ROLE OF F-ACTIN IN THE ACTIVATION OF NA-K+-CL- COTRANSPORT BY FORSKOLIN AND VASOPRESSIN IN MOUSE KIDNEY CULTURED THICK ASCENDING LIMB CELLS()

The influence of microtubules and F-actin on Na+-K+-Cl- cotransport wa s investigated in cultured cells derived from outer-medullary thick as cending limb tubules microdissected from the mouse kidney. The culture d cells contained Tamm-Horsfall protein, produced cAMP in response to dD-arginine vasopressin (dD-AVP), isoproterenol, prostaglandin E(2) an d forskolin (FK), and exhibited an ouabain-resistant furosemide-sensit ive (Or-Fs) component of Rb-86(+) influx mediated by the Na+-K+-Cl- co transporter. Both FK and dD-AVP stimulated the Or-Fs component of Rbinflux. Neither agent altered the tubulin and cytokeratin networks nor the shape of the tight junction using a specific anti-ZO-1 antibody. In contrast, they did induce a marked redistribution of F-actin to the periphery of the cells delineating the tight junctions. Preincubation of the cells with nocodazole, to disrupt microtubules, did not alter the FK-or dD-AVP-elicited Or-Fs Rb+ influx. In contrast, phalloidin an d NBD-phallicidin, which stabilize F-actin, markedly impaired the stim ulation of Na+-K+-Cl(-)cotransport by FK or dD-AVP, without affecting the Na+-K+ ATPase pumps and the rate constant of Cl-36(-) and Rb-86(+) efflux. These results strongly suggested that cAMP-stimulated Na+-K+- Cl- cotransport is linked to F-actin in renal TAL cells.