Recovery of Ca²⁺ pools and growth in Ca²⁺ pool-depleted cells is mediated by specific epoxyeicosatrienoic acids derived from arachidonic acid

Depletion of Ca²⁺ pools using the irreversible C^a2+ 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 α- 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.