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Effects of Ionophores X537A and A23187 and Calcium-Free Medium on Corneal Endothelial Morphology

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Effects of Ionophores X537A and A23187 and Calcium-Free Medium on Corneal Endothelial Morphology Effects of ionophores X537A and A23187 and calcium-free medium on corneal endothelial morphology Michael E. Stern, Henry F. Edelhauser, HarlanJ. Pederson, and William D. Staatz Past studies have shown that apical junctional complexes (AJCs) of corneal endothelial cells break down in the presence of a Ca++-free medium. The purpose of this study xoas to examine the ability of Ca++ ionophores to maintain the AJCs in the Ca++-free media in both isolated perfused corneas and cultured endothelial cells. In addition, the ability of disintegrated AJCs to re-form ivhen the endothelium is returned to a medium containing calcium was also exam- ined. Rabbit corneas were mounted in an in vitro specular microscope and perfused with a Ca++-free medium, or a Ca++-free medium containing 10~5M X537A or A23187 calcium ionophore. Also, confluent monolayer cultures of bovine corneal endothelial cells were placed in a Ca++-free medium or a Ca++-free medium containing IO~5M X537A or A23187 Ca++ ionophore and incubated for selected time periods. When junctional breakdown occurred, one cornea or culture plate was fixed for scanning and transmission electron microscopy (SEM and TEM), and the other was returned to a medium containing Ca++ and subsequently fixed for SEM and TEM. Both isolated perfused and cultured corneal endothelial cell AJCs exhibited marked disintegration in the presence of a Ca++-free medium. The presence of an ionophore in the medium prevented AJC disintegration in the isolated perfused cornea and delayed it in the cultured cells. When returned to a medium containing Ca++, the corneas that had been per- fused with Ca++-free medium containing an ionophore re-formed the junctions sooner than did those that had been perfused with a Ca++-free medium alone. These results suggest that the ionophores may be capable of mobilizing intracellular calcium to protect the AJCs. Key words: calcium, corneal endothelium, corneal swelling, endothelial junctions, ionophores A23187, X537A he maintenance of corneal endothelial tant to prevent corneal opacity. The AJCs are apical junctional complexes (AJCs) is impor- composed of the adjacent cell membranes supported by microfilaments along the apical border. There are only two instances under From the Departments of Physiology and Ophthalmol- experimental conditions where AJCs have ogy, The Medical College of Wisconsin, Milwaukee, been reported to break down. Kaye and as- and The Research Service, Veterans Administration 1 2 Medical Center, Wood (Milwaukee), Wise. sociates ' reported that the corneal endo- Supported in part by NEI research grant EY00933, thelial AJCs undergo a progressive disinte- ++ Training Grant T32-EY007016, Ophthalmic Research gration during perflision with a Ca -free Center Grant IP-30-EY-01931, and the Veterans Ad- medium, and re-formation of the AJCs occurs ministration. with the return of calcium to the medium. Submitted for publication May 16, 1980. Reprint requests to: H. F. Edelhauser, Ph.D., Depart- The second case of junctional breakdown oc- ment of Physiology, The Medical College of Wiscon- curs if the endothelial cells are perfused (15 sin, P.O. Box 26509, Milwaukee, Wise. 53226. to 45 min) with a bicarbonate Ringer's with- 497 Downloaded from iovs.arvojournals.org on 09/28/2021 Invest. Ophthalmol. Vis. Sci. 498 Stern et al. April 1981 out glucose but containing 4 X 10 4M di- normal cell cytokinesis and morphogenesis. amide. This causes thiol oxidation of the in- It has been suggested that intracellular Ca++ tracellular glutathione, with resulting endo- interacts with the apical microfilament net- thelial cell junctional breakdown and marked work in the control of cytokinesis.17 corneal swelling.3 Since past studies have shown that the The effects of many ions on cell function AJCs of corneal endothelial cells undergo a have been investigated with the use of progressive disintegration in a calcium-free ionophores (lipophilic antibiotics), which can medium, the purpose of this study was to complex cations and facilitate their move- examine tha ability of calcium ionophores to ment across membranes. Ionophores are maintain corneal endothelial AJCs in a calci- compounds of moderate molecular weight um-free medium. In addition, the ability of (approximately 200 to 2000) capable of form- disintegrated AJCs to re-form when the en- ing lipid-soluble complexes with polar cat- dothelium is returned to a medium contain- ions. They allow very rapid diffusion of cat- ing calcium was also examined. ions across lipid barriers without requiring participation of any lipid-associated proteins. Methods As typical carboxylic ionophores, X537A and New Zealand white rabbits (2 to 3 kg) were sac- A23187 have one carboxyl group per mole- rificed, and their corneas were isolated and cule, situated at the end of a chain that is held mounted for endothelial perfusion with the in in a ringlike conformation by head-to-tail hy- vitro specular microscope.18 The corneal en- drogen bonding. Two ionophore molecules dothelia were perfused at a constant rate of 20.6 are thought to envelop one cation during fil/min with a Harvard infusion pump, with tem- transport across membranes.4"6 During the perature and pressure being maintained at 37° C and 15 ± 2 mm Hg, respectively. The corneal epi- complexing of the ionophores with a divalent thelium in all experiments was intact and covered cation, an electroneutral exchange occurs in + with silicone oil (No. 20; Dow Corning, Midland, which the ionophore releases 2 H ions. Mich.). Measurements of corneal thickness were When the complex traverses the membrane taken every 15 min. + and releases the divalent cation, 2 H ions The corneal endothelial cells were perfused 7 are picked up. with either (1) a calcium-free glutathione bicar- Past studies have shown that calcium bonate Ringer's containing NaCl (6.521 gm/L), ionophores (A23187, X537A) alter calcium KC1 (0.358 gm/L), MgCl2 (0.159 gm/L), NaH2PO4 flux across cell membranes in erythrocytes,8 (0.103 gm/L), NaHCO3 (2.454 gm/L), glucose (0.90 gm/L), and reduced glutathione (0.92 gm/L) and they have been shown to increase chlo- 9 or (2) the same calcium-free medium containing ride transport in cornea. When ionophores 1 x 10~5M calcium ionophore (X537A or A23187). are perfused to the mucosal surface of the The calcium ionophore was first solubilized with rabbit colon, the tissue has been shown to dimethyl sulfoxide (DMSO). The final concentra- change from active Cl~ absorption to Cl~ se- tion of DM SO added to the perfusion medium was cretion.6' 10 In cardiac muscle, ionophores in- 0.5% to 1% DMSO. DMSO was selected over crease Ca++ available for contraction as well ethanol because it has been used for corneal as modulate K+ permeability in Purkinje cryopreservation, and the endothelium was shown fibers.11 Calcium ionophores have also been to tolerate up to 7.5% DMSO without altered 19 reported to release Ca++ from sarcoplasmic physiological function. Isolated control corneas reticulum in skeletal muscle12' 13 and to carry perfused with 0.5% to 1% DMSO in GBR for 3 hr + H were found to increase in thickness at 55 ± 4 Na across muscle fiber membranes. juWhr (n = 4) due to the osmotic effect of DMSO. Ionophores have also been used to prevent In the initial perfusion studies, six pairs of rab- butyrate-induced moqjhological changes in bit corneas were mounted in the specular micro- 15 cultured mammalian cells. In a recent scope and perfused for 1.25 hr. One of the corneas 16 study, Conrad and Davis have shown that was perfused with a calcium-free medium, and its Ca++ ionophore can alter the bound/ mate perfused with a calcium-free medium con- ionizable calcium ratio in snail eggs and affect taining 1 x 1O"5M X537A ionophore. Downloaded from iovs.arvojournals.org on 09/28/2021 Volume 20 Number 4 Calcium ionophores and endothelial junctions 499 Fig. 1. A, Specular micrograph of corneal endothelium perfused for 1.25 hr with Ca++-free medium containing 1% DMSO. Black areas in the pattern and widened junctions indicate cell separation. (Calibration bar = 50 /u-m.) B, Scanning electron micrograph of the same cornea. The cells are separating at the junctions. (Bar = 50 jum.) C, Transmission electron micrograph of the same cornea with the microplicae disengaged along the lateral intercellular space. (Bar = 1 (Mm.) D, Specular micrograph of the corneal endothelium perfused for 2 hr with a Ca++-free medium plus 1% DMSO and 1 X 10"5M X537A calcium ionophore. (Bar = 50 /iin.) E, Scanning election micrograph of the same cornea showing junctional maintenance as well as an increase in the number of microvilli protruding from the surface. (Bar = 50 jum.) F, Transmission electron micrograph of the same cornea confirming the intact junctional complex; however, cells have become vacuolated. (Bar = 1 ^m.) The reversibility of AJC disintegration was stud- min, one of the corneas from each pair was fixed ied first by perfusing, for 90 min, 10 pairs of cor- for scanning electron microscopy (SEM) and neal endothelia with a calcium-free medium and 6 transmission electron microscopy (TEM). The pairs of corneal endothelia for each ionophore with calcium-free and the calcium-free plus ionophore a calcium-free medium containing 1 X 10"5M perfusion medium from the other paired cornea ionophore (X537A or A23187). At the end of 90 was replaced with a GBR solution containing 1.2 Downloaded from iovs.arvojournals.org on 09/28/2021 Invest. Ophthalmol. Vis. Sci. 500 Stern et al. April 1981 Fig. 2. For legend see facing page. Downloaded from iovs.arvojournals.org on 09/28/2021 Volume 20 Number 4 Calcium ionophores and endothelial junctions 501 mM Ca++ with either 0.5% or 1% DMSO, and the ++ perfusion was continued for an additional 90 min.
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