Effects of Evans Blue and Amiloride on Anti-Diuretic Hormone (ADH)-Induced Sodium Transport Across Frog (Rana hexadactyla) Skin

Abstract

The epithelial sodium channel (ENaC) has four subunits, namely α (alpha), β (beta), γ (gamma) and δ (delta). The functional ENaC is formed by the combination of either αβγ or δβγ subunits. The aim of the present study is to determine the combination of ENaC subunits predominant on the apical side of the frog skin, and the effect of ADH on sodium transport though these two ENaCs subunit combinations. The ventral abdominal skin of the frog, Rana hexadactyla was mounted in an Ussing-type chamber. The voltage-clamp method was performed to measure the ionic transport across the frog skin with normal Ringer solution (NR) on both sides. Evans blue (300 µM) and amiloride (100 µM) were added to the NR on the apical side and ADH (40 nM) was added on the serosal side. Statistical significance was analyzed by Student's paired t-test and repeated-measures ANOVA, P < 0.05 was considered significant. This study suggests that the ENaC of the frog skin consist of both αβγ and δβγ subunit combinations on the apical side. Though both types of subunit combination are present, the αβγ type was found to be more common than δβγ. ADH increases the sodium transport across the frog skin. The effect of ADH on sodium transport is achieved through the combination of δ-subunits, not through the combination of a-subunits in the skin of Pana hexadactyla.