Chloride transport across isolated opercular epithelium of killifish: a membrane rich in chloride cells.

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.

Inhibition of tumor promoter-mediated processes in mouse skin and bovine lens by caffeic acid phenethyl ester.

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.

Stimulation of ion transport by ascorbic acid through inhibition of 3':5'-cyclic-AMP phosphodiesterase in the corneal epithelium and other tissues.

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.

Stimulation of chloride transport by fatty acids in corneal epithelium and relation to changes in membrane fluidity.

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.

Control of Cl- transport in the operculum epithelium of Fundulus heteroclitus: long- and short-term salinity adaptation.

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.

Glucose transport across ocular barriers of the streptozotocin-diabetic rat.

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.

Leukotriene modulation of chloride transport in frog cornea.

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).

Effects of ouabain and furosemide on transepithelial electrical parameters of the isolated shark ciliary epithelium.

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.

A Na/H exchange mechanism in apical membrane vesicles of the retinal pigment epithelium.

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.

Onset of changes in glucose transport across ocular barriers in streptozotocin-induced diabetes.

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.

Decrease of intracellular chloride activity by furosemide in frog retinal 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.

A Na+/Ca2+ exchange mechanism in apical membrane vesicles of the retinal pigment epithelium.

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.

Histochemical demonstration of carbonic anhydrase in gills and opercular epithelium of seawater- and freshwater-adapted killyfish (Fundulus heteroclitus).

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.