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.

Urea and water permeability in the ureotelic gulf toadfish (Opsanus beta).

The permeability of toadfish gills and skin to urea and water has been measured in order to investigate the mechanisms behind the pulsatile excretion of urea previously described in this species. A perfused gill preparation was used in the gill studies and isolated pieces of skin mounted in an Ussing chamber in the skin studies. Simultaneously, urea and water permeability was measured in vivo in free swimming fish. In vivo the nonpulsing urea permeability was exceptionally low compared to other teleosts, while the tritiated water permeability was similar to that of other teleosts. The urea permeability increased 30-fold during a pulse while water permeability stayed unaffected. Compared to in vivo, tritiated water permeability was approximately 50% lower in the gills and the skin when measured directly in the isolated preparations. The urea permeability was almost identical between the three preparations. Four out of 20 perfused gill preparation showed a spontaneous urea pulse during perfusion. Several treatments were tested to elicit the pulse artificially but without success. Hormones and drugs tested were: arginine-vasotocin (AVT), 10(-10) M; adrenaline, 10(-7) M; isoprenaline, 10(-5) M; acetylcholine, 10(-7) and 10(-6) M; serotonin, 10(-7) and 10(-6) M; adenosine, 10(-6) M; cortisol, 10(-7) M; and combinations of AVT, adrenaline, and cortisol. Adrenaline and isoprenaline increased tritiated water permeability without affecting urea permeability. Gradually increasing the ammonia levels in the perfusate from 0.1 mM to 1.6 mM caused a slight increase in water permeability but a marked and progressive increase in urea permeability. No indications of an ammonia trapping mechanism in the gills were found. There was no effect of AVT (10(-10) mol l-1) in the urea permeability of the skin preparation while cortisol (10(-7) M) led to a modest increase in urea permeability. Based on a comparison between the in vivo and in vitro preparations used here, we conclude that the urea pulse in a urea-pulsing toadfish occurs through the gills and not the skin. We still do not know which internal mechanism or signal triggers the urea pulse in the toadfish.

Chloride cells and osmoregulation.

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.

Chloride transport activation by plasma osmolarity during rapid adaptation to high salinity of Fundulus heteroclitus.

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.

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.

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.

Ca2+/Na+ exchanger and Na+,K+ 2Cl- cotransporter in lens fiber plasma membrane vesicles.

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)

Study of the Ca2+/Na+ exchange mechanism in vesicles isolated from apical membranes of lens epithelium of spiny dogfish (Squalus acanthias) and bovine eye.

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)

A Na+/H+ exchanger and its relation to oxidative effects in plasma membrane vesicles from lens fibers.

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.

Lens opacification and H2O2 elevation induced by a tumor promoter.

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.

Characterization of BADS-binding proteins in epithelial plasma membranes.

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.

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.

Fluorescent stilbene (BADS) binding proteins in anion-transporting epithelia.

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.

Mechanically stripped pigmented and non-pigmented epithelium of the shark ciliary body: morphology and transepithelial electrical properties.

Sections of intact ciliary epithelium and mechanically stripped non-pigmented (NPE) and pigmented (PE) cell layers of adult sharks (Squalus acanthias) were mounted in Ussing-type chambers (area 0.1 cm2). Addition of 10(-5) M forskolin to the aqueous side of intact epithelium significantly increased short-circuit current (Isc) within 15 min and a maximum of approx. 30 microA cm-2 was reached after 45-60 min. Transepithelial potential difference (V) increased from -0.8 mV (aqueous side negative as compared with blood/stromal side) to -1.5 mV, whereas resistance (R) was unchanged (50 omega cm2). Forskolin was without effect when applied to the blood side. In stripped PE preparations (R 15 omega cm2), 10(-5) M forskolin applied to the apical side induced a qualitatively similar change of Isc and V compared with the intact tissue. The forskolin-induced effects were fully reversed by 10(-4) M bumetanide and were not dependent on pretreatment of the tissue with 10(-3) M BaCl2. In stripped NPE preparations resistance was usually less than 10 omega cm2 and was not stable. This is consistent with the morphologic observation that although tight junctions were still demonstrable in stripped NPE cells, the apical membranes were damaged. In preparations taken for light and electron microscopy the stripped PE layer revealed intact epithelial cells. In particular, the basal thirds of the stripped PE cells were in very close contact with each other. These attachment zones may have the appearance of tight junctions. Thus the PE cells of the shark ciliary epithelium can be successfully isolated for transepithelial transport studies. The adenylate cyclase system is present in PE cells, and transepithelial transport of chloride may be regulated by intracellular cAMP.

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.

Effect of pH on chloride absorption in the flounder intestine.

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.

Intracellular voltage recordings in the opercular epithelium of Fundus heteroclitus.

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.

Effect of a chloride channel blocker on bullfrog corneal epithelium.

The effect of the chloride channel blocker, diphenylamine-2-carboxylate (DPC), was investigated on the bullfrog cornea epithelium using microelectrodes. Perfusion of 2 X 10(-4) M DPC on the tear side resulted in a 44% reduction of the corneal transepithelial potential difference (TEP) and a 9% reduction in corneal conductance. The cornea epithelial cells hyperpolarized 7.2 +/- 1.5 mV, and the apical fractional resistance increased 35% (n = 8 tissues). The basolateral potential difference depolarized 5.5 +/- 1.8 mV. The relative apical conductance decreased 29%, whereas relative changes of basolateral membranes increased 56% with prolonged DPC exposure, indicating that the DPC inhibition was acting at the apical cell membrane. DPC-treated corneas could be stimulated with isoproterenol (10(-6) M), indicating that the DPC inhibition could be reversed with the stimulation of chloride conductance. The DPC inhibitory effect was dependent on basolateral chloride. Intracellular chloride activity increases with DPC, indicating that the integrity of the basolateral chloride entry mechanism was not altered. The DPC inhibitory action was due to a reduction in apical chloride conductance.

Effect of atriopeptin II on isolated opercular epithelium of Fundulus heteroclitus.

The effect of atriopeptin II (ANF) on the in vitro opercular epithelium was investigated by use of short-circuit current techniques. Serosal addition of ANF stimulates chloride secretion (short-circuit current) 19% above control values with a 7% increase in tissue conductance. Mucosal addition of ANF to the opercular epithelium was without effect. The ANF stimulation of the current was dose dependent with a maximum at 10(-7) M. The addition of ANF had no effect on the current or the conductance of opercular epithelia bathed in Cl--free Ringer. The opercular current could be stimulated above the ANF response by isoproterenol (10(-6) M). Pretreatment of the opercular epithelium with propranolol (10(-5) M) did not inhibit the stimulation of the short-circuit current by ANF but did inhibit the isoproterenol response indicating that the ANF stimulatory activity was independent of the beta-adrenergic receptors. The ANF-stimulated short-circuit current was found in operculi pretreated with tetrodotoxin (10(-6) or 10(-5) M) or diltiazem (10(-4) M) indicating the ANF response was not due to nerve stimulation. Pretreatment of opercular tissue with dibutyryl adenosine 3',5'-cyclic monophosphate, 8-bromoadenosine 3',5'-cyclic monophosphate, or 8-bromoguanosine 3',5'-cyclic monophosphate (10(-4) M) had no effect on the ANF stimulatory response. Opercular epithelia from short-term freshwater-adapted killifish also showed the ANF-induced response. The stimulation of chloride secretion in Fundulus heteroclitus chloride cells by ANF may have a role in teleost ion regulation.

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.