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 examined. 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 perfused 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

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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.

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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

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Fig. 2. For legend see facing page.

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mM Ca++ with either 0.5% or 1% DMSO, and the ++ perfusion was continued for an additional 90 min. 300 -- f GBR -Co +.5% DMSO The initial perfusion was conducted for 90 min because at this time period all cells show junc- r tional breakdown when perfused with a calcium- free medium. — At selected times during perfusion, specular d200 - photomicrographs of the endothelia were taken, /** and at the end of the experiment the corneas were ^t*GBR + CO-H-+ .5% DMSO fixed for SEM and TEM in 3% glutaraldehyde in ui u GBRcX537AvJEZ phosphate buffer (pH 7.2, 330 mOsm). For TEM, 8 GBRc A23I87-^ pieces from each half of each cornea were flat- embedded in a low-viscosity epoxy resin. For SEM, hot acetone was used to wash the resin from the endothelial surface of the other half of each i I cornea before polymerization.20 After polymeriza- 0 1 2 3 TIME(HRS) ++ tion, the specimens were sputter-coated with car- Co FREE MEDIA bon and gold-palladium metal. Fig. 3. Effect of Ca++-free medium and Ca++ For the tissue culture studies, bovine corneal ionophore on corneal swelling. Corneal endothelia endothelial cells were grown to confluency in perfused in Ca++-free medium and a Ca++-free Dulbecco's modified Eagle's medium (DME) con- medium plus ionophore increase in thickness at 52 taining 10% calf serum, 5% dextran, and the an- /xm/hr during the first 90 min. After 90 min in a 21 tibiotics fungizone and gentamicin. Cultures Ca++-free medium, endothelial junctional break- were supplemented every second day with fibro- down occurred, and the corneal swelling rate in- blast growth factor (100 ng/ml) prepared from beef creased to 156 /xm/hr. Further corneal swelling 22 brain. The DME was replaced by either calci- was prevented if Ca++ was added to the medium um-magnesium-free phosphate-buffered saline after 90 minutes. Partial junctional breakdown oc- 5 (CMF) (Gibco) or CMF containing 1 x 10~ M curred in the A23187-perfused corneas (A), and no X537A or A23187 calcium ionophores dissolved in junctional breakdown occurred in the X537A- 0.5% DMSO. Phase-contrast photomicrographs perfused corneas (A) after 90 min. Removal of the were taken at 0, 1, 5, 15, 30, and 60 min after the ionophores and replacement with a Ca++ medium change of medium. At 60 min the cells were fixed prevented a further increase in swelling rate and for SEM with 2.7% glutaraldehyde in sodium junctional re-formation occurred. Each point is cacodylate containing 7% sucrose (319 mOsm), pH the mean of 10 pairs for Ca++-free and six pairs for 7.4, at room temperature, rinsed three times in each ionophore perfusion. The shaded area is the cacodylate-buffered sucrose, and then postfixed in S.E.M. GBR, Glutathione bicarbonate Ringer. 2% OsO4 for 1 hr. Samples were dehydrated

Fig. 2. A, Phase-contrast photomicrograph and, B, scanning electron photomicrograph of control culture of bovine corneal endothelial cells. (Both calibration bars = 50 /im.) C, Pho- tomicrograph of bovine corneal endothelial cells suffused in Ca++, Mg++-free medium for 60 min. The junctions between cells are disrupted except for a small number of junctional bridges between cells. (Bar = 50 fim.) D, Scanning election micrograph of the same tissue culture plate. The cells have become flattened spheroids, and the junctional bridges appear in disar- ray. (Bar = 50 /Am.) E, Bovine corneal endothelial cells suffused with Ca++, Mg++-free medium containing 1 x 10~5M A23187 calcium ionophore. The cell junctions are broken, but a more orderly appearance exists. (Bar = 50 fim.) F, Scanning electron micrograph of the same tissue culture plate illustrating junctional connections between cells. (Bar = 50 fxm.) G, Bovine corneal endothelial cells suffused with Ca++, Mg++-free medium containing 1 X 10"3M X537A calcium ionophore. The junctions between cells are maintained and the cells are vacuolated. (Bar = 50 fxm.) H, Scanning electron micrograph of the same tissue culture plate reveals that the cells have maintained a more normal flattened appearance and the junctions between cells are better maintained. (Bar = 50 /xni.)

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Fig. 4. A, Scanning electron micrograph of a cornea perfused with Ca++-free medium + 0.5% DMSO for 1.75 hr. Junctional breakdown has occurred, and the endothelial cells have a "cobblestone" appearance. (Calibration bar = 50/xm.) B, Transmission electron micrograph of the same cornea. Junctional breakdown is confirmed, and microplicae are evident. (Bar = 1 fxm.) C, Scanning electron micrograph of paired cornea which was returned to Ca++ medium + 0.5% DMSO for 3.25 hr. The junctions have re-formed, and a normal monolayer appearance is seen. (Bar = 50 /xm.) D, Transmission electron micrograph of the same cornea shows re- formed junction (arrow), and the cell is normal in appearance. (Bar = 1 fxm.)

through graded alcohols, exchanged with liquid aration was further confirmed by SEM (Fig. CO2, and critical point-dried. Pieces of culture 1, B) and TEM (Fig. 1, C). The addition of and dish were mounted on stubs and sputter- 1 x 10~5M X537A ionophore to the calcium- coated with gold-palladium. free medium prevented junctional breakdown, and an intact endothelial cell pattern Results was present (Fig. 1, D and £). With TEM it Comparison between effects of calcium-free can be observed that the microfilaments medium and medium plus ionophores on junc- along the apical border were disrupted after tional morphology- Perfusion of rabbit cor- perfusion with a calcium-free medium (Fig. neal endothelia for 2 hr with a calcium-free 1, C) but were maintained with calcium medium containing 0.5% or 1% DMSO re- ionophore (Fig. 1, F). In the presence of sulted in progressive endothelial cell junc- the ionophore, the microfilaments appeared tional breakdown (Fig. 1, A). The junctions condensed and the junctional complexes between endothelial cells formed black areas were intact. as viewed with the specular microscope, in- Tissue culture: effects of calcium-free me- dicative of junctional breakdown. Cell sep- dium and ionophores on junctional mor-

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Fig. 5. A, Scanning electron micrograph of a cornea perfused with Ca++-free medium + 1 X 10~5M A23187 for 90 min. Most of the junctions between endothelial cells remain intact. (Calibration bar = 50 /Am.) B, Transmission electron micrograph of the same cornea shows swollen cells with a large amount of condensed rough endoplasmic reticulum in the junctional area and some degree of junctional complex breakdown. (Bar = 1 /Am.) C, Scanning electron micrograph of cornea perfused with Ca++-f'ree medium + 1 x 10~5M A23187 then returned to Ca++ + 0.5% DMSO without ionophore for 60 min. All the junctions now appear to be intact. (Bar = 50 /Am.) D, Transmission electron micrograph of the same cornea shows an intact junction and a normal-appearing endothelial cell. (Bar = 1 /Am.)

phology. In contrast to a control culture (Fig. swelling and junctional morphology. When 2, A and B), suffusion for 60 min in CMF the endothelium was perfused with a cal- medium severely disrupted the AJCs, and cium-free medium + 0.5% DMSO, the cor- the cells rounded up and became partially neas swelled at a rate of 52 /utm/hr over 90 detached from the substrate. However, a min (Fig. 3). This is due to the presence of sparse, irregular array of cytoplasmic strands DMSO. Corneas perfused with GBR and existed between cells {Fig. 2, C and D). The Ca++ + 0.5% DMSO exhibited the same addition of ionophore A23187 to the suffusion swelling rate; however, if corneas were per- medium allowed more of the intercellular fused with GBR and Ca++ without DMSO, bridges, but junctional breakdown still oc- there was no swelling over 90 min. After 90 curred (Fig. 2, E and F). By comparison, min, the calcium-free medium caused the ionophore X537A maintained the AJCs when endothelial cells to separate at their junc- added to the suffusion medium, and the tions, giving them a cobblestone appearance endothelial cells appear as a nearly confluent (Fig. 4, A). With TEM, the endothelial cells monolayer (Fig. 2, G and H), revealed apical junctional breakdown (Fig. 4, Effect of calcium-free medium on corneal B). A further perfusion of the corneal en-

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Fig. 6. A, Scanning electron micrograph of a cornea perfused with Ca++-free medium 4- 1 x 10~sM X537A for 90 inin. The junctions have been maintained, but an increase in the number of microvilli protruding from the surface can be observed. (Calibration bar = 50 /u,m.) B, Transmission electron micrograph of the same cornea shows the intact junction (arrow) and protruding microvilli. Note the vacuolization of the cell. (Bar = 1 /u,m.) C, Scanning electron micrograph of the paired cornea after 90 min with Ca++-free medium + 1 x 10~5M X537A followed by Ca++ + 0.5% DMSO without ionophore for 60 min. The number of protruding microvilli is closer to normal, and the junctions are intact. (Bar = 50 /am.) D, Transmission electron micrograph of the same cornea shows an intact junction and fewer vacuoles in the cell. (Bar = 1 fim.)

dothelium with a calcium-free medium for an Reversal of morphological and corneal additional 90 min resulted in an increase in swelling effects of calcium-free medium and the corneal swelling rate to 156 /xm/hr (Fig. ionophores. Corneas with their endothelium 3) and complete endothelial cell separation perfused with a calcium-free medium contain- from each other. The addition of 1.2 raM ing calcium ionophore (1 x 10~5M A23187 or Ca++ to the medium prevented a further in- X537A) also swelled at a rate of 52 jotm/hr crease in swelling rate (Fig. 3). Examination during the first 90 min (Fig. 3). of the endotheliurn with SEM shows that After 90 min perfusion with A23187, the with the addition of calcium, the endothelial majority of apical junctions remained intact, junctions re-formed, and a complete mono- although random junctional breakdown oc- layer existed (Fig. 4, C). With TEM it can be curred (Fig. 5, A and B). After 90 min, the observed that the endothelial cells were flat medium was changed to GBR + Ca++ + and the apical junctional complex has re- 0.5% DMSO without the ionophore, and the formed (Fig. 4, D). swelling rate did not change (Fig. 3). How-

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ever, SEM revealed that all disrupted junc- and plasma membrane. These two iono- tions re-formed, and a normal monolayer was phores have been used extensively in past once again observed (Fig. 5, C and D). studies to make plasma membranes perme- Endothelial cell perfusion with a Ca++-free able to exogenous Ca++ and to release stored medium containing 10~5M X537A also re- Ca++ from such intracellular membranous sulted in a swelling rate of 52 /xm/hr; how- organelles as sarcoplasmic reticulum24 and ever, no junctional breakdown was present, mitochondria. 25~27 but an increase in the number of microvilli Both the endothelial perfusion study and protruding from the endothelial surface oc- the suffused endothelial cells in tissue culture curred (Fig. 6, A). Upon TEM examination, have shown that X537A has a greater protec- the endothelial junctions were intact, and the tive effect on the AJCs than A23187. Iono- cell cytoplasm was vacuolated (Fig. 6, B). phore X537A is known to transport Ca++ With the removal of the calcium ionophore across the outer plasma membrane but dis- and the addition of calcium to the medium, plays equal ability to transport Mg++ and the no changes occurred in the swelling rate (Fig. monovalent cations Na+ and K+.7 Ionophore 3). After a continued perfusion for an addi- A23187, on the other hand, transports Ca++ tional 90 min, SEM revealed that the junc- and Mg++ to equal degrees but at pH 7.4 tions remained intact and that the number of does not transport detectable amounts of + + 4 microvilli protruding from the membrane Na and K . Because the perfusion medi- surface was reduced (Fig. 6, C). This can um in these studies was buffered at pH 7.4, further be supported with TEM illustrating ionophore A23187 probably induced large the intact endothelial junctions and the lack changes in intracellular concentrations of ++ ++ of cell vacuolization (Fig. 6, D). Ca and Mg but only small changes in the concentrations of Na+ and K+. Since our Discussion studies have shown X537A ionophore to be The present report confirms the previous more effective in junctional preservation, ++ studies by Kaye and associates,1' 2> 23 who Ca ion may not be the sole ion species in- showed that perfusion of the corneal en- volved in junctional complex formation or dothelium with a calcium-free medium will maintenance. cause the endothelial cell junctions to dis- Since it has been shown in other cells that rupt, resulting in corneal swelling. These there are three types of cytoplasmic compo- studies have also shown that returning cal- nents (actin filaments, intermediate fila- cium to the medium will enable the junctions ments, and microtubules) that control its to re-form and prevent an increase in the cytoskeletal and contractile function,28 it is corneal swelling rate. The dynamic effect not surprising for calcium to play a major role of intracellular calcium on endothelial cell in the maintenance of the corneal endothelial junctions has also been shown with the use of cells. It has been reported that calcium regu- specific calcium ionophores. We have shown lates cytoplasmic contractility in almost all that the ionophores A23187 and X537A delay nonmuscle cells28; however, the mechanism and/or prevent endothelial cell junctional that mediates it has yet to be described. In- breakdown in a calcium-free medium in both termediate filaments serve as cytoskeletal an in vitro perfusion and in confluent mono- struts for cells, and they can also serve as layer cultures of bovine corneal endothelial constituents of adhesion organelles, desmo- cells. somes, hemidesmosomes, and other junc- 29 The simplest interpretation of these results tional complexes. Intermediate filaments is that the ionophore prevents junctional may also interact with actin filaments—best breakdown by causing an increase in intra- illustrated in the construction of the apical cellular calcium by mobilization from the ends of gut epithelial cells, where bundles of mitochondria and/or endoplasmic reticulum actin filaments from microvilli insert into the

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network of tonofilaments in the terminal cally induced by DMSO. When the cells be- web.29 come separated down to Descemet's mem- Past studies by Allison,30 Bitensky et al.,31 brane, which itself is not a barrier to bulk and Gillespie32 have shown that augmented fluid flow, the swelling rate increases mark- local intracytoplasmic concentration of free edly. If the cornea is returned to a Ca++- Ca++ would be expected to promote de- containing medium in time, the barrier func- polymerization of microtubules and contraction is re-established, and further increases tion of the microfilaments; sequestration of in corneal thickness are prevented. Kaye et Ca++ and diminution of its local level would al.2 reported, as we have shown, that junc- favor assembly of microtubules and relaxation tional integrity was restored and also that the of contractility. Thus it could be proposed permeability coefficients were lowered; how- that in the corneal endothelium, when per- ever, they also found some deswelling to be fused with a calcium-free medium, the in- present. It must be noted, however, that tracytoplasmic free Ca++ could be increased, their studies differed from the present one in leading to contraction of the cytoplasmic mi- a number of possibly important ways. They crofilaments; and when Ca++ ionophore is used the Mishima perfusion chamber, the added to a Ca++-free medium, greater se- Mishima + Kudo modified KEI medium, questration of Ca++ would occur, maintaining and carried out their perfusions for up to 7 the endothelial cell microfilament network in hr.J The swelling curves presented in the the relaxed state, with the endothelial junc- present study might have reversed if the per- tions intact. This effect may be mediated fusion had been maintained for a longer time, through cyclic nucleotides, since it has been particularly since the curves of Kaye et al.2 shown in many cell types that increased show a plateau for 45 to 90 min prior to rever- levels of cyclic AMP can bring about an in- sal. Removal of DMSO would also result in a creased stability of the cytoplasmic micro- deswelling. The data expressed in both of tubules.33"35 these studies suggest that reversal (return to In light of the fact that the endothelial mi- a medium with calcium) at this time may be crovilli contain microfilaments, which is not necessary for the morphologic re-establish- unreasonable in comparison to other cells,28 ment of the junctional barrier before pump the increased number protruding into the activity can be observed. aqueous after treatment with ionophore If the endothelium is perfused with a solu- X537A might be related to a loss of mem- tion containing cytochalasin (1 to 40 /oig/ml), brane Ca++ to the junctional area, allowing the swelling rate increases proportionately to the breakdown of the cytoskeletal network. the dosage. The microfilament network is Therefore, when the corneas are returned to disrupted, as in a Ca++-free perfusion, but GBR with Ca++, the microvilli flatten into the junctions are maintained.23 The corneal the membrane surface, and the cytoskeletal endothelium is not unique in this manner, for network of the membrane is restored. It is cytochalasin in other cell types causes the possible that the irreversibility of extreme microfilaments to retract, but most cell-to- junctional disintegration, which is accom- cell adhesions remain intact.28 We have now panied by radical changes in cell shape, may shown that junctions are also maintained in a be due to total disruption of the cytoskeletal Ca++-free medium in the presence of a cal- network. Such is the case after 3 hr perfusion cium ionophore. Our experiments suggest in a calcium-free medium or 45 min perfusion that the ionophore is able to alter the intra- of 1 x 10"4M diamide.3 cellular bound/free ratio of calcium and that The swelling curves presented in this the level of ionic cytoplasmic Ca++ aids in study indicate that as long as the endotheli- junctional complex formation. In comparison um is able to maintain its barrier function in a to our studies, Sauk36 demonstrated that al- Ca++-free medium, the rate of swelling re- teration of cellular Ca++ by A23187 resulted mains constant and is limited to that osmoti- in a biphasic change in melanoma cell shape;

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however, the changes were dependent on ex- secretion in rabbit ileal mucosa: relation to actions of tracellular Ca++, as evidenced by ineffective- cyclic 3',5'-AMP and carbamylcholine. J Membr Biol 35:159, 1977. ness of the ionophore in a Ca++-deficient 7. Pressman BC: Biological applications of ionophores. medium. Perfusion of a number of tissues Annu Rev Biochem 45:501, 1976. with media containing A23187 was found to 8. Edmondson JW and Li TK: The effects of ionophore release Ca++ but did not increase levels of A23187 on erythrocytes: relationship of ATP and cyclic AMP, although levels of cyclic GMP 2,3-diphosphoglycerate to calcium-binding capacity. did increase. This action of A23187 has been Biochim Biophys Acta 443:106, 1976. ++ 6> 37 9. Candia O, Montorerno R, and Podos S: Effect of the found to be Ca -dependent. Whether ionophore A23187 on chloride transport across iso- calcium ionophore affects corneal endothelial lated frog cornea. Am J Physiol 233:F94, 1977. cell cyclic AMP is presently being deter- 10. Frizzell R: Active chloride secretion by rabbit mined in our laboratory. colon: calcium dependent stimulation by ionophore In this study, we have shown that calcium A23187. J Membr Biol 35:175, 1977. 11. Gelles J: Use of calcium ionophores to determine ionophores are able to maintain the corneal the effects of intracellular calcium on the action po- endothelial AJCs by, presumably, releasing tential of canine cardiac Purkinje fibers. Circ Res intracellular stores of calcium. The two iono- 41:94, 1977. phores, X537A and A23187, appear to have 12. Scarpa A, Baldassare J, and Inesi G: The effect of different effects on the corneal endothelial calcium ionophores on fragmented sarcoplasmic reticulum. J Gen Physiol 60:735, 1972. cells. X537A is less specific for calcium yet 13. Hainaut K and Desmedt J: Calcium ionophore maintains the junctions better than does A23187 potentiates twitch and intracellular calcium A23187, although the cells are highly vac- release in single muscle fibers. Nature 252:407, uolated. A23187 is more specific for calcium, 1974. and although the cells look very normal, it is 14. Devore D and Nastuk W: Effects of calcium ionophore X537A on frog skeletal muscle. Nature not as effective in maintaining the AJCs. 253:644, 1975. These effects are seen in cultured bovine 15. Henneberry RC, Fishman PH, and Freese E: Mor- corneal endothelial cells as well as in in vitro phological changes in cultured mammalian cells: perfused rabbit endothelial cells. prevention by calcium ionophore A23187. Cell 5:1, 1975. 16. Conrad G and Davis S: Polar lobe formation and REFERENCES cytokinesis in fertilized eggs of llyanassa obsoleta. +2 1. Kaye GI, Mishima S, Cole JD, et al: Studies on the III. Large bleb formation caused by Sr , ionophore cornea. VII. Effects of perfusion with a Ca++-free X537A and ionophore A23187, and compound medium on the endothelium. INVEST OPHTHALMOL 48-80. Dev Biol 74:152, 1980. 7:53, 1968. 17. Bernfield M and Wessells N: Intra- and extracellular 2. Kaye GI, Hoefle FB, and Donn A: Studies on the control of epithelial morphogenesis. Dev Biol Suppl cornea. VIII. Reversibility of the effects of in vitro 4:195, 1970. perfusion of the rabbit corneal endothelium with 18. McCarey B, Edelhauser H, and Van Horn D: Func- calcium-free medium. INVEST OPHTHALMOL 12:98, tional and structural changes in the corneal en- 1973. dothelium during in vitro perfusion. INVEST OPH- 3. Edelhauser H, Van Horn D, Miller P, and Pederson THALMOL 12:410, 1973. H: Effect of thiol-oxidation of glutathione with 19. Kaufman HE and Capella JA: Preserved corneal tis- diamide on corneal endothelial function, junctional sue for transplantation. J Cryo-Surg 1:125, 1968. complexes, and microfilaments. J Cell Biol 68:567, 20. Cleveland PH and Schneider CW: A simple method 1976. of preserving tissue for scanning electron micros- 4. Pfeiffer DR and Lardy HA: Ionophore A23187: the copy. Vision Res 9:1401, 1969. effect of H+ concentration on complex formation 21. Gospodarowicz D, Bialecki H, and Greenburg G: with divalent and monovalent cations and the dem- Purification of the fibroblast growth factor activity onstration of K+ transport in mitochondria mediated from bovine brain. J Biol Chem 253:3736,' 1978. by A23187. Biochemistry 15:935, 1976. 22. Gospodarowicz D, Mesher A, and Birdwell C: 5. Deber CM and Pfeiffer DR: Ionophore A23187. So- Stimulation of corneal endothelial cell proliferation lution conformation of the calcium complex and free by fibroblast and epidermal growth factors. Exp Eye acid deduced from proton and carbon-13 nuclear Res 25:75, 1977. magnetic resonance studies. Biochemistry 15:132, 23. Kaye G, Fenoglio C, Hoefle F, and Fischbarg J: 1976. Studies on the cornea. IX. Physiologic and mor- 6. Bolton J and Field M: Ca ionophore-stimulated ion phologic effects of cytochalasin B on endothelium of

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