Robert Nielsen

Coupled transepithelial sodium and potassium transport across isolated frog skin: effect of ouabain, amiloride and the polyene antibiotic filipin.

SummaryAddition of the polyene antibiotic filipin (50 μm) to the outside bathing solution (OBS) of the isolated frog skin resulted in a highly significant active outward transport of K+ because filipinper se increases the nonspecific Na+ and K+ permeability of the outward facing membrane. The K+ transport was calculated from the chemically determined changes in K+ concentrations in the solution bathing the two sides of the skin. The active transepithelial K+ transport required the presence of Na+ in the OBS, but not in the inside bathing solution (IBS), and it was inhibited by the Na+, K+-ATPase inhibitor ouabain. The addition of Ba++ to the IBS in the presence of filipin in the OBS resulted in an activation of the transepithelial K+ transport and in an inhibition of the active Na+ transport. This is in agreement with the notion that Ba++ decreases the passive K+ permeability of the inward facing membrane. In the presence of amiloride (which blocks the specific Na permeability of the outward facing membrane) and Ba++ there was a good correlation between the active Na+ and K+ transport. It is concluded that the active transepithelial K+ transport is carried out by a coupled electrogenic Na−K pump, and it is suggested that the pump ratio (Na/K) is 1.5.

Prostaglandin E2-stimulated glandular ion and water secretion in isolated frog skin (Rana esculenta).

SummaryProstaglandins are known to stimulate the active sodium absorption in frog skin. In this paper it is shown that prostaglandin E2 (PGE2) stimulates an active secretion of Cl−, Na+, and K+ from the skin glands inRana esculenta. The active Cl secretion is enhanced more than the Na and K secretion. Therefore, in skins where the Na absorption is inhibited by amiloride, the addition of PGE2 results in an increase in the short-circuit current (SCC). The PGE2-stimulated Cl secretion could be inhibited by the presence of ouabain or furosemide in the basolateral solution or diphenylamine-2-carboxylate in the apical solution. The PGE2-stimulated Cl secretion was enhanced by the phosphodiesterase inhibitor, theophylline, indicating that the effect of PGE2 was caused by an increase in the intracellular cAMP level in the gland cells. The calcium ionophore A23187, which increases the PGE2 synthesis in frog skin, stimulated the glandular Cl secretion. This secretion could be blocked by the prostaglandin synthesis inhibitor indomethacin, indicating that A23187 acts by increasing the prostaglandin synthesis and not by a direct action of Ca2+ ionsper se. The net water flow (Jw) and the Cl secretion were measured simultaneously under the conditions outlined above. The stimulation, inhibition, and the time-course of the outward-directedJw were similar to the change observed for the Cl secretion. These results show that PGE2 stimulates a glandular secretion of Cl and water in frog skin, probably by increasing the cAMP level in the gland cells.

Correlation Between Transepithelial Na+ Transport and Transepithelial Water Movement Across Isolated Frog Skin (Rana esculenta)

AbstractIn the present work the coupling under short-circuited conditions between the net Na+-influx across isolated frog skin and the transepithelial transport of water was examined i.e., the short-circuit current (Isc) and the transepithelial water movement (TEWM) were measured simultaneously. It has been shown repeatedly that the Isc across isolated frog skin is equal to the net transepithelial Na+ transport. Furthermore the coupling between transepithelial uptake of NaCl under open-circuit conditions and TEWM was also measured. The addition of antidiuretic hormone (AVT) to skins incubated under short-circuited conditions resulted in an increase in the Isc and TEWM. Under control conditions Isc was 9.14 ± 2.43 and in the presence of AVT 45.9 ± 7.3 neq cm−2 min−1 (n= 9) and TEWM changed from 12.45 ± 4.46 to 132.8 ± 15.8 nL cm−2 min−1. The addition of the Na+ channel blocking agent amiloride resulted in a reduction both in Isc and TEWM, and a linear correlation between Isc and TEWM was found. The correlation corresponds to that 160 ± 15 (n= 7) molecules of water follow each Na+ across the skin. In another series of experiments it was found that there was a linear correlation between Isc and the increase in apical osmolarity needed to stop the TEWM. The data presented indicate that the observed coupling between the net transepithelial Na+ transport and TEWM is caused by local osmosis.

Effect of ouabain, amiloride, and antidiuretic hormone on the sodium-transport pool in isolated epithelia from frog skin (Rana temporaria)

SummaryWhen tracer Na+ is added to the solution bathing the apical side of isolated epithelia the observed transepithelial tracer influx increases with time until a steady state is reached. The build-up of the tracer flux follows a single exponential course. The halftime for this build-up under control conditions was 0.92 ±0.06 min, and in the presence of ouabain 4.51±0.7 min. It is shown that the calculated Na+-transport pool is located in the cells. The Na+-transport pool under control conditions was 35.6 ±3.4 nmol/cm2, which corresponds to an intracellular Na+ concentration of 7.9mm. Activation of the active Na+ transport by addition of antidiuretic hormone resulted in a highly significant increase in the Na+ transport pool, and inhibition of the transcellular Na+ transport with amiloride resulted in a decrease in the Na+-transport pool.Furthermore, the active Na+ transport increased along anS-shaped curve with increasing intracellular Na+ concentration (Na+-transport pool). The Na+ pump was found to be half saturated at an intracellular Na+ concentration of 12.5mm.

Effect of the polyene antibiotic filipin and the calcium ionophore A23187 on sodium transport in isolated frog skin (Rana temporaria)

SummaryAddition of filipin (50 μm) to the inside bathing solution of the frog skin resulted in a transient increase in the active sodium transport [measured as short-circuit current (SCC)]. The filipin-induced increase in the SCC required the presence of calcium. The calcium ionophore A23187 (4 μm) also induced a transient increase in the SCC. After the activation of the SCC by A23187, the SCC could not be activated by filipin. This indicates that the polyene antibiotic filipin acts as a calcium ionophore. Higher concentrations (40 μm) of A23187 resulted in a shrinking of the cells in the transporting cell layer. A23187 also increased the potassium-42 exchange in the isolated epithelium. It is suggested that calcium inophores enhanced the intracellular calcium concentration; this increase in the calcium concentration resulted in an increase in the potassium permeability of the inward-facing membrane. The increase in the potassium permeability might explain the observed increase in the SCC.

The effect of polyene antibiotics on the aldosterone induced changes in sodium transport across isolated frog skin

Abstract It is shown that: (1) The isolated stratum corneum has a very low electrical resistance. 1. (2) The passive sodium flux through the isolated s. corneum is 20–100 times higher than the active sodium transport. 2. (3) Polyene antibiotics (amphotericin B and filimarisin) abolish the aldosterone induced inhibition of the active sodium transport. 3. (4) Aldosterone induces a recovery in the electrical potential across skins where it had been reduced previously by the addition of a polyene antibiotic. 4. (5) An hypothesis is presented to explain these observations.

An increase in [Ca2+]i activates basolateral chloride channels and inhibits apical sodium channels in frog skin epithelium.

Abstract The aim of this study was to investigate the mechanisms by which increases in free cytosolic calcium ([Ca2+]i) cause a decrease in macroscopic sodium absorption across principal cells of the frog skin epithelium. [Ca2+]i was measured with fura-2 in an epifluorescence microscope set-up, sodium absorption was measured by the voltage-clamp technique and cellular potential was measured using microelectrodes. The endoplasmic reticulum calcium-ATPase inhibitor thapsigargin (0.4 μM) increased [Ca2+]i from 66 ± 9 to 137 ± 19 nM (n = 13, P = 0.002). Thapsigargin caused the amiloride-sensitive short circuit current (Isc) to drop from 26.4 to 10.6 μA cm–2 (n = 19, P<0.001) concomitant with a depolarization of the cells from –79 ± 1 to –31 ± 2 mV (n = 18, P<0.001). Apical sodium permeability (PaNa) was estimated from the current/voltage (I/V) relationship between amiloride-sensitive current and the potential across the apical membrane. PaNa decreased from 8.01·10–7 to 3.74·10–7 cm s–1 (n = 7, P = 0.04) following an increase in [Ca2+]i. A decrease in apical sodium permeability per se would tend to decrease Isc and result in a hyperpolarization of the cell potential and not, as observed, a depolarization. Serosal addition of the chloride channel inhibitors 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS), diphenylamine-2-carboxylate (DPC), indanyloxyacetic acid 94 (IAA-94) and furosemide reversed the depolarization induced by thapsigargin, indicating that chloride channels were activated by the increase in [Ca2+]i. This was confirmed in wash-out experiments with 36Cl where it was shown that thapsigargin increased the efflux of chloride from 32.49 ± 5.01 to 62.63 ± 13.3 nmol·min–1 cm–2 (n = 5, P = 0.04). We conclude that a small increase in [Ca2+]i activates a chloride permeability and inhibits the apical sodium permeability. The activation of chloride channels and the closure of apical sodium channels will tend to lower the macroscopic sodium absorption.

Prostaglandin E2 enhances the sodium conductance of exocrine glands in isolated frog skin (Rana esculenta).

SummaryProstaglandins are known to stimulate the active transepithelial Na+ uptake and the active secretion of Cl− from the glands of isolated frog skin. In the present work the effect of prostaglandin E2 (PGE2) on the glandular Na+ conductance was examined. In order to avoid interference from the Na+ uptake and the glandular Cl− secretion the experiments were carried out on skins where the Cl− secretion was inhibited (the skins were bathed in Cl− Ringers solution in the presence of furosemide, or in NO3− Ringers solution), and the active Na+ uptake was blocked by the addition of amiloride. Transepithelial current, water flow and ion fluxes were measured. A negative current was passed across the skins (the skins were clamped at −100 mV, basolateral solution was taken as reference). When PGE2, was added to the skins under these experimental conditions, the current became more negative; this was mainly due to an increase in the Na+ efflux. Together with the increase in Na+ efflux a significant increase of the water secretion was observed. The water secretion was coupled to the efflux of Na+, and when one Na+ was pulled from the basolateral to the apical solution via this pathway 230 molecules of water follwed. From the data presented it is suggested that this pathway for Na+ is confined to the exocrine glands.

Small transepithelial osmotic gradients affect apical sodium permeability in frog skin

The aim of the present study was to investigate the effects of small, unilateral changes in solution osmolarity on active sodium transport and cellular electrophysiological parameters in frog skin. The active sodium transport across the skin was measured as the amiloride-sensitive short-circuit current (Isc) and cellular potential was monitored with microelectrodes, while small (±20 mOsm) osmotic gradients were imposed on the skin. Increasing the osmolarity of the apical bathing solution (or decresing the osmolarity of the basolateral solution) increased ISC, lowered tissue resistance (R), depolarized the cellular potential and decreased the fractional resistance of the apical membrane, which indicates an increased apical sodium permeability. Conversely, a similar increase in basolateral osmolarity (or a decrease in apical osmolarity) lowered the Isc, increased R, hyperpolarized the cells and increased the fractional resistance of the apical membrane, indicating a decrease in apical sodium permeability. The results indicate that the osmotic gradient across the skin, rather than solution osmolarity as such, is responsible for the observed changes in Isc and apical sodium permeability after small osmotic perturbations.

Effect of amiloride, ouabain and Ba++ on the nonsteady-state Na−K pump flux and short-circuit current in isolated frog skin epithelia

SummaryEffect of amiloride, ouabain, and Ba++ on the nonsteady-state Na−K pump flux and short-circuit current in isolated frog skin epithelia.The active Na+ transport across isolated frog skin occurs in two steps: passive diffusion across the apical membrane of the cells followed by an active extrusion from the cells via the Na+−K+ pump at the basolateral membrane. In isolated epithelia with a very small Na+ efflux, the appearing Na+-flux in the basolateral solution is equal to the rate of the pump, whereas the short-circuit current (SCC) is equal to the active transepithelial Na+ transport. It was found that blocking the passive diffusion of Na+ across the apical membrane (addition of amiloride) resulted in an instantaneous inhibition of the SCC (the transepithelial Na+ transport, whereas the appearing flux (the rate of the Na+−K+ pump) decreased with a halftime of 1.9 min. Addition of the Na+−K+ pump inhibitor ouabain (0.1mm) resulted in a faster and bigger inhibition of the appearing flux than of the SCC. Thus, by simultaneous measurement of the SCC and the appearing Na+ flux one can elucidate whether an inhibitor exerts its effect by inhibiting the pump or by decreasing the passive permeability. Addition of the K+ channel inhibitor Ba++, in a concentration which gave maximum inhibition of the SCC, had no effect on the appearing flux (the rate of the Na−K pump) in the first 2 min, although the inhibition of the SCC was already at its maximum.It is argued that in the short period, where the Ba++-induced inhibition of SCC is at its maximum and the appearing flux in unchanged, the decrease in the SCC (ΔSCC) is equal to the net K+ flux via the Na+−K+ pump, and the coupling ratio (β) of the Na+−K+ pump can be calculated from the following equation β=SCCt=0/ΔSCC where SCCt=0 is the steady-state SCC before the addition of Ba++.