Microfilament network is needed for the endocytosis of water channels and not for apical membrane insertion upon vasopressin action

In the current study, a novel role for the microfilaments in vasopressin-induced water transport in toad urinary bladders, a popular model for the mammalian collecting duct, was established. Vasopressin-induced water transport was not affected by cytochalasin D (CD, 20 μM) or letrunculin 15 (Lat B, 0.5-2 μM), microfilament-disrupting reagents, suggesting that the initial trafficking of vesicles containing water channels end insertion of membranes into the epical membrane are microfilament-independent. After the removal of vasopressin, bladders treated with CD or Lat B continued to transport water at least 2-3-fold greater than those that received the vehicle. Furthermore, the enhanced water transport was inhibited by HgCl₂ (1 mM), a potent inhibitor of water channel-mediated water flow, suggesting that the enhanced water flow was through water channels. In addition, Lat B and CD inhibited vasopressin-induced endocytosis of horseradish peroxidase (HRP), a fluid endocytotic marker. These results suggested that although microfilaments are not needed for the initial trafficking of water channels to the epical side, the microfilament network is essential for the retrieval of water channels following their insertion into epical membranes.