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

所有哺乳动物都通过调节水摄取(即口渴)和尿液水分流失的机制，将体液的渗透压维持在非常有限的范围内。尿量主要由加压素(VP)调节，加压素由下丘脑(MNC)的大细胞神经分泌细胞释放。渗透压的急剧增加通过使单核细胞收缩来兴奋它们，导致机械敏感的TRPV1通道N末端变体(Deltan TRPV1)的激活，从而增加电活动。持续的渗透压升高导致MNC结构和功能的改变，包括细胞肥大，MNC表面许多通道和受体密度的快速增加，以及许多基因表达的增加。我们已经发表的数据表明，渗透压的增加触发了MNC中磷脂酶C(PLC)的激活，而抑制PLC既抑制了MNC肥大，也抑制了Deltan TRPV1通道的渗透激活。因此，PLC可能在MNC渗透敏感性的两个阶段中发挥关键作用。我们还证明了PLC的渗透激活是钙依赖的，这使得我们假设负责的异构体是钙依赖的PLC Delta家族的成员。我们有未发表的数据表明，缺乏PLC Delta1的转基因小鼠在对缺水做出适当反应的能力方面表现出戏剧性的缺陷。我们假设，未能增加VP的释放使尿液在本应被抑制的时候产生。此外，我们有证据表明，在PLC Delta1 KO小鼠的MNC中，Deltan TRPV1电流的渗透激活几乎完全被抑制，这可能部分是由于抑制了PLC介导的肌动蛋白细胞骨架的增强。最后，我们有证据表明渗透压持续升高导致Deltan TRPV1通道移位到MNC质膜，这可能是MNC适应渗透压持续升高的重要部分。因此，我们建议检验以下四个假设：1)PLC Delta1 KO小鼠的渗透调节缺陷是由渗透压诱发的VP释放不足介导的；2)PLC Delta1介导渗透诱导的快速钙依赖的PLC激活；3)PLC Delta1 KO小鼠Deltan TRPV1通道的渗透调节减弱，渗透诱发的MNC放电变化将减少；4)渗透压增加触发Deltan TRPV1通道移位到MNC细胞膜。我们将在大鼠、PLC Delta1 KO和野生型小鼠身上使用电生理学、免疫细胞化学和药理学方法来验证这些假说。拟议的实验将阐明PLC途径在MNC渗透敏感性的急性和持续阶段中的作用。这些实验将对我们理解渗透调节机制的长期目标做出重要贡献。

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 We have unpublished data showing that transgenic mice lacking PLC delta1 show a dramatic deficiency in their ability to respond appropriately to water deprivation.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.