INFLUENCE OF FILTER SUPPORTS ON TRANSPORT CHARACTERISTICS OF CULTUREDA6 KIDNEY-CELLS

Amphibian A6 kidney cells grown on Anocell filters developed a transep ithelial potential difference of 37 mV, a short-circuit current (I(sc) ) of 8 muA/cm2, and a resistance of 5 kOMEGA.cm2. Other observations s uggested a viable arginine vasopressin (AVP) V2 receptor-second messen ger pathway in these cells: 1) AVP increased both an amiloride-sensiti ve I(sc) and adenosine 3',5'-cyclic monophosphate (cAMP) formation, an d 2) scanning electron micrographs of A6 cells cultured on Anocell and ICN Cellagen filters demonstrated increased microvilli formation on t he apical surface after AVP action. However, osmotic water flow (J(v)) across A6 cells on filter supports was not altered by either AVP or t he permeable cAMP analogue dibutyryl cAMP (osmotic permeability coeffi cient = 2.5 x 10(-3) cm/s). Diffusional water flow (J(dw)) measured ac ross A6 cells on Anocell filters using tritiated water (THO) ranged fr om 6 to 8 mul . min-1 . cm-2. Neither AVP nor the membrane-permeabiliz ing agents amphotericin B and digitonin were able to enhance unidirect ional THO fluxes, although amphotericin B increased the I(sc). These r esults suggested that there was an unknown barrier in series with the A6 cells limiting water flow. THO fluxes across filter supports, witho ut an associated cellular monolayer, poe J(dw) values in the range 7-3 0 mul . min-1 . cm-2. J(v) across the bare filter support was in the r ange of 0.3-1.5 mul . min-1 . cm-2, similar to that measured in the pr esence of an A6 monolayer. These observations suggest that the filter may be rate limiting for transepithelial water flow. Chloride fluxes a cross Anocell filters showed a stable value of 5 mueq . h-1 . cm-2. Th ese observations exhibit the limitations of filter supports in the stu dy of transport phenomena in cultured cells.