Cyclic AMP stimulation of Cl- secretion by the opercular epithelium: the basolateral chloride uptake mechanism.

The isolated, short-circuited opercular epithelium of Fundulus heteroclitus, secretes Cl- by a mechanism dependent on the presence of serosal Na+ and inhibited by bumetanide and furosemide. Under serosal Na+-free conditions the active Cl- secretion is abolished. However, subsequent elevations of intracellular cyclic AMP (cAMP) levels with isoproterenol or forskolin stimulated Cl- secretion markedly. This stimulation was unaffected by SITS, DIDS, methazolamide, and HCO-3-free solutions, but was blocked by furosemide and bumetanide. Determinations of relative intracellular 36Cl- levels showed a Na+ dependence of intracellular 36Cl- in epithelia not stimulated by isoproterenol and a Na+ independence of intracellular 36Cl- in isoproterenol stimulated epithelia. In both conditions, the intracellular 36Cl- was bumetanide sensitive. The results indicate that cAMP stimulation of Cl- secretion can occur by a Na+-independent, loop diuretic-inhibitable mechanism, which may be operative even in the presence of Na+. Whether this is a separate Cl- uptake mechanism or a cAMP-induced alteration in the normal Na+-dependent mechanism could not be determined. In either instance, an alternative to the Na+ gradient as a source of energy for Cl- uptake into the cell across the basolateral membrane is required.

Cyclic AMP stimulation of Cl- secretion by the opercular epithelium: the apical membrane chloride conductance.

Chloride secretion (Isc) by the opercular epithelium of the teleost, Fundulus heteroclitus, is stimulated by elevations in intracellular cyclic AMP (cAMP) elicited by beta-adrenergic agonists, such as isoproterenol, and is accompanied by a small but significant increase in the transepithelial conductance (Gt). Cupric ions (Cu2+) have been shown to block the apical membrane Cl- channels in this epithelium, leading to a reduction in both the Isc and Gt (Degnan, '85). In the present studies, the effects of Cu2+ on cAMP-elevated and cAMP-depleted epithelia were observed to define the actions of cAMP in this stimulatory process. At a concentration of 5 X 10(-4) M in the mucosal solution, Cu2+ inhibited the Isc 79.8% and reduced the Gt 39.2%. Isoproterenol produced an attenuated stimulation of the Isc in these tissues compared to untreated controls, but had no effect on the Gt. In tissues bathed bilaterally with Cl- -free Ringer, the Isc was virtually abolished and the Gt was reduced 37.0%; neither Cu2+ nor isoproterenol had any effects on the Isc or Gt under this condition. Simultaneous 2 2Na and 3 6 Cl unidirectional flux determinations indicated that the only effects of both isoproterenol and Cu2+ were on the active Cl- secretory flux. An inhibitor of adenylate cyclase, 2',5' dideoxyadenosine (DDA), reduced the Isc and Gt 39.8% and 20.8% respectively. This inhibitor had no additional effects in Cu2+ -treated tissues and the action of Cu2+ on the Gt was reduced in DDA-treated tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of K+ and Cl- conductances in chloride secretion by the opercular epithelium.

The opercular epithelium of the teleost Fundulus heteroclitus, when mounted in a lucite chamber under short-circuited conditions, secretes Cl- at a rate equivalent to the short-circuit current (Isc). The transepithelial Na+ movements are passive and proceed by a paracellular pathway. The addition of 2 X 10(-3) M Ba2+ to the serosal bathing solution inhibited the Isc 76.8% with no effect on the transepithelial conductance (Gt). The addition of 5 X 10(-4) M Cu2+ to the mucosal bathing solution inhibited the Isc 79.6% and reduced the Gt 35.6%. These inhibitory effects of Ba2+ and Cu2+ on the Isc were initiated within 1 minute after exposure with maximum effects occurring within 20 and 30 minutes, respectively. Simultaneous 36Cl- and 22Na+ unidirectional fluxes were performed on paired epithelia from the same fish. Serosal Ba2+ and mucosal Cu2+ inhibited the Cl- secretory flux 30.2 and 58.9%, respectively. The resulting net Cl- flux after inhibition was not significantly different from the mean measured Isc. Neither ion had significant effects on the Cl- influx (mucosa to serosa) or the unidirectional Na fluxes. These results indicated that the effects of both Ba2+ and Cu2+ were most likely exclusive to the transcellular Cl- pathway. Ba2+ is proposed to inhibit Cl- secretion by blocking the basolateral K+ channels, depolarizing the cell, and reducing the electrochemical driving force for Cl- across the apical membrane. Cu2+ is proposed to inhibit Cl- secretion by blocking the apical membrane Cl- channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Converging adrenergic and cholinergic mechanisms in the inhibition of Cl secretion in fish opercular epithelium.

The rate of Cl secretion (Isc) by the opercular epithelium of Fundulus heteroclitus is stimulated by elevations in cyclic AMP (cAMP) levels elicited via beta 1-adrenergic receptor activation, and inhibited by both alpha 2-adrenergic and muscarinic cholinergic receptor activation via mechanisms presently unknown. A comparison of these two inhibitory responses was made using clonidine, an alpha 2-adrenergic agonist, and acetylcholine (ACh), a cholinergic agonist. The dose required for maximum inhibition was 100 times greater for ACh, but in all other respects the responses elicited by both agonists were statistically indistinguishable. Adrenergic antagonists did not diminish the ACh inhibition, and cholinergic antagonists did not diminish the clonidine inhibition, indicating that the two receptor types were distinct from each other. In control tissues and tissues pretreated with agents that increase cAMP levels (isoproterenol, IBMX, forskolin), both ACh and clonidine had no effects on cyclic AMP levels, indicating an inhibitory mechanism independent of adenylate cyclase. Neither Ca-free media nor a variety of calcium antagonists diminished the ACh or clonidine inhibitions. These results suggest that the alpha 2-adrenergic and muscarinic cholinergic pathways converge into a common pathway to inhibit Cl secretion by a mechanism not involving adenylate cyclase or the mobilization of either extracellular or intracellular calcium stores.

The sodium and chloride dependence of chloride secretion by the opercular epithelium.

The effects of ion substitutions on the Cl- secretion rate and tissue conductance of isolated short-circuited opercular epithelia from sea-water-adapted Fundulus heteroclitus were investigated. Serosal Na+ substitution had the same effect on the Cl- secretion rate that serosal Cl- substitution had on the active component of the Cl- efflux. This similarity indicated a 1:1 Na-Cl requirement for active Cl- secretion across this epithelium, which supports the proposal of a coupled NaCl uptake mechanism at the serosal membrane of Cl- secretory epithelia. Mucosal Na+ and Cl- substitutions appeared to inhibit completely the active Cl- secretory flux. The reductions in the tissue conductance with mucosal ion substitutions suggested that this effect can be attributed to a blocking of the apical membrane Cl- conductance. These mucosal ion effects suggested a possible direct regulatory influence of the external salinity on the Cl- secretion rate and tissue conductance, which provide alternative explanations for observations with the teleost gill epithelium.

cAMP-mediated regulation of chloride secretion by the opercular epithelium.

Catecholamine regulation of the Cl- secretion rate (short-circuit current, Isc) and adenosine 3',5'-cyclic monophosphate (cAMP) levels of the opercular epithelium was investigated by using 3-isobutyl-1-methylxanthine (IBMX), forskolin, and adrenergic agonists. In this tissue alpha-adrenergic agonists inhibit, and beta-adrenergic agonists stimulate, the Isc (J. Physiol. London 294: 483-495, 1979). IBMX and forskolin stimulated the Isc 125 and 85%, respectively, and simultaneously produced 2.5- and 70.0-fold elevations in the cAMP levels, respectively. These findings confirm previous observations demonstrating that stimulation of the Isc in this tissue is mediated by elevations in cAMP (J. Comp. Physiol. B 145: 29-35, 1981). Isoproterenol, a beta-agonist, had no effect on the Isc of either IBMX- or forskolin-stimulated tissues but increased the cAMP level an additional 5.8-fold in IBMX-stimulated tissues. Clonidine, an alpha-agonist, inhibited the Isc in IBMX-stimulated tissues only and had no effect on cAMP levels in either IBMX- or forskolin-stimulated tissues. These findings demonstrate that catecholamine-induced inhibition of the Isc can occur while the cAMP level remains elevated, indicating that this effect is not mediated by lowering cAMP levels. This observation is strong evidence for a cAMP-independent mechanism for catecholamine-induced inhibition of Cl- secretion in the opercular and similar epithelia.

Identification and quantification of mitochondria-rich cells in transporting epithelia.

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.

A model for transepithelial ion transport across the isolated retinal pigment epithelium of the frog.

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.

Passive sodium movements across the opercular epithelium: the paracellular shunt pathway and ionic conductance.

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

Open-circuit sodium and chloride fluxes across isolated opercular epithelia from the teleost Fundulus heteroclitus.

1. The Na+ and Cl- fluxes across opercular epithelia from sea water-adapted Fundulus heteroclitus were measured in vitro under open-circuit conditions while bathed on the mucosa with sea water and the serosa with Ringer solution. 2. The mean predicted Na+ flux ratio was 0.94 +/- 0.08 and the observed ratio was 1.14 +/- 0.12 (n = 15; mean +/- S.E. of mean). The difference in these means was not significant (P greater than 0.20). The mean predicted Cl- flux ratio was 11.4 +/- 0.9 and the mean observed ratio was 1.38 +/- 0.27 (n = 10). The difference in these means was significant (P less than 0.001). 3. Ouabain, at 10(-6) M in the serosal solution, produced a significant (P less than 0.01) reduction in the Na+ efflux while having no significant (P greater than 0.40) effect on the Na+ influx. The agreement between the predicted (1.70 +/- 0.14) and observed (1.72 +/- 0.18) Na+ flux ratios after ouabain treatment suggested that this effect could be completely attributed to the depolarization of the epithelium secondary to ATPase inhibition. 4. beta-adrenergic activation by isoprenaline stimulated the Cl- efflux 24.2% and alpha-adrenergic activation by noradrenaline inhibited the Cl- efflux 66.5%. These changes occurred oppositely to those predicted by the changes in the electrical gradient produced by these agents, while the changes in the Cl- influxes corresponded to the electrical changes. Short-circuit experiments confirmed these effects on the Cl- efflux and the lack of effects on the Cl- influx. 5. The results suggested that Na+ was near theromodynamic equilibrium and that the unidirectional fluxes were passive. The effects of alpha- and beta-adrenergic activation suggested that the active Cl- secretion may be antagonistically regulated by catecholamines.

Active chloride transport in the in vitro opercular skin of a teleost (Fundulus heteroclitus), a gill-like epithelium rich in chloride cells.

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).

The electrical properties and active ion transport across the urinary bladder of the urodele, Amphiuma means.

1. The electrical properties and the active transport processes of the isolated urinary bladder of the urodele, Amphiuma means, were studied by mounting this tissue as a flat sheet between two halves of a lucite chamber. The mean transepithelial potential difference was 70-2 +/- 2-3 mV (serosa positive), the mean short-circuit current was 10-9 +/- 0-5 micrionA/mg of dry weight and the mean transepithelial d.c. resistance was 6540 +/- 374 omega mg of dry weight. 2. The short-circuit current (Isc) accounted for 92% of the net 22Na+ flux from the mucosa to the serosa. The difference resulted from a transport of 36Cl- in the same direction as sodium. 3. The active sodium transport exhibited typical saturation kinetics, having a Km of 15-4 m-equiv/l. and approaching zero order at 60-70 m-equiv/l. The transepithelial potential difference increased linearly with the log of the mucosal sodium concentration at a rate of 50-3 mV per tenfold concentration change. 4. In the absence of the major anions (HCO3- and Cl-) from the bathing solutions, the electrical properties and the sodium influx decreased to less than 40% of their control values. The presence of only one of these two anions in the serosal bathing solution was sufficient to maintain these parameters. 5. Amiloride (10(-5)M) and ouabain (10(-6)M) inhibited the sodium transport 97% and 85% respectively. Amphotericin B (10(-6)M) stimulated the sodium transport 47%. Furosemide (10(-3)M) inhibited the chloride transport 43%. The sodium transport was insensitive to the action of two enurohypophyseal peptides tested, lysine-vasotocin and pitocin.

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.