Electrogenic Cl- Absorption by Amphiuma Small Intestine: Dependence on Serosal Na+ from Tracer and Cl- Microelectrode Studies

The Na+ requirement for active, electrogenic Cl- absorption by Amphiuma small intestine was studied by tracer techniques and double-barreled Cl- -sensitive microelectrodes. Addition of Cl- to a Cl- -free medium bathing in vitro intestinal segments produced a saturable (Km = 5.4 mM) increase in short-circuit current (ISC) which was inhibitable by 1 mM SITS. The selectivity sequence for the anion-evoked current was Cl- = Br- greater than SCN- greater than NO-3 greater than F- = I-. Current evoked by Cl- reached a maximum with increasing medium Na concentration (KM = 12.4 mM). Addition of Na+, as Na gluconate (10 mM), to mucosal and serosal Na+-free media stimulated the Cl- current and simultaneously increased the absorptive Cl- flux (JCl m----s) and net flux ( JClnet ) without changing the secretory Cl- flux ( JCls ----m). Addition of Na+ only to the serosal fluid stimulated JClm ----s much more than Na+ addition only to the mucosal fluid in paired tissues. Serosal DIDS (1 mM) blocked the stimulation. Serosal 10 mM Tris gluconate or choline gluconate failed to stimulate JClm ----s. Intracellular Cl- activity ( aiCl ) in villus epithelial cells was above electrochemical equilibrium indicating active Cl- uptake. Ouabain (1 mM) eliminated Cl- accumulation and reduced the mucosal membrane potential (psi m) over 2 to 3 hr. In contrast, SITS had no effect on Cl- accumulation and hyperpolarized the mucosal membrane. Replacement of serosal Na+ with choline eliminated Cl- accumulation while replacement of mucosal Na+ had no effect. In conclusion by two independent methods active electrogenic Cl- absorption depends on serosal rather than mucosal Na+. It is concluded that Cl- enters the cell via a primary (rheogenic) transport mechanism. At the serosal membrane the Na+ gradient most likely energizes H+ export and regulates mucosal Cl- accumulation perhaps by influencing cell pH or HCO-3 concentration.