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 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 alpha-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 microM 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.
Graber
M N
MN
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Alfonso
A
A
Gill
D L
DL
D015126:8,11,14-Eicosatrienoic Acid / Q000378:metabolism*; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D002118:Calcium / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002453:Cell Cycle / Q000502:physiology*; D002455:Cell Division; D002478:Cells, Cultured; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D009637:Masoprocol / Q000494:pharmacology; D008797:Metyrapone / Q000494:pharmacology; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011335:Proadifen / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D019284:Thapsigargin / Q000494:pharmacology
D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D007211:Indoles; D016718:Arachidonic Acid; D019284:Thapsigargin; D009637:Masoprocol; D011335:Proadifen; D000252:Calcium-Transporting ATPases; D015126:8,11,14-Eicosatrienoic Acid; D002118:Calcium; C000543:cyclopiazonic acid; D008797:Metyrapone