Functional asymmetry of phosphate transport and its regulation in opossum kidney cells: Phosphate transport

Abstract

The polarized distribution of phosphate (Pi) transport systems in a continuous renal cell line derived from opossum kidney (OK) was measured in monolayers grown on permeant filter support. When cultured on collagen-coated nitrocellulose filters, OK cells formed tight, functionally polarized monolayers. Three Pi transport systems were identified in these monolayers: one apical sodium (Na)-dependent system and two systems on the basolateral surface, one Na-dependent and one Na-independent. The apical system was high-affinity (Km = 0.4 mM Pi), low-capacity (Jmax = 1100 pmol Pi/mg protein per minute) with a Na:Pi stoichiometry greater than 1 (n = 3) and a high interaction coefficient (KNa = 105 mM Na). On the basolateral surface the Na-independent system comprised about 30% of the total Pi transport at this surface. Both basolateral systems were of low affinity (Km: Na-independent, 2.6 mM; Na-dependent, 5.2 mM) and high capacity (Jmax: Na-independent, 2100; Na-dependent, 2400 pmol/mg protein per minute). The basolateral Na-dependent system had a Nai stoichiometry of 1 and a relatively low interaction coefficient (KNa = 25 mM Na). Only the basolateral Na-independent system was inhibitable by 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). These results are compatible with a net vectorial transcellular transport of Pi from the apical through the basolateral cell surfaces. The presence of a basolateral Na-dependent system may reflect additional metabolic requirements that cannot be met only by apical influx. Taken together, these results demonstrate the ability to grow cell monolayers successfully, displaying polarized transport activities similar to in situ.