Forskolin increases apical sodium conductance in cultured toad kidney cells (A6) by stimulating membrane insertion

Abstract

The role of membrane traffic in the stimulation of apical Na+ permeability caused by increases in cytoplasmic cyclic AMP was assessed by measuring the effects of forskolin on transepithelial capacitance (CT), transepithelial conductance (GT), and short-circuit current (Isc) in A6 cultured toad kidney cells. Apical water permeability was probed by recording cell volume changes after reducing the osmolality of the apical bath. We found that forskolin does not increase the osmotic water permeability of the apical membrane of A6 cells, and thus does not stimulate the insertion of water channels. Comparison of the effects of forskolin and insulin on Na+ transport demonstrated that both agents produce reversible increases in CT, GT and Isc. GT and CT increased proportionally during the rising phase of the insulin response. However, a non-linear relationship between both parameters was recorded when forskolin was given in NaCl Ringer's solution. The relationship between CT and GT became linear after the effects of forskolin on Cl- conductances were eliminated by substituting Cl- by an impermeant anion. In contrast, in Cl--containing Na+-free solutions, the non-linearity became more pronounced. Successive additions of insulin and forskolin caused additive increases in CT. Because increases in CT and Na+ transport occurred in the absence of stimulation of water permeability and increases of CT and GT were directly proportional when Na+ was the major permeating ion across the apical membrane, we suggest that the increase in apical Na+ permeability in the presence of either forskolin or insulin is due to the insertion of channels residing in intracellular pools. In contrast, the increased Cl- permeability caused by forskolin may be related to the activation of channels already present in the membrane.

Role of SGK in hormonal regulation of epithelial sodium channel in A6 cells

“…There is increasing evidence that insulin and AVP control the number of ENaC channels in the plasma membrane rather than their open probability (3,7,16,24). SGK has also been shown to increase ENaC density in the plasma membrane of Xenopus oocytes (2,6,15), although a recent report suggested that SGK might in addition increase the open probability of ENaC (31).…”

Ankyrin G Expression Regulates Apical Delivery of the Epithelial Sodium Channel (ENaC)

“…Transepithelial resistance was measured by applying a 2-mV bipolar pulse and calculated using Ohm's law. In some experiments, I sc and total membrane capacitance were measured simultaneously with an automated voltage clamp system (designed by W. Van Driesche, KU Leuven, Leuven, Belgium) as described previously (42,43). Briefly, the equipment were comprised of two digital signal processing boards: one that recorded I sc and one that recorded epithelial capacitance (C T ).…”

Gastric inhibitory peptide, serotonin, and glucagon are unexpected chloride secretagogues in the rectal gland of the skate (Leucoraja erinacea)

“…The chambers were gassed with 95% O2-5% CO2 and kept at 18°C to maintain the pH of 7.4 -7.6. The voltage clamp and the recording system to acquire data were designed and constructed by W. Van Driessche (Katholieke Universiteit, Leuven, Belgium) and have been previously described (7,12,40). Membranes that reached a baseline Isc between 0 and 20 A/cm 2 and showed a steady conductance below 20 mS were used for experiments.…”