TY - JOUR

T1 - Proton pump activity is required for active uptake of chloride in isolated amphibian skin exposed to freshwater

AU - Jensen, Lars Jørn

AU - Willumsen, Niels J.

AU - Larsen, Erik Hviid

N1 - Keywords Active Cl– uptake Æ Proton pump activity Æ V-ATPase Æ Anion exchanger Æ Amphibian skin

PY - 2002

Y1 - 2002

N2 - Net proton secretion and unidirectional chloride fluxes were measured in isolated skin of toads (Bufo bufo) and frogs (Rana esculenta) mounted in an Ussing chamber and exposed to a Ringer's solution on the serosal side and a freshwater-like solution (1-3 mM Cl-) on the external side. Active proton secretion was 34.2-2.0 pmol·cm-2·s-1 (n=18) in frog skin, and 16.7-1.7 pmol·cm-2·s-1 (n=10) in toad skin. Proton secretion by toad skin was dependent on the transepithelial potential (VT), and an amiloride-insensitive short-circuit current was stimulated by exogenous CO2/HCO3-, indicating the presence of a rheogenic proton pump. Cl- influx was 37.4-7.5 pmol·cm-2·s-1 (n=14) in frog skin and 19.5-3.5 pmol·cm-2·s-1 (n=11) in toad skin. In toad skin, the mean Cl- flux ratio was larger than expected for simple electro-diffusion. In 8 of 11 sets of paired skins, influx was greater than the efflux indicating active uptake of Cl-. Cl- influx in toad skin was unaffected by large perturbations (100-150 mV) of VT, which was accomplished by adding amiloride to the outer bath under open circuit conditions. A component of the Cl- efflux seemed to be dependent on VT. 4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS; 0.3 mM or 1.3 mM) inhibited Cl- influx and, surprisingly, increased Cl- efflux in toad skin. Influx and efflux of Cl- in toad skin were highly dependent on the external [Cl-] in the freshwater range (0.1-4 mM). 36Cl- influx decreased whereas the total Cl- efflux increased as a function of external [Cl-]. These data indicate the presence of a DIDS-sensitive, electroneutral carrier mechanism with an external binding site for Cl-. Ethoxzolamide (100 µM), an inhibitor of carbonic anhydrase, reduced proton secretion and Cl- influx in frog skin. Concanamycin A (0.1-10 µM), a specific vacuolar-type proton pump (V-ATPase) inhibitor, significantly reduced proton secretion in frog skin. In addition, concanamycin A (1 µM) significantly reduced Cl- influx in frog skin. We suggest that the active proton secretion and Cl- influx are coupled. We hypothesise that an apical V-ATPase is capable of energising active Cl- uptake in fresh water by creating a favourable gradient for an apical HCO3- exit in exchange for external Cl-. The data also suggest that a carbonic anhydrase activity provides H+ and HCO3- for apically co-expressed proton pumps and Cl-/HCO3- exchangers.

AB - Net proton secretion and unidirectional chloride fluxes were measured in isolated skin of toads (Bufo bufo) and frogs (Rana esculenta) mounted in an Ussing chamber and exposed to a Ringer's solution on the serosal side and a freshwater-like solution (1-3 mM Cl-) on the external side. Active proton secretion was 34.2-2.0 pmol·cm-2·s-1 (n=18) in frog skin, and 16.7-1.7 pmol·cm-2·s-1 (n=10) in toad skin. Proton secretion by toad skin was dependent on the transepithelial potential (VT), and an amiloride-insensitive short-circuit current was stimulated by exogenous CO2/HCO3-, indicating the presence of a rheogenic proton pump. Cl- influx was 37.4-7.5 pmol·cm-2·s-1 (n=14) in frog skin and 19.5-3.5 pmol·cm-2·s-1 (n=11) in toad skin. In toad skin, the mean Cl- flux ratio was larger than expected for simple electro-diffusion. In 8 of 11 sets of paired skins, influx was greater than the efflux indicating active uptake of Cl-. Cl- influx in toad skin was unaffected by large perturbations (100-150 mV) of VT, which was accomplished by adding amiloride to the outer bath under open circuit conditions. A component of the Cl- efflux seemed to be dependent on VT. 4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS; 0.3 mM or 1.3 mM) inhibited Cl- influx and, surprisingly, increased Cl- efflux in toad skin. Influx and efflux of Cl- in toad skin were highly dependent on the external [Cl-] in the freshwater range (0.1-4 mM). 36Cl- influx decreased whereas the total Cl- efflux increased as a function of external [Cl-]. These data indicate the presence of a DIDS-sensitive, electroneutral carrier mechanism with an external binding site for Cl-. Ethoxzolamide (100 µM), an inhibitor of carbonic anhydrase, reduced proton secretion and Cl- influx in frog skin. Concanamycin A (0.1-10 µM), a specific vacuolar-type proton pump (V-ATPase) inhibitor, significantly reduced proton secretion in frog skin. In addition, concanamycin A (1 µM) significantly reduced Cl- influx in frog skin. We suggest that the active proton secretion and Cl- influx are coupled. We hypothesise that an apical V-ATPase is capable of energising active Cl- uptake in fresh water by creating a favourable gradient for an apical HCO3- exit in exchange for external Cl-. The data also suggest that a carbonic anhydrase activity provides H+ and HCO3- for apically co-expressed proton pumps and Cl-/HCO3- exchangers.

U2 - 10.1007/s00360-002-0276-x

DO - 10.1007/s00360-002-0276-x

M3 - Journal article

VL - 172

SP - 503

EP - 511

JO - Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology

JF - Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology

SN - 0174-1578

ER -