title: Calcium influx factor from cytochrome P-450 metabolism and secretion-like coupling mechanisms for capacitative calcium entry in corneal endothelial cells.
abstract: Notwithstanding extensive efforts, the mechanism of capacitative calcium entry (CCE) remains unclear. Two seemingly opposed theories have been proposed: secretion-like coupling (Patterson, R. L., van Rossum, D. B., and Gill, D. L. (1999) Cell 98, 487-499) and the calcium influx factor (CIF) (Randriamampita, C., and Tsien, R. Y. (1993) Nature 364, 809-814). In the current study, a combinatorial approach was taken to investigate the mechanism of CCE in corneal endothelial cells. Induction of cytochrome P-450s by beta-naphthoflavone (BN) enhanced CCE measured by Sr(2+) entry after store depletion. 5,6-Epoxyeicosatrienoic acid (5,6-EET), a proposed CIF generated by cytochrome P-450s (Rzigalinski, B. A., Willoughby, K. A., Hoffman, S. W., Falck, J. R., and Ellis, E. F. (1999) J. Biol. Chem. 274, 175-182), induced Ca(2+) entry. Both BN-enhanced CCE and the 5,6-EET-induced Ca(2+) entry were inhibited by the CCE blocker 2-aminoethoxydiphenyl borate, indicating a role for cytochrome P-450s in CCE. Treatment with calyculin A (CalyA), which causes condensation of cortical cytoskeleton, inhibited CCE. The actin polymerization inhibitor cytochalasin D partially reversed the inhibition of CCE by CalyA, suggesting a secretion-like coupling mechanism for CCE. However, CalyA could not inhibit CCE in BN-treated cells, and 5,6-EET caused a partial activation of CCE in CalyA-treated cells. These results further support the notion that cytochrome P-450 metabolites may be CIFs. The vesicular transport inhibitor brefeldin A inhibited CCE in both vehicle- and BN-treated cells. Surprisingly, Sr(2+) entry in the absence of store depletion was enhanced in BN-treated cells, which was also inhibited by 2-aminoethoxydiphenyl borate. An integrative model suggests that both CIF from cytochrome P-450 metabolism and secretion-like coupling mechanisms play roles in CCE in corneal endothelial cells.
authors.affiliation: School of Optometry, Indiana University, Bloomington, Indiana 47405, USA.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Xie
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Changbin
authors.initials: C
authors.lastname: Zhai
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Bonanno
mesh_terms: D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001896:Boron Compounds / Q000494:pharmacology; D020126:Brefeldin A / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D003315:Cornea / Q000187:drug effects / Q000378:metabolism; D015638:Cytochalasin D / Q000378:metabolism / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D004728:Endothelium, Corneal / Q000166:cytology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008387:Marine Toxins; D008954:Models, Biological; D010080:Oxazoles / Q000378:metabolism; D011487:Protein Conformation; D013324:Strontium / Q000378:metabolism; D013997:Time Factors; D019324:beta-Naphthoflavone / Q000494:pharmacology
chemical_list: D001896:Boron Compounds; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D020126:Brefeldin A; D015638:Cytochalasin D; D019324:beta-Naphthoflavone; C059041:calyculin A; C040776:5,6-epoxy-8,11,14-eicosatrienoic acid; D000255:Adenosine Triphosphate; D003577:Cytochrome P-450 Enzyme System; C109986:2-aminoethoxydiphenyl borate; D015126:8,11,14-Eicosatrienoic Acid; D002118:Calcium; D013324:Strontium