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

Abstract

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.

Living with a leaky skin: upregulation of ion transport proteins during sloughing

“…In addition, an increase in subunit abundance does not always indicate an increase in functional activity of the whole protein (Sardella and Kültz, 2009;Reilly et al, 2011). For example, the ENaC subunits (α-, β-and γ-subunits) alone cannot induce amiloride-sensitive currents (Canessa et al, 1994), and in bullfrog (Rana catesbeiana) tadpoles, ENaC is expressed in the skin but not in a functional state (no amiloride-blockable Na + transport present) until metamorphosis (Takada et al, 2006).…”

Frog skin epithelium: Electrolyte transport and chytridiomycosis

“…The electroneutral, absorption of NaCl generates an osmotic driving force that facilitates uptake of water across the skin. The molecular identity of these Na + -selective channels was not confirmed for almost 40 years after Ussing’s studies (Canessa et al, 1994, Takada et al, 2006). …”

Immunolocalization and mRNA expression of the epithelial Na+ channel α-subunit in the kidney and urinary bladder of the marine toad, Bufo marinus, under hyperosmotic conditions

“…In addition, the molecular mass of 84-kDa protein is reduced to 72-kDa by digestion treatment with PNGase F. The 72-kDa molecular mass derived by SDS-PAGE is consistent with the molecular mass predicted on the basis of the amino-acid sequence of the Bufo ENaC α-subunit. Recently, Takada et al (2006) have reported an 82-kDa band immunostained in extracts from the skin and kidney of Rana catesbeiana by means of an antibody raised against the Rana ENaC α-subunit. The 82-kDa band is presumed to be a glycosylated form, like the glycosylated 85-kDa band reported in A6 cells (Alvarez de la Rosa et al 2002).…”

“…In amphibians, cDNAs of ENaC subunits have recently been isolated and characterized in the A6 renal cells of Xenopus laevis and in the skin of Rana catesbeiana (Puoti et al 1995;Jensik et al 2002). Immunohistochemically, the presence of ENaC has been indicated in toad urinary bladder (Kleyman et al 1994) and frog skin (Takada et al 2006). However, the distribution and cellular localization of ENaC in amphibian renal nephrons have not been addressed by immunohistochemistry, although the presence of electrical characteristics of amiloride-sensitive Na + channels has been suggested in Ambystoma distal nephron and toad collecting duct (Stoner et al 1995;Møbjerg et al 2004).…”