Chloride conductance in amphibian skin: regulatory control in the skin of Rana pipiens.

Chloride conductance across the isolated skin of Rana pipiens shows a voltage-activated component (G(Cl)(V)) which requires the presence of mucosal Cl. G(Cl)(V) is normally low or dormant. It is stimulated by elevated intracellular cAMP, irrespective whether originating from application of ss-adrenergic agonists (isoproterenol), stimulators of the adenylyl-cyclase (forskolin), inhibitors of the phosphodiesterases (isobutyl-methyl-xanthine) or membrane-permeable cAMP analogues (CPT-cAMP). Baseline G(Cl) under inactivating conditions increases also with cAMP dose-dependently. The data indicate that cAMP is a central regulator of the passive, conductive chloride transport across amphibian skin.

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.

Differential processing of provasotocin: relative increase of hydrin 2 (vasotocinyl-Gly) in amphibians able to adapt to an arid environment.

Hydrin 2 (vasotocinyl-Gly) is an intermediate in pro-vasotocin processing found, along with vasotocin, only in the neurohypophysis of anuran amphibians. It increases cutaneous water permeability in the frog and is likely involved in neuroendocrine control of osmoregulation. The relative amounts of vasotocin and hydrin 2 stored in neurohypophysis have been measured on the one hand in amphibian species known not to adapt in dry areas, on the other hand in two species, Bufo regularis (Africa) and Bufo viridis (Near-East) able to survive in an arid environment. In the first group, the proportions of the two peptides are approximately equal whereas in the two toads the molar ratio hydrin 2 to vasotocin reaches 2. The ratio does not appear to vary significantly when these toads are either submitted to dehydration or placed in saline solutions. Predominance of hydrin 2 suggests an adaptive decrease of the activity of the alpha-amidating enzymatic system involved in the conversion of vasotocinyl-Gly into mature amidated vasotocin.

Digitalis-like compounds in the toad Bufo viridis: tissue and plasma levels and significance in osmotic stress.

Digitalis-like compounds (DLC), constituents of animal tissues, are possible regulators of the Na+, K(+)-ATPase implicated in water and salt homeostasis. The distribution of DLC in the toad (Bufo viridis) was determined following methanol extraction and partial purification. DLC highest levels were found in the skin but it was also detected in the plasma and many internal organs. Short term (hours) exposure of the toad to hypertonic shock (1.5% NaCl) induced an increase in plasma osmolarity due to an increase in Na+ and Cl- levels. This treatment induced a transient, three fold, increase of DLC levels in the brain and transient reduction of its levels in the ventral skin. Acclimation of the toads to burrowing conditions for six weeks resulted in an increase in plasma osmolarity due to a large increase in plasma urea with a small increase in ion concentrations. Under these conditions DLC levels in the dorsal skin increased by 100% without alteration of its levels in the plasma, brain and ventral skin. DLC levels in the toad brain of control animals, showed a significant dependence on season, being highest in the summer and lowest in the winter. DLC levels in the skin peaked in May while the levels in the plasma were season independent. The changes in DLC levels induced by the short- as well as long-term perturbations in the animal environmental salinity together with the seasonal differences suggest that DLC in the toad is involved in water and salt homeostasis of these animals, but may also participate in other unknown functions.

The ecological significance of burrowing behaviour in the toad (Bufo viridis).

The burrowing behaviour of the toad Bufo viridis was studied in the laboratory on soil containing 11-13% moisture. These toads usually excavate shallow, temporary burrows from which they emerge intermittently. Time spent subsurface increases with rising ambient temperature in the range of 18-30° C. In these conditions, water loss is effectively reduced and a new water balance is achieved. On return to water access, after two months on soil, the toads rapidly regain only that amount of water lost previously. It is concluded that this temperature-sensitive burrowing behaviour is an important factor in these animals' successful survival in semi-arid conditions.

Lectin binding patterns in amphibian skin epithelium.

Seven lectins (PNA, DBA, WGA, UEA-I, RCA, SBA, Con A) were used to localize glycoconjugates in the skin of 10 species of Amphibia, 7 anurans (Bufo marinus, Bufo bufo, Rana ridibunda, Rana pipiens, Hyla arborea, Pelobates syriacus and Xenopus laevis) and 3 urodeles (Salamandra salamandra, Triturus vulgaris and Ambystoma mexicanum). It was found that every lectin has a specific binding pattern in the skin of each species. No common pattern could be established, either among frogs or toads, nor for a particular lectin. Each lectin bound specifically and selectively to a particular epithelial component, which differed from one species to the other. A number of lectins showed selective binding to mitochondria-rich cells, but, again, a pattern in positivity could not be found. It is concluded that lectin histochemistry does correlate with cellular function. Our data can be applied in studies of epithelium and skin development, and of changes that occur during adaptation to the environment by amphibian species.

Renal response of euryhaline toad (Bufo viridis) to acute immersion in tap water, NaCl, or urea solutions.

Green toads (Bufo viridis) were acclimated to either tap water, 230 mOsmol NaCl kg-1 H2O (saline), 500 mOsmol NaCl kg-1 H2O (high saline), or 500 mmol L-1 urea. Renal functions for each acclimation group were studied on conscious animals that had one ureter chronically catheterized. Reciprocal immersion of tap-water- and saline-acclimated toads in the opposite solution did not stress the animals osmotically, and plasma osmolality increased or decreased by no more than 15%. However, urine osmolality and ionic composition changed immediately and profoundly on exposure to the other solution. Exposure of tap-water-acclimated toads to saline decreased urine flow by 30%, whereas the reciprocal immersion led to an increase of 30%. Immersion of tap-water-acclimated toads in high saline led to immediate cessation of urine flow, whereas immersion of 500 NaCl- or urea-acclimated toads in tap water led to a large increase in urine flow, with an overshoot that lasted 10 h (as a result of either salt or urea diuresis). Urine flow then stabilized at a level 5-6 times higher than the value attained at high-salt environment. On immersion of 500 urea-acclimated toads in 500 NaCl, urine flow doubled, accompanied by a change in ion composition, without change in the osmolality. In all experimental conditions, plasma potassium concentration was maintained within a narrow range. The results show that the toad's kidneys contributed efficiently both to osmo- and ionoregulation in a wide range of ambient solutions.

Specific self-antigen-driven immune response in pericardial effusion as an isolated GVHD manifestation.

Pericardial effusion and cardiac tamponade have been described as GVHD manifestations in the post transplant period. Direct evidence of GVHD-related TCR or B-cell receptor clones in patients with pericardial effusion has never been described. Using several methods, including FACS and spectratyping analysis to assess T- and B-cell clonality and to quantify TCR excision circles to assess newly thymus-derived T cells, we were able to show expansion of oligoclonal T-cell populations and the possible presence of early/premature B cells in the pericardial effusion but not in peripheral mononuclear cells. This may explain the presentation of an isolated GVHD manifestation.

Review: intravenous immunoglobulin therapy and thromboembolic complications.

Intravenous immunglobulin (IVIg) is used to treat a number of immune-deficiences and autoimmune diseases. Safety concerns related to a number of reported thromboembolic complications prompted us to review the literature. These complications happened mainly in individuals that had risk factors for thromboembolism, like advanced age, previous thromboembolic diseases, bed-ridden, and in individuals in which high doses or high infusion rates of IVIg were administered. The mechanism responsible for these events seems to be a rise in plasma viscosity that can trigger a thromboembolic event, especially in cases in which there is an underlying circulation impairment. Complications can be minimized by using IVIg only in clear-cut indications, weighting risk versus benefit in patients who are at high risk for thromboembolism and by sticking to carefully monitored slow infusion rates. IVIg for the treatment of autoimmune disorders should be administered as a five-day course of 2 g/kg of body weight. Each daily dose of 400 mg/kg should be given in not less than eight hours.

The effect of salt acclimation of the water uptake and osmotic permeability of the skin of the toad (Bufo viridis, L.).

1. Water uptake in vivo, and water fluxes across the isolated skin were studied in salt (NaCl) acclimated toads. 2. Water uptake of acclimated toads maintained in the solution of acclimation, decreased with the environmental salinity. 3. The osmotic water permeability (Pos) of the skin increased upon salt (NaCl) acclimation, both in vivo and in vitro. 4. Pos of the skin of toads acclimated to non-permeant solutes such as sucrose (230 mmol/l) or mannitol (400 nmol/l), was greatly reduced. 5. Oxytocin (syntocinon) increased the Pos both in tap water and salt acclimated toads. In high salt (greater than 200 mmol/l NaCl) acclimated toads however, the increased Pos and water flux at larger osmotic gradients, could not be stimulated further by the hormone. 6. The adaptive nature of the selective changes in the permeability properties of the skin under salt acclimation conditions is discussed.

Changes in ionic conductances and in sensitivity to amiloride during the natural moulting cycle of toad skin (Bufo viridis, L.).

The resistance of the apical membranes of toad skin (Bufo viridis) was measured during its natural moulting cycle using a fast flow technique. The skin behaved in all periods of the moulting cycle as a nearly perfect sodium electrode. In the presence of amiloride (10(-4)M), the total resistance of the same skin was identical with solutions which contained either sodium or potassium. The resistance of the skin with potassium was sensitive to amiloride in the period just after moulting. The resistance of skins which were made shunted by treating them with urea on the outside area was insensitive to amiloride in solutions containing potassium; a small effect was still observed with sodium. It is suggested that the transient sensitivity to amiloride, with potassium, is the result of differentiation of the sodium specific sites at the apical membranes of the skin.