Jose A. Zadunaisky

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 × 105 cells/cm2. This epithelium can be isolated, mounted in a Lucite chamber, and its ion transport properties studied with the short‐circuit current technique.

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, chloridecell-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 10−4m and 10−4m 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 10−4m 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. Freshwater-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.

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

SummaryThe unidirectional Na+, Cl−, and urea fluxes across isolated opercular epithelia from seawater-adaptedFundulus heteroclitus were measured under different experimental conditions. The mean Na+, Cl−, and urea permeabilities were 9.30×10−6 cm·sec−1, 1.24×10−6 cm·sec−1, and 5.05×10−7 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 a 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 or 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 extracellularly (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.

Membrane potentials and intracellular chloride activity in the ciliary body of the shark

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·l−1) decrease the potential, and furosemide (10−4 mol·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·l−1) was significantly higher than could be predicted from the equilibrium distribution (26 mmol·l−1) across the cell membranes. When furosemide was applied to the aqueous side of the epithelium, intracellular chloride activity decreased to 35 mmol·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.

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.

Glucose transport into the ocular compartments of the rat

An exchange model was developed for the study of the transfer between a central plasma compartment and the aqueous and vitreous compartments in the rat eye. The solutes investigated include sucrose, urea, 3-O-methyl- d -glucose, d -glucose and l -glucose. For the substances studied, rate parameters were used which enabled ocular concentration functions to be evaluated from various plasma functions. Rate transfer constants were evaluated from plasma functions and compartments significantly faster than does urea. d -glucose is transported six times faster than l -glucose, indicating that the transport mechanism is stereospecific. The saturability of the d -glucose transport system was explored by elevating the plasma concentration of unlabeled d -glucose. The system was found to be saturable. A Michaelis-Menten analysis of transport vs. Plasma concentration revealed the Km to be 6·08 m m and 4·64 m m and the Vm to be 0·0225 and 0·0238 mmoles/min (for the aqueous and vitreous repectively). Relative high glucose concentrations (50 m m ) were required for saturation. We conclude that transport of glucose into the ocular fluids is consistent with a carrier-mediated facilitated diffusion mechanism which is stereospecific and saturable.

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.

Displacement of alpha- and beta-radioligands by specific adrenergic agonists in rat pancreatic islets.

Secretion of insulin is increased by beta-adrenergic agonists and inhibited by alpha-adrenergic agonists. However, administration of epinephrine, which acts on both types of receptors, inhibits insulin secretion. A preliminary study using [3H]-dihydroergocryptine and [3H]-dihydroalprenolol as the respective alpha- and beta-receptor binding ligands, surprisingly revealed a preponderance of beta-binding sites in normal rat pancreatic islets. The present study, using displacement by epinephrine, norepinephrine, isoproterenol and clonidine validated the use of these radioligands as appropriate for specific receptor binding in pancreatic islet cells. The islets were found to have 55 fmol/mg protein of alpha-adrenergic receptor sites and 170 fmol/mg protein of beta-receptor sites. The affinity of both alpha- and beta-receptors for epinephrine was similar, as judged by the displacement of either radioligand, thus ruling out a preferential affinity of alpha-receptor binding as an explanation for the alpha-inhibition of insulin secretion. The data on radioligand displacement by clonidine indicate that the alpha-receptor is of the alpha 2-type.

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.

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.