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.
| Willoughby, Karen A; Kleindienst, Andrea; Muller, Christian et al. (2004) S100B protein is released by in vitro trauma and reduces delayed neuronal injury. J Neurochem 91:1284-91 |
| Chen, Tao; Willoughby, Karen A; Ellis, Earl F (2004) Group I metabotropic receptor antagonism blocks depletion of calcium stores and reduces potentiated capacitative calcium entry in strain-injured neurons and astrocytes. J Neurotrauma 21:271-81 |
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| Rzigalinski, B A; Willoughby, K A; Hoffman, S W et al. (1999) Calcium influx factor, further evidence it is 5, 6-epoxyeicosatrienoic acid. J Biol Chem 274:175-82 |
