Hallpike & Cairns VP V 1 V 2 R V 2 R V 2 R The membranous labyrinth is composed of the endo and perilymphatic spaces. The endolymphatic space is filled with endolymph, a K-rich and positively polarized fluid, whereas the surrounding perilymphatic space is filled with perilymph, with a composition similar to the usual extracellular fluid. The homeostasis of the inner ear fluid is essential for maintenance of audio-vestibular function. Since the discovery of aquaporin water channels , it has become known that these channels play a crucial role in inner ear fluid homeo-stasis. In this paper, experimental evidence to support the above-mentioned hypothesis will be presented mainly based on our recent studies.
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 microM) or latrunculin B (Lat B, 0.5-2 microM), microfilament-disrupting reagents, suggesting that the initial trafficking of vesicles containing water channels and insertion of membranes into the apical 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 apical side, the microfilament network is essential for the retrieval of water channels following their insertion into apical membranes.
Analysis of the signal transduction cascade of vasopressin-induced increase in intracellular Ca2+ concentration ([Ca2+]i) in LLC-PK1 cells was performed. First, a comparison of the effect of vasopressin on [Ca2+]i in LLC-PK1 cells with that produced in rat hepatocytes was performed [an intracellular mobilizing mechanism involving a V1 receptor coupled to the production of inositol 1,4,5-trisphosphate (IP3)]. Second, the effect of known inhibitors of intracellular Ca2+ mobilization on vasopressin Ca2+ response in LLC-PK1 cells was studied. Vasopressin induced a transient increase in [Ca2+]i in both LLC-PK1 cells and hepatocytes. In contrast to the single [Ca2+]i spike seen in LLC-PK1 cells, vasopressin induced an average of two to three [Ca2+]i spikes in hepatocytes. The V1 antagonist (Pmp1-O-Me-Tyr2-[Arg8]vasopressin, 1 microM) abolished vasopressin Ca2+ response in both cell types. Inhibitors of intracellular Ca2+ mobilization, thapsigargin (5 microM) and U-73122 (3 microM), abolished the Ca2+ response by vasopressin in LLC-PK1 cells. The results suggest that vasopressin-induced increase in [Ca2+]i in LLC-PK1 cells is mediated via a V1-like receptor and involves the mobilization of intracellular Ca2+ through an IP3- or thapsigargin-sensitive Ca2+ pool.
The involvement of protein kinase C (PKC) in vasopressin‐induced effects on renal water reabsorption is still unresolved. Activation of PKC can be detected by its translocation from the cytosol (C) to the plasma membrane (PM). In LLC‐PK1 cells, the redistribution of PKCα, a predominant isoform of PKC detected, was studied utilizing western blotting after stimulation with vasopressin. Vasopressin (100 mU/ml) failed to induce a translocation of PKCα from the C to the PM. By contrast, phorbol myristate acetate (PMA, 200 nM), a potent activator of PKC, induced a relocalization of PKCα from the C to the PM. After 2 hours of treatment of cells with PMA, PKCα was predominantly detected in the PM and absent from the C. These results suggest that the signal transduction pathway of vasopressin in LLC‐PK1 cells does not involve PKCα activation and translocation.
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