Glycoconjugate localization in larval and adult skin of the bullfrog, Rana catesbeiana: a lectin histochemical study

This study investigates whether or not the distribution of specific glycoconjugates within the skin is related to the regulation of water balance in the aquatic larvae and semiaquatic adults of the bullfrog, Rana catesbeiana. A lectin histochemical study was carried out on paraffin sections of dorsal and ventral skin from tadpoles in representative stages as well as from adult frogs. Sections were stained with the following horseradish peroxidase (HRP)-conjugated lectins, which bind to specific terminal sugar residues of glycoconjugates: UEA 1 for alpha-L-fucose, SBA for N-acetyl-D-galactosamine, WGA for N-acetyl-B-D-glucosamine, and PNA for beta-galactose. Results indicate that lectins serve as markers for specific skin components (e.g., a second ground substance layer within the dermis was revealed by positive UEA 1 staining). Moreover, each lectin has a specific binding pattern that is similar in dorsal and ventral skin; the larval patterns change as the skin undergoes extensive histological and physiological remodeling during metamorphic climax. These findings enhance our understanding of glycoconjugates and their relationship to skin structure and function-in particular, to the regulation of water balance in R. catesbeiana.     

Lymphomyeloid organs of amphibia. II. Vasculature in larval and adult Rana catesbeiana

There were no lymphatic vessels and lymph nodes demonstrable in adult and larval Rana catesbeiana by a method that adequately demonstrated the same in mammals. Although the parenchymal arrangement in the lymphomyeloid organs is not exactly the same as in mammalian hemal nodes, nonetheless the vascular patterns of the lymph glands and jugular bodies are prima facie evidence that they function as blood-filtering organs among other probable functions. The vascular pattern of the lymph gland is that of a rete mirabile, particularly a venous portal system, inasmuch as the afferent and efferent vessels are venous in character and interposed between them is a labyrinth of sinusoids. This is not the case, though, in the adult organs. The vascular pattern of the jugular bodies is very much like the spleen, viz., artery-capillary-sinusoid-vein sequence. It is doubtful, however, if the propericardial and procoracoid bodies ever filter blood, because the smallest blood vessels in them are capillary in type Because of the absence of a well-defined capsule in some parts of the propericardial body, similarly to lymphoid follicles, especially in the mammalian gastrointestinal tract, it is probable that it filters tissue fluid. The last two organs are apparently mainly blood cell-forming organs. It is inferred from the vascular connections of the larval and the adult lymphomyeloid organs that they are not genetically related. This aspect was analyzed from earlier developmental data, but actual follow-up of the larval organs to the adult stage is still in progress.     

Lympho-myeloid organs of Amphibia. I. Appearance during larval and adult stages of Rana catesbeiana

The bullfrog, Rana catesbeiana, possesses lympho-myeloid and epithelial structures that are morphologically similar in some respects to lymph nodes of mammals. These organs are present during the entire life cycle of the frog, however, the structures that are present during larval stages do not appear to be morphological precursors of adult organs. According to certain terms used previously by other investigators, two major organs are present throughout the larval stages: the lymph gland and the ventral cavity body. In the adult, the jugular body, the epithelial body, the precoracoid and propericardial bodies are found in the ventral neck region in contrast to the lateral and ventral arrangement of the lymph gland and ventral cavity body in larvae. The function of these organs is not known but it is believed that they play a role in the production of certain blood cells, particularly lymphocytes, and they may be involved in some aspects of the differentiation and maintenance of the immune response capacity.     

Ion transport across the skin of a larval salamander

The transport characteristics of the skin of neotenic Ambystoma tigrinum were investigated using ion substitution and circuit analysis. When bathed with sodium Ringer solution on both sides, a transepithelial potential of up to 50 mV (inside positive) and a short-circuit current (I_sc) of up to 10 μA/cm² were observed. When amiloride was added or Na⁺ was replaced by tetramethylammonium in the apical solution, I_sc was decreased from 3.7 ± 0.4 to 1.5 ± 0.2 μA/cm² (n = 10). When K⁺ replaced Na⁺, there was a smaller change in I_sc from 5.8 ± 0.6 to 3.7 ± 0.5 μA/cm² (n = 10). Although barium had no effect when added to 100 K Ringer on normal skin, it inhibited I_sc on skins taken from K⁺-loaded animals. Nystatin caused substantial increases in I_sc with either Na⁺ or K⁺ as the dominant cation in the apical solution. Current voltage analysis using amiloride was used to estimate the resistances and electromotive forces (EMF) associated with ion transport. The EMF for ion transport was partially dependent on K⁺ in the basolateral solution and it was similar to that observed in other epithelia. The resistance of the transport pathway was high, consistent with the low I_sc. These results suggest that there is an amiloride-sensitive Na⁺ channel in parallel with a small K⁺ conductance in the apical membrane of this preparation.     

Apoptosis in larval and frog skin of Rana pipiens,R. catesbeiana,and Ceratophrys ornata

Apoptosis (programmed cell death) occurs during normal development of anurans in organs such as gills, gut, and tail. For example, apoptotic cells have been reported in the luminal epithelium along the length of the digestive tract of both larvae and frogs; however, timing of the peak number of such cells varies in different species. The purpose of the present study was to ascertain whether apoptosis also varies by species during metamorphic restructuring of the skin (as larval epithelium is replaced by adult epidermis). To determine this, cross‐sections of dorsal skin from representative larval stages and frogs of Rana pipiens, R. catesbeiana, and Ceratophrys ornata were incubated with monoclonal antibody against active caspase‐3, one of the main enzymes in the apoptotic cascade. We observed apoptotic cells in the epidermis of the skin of the three species and found that such cells were more numerous in larval stages than in frogs and more abundant in the two ranid species than in C. ornata. These results contribute to our understanding of metamorphic changes in anuran skin. J. Morphol. 275:51–56, 2014. © 2013 Wiley Periodicals, Inc.     

A fluctuation analysis study of the development of amiloride-sensitive Na+ transport in the skin of larval bullfrogs (Rana catesbeiana)

In the presence of the Na+ -channel blocker amiloride, the short-circuit current across the skins of bullfrog tadpoles in metamorphic stages XIX-XXIV was subjected to fluctuation analysis. The resulting power spectra contained a Lorentzian component of which the plateau value (S0) decreased while the corner frequency (fc) increased as the mucosal amiloride concentration was increased from 0.5 to 24 microM. From the linear relationship between the fc values and the amiloride concentrations it was possible to determine the binding (k'01) and unbinding (k10) constants for amiloride to its receptor on the Na+ channel. With these parameters as well as short-circuit current and S0 values, the current through the individual Na+ channels (i) was calculated (average 0.58 pA). It did not increase significantly during late metamorphosis. The density of Na+ channels (M) in the apical membrane, on the other hand, increased significantly. It would appear that the increase in short-circuit current which occurs at this time is due primarily to an increase in amiloride-blockable Na+ channels. Unexpectedly, a Lorentzian component could be fitted to power spectra in amiloride-treated skins (stages XIX-XXI) which showed no amiloride-sensitive short-circuit current. Moreover, the typical increase in fc with the amiloride concentration did not occur in these animals.     

Modification of silver-enhanced sodium transport across toad skin

1. Silver stimulated short-circuit current and transepithelial potential difference. 2. Cysteine inhibited the silver-induced short-circuit current. 3. There was a dose-response inhibition of silver-induced short-circuit current by cysteine. 4. The silver-induced short-circuit current is carried by a net active sodium transfer from the outside to the inside bathing solution.     

Effect of 5-hydroxytryptamine on sodium transport across bullfrog skin

Summary 5-hydroxytryptamine, when present in the solution bathing the inside surface of bullfrog skin at concentrations of 0.25–25.0 mM, reduced both electrical potential difference and short-circuit current across the skin. The magnitude of reduction in potential difference and short-circuit current was dependent on 5 HT concentration. Reduction in sodium influx entirely accounted for the reduction in short-circuit current. Preliminary evidence suggested a competition between 5 HT and vasopressin in the production of their effects on sodium transport across the skin, while high Ca⁺⁺ concentrations and 5 HT seemed to act independently of each other.Dr. Henry C. and Bertha H. Buswell Fellow.     

Active transport of chloride across skin isolated from Rana esculenta]

The effect of SITS, ouabain and acetazolamide on potential difference (E) and short-circuit current (ccc) across isolated frog skin after Amiloride treatment have been investigated. It has been found that Amiloride (3.5 . 10(-5)M) reverses the E and ccc values. After Amiloride treatment, with the use of SITS (6 . 10(-4)M) positive values of E and ccc have been measured while Ouabain (10(-4)M) only slightly reduces the reversed E and ccc values. On the other hand the effect of Acetazolamide (6 . 10(-4)M) is additional to the effect of SITS. It is suggested that the frog skin possesses an active transport for Cl-, as demonstrated by the use of Amiloride, and that it is carried out by two distinct mechanism: the first SITS and Acetazolamide sensible, the second one sensible to Strofantine.     

Effect of aminoglycoside antibiotics on sodium transport across the Rana esculenta urinary bladder

The aminoglycoside antibiotics inhibit the short circuit current (s.c.c.) in the urinary bladder of Rana esculenta. This effect is due to a direct inhibition on the sodium pump; ADH or db-AMPc are not able to reverse the inhibition of the sodium current, but are effective in stimulating the urea fluxes in the presence of antibiotics. In correlation with the number of the NH+2 groups in the antibiotic molecule there is a different pattern of inhibitory effect, i.e. neomycin greater than gentamycin greater than dihydrostreptomycin greater than kanamycin greater than spectinomycin.     

Interactive effects of ethanol and silver on sodium transport across toad skin

Both ethanol and silver ions have been shown to affect ion transport across various epithelia. This investigation was principally undertaken to further define mechanisms of silver ions and ethanol, and their possible interactions, on sodium transport across toad skin. Isolated toad skin, mounted between identical oxygenated amphibian bicarbonate Ringer solutions, maintained stable transepithelial potential differences (serosa positive) and short-circuit currents for several hours at 25 degrees C. It was observed that (1) ethanol inhibited the active transcellular component of sodium absorption and this effect was reversible; (2) inhibition of sodium transport by ethanol was directly proportional to the applied concentration; (3) pretreatment with silver ions prevented any ethanol effects; and (4) pretreatment with ethanol prevented any silver ion effects. It was concluded from these results that ethanol induced its inhibitory effects on membrane phospholipids thereby perturbing the function of a sulfhydryl ligand, while silver ion or silver chloride complex binding to this ligand would maintain its function in sodium transport despite the presence of ethanol.