Bullfrog Skin Research Articles

Larval bullfrog skin lacks amiloride-blockable epithelial transport because α-ENaC is located within intracellular vesicles in epidermal apical cells and not in the apical plasma membrane

The epithelial Na channel (ENaC) plays an essential role in sodium transport across epithelia such as adult frog skin. Transport across the skin, measured as short-circuit current (SCC), is blocked by amiloride. Bullfrog alpha-ENaC (α-fENaC) is expressed in adult bullfrog skin, and the SCC across this skin is blocked by amiloride. In contrast, an amiloride-blockable SCC is not detected in larval bullfrog skin, even though it expresses α-fENaC. We examined the subcellular localization of α-ENaC in such larval and adult skins. Immunofluorescent and immunoelectron microscopy of apical cells in the larval epidermis revealed α-fENaC localization within intracellular vesicles, but not in the plasma membrane. In contrast, in adult skin α-fENaC was localized to the apical-side membrane and to intracellular vesicles in Stratum granulosum cells. This may support the view that amiloride-blockable SCC is absent from larval skin, but is present in adult skin.

Localization of ENaC subunit mRNAs in adult bullfrog skin

Adult amphibian skin has served as a model for the investigation of Na+-transporting epithelia, such as mammalian renal tubules. The amiloride-blockable epithelial Na+ channel (ENaC), which is located in the apical membrane of the outer living cell layer, regulates Na+ transport across the epithelium. ENaC is thought to develop during the terminal differentiation of epidermal cells, but the details are unclear. Here, we used in situ hybridization to examine the localization of the ENaC subunit mRNAs in skin of adult bullfrogs, to clarify the development of ENaC. We found that α-ENaC mRNA was expressed within the cells of the Stratum granulosum, the Stratum spinosum, and the Stratum germinativum, while β-ENaC mRNA was expressed within the cells of the S. granulosum and the S. spinosum. However, we could not detect expression of γ-ENaC mRNA, possibly for technical reasons. α- and β-ENaC mRNAs, at least, were present in the sub-apical cells, in which ENaC protein is not necessary for amphibian skin to possess its Na+-transport function. Our results may mean that the sub-apical cells are already producing the ENaC subunit mRNAs prior to the final step in their differentiation.

Expression of genes encoding antimicrobial peptides in the Harderian gland of the bullfrog Lithobates catesbeianus

The Harderian gland is an orbital gland found in many tetrapod species that possess a nictitating membrane. While the main role of the Harderian gland is lubrication of the eyeballs, numerous other functions are attributed to this gland. In amphibians, mast cells have been detected in the Harderian gland, suggesting that the gland is involved in the host's system of innate immunity defending against microbial invasions. Using reverse-transcription polymerase chain reaction, we cloned from the bullfrog Harderian gland total RNA preparations, cDNAs encoding biosynthetic precursors for the antimicrobial peptides temporin-CBa (FLPIASLLGKYL-NH 2), previously isolated from an extract of bullfrog skin, and chensirin-2CBa (IIPLPLGYFAKKP) that contained the amino acid substitution Thr 13 → Pro compared with chensirin-2 from the Chinese brown frog, Rana chensinensis. By means of in situ hybridization using digoxigenin-labeled cRNA probes for preprotemporin-CBa and preprochensirin-2CBa, we have demonstrated for the first time in an amphibian the presence of mRNAs encoding these two precursors in the cytoplasm of the glandular cells in the bullfrog Harderian gland.

Stretch-activated cation channel from larval bullfrog skin

Cell-attached patches from isolated epithelial cells from larval bullfrog skin revealed a cation channel that was activated by applying suction (-1 kPa to -4.5 kPa) to the pipette. Activation was characterized by an initial large current spike that rapidly attenuated to a stable value and showed a variable pattern of opening and closing with continuing suction. Current-voltage plots demonstrated linear or inward rectification and single channel conductances of 44-56 pS with NaCl or KCl Ringer's solution as the pipette solution, and a reversal potential (-V(p)) of 20-40 mV. The conductance was markedly reduced with N-methyl-D-glucamide (NMDG)-Cl Ringer's solution in the pipette. Neither amiloride nor ATP, which are known to stimulate an apical cation channel in Ussing chamber preparations of larval frog skin, produced channel activation nor did these compounds affect the response to suction. Stretch activation was not affected by varying the pipette concentrations of Ca(2+) between 0 mmol l(-1) and 4 mmol l(-1) or by varying pH between 6.8 and 8.0. However, conductance was reduced with 4 mmol l(-1) Ca(2+). Western blot analysis of membrane homogenates from larval bullfrog and larval toad skin identified proteins that were immunoreactive with mammalian TRPC1 and TRPC5 (TRPC, canonical transient receptor potential channel) antibodies while homogenates of skin from newly metamorphosed bullfrogs were positive for TRPC1 and TRPC3/6/7 antibodies. The electrophysiological response of larval bullfrog skin resembles that of a stretch-activated cation channel characterized in Xenopus oocytes and proposed to be TRPC1. These results indicate this channel persists in all life stages of anurans and that TRP isoforms may be important for sensory functions of their skin.

P.251 Assessment of facial asymmetry

Ultra-high pressure technology has attracted a great deal of attention in recent years, and has been widely used in food science, medicine, and other fields. This study aimed to determine the effect of ultra-high pressure on the structure and properties of collagen. Native collagen extracted from bullfrog skin was processed under different ultra-high pressure treatment conditions (300, 400, and 500 MPa). Then systematic analysis of the molecular structures and properties of the samples after ultra-high pressure treatment were performed. It was found that the conformation of collagen molecules could be adjusted by ultra-high pressure treatment, and this regulation was closely related to the level of treatment pressure. A possible mechanism of the impact of ultra-high pressure on the collagen molecular structures was speculated according to the experimental results. At low pressure levels (300–400 MPa), the pressure perpendicular to collagen axis dominates and leads to a tightening of the triple helix structure of collagen, while the pressure parallel to collagen axis is dominant and the triple helix tends to dissociate like a zipper at high pressure levels (> 400 MPa). These structural changes would simultaneously result in various changes to thermal stability, self-assembly properties, and antigenicity of collagen.

Vehicle effects on in vitro transdermal absorption of sevoflurane in the bullfrog, Rana catesbeiana

The experimental objectives were to identify a vehicle which produces a homogenous formulation when combined with the anesthetic solution sevoflurane and understand the dermal absorption of sevoflurane in silastic membranes and amphibian skin in vitro utilizing a flow-through diffusion system. Seven vehicles were evaluated in varying ratios with 5 formulations resulting in the desired homogenous consistency for practical application. Sevoflurane diffusion across silastic membranes was influenced by pluronic/lecithin organogel (PLO), pluronic F 127 20% gel, and sterile lube. Flux and permeability across silastic membranes were significantly greater in sterile lube than in the other formulations. While no significant vehicle effects were observed in bullfrog skin, the flux-time profiles suggest that sevoflurane diffusion in bullfrog skin may be positively influenced by PLO. Future in vivo studies are required to assess sevoflurane retention after removal of these formulations to more accurately control the plane of anesthesia in amphibians.

Isolation and Characterization of Collagen from Skin of Bullfrog, Rana catesbeiana Shaw

In order to utilize skin of bullfrog (Rana catesbeiana Shaw) as an alternative source of collagen, we investigated and compared biochemical and physical properties of collagens isolated from bullfrog skin. Two kinds of collagen (BSASC; bullfrog skin acid-soluble collagen and BSPSC; bullfrog skin pepsin-solubilized collagen) were isolated by subsequent treatments with acetic acid and pepsin. The amounts of skin collagen isolated in the subsequent treatments were 7.3% BSASC and 18.2% BSPSC on the basis of lyophilized bullfrog skin weight, respectively. According to the electrophoretic pattern and CM-cellulose column chromatogram, the BSASC has the chain composition of <TEX>${\alpha}1{\alpha}2{\alpha}3$</TEX> heterotrimer, and the BSPSC consists of two <TEX>${\alpha}$</TEX> chains of <TEX>${\alpha}1{\alpha}2$</TEX>. In addition, the denaturation temperatures of all collagens tested were ranged from <TEX>$30^{\circ}C\;to\;38^{\circ}C$</TEX>. This study suggests that there is a possibility to use bullfrog skin collagen as an alternative source of collagen for industrial purposes, and subsequently it may increase the economical value of the bullfrog.

Free radical scavenging activity of a novel antioxidative peptide purified from hydrolysate of bullfrog skin, Rana catesbeiana Shaw

In the present study, a peptide having antioxidant properties was isolated from bullfrog skin protein, Rana catesbeiana Shaw. Bullfrog skin protein was hydrolyzed using alcalase, neutrase, pepsin, papain, α-chymotrypsin and trypsin. Antioxidant activities of respective hydrolysates were evaluated using lipid peroxidation inhibition assay and direct free radical scavenging activity by using electron spin resonance (ESR) spectrometer. Among hydrolysates, alcalase derived hydrolysate exhibited the highest antioxidant activities than those of other enzyme hydrolysates. In order to purity a peptide having potent antioxidant properties, alcalase hydrolysate was separated using consecutive chromatographic methods on a Hiprep 16/10 DEAE FF anion exchange column, Superdex Peptide 10/300 GL gel filtration column and highan octadecylsilane (ODS) C18 reversed phase column. Finally, a potent antioxidative peptide was isolated and its sequence was identified to be LEELEEELEGCE (1487 Da) by Q-TOF ESI mass spectroscopy. This antioxidant peptide from bullfrog skin protein (APBSP) inhibited lipid peroxidation higher than that of α-tocopherol as positive control and efficiently quenched different sources of free radicals: DPPH radical (IC 50 = 16.1 μM), hydroxyl radical (IC 50 = 12.8 μM), superoxide radical (IC 50 = 34.0 μM) and peroxyl radical (IC 50 = 32.6 μM). Moreover, MTT assay showed that this peptide does not exert any cytotoxicity on human embryonic lung fibroblasts cell line (MRC-5).

Prolactin increases Na + transport across adult bullfrog skin via stimulation of both ENaC and Na +/K +-pump

PRL is involved in osmoregulation in lower vertebrates. Its serum concentration starts to increase during the metamorphosis of bullfrog tadpoles. Adult bullfrog skin transports Na + from the apical to the basolateral side across the skin. PRL is involved in the regulation of this transport. We investigated the effect of ovine PRL on the epithelial Na + channel (ENaC), Na +/K +-pump, and basolateral K + channels, which regulate Na + transport across adult bullfrog skin, by measuring the short-circuit current (SCC). At 0.1 μg/ml, PRL had no effect on the SCC. PRL (1 μg/ml) was sufficient to stimulate the SCC since 1 and 10 μg/ml of PRL each increased SCC 1.8-fold. Current-fluctuation analysis revealed that PRL (10 μg/ml) increased the density of active ENaC almost 1.8-fold. The effect of PRL on the Na +/K +-pump was investigated using apically nystatin-permeabilized skin with Ca-free Na-Ringers’ solution on each side. PRL (10 μg/ml) increased SCC in this condition around 1.1-fold, suggesting that PRL stimulates the Na +/K +-pump [although PRL (1 μg/ml) had no effect on this SCC]. The effect of PRL on basolateral K + channels was investigated using apically nystatin-permeabilized skin with high-K Ringer’s solution on the apical side. PRL (10 μg/ml) had no effect on the SCC, suggesting that PRL does not affect basolateral K + channels. Thus, although PRL stimulates the Na +/K +-pump, this effect probably contributes less than that on ENaC to the regulation of Na + transport across adult bullfrog skin.

Larval bullfrog skin expresses ENaC despite having no amiloride-blockable transepithelial Na+ transport

Amiloride-blockable Na(+) transport, measured as an amiloride-blockable short-circuit current (Am-SCC), is mediated by the epithelial Na(+) channel (ENaC). Am-SCC is not normally present in bullfrog tadpole skin, but when such skin is cultured with corticoids an amiloride-blockable Na transport appears. Prolactin (PRL) inhibits its corticoid-induced development. Using specific PCR primers for adult frog ENaC and RT-PCR, we investigated whether corticoids can induce all three ENaC subunits, and whether this expression of ENaC subunit(s) can be blocked by adding PRL with the corticoids. We found that (1) the sequences of the RT-PCR products obtained using primers for alpha-ENaC were identical between larval and adult skins, (2) the mRNAs for all three ENaC subunits were expressed in larval skin under normal conditions despite no amiloride-blockable Na(+) transport being detectable, (3) all three subunits were expressed in larval skins whether they were cultured with corticoids (amiloride-blockable Na transport present) or with corticoids supplemented with PRL (no amiloride-blockable Na transport present). An antibody against a peptide from the alpha-ENaC of adult bullfrog was localized to the apical cells of both larval and adult skins. Since no amiloride-blockable Na transport exists across larval skin under these conditions, these results suggest that ENaC protein was expressed prior to the onset of transport. ENaC may be in the plasma membrane in an inactivated form or, alternatively, within vesicles waiting to be inserted.

Sustained release of BSA from a novel drug delivery matrix -- bullfrog skin collagen film.

A novel drug delivery system, bullfrog skin collagen film, was employed to release bovine serum albumin (BSA). The biophysical properties of bullfrog skin collagen film used were evaluated by thermal denaturation temperature and percentage water loss. The film obtained exhibited good stability and can be taken as a good candidate for drug delivery matrix. Compared with conventional collagen from molecular weight and amino acid composition, bullfrog skin collagen showed significant differences. For safety considerations, bullfrog skin collagen has great advantages. The release kinetics of BSA from bullfrog skin collagen film was measured in vitro. The results indicated bullfrog skin collagen film could be used for sustained release of BSA. Thus, bullfrog skin collagen film can be taken as a novel drug delivery system.

Studies on bullfrog skin collagen

Pepsin-soluble collagens were prepared from bullfrog skin and partially characterized. This study revealed interesting differences in the frog skin collagen when compared with known vertebrate collagens. This may be attributed to the position of the amphibians in the vertebrate hierarchy. Therefore, detailed investigations on various physicochemical properties, such as molecular weight, amino acid composition, denaturation temperature, UV–Vis and IR spectra of bullfrog skin collagen were carried out. The study confirms the structural relationship of collagen to habitat and function. Moreover bullfrog skin has potential as an alternative source of collagen to calf skin and bone for use in various fields.

Growth hormone is a weaker candidate than prolactin for the hormone responsible for the development of a larval-type feature in cultured bullfrog skin

Prolactin (PRL) has, for some years, been considered to be the 'juvenile hormone' in amphibians. Recently, growth hormone (GH) has been proposed as another candidate, because in the larval stages the expression of the mRNA GH is high but it is downregulated in the climax stages of metamorphosis or following treatment with thyroid hormone. In the present study, we investigated whether GH promotes the development of one particular larval-type feature of bullfrog tadpole skin in vitro. The amiloride-, acetylcholine- and ATP-stimulated short-circuit current (SCC) is a physiological marker of larval-type bullfrog skin. These types of ligand-stimulated SCC (1) developed when EDTA-treated tadpole skin was cultured with corticoids supplemented with PRL or GH and (2) were not significantly different between skin cultured with PRL and intact tadpole skin. However, the amiloride-induced SCC response in skin cultured with GH differed in its kinetics from that of the intact (control) tadpole. On this basis, PRL seems a better candidate than GH for the juvenile hormone, at least with regard to the development of amiloride-stimulated non-selective cation channels.

Basolateral Cl- channels in the larval bullfrog skin epithelium.

The addition of 150 U/ml nystatin to the mucosal surface of isolated skin from larval bullfrogs increases apical membrane permeability and allows a voltage clamp to be applied to the basolateral membrane. With identical Ringer's solutions bathing either side of the tissue the short-circuit current (I(SC)) averaged 7.60+/-0.78 micro A/cm2, and this current could be increased or decreased by imposing a Cl- concentration gradient. Fluctuation analysis of the I(SC) gave power spectra that could be fit with low- and high-frequency Lorentzian functions having corner frequencies of 1.48+/-0.06 Hz and 48.5+/-11.4 Hz, respectively. The Lorentzian plateau was minimal at the lowest I(SC) and increased as the I(SC) became greater in the positive or negative direction. Current-voltage plots with identical Ringer's on either side of the tissue showed a pattern of outward rectification. Cell attached patches of cells isolated from the skin with collagenase-trypsin treatment showed spontaneous channel activity with a conductance of 20.9 pS at a pipette potential, -Vp=20 mV. Current-voltage plots of single channels showed a similar pattern of rectification to that of the intact skin, and partial replacement of Cl- by gluconate in the pipette solution shifted the reversal potential from zero to about 40 mV, which is close to the expected shift of the reversal potential of the chloride current through a Cl- selective ion channel. These results suggest that the basolateral Cl- conductance of the larval skin is mediated by a channel with properties that resemble a volume-sensing outward-rectifier anion channel that has been described in a variety of cell types

Cloned bullfrog skin sodium (fENaC) and xENaC subunits hybridize to form functional sodium channels.

For many years the adult frog skin has been used as a model system to study transepithelial sodium transport. In other sodium transporting tissues the three homologous subunits of the sodium channel have been cloned. The aim of this study was to clone and characterize the amiloride inhibitable sodium channel (ENaC) in adult bullfrog (Rana catesbeiana) skin. Three transcripts corresponding to the alpha, beta, and gamma subunits of ENaC were cloned and sequenced. Co-expression of all three in Xenopus oocytes yielded a functional frog sodium channel (fENaC). Amiloride sensitivity and current voltage relationships suggested that its characteristics were similar to other ENaCs. Subunits from the Xenopus sodium channel (xENaC) and fENaC were combined in all possible triplets. Although functional amiloride inhibitable sodium channels were formed in every case, the amiloride sensitivities were not identical. Subunit combination studies suggested that the alpha subunit made a major contribution to amiloride sensitivity but interactions of beta and gamma were also seen. When the amiloride sensitivities of intact skin from adult R. catesbeiana and Xenopus laevis were compared, Rana also had a consistently higher affinity. Comparison of fENaC and xENaC sequences may provide insight into which amino acids beyond those already identified are critical for amiloride binding.

Cu2+ reveals amiloride-induced activation and blocking of non-selective cation channel in larval bullfrog skin.

The non-selective cation channel (NSCC) in the larval bullfrog skin contributes to the short-circuit current (SCC) across the skin. The effects of amiloride and acetylcholine on the SCC were examined in the presence or absence of Cu2+ to determine whether the amiloride binding site mediating activation or that mediating inhibition of the channel is blocked by Cu2+ and whether amiloride and acetylcholine share a common binding site on the NSCC. The skin of tadpoles raised in aldosterone was examined with K-Ringer present on the apical side to potentiate the SCC. Amiloride (10(-4) M) transiently increased SCC in the absence of Cu2+. Apical application of 500 microM Cu2+ increased the SCC. In the presence of Cu2+, amiloride decreased the SCC. In contrast, acetylcholine (1 mM) transiently increased SCC whether Cu2+ was present or not. These results suggest that there are two binding sites for amiloride on the NSCC, whereby the site that activates the channel is blocked by Cu2+ while the site that inhibits it is not, and that the binding sites for acetylcholine and amiloride may be different.

Possible role of aldosterone and T(3) in development of amiloride-blockable SCC across frog skin in vivo.

There are inconsistencies between the in vitro and in vivo effects of thyroid hormone and aldosterone (Aldo) on the development of an amiloride-blockable short-circuit current (SCC) across bullfrog skin [Takada, M., H. Yai, and K. Takayama-Arita. Am. J. Physiol. 268 (Cell Physiol. 37): C218-C226, 1995]. To address this issue, tadpoles were raised in Aldo + T(3). An amiloride-blockable SCC developed across the skin before forelimbs appeared. Noise analysis of the characteristics (single-channel current, blocking and unblocking rate coefficients, and apparent dissociation constant) of this amiloride-blockable Na(+) channel showed that it really was of the adult type. A similar SCC developed at stage XIX in the skin of tadpoles raised with Aldo alone. These results strongly support our hypothesis that the crucial hormone in the development of this SCC is Aldo but that a suppression mechanism attenuates its effect on SCC development until it is removed by the increase in the serum concentration of thyroid hormone (which starts at stages XVIII-XIX in vivo).

Ranatuerins: Antimicrobial Peptides Isolated from the Skin of the American Bullfrog,Rana catesbeiana

Nine peptides, termed ranatuerins 1-9, with antimicrobial activity towardsStaphylococcus aureus,were isolated from an extract of the skin of the adult American bullfrog,Rana catesbeiana.In common with other cytolytic peptides from Ranid frogs, (e.g. ranalexin, gaegurins, brevinins), ranatuerins 1 and 4 contain an intramolecular disulfide bridge forming a heptapeptide ring whereas in ranatuerins 2 and 3 the disulfide bridge forms a hexapeptide ring. The structurally related ranatuerins 5-9 comprise 12 - 14 amino acids and show sequence similarity towards the hemolytic peptides A1 and B9 previously isolated from the skin ofRana esculenta.Of the peptides purified, ranatuerin 1 (SMLSVLKNLGKVGLG FVACKINKQC) showed the broadest spectrum of antimicrobial action with inhibitory activity againstS. aureus, Escherichia coliandCandida albicans.

In vivoTreatment of Bullfrog Tadpoles with Aldosterone Potentiates ACh-Receptor Channels, but not Amiloride-Blockable Na+Channels in the Skin

Amiloride-blockable Na(+) channels participate in active Na(+) transport across adult, but not larval, bullfrog skin. Their development is induced in vitro by culturing the tadpole skin with aldosterone. When tadpoles were raised in aldosterone (5 x 10(-7) M) for 2 weeks, however, neither development of such channels nor localization of antigen A, a marker of adult-type epidermis, was seen, the skin still being of the larval type. In contrast, aldosterone treatment did potentiate (by a factor of two) the activity of the acetylcholine receptor (ACh-receptor) channel, a functional marker of larval-type skin. The short-circuit current (SCC) across the skin, far from being inhibited by amiloride, was stimulated by both amiloride and ACh. The nystatin-stimulated SCC was about twice its control amplitude, suggesting that the aldosterone treatment also potentiated the activity of the Na(+) pump.

Apical regulation of nonselective cation channels by ATP in larval bullfrog skin

The apical membrane of larval bullfrog skin contains a nonselective cation channel that can be activated by apically applied amiloride and acetylcholine. In our search for other ligands that might activate this channel, ATP and other purinergics were tested. When ATP (10-1,000 microM) was added to the apical side of tadpole skin mounted in a modified Ussing chamber, there was a transient increase in short circuit current (Isc). The increase in Isc occurred with either Na or K as the dominant cation in the apical solution. The response was larger in a calcium-free Ringer. ADP and AMP had similar but smaller effects than ATP. Adenosine and UTP were without effect. The ATP response was blocked by W-7, atropine, curare, diltiazem, and suramin. These blockers also inhibit amiloride stimulation of Isc, suggesting that ATP activates a related transport pathway. Studies with analogs of ATP suggest that the ATP binding site in tadpole skin has characteristics in common with the P2x receptor found in other tissues. These results demonstrate that in addition to amiloride and acetylcholine, ATP stimulates cation transport at the apical membrane of larval amphibian skin epithelia.