Anion-sensitive Sodium Conductance in the Apical ... - BioMedSearch
Anion-sensitive Sodium Conductance in the Apical Membrane of Toad Urinary Bladder J A V I E R N A R V A R T E and A R T H U R L. FINN From the Departmentof Medicineand the Department of Physiology,Universityof North Carolina School of Medicine, Chapel Hill, North Carolina 27514 ABSTRACT Membrane potentials and the electrical resistance of the cell membranes and the shunt pathway of toad urinary bladder epithelium were measured using microelectrode techniques. These measurements were used to compute the equivalent electromotive forces (EMF) at both cell borders before and after reductions in mucosal C1- concentration ([C1]m). The effects of reduction in [C1]m depended on the anionic substitute. Gluconate or sulfate substitutions increased transepithelial resistance, depolarized membrane potentials and EMF at both cell borders, and decreased cell conductance. Iodide substitutions had opposite effects. Gluconate or sulfate substitutions decreased apical Na conductance, whereas iodide replacements increased it. When gluconate or sulfate substitutions were brought about in the presence of amiloride in the mucosal solution, apical membrane potential and EMF hyperpolarized with no significant changes in basolateral membrane potential or EMF. It is concluded that: (a) apical Na conductance depends, in part, on the anionic composition of the mucosal solution, (b) there is a CI- conductance in the apical membrane, and (c) the electrical communication between apical and basolateral membranes previously described is mediated by changes in the size of the cell Na pool, most likely by a change in sodium activity. Singer a n d C i v a n (1971) showed t h a t CI substitutions in the solutions b a t h i n g t o a d u r i n a r y b l a d d e r resulted in significant changes in mucosa-to-serosa s o d i u m flux a n d in the electrical p a r a m e t e r s o f the tissue. T h e ability o f anions to m o d i f y N a t r a n s p o r t was related to the position o f the a n i o n in the lyotropic series. H o w e v e r , it c o u l d not be established w h e t h e r the anions were acting on s o d i u m e n t r y t h r o u g h the apical m e m b r a n e or u p o n a c t i v e ' N a extrusion at the basolateral side (Singer a n d Civan, 1971). In addition, it has been c o n c l u d e d from studies o f transepithelial fluxes o f isotopes that the m o v e m e n t s o f C I - across the tissue are passive a n d p a r a c e l l u l a r (Saito et al., 1974; Finn a n d Bright, 1978) a n d from e x p e r i m e n t s involving ~CI e q u i l i b r a t i o n b e t w e e n cellular a n d e x t r a c e l l u l a r c o m p a r t m e n t s that little or no chloride enters the cell from the mucosal m e d i u m ( M a c K n i g h t , 1977). O n the o t h e r h a n d , an active mucosa-to-serosa CI flux was f o u n d w h e n K was r e m o v e d from the j. GEN. PHYSIOL.9 The Rockefeller University Press - 0022-1295/80/07/0069/13 $1.00 Volume 76 July 1980 69-81
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serosal m e d i u m (Finn et al., 1967) a n d when maneuvers that included removing Na or a d d i n g o u a b a i n or amiloride to the solutions b a t h i n g toad urinary bladders (Soboslai et al., 1977) were carried out; thus, at least u n d e r those conditions, CI- p r e s u m a b l y enters the cells. W e have recently shown that the apical electromotive force in toad urinary b l a d d e r is a direct function of sodium selectivity, indicating that the apical m e m b r a n e is permeable to ions other t h a n Na + (Narvarte a n d Finn, 1980). In this paper, we report the effect of mucosal chloride substitutions in the mucosal m e d i u m on the electrical parameters of toad u r i n a r y bladder. O u r results show that there is a C I - c o n d u c t a n c e in the mucosal m e m b r a n e a n d that apical sodium cond u c t a n c e is sensitive to the anionic composition of the mucosal m e d i u m . T h e y also suggest t h a t the c o m m u n i c a t i o n between apical a n d basolateral membranes previously described (Reuss a n d Finn, 1975 b) is m e d i a t e d by changes in cell Na activity or some other function of the size of the sodium transport pool. METHODS
Mexican toads (Bufo marinus) were obtained from the Charles P. Chase Co. (Miami, Fla.), the Pet Farm (Miami, Fla.), or the Jacques Well Co. (Rayne, La) and kept in running tap water. Bladders were excised and mounted mucosal side upward in a Lucite chamber, as previously described (Reuss and Finn, 1974), with Ringer's solution bathing both sides of the tissue. A nylon mesh was used to support the serosal side of the bladder, and a small negative pressure was applied to the lower half of the chamber to change continuously the bathing solution and keep the tissue against its support.
Solutions Standard Ringer's solution had the following composition (mM): NaCI 110, KCI 2.5, NaHCO3 2.4, CaCI2 0.9, glucose 5.5, pH about 8.5, gassed with room air. CI was replaced with sulfate, gluconate, or iodide. Sucrose was used to keep constant osmolality in sulfate replacements. Amiloride (a gift from Merck, Sharp and Dohme Research Laboratories, West Point, Pa.) was dissolved in the Ringer's solution to a final concentration of 10-5 o r 10 -4 M.
Electrical Measurements (a) Transepithelial potential (Vr~) and resistance ( R t ) . Vrm was measured with a highimpedance electrometer connected to both bathing media with Ag-AgCI pellets and agar-Ringer bridges. Corrections for liquid junction potentials were made. Rt was measured from the change in V~ produced by a transepithelial DC pulse of 2.5 to 17 pA/cm 2. (b) Cell membrane potential. Apical (Vmc) and basolateral (V~) membrane potentials were measured with glass microelectrodes prepared by pulling glass tubing (1 mm o.d., 0.6 mm i.d.) threaded with fiber glass and filled with 4 M potassium acetate. The microelectrodes were beveled to tip resistances of 10-30 Mf~ and tip potentials were
69
70
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
9 VOLUME
76 9 1980
serosal m e d i u m (Finn et al., 1967) a n d when maneuvers that included removing Na or a d d i n g o u a b a i n or amiloride to the solutions b a t h i n g toad urinary bladders (Soboslai et al., 1977) were carried out; thus, at least u n d e r those conditions, CI- p r e s u m a b l y enters the cells. W e have recently shown that the apical electromotive force in toad urinary b l a d d e r is a direct function of sodium selectivity, indicating that the apical m e m b r a n e is permeable to ions other t h a n Na + (Narvarte a n d Finn, 1980). In this paper, we report the effect of mucosal chloride substitutions in the mucosal m e d i u m on the electrical parameters of toad u r i n a r y bladder. O u r results show that there is a C I - c o n d u c t a n c e in the mucosal m e m b r a n e a n d that apical sodium cond u c t a n c e is sensitive to the anionic composition of the mucosal m e d i u m . T h e y also suggest t h a t the c o m m u n i c a t i o n between apical a n d basolateral membranes previously described (Reuss a n d Finn, 1975 b) is m e d i a t e d by changes in cell Na activity or some other function of the size of the sodium transport pool. METHODS
Mexican toads (Bufo marinus) were obtained from the Charles P. Chase Co. (Miami, Fla.), the Pet Farm (Miami, Fla.), or the Jacques Well Co. (Rayne, La) and kept in running tap water. Bladders were excised and mounted mucosal side upward in a Lucite chamber, as previously described (Reuss and Finn, 1974), with Ringer's solution bathing both sides of the tissue. A nylon mesh was used to support the serosal side of the bladder, and a small negative pressure was applied to the lower half of the chamber to change continuously the bathing solution and keep the tissue against its support.
Solutions Standard Ringer's solution had the following composition (mM): NaCI 110, KCI 2.5, NaHCO3 2.4, CaCI2 0.9, glucose 5.5, pH about 8.5, gassed with room air. CI was replaced with sulfate, gluconate, or iodide. Sucrose was used to keep constant osmolality in sulfate replacements. Amiloride (a gift from Merck, Sharp and Dohme Research Laboratories, West Point, Pa.) was dissolved in the Ringer's solution to a final concentration of 10-5 o r 10 -4 M.
Electrical Measurements (a) Transepithelial potential (Vr~) and resistance ( R t ) . Vrm was measured with a highimpedance electrometer connected to both bathing media with Ag-AgCI pellets and agar-Ringer bridges. Corrections for liquid junction potentials were made. Rt was measured from the change in V~ produced by a transepithelial DC pulse of 2.5 to 17 pA/cm 2. (b) Cell membrane potential. Apical (Vmc) and basolateral (V~) membrane potentials were measured with glass microelectrodes prepared by pulling glass tubing (1 mm o.d., 0.6 mm i.d.) threaded with fiber glass and filled with 4 M potassium acetate. The microelectrodes were beveled to tip resistances of 10-30 Mf~ and tip potentials were
