in fatty acid metabolism and the concomitant cerebrovascular consequences of increased oxygen radical production following brain injury. The proposed studies will address these problems and are consistent with our long term goal of elucidating chemical mediators of, and therapeutic agents for, brain injury.

DESCRIPTION: (Adapted from the application) Recent evidence has suggested that Calcium Influx Factor (CIF), an unidentified factor which signals influx of extracellular calcium in response to depletion of intracellular calcium stores, is 5,6-epoxyeicosatrienoic acid (5,6-EET), a P450 monooxygenase metabolite of arachidonic acid (AA). The proposed studies will further test the hypothesis that P450 metabolites of AA formed upon depletion of Ca2+ stores induce influx of extracellular Ca2+, and thus influence cell function and intercellular signaling. The hypothesized relationships will be examined in cultured astrocytes, endothelial cells and vascular smooth muscle (VSM) cells as well as in anesthetized rats. They will determine whether agonist-or thapsigargin-induced store depletion activates phospholipase A2, liberates AA and induces 5,6-EET formation with subsequent influx of Ca2+. They will also elucidate whether 5,6-EET is released from stimulated astrocytes and endothelium and acts as a paracrine signal for VSM causing activation of Ca2+-dependent K+ channels, hyperpolarization and relaxation. The hypothesized relationships will be tested in cultured cells using microspectrofluorometry with the calcium indicator Fura-2, radiolabeled tracers, HPLC, gas chromatography/mass spectrometry and patch clamping. Tests of applicability to the control of the cerebral circulation will employ the in vivo cranial window technique, microscopy and laser-Doppler flowmetry. These studies will provide important basic information relevant to the role of P450 eicosanoids in the regulation of intra- and intercellular relationships of many cell types, including those which control brain blood flow.