The role of the Ca2+-dependent phospholipase C delta1 isoform in neuronal osmosensitivity

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All mammals maintain the osmolality of their body fluids to within very narrow limits through mechanisms that regulate water intake (i.e. thirst) and the loss of water in urine. Urine volume is regulated primarily by vasopressin (VP), which is released from the magnocellular neurosecretory cells of the hypothalamus (MNCs). Acute increases in osmolality excite MNCs by causing them to shrink, leading to the activation of a mechanosensitive TRPV1 channel N-terminal variant (DeltaN TRPV1), which increases electrical activity. Sustained increases in osmolality lead to a structural and functional transformation of MNCs that includes cellular hypertrophy, rapid increases in the density of many channels and receptors on the MNC surface, and an increase in the expression of many genes. We have published data showing that increases in osmolality trigger activation of phospholipase C (PLC) in MNCs and that inhibition of PLC suppresses both MNC hypertrophy and the osmotic activation of DeltaN TRPV1 channels. PLC may therefore play key roles in both phases of MNC osmosensitivity. We also demonstrated that the osmotic activation of PLC is Ca2+-dependent, which led us to hypothesize that the isoform responsible is a member of the Ca2+-dependent PLC delta family. We have unpublished data showing that transgenic mice lacking PLC delta1 show a dramatic deficiency in their ability to respond appropriately to water deprivation. We hypothesize that the failure to increase VP release enables urine production when it should be suppressed. Furthermore, we have evidence that osmotic activation of DeltaN TRPV1 currents is almost completely suppressed in MNCs from PLC delta1 KO mice and that this may be due in part to the suppression of PLC-mediated enhancement of the actin cytoskeleton. Lastly, we have evidence that sustained increases in osmolality cause the translocation of DeltaN TRPV1 channels to the MNC plasma membrane, which may be an important part of the MNC adaptation to sustained increases in osmolality. We therefore propose to test the following four hypotheses: 1) that the defective osmoregulation in PLC delta1 KO mice is mediated by a deficiency in osmotically-evoked release of VP, 2) that PLC delta1 mediates the rapid osmotically-induced Ca2+-dependent activation of PLC, 3) that osmotic regulation of DeltaN TRPV1 channels will be diminished and osmotically-evoked changes in MNC firing will be lessened in PLC delta1 KO mice, and 4) that increased osmolality triggers the translocation of DeltaN TRPV1 channels to the MNC cell membrane. We will test these hypotheses using electrophysiological, immunocytochemical, and pharmacological approaches in rats and in PLC delta1 KO and wild type mice. The proposed experiments will elucidate the role of the PLC pathway in both the acute and sustained phases of MNC osmosensitivity. These experiments will make an vital contribution to our long-term goal of understanding of the mechanisms underlying osmoregulation.