Volume 272, Number 47, Issue of November 21, 1997 pp. 29546-29553
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Recovery of Ca²⁺ Pools and Growth in Ca²⁺ Pool-depleted Cells Is Mediated by Specific Epoxyeicosatrienoic Acids Derived from Arachidonic Acid

(Received for publication, July 1, 1997, and in revised form, September 4, 1997)

From the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201

Depletion of Ca²⁺ pools using the irreversible Ca²⁺ pump blocker, thapsigargin, induces DDT₁MF-2 smooth muscle cells to enter a stable nonproliferative state. Reversal of this state can be mediated by high (20%) serum treatment, which induces new Ca²⁺ pump protein, return of Ca²⁺ pools, and reentry of cells into the cell cycle; the effect of serum can be mimicked by the essential fatty acids (EFA), arachidonic, linoleic, and -linolenic acids (Graber, M.N., Alfonso, A., and Gill, D.L., (1996) J. Biol. Chem. 271, 883-888). The possible requirement for EFA metabolism in inducing recovery of Ca²⁺ pool-depleted growth-arrested cells was investigated. Neither cyclooxygenase or lipoxygenase inhibitors had any effect on arachidonic acid-induced growth recovery of thapsigargin-treated cells. In contrast, the cytochrome P-450 epoxygenase inhibitors, SKF525A and metyrapone, substantially reduced arachidonic acid-induced recovery of growth while having minimal effects on control cell growth. Both epoxygenase inhibitors completely prevented the arachidonic acid-induced recovery of bradykinin-releasable Ca²⁺-pumping pools, whereas cyclooxygenase and lipoxygenase inhibitors had no effect. The effectiveness of the four cytochrome P-450 metabolites of arachidonic acid on recovery of Ca²⁺ pools were compared; 8,9- and 11,12-epoxyeicosatrienoic acid (EET) at 1.5 µM were completely effective in recovering agonist-sensitive Ca²⁺ pools, whereas the 5,6- and 14,15-EETs were without effect. SKF525A did not block the action of 8,9- or 11,12-EET indicating further P-450 metabolism was not required. Hydration of the active EET molecules prevented Ca²⁺ pool recovery since the dihydroxy-derivatives of both 8,9- and 11,12-EET were ineffective. The specificity of effectiveness among EET molecules for subsequent resumption of growth of thapsigargin-treated cells was the same as for Ca²⁺ pool recovery. Significantly, the P-450 inhibitors, SKF525A and metyrapone, both prevented the action of 20% serum in inducing recovery of thapsigargin-treated cells, whereas cyclooxygenase and lipoxygenase inhibitors were ineffective, indicating that EFAs are the active component within serum that is responsible for recovery of Ca²⁺ pool-depleted cells. The specific action of EETs in mediating recovery of Ca²⁺ pools and growth of thapsigargin-treated cells represents not only a novel action of epoxygenase products from EFAs, but also a potentially significant new signaling pathway that may effect translational control and regulate transition from a stationary to proliferative growth state.

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