We have investigated the possibility that the mitochondria-rich (MR) cells participate in sodium and proton transport, when the frog skin epithelium is bathed on its apical side with solutions of low Na+ concentration, by comparing transport rates with morphological observations (MR cell number and MR cell pit surface area). Frogs were adapted to various salinities or the isolated skins were treated with the following hormones, deoxycorticosterone acetate (DOCA), arginine vasotocin (AVT) and oxytocin in order to modify the transport of sodium and hydrogen ions. Adaptation of the frogs (either 3-4 days or 7-10 days) to distilled water, NaCl (50 mmol/l), KCl (50 mmol/l) or Na2SO4 (25 mmol/l) solutions modified the Na+ transport rate and the morphology of the epithelium. The highest Na+ transport rates were found for the animals adapted to the Na+ free solutions and were correlated with an increase in the total MR cell pit surface area (number of MR cells x individual cell pit-surface area). The KCl adaptated group showed the largest increase in sodium and proton transport and also presented a metabolic acidosis as reflected by plasma acidification (pCO2 increase and HCO3- decrease). Proton secretion and sodium absorption were also found to be stimulated by either serosal DOCA addition (10(-6) M) or during acidification of the epithelium by serosally applied CO2. Na+ transport was enhanced by AVT (10(-6) M) or oxytocin (100 mU/ml) when the skin was bathed on its apical side with a high Na+ containing solution (115 mmol/l), whereas these hormones did not exert any effect on Na+ transport when the apical solution was low in Na+ (0.5 mmol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the relationship between H+ secretion (JH), Na+ absorption (JNa), and urea transport (Ju) in skin of frogs (Rana esculenta) adapted to running tap water, NaCl (100 mM), and KCl (100 mM). In addition, cell morphological changes, particularly in the mitochondria-rich cells (MRC), were followed. NaCl adaptation stimulated an active Ju, reduced JNa and JH, and caused a decrease in the apical surface of MRC. After KCl adaptation, JNa and JH were increased and highly correlated, with a twofold increase in Ju, whereas the numerous MRC developed infoldings on their apical membranes. No correlation was found between JH and Ju. Clamping the skins in a range of +/- 50 mV or changing the external pH from 7.4 to 5.4 (at high cellular buffering power) had no effect on Ju. Depolarization of the basolateral membranes (serosal KCl-Ringer) had no effect on Ju. Ju was reversibly blocked by acidification of the cells by oxygen-free solution and sulfhydryl reagents (Hg2+, p-chloromercuribenzenesulfonic acid, and N-ethylmaleimide). Diethylstilbestrol, a proton transport blocker, had no effect on Ju. Apical addition of amiloride and derivatives (phenamil and ethylisopropyl amiloride) reversibly blocked Ju, whereas ouabain had no effect. We conclude that a cation (Na+ or H+)-dependent process is unlikely to exist in R. esculenta skin. A primary active transport in a two-step process is the simplest hypothesis to account for the energy-dependent Ju that develops in NaCl-adapted frogs.

We have compared the response of proton and water transport to oxytocin treatment in isolated frog skin and urinary bladder epithelia to provide further insights into the nature of water flow and H ÷ flux across individual apical and basolateral cell membrances. In isolated spontaneous sodium-transporting frog skin epithelia, lowering the pH of the apical solution from 7.4 to 6.4, 5.5, or 4.5 produced a fall in pH~ in principal cells which was completely blocked by amiloride (50 ~.M), indicating that apical Na ÷ channels are permeable to protons. When sodium transport was blocked by amiloride, the H ÷ permeability of the apical membranes of principal cells was negiligible but increased dramatically after treatment with antidiuretic hormone (ADH). In the latter condition, lowering the pH of the apical solution caused a voltage-dependent intracellular acidification, accompanied by membrane depolarization, and an increase in membrane conductance and transepithelial current. These effects were inhibited by adding Hg '÷ (100 o.M) or dicydohexylcarbodiimide (DCCD, to the apical bath. Net titratable H ÷ flux across frog skin was increased from 30 -+ 8 to 115 _ 18 neq-h-l-cm -~ (n = 8) after oxytocin treatment (at apical pH 5.5 and serosal pH 7.4) and was completely inhibited by DCCD (10 -5 M). The basolateral membranes of the principal cells in frog skin epithelium were found to be spontaneously permeable to H ÷ and passive electrogenic H ÷ transport across this membrane was not affected by oxytocin. Lowering the pH of the basolateral bathing solution (pHb) produced an intraceUular acidification and membrane depolarization (and an increase in conductance when the normal dominant K ÷ conductance of this membrane was abolished by Ba 2÷ 1 mM). These effects of low pH b were blocked by micromolar concentrations of heavy metals (Zn 2÷, Ni ~÷, Co 2÷, Cd ~+, and Hg~+). 750THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 97. 1991 (50 mU/ml) produced a transepithelial current (3 p~A'cm -~ at pH b 5.5) which was blocked by 100 I~M of rig z+, Zn 2+, or Ni 2+ at the basolateral side, and by DCCD (10 -~' M) or Hg 2+ (100 p~M) from the apical side. The net hydroosmotic water flux (JH~o) induced by oxytocin in frog bladder sacs was blocked by inhibitors of H+-adenosine triphosphatase (ATPase). Diethylstilbestrol (DES 10 -5 M), oligomycin (10 -~ M), and DCCD (10 -~ M) preventedJH~o when present in the lumen. These effects cannot be attributed to inhibition of metabolism since cyanide (10 -4 M), or 2-deoxyglucose (10 -3 M) had no effect onJ~o. DCCD and oligomycin, which are known to interact with the F o proton channel of F~-F o H+-ATPase also appear to act directly on the apical water channel in glutaraldehyde-fixed bladders. The sulfhydryl group reagent n-ethylmaleimide (10 -4 M) which interacts with the F~-Fo-type H+-ATPase in renal epithelia as well as vacuolar H+-ATPase produced an 85% inibition of JH~o. Oxytocin failed to activate a net water flux when Zn 2÷ (100 ~M) or Hg z+ (100 ~M) were present in the serosal solution, ...

The relationship linking Na+ and H+ transports and exocytosis/endocytosis located in the apical membranes of the frog skin epithelium was investigated under various conditions of ion transport stimulation. The exocytosis process, indicating insertion of intracellular vesicles, which were preloaded with fluorescent FITC-dextran (FD), was measured by following the FD efflux in the apical bathing solution. Na+ transport stimulators such as serosal hypotonic shock (replacement of serosal Ringer solution by half-Ringer or 4/5-Ringer), apical PCMPS (10(-3) M) and amphotericin-B (20 micrograms/ml), were also found to stimulate the exocytotic rates of FD. Acidification of the epithelium by CO2 or post NH4 load, conditions which increase the proton secretion also stimulated the FD release in the apical bathing solution. On the other hand, alkalization of the epithelial cells increased the endocytosis rate. Hypotonic shock, acid load and PCMPS induced an increase in cell calcium which is probably the signal within the cell for exocytosis. In addition, quantitative spectrofluorimetric measurements of F-actin content after rhodamine-phalloidin staining, indicated a decrease in the F-actin content as a result of cell acidosis, hypotonic conditions and amphotericin additions. It is proposed that the insertion/retrieval of intracytoplasmic vesicles containing H+ pumps plays a key role in the regulation of proton secretion in tight epithelia. In addition, it is suggested that cytoskeleton depolymerization of F-actin filaments facilitates H+ pump insertion. A comparable working hypothesis for the control of Na+ transport is proposed.

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