Mechanism of Action of Anti Diuretic Hormone (ADH) On Transport across Frog-Skin

INTRODUCTION: The frog-skin is an ideal model to understand the various ion channels and transporters of the epithelium, which are involved in various ionic transport mechanisms to maintain intracellular ionic and osmotic concentration to perform vital functions of cells. They are studied by using specific blockers or stimulators of specific ionic conductance. AIM OF THE STUDY: To study the effect of ADH on transepithelial and transmembrane transport in frog skin. OBJECTIVES: A. Intact and nystatin-permeabilized frog skins. 1. Intact skin: An ionic transport activity. 2. Action of ADH on intact skin. 3. Effect of ADH on nystatin-permeabilized skin bathed in apical KGS -1 solution. 4. Effect of ADH on nystatin-permeabilized skin bathed in apical KR solution (Sodium -free solutions). 5. Effect of ADH on nystatin-permeabilized skin bathed in SGS (chloride-free solutions). 6. Comparison of nystatin effect in apical KGS-1 and KR solutions. 7. Comparison of effect of ADH on nystatin-permeabilized skin bathed in KGS-1 and KR solutions. B. Action of amiloride and ouabain on frog skin and the lack of effect of ADH subsequently. 8. Action of amiloride on intact skin and the lack of effect of ADH subsequently. 9. Action of ouabain on the intact skin and the lack of effect of ADH subsequently. 10. Effect of ouabain in nystatin-permeabilized frog-skin (KGS-1). MATERIALS AND METHODS: The method used to study the effect of ADH on frog skin was Electrophysiology. ELECTROPHYSIOLOGY: The short circuit current (SCC) and current-voltage relations were measured by an electrophysiological method. For this study on frog skin the following materials were used. 1. A modified Ussing chamber. 2. Chloride coated Silver electrodes. 3. Salt bridges. 4. A computer-based voltage clamp circuit. RESULTS: ADH actions on intact and nystatin-permeabilized frog skins and a simplistic understanding of the circuit model for frog-skin. Frog skin has been used extensively as a model for studying transepithelial transport. The ventral skin of frogs is equipped to absorb water and salt from the surroundings. Hans Ussing states that the apical membrane of the frog skin is water tight. Nevertheless, the apical membrane can express aquaporins and has vasotocin (equivalent of mammalian ADH) receptors. There is evidence that aquaporins are expressed in the apical membrane of ventral skin of frogs. Though found constitutively on the basolateral membrane, aquaporins get expressed in the apical membrane with vasotocin stimulation. In this study, we report our observations on the role of some transporters and channels in altering the electrophysiological properties of the ventral skin of frogs belonging to the species Rana hexadactyla, using voltage-clamp experiments. We have used ADH, amiloride and ouabain in addition to ion substitution to understand the circuit model of the frog-skin. SUMMARY AND CONCLUSION: Now considering the actions of ADH, the following results are important: A. INTACT FROG SKIN: 1. ADH makes the TEPD more negative, increases the SCC, but does not change the conductance in the intact skin. 2. It follows that ADH increases sodium transport without affecting the conductance. It can be argued therefore that ADH in this case does not have a direct effect on ENaC, but increases sodium transport by augmenting the sodiumpotassium pump or, augmenting the action of an electrogenic sodium transporter on the apical membrane. 3. After imposition of conditions that block sodium transport, namely amiloride and ouabain, ADH has no further action. This observation is more in line of augmentation of sodium pump activity in the basolateral membrane which is dependent on incoming sodium. B.NYSTATIN PERMEABILISED FROG SKIN: 4. The next step in building a circuit model for the frog-skin was to add to the data, observations in the nystatin-permeabilized skin where the apical membrane is permeabilized and therefore electrically non-existent and the only membrane capable of creating a potential difference in our model is the basolateral membrane. 5. In the nystatin-permeabilized skin, TEPD is essentially transmembrane PD across the basolateral membrane. 6. The conductance per se of the nystatin-permeabilized skin is higher in KR (chloride-rich) solution on the apical side as compared to the chloride-poor KGS: 1 solution (with only 20 mM/L chloride) corroborates the idea that there may be a chloride-absorptive conductance on the basolateral membrane