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Localization of ENaC subunit mRNAs in adult bullfrog skin
Acta Histochemica 114 (2012) 172–176
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Localization of ENaC subunit mRNAs in adult bullfrog skin Yuko Kaneko a , Kayo Fujimaki-Aoba a , Shu-Ichi Watanabe a , Shigeru Hokari b , Makoto Takada a,∗ a b
Department of Physiology, Faculty of Medicine, Saitama Medical University, 38 Morohongo, Moroyama, Iruma-gun, Saitama 350-0495, Japan Department of Biochemistry, Faculty of Medicine, Saitama Medical University, 38 Morohongo, Moroyama, Iruma-gun, Saitama 350-0495, Japan
a r t i c l e
i n f o
Article history: Received 4 January 2011 Received in revised form 11 February 2011 Accepted 16 February 2011
Keywords: Epidermis In situ hybridization ENaC Epithelial Na+ transport Frog skin
a b s t r a c t 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 ␣-ENaC mRNA was expressed within the cells of the Stratum granulosum, the Stratum spinosum, and the Stratum germinativum, while -ENaC mRNA was expressed within the cells of the S. granulosum and the S. spinosum. However, we could not detect expression of ␥-ENaC mRNA, possibly for technical reasons. ␣- and -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. © 2011 Elsevier GmbH. All rights reserved.
Introduction The epidermis of adult bullfrog skin is composed of about 5–7 layers of epithelial cells. The innermost layer is the Stratum germinativum, followed by the Stratum spinosum and Stratum granulosum, and the outermost layer is the Stratum corneum. The cells of the S. germinativum differentiate to form the cells of the S. corneum by programmed terminal differentiation, while the cells of the S. granulosum are considered to be the most external living cell layer. Functionally, the principal cells form a syncytium, with the apical membrane of the S. granulosum representing the outer barrier and the basolateral membranes of the other cell layers collectively forming the basolateral membrane (Robinson and Heintzelman, 1987). Adult bullfrog skin is known to have an osmoregulatory function and it actively transports Na+ across its epithelial cells from the apical to the basolateral side (Ussing and Zerahn, 1951). Na+ transport is mediated both by passive entry, through the amiloride-blockable epithelial Na+ channel (ENaC; located in the apical membrane), and by active secretion via the Na+ /K+ -pump (Na+ , K+ -ATPase; located in the basolateral membrane) (Koefoed-Johnsen and Ussing, 1958; Farquhar and Palade, 1964; Aceves and Erlij, 1971; MartinezPalomo et al., 1971; Mills et al., 1977; Benos et al., 1979; Cox and Alvarado, 1979; DiBona and Mills, 1979; Takada and Hayashi, 1981).
∗ Corresponding author. E-mail address: [email protected] (M. Takada). 0065-1281/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2011.02.008
This two-part system is constructed during the terminal differentiation of epidermal cells, in which the cells of the S. germinativum give rise to the cells of the S. granulosum. The role of ENaC in the skin may be involved, not only in Na+ reabsorption, but also in cell-volume regulation during epidermal morphogenesis (RoudierPujol et al., 1996). An ENaC consists of three subunits: namely ␣-, -, and ␥-ENaC (Canessa et al., 1994; Kellenberger and Schild, 2002). The ENaC of bullfrog skin has been cloned, and all three subunits are present in the skin (Jensik et al., 2002). When we examined the localization of ␣-ENaC protein by immunocytochemistry (Takada et al., 2006), we found it to be localized to the apical-side membrane of cells in the S. granulosum. Actually, this could be predicted from electrophysiological studies showing that amiloride inhibits the short-circuit current (SCC) by producing current fluctuations in single channels (Lindemann and Van Driessche, 1977; Takada et al., 1999). We found no ENaC expression in other cells, such as the cells of the S. spinosum or S. germinativum. Since the localization of ENaC subunit mRNAs in amphibian skin has not previously been investigated, it is unknown whether such mRNAs might be expressed in the cells of the S. granulosum alone, as ␣-ENaC protein is, or if they are also in other epidermal cells, which have not yet differentiated to form S. granulosum cells. Consequently, the questions we addressed were (a) whether the messages for the ENaC subunits appear when the cells of the S. granulosum are differentiating, and are then translated and inserted into the apical membrane, or (b) whether those messages are transcribed during the process of differentiation of the epithelial cells when, by
Y. Kaneko et al. / Acta Histochemica 114 (2012) 172–176
cell division, germinative cells give rise to spinous and finally to granular cells. In this study we used in situ hybridization to examine the localization of ENaC subunit mRNAs in the epidermis of adult bullfrog skin. We found that ␣- and -ENaC mRNAs were developed in the sub-apical cells, in which ENaC protein is not necessary for the Na+ -transport function of amphibian skin.
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Materials and methods
alkaline phosphatase antibody (Roche Diagnostics, 1:5000) in 1% (w/v) Blocking Reagent (Roche Diagnostics) in maleic acid buffer for 30 min at RT. After the antibody reaction, the membranes were washed twice with 1 mol/L maleic acid/0.15 mol/L NaCl/0.3% Tween 20 (pH 7.5) for 15 min at RT. After being treated with 100 mmol/L Tris–HCl (pH 9.5)/100 mmol/L NaCl for 3 min at RT, the membranes were incubated with NBT/BCIP (Roche Diagnostics, 1:50) in Tris–HCl (pH 9.5)/100 mmol/L NaCl in the dark at RT (for 24–30 h).
Animals and sample preparation
In situ hybridization
Adult bullfrogs (Lithobates catesbeianus, formerly called Rana catesbeiana) were supplied by a local animal supplier (Misato, Saitama, Japan), and were raised in shallow water. They were anesthetized by an intrathecal injection of 0.5 mL urethane solution (0.25 g/mL), then double-pithed (brain and spinal cord). The ventral skin was dissected out. All experiments were approved by the Animal Research Committee of Saitama Medical University, Japan. All procedures involving animals and their care were performed in conformity with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences (Physiological Society of Japan; revised 2003).
Dissected skins were fixed in 4% paraformaldehyde for 1–2 h at 4 ◦ C. The samples were equilibrated in 10 and 15% sucrose in phosphate buffered saline (PBS) for 2 h each, and in 20% sucrose in PBS overnight at 4 ◦ C. Following equilibration, the samples were immersed in O.C.T. compound (Sakura Finetek Co., Tokyo, Japan) for 2 h at 4 ◦ C, and then embedded in fresh O.C.T. compound, and finally frozen. Sections were cut at a thickness of 12 m using a cryostat, mounted on MAS-GP type A-coated slides (Matsunami Glass Inc., Osaka, Japan), air-dried, and used for in situ hybridization. Air-dried sections were rinsed in PBT [PBS (pH 7.4), DEPCtreated/0.1% Tween 20], digested with 10 g/mL proteinase K in PBT for 15 min at 37 ◦ C, refixed in 4% paraformaldehyde in PBT for 30 min at RT, treated with 2 mg/mL glycine in PBT for 10 min at RT, and rinsed twice in PBT for 5 min each at RT. After rinsing, the sections were treated with 0.1 mol/L triethanolamine (TEA), then with 0.5% acetic anhydride in 0.1 mol/L TEA for 10 min each at RT, and washed four times in PBT for 5 min each at RT. The sections were then incubated in 2× SSC/50% formamide/0.1% Tween 20 for 30 min at RT, and then with 0.7 g/mL DIG RNA probe in a hybridization mixture [1 mg/mL Escherichia coli tRNA, 20 mmol/L Tris–HCl (pH 8.0), 2.5 mmol/L EDTA (pH 8.0), 1× Denhard’s solution, 300 mmol/L NaCl, 10% dextran sulfate, and 50% formamide] overnight at 50 ◦ C. After hybridization, the tissue sections were washed first in 2× SSC/50% formamide/0.1% Tween 20 for 30 min at 45 ◦ C, then twice in 10 mmol/L Tris–HCl (pH 8.0)/500 mmol/L NaCl for 5 min each at RT, and treated with 20 g/mL RNase A in 10 mmol/L Tris–HCl (pH 8.0)/500 mmol/L NaCl for 30 min at 37 ◦ C. The tissue sections were then washed once with 2× SSC/50% formamide/0.1% Tween 20 for 30 min at 45 ◦ C, once with 1× SSC/50% formamide/0.1% Tween 20 for 30 min at 45 ◦ C, once with 1× SSC/50% formamide/0.1% Tween 20 for 30 min at RT, and six times with 100 mmol/L Tris–HCl (pH 7.5)/150 mmol/L NaCl/0.1% Tween 20 for 15 min each at RT. They were then incubated with 1% (w/v) Blocking Reagent (Roche Diagnostics) in 100 mmol/L maleic acid (pH 7.5)/150 mmol/L NaCl for 30 min at RT, and then with sheep antiDIG alkaline phosphatase antibody (Roche Diagnostics, 1:2000) in 100 mmol/L Tris–HCl (pH 7.5)/150 mmol/L NaCl/0.1% Tween 20 overnight at 4 ◦ C. The sections were washed six times with 100 mmol/L Tris–HCl (pH 7.5)/150 mmol/L NaCl/0.1% Tween 20 for 15 min each at RT, then treated twice with 100 mmol/L Tris–HCl (pH 9.5)/100 mmol/L NaCl/50 mmol/L MgCl2 /2 mmol/L levamisole for 5 min each at RT. After mounting with NBT/BCIP (Roche Diagnostics, 1:25) in 100 mmol/L Tris–HCl (pH 9.5)/100 mmol/L NaCl/50 mmol/L MgCl2 /2 mmol/L levamisole/0.1% Tween 20/9.5% polyvinyl alcohol, sections were incubated in the dark at 30 ◦ C (for 24–30 h). After detection of the DIG signals, the sections were dehydrated, cleared, and mounted with New Entellan (Merck, Readington Township, NJ, USA). Brightfield images of the tissue sections were acquired using a light microscope (Power BX BX-60; Olympus, Tokyo, Japan), a color CCD camera (DXM-1200F; Nikon, Tokyo, Japan), and a personal computer (Dell Optiplex GX 260; Dell Inc., Round Rock, TX, USA). The digital images were processed using Adobe PhotoShop
Preparation of digoxigenin-labeled RNA probes The epidermal sides of adult skins were scraped with a glassedge to collect epithelial cells. Total RNA or poly(A)+ RNAs (mRNAs) were isolated from these cells. Total RNA was prepared by the method of Chomczynski and Sacchi (1987). Poly(A)+ RNAs were isolated using a FastTrack Kit (Invitrogen, Carlsbad, CA, USA). We prepared digoxigenin (DIG)-labeled RNA probes specific for Rana ␣-ENaC, Rana -ENaC, Rana ␥-ENaC (Jensik et al., 2002), and Rana adult keratin (RAK; type I keratin: Suzuki et al., 2001). The RNA probes recognized nucleotides ␣: 113–522, : 76–578, ␥: 222–918, and k: 209–1177 (GenBank numbering; GenBank accession nos. AF514844, AF514845, AF514846, and AB050955, respectively) of the Rana ␣-ENaC, Rana -ENaC, Rana ␥-ENaC, and RAK, respectively. The preparation of the RNA probes was carried out essentially as in our previous studies (Hirota et al., 1999; Kaneko et al., 1999). DIG-labeled antisense/sense RNA probes (DIG RNA Labeling Kit SP6/T7, Roche Diagnostics) were purified using a quick-spin column (G-50 Sephadex Columns for Radiolabeled RNA Purification; Roche Diagnostics, Penzberg, Germany). Northern blot analysis Poly(A)+ RNAs (0.1–0.2 g/lane) and RNA ladder (DynaMarker prestained, BioDynamics Laboratory Inc., Tokyo, Japan) were electrophoresed in a 1% agarose gel containing 0.22 mol/L formaldehyde in MOPS buffer (20 mmol/L 3-N-morpholinol propane sulfonic acid, 5 mmol/L sodium acetate, 1 mmol/L EDTA, 240 mmol/L formaldehyde, pH 7.0) and transferred to a Nytran SuPerCharge membrane (GE Healthcare, Little Chalfont, Bucks., UK). The Nytran membrane was cut into separate lanes, and each blot was hybridized with one of the DIG-labeled RNA probes (see Results) (final probe concentration: 100 ng/mL) in 1× DIG Easy Hyb (Roche Diagnostics) at 68 ◦ C overnight. The membranes were washed with 2× SSC/0.1% SDS for 2× 5 min at room temperature (RT), with 0.1× SSC/0.1% SDS for 2× 15 min at 68 ◦ C, and with 1 mol/L maleic acid/0.15 mol/L NaCl (pH 7.5)/0.3% Tween 20 for 2 min at room temperature (RT). To detect the hybridization signals for the DIG-labeled probes, the membranes were then incubated with 1% (w/v) Blocking Reagent (Roche Diagnostics) in maleic acid buffer (100 mmol/L maleic acid, 150 mmol/L NaCl, pH 7.5) for 30 min at RT, and then with sheep anti-DIG
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employed for in situ hybridization worked well (Fig. 2e). The ␣ENaC mRNA was located in the cells of the S. granulosum and in many cells of the S. spinosum (Fig. 2a), and also in a few cells in the S. germinativum (arrows in Fig. 2a). -ENaC mRNA was mainly localized to the cells of the S. granulosum, although a number of cells in the upper part of the S. spinosum also expressed this mRNA. However, no -ENaC mRNA signal was detected in the cells of the S. germinativum (Fig. 2c). The signals for the ␣- and -ENaC mRNAs were particularly evident near the nucleus (Fig. 2a and c). Non-specific staining was observed in the S. corneum and dermis (Fig. 2a–f). We were unable to detect a signal for ␥-ENaC mRNA by in situ hybridization using any of the probes we made (data not shown).
Discussion
Fig. 1. Northern blot analysis of mRNA expressions of ENaC subunits and adult Rana keratin (RAK). Lanes 2–4 contain 0.1–0.2 g of mRNA from bullfrog epidermis. A Nytran membrane was cut into separate lanes, and the blots were then hybridized with dissimilar DIG-labeled RNA probes (lane 2, Rana ␣-ENaC; lane 3, Rana -ENaC; lane 4, RAK). Arrows show signals. We performed Northern blotting three times with different RNA samples and obtained similar results each time. Marker, RNA ladder.
CS4 (Adobe Systems Inc., San Jose, CA, USA), with image brightness and contrast being adjusted. Chemicals Levamisole, 3-N-morpholinol propane sulfonic acid, polyvinyl alcohol, TEA, and Tween 20 were purchased from Sigma–Aldrich Chemicals (St. Louis, MO, USA). E. coli t-RNA and proteinase K were from Roche Diagnostics (Penzberg, Germany), dextran sulfate from Eppendorf (Hamburg, Germany), and agarose from Takara Bio (Shiga, Japan). RNA ladder (DynaMarker) was from BioDynamics Laboratory (Tokyo, Japan) and RNase A was from Qiagen (Dusseldorf, Germany). Other chemicals were from Wako Chemicals (Osaka, Japan), unless otherwise described in the text. Results To confirm the specificity of the DIG-labeled RNA probes and to examine the expression of each mRNA in adult bullfrog skin, Northern blot hybridization was performed (Fig. 1). First, total RNAs were used as samples for Northern blotting. The signals for ENaC subunit mRNAs were weak (data not shown), which we thought might be due to their concentrations being lower than those of other RNAs. Next, therefore, we used poly A+ RNAs as the samples. A Nytran membrane was cut into separate lanes, and the blots were hybridized with different DIG-labeled RNA probes (probes for ␣-, -, and ␥-ENaC, or RAK, respectively). A 2281 bp message for ␣ENaC, a 2745 bp message for -ENaC, and a 1700 bp message for RAK were detected, suggesting that our probes were working well for the mRNAs of ␣- and -ENaC, and RAK. We were unable to detect a ␥-ENaC signal (expected to be 2497 bp) (data not shown), and although we made other probes using other sequences in further attempts to detect ␥-ENaC mRNA, we again failed to detect the signal (data not shown). The localization of ␣-, -, and ␥-ENaC, and RAK mRNAs in the adult skin was examined by in situ hybridization. Antisense and sense probes were hybridized with sections of the skin. The mRNA of RAK was detected in cells in the S. germinativum, as previously described by Suzuki et al. (2001), indicating that the method we
In this study, we have shown the localization of the ␣- and ENaC subunit mRNAs in adult bullfrog skin by the use of in situ hybridization. We found that ␣-ENaC mRNA was expressed within the cells of the S. granulosum, the S. spinosum, and the S. germinativum, while -ENaC mRNA was expressed within the cells of the S. granulosum and the S. spinosum. In contrast, we were unable (possibly for technical reasons, see below) to detect expression of ␥-ENaC mRNA in the epidermis. Roudier-Pujol et al. (1996) found, by using in situ hybridization, that ␣-, -, and ␥-ENaC mRNAs were expressed in all epidermal layers in the adult rat skin, a tissue that is not osmoregulatory in mammals. They discussed the possibility that ENaC in that skin may play a role in the control of cell volume during epidermal proliferation. It is possible, therefore, that the role of ENaC in amphibian skin may not be solely to reabsorb Na+ , but may also be involved in cell-volume regulation during epidermal morphogenesis. They further showed that the mRNA expression of -ENaC was lower than those of the ␣- and ␥-ENaC subunits. According to Oda et al. (1999), ␣-, -, and ␥-ENaC mRNAs are all expressed in keratinocytes throughout the adult human epidermis, although the ␥-ENaC signal is low. Roudier-Pujol et al. (1996) also reported that the abundance of ENaC mRNAs seemed to differ between epidermal cells; that is, the abundance seems to be lower in basal keratinocytes than in differentiated suprabasal keratinocytes. On the basis of the above results, we might have anticipated that all layers of the epidermis in adult bullfrog skin would express ␣- and -ENaC mRNAs. Actually, what we found was that cells expressing ␣-ENaC mRNA were predominantly in the S. spinosum and S. granulosum, with only a very few in the S. germinativum, whereas cells expressing -ENaC mRNA were detected only in the upper S. spinosum and in the S. granulosum. Previously, by applying RT-PCR methods to the whole adult bullfrog skin, we detected signals for the mRNAs of all three ENaC subunits (Takada et al., 2006). However, in the present study the detection of the ␥-ENaC mRNA signal proved to be difficult by either Northern blotting or in situ hybridization (data not shown). Possibly, the quantity of ␥-ENaC mRNA might be too low for Northern blotting and in situ hybridization, or the probe used to try to detect ␥-ENaC mRNA was not suitable. Consequently, we cannot exclude the possibility that in adult bullfrog skin, ␣-, -, and ␥-ENaC mRNAs are all expressed at low levels throughout the epidermis. Furthermore, it is well known that the SCC across amphibian skin can vary considerably depending on such factors as the amphibian species, the season of the year, the animal’s diet, and the ionic composition of the external environment (Takada and Komazaki, 1988; Takada, 1989; Takada et al., 1998, 2011). The bullfrogs used in the present study were raised in tapwater, so one speculation is that if we had raised them in a solution containing higher concentrations of electrolytes, this might have increased the expression of ␥-ENaC mRNA sufficiently for us to detect it by the present methods. Addi-
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Fig. 2. Localization of ␣- and -ENaC, and RAK (Rana adult keratin) mRNAs in the normal adult bullfrog skin was examined by in situ hybridization. Photomicrographs of vertical sections of such skin hybridized with specific RNA probe for ␣- or -subunit of ENaC, or RAK. (a and b) ␣-ENaC; (c and d) -ENaC; (e and f) RAK. (a, c, and e) Antisense probe; (b, d, and f) sense probe. Hybridization signal for ␣-ENaC was detected in both the Stratum granulosum and the Stratum spinosum. In addition, a few cells in the Stratum germinativum expressed ␣-ENaC (arrows in a). Hybridization signal for -ENaC was detected in both the S. granulosum and the upper S. spinosum. Hybridization signal for RAK was detected in the S. germinativum. Non-specific staining was observed in the Stratum corneum and the dermis (a–f). Str. corn., Stratum corneum; Str. gran., Stratum granulosum; Str. spino., Stratum spinosum; Str. germ., Stratum germinativum; SC, cornified cells; GC, granular cells; SP, spinosal cells; SG, germinative cells. We performed in situ hybridization experiments thirteen separate times (about 40–50 sections per probe; total, eight frogs), and obtained similar results each time. Bar = 20 m.
tional experiments, including single-cell RT-PCR applied to slices of bullfrog skin, will be needed to resolve these issues. Co-expression of all three ENaC subunits is necessary for maximal channel activity; however, a small current can flow when ␣-ENaC is expressed either alone or paired with - or ␥-ENaC in Xenopus oocytes or MDCK cells (McNicholas and Canessa, 1997; Ishikawa et al., 1998). Therefore, even if expression of ␥-ENaC is low, as our data may suggest, the ENaC channel may be able to participate in Na+ transport across adult bullfrog skin. As described in the Introduction, ENaC protein needs to be expressed in cells of the S. granulosum, but not necessarily in other cell layers, for it to participate in Na+ transport. Previously, we found that we could detect an expression of ␣-ENaC protein only within S. granulosum in adult bullfrog skin (Takada et al., 2006). On the other hand, both ␣- and -ENaC mRNAs were found here to be expressed not only in the S. granulosum, but also in at least the upper S. spinosum (Fig. 2a and c). It seems unlikely that the ␣- and -ENaC mRNAs localized in the S. spinosum are involved in Na+ transport, and what function, if any, they might perform in that layer remains unknown. One question might be: “Do the cells of the Stratum spinosum produce ENaC mRNA without ENaC protein expression, or with only a small amount of the protein, before they differentiate to become cells of the Stratum granulosum?” Further research will be necessary to elucidate the role(s) of the ENaC mRNAs expressed in the S. spinosum. In conclusion, our results demonstrate that ␣- and -ENaC mRNAs are present in the sub-apical cells of adult bullfrog skin. It is known that ENaC protein does not need to be present in such cells for the skin to perform its Na+ -transport function, and so one interpretation is that sub-apical cells are already producing ENaC subunit mRNAs prior to the final step in their differentiation.
However, we cannot exclude such mRNAs performing some, as yet unknown, functions in sub-apical cells. Acknowledgment This research was supported in part by a Saitama Medical University Internal Grant (No. 21-1-2-11). References Aceves J, Erlij D. Sodium transport across the isolated epithelium of the frog skin. J Physiol 1971;212:195–210. Benos DJ, Mandel LJ, Balaban RS. On the mechanism of the amiloride–sodium entry site interaction in anuran skin epithelia. J Gen Physiol 1979;73:307–26. Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, et al. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 1994;367:463–7. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem 1987;162:156–9. Cox TC, Alvarado RH. Electrical and transport characteristics of skin of larval Rana catesbeiana. Am J Physiol 1979;237:R74–9. DiBona DR, Mills JW. Distribution of Na+ -pump sites in transporting epithelia. Fed Proc 1979;38:134–43. Farquhar GM, Palade GE. Functional organization of amphibian skin. Proc Natl Acad Sci USA 1964;51:569–77. Hirota K, Kaneko Y, Matsumoto G, Hanyu Y. Cloning and distribution of a putative tetrodotoxin-resistant Na+ channel in newt retina. Zool Sci 1999;16:587–94.
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Ishikawa T, Marunaka Y, Rotin D. Electrophysiological characterization of the rat epithelial Na+ channel (rENaC) expressed in MDCK cells: effects of Na+ and Ca2+ . J Gen Physiol 1998;111: 825–46. Jensik PJ, Holbird D, Cox TC. Cloned bullfrog skin sodium (fENaC) and xENaC subunits hybridize to form functional sodium channels. J Comp Physiol B 2002;172:569–76. Kaneko Y, Matsumoto G, Hanyu Y. Pax-6 expression during retinal regeneration in the adult newt. Dev Growth Differ 1999;41:723–9. Kellenberger S, Schild L. Epithelial sodium channel/degenerin family of ion channels: a variety of functions for a shared structure. Physiol Rev 2002;82:735–67. Koefoed-Johnsen V, Ussing HH. The nature of the frog skin potential. Acta Physiol Scand 1958;42:298–308. Lindemann B, Van Driessche W. Sodium-specific membrane channels of frog skin are pores: current fluctuations reveal high turnover. Science 1977;195:292–4. Martinez-Palomo A, Erlij D, Bracho H. Localization of permeability barriers in the frog skin epithelium. J Cell Biol 1971;50: 277–87. McNicholas CM, Canessa CM. Diversity of channels generated by different combinations of epithelial sodium channel subunits. J Gen Physiol 1997;109:681–92. Mills JW, Ernst SA, DiBona DR. Localization of Na+ -pump sites in frog skin. J Cell Biol 1977;73:88–110. Oda Y, Imanzahrai A, Kwong A, Komuves L, Elias PM, Largman C, et al. Epithelial sodium channels are upregulated during epidermal differentiation. J Invest Dermatol 1999;113: 796–801. Robinson DH, Heintzelman MB. Morphology of ventral epidermis of Rana catesbeiana during metamorphosis. Anat Rec 1987;217:305–17.
Roudier-Pujol C, Rochat A, Escoubet B, Eugene E, Barrandon Y, Bonvalet JP, et al. Differential expression of epithelial sodium channel subunit mRNAs in rat skin. J Cell Sci 1996;109:379–85. Suzuki K, Sato K, Katsu K, Hayashita H, Kristensen DB, Yoshizato K. Novel Rana keratin genes and their expression during larval to adult epidermal conversion in bullfrog tadpoles. Differentiation 2001;68:44–54. Takada M. Seasonal variations in the effect of prolactin on the short-circuit current across bullfrog skin. Gen Comp Endocrinol 1989;75:413–6. Takada M, Fujimaki-Aoba K, Hokari S. Vasotocin- and mesotocininduced increase in short-circuit current (SCC) across tree frog skin. J Comp Physiol B 2011;181:239–48. Takada M, Hayashi H. Interaction of cadmium, calcium, and amiloride in the kinetics of active sodium transport through frog skin. Jpn J Physiol 1981;31:285–303. Takada M, Komazaki S. Effect of prolactin on transcutaneous Na transport in the Japanese newt, Cynopus pyrrhogaster. Gen Comp Endocrinol 1988;69:141–5. Takada M, Shiibashi M, Kasai M. Possible role of aldosterone and T3 in development of amiloride-blockable SCC across frog skin in vivo. Am J Physiol Regul Integr Comp Physiol 1999;277:R1305–12. Takada M, Shimomura T, Hokari S, Jensik PJ, Cox TC. Larval bullfrog skin expresses ENaC despite having no amilorideblockable transepithelial Na+ transport. J Comp Physiol B 2006;176:287–93. Takada M, Yai H, Komazaki S. Effect of calcium on development of amiloride-blockable Na+ transport in axolotl in vitro. Am J Physiol Regul Integr Comp Physiol 1998;275:R69–75. Ussing HH, Zerahn K. Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiol Scand 1951;23:110–27.
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Acta Histochemica journal homepage: www.elsevier.de/acthis
Short communication
Localization of ENaC subunit mRNAs in adult bullfrog skin Yuko Kaneko a , Kayo Fujimaki-Aoba a , Shu-Ichi Watanabe a , Shigeru Hokari b , Makoto Takada a,∗ a b
Department of Physiology, Faculty of Medicine, Saitama Medical University, 38 Morohongo, Moroyama, Iruma-gun, Saitama 350-0495, Japan Department of Biochemistry, Faculty of Medicine, Saitama Medical University, 38 Morohongo, Moroyama, Iruma-gun, Saitama 350-0495, Japan
a r t i c l e
i n f o
Article history: Received 4 January 2011 Received in revised form 11 February 2011 Accepted 16 February 2011
Keywords: Epidermis In situ hybridization ENaC Epithelial Na+ transport Frog skin
a b s t r a c t 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 ␣-ENaC mRNA was expressed within the cells of the Stratum granulosum, the Stratum spinosum, and the Stratum germinativum, while -ENaC mRNA was expressed within the cells of the S. granulosum and the S. spinosum. However, we could not detect expression of ␥-ENaC mRNA, possibly for technical reasons. ␣- and -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. © 2011 Elsevier GmbH. All rights reserved.
Introduction The epidermis of adult bullfrog skin is composed of about 5–7 layers of epithelial cells. The innermost layer is the Stratum germinativum, followed by the Stratum spinosum and Stratum granulosum, and the outermost layer is the Stratum corneum. The cells of the S. germinativum differentiate to form the cells of the S. corneum by programmed terminal differentiation, while the cells of the S. granulosum are considered to be the most external living cell layer. Functionally, the principal cells form a syncytium, with the apical membrane of the S. granulosum representing the outer barrier and the basolateral membranes of the other cell layers collectively forming the basolateral membrane (Robinson and Heintzelman, 1987). Adult bullfrog skin is known to have an osmoregulatory function and it actively transports Na+ across its epithelial cells from the apical to the basolateral side (Ussing and Zerahn, 1951). Na+ transport is mediated both by passive entry, through the amiloride-blockable epithelial Na+ channel (ENaC; located in the apical membrane), and by active secretion via the Na+ /K+ -pump (Na+ , K+ -ATPase; located in the basolateral membrane) (Koefoed-Johnsen and Ussing, 1958; Farquhar and Palade, 1964; Aceves and Erlij, 1971; MartinezPalomo et al., 1971; Mills et al., 1977; Benos et al., 1979; Cox and Alvarado, 1979; DiBona and Mills, 1979; Takada and Hayashi, 1981).
∗ Corresponding author. E-mail address: [email protected] (M. Takada). 0065-1281/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2011.02.008
This two-part system is constructed during the terminal differentiation of epidermal cells, in which the cells of the S. germinativum give rise to the cells of the S. granulosum. The role of ENaC in the skin may be involved, not only in Na+ reabsorption, but also in cell-volume regulation during epidermal morphogenesis (RoudierPujol et al., 1996). An ENaC consists of three subunits: namely ␣-, -, and ␥-ENaC (Canessa et al., 1994; Kellenberger and Schild, 2002). The ENaC of bullfrog skin has been cloned, and all three subunits are present in the skin (Jensik et al., 2002). When we examined the localization of ␣-ENaC protein by immunocytochemistry (Takada et al., 2006), we found it to be localized to the apical-side membrane of cells in the S. granulosum. Actually, this could be predicted from electrophysiological studies showing that amiloride inhibits the short-circuit current (SCC) by producing current fluctuations in single channels (Lindemann and Van Driessche, 1977; Takada et al., 1999). We found no ENaC expression in other cells, such as the cells of the S. spinosum or S. germinativum. Since the localization of ENaC subunit mRNAs in amphibian skin has not previously been investigated, it is unknown whether such mRNAs might be expressed in the cells of the S. granulosum alone, as ␣-ENaC protein is, or if they are also in other epidermal cells, which have not yet differentiated to form S. granulosum cells. Consequently, the questions we addressed were (a) whether the messages for the ENaC subunits appear when the cells of the S. granulosum are differentiating, and are then translated and inserted into the apical membrane, or (b) whether those messages are transcribed during the process of differentiation of the epithelial cells when, by
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cell division, germinative cells give rise to spinous and finally to granular cells. In this study we used in situ hybridization to examine the localization of ENaC subunit mRNAs in the epidermis of adult bullfrog skin. We found that ␣- and -ENaC mRNAs were developed in the sub-apical cells, in which ENaC protein is not necessary for the Na+ -transport function of amphibian skin.
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Materials and methods
alkaline phosphatase antibody (Roche Diagnostics, 1:5000) in 1% (w/v) Blocking Reagent (Roche Diagnostics) in maleic acid buffer for 30 min at RT. After the antibody reaction, the membranes were washed twice with 1 mol/L maleic acid/0.15 mol/L NaCl/0.3% Tween 20 (pH 7.5) for 15 min at RT. After being treated with 100 mmol/L Tris–HCl (pH 9.5)/100 mmol/L NaCl for 3 min at RT, the membranes were incubated with NBT/BCIP (Roche Diagnostics, 1:50) in Tris–HCl (pH 9.5)/100 mmol/L NaCl in the dark at RT (for 24–30 h).
Animals and sample preparation
In situ hybridization
Adult bullfrogs (Lithobates catesbeianus, formerly called Rana catesbeiana) were supplied by a local animal supplier (Misato, Saitama, Japan), and were raised in shallow water. They were anesthetized by an intrathecal injection of 0.5 mL urethane solution (0.25 g/mL), then double-pithed (brain and spinal cord). The ventral skin was dissected out. All experiments were approved by the Animal Research Committee of Saitama Medical University, Japan. All procedures involving animals and their care were performed in conformity with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences (Physiological Society of Japan; revised 2003).
Dissected skins were fixed in 4% paraformaldehyde for 1–2 h at 4 ◦ C. The samples were equilibrated in 10 and 15% sucrose in phosphate buffered saline (PBS) for 2 h each, and in 20% sucrose in PBS overnight at 4 ◦ C. Following equilibration, the samples were immersed in O.C.T. compound (Sakura Finetek Co., Tokyo, Japan) for 2 h at 4 ◦ C, and then embedded in fresh O.C.T. compound, and finally frozen. Sections were cut at a thickness of 12 m using a cryostat, mounted on MAS-GP type A-coated slides (Matsunami Glass Inc., Osaka, Japan), air-dried, and used for in situ hybridization. Air-dried sections were rinsed in PBT [PBS (pH 7.4), DEPCtreated/0.1% Tween 20], digested with 10 g/mL proteinase K in PBT for 15 min at 37 ◦ C, refixed in 4% paraformaldehyde in PBT for 30 min at RT, treated with 2 mg/mL glycine in PBT for 10 min at RT, and rinsed twice in PBT for 5 min each at RT. After rinsing, the sections were treated with 0.1 mol/L triethanolamine (TEA), then with 0.5% acetic anhydride in 0.1 mol/L TEA for 10 min each at RT, and washed four times in PBT for 5 min each at RT. The sections were then incubated in 2× SSC/50% formamide/0.1% Tween 20 for 30 min at RT, and then with 0.7 g/mL DIG RNA probe in a hybridization mixture [1 mg/mL Escherichia coli tRNA, 20 mmol/L Tris–HCl (pH 8.0), 2.5 mmol/L EDTA (pH 8.0), 1× Denhard’s solution, 300 mmol/L NaCl, 10% dextran sulfate, and 50% formamide] overnight at 50 ◦ C. After hybridization, the tissue sections were washed first in 2× SSC/50% formamide/0.1% Tween 20 for 30 min at 45 ◦ C, then twice in 10 mmol/L Tris–HCl (pH 8.0)/500 mmol/L NaCl for 5 min each at RT, and treated with 20 g/mL RNase A in 10 mmol/L Tris–HCl (pH 8.0)/500 mmol/L NaCl for 30 min at 37 ◦ C. The tissue sections were then washed once with 2× SSC/50% formamide/0.1% Tween 20 for 30 min at 45 ◦ C, once with 1× SSC/50% formamide/0.1% Tween 20 for 30 min at 45 ◦ C, once with 1× SSC/50% formamide/0.1% Tween 20 for 30 min at RT, and six times with 100 mmol/L Tris–HCl (pH 7.5)/150 mmol/L NaCl/0.1% Tween 20 for 15 min each at RT. They were then incubated with 1% (w/v) Blocking Reagent (Roche Diagnostics) in 100 mmol/L maleic acid (pH 7.5)/150 mmol/L NaCl for 30 min at RT, and then with sheep antiDIG alkaline phosphatase antibody (Roche Diagnostics, 1:2000) in 100 mmol/L Tris–HCl (pH 7.5)/150 mmol/L NaCl/0.1% Tween 20 overnight at 4 ◦ C. The sections were washed six times with 100 mmol/L Tris–HCl (pH 7.5)/150 mmol/L NaCl/0.1% Tween 20 for 15 min each at RT, then treated twice with 100 mmol/L Tris–HCl (pH 9.5)/100 mmol/L NaCl/50 mmol/L MgCl2 /2 mmol/L levamisole for 5 min each at RT. After mounting with NBT/BCIP (Roche Diagnostics, 1:25) in 100 mmol/L Tris–HCl (pH 9.5)/100 mmol/L NaCl/50 mmol/L MgCl2 /2 mmol/L levamisole/0.1% Tween 20/9.5% polyvinyl alcohol, sections were incubated in the dark at 30 ◦ C (for 24–30 h). After detection of the DIG signals, the sections were dehydrated, cleared, and mounted with New Entellan (Merck, Readington Township, NJ, USA). Brightfield images of the tissue sections were acquired using a light microscope (Power BX BX-60; Olympus, Tokyo, Japan), a color CCD camera (DXM-1200F; Nikon, Tokyo, Japan), and a personal computer (Dell Optiplex GX 260; Dell Inc., Round Rock, TX, USA). The digital images were processed using Adobe PhotoShop
Preparation of digoxigenin-labeled RNA probes The epidermal sides of adult skins were scraped with a glassedge to collect epithelial cells. Total RNA or poly(A)+ RNAs (mRNAs) were isolated from these cells. Total RNA was prepared by the method of Chomczynski and Sacchi (1987). Poly(A)+ RNAs were isolated using a FastTrack Kit (Invitrogen, Carlsbad, CA, USA). We prepared digoxigenin (DIG)-labeled RNA probes specific for Rana ␣-ENaC, Rana -ENaC, Rana ␥-ENaC (Jensik et al., 2002), and Rana adult keratin (RAK; type I keratin: Suzuki et al., 2001). The RNA probes recognized nucleotides ␣: 113–522, : 76–578, ␥: 222–918, and k: 209–1177 (GenBank numbering; GenBank accession nos. AF514844, AF514845, AF514846, and AB050955, respectively) of the Rana ␣-ENaC, Rana -ENaC, Rana ␥-ENaC, and RAK, respectively. The preparation of the RNA probes was carried out essentially as in our previous studies (Hirota et al., 1999; Kaneko et al., 1999). DIG-labeled antisense/sense RNA probes (DIG RNA Labeling Kit SP6/T7, Roche Diagnostics) were purified using a quick-spin column (G-50 Sephadex Columns for Radiolabeled RNA Purification; Roche Diagnostics, Penzberg, Germany). Northern blot analysis Poly(A)+ RNAs (0.1–0.2 g/lane) and RNA ladder (DynaMarker prestained, BioDynamics Laboratory Inc., Tokyo, Japan) were electrophoresed in a 1% agarose gel containing 0.22 mol/L formaldehyde in MOPS buffer (20 mmol/L 3-N-morpholinol propane sulfonic acid, 5 mmol/L sodium acetate, 1 mmol/L EDTA, 240 mmol/L formaldehyde, pH 7.0) and transferred to a Nytran SuPerCharge membrane (GE Healthcare, Little Chalfont, Bucks., UK). The Nytran membrane was cut into separate lanes, and each blot was hybridized with one of the DIG-labeled RNA probes (see Results) (final probe concentration: 100 ng/mL) in 1× DIG Easy Hyb (Roche Diagnostics) at 68 ◦ C overnight. The membranes were washed with 2× SSC/0.1% SDS for 2× 5 min at room temperature (RT), with 0.1× SSC/0.1% SDS for 2× 15 min at 68 ◦ C, and with 1 mol/L maleic acid/0.15 mol/L NaCl (pH 7.5)/0.3% Tween 20 for 2 min at room temperature (RT). To detect the hybridization signals for the DIG-labeled probes, the membranes were then incubated with 1% (w/v) Blocking Reagent (Roche Diagnostics) in maleic acid buffer (100 mmol/L maleic acid, 150 mmol/L NaCl, pH 7.5) for 30 min at RT, and then with sheep anti-DIG
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employed for in situ hybridization worked well (Fig. 2e). The ␣ENaC mRNA was located in the cells of the S. granulosum and in many cells of the S. spinosum (Fig. 2a), and also in a few cells in the S. germinativum (arrows in Fig. 2a). -ENaC mRNA was mainly localized to the cells of the S. granulosum, although a number of cells in the upper part of the S. spinosum also expressed this mRNA. However, no -ENaC mRNA signal was detected in the cells of the S. germinativum (Fig. 2c). The signals for the ␣- and -ENaC mRNAs were particularly evident near the nucleus (Fig. 2a and c). Non-specific staining was observed in the S. corneum and dermis (Fig. 2a–f). We were unable to detect a signal for ␥-ENaC mRNA by in situ hybridization using any of the probes we made (data not shown).
Discussion
Fig. 1. Northern blot analysis of mRNA expressions of ENaC subunits and adult Rana keratin (RAK). Lanes 2–4 contain 0.1–0.2 g of mRNA from bullfrog epidermis. A Nytran membrane was cut into separate lanes, and the blots were then hybridized with dissimilar DIG-labeled RNA probes (lane 2, Rana ␣-ENaC; lane 3, Rana -ENaC; lane 4, RAK). Arrows show signals. We performed Northern blotting three times with different RNA samples and obtained similar results each time. Marker, RNA ladder.
CS4 (Adobe Systems Inc., San Jose, CA, USA), with image brightness and contrast being adjusted. Chemicals Levamisole, 3-N-morpholinol propane sulfonic acid, polyvinyl alcohol, TEA, and Tween 20 were purchased from Sigma–Aldrich Chemicals (St. Louis, MO, USA). E. coli t-RNA and proteinase K were from Roche Diagnostics (Penzberg, Germany), dextran sulfate from Eppendorf (Hamburg, Germany), and agarose from Takara Bio (Shiga, Japan). RNA ladder (DynaMarker) was from BioDynamics Laboratory (Tokyo, Japan) and RNase A was from Qiagen (Dusseldorf, Germany). Other chemicals were from Wako Chemicals (Osaka, Japan), unless otherwise described in the text. Results To confirm the specificity of the DIG-labeled RNA probes and to examine the expression of each mRNA in adult bullfrog skin, Northern blot hybridization was performed (Fig. 1). First, total RNAs were used as samples for Northern blotting. The signals for ENaC subunit mRNAs were weak (data not shown), which we thought might be due to their concentrations being lower than those of other RNAs. Next, therefore, we used poly A+ RNAs as the samples. A Nytran membrane was cut into separate lanes, and the blots were hybridized with different DIG-labeled RNA probes (probes for ␣-, -, and ␥-ENaC, or RAK, respectively). A 2281 bp message for ␣ENaC, a 2745 bp message for -ENaC, and a 1700 bp message for RAK were detected, suggesting that our probes were working well for the mRNAs of ␣- and -ENaC, and RAK. We were unable to detect a ␥-ENaC signal (expected to be 2497 bp) (data not shown), and although we made other probes using other sequences in further attempts to detect ␥-ENaC mRNA, we again failed to detect the signal (data not shown). The localization of ␣-, -, and ␥-ENaC, and RAK mRNAs in the adult skin was examined by in situ hybridization. Antisense and sense probes were hybridized with sections of the skin. The mRNA of RAK was detected in cells in the S. germinativum, as previously described by Suzuki et al. (2001), indicating that the method we
In this study, we have shown the localization of the ␣- and ENaC subunit mRNAs in adult bullfrog skin by the use of in situ hybridization. We found that ␣-ENaC mRNA was expressed within the cells of the S. granulosum, the S. spinosum, and the S. germinativum, while -ENaC mRNA was expressed within the cells of the S. granulosum and the S. spinosum. In contrast, we were unable (possibly for technical reasons, see below) to detect expression of ␥-ENaC mRNA in the epidermis. Roudier-Pujol et al. (1996) found, by using in situ hybridization, that ␣-, -, and ␥-ENaC mRNAs were expressed in all epidermal layers in the adult rat skin, a tissue that is not osmoregulatory in mammals. They discussed the possibility that ENaC in that skin may play a role in the control of cell volume during epidermal proliferation. It is possible, therefore, that the role of ENaC in amphibian skin may not be solely to reabsorb Na+ , but may also be involved in cell-volume regulation during epidermal morphogenesis. They further showed that the mRNA expression of -ENaC was lower than those of the ␣- and ␥-ENaC subunits. According to Oda et al. (1999), ␣-, -, and ␥-ENaC mRNAs are all expressed in keratinocytes throughout the adult human epidermis, although the ␥-ENaC signal is low. Roudier-Pujol et al. (1996) also reported that the abundance of ENaC mRNAs seemed to differ between epidermal cells; that is, the abundance seems to be lower in basal keratinocytes than in differentiated suprabasal keratinocytes. On the basis of the above results, we might have anticipated that all layers of the epidermis in adult bullfrog skin would express ␣- and -ENaC mRNAs. Actually, what we found was that cells expressing ␣-ENaC mRNA were predominantly in the S. spinosum and S. granulosum, with only a very few in the S. germinativum, whereas cells expressing -ENaC mRNA were detected only in the upper S. spinosum and in the S. granulosum. Previously, by applying RT-PCR methods to the whole adult bullfrog skin, we detected signals for the mRNAs of all three ENaC subunits (Takada et al., 2006). However, in the present study the detection of the ␥-ENaC mRNA signal proved to be difficult by either Northern blotting or in situ hybridization (data not shown). Possibly, the quantity of ␥-ENaC mRNA might be too low for Northern blotting and in situ hybridization, or the probe used to try to detect ␥-ENaC mRNA was not suitable. Consequently, we cannot exclude the possibility that in adult bullfrog skin, ␣-, -, and ␥-ENaC mRNAs are all expressed at low levels throughout the epidermis. Furthermore, it is well known that the SCC across amphibian skin can vary considerably depending on such factors as the amphibian species, the season of the year, the animal’s diet, and the ionic composition of the external environment (Takada and Komazaki, 1988; Takada, 1989; Takada et al., 1998, 2011). The bullfrogs used in the present study were raised in tapwater, so one speculation is that if we had raised them in a solution containing higher concentrations of electrolytes, this might have increased the expression of ␥-ENaC mRNA sufficiently for us to detect it by the present methods. Addi-
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Fig. 2. Localization of ␣- and -ENaC, and RAK (Rana adult keratin) mRNAs in the normal adult bullfrog skin was examined by in situ hybridization. Photomicrographs of vertical sections of such skin hybridized with specific RNA probe for ␣- or -subunit of ENaC, or RAK. (a and b) ␣-ENaC; (c and d) -ENaC; (e and f) RAK. (a, c, and e) Antisense probe; (b, d, and f) sense probe. Hybridization signal for ␣-ENaC was detected in both the Stratum granulosum and the Stratum spinosum. In addition, a few cells in the Stratum germinativum expressed ␣-ENaC (arrows in a). Hybridization signal for -ENaC was detected in both the S. granulosum and the upper S. spinosum. Hybridization signal for RAK was detected in the S. germinativum. Non-specific staining was observed in the Stratum corneum and the dermis (a–f). Str. corn., Stratum corneum; Str. gran., Stratum granulosum; Str. spino., Stratum spinosum; Str. germ., Stratum germinativum; SC, cornified cells; GC, granular cells; SP, spinosal cells; SG, germinative cells. We performed in situ hybridization experiments thirteen separate times (about 40–50 sections per probe; total, eight frogs), and obtained similar results each time. Bar = 20 m.
tional experiments, including single-cell RT-PCR applied to slices of bullfrog skin, will be needed to resolve these issues. Co-expression of all three ENaC subunits is necessary for maximal channel activity; however, a small current can flow when ␣-ENaC is expressed either alone or paired with - or ␥-ENaC in Xenopus oocytes or MDCK cells (McNicholas and Canessa, 1997; Ishikawa et al., 1998). Therefore, even if expression of ␥-ENaC is low, as our data may suggest, the ENaC channel may be able to participate in Na+ transport across adult bullfrog skin. As described in the Introduction, ENaC protein needs to be expressed in cells of the S. granulosum, but not necessarily in other cell layers, for it to participate in Na+ transport. Previously, we found that we could detect an expression of ␣-ENaC protein only within S. granulosum in adult bullfrog skin (Takada et al., 2006). On the other hand, both ␣- and -ENaC mRNAs were found here to be expressed not only in the S. granulosum, but also in at least the upper S. spinosum (Fig. 2a and c). It seems unlikely that the ␣- and -ENaC mRNAs localized in the S. spinosum are involved in Na+ transport, and what function, if any, they might perform in that layer remains unknown. One question might be: “Do the cells of the Stratum spinosum produce ENaC mRNA without ENaC protein expression, or with only a small amount of the protein, before they differentiate to become cells of the Stratum granulosum?” Further research will be necessary to elucidate the role(s) of the ENaC mRNAs expressed in the S. spinosum. In conclusion, our results demonstrate that ␣- and -ENaC mRNAs are present in the sub-apical cells of adult bullfrog skin. It is known that ENaC protein does not need to be present in such cells for the skin to perform its Na+ -transport function, and so one interpretation is that sub-apical cells are already producing ENaC subunit mRNAs prior to the final step in their differentiation.
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