Recovery of Ca2+ Pools and Growth in Ca2+Pool-depleted Cells Is Mediated by Specific Epoxyeicosatrienoic Acids Derived from Arachidonic Acid
1997; Elsevier BV; Volume: 272; Issue: 47 Linguagem: Inglês
10.1074/jbc.272.47.29546
ISSN1083-351X
AutoresMatthew N. Graber, Amparo Alfonso, Donald L. Gill,
Tópico(s)Eicosanoids and Hypertension Pharmacology
ResumoDepletion 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 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. 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 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. Cytosolic Ca2+ signals activate numerous rapid cellular responses including contraction, secretion, and excitation; Ca2+ signals also mediate control over longer term responses such as cell division and growth (1Berridge M.J. Biochem. J. 1993; 312: 1-11Crossref Scopus (1046) Google Scholar, 2Gill D.L. Waldron R.T. Rys-Sikora K.E. Ufret-Vincenty C.A. Graber M.N. Favre C.J. Alfonso A. Biosci. Rep. 1996; 16: 139-157Crossref PubMed Scopus (68) Google Scholar). A significant source of Ca2+ for these signals is the Ca2+ stored within intracellular pools, which can be released through the activation of intracellular release Ca2+ channels (1Berridge M.J. Biochem. J. 1993; 312: 1-11Crossref Scopus (1046) Google Scholar, 2Gill D.L. Waldron R.T. Rys-Sikora K.E. Ufret-Vincenty C.A. Graber M.N. Favre C.J. Alfonso A. Biosci. Rep. 1996; 16: 139-157Crossref PubMed Scopus (68) Google Scholar, 3Putney Jr., J.W. Bird G. St J. Trends Endocrinol. Metab. 1994; 5: 256-260Abstract Full Text PDF PubMed Scopus (32) Google Scholar). Intracellular pools exist within the endoplasmic reticulum or subfractions thereof (1Berridge M.J. Biochem. J. 1993; 312: 1-11Crossref Scopus (1046) Google Scholar, 2Gill D.L. Waldron R.T. Rys-Sikora K.E. Ufret-Vincenty C.A. Graber M.N. Favre C.J. Alfonso A. Biosci. Rep. 1996; 16: 139-157Crossref PubMed Scopus (68) Google Scholar, 3Putney Jr., J.W. Bird G. St J. Trends Endocrinol. Metab. 1994; 5: 256-260Abstract Full Text PDF PubMed Scopus (32) Google Scholar, 4Gill D.L. Ghosh T.K. Bian J. Short A.D. Waldron R.T. Rybak S.L. Adv. Second Messenger Phoshoprotein Res. 1992; 26: 265-308PubMed Google Scholar) and accumulate Ca2+ via the action of Ca2+ pumps of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) 1The abbreviations used are: SERCA, sarcoplasmic/endoplasmic reticulum Ca2+ ATPase; EET, epoxyeicosatrienoic acid; EFA, essential fatty acid; DHT, dihydroxyeicosatrienoic acid; DMEM, Dulbecco's modified Eagle's medium; InsP3, inositol 1,4,5-trisphosphate; NDGA, nordiydroguaiuretic acid; BSA, bovine serum albumin. 1The abbreviations used are: SERCA, sarcoplasmic/endoplasmic reticulum Ca2+ ATPase; EET, epoxyeicosatrienoic acid; EFA, essential fatty acid; DHT, dihydroxyeicosatrienoic acid; DMEM, Dulbecco's modified Eagle's medium; InsP3, inositol 1,4,5-trisphosphate; NDGA, nordiydroguaiuretic acid; BSA, bovine serum albumin. family, which are widely distributed within the endoplasmic reticulum of most cells (5Lytton J. Westlin M. Hanley M.R. J. Biol. Chem. 1991; 266: 17067-17071Abstract Full Text PDF PubMed Google Scholar,6Maclennan D.H. Toyofuku T. Lytton J. Ann. N. Y. Acad. Sci. 1992; 671: 1-10Crossref PubMed Scopus (54) Google Scholar). In addition to serving as a source of Ca2+ for the generation of cytosolic Ca2+ signals, the Ca2+accumulated within pools may effect control over a number of cellular functions. Thus, the intraluminal Ca2+ level appears to be the primary trigger for activating Ca2+ influx channels in the plasma membrane following Ca2+ pool release (1Berridge M.J. Biochem. J. 1993; 312: 1-11Crossref Scopus (1046) Google Scholar, 2Gill D.L. Waldron R.T. Rys-Sikora K.E. Ufret-Vincenty C.A. Graber M.N. Favre C.J. Alfonso A. Biosci. Rep. 1996; 16: 139-157Crossref PubMed Scopus (68) Google Scholar, 3Putney Jr., J.W. Bird G. St J. Trends Endocrinol. Metab. 1994; 5: 256-260Abstract Full Text PDF PubMed Scopus (32) Google Scholar, 7Putney Jr., J.W. Bird G. St J. Endocr. Rev. 1993; 14: 610-631Crossref PubMed Scopus (483) Google Scholar). Intraluminal Ca2+ levels also appear to exert control over fundamental endoplasmic reticulum functions including the folding, processing, and assembly of proteins (8Sambrook J.F. Cell. 1990; 61: 197-199Abstract Full Text PDF PubMed Scopus (237) Google Scholar, 9Kuznetsov G. Brostrom M.A. Brostrom C.O. J. Biol. Chem. 1992; 267: 3932-3939Abstract Full Text PDF PubMed Google Scholar, 10Lodish H.F. Kong N. Wikström L. J. Biol. Chem. 1992; 267: 12753-12760Abstract Full Text PDF PubMed Google Scholar). These actions may be mediated by intraluminal Ca2+ binding proteins, which can function as molecular chaperones (11Milner R.E. Famulski K.S. Michalak M. Mol. Cell. Biochem. 1992; 112: 1-13Crossref PubMed Scopus (97) Google Scholar, 12Nigam S.J. Goldberg A.L. Ho S. Rohde M.F. Bush K.T. Sherman M.Y. J. Biol. Chem. 1994; 269: 1744-1749Abstract Full Text PDF PubMed Google Scholar).In recent studies we have shown that the content of agonist-sensitive Ca2+ pools also exerts a profound effect upon the ability of cells to progress through the cell cycle (2Gill D.L. Waldron R.T. Rys-Sikora K.E. Ufret-Vincenty C.A. Graber M.N. Favre C.J. Alfonso A. Biosci. Rep. 1996; 16: 139-157Crossref PubMed Scopus (68) Google Scholar, 4Gill D.L. Ghosh T.K. Bian J. Short A.D. Waldron R.T. Rybak S.L. Adv. Second Messenger Phoshoprotein Res. 1992; 26: 265-308PubMed Google Scholar, 13Ghosh T.K. Bian J. Short A.D. Rybak S.L. Gill D.L. J. Biol. Chem. 1991; 266: 24690-24697Abstract Full Text PDF PubMed Google Scholar, 14Short A.D. Bian J. Ghosh T.K. Waldron R.T. Rybak S.L. Gill D.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4986-4990Crossref PubMed Scopus (247) Google Scholar, 15Waldron R.T. Short A.D. Meadows J.J. Ghosh T.K. Gill D.L. J. Biol. Chem. 1994; 269: 11927-11933Abstract Full Text PDF PubMed Google Scholar, 16Waldron R.T. Short A.D. Gill D.L. J. Biol. Chem. 1995; 270: 11955-11961Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 17Waldron R.T. Short A.D. Gill D.L. J. Biol. Chem. 1997; 272: 6440-6447Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Experiments reveal that the SERCA pump inhibitors thapsigargin (18Thastrup O. Cullen P.J. Drrbak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (2986) Google Scholar) and 2,5-di-tert-butylhydroquinone (19Moore G.A. McConkey D.J. Kass G.E.N. O'Brien P.J. Orrenius S. FEBS Lett. 1987; 224: 331-336Crossref PubMed Scopus (164) Google Scholar) deplete intracellular Ca2+ pools and cause the accompanying entry of DDT1MF-2 smooth muscle cells into a stable quiescent G0-like growth state (13Ghosh T.K. Bian J. Short A.D. Rybak S.L. Gill D.L. J. Biol. Chem. 1991; 266: 24690-24697Abstract Full Text PDF PubMed Google Scholar, 14Short A.D. Bian J. Ghosh T.K. Waldron R.T. Rybak S.L. Gill D.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4986-4990Crossref PubMed Scopus (247) Google Scholar). Although growth-arrested, these Ca2+ pool-depleted cells remain intact and viable, and maintain normal cellular and subcellular morphology and mitochondrial function for up to one week (13Ghosh T.K. Bian J. Short A.D. Rybak S.L. Gill D.L. J. Biol. Chem. 1991; 266: 24690-24697Abstract Full Text PDF PubMed Google Scholar, 14Short A.D. Bian J. Ghosh T.K. Waldron R.T. Rybak S.L. Gill D.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4986-4990Crossref PubMed Scopus (247) Google Scholar). Whereas SERCA pump inhibition by thapsigargin is essentially irreversible (20Bian J. Ghosh T.K. Wang J.C. Gill D.L. J. Biol. Chem. 1991; 266: 8801-8806Abstract Full Text PDF PubMed Google Scholar, 21Sagara Y. Inesi G. J. Biol. Chem. 1991; 266: 13503-13506Abstract Full Text PDF PubMed Google Scholar), we previously demonstrated that treatment of thapsigargin-arrested cells with high (20%) serum induces reappearance of Ca2+ pools and activation of a transition of cells back into the cell cycle (14Short A.D. Bian J. Ghosh T.K. Waldron R.T. Rybak S.L. Gill D.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4986-4990Crossref PubMed Scopus (247) Google Scholar,15Waldron R.T. Short A.D. Meadows J.J. Ghosh T.K. Gill D.L. J. Biol. Chem. 1994; 269: 11927-11933Abstract Full Text PDF PubMed Google Scholar). High serum induces expression of new functional Ca2+pump protein within 1–3 h and the reappearance of agonist-releasable Ca2+ pools within 6 h (15Waldron R.T. Short A.D. Meadows J.J. Ghosh T.K. Gill D.L. J. Biol. Chem. 1994; 269: 11927-11933Abstract Full Text PDF PubMed Google Scholar). Cells begin to enter the S phase 16 h later and thereafter continue to divide normally (14Short A.D. Bian J. Ghosh T.K. Waldron R.T. Rybak S.L. Gill D.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4986-4990Crossref PubMed Scopus (247) Google Scholar).Recently we revealed that each of the essential fatty acids, arachidonic acid, linoleic acid, and linolenic acid (22Graber M.N. Alfonso A. Gill D.L. J. Biol. Chem. 1996; 271: 883-888Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), can mimic the action of high serum treatment on Ca2+ pool-depleted growth-arrested cells, inducing reappearance of agonist-sensitive Ca2+ pools and re-entry of cells into the cell cycle. It was shown that either high serum or EFA treatment can induce recovery of Ca2+ pools, the actions of both being dependent upon protein synthesis (22Graber M.N. Alfonso A. Gill D.L. J. Biol. Chem. 1996; 271: 883-888Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Experiments indicated that the EFAs within serum could be the component responsible for stimulating pool reappearance and growth recovery. Thus, the EC50 values for the action of each of the three EFAs on growth induction of thapsigargin-arrested cells were similar, approximately 5 μm, corresponding with the total EFA concentration present in the 20% serum treatment conditions (22Graber M.N. Alfonso A. Gill D.L. J. Biol. Chem. 1996; 271: 883-888Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Phospholipase A2 inhibitors did not inhibit high serum-induced recovery suggesting that serum does not stimulate phospholipase A2-induced formation of free EFAs. We postulated that the EFAs themselves may not be the agents directly involved in recovery, but instead, active metabolite(s) of these lipid species might be responsible. This was supported by studies showing that the nonmetabolizable analogue of arachidonic acid, 5,8,11,14-eicosatetraynoic acid (ETYA), was unable to induce recovery (22Graber M.N. Alfonso A. Gill D.L. J. Biol. Chem. 1996; 271: 883-888Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). ETYA has been shown to mimic the actions of arachidonic acid in systems where metabolism of the fatty acid is not required (23Tobias L.D. Hamilton J.G. Lipids. 1978; 14: 181-193Crossref Scopus (118) Google Scholar, 24Damron D.S. Bond M. Circ. Res. 1993; 72: 376-386Crossref PubMed Google Scholar, 25Kohout T.A. Rogers T.B. J. Biol. Chem. 1995; 270: 20432-20438Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Additionally, ETYA is an effective blocker of the entry of EFAs into each of the pathways through which eicosanoids are formed, including the cyclooxygenase, lipoxygenase, and epoxygenase (or monooxygenase) pathways (26Cunningham F.M. Lipid Mediators. Academic Press, London1994Google Scholar). Our observations that ETYA is effective in blocking arachidonic acid-induced recovery provided further support for the possible requirement for metabolism of arachidonic acid in inducing cell recovery (22Graber M.N. Alfonso A. Gill D.L. J. Biol. Chem. 1996; 271: 883-888Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar).The metabolism of EFAs within cells is complex, and at least three major pathways are known (27Smith W.L. Biochem. J. 1989; 259: 315-324Crossref PubMed Scopus (763) Google Scholar). It was important to determine whether metabolism of EFAs through one of these pathways was required for the activation of recovery of pools and cell growth following Ca2+ pool emptying. In addition, it was important to try to identify whether any of the many products of metabolism might be active in mediating this response. Presented here are studies examining the role of each of the three major pathways of eicosanoid synthesis in mediating the action of EFAs. The results indicate that the cytochrome P-450 epoxygenase pathway is required to give rise to active products. Studies further reveal that the effect is induced by only two specific epoxyeicosatrienoic acid metabolites of arachidonic acid and not observed with other cytochrome P-450 metabolites or mimicked by products from other eicosanoid pathways. The results also provide evidence indicating that the EFAs contained within serum are indeed the active components mediating serum-induced recovery of Ca2+pool-depleted cells. The results not only provide further information on the link between Ca2+ pools and cell growth but also shed light on a possible new signaling pathway controlling transition from a stationary to proliferative growth state.RESULTS AND DISCUSSIONThe intracellular Ca2+ pump blockers, thapsigargin, 2,5-di-tert-butylhydroquinone, and cyclopiazonoic acid, induce profound changes in the growth of DDT1MF-2 smooth muscle cells in culture (2Gill D.L. Waldron R.T. Rys-Sikora K.E. Ufret-Vincenty C.A. Graber M.N. Favre C.J. Alfonso A. Biosci. Rep. 1996; 16: 139-157Crossref PubMed Scopus (68) Google Scholar, 4Gill D.L. Ghosh T.K. Bian J. Short A.D. Waldron R.T. Rybak S.L. Adv. Second Messenger Phoshoprotein Res. 1992; 26: 265-308PubMed Google Scholar). In previous studies, we described how Ca2+ pool emptying with these agents induced entry of the normally rapidly dividing DDT1MF-2 cells into a growth-arrested state (13Ghosh T.K. Bian J. Short A.D. Rybak S.L. Gill D.L. J. Biol. Chem. 1991; 266: 24690-24697Abstract Full Text PDF PubMed Google Scholar, 14Short A.D. Bian J. Ghosh T.K. Waldron R.T. Rybak S.L. Gill D.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4986-4990Crossref PubMed Scopus (247) Google Scholar, 15Waldron R.T. Short A.D. Meadows J.J. Ghosh T.K. Gill D.L. J. Biol. Chem. 1994; 269: 11927-11933Abstract Full Text PDF PubMed Google Scholar). After treatment of cells with pump blockers, cells can progress through S phase before entering a stable G0-like quiescent growth state (13Ghosh T.K. Bian J. Short A.D. Rybak S.L. Gill D.L. J. Biol. Chem. 1991; 266: 24690-24697Abstract Full Text PDF PubMed Google Scholar). In this state, the cells remain viable with normal morphology and mitochondrial function for up to 1 week; protein synthesis continues but at a substantially reduced rate, approximately 20% of that in normal dividing cells (13Ghosh T.K. Bian J. Short A.D. Rybak S.L. Gill D.L. J. Biol. Chem. 1991; 266: 24690-24697Abstract Full Text PDF PubMed Google Scholar,14Short A.D. Bian J. Ghosh T.K. Waldron R.T. Rybak S.L. Gill D.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4986-4990Crossref PubMed Scopus (247) Google Scholar). Thapsigargin induces an essentially irreversible blockade of intracellular Ca2+ pump activity (20Bian J. Ghosh T.K. Wang J.C. Gill D.L. J. Biol. Chem. 1991; 266: 8801-8806Abstract Full Text PDF PubMed Google Scholar, 21Sagara Y. Inesi G. J. Biol. Chem. 1991; 266: 13503-13506Abstract Full Text PDF PubMed Google Scholar, 31Davidson G.A. Varhol R.J. J. Biol. Chem. 1995; 270: 11731-11734Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar); a brief (30-min) treatment of cells with thapsigargin results in permanently emptied pools being retained in nondividing cells for 7 days even if the cells are maintained in thapsigargin free medium (13Ghosh T.K. Bian J. Short A.D. Rybak S.L. Gill D.L. J. Biol. Chem. 1991; 266: 24690-24697Abstract Full Text PDF PubMed Google Scholar). The observation that treatment of the pool-depleted growth-arrested cells with high serum (20%) could rescue the cells (14Short A.D. Bian J. Ghosh T.K. Waldron R.T. Rybak S.L. Gill D.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4986-4990Crossref PubMed Scopus (247) Google Scholar) lead to a search for active components within serum responsible for this effect. We determined that the essential fatty acids, linolenic acid, linoleic acid, and arachidonic acid, each were able to induce similar recovery of Ca2+ pools and the resumption of growth (22Graber M.N. Alfonso A. Gill D.L. J. Biol. Chem. 1996; 271: 883-888Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The levels of free EFAs present in effective serum concentrations (approximately 5 μm) were close to the EC50 values for EFAs in inducing recovery, suggesting, but not proving, that EFAs might be the active component within serum inducing recovery. This view was supported by the lack of effect of phospholipase A2inhibitors on serum-induced recovery indicating that serum does not activate breakdown of cellular phospholipids, but more likely supplies the fatty acids directly. The observation that the nonmetabolizable structural analogue of arachidonic acid, ETYA, did not mimic the action of arachidonic acid (22Graber M.N. Alfonso A. Gill D.L. J. Biol. Chem. 1996; 271: 883-888Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) provided further support for the view that metabolism of arachidonic acid was required for the recovery-inducing action. Important to investigate was the basis of action of EFA-induced pool recovery and reentry of pool-depleted cells into the cell cycle, specifically, to assess whether metabolism is in fact required, through which pathway(s) active metabolites might be produced, and which of the many possible metabolic products of EFA metabolism might be the active species.Initial experiments were designed to determine the role of essential fatty acid metabolism through dissection of the three major components of the eicosanoid synthesis pathway (26Cunningham F.M. Lipid Mediators. Academic Press, London1994Google Scholar). Studies utilized a range of specific inhibitors of the cyclooxygenase, lipoxygenase, and cytochrome P-450 epoxygenase pathways. Thapsigargin-treated cells were either induced to recover with 100 μm arachidonic acid or pretreated with an eicosanoid synthesis pathway inhibitor followed by arachidonic acid in the continued presence of the inhibitor. All fatty acids and derivatives were utilized in the presence of 1% (w/v) fatty acid free bovine serum albumin, and measurements of cell number were undertaken after 72 h in culture (see "Experimental Procedures").The lipoxygenase and cyclooxygenase pathways have been extensively studied, and the actions of a number of well characterized inhibitors of these pathways were examined for their effects on arachidonic acid-induced growth recovery in Ca2+ pool-depleted growth-arrested cells. Their effects on the growth of untreated control cells were also examined. As shown in Fig.1 A, the cyclooxygenase inhibitors, aspirin (32Vane J. Botting R. FASEB. 1987; 1: 89-96Crossref PubMed Scopus (451) Google Scholar, 33DeWitt D.L. el-Harith E.A. Kraemer S.A. Andrews M.J. Yao E.F. Armstrong R.L. Smith W.L. J. Biol. Chem. 1990; 265: 5192-5198Abstract Full Text PDF PubMed Google Scholar), indomethacin (34Shen T.-Y. Winter C.A. Adv. Drug Res. 1977; 12: 89-98Crossref Google Scholar), NS-398 (35Copeland R.A. Williams J.M. Giannaras J. Nurnberg S. Covington M. Pinto D. Pick S. Trzaskos J.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11202-11206Crossref PubMed Scopus (407) Google Scholar, 36Gierse J.K. Hauser S.D. Creely D.P. Koboldt C. Rangwala S.H. Isakson P.C. Seibert K. Biochem. J. 1995; 305: 479-484Crossref PubMed Scopus (378) Google Scholar), and valeryl salicylate (37Bhattacharyya D.K. Lecomte M. Dunn J. Morgans D.J. Smith W.L. Arch. Biochem. Biophys. 1995; 317: 19-24Crossref PubMed Scopus (99) Google Scholar), in each case did not prevent arachidonic acid-induced growth recovery of thapsigargin-arrested cells. Each of these inhibitors did have a small retarding effect on the growth of control cells and this limited growth-slowing effect was also evident on the growth of pool-depleted cells induced to recover with arachidonic acid.Investigations also examined inhibitors of the second eicosanoid synthesizing pathway, the lipoxygenase pathway. As shown in Fig.1 A, the lipoxygenase inhibitors, 5,8,11-eicosatriynoic acid (ETI) (38Byczkowski J.Z. Ramgoolie P.J. Kulkarni A.P. Int. J. Biochem. 1992; 24: 1691-1695Crossref PubMed Scopus (10) Google Scholar, 39Joseph P. Srinivasan S.N. Kulkarni A.P. Biochem. J. 1993; 293: 83-91Crossref PubMed Scopus (49) Google Scholar) and baicalein (40Kimura Y. Okuda H. Arichi S. Biochim. Biophys. Acta. 1987; 922: 278-286Crossref PubMed Scopus (40) Google Scholar, 41Stern N. Golub M. Nozawa K. Berger M. Knoll E. Yanagawa N. Natarajan R. Nadler J.L. Tuck M.L. Am. J. Physiol. 1989; 257: H434-H443Crossref PubMed Google Scholar), induced little significant change in either control growth of cells or arachidonic acid-induced recovery. Similarly, the dual cyclooxygenase and lipoxygenase inhibitors, BW755c (42Cucurou C. Battioni J.P. Thang D.C. Nam N.H. Mansuy D. Biochemistry. 1991; 30: 8964-8970Crossref PubMed Scopus (65) Google Scholar, 43Oyekan A.O. McGiff J.C. Quilley J. Circ. Res. 1991; 68: 958-965Crossref PubMed Scopus (42) Google Scholar), and octadeca-9,12-diynoic acid (ODYA) (44Nieuwenhuizen W.F. Schilstra M.J. Van der kerk-Van Hoof A. Brandsma L. Veldink G.A. Vliegenthart J.F. Biochemistry. 1995; 34: 10538-10545Crossref PubMed Scopus (13) Google Scholar), were ineffective on control growth or arachidonic acid-induced recovery. These results indicate that the more thoroughly studied eicosanoid synthesizing pathways, the cyclooxygenase and lipoxygenase pathways, are unlikely to be involved in the arachidonic acid induced recovery mechanism.In contrast, experiments utilizing inhibitors of the third eicosanoid pathway, the cytochrome P-450 epoxygenase pathway, consistently indicated that the actions of arachidonic acid on inducing recovery of pool-depleted cells, were dependent on this pathway. As shown in Fig.1 B, the cytochrome P-450 inhibitors, metyrapone (45Fitzpatrick F.A. Murphy R.C. Pharmacol. Rev. 1989; 40: 229-241Google Scholar) and SKF525A (45Fitzpatrick F.A. Murphy R.C. Pharmacol. Rev. 1989; 40: 229-241Google Scholar, 46Kauser K. Clark J.E. Masters B.S. Ortiz de Montellano P.R. Ma Y.H. Harder D.R. Roman R.J. Circ. Res. 1991; 68: 1154-1163Crossref PubMed Scopus (122) Google Scholar), each prevented arachidonic acid-induced growth recovery but had only marginal effects on the growth of control cells. Interestingly, the "classic" lipoxygenase inhibitor, nordiydroguaiuretic acid (NDGA), also prevented growth recovery induced by arachidonic acid. However, there is an interesting consistency in these results; NDGA in addition to acting as a lipoxygenase inhibitor, is also a potent cytochrome P-450 epoxygenase inhibitor (47Agarwal R. Wang Z.Y. Bik D.P. Mukhtar H. Drug Metab. Dispos. 1991; 19: 620-624PubMed Google Scholar, 48Sarubbi D. Quilley J. Eur. J. Pharmacol. 1991; 197: 27-31Crossref PubMed Scopus (7) Google Scholar), therefore the inhibitory effect is consistent with the involvement of only the epoxygenase pathway in arachidonic acid-induced recovery. It is important to note that, whereas each of these cytochrome P-450 inhibitors greatly reduced (by 80–90%) the ability of arachidonic acid to induce growth recovery of quiescent pool-depleted cells, each had little or no effect upon control cell growth. This indicates that there is a specific requirement for arachidonic acid-derived products of the P-450 epoxygenase pathway in mediating the transition from growth arrest back into the cell cycle and that this effect does not reflect a generalized action of these products on cell growth per se.As mentioned above, we previously reported that the nonmetabolizable arachidonic acid analogue, 5,8,11,14-eicosatetraynoic acid (ETYA), is unable to induce growth recovery of pool-depleted cells (22Graber M.N. Alfonso A. Gill D.L. J. Biol. Chem. 1996; 271: 883-888Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). This analogue also acts as an inhibitor of all three eicosanoid synthesizing pathways (23Tobias L.D. Hamilton J.G. Lipids. 1978; 14: 181-193Crossref Scopus (118) Google Scholar, 26Cunningham F.M. Lipid Mediators. Academic Press, London1994Google Scholar, 27Smith W.L. Biochem. J. 1989; 259: 315-324Crossref PubMed Scopus (763) Google Scholar). Experiments revealed that ETYA up to 10 μm did, at least partially inhibit the growth recovery of thapsigargin-arrested cells induced by arachidonic acid (data not shown). However, we also noted that above 10 μm, ETYA has a significant inhibitory effect on normal cell growth therefore it was not possible to effectively gauge its inhibitory action on growth recovery. The basis of the inhibitory action of ETYA on normal cell growth is unknown but may be related to the inhibition of normal cell growth seen with high concentrations (>100 μm) of arachidonic acid, as described earlier (22Graber M.N. Alfonso A. Gill D.L. J. Biol. Chem. 1996; 271: 883-888Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar).From the above experiments it
Referência(s)