NA+ P-I COTRANSPORT ALTERS RAPIDLY CYTOSKELETAL PROTEIN POLYMERIZATION DYNAMICS IN OPOSSUM KIDNEY-CELLS/

We studied with biochemical and immunofluorescent techniques the inter actions between the actin microfilament and tubulin microtubule cytosk eleton and Na+/P-i, co-transport in opossum kidney cells, a line with proximal tubular characteristics. On brief (5 min) incubation of the c ells with a low (0.1 mM) concentration of P-i, a rapid F-actin depolym erization takes place, which fails to occur in cells incubated under s imilar conditions with 1 mM P-i. The disassembly of actin microfilamen ts could be quantitatively expressed as a 33% increase in the ratio of monomeric G-actin to polymerized F-actin (G/F-actin ratio from 0.80+/ -0.03 to 1.06+/-0.06, n = 28, P < 0.01), owing to a significant decrea se in the latter. Under these conditions microfilaments were also mark edly destabilized, as shown by their diminished resistance to graded c ytochalasin B concentrations. In addition, incubation of opossum kidne y cells with low P-i concentrations (0.1 mM) resulted within 5 min in a substantial depolymerization of microtubules, shown by immunofluores cence microscopy and measured as a 70.9+/-6.9% (n = 11, P < 0.01) decr ement by immunoblot analysis. These changes, which occur only when ext racellular P-i concentrations are kept low, seem to be related to a si gnificant increase within 5 min in the rate of cellular Pi uptake by 2 5.5% under these conditions. The shifts in the dynamic equilibria betw een monomeric and polymerized actin and tubulin in response to cellula r P-i uptake were transient, being fully reversible within 30 min. Mor eover, the effect of P-i seemed to be specific because inhibition of i ts uptake by phosphonoformic acid blunted microtubular disassembly mar kedly. In contrast, measurement of P-i uptake in the presence of agent s known to stabilize cytoskeletal structures showed a substantial decr ease with phallacidin, which stabilizes microfilaments, whereas the mi crotubule stabilizer taxol had no apparent effect. These results indic ate that acute alterations in the polymerization dynamics and stabilit y of both microfilaments and microtubules are involved in the modulati on of Na+/P-i co-transport and suggest important cytoskeletal particip ation in proximal tubular transport functions.