Influence of filter supports on transport characteristics of cultured A6 kidney cells

Abstract

Amphibian A6 kidney cells grown on Anocell filters developed a transepithelial potential difference of 37 mV, a short-circuit current (Isc) of 8 microA/cm2, and a resistance of 5 k omega.cm2. Other observations suggested a viable arginine vasopressin (AVP) V2 receptor-second messenger pathway in these cells: 1) AVP increased both an amiloride-sensitive Isc and adenosine 3',5'-cyclic monophosphate (cAMP) formation, and 2) scanning electron micrographs of A6 cells cultured on Anocell and ICN Cellagen filters demonstrated increased microvilli formation on the apical surface after AVP action. However, osmotic water flow (JV) across A6 cells on filter supports was not altered by either AVP or the permeable cAMP analogue dibutyryl cAMP (osmotic permeability coefficient = 2.5 x 10(-3) cm/s). Diffusional water flow (Jdw) measured across A6 cells on Anocell filters using tritiated water (THO) ranged from 6 to 8 microliters.min-1.cm-2. Neither AVP nor the membrane-permeabilizing agents amphotericin B and digitonin were able to enhance unidirectional THO fluxes, although amphotericin B increased the Isc. These results suggested that there was an unknown barrier in series with the A6 cells limiting water flow. THO fluxes across filter supports, without an associated cellular monolayer, gave Jdw values in the range 7-30 microliters.min-1.cm-2. Jv across the bare filter support was in the range of 0.3-1.5 microliters.min-1.cm-2, similar to that measured in the presence of an A6 monolayer. These observations suggest that the filter may be rate limiting for transepithelial water flow. Chloride fluxes across Anocell filters showed a stable value of 5 mu eq.h-1.cm-2. These observations exhibit the limitations of filter supports in the study of transport phenomena in cultured cells.