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 was investigated in cultured cells derived from outer-medullary thick ascending limb tubules microdissected from the mouse kidney. The cultured cells contained Tamm-Horsfall protein, produced cAMP in response to dD-arginine vasopressin (dD-AVP), isoproterenol, prostaglandin E2 and forskolin (FK), and exhibited an ouabain-resistant furosemidesensitive (Or-Fs) component of ⁸⁶Rb⁺ influx mediated by the Na⁺-K⁺-Cl^- cotransporter. Both FK and dD-AVP stimulated the Or-Fs component of Rb⁺ influx. 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 and NBD-phallicidin, which stabilize F-actin, markedly impaired the stimulation of Na⁺-K⁺-Cl^- cotransport by FK or dD-AVP, without affecting the Na⁺-K⁺ ATPase pumps and the rate constant of ³⁶Cl^- and ⁸⁶Rb⁺ efflux. These results strongly suggested that cAMP-stimulated Na⁺-K⁺-Cl^- cotransport is linked to F-actin in renal TAL cells.