Subject(s)
Epithelial Sodium Channels/metabolism , Epithelium/metabolism , Skin/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Sodium/metabolism , Animals , Biological Transport/drug effects , Biological Transport/physiology , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Epithelium/drug effects , Organ Culture Techniques , Ouabain/metabolism , Ouabain/pharmacology , Ranidae , Skin/drug effects , Skin Physiological Phenomena/drug effects , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitorsABSTRACT
The epithelial sodium channel (ENaC) has four subunits, namely α (alpha), ß (beta), γ (gamma) and δ (delta). The functional ENaC is formed by the combination of either αßγ or δßγ 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 αßγ and δßγ subunit combinations on the apical side. Though both types of subunit combination are present, the αßγ type was found to be more common than δßγ. 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.
Subject(s)
Amiloride/pharmacology , Evans Blue/pharmacology , Ranidae/physiology , Skin/drug effects , Skin/metabolism , Vasopressins/pharmacology , Animals , Epithelial Sodium Channels/genetics , Epithelial Sodium Channels/metabolism , Protein Subunits , Sodium/metabolismABSTRACT
The aim of the present study is to study whether the presence of K(+) in bathing media is required for the action of ADH to the ionic transport across the skin in the frog species Rana hexadactyla. lonic transport was measured as transepithelial potential difference (TEPD) and short circuit current (SCC) by using an indigenously developed computer based voltage-clamp technique. Addition of ADH (40 nM) on the serosal side significantly increased the TEPD and SCC with Normal Ringer (NR) on both sides. ADH had no effect subsequent to amiloride (100 µM) pre-treatment, which confirmed the ADH-induced Na(+) transport. Chloride also has a significant role in the development of TEPD. To determine the role of K(+), Potassium-free Ringer (KFR) was placed on both sides; addition of ADH had no effect consequently. Further experiments were carried out to find out which side of K(+) was required for the action of ADH. There was a lack of ADH effect with apical NR and serosal KFR, demonstrating that serosal K(+) is essential to activate Na(+), K(+)- ATPase. Similarly, the ADH effect was lacking with apical KFR and serosal NR that was the novel finding of this study. Due to the concentration gradient, the K(+) was secreted from serosal side to apical side through barium (1 mM) blockable K(+) channel. This study provides evidence that serosal as well as apical K(+) are necessary for the action of ADH.