Calcium Influx Factor from Cytochrome P-450 Metabolism and Secretion-like Coupling Mechanisms for Capacitative Calcium Entry in Corneal Endothelial Cells
2002; Elsevier BV; Volume: 277; Issue: 19 Linguagem: Inglês
10.1074/jbc.m109518200
ISSN1083-351X
AutoresQiang Xie, Yan Zhang, Changbin Zhai, Joseph A. Bonanno,
Tópico(s)Circadian rhythm and melatonin
ResumoNotwithstanding 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 β-naphthoflavone (BN) enhanced CCE measured by Sr2+ 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 Ca2+ entry. Both BN-enhanced CCE and the 5,6-EET-induced Ca2+ 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, Sr2+ 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. 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 β-naphthoflavone (BN) enhanced CCE measured by Sr2+ 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 Ca2+ entry. Both BN-enhanced CCE and the 5,6-EET-induced Ca2+ 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, Sr2+ 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. A wide variety of ligands initiate responses through the process of calcium signaling (1.Berridge M. Nat. Rev. 2000; 1: 11-21Crossref Scopus (4419) Google Scholar). Ligand-induced generation of intracellular calcium signaling involves generation of inositol 1,4,5-triphosphate (IP3) 1The abbreviations used are: IP3inositol trisphosphate2-APBaminoethoxydiphenyl borateBFAbrefeldin ABNβ-naphthoflavoneCalyAcalyculin ACCEcapacitative calcium entryCIFcalcium influx factorCPAcyclopiazonic acidCytDcytochalasin DEETepoxyeicosatrienoic acidERendoplasmic reticulumIP3RIP3receptor(s)PMplasma membraneRyRryanodine receptorSERCAsarco-endoplasmic reticulum Ca2+-ATPaseSOCstore-operated Ca2+ channelTRPtransient receptor potentialAAarachidonic acid(s)1The abbreviations used are: IP3inositol trisphosphate2-APBaminoethoxydiphenyl borateBFAbrefeldin ABNβ-naphthoflavoneCalyAcalyculin ACCEcapacitative calcium entryCIFcalcium influx factorCPAcyclopiazonic acidCytDcytochalasin DEETepoxyeicosatrienoic acidERendoplasmic reticulumIP3RIP3receptor(s)PMplasma membraneRyRryanodine receptorSERCAsarco-endoplasmic reticulum Ca2+-ATPaseSOCstore-operated Ca2+ channelTRPtransient receptor potentialAAarachidonic acid(s) and diacylglycerol by phospholipase C. The binding of IP3 to IP3 receptors (IP3R), which are located in the membrane of the endoplasmic reticulum (ER), activates the intrinsic Ca2+ channel and releases Ca2+ from the ER (the Ca2+ stores) into the cytosol. The release of Ca2+ is closely followed by entry of extracellular Ca2+ into the cytoplasm across the plasma membrane (PM). This process is called "capacitative calcium entry" (CCE) or "store-operated calcium entry" (2.Putney Jr., J.W. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2104) Google Scholar, 3.Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1289) Google Scholar, 4.Putney Jr., J.W. Broad L.M. Braun F.-J. Lievremont J.-P. Bird G.S.J. J. Cell Sci. 2001; 114: 2223-2229Crossref PubMed Google Scholar). The signal for activation of PM Ca2+ channels, termed store-operated Ca2+channels (SOC), appears to be the depletion of the ER Ca2+stores. CCE can be alternatively induced by emptying the Ca2+ store with the use of inhibitors of the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA), which actively transports Ca2+ from the cytosol into the ER (3.Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1289) Google Scholar). inositol trisphosphate aminoethoxydiphenyl borate brefeldin A β-naphthoflavone calyculin A capacitative calcium entry calcium influx factor cyclopiazonic acid cytochalasin D epoxyeicosatrienoic acid endoplasmic reticulum IP3receptor(s) plasma membrane ryanodine receptor sarco-endoplasmic reticulum Ca2+-ATPase store-operated Ca2+ channel transient receptor potential arachidonic acid(s) inositol trisphosphate aminoethoxydiphenyl borate brefeldin A β-naphthoflavone calyculin A capacitative calcium entry calcium influx factor cyclopiazonic acid cytochalasin D epoxyeicosatrienoic acid endoplasmic reticulum IP3receptor(s) plasma membrane ryanodine receptor sarco-endoplasmic reticulum Ca2+-ATPase store-operated Ca2+ channel transient receptor potential arachidonic acid(s) For CCE, answers to two main questions remain elusive: 1) What are the molecular identities of SOCs? and 2) What are the mechanisms linking store depletion to calcium influx? Recently, homologues ofDrosophila transient receptor potential (TRP) channels have been postulated to be SOCs (5.Zhu X. Jiang M. Peyton M. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar, 6.Clapham D.E. Runnels L.W. Strübing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (955) Google Scholar). In the current study, we addressed the second question only. The conformational coupling model, suggesting that IP3Rs activate SOCs/TRPs, was recently favored (7.Kiselyov K. Xu X. Mozhayeva G. Kuo T. Pessah I. Mignery G. Zhu X. Birnbaumer L. Muallem S. Nature. 1998; 396: 478-482Crossref PubMed Scopus (560) Google Scholar, 8.Kiselyov K. Mignery G.A. Zhu M.X. Muallem S. Mol. Cell. 1999; 4: 423-429Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 9.Kiselyov K. Muallem S. Trends Neurosci. 1999; 22: 334-337Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 10.Boulay G. Brown D.M. Qin N. Jiang M. Dietrich A. Zhu M.X. Chen Z. Birnbaumer M. Mikoshiba K. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14955-14960Crossref PubMed Scopus (348) Google Scholar, 11.Ma H.-T. Patterson R.L. Van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (531) Google Scholar, 12.Rosado J.A. Sage S.O. Trends Cardiovasc. Med. 2000; 10: 327-332Crossref PubMed Scopus (53) Google Scholar). However, this model was challenged by the finding that cells lacking IP3R have normal SOC activity (13.Broad L.M. Braun F.-J. Lievremont J.-P. Bird G.S.J. Kurosaki T. Putney Jr., J.W. J. Biol. Chem. 2001; 276: 15945-15952Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 14.Ma H.-T. Venkatachalam K. Li H.-S. Montell C. Kurosaki T. Patterson R.L. Gill D.L. J. Biol. Chem. 2001; 276: 18888-18896Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Furthermore, 2-aminoethoxydiphenyl borate (2-APB), a drug thought to be a specific inhibitor of IP3R, blocks the CCE pathway independently of IP3R (13.Broad L.M. Braun F.-J. Lievremont J.-P. Bird G.S.J. Kurosaki T. Putney Jr., J.W. J. Biol. Chem. 2001; 276: 15945-15952Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 14.Ma H.-T. Venkatachalam K. Li H.-S. Montell C. Kurosaki T. Patterson R.L. Gill D.L. J. Biol. Chem. 2001; 276: 18888-18896Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 15.Ma H.T. Venkatachalam K. Parys J.B. Gill D.L. J. Biol. Chem. 2002; 277: 6915-6922Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 16.Prakriya M. Lewis R.S. J. Physiol. (Lond.). 2001; 536: 3-19Crossref Scopus (424) Google Scholar, 17.Dobrydneva Y. Blackmore P. Mol. Pharmacol. 2001; 60: 541-552PubMed Google Scholar, 18.Putney Jr., J.W. Mol. Interventions. 2001; 1: 84-94PubMed Google Scholar). Alternatively, a secretion-like coupling model was put forward based on the fact that reorganization of the cortical actin cytoskeleton modulates CCE (19.Patterson R.L. van Rossum D.B. Gill D.L. Cell. 1999; 98: 487-499Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). Formation of a tight actin layer subjacent to the PM displaces the cortical ER and inhibits CCE, whereas CCE is not affected by whole cell actin disassembly. The secretion-like coupling model was further supported by a study in Xenopus oocytes, showing that SNAP-25, a component of the vesicle fusion machinery, is required for CCE (20.Yao Y. Ferrer-Montiel A.V. Montal M. Tsien R.Y. Cell. 1999; 98: 475-485Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). However, the secretion-like coupling model was also challenged by a study on the rat basophilic cell line (21.Bakowski D. Glitsch M.D. Parekh A.B. J. Physiol. (Lond.). 2001; 532: 55-71Crossref Scopus (136) Google Scholar), showing that none of the maneuvers that alter the actin cytoskeleton affectsI crac, the best characterized store-operated Ca2+ current. It seems that different cells may possess distinct mechanisms for CCE. A seemingly opposed model suggests that store depletion causes release of a soluble factor called calcium influx factor (CIF), which activates SOCs in the PM. Extraction of CIFs has been documented by several groups (22.Randriamampita C. Tsien R.Y. Nature. 1993; 364: 809-814Crossref PubMed Scopus (790) Google Scholar, 23.Csutora P. Su Z. Kim H.Y. Bugrim A. Cunningham K.W. Nuccitelli R. Keizer J.E. Hanley M.R. Blalock J.E. Marchase R.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 121-126Crossref PubMed Scopus (103) Google Scholar, 24.Trepakova E.S. Csutora P. Hunton D.L. Marchase R.B. Cohen R.A. Bolotina V.M. J. Biol. Chem. 2000; 275: 26158-26163Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). 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Several recent studies provided more evidence for the role of cytochrome P-450s in CCE (29.Rzigalinski B.A. Willoughby K.A. Hoffman S.W. Falck J.R. Ellis E.F. J. Biol. Chem. 1999; 274: 175-182Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 30.Graier W. Simecek S. Sturek M. J. Physiol. (Lond.). 1995; 482: 259-274Crossref Scopus (209) Google Scholar, 31.Hoebel B. Kostner G. Graier W. Br. J. Pharmacol. 1997; 121: 1579-1588Crossref PubMed Scopus (63) Google Scholar). In particular, 5,6-epoxyeicosatrienoic acid (5,6-EET), one of the metabolites of cytochrome P-450 epoxygenases, was suggested to act as a CIF (29.Rzigalinski B.A. Willoughby K.A. Hoffman S.W. Falck J.R. Ellis E.F. J. Biol. Chem. 1999; 274: 175-182Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 30.Graier W. Simecek S. Sturek M. J. Physiol. (Lond.). 1995; 482: 259-274Crossref Scopus (209) Google Scholar). Several recent reviews on the CCE mechanism are available (4.Putney Jr., J.W. Broad L.M. Braun F.-J. Lievremont J.-P. Bird G.S.J. J. Cell Sci. 2001; 114: 2223-2229Crossref PubMed Google Scholar, 12.Rosado J.A. Sage S.O. Trends Cardiovasc. Med. 2000; 10: 327-332Crossref PubMed Scopus (53) Google Scholar, 32.Birnbaumer L. Boulay G. Brown D. Jiang M. Dietrich A. Mikoshiba K. Zhu X. Qin N. Recent Prog. Horm. Res. 2000; 55: 127-161PubMed Google Scholar). The corneal endothelium is a monolayer of cells located at the posterior surface of the cornea. This cell layer plays a critical role in regulating the hydration and transparency of the cornea. Ion and fluid transport by the corneal endothelium control the connective tissue (stroma) hydration (33.Maurice D.M. Giardini A.A. Br. J. Ophthalmol. 1951; 35: 791-797Crossref PubMed Scopus (72) Google Scholar, 34.Sun X.C. Bonanno J.A. Jelamskii S. Xie Q. Am. J. Physiol. 2000; 279: C1648-C1655Crossref PubMed Google Scholar). In corneal endothelial cells, purinergic agonists and the SERCA inhibitor CPA induce CCE (35.Srinivas S.P. Yeh J.C. Ong A. Bonanno J.A. Curr. Eye Res. 1998; 17: 994-1004Crossref PubMed Scopus (41) Google Scholar). CCE is also important in regulating bicarbonate flux across the corneal endothelium, 2Y. Zhang, Q. Xie, and J. A. Bonanno, unpublished observation.2Y. Zhang, Q. Xie, and J. A. Bonanno, unpublished observation. which is essential for the transparency of the cornea. In the current study, the secretion-like coupling and CIF models were examined using a combinatorial approach in corneal endothelial cells. The CCE blocker 2-APB inhibited both β-naphthoflavone (BN)-enhanced CCE and 5,6-EET-induced [Ca2+]i elevation, indicating that cytochrome P-450 metabolites of arachidonic acids (AA) may be CIFs. Treatment with calyculin A (CalyA) inhibited CCE, which was partially reversed by cytochalasin D, suggesting a secretion-like coupling mechanism for CCE. However, CalyA had no effect on BN-enhanced CCE. The vesicular transport inhibitor brefeldin A (BFA) inhibited CCE in both vehicle- and BN-treated cells. Surprisingly, Sr2+entry without store depletion was enhanced in BN-treated cells, which was inhibitable by 2-APB. These results suggest that both the secretion-like coupling and CIF mechanisms play roles in CCE. An integrative model of CCE is discussed. Bovine corneal endothelial cells were cultured to confluence on glass coverslips. Briefly, primary cultures from fresh cow eyes were established in T-25 flasks in 3 ml of Dulbecco's modified Eagle's medium, 10% bovine calf serum with antibiotic antimycotic agents (100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml fungizone); gassed with 5% CO2-95% air at 37 °C; and fed every 2–3 days. These cells were subcultured to coverslips. The cells were transferred to 0.5% serum with Dulbecco's modified Eagle's medium for at least 12 h before experiments. The composition of Ca2+-rich Ringer solution was 157.7 mmNa+, 4 mm K+, 0.61 mmMg2+, 1.4 mm Ca2+, 151.02 mm Cl−, 1 mmHPO42−, 10 mm HEPES, 12.7 mm gluconate−, and 5 mm glucose. Ca2+-free solution was composed of 157.7 mmNa+, 4 mm K+, 0.61 mmMg2+, 148.22 mm Cl−, 1 mm HPO42−, 10 mm HEPES, 12.7 mm gluconate−, 0.5 mm EGTA, and 5 mm glucose. Sr2+solution was made by replacing Ca2+ with Sr2+in Ca2+-rich solution. For all solutions, the osmolarity was adjusted to 300 ± 5 mOsm with sucrose, and the pH was adjusted to 7.5. Fura-2 acetoxymethyl ester was obtained from Molecular Probes (Eugene, OR). Cell culture supplies were obtained from Invitrogen. Calyculin A was purchased from LC Labs (Woburn, MA). 5,6-EET was purchased from Cayman Chemical (Ann Arbor, MI). 5,6-EET was prepared in the same manner as the manufacturer's protocol and as described by Rzigalinskiet al. (29.Rzigalinski B.A. Willoughby K.A. Hoffman S.W. Falck J.R. Ellis E.F. J. Biol. Chem. 1999; 274: 175-182Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). 2-APB and brefeldin A were obtained fromCalbiochem (San Diego, CA). All other chemicals were obtained from Sigma. Stock solutions of the above chemicals were stored desiccated at −20 °C. Intracellular Ca2+([Ca2+]i) was measured with Fura-2, a calcium-sensitive fluorescent dye. The cells on coverslips were loaded with Fura-2 by incubation in Ca2+-rich Ringer containing 5 μm Fura-2/AM for 30 min at room temperature. Then cells were washed for 45 min in Ca2+-rich Ringer without Fura-2. The coverslips were placed in a perfusion chamber designed for an inverted microscope (Diaphot; Nikon). Ca2+ measurements were done at room temperature using a microscope fluorimeter (Photon Technology International, Lawrenceville, NJ). Fura-2 was excited at 340 and 380 nm, whereas fluorescence emission was monitored at 505 nm. Ca2+ measurements are shown as 340/380 nm ratios obtained from groups of 10–15 cells. CCE was studied by two protocols: 1) for Ca2+-rich protocol, CPA was applied in Ca2+-rich solution; the sustained [Ca2+]i elevations after 10-min applications of CPA were used as relative levels of CCE (see Fig. 2 A) (36.Singh B.B. Liu X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 36483-36486Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar,37.Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar) and 2) for Sr2+ add-back protocol, store depletion was achieved by application of 100 μm ATP or 20 μm CPA in Ca2+-free solution. After allowing the [Ca2+]i to peak and then decrease to a steady-state level (normally 6–10 min for ATP and 12–15 min for CPA), 1.4 mm Sr2+ was introduced. Because Sr2+ acts similar to Ca2+ in interactions with Fura-2 (38.Schilling W. Rajan L. Strobl-Jager E. J. Biol. 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Student's t test was used to analyze most of the data, except a two-way analysis of variance was used to analyze the difference between the increase of Sr2+entry after store depletion and the increase of basal Sr2+entry in BN-treated cells; p < 0.05 was considered significant. The means and standard errors are shown in all inset histographs in the figures. Although originally recognized as an IP3R inhibitor, 2-APB has recently been shown to block the CCE pathway independent of the IP3R (13.Broad L.M. Braun F.-J. Lievremont J.-P. Bird G.S.J. Kurosaki T. Putney Jr., J.W. J. Biol. Chem. 2001; 276: 15945-15952Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 14.Ma H.-T. Venkatachalam K. Li H.-S. Montell C. Kurosaki T. Patterson R.L. Gill D.L. J. Biol. Chem. 2001; 276: 18888-18896Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 15.Ma H.T. Venkatachalam K. Parys J.B. Gill D.L. J. Biol. 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To determine whether cytochrome P-450 metabolites are involved in the activation of SOCs in corneal endothelial cells, we first examined whether enhancement of CCE by the cytochrome P-450 inducer BN could be inhibited by the CCE blocker 2-APB. BN is a well known cytochrome P-450 inducer in vascular endothelium (30.Graier W. Simecek S. Sturek M. J. Physiol. (Lond.). 1995; 482: 259-274Crossref Scopus (209) Google Scholar, 48.Pinto A. Abraham N.G. Mullane K.M. J. Pharmacol. Exp. Ther. 1986; 236: 445-451PubMed Google Scholar). Induction of cytochrome P-450s by BN potentiated agonist-induced Ca2+ and Mn2+ influx in cultured endothelial cells from human umbilical veins, whereas intracellular Ca2+ release remained unchanged (30.Graier W. Simecek S. Sturek M. J. Physiol. (Lond.). 1995; 482: 259-274Crossref Scopus (209) Google Scholar). Similarly, BN did not affect agonist-induced intracellular Ca2+ release or base-line fluorescence ratio in corneal endothelial cells (data not shown). Fig. 1 A shows that after store depletion by ATP in the absence of Ca2+, we observed robust Sr2+ entry in BN-treated cells, significantly greater than that in vehicle-treated cells using the Sr2+ add-back protocol. BN also enhanced Ca2+ entry after store depletion using the Ca2+ add-back protocol (data not shown). Fig. 1 A shows that 2-APB significantly inhibited Sr2+entry in vehicle-treated cells. Interestingly, 2-APB also inhibited Sr2+ entry in BN-treated cells to the same level as in vehicle-treated cells (Fig. 1 A, inset, compareCon + 2-APB with BN + 2-APB; p > 0.33). Additionally, less selective CCE blockers La3+ (100 μm) and SKF 96365 (50 μm) completely blocked Sr2+ entry in both vehicle- and BN-treated cells (data not shown), further supporting the role of BN-induced cytochrome P-450 activity in CCE. 5,6-EET, one of the products of cytochrome P-450 metabolism of AA, has been proposed to be a CIF in vascular endothelial cells and astrocytes (29.Rzigalinski B.A. Willoughby K.A. Hoffman S.W. Falck J.R. Ellis E.F. J. Biol. Chem. 1999; 274: 175-182Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 30.Graier W. Simecek S. Sturek M. J. Physiol. (Lond.). 1995; 482: 259-274Crossref Scopus (209) Google Scholar). 5,6-EET-activated Ca2+ channels were also permeable to Mn2+ and Ba2+ and sensitive to Ni2+ and La3+, characteristics of SOCs (30.Graier W. Simecek S. Sturek M. J. Physiol. (Lond.). 1995; 482: 259-274Crossref Scopus (209) Google Scholar). In Fig. 1 B, we tested whether 5,6-EET could induce Ca2+ entry in the absence of store depletion. Although the vehicle had no effect (not shown), 5,6-EET caused a small but significant [Ca2+]i elevation in Ca2+-rich medium. In Ca2+-free medium, 5,6-EET did not affect [Ca2+]i (not shown), indicating that 5,6-EET acts on Ca2+ entry but not Ca2+release from the intracellular Ca2+ stores (29.Rzigalinski B.A. Willoughby K.A. Hoffman S.W. Falck J.R. Ellis E.F. J. Biol. Chem. 1999; 274: 175-182Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 30.Graier W. Simecek S. Sturek M. J. Physiol. (Lond.). 1995; 482: 259-274Crossref Scopus (209) Google Scholar). To test whether 5,6-EET was activating SOCs, we examined the effects of the CCE blocker 2-APB on 5,6-EET-induced [Ca2+]ielevation. Because 2-APB alone could partially release the Ca2+ store and increase [Ca2+]i3 (43.Maruyama T. Kanaji T. Nakade S. Kanno T. Mikoshiba K. J. Biochem. (Tokyo). 1997; 122: 498-505Crossref PubMed Scopus (773) Google Scholar, 49.Gregory R.B. Rychkov G. Barritt G.J. Biochem. J. 2001; 354: 285-290Crossref PubMed Scopus (169) Google Scholar, 50.Missiaen L. Callewaert G. De Smedt H. Parys J.B. Cell Calcium. 2001; 29: 111-116Crossref PubMed Scopus (153) Google Scholar), we pretreated cells with 2-APB until the traces became steady, usually for 5 min, before 5,6-EET was introduced. Consistent with the results on BN-enhanced Sr2+ entry, 2-APB significantly inhibited 5,6-EET-induced Ca2+ entry (Fig. 1 B). Thus, both BN-enhanced CCE and 5,6-EET-induced Ca2+ entry were inhibited by 2-APB, indicating that cytochrome P-450 metabolites may be the chemical messengers for CCE. The elevation of [Ca2+]i caused by 5,6-EET is smaller than that caused by CPA (Fig. 2 A). One possible reason is the labile nature of 5,6-EET. Another possibility is that 5,6-EET may be just one of the metabolites that activate SOC and that the full activation of CCE needs multiple cytochrome P-450 metabolites. In contrast to the CIF model for CCE, a seemingly opposed model suggests that a secretion-like coupling mechanism is required for the activation of SOCs (19.Patterson R.L. van Rossum D.B. Gill D.L. Cell. 1999; 98: 487-499Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). The primary evidence for the secretion-like coupling model was the observation that manipulation of the actin cytoskeleton could modulate CCE. However, these two models may not be mutually exclusive if the key protein/enzyme producing CIFs needs to be "secreted" in the proximity of the PM to exert their effects, especially if the CIFs are ephemeral. To explore this possibility, we first attempted to test whether a secretion-like coupling mechanism exists in corneal endothelial cells. The effects of rearranging the actin cytoskeleton on CCE were examined using cytochalasin D (CytD), which inhibits actin polymerization (51.Fox J.E. Phillips D.R. Nature. 1981; 292: 650-652Crossref PubMed Scopus (142) Google Scholar), and CalyA, which induces translocation of actin to the subplasmalemmal region (19.Patterson R.L. van Rossum D.B. Gill D.L. Cell. 1999; 98: 487-499Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). Cultured corneal endothel
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