Thomas U.L. Biber

The identity of the current carriers in canine lingual epithelium in vitro

Ion transport across the lingual epithelium has been implicated as an early event in gustatory transduction. The fluxes of isotopically labelled Na+ and Cl- were measured across isolated canine dorsal lingual epithelium under short-circuit conditions. The epithelium actively absorbs Na+ and to a lesser extent actively secretes Cl-. Under symmetrical conditions with Krebs-Henseleit buffer on both sides, (1) Na+ absorption accounts for 46% of the short-circuit current (Isc); (2) there are two transcellular Na+ pathways, one amiloride-sensitive and one amiloride-insensitive; (3) ouabain, added to the serosal solution, inhibits both Isc and active Na+ absorption. When hyperosmotic (0.25 M) NaCl is placed in the mucosal bath, both Isc and Na+ absorption increase; net Na+ absorption is at least as much as Isc. Ion substitution studies indicate that the tissue may transport a variety of larger ions, though not as effectively as Na+ and Cl-. Thus we have shown that the lingual epithelium, like other epithelia of the gastrointestinal tract, actively transports ions. However, it is unusual both in its response to hyperosmotic solutions and in the variety of ions that support a transepithelial short-circuit current. Since sodium ion transport under hyperosmotic conditions has been shown to correlate well with the gustatory neural response, the variety of ions transported may likewise indicate a wider role for transport in taste transduction.

Influence of extracellular Cl concentration on Cl transport across isolated skin ofRana Pipiens

SummaryThe effect of changes in Cl concentration in the external and/or serosal bath on Cl transport across short-circuited frog skin was studied by measurements of transepithelial Cl influx (J13Cl) and efflux (J31Cl), short-circuit current, transepithelial potential, and conductance (Gm).J13Cl as well asJ31Cl were found to have a saturating component and a component which is apparently linear with Cl concentration. The linear component ofJ31Cl appears only upon addition of Cl to external medium, and about 3/4 of this component does not contribute toGm. The saturating component ofJ31Cl is only 5% of totalJ31Cl with 115mm Cl in the serosal medium. Replacement of 115mm Cl− in external medium by SO4=, NO3−, HCO3− or I− results in 87–97% reduction ofJ31Cl, whereas replacement with Br− has no effect. As external Cl concentration is raised in steps from 2 to 115mm,J13Cl andJ31Cl increase by the same amount butJ13Cl is persistently 0.15 μeq/cm2 hr larger thanJ31Cl. These results indicate that at least 3/4 of linear components ofJ13Cl andJ31Cl proceed via an exchange diffusion mechanism which seems to be located at the outer cell border. The saturating component ofJ13Cl is involved in active Cl transport in an inward direction, and there is evidence suggesting that Cl uptake across outer cell border, which proceeds against an electrochemical gradient, is electroneutral but not directly linked to Na.

Effect of external cation and anion substitutions on sodium transport in isolated frog skin.

SummaryEffects of changes in external ionic strength, external cation and/or anion substitution on transepithelial influx and efflux of sodium, short-circuit current and on transepithelial potential difference and resistance were studied in isolated frog skin. Active transport of Na was found to be highly dependent on both anionic and cationic composition of external medium. Relative abilities of external monovalent cations to inhibit active Na transport were H>Li>K>Rb>Cs>choline. Relative abilities of external monovalent anions to stimulate active Na transport were I>Br>Cl. Sequences of anion interaction and of resistance changes suggest that anionic stimulation of Na transport is not due to electrical coupling across outer cell membrane. The ability of different anions and cations to alter Na transport suggests that externally located charged groups act as important barriers or filters to ion movement. In addition, the experiments suggest that an increase in ionic strength of external medium has an effect on active transport of Na, a finding that indicates interference of surface charges with Na entry. Directional changes in efflux of Na due to changes in ionic composition of external medium usually paralleled changes in active Na transport. It is possible that the observed relationship between influx and efflux of Na is the result of common pathways and of interaction of the active transport system with Na efflux.

Na+ channel blockers inhibit voltage-dependent intracellular ph changes in principal cells of frog (Rana pipiens) skin

1. The relationship between Va and pHi was studied with double-barrelled microelectrodes in principal cells of frog skin (Rana pipiens) when (i) the transepithelial potential (Vt) was clamped at different values of Vt and (ii) when the pH of the apical solution was altered. 2. Under all conditions examined here, depolarization of Va was associated with an increase in pHi and hyperpolarization of Va was accompanied by a decrease in pHi. However, the changes in the basolateral cell membrane potential occurred, either in the same or opposite direction to that of Va depending on the conditions. 3. The voltage-dependent changes in pHi were not affected by H+ transport inhibitors or the complete removal of Na+, Cl- and HCO3- but were effectively inhibited by the application of amiloride (10(-4) M) or benzamil (10(-6)M) on the apical side. 4. A decrease in pH of the apical solution hyperpolarized Va and decreased pHi, an effect that was significantly attenuated when benzamil was present on the apical side. 5. The results indicate the presence of an H+ and/or OH- conductive pathway in the apical cell membrane of the principal cells. The effect of Na+ channel blockers suggests that this pathway proceeds through the apical Na+ channels.

Potential dependence of unidirectional chloride fluxes across isolated frog skin

Isolated frog skins were voltage clamped at transepithelial potentials (Vt) ranging from -60 mV to 60 mV to measure transepithelial 36Cl- fluxes from the apical to the basolateral bathing solution (J13) and in the opposite direction (J31). The potential dependence of fluxes obtained in Na+-free choline Ringers indicates the presence of conductive and nonconductive components that probably correspond to fluxes through paracellular and cellular pathways, respectively. Rectification of fluxes with reversal of the potential reflects a structural asymmetry, presumably in surface charge density. The data are consistent with a charge density of one negative charge per 280 A2 on the apical side. A new model for passive Cl- transport was developed that includes surface charge asymmetry and specifically accounts for the observed variation of conductance with potential. In normal frog Ringers, J13 was larger than J31 at zero potential (active Cl- transport), J13 rose exponentially with increasing positive potential to reach a maximum at 40 mV (approximately open-circuit), and the predicted partial Cl- conductance exceeded the measured conductance leading to the conclusion that when J13 is largely driven by Na+ transport, much of the coupling occurs via nonconductive pathways. Theophylline stimulates Cl- transport that also occurs via nonconductive pathways as Vt becomes more positive.

Effect of FeCl3 on ion transport in isolated frog skin

SummaryThe effect of addition of FeCl3 to the media bathing the isolated skin ofRana pipiens was studied by measuring short-circuit current, transepithelial potential, and resistance, and by determining the influx and efflux of sodium (J13Na andJ31Na, respectively) and the influx and efflux of chloride (J13Cl andJ31Cl, respectively) across the epithelium. With normal Ringers solution on both sides of the skin, addition of 10−3m FeCl3 to the external medium resulted in nearly complete inhibition of active Na transport (J13Na decreased from 1.30±0.14 to 0.10±0.04 μeq/cm2 hr (N=8)) and in appearance of active chloride transport in outward direction due to an 80% increase inJ31Cl. Average (J31Cl−J13Cl) obtained from means of 8 skins in 6 consecutive control and last 3 experimental periods was −0.17±0.04 and 0.38±0.05 μeq/cm2 hr, respectively. FeCl3 added to external medium also induced substantial net chloride movement in outward direction when external medium contained Na-free choline chloride Ringers or low ionic strength solution. Under the latter condition net Na movement was virtually eliminated by external FeCl3. After addition of FeCl3 to serosal medium there was delayed inhibition ofJ13Na but no change in chloride fluxes. Immediate and profound changes in Na and Cl transport systems seen after external application of FeCl3 indicate charge effects of Fe3+ on surface of apical cell membranes, possibly close to or in ion channels.