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The eurohaline fish, Fundulus heteroclitus, adapts rapidly to enhanced salinity by increasing the ion secretion by gill chloride cells. An increase of approximately 70 mOsm in plasma osmolarity was previously found during the transition. To mimic this in vitro, isolated opercular epithelia of seawater-adapted Fundulus mounted in a modified Ussing chamber were exposed to an increase in NaCl and/or osmolarity on the basolateral side, which immediately increased I(SC). Various Cl(-) channel blockers as well as the K(+) channel blocker Ba(2+) added to the basolateral side all inhibited the steady-state as well as the hypertonic stimulation of I(SC). The exists -agonist isoproterenol stimulates I(SC) in standard Ringer solutions. In contrast, when cell volume was kept at the larger value by simultaneous addition of water, the stimulation with isoproterenol was abolished, suggesting that the key process for activation of the Na(+), K(+), 2Cl(-) cotransporter is cell shrinkage. The protein kinase C (PKC) inhibitor chelerythrine and the myosin light chain kinase (MLCK) inhibitor ML-7 had strong inhibitory effects on the mannitol activation of I(SC), thus both MLCK and PKC are involved. The two specific protein kinase A (PKA) inhibitors H-89 and KT 5720 had no effect after mannitol addition whereas isoproterenol stimulation was completely blocked by H-89. This indicates that PKA is involved in the activation of the apical Cl(-) channel via c-AMP whereas the shrinkage activation of the Na(+), K(+), 2Cl(-) cotransporter is independent of PKA activation. The steady-state Cl(-) secretion was stimulated by an inhibitor of serine/threonine phosphatases of the PP-1 and PP-2A type and inhibited by a PKC inhibitor but not by a PKA inhibitor. Thus, it seems to be determined by continuous phosphorylation and dephosphorylation involving PKC but not PKA. The steady-state Cl(-) secretion and the maximal obtainable Cl(-) secretion were measured in freshwater-adapted fish and in fish retransferred to saltwater. No I(SC) could be measured in freshwater-adapted fish or in the fish within the first 18 h after transfer to saltwater. As evidenced from Western blot analysis using antiserine-antibodies, a heavily serine phosphorylated protein of about 190 kDa was consistently observed in the saltwater-acclimated fish, but was only weakly present in freshwater-acclimated fish. This observation indicates that acclimatization to saltwater stimulates the expression of this 190-kDa protein and/or a serine/threonine kinase, which subsequently phosphorylates the protein.

The chloride cells of the gill secretory epithelium of fish that make the transition from fresh water to sea water adapt to the increased salinity by responding to a rapid signal that stimulates chloride secretion. In this paper, data are presented supporting the view that the transient increase in plasma osmolarity that can be measured during the transition is responsible for the stimulation of chloride secretion. A maximal increase of 65 mOsm in the plasma of Fundulus heteroclitus (the killifish) was found during acclimation to sea water. Similar or greater increases of osmolarity induced by mannitol on the basolateral side of isolated opercular epithelial membranes of the same species of fish containing great numbers of chloride cells produced stimulation of chloride secretion detected as the short circuit current. The shrinkage of the chloride cell activates the Na-K-2Cl cotransporter, and the Na/H exchanger and requires the integrity of apical chloride channels and normal levels of Ca. A Cl/HCO3 exchanger did not participate in this osmotic response to higher salinity. Chloride cell volume responses to osmolarity were studied with imagine and quantitative optics.

Transition from low salt water to sea water of the euryhaline fish, Fundulus heteroclitus, involves a rapid signal that induces salt secretion by the gill chloride cells. An increase of 65 mOsm in plasma osmolarity was found during the transition. The isolated, chloride-cell-rich opercular epithelium of sea-water-adapted Fundulus exposed to 50 mOsm mannitol on the basolateral side showed a 100% increase in chloride secretion, which was inhibited by bumetanide 10(-4) M and 10(-4) M DPC (N-Phenylanthranilic acid). No effect of these drugs was found on apical side exposure. A Na+/H+ exchanger, demonstrated by NH4Cl exposure, was inhibited by amiloride and its analogues and stimulated by IBMX, phorbol esters, and epithelial growth factor (EGF). Inhibition of the Na+/H+ exchanger blocks the chloride secretion increase due to basolateral hypertonicity. A Cl-/HCO3- exchanger was also found in the chloride cells, inhibited by 10(-4) M DIDS but not involved in the hyperosmotic response. Ca2+ concentration in the medium was critical for the stimulation of Cl- secretion to occur. Chloride cell volume shrinks in response to hypertonicity of the basolateral side in sea-water-adapted operculi; no effect was found on the apical side. Fresh-water-adapted fish chloride cells show increased water permeability of the apical side. It is concluded that the rapid signal for adaptation to higher salinities is an increased tonicity of the plasma that induces chloride cell shrinkage, increased chloride secretion with activation of the Na+K+2Cl- cotransporter, the Na+/H+ exchanger and opening of Cl- channels.

Caffeic acid phenethyl ester (CAPE) was isolated from propolis (a product of honeybee hives) that has been used in folk medicine as a potent antiinflammatory agent. CAPE is cytotoxic to tumor and virally transformed but not to normal cells. Our main goal was to establish whether CAPE inhibits the tumor promoter (12-O-tetradecanoylphorbol-13-acetate)-induced processes associated with carcinogenesis. Topical treatment of SENCAR mice with very low doses (0.1-6.5 nmol/topical treatment) of CAPE strongly inhibits the following 12-O-tetradecanoylphorbol-13-acetate-mediated oxidative processes that are considered essential for tumor promotion: (a) polymorphonuclear leukocyte infiltration into mouse skin and ears, as quantified by myeloperoxidase activity; (b) hydrogen peroxide (H2O2) production; and (c) formation of oxidized bases in epidermal DNA, as measured by 5-hydroxymethyluracil and 8-hydroxylguanine. A 0.5-nmol dose of CAPE suppresses the oxidative burst of human polymorphonuclear leukocytes by 50%. At higher doses (1-10 mumol), CAPE inhibits edema and ornithine decarboxylase induction in CD-1 and SENCAR mice. Interestingly, we discovered that 12-O-tetradecanoylphorbol-13-acetate-induced H2O2 production in bovine lenses also is inhibited by CAPE. Cumulatively, these findings point to CAPE as being a potent chemopreventive agent, which may be useful in combating diseases with strong inflammatory and/or oxidative stress components, i.e., various types of cancer and possibly cataract development.

The retinal pigment epithelium (RPE) lying between the neural retina and the choroid, performs as a transport organ for solutes and water between the choriocapillaries and the subretinal space. It also has the function to maintain the microenvironment of photoreceptors including the regulation of calcium ions during light or dark adaptation. In order to further elucidate the transport functions of the RPE, apical membranes were isolated from RPE by differential precipitation with divalent ions. In this work bovine tissues were used as well as elasmobranch tissues. For the latter, we have already purified and characterized membrane vesicles in a previous paper. Na(+)-K(+)-ATPase, alkaline phosphatase, and 5'-nucleotidase, which are marker enzymes of the apical membrane, were highly enriched in the final membrane fraction. The majority of the fraction consists of right side out vesicles. The fluorescent indicator for sodium, SBFI, or the calcium specific indicator, Fura-2, were pre-loaded into the apical membrane vesicles of RPE of either dogfish eyes or bovine eyes. When an outwardly-directed Ca2+ gradient was formed across the vesicular membranes, the Ca2+ influx was also enhanced by 136% for dogfish RPE and 167% for bovine RPE. This Na+ gradient dependent Ca2+ influx was blocked by bepridil, an antiarrhythmic agent which is a Na+/Ca2+ exchanger inhibitor. These results indicate that a Na+/Ca2+ exchanger is present in the apical membrane of bovine and dogfish RPE.

When the Ca(2+)-sensitive fluorescent probe, Fura-2, or the Na(+)-sensitive probe, SBFI, in their cell permeable forms or the Cl(-)-specific probe, SPQ, were incubated with plasma membrane vesicles prepared from dogfish and bovine lenses fibers, there was a selective accumulation of the ion-specific probes within the vesicles. The SBFI and Fura-2 fluorescent excitation ratios of 340 nm to 380 nm (em: 505 nm) in the presence of an outwardly-directed Na+ gradient across the vesicles membrane, indicate that the influx of Ca2+ is increased by 152.5% and 147.4% for dogfish and bovine vesicles, respectively. The Na+ influx into the vesicles is also enhanced by 154.1% for dogfish and 149.1% for bovine lens when an outwardly-directed Ca2+ was present. This stimulation is not affected when either 50 microM valinomycin, or 50 mM K+ is present. The activity of this bidirectional Ca2+/Na+ exchanger could be inhibited by 100 microM bepridil or 200 microM La3+. The entrance behaviour of Cl- as monitored by the SPQ fluorescent signal indicates that, the Cl- influx is Na(+)-dependent. The Cl- influx is stimulated 152.8% and 187.6% for dogfish and bovine lens, respectively, when an inwardly-directed Na+ gradient is present, and is further enhanced when a K+ gradient is also present. The stoichiometry of Na+ to Cl- entering the vesicles was 1:2. This Na+,K+ 2Cl- cotransporter is not affected by 20 microM valinomycin or 50 mM K+. However, the transporter is completely inhibited by 50 microM furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)

The existence of a Na+/H+ exchange mechanism and its role in the regulation of lens fiber cell intracellular pH, as well as the effect of oxidants and antioxidants, have been examined in vesicular preparations from spiny dogfish and bovine eyes. The fluorescent probes, SBFI (sodium-binding benzofuran isophthalate) for Na+ and SNARF-1 (seminaphthorhodafluor-1) for H+, were used to determine fluorescent ratios in a dual-wavelength spectrofluorimeter. The results show that: (1) the plasma membrane vesicles can be purified from lens fiber (52.5% of the vesicles were in the right-side out orientation for the fish eyes and 56.3% for the bovine eyes, show enrichment for the membrane marker activities and have an average size of 0.2-0.5 microns); (2) the influx of Na+ was dependent on an outwardly directed pH gradient with the uptake of Na+ sensitive to 500 microM amiloride but not affected by 50 microM valinomycin and 50 mM K gluconate; (3) when the pHo (extravesicular pH) of the vesicles was set at 8.1 and pHi (intravesicular pH) to 6.1, an inwardly directed Na+ gradient caused an increase in intravesicular pH by about 0.3 pH unit, and in bovine lens fiber vesicles the Na+ influx is highly dependent on the intravesicular pH (or on the pHo/pHi gradient); (4) 50 microM H2O2 increases the Na+/H+ exchange rate by 174% in the vesicles derived from fish lens fibers. Similarly, 100 microM H2O2 stimulates the Na+/H+ exchange rate by 194% in bovine eye fibers. This activation is prevented by preincubation with GSH (reduced glutathione) but not with GSSG (oxidizing glutathione). We conclude that a Na+/H+ exchange mechanism is present in the lens fiber membranes. This exchange possibly regulates intracellular pH and controls the intracellular Na+ concentration. The sensitivity of the Na+/H+ exchanger to the oxidant, hydrogen peroxide (H2O2) and the protection by the antioxidant GSH suggests a possible role for the Na+/H+ exchange mechanism in the formation of cataracts.

Apical membrane vesicles of dogfish and bovine lens epithelium were prepared by differential centrifugation and Mg2+ precipitation. Enrichment of the apical enzyme markers, for Na+,K(+)-ATPase was achieved. Na+,K(+)-ATPase was enriched 25.9-fold and 23.6-fold for dogfish and bovine lens epithelia, respectively, and acid phosphatase was enriched 10.4-fold and 12.6-fold, respectively. There was no enrichment of the cytoplasmic marker, NADH reductase. The majority of the apical membrane vesicles isolated from both dogfish and bovine lens epithelia were oriented right-side out. Electron micrographs of the purified vesicles show the presence of circular sealed vesicles with an average size of approximately 0.2-0.5 microns. Incubation of the vesicles with either the acetoxymethyl ester of SBFI, a new indicator for sodium, or with Fura-2, the calcium-specific indicator, leads to a large accumulation of the de-esterified SBFI or Fura-2 in the lens epithelial apical vesicles as determined by fluorescence measurements. When an outwardly direct Ca2+ gradient is formed across the vesicular membranes, the influx of Na+ is stimulated 77.8 and 63.0% for dogfish and bovine lens epithelia, respectively. When an outwardly directed Na+ gradient is formed across the vesicular membranes, the Ca2+ influx is also greatly enhanced. Both cases indicate that there is a bidirectional Ca2+/Na+ exchanger present in the apical side of the lens epithelial cell. The exchanger is inhibited by 50 microM bepridil or by 200 microM La3+. The stimulatory effects are also observed in membrane vesicles that are 'short-circuited' with valinomycin and high concentrations of K+.(ABSTRACT TRUNCATED AT 250 WORDS)

Incubation of whole bovine lens with 10(-7) M 12-O-tetradecanoylphorbol-13-acetate (TPA) led to the lens opacity within 24 h. The hydrogen peroxide (H2O2) concentration in the whole lens was elevated 4 fold after treatment with either 10(-7) M TPA or 2.5 mM glucose/20 microM glucose oxidase. The lens opacification and H2O2 elevation were TPA dose-dependent. Preincubation of the lens with anti-tumor promoting agents EGCG (epigallocatechin gallate) or Sarp A (sarcophytol A) stopped the TPA-mediated opacification process and suppressed H2O2 elevation.

When a fluorescent stilbene was added to epithelial plasma membrane suspension the emission spectrum showed a broad peak containing overlapping emissions resulting from different adducts. By focusing on a specific emission wavelength a common site having a dissociation constant of approximately 5 microM was calculated in the rat kidney, small intestine, pancreatic islets and shark rectal gland. This binding could be displaced by loop diuretics, (e.g., furosemide with an IC50 of 40 microM), DIDS (ki 1 microM) and thiocyanate. These results pose certain questions such as: (i) whether the evidence for multiple peaks are due to specific interactions representing multiple binding affinities and (ii) whether the binding of stilbene and the observed displacement can be identified on a specific protein. Separating the proteins present in the purified basolateral and brush-border membranes by SDS-PAGE, transfer of these proteins onto nitrocellulose paper and labeling of the nitrocellulose strips by radioactive BADS (4-benzamido-4'aminostilbene-2-2'disulphonic acid) and bumetanide could identify labeled proteins. These experiments showed that whereas some proteins bound either BADS or bumetanide, one protein with a molecular weight of approximately 100 or 130,000 D appeared to bind both. This protein was found on the basolateral membrane in the rat kidney cortex and medulla and the shark rectal gland and in the basolateral and brush-border membranes of the small intestine. Displacement of the protein-bound stilbene by loop diuretics could not be quantitated on the nitrocellulose transfer strips for this protein. Antibodies raised against the cytoplasmic fragment of band 3 reacted with the stilbene-labeled 100-130,000 D proteins indicating sufficient immuno-cross-reactivity between the separate species. These experiments involving binding of BADS and bumetanide and cross-reactivity with the human band 3 antibody suggest that these kilodalton proteins could structurally resemble human band 3.

Gills and operculum of seawater- and freshwater-adapted killyfish (Fundulus heteroclitus) were stained histochemically for carbonic anhydrase (CA). In the seawater-acclimatized specimens, CA was found predominantly in the chloride cells which were considerably larger than in the freshwater-adapted ones. Within these cells, the reaction products were concentrated in the apical parts of the cytoplasm. In contrast, chloride cells of freshwater-adapted fish were not, or only faintly, stained both in gills and opercular epithelium. Reaction products for CA were seen additionally in the cytoplasm of the outer respiratory cells lining the lamellae of gills both in seawater- and freshwater-adapted fish.

Chloride transport occurs at the interface between the internal and external environments of a cell where chloride uptake or efflux is regulated through a variety of mechanisms that involve cotransport of cations, exchange mechanism with anions, or movement through channels. One of these mechanisms, a chloride-bicarbonate exchange found in the human red blood cell, is well characterized and is mediated by a protein commonly known as band 3. To ascertain the presence of this or other mechanisms in epithelia, the sensitivity of epithelial membranes toward stilbenes was examined. Structure function activities of stilbene derivatives with red cell ghosts show that stilbene molecules block anion transport sites. One of these stilbenes, 4-benzamido-4'-aminostilbene-2-2'-disulfonic acid (BADS), chosen for its property of enhanced fluorescence on binding to hydrophobic sites, was used as a probe to examine the presence or absence of similar sites on epithelial membranes. With the use of nonlinear curve fitting, a single class of sites was found for BADS in the rat kidney cortex (1.6 microM), rat kidney medulla (2.1 microM), rat small intestine (2.2 microM), rat pancreatic islets (5.8 microM), frog cornea (4.3 microM), and shark rectal gland (1.5 microM). In the presence of chloride, the affinity for BADS decreased in all tissues except the frog corneal epithelium where it remained unchanged. The binding of BADS could be displaced by loop diuretics (furosemide, bumetanide, and piretanide) and thiocyanate anion in the kidney, intestine, and shark rectal gland; 50% displacement occurred at approximately 40 microM concentrations for furosemide with an order of magnitude less for bumetanide. The near-millimolar concentrations required for the displacement of BADS by loop diuretics indicate that this effect is nonspecific. However, the effect of chloride, thiocyanate, and loop diuretics on the binding of BADS indicates that BADS possibly interacts with an anion site.

The effect of altering cell membrane lipids on ion transport across isolated corneas was studied. Corneas mounted in Ussing-type chambers showed a rapid increase in short-circuit current following treatment with a variety of unsaturated fatty acids of varying chain length and unsaturation. Measurements of membrane fluidity which utilize immunofluorescence labelling of membrane proteins showed corneal epithelial cell membranes to be significantly more fluid following linoleic acid treatment. Uptake studies indicate rapid incorporation of [14C]linoleic acid into corneal cell membranes. Highly unsaturated fatty acids were found to have the greatest ability to stimulate chloride transport. Saturated fatty acids were tested and were found to have no effect on chloride transport at any concentration. It is proposed that unsaturated fatty acids activate chloride transport by increasing membrane lipid fluidity. The relationship of these parameters is discussed in terms of a mobile receptor model. We speculate that an increase in membrane lipid fluidity promotes lateral diffusion of membrane receptor proteins and enzymes, increasing protein-protein interactions within the membrane, ultimately resulting in the enhancement of cyclic AMP synthesis.

In a previous study, measurements were made of facilitated and passive transport of glucose, using [3H]-3-O-methyl-D-glucose and [14C]-L-glucose, respectively, across blood-aqueous and blood-vitreous barriers in long-term streptozotocin-diabetic rats. It was found that passive transport was increased, while facilitated transport was decreased, possibly due to saturation of the transport system. The present study examines the appearance of these changes in glucose transport at various times following streptozotocin (STZ) injection. Passive transport, as indicated by the L-glucose rate constant, began to increase at about 10 days following induction of diabetes, stabilized at the elevated rates at 50-60 days and persisted during the 170-day period of observation. Rate constants for [3H]-3-O-methyl-D-glucose transport decreased within 1 day following induction of diabetes. Prevention of hyperglycemia by insulin treatment upon onset of diabetes prevented the latter changes ruling out a direct effect of STZ. Glucose infusion into normal rats produced a similar decrease in 3-O-methylglucose transport constants suggesting that hyperglycemia was responsible for the early decrease in facilitated transport found in the diabetic rats. It is speculated that increased passive transport of glucose may reflect an early loss in ocular barrier integrity. The later decrease in carrier facilitated transport cannot be explained by hyperglycemia alone and, thus, a loss in carrier function is suggested. Despite a decrease in facilitated transport, absolute glucose entry rates are increased in the diabetic due to elevated plasma glucose, which serves as an inward driving force, due to the significantly increased entry of glucose by the passive route.

The contribution of chloride ion movement and sodium and bicarbonate concentrations to the net current across the isolated choroid-retinal pigment epithelium (RPE) of the bullfrog were studied. The presence of a ouabain-sensitive Na+/K+-pump on the retinal side was confirmed and complete inhibition of this pump with Na+ removal and ouabain treatment abolished nearly all the RPE transepithelial transport and SCC suggesting that all ionic transport was dependent on sodium. It was found that apical to basal (AB) chloride flux accounted for 26 +/- 2% (mean +/- S.E.M.) of the short circuit (SCC). Results suggest that AB bicarbonate and/or basal to apical (BA) hydrogen ion net transport accounts for 38 +/- 2% of the SCC while BA sodium is presumably responsible for the remaining 34% of the SCC. Transport was inhibited by apical administration of known chloride inhibitors. Trans-RPE 36Cl flux measurements indicate that furosemide (10(-4) M) and SITS (10(-3) ) decrease the retinal-choroid flux. Results suggest that net transport of chloride and bicarbonate are independent of each other and additive. It was found that a bicarbonate-free preparation was relatively unaffected by changes in pH (5.5-8.5) indicating that pH has little, if any, effect on sodium or chloride current in this range. A model is presented which is compatible with the various data. It is suggested that along with the apical Na+/K+-ATPase pump, there exists an apical Na+/Cl- -co-transport system which is driven by the established sodium gradient. Moreover, this pump established sodium gradient is postulated to drive a Na+/HCO3- -co-transport system tentatively placed on the retinal side of the RPE.

The leukotriene, LTC4, exerts a stimulatory effect on chloride transport in the frog cornea. In the work described here, the mechanism of action of LTC4 to stimulate chloride transport was studied. In corneas pretreated with indomethacin, the effect of LTC4 was abolished, suggesting the involvement of cyclo-oxygenase products in the response. Incubation of corneas with LTC4 resulted in a significant stimulation in PGE2 synthesis, as determined by TLC-autoradiography and radioimmunoassay. In addition, LTC4 was found to stimulate cAMP synthesis in the cornea, and this stimulation was blocked with indomethacin. PGE2 was previously shown by us to be the dominant cyclo-oxygenase product formed in the frog cornea, and is capable of stimulating cAMP and chloride transport. We suggest that LTC4 stimulation of chloride transport is mediated via activation of the cyclo-oxygenase pathway, resulting in enhanced PGE2 synthesis. Elevated PGE2 levels induce cAMP synthesis, and ultimately, the stimulation of chloride transport. Further, the activation of cyclo-oxygenase was found to be dependent on phospholipase A2 activity. This was shown by the inhibition of the LTC4 effect in the presence of quinacrine. Similarly, inhibition of the LTC4 effect in the presence of trifluoperazine suggests that cyclo-oxygenase activation by LTC4 may be mediated via calmodulin. We have previously demonstrated that the frog cornea has the biosynthetic capacity to produce LTC4. Therefore LTC4 may function as an endogenous regulator of chloride transport in this tissue.

The opercular epithelium of Fundulus heteroclitus contains typical gill chloride-secreting cells at the high density of 4 X 10(5) cells per square centimeter. When isolated, mounted as a membrane, and short-circuited, it actively transports chloride ions from the blood side to the seawater side of the preparation. This preparation offers a useful approach to the study of osmoregulation in bony fishes.

The transport kinetics across the plasma-ocular barriers of labeled molecules including urea, D-glucose, 3-0-methyl-D-glucose, L-glucose, and sucrose were studied in adult RCS rats and compared to control albino Sprague-Dawleys (SD). Results indicate that substances that passively cross the plasma-aqueous and plasma-vitreous barriers (urea, sucrose, L-glucose) do so more readily in the RCS rat especially via the trans-retinal route. By contrast, D-glucose, which penetrates the ocular barriers of the control rat by a carrier-mediated mechanism was found to cross the ocular barriers of the RCS rat at a reduced rate. Thus, the ocular barriers of the RCS rat with well developed retinal degeneration demonstrate an increase in passive permeability and a reduction in ability to perform their membrane transport function (D-glucose). The permeability defect was more pronounced in the blood-vitreous barrier than in the blood-aqueous barrier.

The retinal pigment epithelium (RPE) interposed between the vascular system of the choroid and the neural retina performs a variety of functions essential for vision. In order to further elucidate the transport functions of the RPE, apical membranes were isolated from the RPE of the dogfish (Squalus acanthias) by differential precipitation with calcium. Na-K-ATPase, an apical marker enzyme in this tissue, was enriched 15-fold in the final membrane fraction. About 50% of the membranes form right-side-out vesicles in which the membrane has retained its in vivo orientation. Sodium uptake into these vesicles as determined by a rapid filtration method was stimulated 37% by the presence of a proton gradient across the membrane (pHi = 6.1, pHo = 8.1). The stimulation was also observed in membrane vesicles "short-circuited" with valinomycin and K. The pH gradient-dependent sodium uptake but not the uptake in the absence of a pH gradient was completely inhibited by 5 X 10(-4) M amiloride, and 56% inhibition was found at 10(-5) M amiloride. The uptake of 22Na was also strongly decreased in the presence of nonradioactively labelled sodium and lithium; potassium was without effect. pH gradient dependence, amiloride sensitivity, saturability and cation specificity of the sodium flux indicate the presence of a Na/H exchanger in the apical membrane of the retinal pigment epithelium. The presence of the Na/H exchange process might have important implications for the control of pH in the subretinal space, optimum intracellular pH of the RPE and the triggering of other functions of the RPE.

1. The opercular epithelium lining the inside of the gill chamber of the killifish, Fundulus heteroclitus, contains Cl(-) cells, identical in fine structure to gill Cl(-) cells, at the high density of 4 x 10(5) cells/cm(2). This epithelium can be isolated, mounted in a Lucite chamber, and its ion transport properties studied with the short-circuit current technique.2. The isolated opercular epithelia of seawater-adapted fish, when bathed on both sides with Ringer and gassed with 100% O(2), displayed a mean short-circuit current of 136.5 +/- 11.1 muA/cm(2), a mean transepithelial potential difference of 18.7 +/- 1.2 mV (blood side positive), and a mean transepithelial d.c. resistance of 173.7 +/- 12.1 Omega.cm(2) (mean +/- S.E. of mean; n = 64).3. The transepithelial potential difference across the opercular epithelia of seawater-adapted fish was dependent on both Na(+) and Cl(-) in the bathing solutions and increased linearly with increasing Cl(-) concentrations with a slope of 28.3 +/- 2.1 mV/tenfold concentration change. The short-circuit current was Na(+) dependent and increased linearly with increasing Cl(-) concentrations with no evidence of saturation kinetics below 142.5 m-equiv/l.4. When the short-circuited epithelia of seawater-adapted fish, bathed on both sides with Ringer, was gassed with 100% O(2) the mean Cl(-) blood side to seawater side flux was 211.7 +/- 27.1 muA/cm(2) and the mean Cl(-) seawater side to blood side flux was 48.9 +/- 10.0 muA/cm(2). This resulted in a net Cl(-) blood side to seawater side flux of 162.8 muA/cm(2) which was not statistically different (P > 0.70) from the mean short-circuit current of 158.6 +/- 16.3 muA/cm(2) for these flux studies. The mean Na(+) blood side to seawater side flux was 32.2 +/- 3.3 muA/cm(2) and the mean Na(+) seawater side to blood side flux was 34.8 +/- 4.1 muA/cm(2), resulting in no significant (P > 0.20) net flux of this cation. Similar results were obtained with short-circuited epithelia of seawater-adapted fish when bathed on both sides with Ringer and gassed with 95% O(2)/5% CO(2).5. Ouabain (10(-5)M), furosemide (10(-3)M), thiocyanate (10(-2)M), adrenaline (10(-6)M), and anoxia (100% N(2)) decreased the short-circuit current 92.7, 85.0, 45.3, 62.6, and 83.3% respectively. Theophylline (10(-4)M) stimulated the short-circuit current 54.9%. Increasing the HCO(3) (-) concentration in the bathing solutions had a stimulatory effect on the short-circuit current and the potential difference across epithelia from seawater-adapted fish.6. The opercular epithelia of freshwater-adapted F. heteroclitus, when bathed on both sides with Ringer, displayed a mean short-circuit current of 94.1 +/- 10.4 muA/cm(2), a mean transepithelial potential difference of 14.8 +/- 1.9 mV (blood side positive), and a mean d.c. resistance of 169.0 +/- 14.0 Omega.cm(2) (mean +/- S.E. of mean; n = 20). Isotope flux studies across these short-circuited epithelia revealed a net Cl(-) blood side to freshwater side flux of 95.2 +/- 16.1 muA/cm(2) and no significant net flux of Na(+).7. The opercular epithelia of 200% seawater-adapted F. heteroclitus, when bathed on both sides with Ringer, displayed a mean short-circuit current of 33.5 +/- 8.5 muA/cm(2), a mean transepithelial potential difference of 10.5 +/- 2.5 mV (blood side positive), and a mean transepithelial d.c. resistance of 440.7 +/- 62.6 Omega.cm(2) (mean +/- S.E. of mean n = 18). Isotope flux studies across these short-circuited epithelia revealed a net Cl(-) blood side to seawater side flux of 96.2 +/- 51.5 muA/cm(2) and a net Na(+) blood side to seawater side flux of 65.3 +/- 28.6 muA/cm(2).

A fluorescence receptor binding assay, based upon the high-affinity beta-adrenergic receptor antagonist propranolol, is utilized to probe the microenvironment of the antagonist-receptor complex in the frog (Rana catesbeiana) erythrocyte membrane. The technique of steady-state fluorescence depolarization is applied to the propranolol-receptor complex, allowing quantitation of the rotational relaxation time of the complex. It is found that the complex is dynamically constrained at 20 degrees C. However, in the temperature range 6-10 degrees C a sharp reversible release of constraint is observed. It is further demonstrated that the addition of drugs that are known to specifically disrupt the cytoskeleton (colchicine, vincristine, and vinblastine) causes a similar but irreversible release of constraint at 20 degrees C. Cytochalasin B has a much smaller influence on the rotational mobility of the propranolol-receptor complex than do the other drugs that disrupt the cytoskeleton. Amphotericin B is without effect on the rotational constraint of the complex. Binding of the antagonist [3H]dihydroalprenolol is not influenced by colchicine. A model is proposed which postulates that cytoskeletal elements are linked to the antagonist-receptor complex. Antagonist binding does not result in cytoskeletal release, whereas agonist binding is postulated to lead to dissociation of the agonist-receptor complex from the cytoskeleton, thereby activating adenylate cyclase.

Fluorescent dyes specific for mitochondria have become important tools in the study of transporting epithelia. These dyes permit the localization and quantification of mitochondria-rich (MR) cells in these epithelia. To determine the specificity of the dye, dimethylaminostyrylmethylpyridiniumiodine ( DASPMI ), we combined fluorescence microscopy of this dye with the ultrastructural localization of the mitochondrial enzyme, cytochrome oxidase. Labeled cells were traced from the fluorescence-microscopic to the electron-microscopic level by devising several novel technical procedures. This new methodology assures a critical assessment of the specificity of fluorescent mitochondrial dyes in heterogeneous epithelia. Confirmation of DASPMI specificity allows the unequivocal identification of MR chloride cells in two epithelia in the head region of Fundulus heteroclitus and validates linear regression analysis of chloride cell number and short-circuit current in this species. In addition, this method provides a permanent, flat whole mount of labeled cells for morphological studies with the light microscope and with the scanning and transmission electron microscopes.

We demonstrate that arachidonic acid (AA) stimulation of chloride transport across frog cornea is mediated via two independent pathways: (1) stimulation of prostaglandins and cAMP synthesis, and (2) a direct physical change in the membrane produced by substitution of different phospholipid acyl chains. AA is well known as a precursor in the synthesis of prostaglandins, which have been shown to stimulate cAMP synthesis and chloride transport in frog cornea. We show that frog cornea can convert exogenous AA to PGE2, but that in the presence of 10(-5) M indomethacin both the conversion to PGE2 and stimulation of cAMP are completely blocked. However, with indomethacin the action of AA to stimulate chloride transport (as measured by SCC) remains, but peak height of the response is reduced to 57% of that found when AA alone is given. Similarly, we show that propranolol completely blocks cAMP stimulation, but stimulation of SCC is reduced to 45% of the original response. Therefore, cAMP appears to be responsible for roughly half of the observed stimulation in SCC. By gas chromatographic analysis we show that significant quantities of AA can rapidly substitute into membrane phospholipids of corneal epithelium and L929 cells following the addition of AA to the medium. Modification of membrane phospholipid structure can affect membrane viscosity, membrane-bound enzyme activity, and the distribution and lateral mobility of integral proteins. It seems likely that such alterations in the properties of the membrane may modulate the rate of chloride transport, and this may constitute the second mechanism. Upon addition of AA, both mechanisms appear to stimulate chloride transport simultaneously, and are apparently additive. We show that prolonged exposure to AA results in a large incorporation of AA into phospholipid and consequently, a perturbation in the ratio of unsaturated to saturated fatty acids. We also find evidence of a compensatory cellular mechanism that alters the ratio of endogenously synthesized fatty acids and tends to reduce the membrane-perturbing effect of AA.U

Previous studies utilizing the fluorescence of propanolol as a probe for the beta-adrenergic receptor showed that this receptor is motionally constrained. To further study the beta-adrenergic receptor in situ we have reinserted rhodamine-labeled beta-receptors into cell membranes. This report presents documentation of their insertion and physiologic viability. Beta-receptors were purified by affinity chromatography (10,000-fold), then fluorescently labeled with tetramethyl rhodamine isothiocyanate, repurified (55,000-fold) and incubated with rat pancreatic islet cells. The binding of 3H-dihydroalprenolol by these cells was increased from a Bmax of 168 +/- 2 to 309 +/- 20 fmol/mg protein with no change in Kd. Various treatments which remove peripheral membrane proteins, e.g. NaOH, lithium diiodosalicylate, and trypsinization, did not alter binding by the cells with inserted receptors indicating that the receptors inserted into cell membranes. In islet cells treated with Koshland's reagent I, beta-adrenergic binding was completely abolished, but following incubation with isolated beta-receptors, the cells bound beta-adrenergic radioligand with a Bmax of 100 fmol/mg protein, indicating functionality on the part of the inserted receptors. Furthermore, insertion of isolated receptors into frog erythrocytes resulted in increased production of cAMP in response to added isoproterenol. In pancreatic islet cells, incubation with labeled receptors caused the fluorescence to shift in wavelength with increased intensity indicating a shift from an aqueous to a lipid environment, probably into the membrane. Using fluorescence (P), it was found that the inserted receptors became motionally constrained to a P of 0.38 (limiting Po = 0.42) during the first 15 min, remaining so for at least 2 hr. Colchicine (5 micrograms/ml) caused a decrease in P to 0.18 indicating release of constraint. Isoproterenol (10(-5)M) caused a rapid decrease to P = 0.15. This effect was blocked by propranolol. Propranolol itself (10(-5) M) had no effect. These results indicate that the labeled receptors rapidly insert into cell membranes and also support the view that agonist activation of the receptor causes an increase in receptor mobility, presumably due to release of constraint from cytoskeleton elements.

Sections of the ciliary epithelium of adult sharks (Squalus acanthias) were mounted in Ussing-type chambers (area 0.2 cm2) for measurements of transepithelial potential difference (PD), short circuit current (SCC) and calculation of transepithelial resistance (R). In 15 preparations PD was aqueous side negative (-0.51 +/- 0.12 mV; SCC 18.3 +/- 2.5 microA cm-2; R 30.7 +/- 3.1 Ohm cm2). However, in 15 other preparations incubated in identical Ringer's solution PD was aqueous side positive (0.53 +/- 0.09 mV; SCC -19.6 +/- 2.3 microA cm-2; R 27.9 +/- 2.8 Ohm cm2). 10(-5) M ouabain or 10(-4) M furosemide were applied either to the aqueous or blood side of the isolated ciliary epithelium at transepithelial negative or positive PD. When the transepithelial PD was positive on the aqueous side ouabain decreased PD and SCC within 15 to 45 min. When the spontaneous PD was negative both PD and SCC decreased when ouabain was applied to the blood side. When the drug was given to the aqueous side a biphasic response (first stimulation, then inhibition) of PD and SCC was observed. Furosemide when given to the blood side (with aqueous side PD positive) or to the aqueous side (with aqueous side PD negative) decreased PD and SCC. However, a transient stimulation of both electrical parameters was observed when furosemide was applied to either the blood side (with aqueous side PD negative) or to the aqueous side (with aqueous side PD positive). The polarity and magnitude of PD and SCC probably depend on the relative activity of sodium and chloride pumps across the cell membranes of the non-pigmented and/or pigmented cell layer. However, additional transport mechanisms cannot be excluded.

Opercular epithelial cells of Fundus heteroclitus were investigated using conventional microelectrodes. The area of interest was the cells lining the inside of the opercular epithelium closest to the gill arches, an area with a high density of chloride cells. Only one cell type could be discerned from the values of 60 opercular cells measured with the opercular epithelium in open circuit conditions. A mean apical voltage of -18.0 +/- 0.6 mV was observed with intracellular values ranging from -10 to -30 mV. The predicted intracellular chloride content was 59 mM/liter. Apical fractional resistance (faR) was 0.78 +/- 0.02. The intracellular potential measurements were typically difficult to maintain for extended periods (longer than 3 min). The opercular cells depolarize with serosal isoproterenol treatment (10(-6) M) corresponding to the increase in opercular transepithelial potential. The opercular cell apical fR decreased with isoproterenol treatment. These data indicate the observed opercular cells were involved in opercular chloride transport.

Ascorbic acid stimulates active transport of Cl-minus by the isolated intact cornea. The effect is not present in corneas previously stimulated by the theophylline, an inhibitor of 3':5"-cyclic-AMP phosphodiesterase (EC 3.1.4.17), and vice versa, theophylline has no action after stimulation with ascorbic acid. This indicated inhibition of 3':5'-cyclic-AMP phosphodiesterase by ascorbic acid. Assay of phosphodiesterase using 3-H-labeled cyclid AMP of frog and rabbit corneal epithelial homogenates showed an inhibitory effect of ascorbic acid. Concentration of 5 mM produced 16% inhibition with 20 mM producing 46%. This compares with 58% inhibition by theophylline at 5 mM. Phosphodiesterase activity is mostly soluble in frog corneal epithelium but in rabbit 45% is particulate. Soluble and particulate fractions are inhibited by ascorbate, but in rabbits greater inhibition (50%) was observed in the particulate fraction than in the soluble fraction. Other tissues showed inhibition also: frog retina 12%, rat brain (caudate nucleus) 48%, rabbit brain 14%, rabbit liver 16%. It is concluded that ascorbate produces an increase in cyclic AMP content of corneal epithelium and other tissues by inhibition of 3':5'-cyclic-AMP phosphodiesterase. This action may be one of the main functions of the high ascorbic acid content of ocular tissues and explain some of the effects of high dosis of ascorbate in other systems.

Evidence is presented demonstrating that in the dogfish, Squalus acanthias, D-glucose is transported from the blood to the ocular fluid compartments by a mechanism that is consistent with carrier-facilitated transport. Across the dogfish aqueous barrier D-glucose is transported 8.4 times faster than L-glucose, whereas this D/L transport ratio is only 5.5 in the rat. Similarly, the D/L transport ratio is 12.7 across the dogfish vitreous barrier and only 6.1 across this barrier in the rat. It is estimated that per unit area of vitreous barrier, the rat transports D-glucose faster than the dogfish but also has a greater passive permeability, as measured by L-glucose, and transport is therefore less efficient. Urea transport was found to be faster than L-glucose transport, suggesting a transport mechanism for urea other than simple diffusion. The sectional analysis of frozen vitreous humor indicated that the lens of the dogfish is supplied with D-glucose from both the ciliary epithelium and across the retinal pigment epithelium, while the retina receives D-glucose primarily across the pigment epithelium.

We found that intracellular chloride activity in frog retinal pigment epithelium (RPE) was significantly higher than predicted from the equilibrium distribution across the cell membranes. When transepithelial chloride transport was inhibited by furosemide intracellular chloride activity decreased and approached electrochemical equilibrium. These data indicate that RPE possesses an active, furosemide-sensitive chloride transport across the apical membrane.

The nature of the propranolol binding site of the beta-adrenergic receptor has been examined by utilizing the intrinsic fluorescence of propranolol as a probe. Additionally, the spatial relationship between the propranolol binding site and membrane tryptophan has been examined by utilizing I-quenching of intrinsic tryptophan fluorescence, chemical modification of membrane tryptophan, and singlet-singlet energy transfer between membrane-bound propranolol and tryptophan. Propranolol, at concentrations consistent with specific beta-receptor binding, protected approximately 42% of the membrane tryptophan fluorescence from I-quenching. Further, in the presence of propranolol, the apparent quenching constant (kq) was altered from 3.6 to 21.8 M-1. Reaction of the membrane fragments with 2-hydroxy-5-nitrobenzyl bromide (Koshland's reagent I) in the presence and absence of propranolol indicated that low concentrations of propranolol protected approximately 45% of the membrane tryptophan from the reagent. The singlet-singlet energy transfer from tryptophan to propranolol was determined by sensitized emission. The distance between these two species was found to be less than 20 A. These results have been interpreted to indicate that propranolol, when bound to the beta-adrenergic receptor, is situated such that its naphthyl moiety is inserted into a tryptophan-rich hydrophobic pocket of the receptor.

Chloride absorption in the small intestine of the winter flounder, Pseudopleuronectes americanus, is reported to be sensitive to ambient pH. We studied this sensitivity in isolated stripped intestinal mucosa mounted in modified Ussing chambers. Unidirectional 36Cl fluxes (JClm----s, JCls----m) were measured under short-circuited conditions in bathing solutions containing various combinations of HCO3- (0-20 mM), partial pressure of CO2 (0-36 mmHg), and pH (6.77-7.85). We found that JClm----s, net 36Cl flux (JClnet), and short-circuit current (Isc) increased and JCls----m decreased predominately in response to increases in bathing solution pH. There was a linear relationship between pH and both JClnet (r = 0.92, P less than 0.01) and Isc (r = 0.96, P less than 0.005) between pH 6.77 and 7.74. The pH effect was completely reversible, did not require either CO2 or HCO3-, and was not affected by the presence of mucosal barium at 1 mM. Mucosal bumetanide (0.1 mM) completely inhibited the pH effect. These data suggest that the process by which Cl- is absorbed in the flounder intestine is sensitive to pH. The data do not indicate whether pH affects Na+-K+-2Cl- cotransport or a Cl- transport pathway in series with this process. The direction of Cl- absorption in response to pH contrasts with inverse relation of pH and Cl- absorption in mammalian small intestine.

The new calcium antagonist, nisoldipine, inhibited short circuit current and transcorneal potential difference in the isolated frog cornea (a chloride transporting epithelium). Transepithelial resistance increased. The effects of nisoldipine were dose-dependent. Nisoldipine did not change the stimulation of short circuit current induced by epinephrine. Intracellular membrane potential across both the apical and basolateral cell membrane were decreased by nisoldipine. The data indicate that intracellular calcium seemed to modulate chloride transport of the cornea by changing cell membrane permeability.

The transport kinetics across the plasma-aqueous and plasma-vitreous barriers were studied in normal and long-term streptozotocin-diabetic rats, using trace amounts of [14C]-L-glucose and [3H]-3-O-methyl-D-glucose. The former is passively transported while the latter uses the same transport-facilitating system as D-glucose. Transport rates of L-glucose were significantly higher in the diabetic rats, with ocular entry rates from the plasma being increased by 69% across both barriers. Thus, the data indicate that in experimental diabetes the passive permeability of the blood-ocular barriers is significantly increased. By contrast, calculated transport rate constants for 3-O-methyl-D-glucose, when adjusted for the hyperglycemia and the increased passive glucose movement, are not altered in the diabetic animal. Nevertheless, there is actually more mass D-glucose movement due to the prevailing hyperglycemia. The present study suggests that although streptozotocin diabetes alters plasma-ocular glucose transport, there is no direct impairment of glucose carrier function. Alterations in transport occurred at both ocular barriers, suggesting that involvement is general and that both the retinal pigment epithelium and the ciliary epithelium may be affected by the diabetes. It is unknown whether the increase in passive movement is related to the prevailing hyperglycemia or to insulin deficiency or other unknown factors.

Addition of adenosine (10-7 to 10-4 M) to the tear side of isolated corneas (Rana catesbeiana) produced a rapid, sustained increase in short-circuit current, potential difference, and radioisotopic chloride net flux. The increased net chloride flux accounted for the increased short-circuit current. Adenosine, a known activator of adenyl cyclase in other tissues, exerted its effects on chloride transport through a receptor different from the one described for epinephrine and prostaglandins in the corneal epithelium. Propranolol inhibited the epinephrine response but not the adenosine effect. Dipolyphloretin phosphate inhibited prostaglandin responses but did not affect the adenosine stimulation of chloride transport. Adenine and/or ribose, parts of the adenosine molecule, had no stimulatory effect, but 5'-AMP had a partial effect. The activation of the chloride pump with DBcAMP blocked the response to adenosine. Adenosine interacted with the effects of theophylline. Adenosine, a naturally occurring molecule, stimulated chloride transport by activation of adenyl cyclase through a separate membrane receptor in the corneal eqithelium.

The unidirectional Na+, Cl-, and urea fluxes across isolated opercular epithelia from seawater-adapted Fundulus heteroclitus were measured under different experimental conditions. The mean Na+, Cl0, and urea permeabilities were 9.30 x 10(-6) cm . sec-1, 1.24 x 10(-6) cm . sec-1, and 5.05 x 10(-7) cm . sec-1, respectively. The responses of the unidirectional Na+ fluxes and the Cl- influx (mucosa to serosa) to voltage clamping were characteristic of passively moving ions traversing only one rate-limiting barrier. The Na+ conductance varied linearly with, and comprised and mean 54% of, the total tissue ionic conductance. The Cl- influx and the urea fluxes were independent of the tissue conductance. Triaminopyrimidine (TAP) reduced the Na+ fluxes and tissue conductance over 70%, while having no effect on the Cl- influx of urea fluxes. Mucosal Na+ substitution reduced the Na+ permeability 60% and the tissue conductance 76%, but had no effect on the Cl- influx or the urea fluxes. Both the Na+ and Cl- influxes were unaffected by respective serosal substitutions, indicating the lack of any Na+/Na+ and Cl-/Cl- exchange diffusion. The results suggest that the unidirectional Na+ fluxes are simple passive fluxes proceeding extracelluarly (i.e., movement through a cation-selective paracellular shunt). This pathway is dependent on mucosal (external) Na+, independent of serosal (internal) Na+, and may be distinct from the transepithelial Cl- and urea pathways.U

We have found that membrane potential in the isolated ciliary epithelium of the shark, Squalus acanthias, is -53 mV. High extracellular potassium or ouabain (10(-5) mol X l-1) decrease the potential, and furosemide (10(-4) mol X l-1) hyperpolarizes it. There is no difference in membrane potential between the cells of the non-pigmented and pigmented layers. Intracellular chloride activity (64 mmol X l-1) was significantly higher than could be predicted from the equilibrium distribution (26 mmol X l-1) across the cell membranes. When furosemide was applied to the aqueous side of the epithelium, intracellular chloride activity decreased to 35 mmol X l-1 and approached electrochemical equilibrium. The data indicate that the ciliary epithelium possesses an active, furosemide-sensitive chloride transport mechanism which could be a Na-Cl or a 1 Na-1 K-2 Cl symport.

The action of the ichthyotoxic secretion of the Red Sea flatfish Pardachirus marmoratus and its derived toxin, pardaxin, was examined in the dogfish shark (Squalus acanthias). Pardaxin was more toxic when administered to the bathing medium than when injected into a dorsal artery and it transiently diminished the spiracular rate and caused a severe struggling response in the adult shark only when administered to the head region of the shark. Pardaxin caused a transient leakage to urea and sodium between the shark and the seawater. In the isolated perfused rectal gland pardaxin irreversibly reduced the rate of chloride secretion and concentration gradient of urea between perfusate and rectal gland fluid. In addition, ultrastructural studies on the rectal gland showed that ionic lanthanum penetrated the tight junctions and foci of cell necrosis were observed. These studies indicate that in shark the gills are the most probable target of the toxicity of pardaxin.

The present study has identified the metabolites of arachidonic acid (AA) formed by the isolated frog cornea and has shown that this tissue is capable of the biosynthesis of both lipoxygenase and cyclo-oxygenase pathway metabolites. The cyclo-oxygenase (CO) metabolites found in greatest abundance were the prostaglandins E2 and F2a (PGE2 and PGF2a). The major lipoxygenase (LO) pathway metabolite found by thin-layer chromatography (TLC) was leukotriene B4 (LTB4), whereas leukotriene C4 (LTC4) biosynthesis was detected by radioimmunoassay. These leukotrienes (LTs) are highly potent modulators of active chloride transport, since incubation with LTC4 (10(-7)-10(-9) M) resulted in a dose-dependent stimulation of both the Cl- originated short-circuit current (SCC) and potential difference (PD). In contrast, LTB4 (10(-7)-10(-9) M) inhibited both of these parameters. Both LTC4 and LTB4 exerted these effects only when applied to the endothelial side. Preincubation with the leukotriene receptor antagonist, FPL 55712 completely blocked the response to LTC4, indicating that the action of LTC4 in the cornea is receptor-mediated. In this report the authors show that LTB4 and LTC4 are formed by the cornea and that they are potent modulators of the SCC and PD in chamber experiments. The possibility exists that LTB4 and LTC4 may function as endogenous regulators of net Cl- transport in the cornea, since the addition of these metabolites resulted in a dose-dependent stimulation (with LTC4), and inhibition (with LTB4), of both the short-circuit current (SCC) and potential difference (PD).

Acetylcholinesterase (AChE) has been localized in the rabbit cornea by light and electron microscopy histochemical techniques. In the stroma, the enzyme is concentrated in nerves. In the epithelium, the enzyme is concentrated in intercellular spaces devoid of nerves. The morphologic appearance of the enzyme staining by light and electron microscopy in the epithelium is similar; consequently, the staining demonstrated with light microscopy examination does not always represent epithelial nerves. A significant portion of corneal acetylcholinesterase therefore appears unrelated to nerves. Considerably smaller deposits of enzyme reaction product were present in cells in every layer of the cornea, using electron microscopy histochemistry; they were not identified by light microscopy.