Glucocorticoid induction of the glaucoma gene MYOC in human and monkey trabecular meshwork cells and tissues.

PURPOSE: To examine the intracellular and extracellular expression of myocilin in the human and primate trabecular meshwork (TM) in the presence and absence of glucocorticoids. METHODS: Myocilin expression was examined in cultured human TM cells by Northern blot analysis and myocilin antibody-mediated immunoprecipitation. Myocilin expression was quantified using high-resolution two-dimensional polyacrylamide gel electrophoresis of radiolabeled proteins from human TM cells, TM tissue explants, and perfused human anterior segments cultured with and without dexamethasone (DEX) for 14 to 21 days, as well as TM tissue from pigtailed monkeys treated orally for 1 year with cortisone acetate. Immunofluorescence with anti-myocilin antibodies was used to localize cellular and extracellular expression of myocilin in cultured human TM cells. RESULTS: Glucocorticoid treatment caused a significant induction of myocilin mRNA, a tetrad of cell-associated proteins, and 8 to 20 secreted proteins (molecular mass [M(r)] 56 and 59 kDa and isoelectric point [pI] 5.2 and 5.3) in some, but not all the cultured human TM cells and explanted tissues. Western immunoblot analysis using anti-myocilin peptide antibodies identified these proteins as encoded by the MYOC gene. There was significant induction of the myocilin proteins in three perfusion-cultured human eyes, in which DEX-induced elevated intraocular pressure developed. Monkeys treated 1 year with cortisol acetate showed steroid glaucoma-like morphologic changes in the TM that correlated with the induction of myocilin in the TM. Immunofluorescence analysis of cultured TM cells localized myocilin intracellularly in discrete perinuclear and cytoplasmic vesicular deposits as well as extracellularly on the cell surface associated with the extracellular matrix. In several DEX-treated TM cell lines, there were significant levels of myocilin secreted into the media. Enzymatic deglycosylation of proteins in the TM media converted the higher molecular weight isoforms of myocilin (approximately 57 kDa) to the lower molecular weight isoforms ( approximately 55 kDa). CONCLUSIONS: Although the function of myocilin is unknown, induction of these TM proteins was found in eyes in which glucocorticoid-induced ocular hypertension developed. Therefore, myocilin may play an important pathogenic role in ocular hypertension in addition to its role in certain forms of POAG.

The eicosanoid, 15-(S)-HETE, stimulates secretion of mucin-like glycoprotein by the corneal epithelium.

PURPOSE: The eicosanoid, 15-(S)-hydroxyeicosa-5Z, 8Z-11Z, 13E-tetraenoic acid (15-(S)-HETE), is known to stimulate production of mucin glycoprotein by airway epithelium. This study investigated the effect of 15-(S)-HETE on the mucin glycoprotein secretion by the corneal epithelium. METHODS: To determine the effect of dose, corneas of anesthetized New Zealand White rabbits were treated with 50, 500, or 5,000 nM 15-(S)-HETE in artificial tears for 120 minutes. To determine the time to onset of the response, corneas were treated with 500 or 1,000 nM 15-(S)-HETE in balanced salt solution for periods ranging from 5 to 120 minutes. Corneas were fixed for electron microscopy in fixative containing 0.5% cetylpyridinium chloride (CPC) to stabilize the layer of mucin-like glycoprotein on the corneal surface. The mucin layer thickness was measured by image analysis of electron micrographs. RESULTS: The layer of CPC-fixed mucin-like glycoprotein on the surface of control corneas was 0.46 +/- 0.04 microm thick. After treatment with 15-(S)-HETE, the thickness of the mucin layer increased to 0.64 +/- 0.1 microm at 50 or 5,000 nM HETE and as much as 1.02 +/- 0.2 microm in response to 500 nM HETE. Mucin thickness reached a statistical maximum of 0.59 +/- 0.1 microm after only 5 minutes of exposure to 500 or 1,000 nM HETE. CONCLUSIONS: Exposure of the cornea to 15-(S)-HETE causes a rapid-onset increase in the thickness of a layer of mucin-like glycoprotein on the surface of the corneal epithelium. This supports previous reports that corneal epithelial cells produce mucin and suggests that treatment with topical 15-(S)-HETE may be effective in treating ocular surface mucin deficiency in dry eye syndrome.

Inhibition of dexamethasone-induced cytoskeletal changes in cultured human trabecular meshwork cells by tetrahydrocortisol.

PURPOSE: To determine the cellular mechanism of action of the intraocular pressure (IOP) lowering steroid tetrahydrocortisol (THF). METHODS: Tetrahydrocortisol was evaluated for glucocorticoid antagonist activity using in vitro and in vivo assays. Systemically administered THF was evaluated for its ability to inhibit dexamethasone-induced body weight loss and systemic hypertension in rats. In vitro receptor antagonism was tested using the supernatant fraction of IM9 cells as the source of soluble glucocorticoid receptor in 3H-dexamethasone displacement binding assays. In addition, six different primary human trabecular meshwork (TM) cell lines were cultured for 0 to 14 days in the absence or presence of dexamethasone (10(-7) M) and/or THF (10(-6) to 10(-8) M). The effects of these steroids on the TM cytoskeleton were determined by epifluorescent microscopy and by transmission electron microscopy. RESULTS: Tetrahydrocortisol was unable to inhibit the dexamethasone (DEX)-induced systemic hypertension and decrease in body mass in rats and was unable to displace 3H-DEX from the soluble human glucocorticoid receptor. However, THF inhibited the DEX-induced formation of cross-linked actin networks in cultured human TM cells in a progressive and dose-dependent manner (IC50 = 5.7 x 10(-7) M). Dexamethasone caused changes in the TM cell microtubules that were reversed partially by concomitant treatment with THF. Tetrahydrocortisol alone appeared to increase microfilament bundling in TM cells. CONCLUSIONS: Tetrahydrocortisol was not a glucocorticoid antagonist at the level of the classical glucocorticoid receptor and did not appear to antagonize systemically mediated glucocorticoid activity in the rat. Tetrahydrocortisol inhibited DEX-induced changes in the TM microfilaments and microtubules. These results may explain partially the IOP lowering activity of THF because glucocorticoid-mediated changes in the TM cytoskeleton have been proposed to be involved in the generation of ocular hypertension.

Comparison of SEM processing methods for cultured human lens epithelial cells grown on flat and microcarrier bead substrates.

The increasing importance of in vitro models has presented new challenges in SEM processing techniques. The present study has evaluated the quality of preservation of cultured human lens epithelial cells processed by critical point, Peldri II, and tert-butyl alcohol drying. Specimens processed by critical point drying produced specimens with severe cracking of cell processes and microcracks across cell membrane surfaces. Peldri II and tert-butyl alcohol drying eliminated breakage of the filopodia and lamellipodia as well as eliminating the microcracks across the apical membrane surface. The morphology of lens epithelial cells grown on Cytodex 3 beads appeared rounded with convoluted membrane surfaces. These morphological features were present for cells processed by all three methods. Cytodex 3 beads were subsequently shown to shrink 52% in diameter during dehydration, which results in an 89% reduction in volume for the bead. Cells grown on Biosilon beads, which do not shrink, had a morphology similar to the cells grown on a flat substrate. These results indicate that Peldri II and tert-butyl alcohol drying offer an attractive alternative to critical point drying when preparing cultured cells for SEM. Interpretation of cultured cell morphology must consider shrinkage of the substrate material as a possible contributor to artifact.

Effects of preservative-free artificial tear solutions on corneal epithelial structure and function.

OBJECTIVES: To test the efficacy of a bicarbonate-containing artificial physiologic tear solution (solution PT) in providing an environment in which the damaged corneal epithelium can recover its normal barrier function and to compare this solution with other available artificial tears. Also, to investigate the effects on the corneal mucin layer and epithelial ultrastructure. METHODS: The corneal epithelial permeability of anesthetized rabbits was increased by exposure to 0.1% benzalkonium chloride. The corneas were then exposed to solution PT, with or without bicarbonate, or one of four commercially available artificial tear solutions for 1.5 hours, followed by a 5-minute exposure to 5(6)-carboxyfluorescein. Frozen sections of the corneas were examined by fluorescence microscopy. The fluorescence intensity (FI) of the epithelium was measured by image analysis. Undamaged corneas exposed to tear solutions were examined by transmission electron microscopy after fixation of the mucin layer with cetylpyridinium chloride. RESULTS: The FI of corneas damaged by benzalkonium chloride was increased threefold above those of undamaged controls. Damaged corneas treated with either of two commercial isotonic tear solutions partially recovered their barrier function, but the FI did not reach control levels. Corneas treated with hypotonic solutions containing ethylenediaminetetraacetic acid (EDTA) did not recover. In contrast, the FI of corneas treated with solution PT returned to control levels. This effect was lost in the absence of bicarbonate. Solution PT and the two isotonic solutions maintained normal corneal ultrastructure and mucin layer. Lack of bicarbonate in solution PT resulted in focal damage to superficial epithelial cells, whereas the EDTA-containing solutions destroyed the first two cell layers and reduced the mucin thickness. CONCLUSIONS: Bicarbonate-containing solution PT is superior to the other tear solutions tested in promoting recovery of the damaged corneal epithelial barrier and maintaining normal ultrastructure. The presence of bicarbonate appears to be essential to this process.

Dexamethasone-induced ocular hypertension in perfusion-cultured human eyes.

PURPOSE: Glucocorticoid administration can lead to the development of ocular hypertension and corticosteroid glaucoma in a subset of the population through a decrease in the aqueous humor outflow facility. The purpose of this study was to determine whether glucocorticoid treatment can directly affect the outflow facility of isolated, perfusion-cultured human eyes. METHODS: The anterior segments of human donor eyes from regional eye banks were placed in a constant flow, variable pressure perfusion culture system. Paired eyes were perfused in serum-free media with or without 10(-7) M dexamethasone for 12 days. Intraocular pressure was monitored daily. After incubation, the eyes were morphologically characterized by light microscopy, transmission and scanning electron microscopy, and scanning laser confocal microscopy. RESULTS: A significant increase in intraocular pressure developed in 13 of the 44 pairs of eyes perfused with dexamethasone with an average pressure rise of 17.5 +/- 3.8 mm Hg after 12 days of dexamethasone exposure. The contralateral control eyes, which did not receive dexamethasone, maintained a stable intraocular pressure during the same period. The outflow pathway of the untreated eyes appeared morphologically normal. In contrast, the dexamethasone-treated hypertensive eyes had thickened trabecular beams, decreased intertrabecular spaces, thickened juxtacanalicular tissue, activated trabecular meshwork cells, and increased amounts of amorphogranular extracellular material, especially in the juxtacanalicular tissue and beneath the endothelial lining of the canal of Schlemm. The dexamethasone-treated nonresponder eyes appeared to be morphologically similar to the untreated eyes, although several subtle dexamethasone-induced morphologic changes were evident. CONCLUSION: Dexamethasone treatment of isolated, perfusion-cultured human eyes led to the generation of ocular hypertension in approximately 30% of the dexamethasone-treated eyes. Steroid treatment resulted in morphologic changes in the trabecular meshwork similar to those reported for corticosteroid glaucoma and open angle glaucoma. This system may provide an acute model in which to study the pathogenic mechanisms involved in steroid glaucoma and primary open angle glaucoma.

Cytoskeletal changes in cultured human glaucoma trabecular meshwork cells.

PURPOSE: Glucocorticoid treatment of cultured human trabecular meshwork (TM) cells has been shown to reorganize actin microfilaments into cross-linked actin networks (CLANs) and to alter TM cell function. The purpose of the present study is to determine whether similar microfilament structural changes occur in TM cells derived from glaucoma patients. METHODS: The microfilament structures of nine different TM cell cultures, derived from four glaucoma patients and from five normal subjects, was examined by epifluorescent microscopy and by whole-mount transmission electron microscopy. The cells were cultured in the absence and presence of dexamethasone (DEX) for 7-14 days. RESULTS: The normal TM cell lines and glaucoma cell line TM13 had relatively low levels of cross-linked actin networks in the absence of exogenously added glucocorticoids. In contrast, the glaucoma cell lines TM23, TM36C, and TM48D had high levels of CLANs without the addition of dexamethasone. The addition of 10 M DEX to the culture medium significantly increased CLAN expression in all normal as well as glaucoma TM cell lines. CONCLUSION: CLAN formation in glaucoma TM cells or in TM cells exposed to glucocorticoids may be a factor contributing to the generation of ocular hypertension.

Glucocorticoid-induced formation of cross-linked actin networks in cultured human trabecular meshwork cells.

PURPOSE: To determine the effects of glucocorticoid treatment on the microfilament structure of cultured human trabecular meshwork cells. Topical or systemic administration of glucocorticoids can lead to the development of ocular hypertension and to the development of vision loss, which is clinically similar to primary open angle glaucoma. However, the mechanism(s) by which glucocorticoids cause ocular hypertension is not well defined. Alterations in the trabecular meshwork, the site of drainage of aqueous humor from the eye, have been linked to the development of ocular hypertension. METHODS: Human trabecular meshwork cells were cultured in the presence and absence of glucocorticoids for 0 to 21 days. The microfilament organization of the cultured trabecular meshwork cells was examined by epifluorescent and transmission electron microscopy. RESULTS: Glucocorticoids caused a progressive change in the organization of microfilaments in the trabecular meshwork cells, but not in other cultured ocular cells. By fluorescence microscopic analysis, the actin stress fibers found in control trabecular meshwork cells were reorganized on treatment with glucocorticoids into cross-linked actin networks that resembled geodesic-dome-like polygonal lattices. The cross-linked actin networks were reversible on withdrawal of the glucocorticoid treatment. Dose-response data for dexamethasone, relative ranking of activity with glucocorticoid potency, and partial inhibition with glucocorticoid antagonists all suggest the involvement of the trabecular meshwork glucocorticoid receptor in cross-linked actin network formation. The reorganization of the trabecular meshwork cytoskeleton alters cell function because glucocorticoid treatment of cultured trabecular meshwork cells also inhibited trabecular meshwork cell migration and proliferation. CONCLUSION: The steroid-induced alteration in trabecular meshwork cytoskeleton may be an important factor in the development of steroid-induced ocular hypertension and may play a role in the ocular hypertension associated with primary open angle glaucoma.

Dexamethasone induced ultrastructural changes in cultured human trabecular meshwork cells.

Glucocorticoid-induced ocular hypertension has been demonstrated in both animals and humans. It is possible that glucocorticoid-induced changes in trabecular meshwork (TM) cells are responsible for this hypertension. In order to elaborate further the effect of glucocorticoids on the trabecular meshwork, the ultrastructural consequences of dexamethasone (DEX) treatment were examined in three different human TM cell lines. Confluent TM cells were treated with 0.1 microM of DEX for 14 days, and then processed for light, epifluorescent microscopy or transmission electron microscopy (TEM). The effect of DEX treatment on TM cell and nuclear size was quantified using computer assisted morphometrics. Morphometric analysis showed a significant increase in both TM cell and nuclear size after 14 days of DEX treatment. Epifluorescent microscopy of rhodamine-phalloidin stained, control TM cells showed the normal arrangement of stress fibers. In contrast, DEX-treated TM cells showed unusual geodesic dome-like cross-linked actin networks. Control TM cells had the normal complement and arrangement of organelles as well as electron dense inclusions and large vacuoles. DEX-treated TM cells showed stacked arrangements of smooth and rough endoplasmic reticulum, proliferation of the Golgi apparatus, pleomorphic nuclei and increased amounts of extracellular matrix material. The DEX-induced alterations observed in the present study may be an indication of the processes that are occurring in the in vivo disease process.

Collagen shields exacerbate ulceration of alkali-burned rabbit corneas.

OBJECTIVE: To determine the impact of collagen shields on ulceration of rabbit corneas after alkali burn. METHODS: After a 60-second 2N sodium hydroxide burn to rabbit corneas, 24-hour collagen shields were placed on the corneas daily for 21 days; control corneas did not receive collagen shields. The extent of corneal ulceration was documented daily for 21 days by slit-lamp examination of treated and control eyes. Three separate studies were performed using collagen shields from two commercial sources. RESULTS: In the three studies, corneas in the collagen shield-treated eyes began to ulcerate sooner than those in the control group; the corneas in collagen shield-treated eyes also began to perforate sooner. At 21 days after alkali injury, the mean (+/- SE) corneal ulceration score in the collagen shield-treated rabbits was 4.1 +/- 0.17 (descemetocele formation) compared with 2.7 +/- 0.28 (midstromal ulceration) in controls. This difference was significant at P < .005. CONCLUSION: Collagen shield treatment results in marked acceleration of corneal ulceration and perforation after alkali injury.

An electron microscopic and nuclear magnetic resonance spectroscopic evaluation of rabbit corneal epithelial wound healing.

Phosphorus-31 (31P) nuclear magnetic resonance (NMR) spectroscopy is a powerful method that can be used to measure levels of phosphorus metabolites in living tissue. To determine whether NMR measurements adequately describe and quantify the wound-healing process in corneal epithelium, 31P NMR spectroscopic and electron microscopic results were correlated. Dutch belted rabbit corneal epithelium was removed with a limbus-to-limbus scrape wound and sampled from 0-30 days postwounding. Four corneas were pooled for each NMR determination of corneal adenosine triphosphate (ATP). A minimum of three animals from each time point were processed for transmission and scanning electron microscopy (EM). Similar to previous studies, migrating cells completely closed the defect by 4 days postwounding. The epithelium continued to increase in thickness and hemidesmosome density was similar qualitatively to that in control animals by 10 days postwounding. The 31P NMR ATP values showed an increase toward control values after an initial 70-75% decrease, returned to within 90-95% of nonwounded values by 10 days, and returned to control values by 30 days. After these experimental values were obtained, the predictive value of the model was tested using platelet-derived growth factor (PDGF)-treated corneas. The PDGF-treated corneas showed NMR and EM results similar to day-10 untreated corneas by 5 days after wounding. These results demonstrated the feasibility of using 31P NMR spectroscopic measurements of ATP levels as a nondestructive technique for quantifying corneal epithelial wound healing.

Rabbit corneal epithelial wound repair: tight junction reformation.

Previous studies have shown that the corneal epithelium will close a limbus to limbus scrape wound in four to five days, but requires 10 days to become firmly attached to the stroma. In order to determine if the restoration of the corneal epithelial barrier follows a similar sequence, we have used freeze-fracture to study tight junction reformation in a rabbit epithelial wound model. Dutch-belted rabbit corneal epithelium was removed with a limbus to limbus scrape wound and sampled from 0 to 30 days post-wounding. A minimum of 3 animals from each time point were processed for electron microscopy and freeze-fracture. Freeze-fracture showed that the cells at the wound margin had a reduction in the number of intramembrane particles on their apical surface. In areas adjacent to the wound edge, fragments of tight junctions were first observed on two days post-wounding specimens. The junctions became progressively more complex in the area behind the wound edge until wound closure at four days when the junctions were also present in the central region of the cornea. The maturation of the junctions continued and at five days after surgery they resembled control junctions. This sequence suggests that the the establishment of morphologically mature tight junctions may be necessary before the corneal epithelium can firmly reattach to the stroma.

Maturation of the corneal endothelial tight junction.

Apical tight junctional formation of the rabbit corneal endothelium was examined by freeze-fracture analysis and measurement of paracellular permeability to 5(6)-carboxyfluorescein. Freeze-fracture analysis indicated that apical tight junction formation of the rabbit corneal endothelium is a dynamic process. At birth, there are few tight junctional strands present and a minimal barrier for paracellular diffusion. As the rabbit matures, a more complex network of anastomosing tight junctional strands begins to encircle the cell perimeter under the apical folds. However, even in the mature animal (3 months), there are discontinuities and free ends in the network, thus suggesting that the barrier is not complete even at this stage. Paracellular permeability measurements using 5(6)-carboxyfluorescein as a tracer corroborate these anatomic findings. Endothelial paracellular flux measurements steadily decrease as the rabbit matures from birth to young adult. This indicates that the tight junctional network is increasing in complexity and progressively limiting the flow of substances through the intercellular space.

Corneal endothelial junctions and the effect of ouabain.

Paired rabbit corneas were perfused in vitro for endothelial permeability (Pac) determination with glutathione bicarbonate Ringer's solution (GBR) and GBR plus ouabain (10(-4) M). Results indicated no difference in Pac between the two groups (3.39 vs 3.67, respectively) despite significantly greater stromal swelling in the group perfused with ouabain. Freeze-fracture microscopy of similarly perfused corneas revealed intact tight junctional complexes in both groups, although the tight junctional complex of perfused corneas appeared less organized than that of freshly enucleated, nonperfused controls. Gap junctions were abundant as observed in freeze-fracture replicas of GBR-perfused endothelium, and appeared to be decreased or absent in ouabain-perfused endothelium. These results indicate that corneal endothelial tight junctions are unaffected by perfusion with ouabain, whereas gap junctions appear to be lost. The permeability and freeze-fracture data reaffirms the importance of tight junctions as permeability barriers and indicates that gap junctions are not of primary importance for maintenance or control of the corneal endothelial barrier.

Moderate Fuchs' endothelial dystrophy ATPase pump site density.

The Na+, K+-ATPase pump site density on corneal endothelial cells from Fuchs' endothelial dystrophy corneas has been shown to be drastically decreased in end-stage disease (McCartney et al, Invest Ophthalmol Vis Sci 28:1955, 1987) and significantly increased in the early stages (Geroski et al, Ophthalmology 92:759, 1985) as compared to normal endothelium. In order to provide values for corneas between these two extremes, eye bank corneas from donors with no evidence of corneal edema but with guttata across the extent of the cornea were processed for autoradiography as well as immunohistochemistry. Pump site density was increased compared to end-stage disease but was less than values reported for either functional tissue or early stage disease. Similarly, immunohistochemistry results showed the amount of Na+, K+-ATPase antibody localization to be increased in respect to end-stage disease, but reduced as compared to functional tissue. These results suggest that pump site density on endothelial cells affected with Fuchs' endothelial dystrophy follows a gradual decline towards end-stage disease values as opposed to a sudden sharp deterioration after an initial increase.

A correlative electron microscopic and freeze-fracture examination of cat corneal endothelial wound repair.

In an attempt to create a model for sustained corneal edema in humans, the present study has examined wounded cat corneal endothelium. Small central (7 mm) wounds or large 90 percent debridement wounds were created with an olive tipped cannula and corneas sampled from 1 to 75 days post-wounding were processed for light and transmission electron microscopy and freeze-fracture. In small wounds, wound closure was complete by 14 days and corneal edema was absent. During wound closure, leading edge cell membranes had decreased intramembrane particles, numerous vesicle fusion sites and lacked cell junctions. Endothelium behind the wound margin was multilayered with fragmented cell junctions. After wound closure, endothelium returned to the morphology of non-wounded endothelium except that an abnormal posterior collagenous layer (PCL) was present. Wound closure was greatly retarded in large wounds and corneas remained edematous at 75 days. The morphology of the endothelial cells was similar to that in small wounds except for the presence of large multinucleated cells and a thicker PCL. These large wound findings are similar to those observed in chronically stressed dysfunctional human corneal endothelium and in this animal model may represent a similar response to injury.

ATPase pump site density in human dysfunctional corneal endothelium.

Proper corneal hydration is maintained by a Na, K-ATPase pump located in the lateral membranes of the endothelial cells. In dysfunctional corneas this pumping action appears to break down as the corneas become edematous. In order to provide quantitative and qualitative data on the Na, K-ATPase pump site density on dysfunctional and functional human corneal endothelial cells, the present study has employed both autoradiographic and histochemical techniques. Computer-assisted morphometrics and statistical analysis showed that there was a significant reduction (P less than 0.001) in 3H-ouabain binding, and thus ATPase pump sites, in the three types of corneas (Fuchs' endothelial dystrophy, aphakic and pseudophakic bullous keratopathy) with dysfunctional endothelia as compared to both types of corneas (eye bank, keratoconus) with functional endothelial cells. There were no significant differences amongst the dysfunctional types or between the two functional types of corneal endothelial cells in respect to density of silver grains. Histochemical staining for ATPase showed less p-nitro-phenylphosphatase histochemical reaction product present on dysfunctional endothelial lateral membranes than in the functional cells.

Immunohistochemical localization of ATPase in human dysfunctional corneal endothelium.

Previous light microscopic autoradiographic results from our laboratory have shown a quantitative decrease in ATPase pump site density in dysfunctional corneal endothelium. In order to develop antibody techniques to correlate these findings with electron microscopic localization of pump sites, three types of corneas with dysfunctional endothelium (Fuchs' endothelial dystrophy, aphakic and pseudophakic bullous keratopathy) and two types of corneas with functional endothelium (age matched eye bank and keratoconus) were obtained at the time of transplant surgery. Corneas were fixed, frozen and cryostat sections were incubated in a rabbit kidney ATPase primary antibody followed by incubation in an HRP-labeled secondary antibody. Functional endothelia showed dense antibody labeling along the lateral cell membranes and there was a substantial reduction in labeling along the lateral membranes of dysfunctional endothelia. Positive tissue controls of rabbit kidney showed HRP reaction product in the convoluted tubules. Control tissue, incubated in either non-immune serum or primary antibody preabsorbed with ATPase, confirmed the specificity by having diminished or no reaction product. These results provide immunohistochemical confirmation of our autoradiographic data showing a quantitative reduction in pump site density on dysfunctional corneal endothelium.