Uri Katz

The voltage-dependent chloride current conductance of toad skin is localized to mitochondria-rich cells

The chloride current across the isolated epithelium from saline-acclimated Bufo viridis toads was studied using the extracellular vibrating probe technique. Local peak current densities varying between 5 and 100 microA/cm2 were recorded over subpopulation of mitochondria-rich cells, but never over granulosum cells. These local transepithelial currents had characteristics similar to the activated chloride current observed in the whole skin (Katz, U. and Larsen, E.H. (1984) J. Exp. Biol. 109, 353-371). Replacement of the apical Ringer with chloride-free (nitrate) ringer resulted in reversible reduction in the current at the mitochondria-rich cells. It is concluded that the mitochondria-rich cells are the principal site of passive chloride conductance across the epithelium.

Effect of salt acclimation on digitalis-like compounds in the toad

Digitalis-like compounds (DLC) were shown to be a normal constituent of the skin and plasma of toads. In order to assess the possible physiological role of these compounds in the toad, their levels were determined in the brain, plasma and skin following acclimation in different NaCl solutions. We demonstrate that an increase in salt concentrations in the animal medium from 0 to 1.2% decreased the levels of DLC in the brain by 50% without altering significantly its levels in the plasma and skin. An increase in medium salt concentration to 1.5% resulted in a 50% increase of DLC levels in the skin without changing its levels in the plasma or brain. These results suggest that skin and brain DLC may participate in the long-term salt and water homeostasis in the toad, while the plasma compound either participates in the short-term regulations of salt and water homeostasis or have some other, unknown, function.

Water retention and plasma and urine composition in toads (Bufo viridis Laur.) under burrowing conditions

SummaryOsmoregulation in the terrestrial toad,Bufo viridis, was studied under burrowing conditions in the laboratory. The toads can live for over 3 months burrowed in soil containing 9–10% moisture, maintaining constant body volume due to a large increase in the plasma osmolality, contributed mainly by urea. Water content of the tissues remains constant. Relatively large volumes of urine are stored in the urinary bladder during water restriction. The osmolality of the urine does not exceed that of the plasma. Urea uptake across the skin was measured in vitro and was greatly elevated in skins from the burrowed toads. The increase in plasma osmolality enables greater water absorption from the soil under water restricted conditions while the water content of the tissues is maintained constant since cell membranes are highly permeable to urea. It is concluded that the urea accumulating ability and urea tolerance form the basis for both the terrestriality and salt adaptability of this and other amphibian species.

The Role of Amphibian Epidermis in Osmoregulation and Its Adaptive Response to Changing Environment

The epidermis forms the major barrier separating the external surface of animals from their surroundings. However, while its major role in most vertebrates is to provide protection and insulation, it carries out a unique function in Amphibia in the regulation of the water content and osmotic pressure of the body fluids. Overton (1904) in his Thirty nine Theses on the Water Economy of Amphibia observed that when a dehydrated frog is returned to water it will absorb water through the skin until the original water content has been restored, when the uptake slows down and urine output ensues. Amphibia, which were the first class among vertebrates to invade land, are dependent on the availability of free water to a greater or lesser extent, particularly for reproduction. They do not usually drink, but, rather they absorb the water through their skin, which is permeable to salt and also participates in respiration (Krogh 1939; Randall et al. 1981).

Mitochondria-rich cells in anuran Amphibia: chloride conductance and regional distribution over the body surface

The distribution and density (D(mrc)) of mitochondria-rich cells (MR cells) in skin epithelium, were determined over the whole body surface in nine species of anuran Amphibia that live in a variety of habitats. It was found that the more terrestrial species (beginning with Hyla arborea) have a higher density of MR cells in their pelvic region. In the skin of aquatic (Xenopus laevis) or fossorial (Pelobates syriacus) species, D(mrc) is evenly distributed over the whole body surface. In dorsal skin pieces of H. arborea that lack detectable MR cells, transepithelial voltage activation did not induce Cl(-) conductance as it did in ventral pieces. Skins from Bufo viridis and X. laevis, both have MR cells in their skin, differ markedly in their biophysical properties: a Cl(-) specific current conductance is predominant in the skin epithelium of B. viridis, and is absent in X. laevis. In the latter, anionic conductance is due to glandular secretion. The biophysical properties cannot therefore be related solely to the presence or density of MR cells. Mitochondria-rich cells are sites of Cl(-) conductance across the skin of those amphibians that show this property, but must have different function(s) in other species. It is suggested that the specific zonal distribution of MR cells in the species that were examined in this study could be due to ion exchange activity and water conservation in more terrestrial environments.

Localization of a Band 3-related protein in the mitochondria-rich cells of amphibian skin epithelium

Based on immunoblotting procedure, the isolated epithelium of amphibian skin was found to contain a 180 kDa protein which cross‐reacts with a polyclonal antiserum raised against human erythrocyte Band 3. Immunoperoxidase and immunofluorescence staining techniques indicated that the Band 3‐related protein was localized in the mitochondria‐rich cells (MRC) of this epithelium, with characteristic apical labelling pattern. Our findings show that the putative apical anion exchanger of the MRC is immunologically related to the band 3 multigenic family, which catalyzes Cl‐HCO3 − transmembranous exchange. It thus suggests a molecular basis for the role played by these cells in the transepithelial Cl pathway and acid‐base regulation.

The route of passive chloride movement across amphibian skin: localization and regulatory mechanisms.

Transepithelial Cl(-) conductance (G(Cl)) in amphibian skin can be activated in several species by serosa positive potentials. Mitochondria-rich cells (MRC) or tight junctions (TJ) between the epithelial cells are possible sites for this pathway. The properties and the techniques used to investigate this pathway are reviewed in the present paper. In situ techniques are preferable, since specific properties of the MRC are apparently not maintained in isolated cells. Volume measurements and electronprobe microanalysis of intracellular ions suggest the localization of voltage-activated G(Cl) to MRC. G(Cl) correlates poorly with the density of MRC. The vibrating voltage probe allows quantitative correlation of the local Cl(-) current through morphologically identified structures and the transepithelial Cl(-) current. Our analysis shows that 80% of the voltage-activated Cl(-) current is accounted for by current through MRC or their immediate vicinity. The activation patterns of this current and the inhibition by the alpha(1)-adrenergic agonist, epinephrine, conform to those of the transepithelial current. However, less than 20% of the MRC are active at a certain moment and the activity is spontaneously variable with time. The molecular nature of this pathway, physiological control mechanisms and their relation to the temporal activity of MRC remain to be studied.

Mechanisms of hyperosmotic acclimation in Xenopus laevis (salt, urea or mannitol)

The acclimation of the clawed toad Xenopus laevis to hyperosmotic solutions of NaCl (balanced solution of sea salt), urea or mannitol was studied. The animals could not be acclimated to salt solutions more concentrated centrated than 400 mosm·l-1. Urea was tolerated till 500 mmol·l-1. Plasma osmolality was always hyperosmotic to the environmental solution, but with diminished osmotic gradient at the highest tolerated solutions. Plasma urea concentration approached 90 mmol·l-1, similar in the three solutions of acclimation. Urine volume was very small under all conditions. Serum aldosterone and corticosterone did not differ significantly, although there was a slight tendency towards lower aldosterone in the NaCl solution. In vivo water uptake in tap water acclimated animals was very small, and was higher in the other groups. Only the salt- and urea-acclimated, but not the tap water and mannitol-acclimated groups responded with a clear increase following injection of oxytocin or theophylline. In vitro urea fluxes were similar and invariable in both directions under all conditions. No significant effect of theophylline was observed. Sodium transport measured by the short-circuit technique in vitro was lower in salt- and mannitol-acclimation conditions, and was stimulated significantly under all conditions in response to serosal oxytocin or theopylline. It is concluded that Xenopus laevis can osmoregulate at a limited range of external solutions. It is limited in the increase of its plasma urea concentration; the transport properties of the skin do not change very much upon acclimation, except for the hydroosmotic response to oxytocin.

Tissue osmolytes and body fluid compartments in the toad Bufo viridis under simulated terrestrial conditions

SummaryToads (Bufo viridis) were kept on soil without access to free water (simulated terrestrial conditions) for over 12 weeks. Body water compartments were estimated using the dilution method (inulin and Evans Blue). They were found to remain fairly constant after a period of adjustment which lasted 1–2 weeks. In particular, plasma volume was closely controlled. Plasma osmolarity increased to over 1000 mOsm · 1−1 accompanying a large increase in its urea concentration. NaCl also increased, while potassium remained constant. Tissue (liver and skeletal muscle) water content did not change much and electrolytes were kept constant. Tissue water urea concentration seemed to equilibrate with that of the plasma. Urine osmolarity, which was hypotonic during water access, became nearly isosmotic and correlated with the plasma following transfer onto soil. Urine urea concentration correlated with the plasma in the terrestrial conditions, potassium was greatly elevated, sodium increased to some extent, and chloride hardly changed. The efficient osmoregulatory mechanisms for the control of distribution of body water sustain normal physiological functions.

α1-Adrenoceptors antagonize activated chloride conductance of amphibian skin epithelium

Abstract The effects of adrenoceptor agonists on the transepithelial Cl– conductance (GCl) in the skin of several amphibian species, both toads and frogs, were studied. Epinephrine (Epi) from the serosal side selectively and reversibly inhibited the voltage-activated GCl in toad skin and the short-circuit GCl in frog skin. The main effects of activation of the adrenoceptors must reside in the skin epithelium and not in the glands, since measurements were made both from intact skins and split epithelia with essentially the same results. Effective concentrations of Epi were variable among individual tissues. GCl was reduced to 34±17% (n=46) with 1 µmol/l Epi, but in some tissues 0.1 µmol/l inhibited more than 80% of GCl, whereas some preparations were little influenced at >3 µmol/l Epi. The affected receptor type was identified by the use of the α1-agonist phenylephrine, which mimicked the response of Epi at concentrations above 30 µmol/l, whereas the α2-agonists xylazine and iodoclonidine had no effect at supramaximal concentrations. Prazosin, a specific α1-antagonist, reduced or eliminated the inhibition by Epi, but the response pattern suggests a low affinity. The α2-antagonist yohimbine, at concentrations ≤0.3 µmol/l, had a minimal effect, but reduced the inhibition by Epi at concentrations of 1–10 µmol/l. This might indicate affinity to α1-adrenoceptors in amphibian skin. Activation of β-adrenoceptors by isoproterenol (0.1–5 µmol/l) led to a transient increase of the baseline inactivated component of GCl with a slight reduction of the voltage-activated GCl at the higher concentrations, but the inhibitory effect of Epi was not altered. Epi, on the other hand, neither prevented nor reversed the induction of a voltage-insensitive GCl in toad skin caused by application of cAMP at supramaximal concentrations (>100 µmol/l CPT-cAMP). Preincubation of the serosal medium with Ca2+-free solution (in the presence of 2 mmol/l EGTA) for extended periods of time (>30 min) eliminated the response to Epi. It is concluded that α1-adrenoceptors participate in the physiological control of voltage-activated Cl– conductance in amphibian skin epithelium via modulation of intracellular Ca2+, presumably by efflux from intracellular stores.