Activation of Ionic Channels by Deoxycholate in Frog and Human Cell Lines

Abstract

summary Humans, after extensive ileal resection, frequently suffer from diarrhoea, which may be due to an increased delivery of deoxycholate (DOC) to the large intestine. In the frog skin the addition of DOC (0·5 mÒ) to the apical side induced the activation of amiloride-sensitive Na¤ channels and an increase in the unidirectional Cl¦ fluxes. Here we used two established cell lines (AÜ and Cacoµ) to study the effect of DOC on ion channels at cell and membrane level using the patch-clamp technique. In AÜ cells subcultured directly on Petri dishes and studied in the whole-cell configuration, DOC induced an increase in cell conductance of 110·3 ± 4 pS pF¢ (N = 8) which was reduced to 89 ± 14 pS pF¢ (N = 8) by the addition of DIDS (0·5 mÒ). The absolute values of these two effects were not statistically different (P < 0·2). In Cacoµ cells, the addition of DOC (0·5 mÒ) induced, after 1 min, an increase in cell conductance of 583 ± 16 pS pF¢ (N = 8) which was reduced to 560·4 ± 16 pS pF¢ (N = 8) by DIDS (0·5 mÒ) and N_phenylanthranilic acid (DPC; 0·5 mÒ). The two values were not statistically different (P < 0·4). In Cacoµ cells subcultured under the same conditions, DOC induced an increase in cell conductance of 1710 ± 64 pS pF¢ (N = 6). Subsequent addition of amiloride (0·1 mÒ) reduced the cell conductance to 1558 ± 33 pS pF¢ (N = 6). These two mean values were statistically different allowing for an error of the second kind < 0·05. In cells in which DOC produced a conductance increase of 1010 ± 10 pS pF¢, gadolinium (0·5 mÒ) induced a fall in cell conductance of 1800 ± 10 pS pF¢. In Cacoµ cells, addition of DOC (0·5 mÒ) to the bath reversibly induced the appearance of or an increase in channel activity in patches studied in cell-attached and excised inside-out configuration. In inside-out experiments (N = 13) DOC (0·5 mÒ) induced the appearance of channel activity with conductances and reversal potentials (Er) of 27·7 ± 1·9 pS and 0·8 ± 5·7 mV, respectively. In cell-attached patches (N = 13) these values were 24·9 ± 4·4 pS and −18·1 ± 6·4 mV. In excised inside-out patches from Cacoµ cells, subjected to electrochemical gradients for Na¤, K¤ and Cl¦, (+85, −85 and 0 mV, respectively), addition of DOC also induced an increase in the baseline conductance and a shift in the reversal potential from values around +25 mV to values around 0 mV. Bile salts activated both anionic and cationic channels and did not require the presence of intracellular factors for these effects. We suggest that they act at the membrane level.introduction The occurrence of diarrhoea in humans after extensive ileal resections (Hofmann, 1967;Hofmann & Poley, 1969) has been attributed to the action of products resulting from bacterial degradation of bile acids in the colon. Such products would inhibit absorption or even stimulate secretion in the large intestine. The addition of deoxycholate (DOC) to the apical side of frog skin stimulated transepithelial sodium transport (Lima et al. 1996) by activating amiloride-sensitive sodium channels. In the...

Excitatory Neurotoxic Properties of Pontamine Sky Blue Make It a Useful Tool for Examining the Functions of Focal Brain Parts

“…Synthetic detergents (Triton X-100 and ␤-octyl glucoside), however, reduce bilayer stiffness and reversibly shift the inactivation to negative potentials. Moreover, Mauricio and Ferreira [35] demonstrated that a bile salt, deoxycholate, activates both anionic and cationic channels in cultured cell lines from the frog kidney (A6) and from human colonic adenocarcinoma (Caco2). PSB has been presumed to have an almost planar conformation [12], which is well known to be the conformation of bile salts.…”