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分摄入（即口渴）和尿液中水分流失的机制将体液的渗透压浓度维持在非常狭窄的范围内。尿量主要由下丘脑大细胞神经分泌细胞（MNCs）释放的加压素（VP）调节。渗透压浓度的急性增加通过使它们收缩来刺激MNC，导致机械敏感性TRPV 1通道N-末端变体（DeltaN TRPV 1）的激活，这增加了电活动。渗透压摩尔浓度的持续增加导致MNC的结构和功能转化，包括细胞肥大、MNC表面上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，渗透压摩尔浓度的增加触发激活的磷脂酶C（PLC）在MNC和PLC抑制抑制MNC肥大和渗透激活DeltaN TRPV 1通道。因此，PLC可能在MNC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Ca 2+依赖性的，这使我们假设负责的同种型是Ca 2+依赖性PLC δ家族的成员。我们有未发表的数据表明，缺乏PLC delta 1的转基因小鼠在对缺水作出适当反应的能力方面表现出明显的缺陷。我们推测，未能增加VP释放使尿液生产时，它应该被抑制。此外，我们有证据表明，渗透激活的DeltaN TRPV 1电流几乎完全抑制PLC delta 1基因敲除小鼠的MNCs，这可能是由于部分抑制PLC介导的增强肌动蛋白细胞骨架。最后，我们有证据表明，渗透压的持续增加导致DeltaN TRPV 1通道易位到MNC质膜，这可能是MNC适应渗透压持续增加的重要部分。因此，我们建议检验以下四个假设：1）PLC δ 1 KO小鼠中有缺陷的VP调节是由垂体诱发的VP释放缺陷介导的，2）PLC δ 1介导快速的垂体诱导的PLC的Ca 2+依赖性激活，3）DeltaN TRPV 1通道的渗透调节将减弱，并且是可逆的-在PLC delta 1 KO小鼠中诱发的MNC放电变化将减少，以及4）增加的渗透压引发DeltaN TRPV 1通道易位至MNC细胞膜。 我们将在大鼠和PLC delta 1 KO和野生型小鼠中使用电生理学、免疫细胞化学和药理学方法来测试这些假设。所提出的实验将阐明PLC途径在急性和持续阶段的MNC β-内酰胺敏感性的作用。 这些实验将为我们理解神经元调控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 We have unpublished data showing that transgenic mice lacking PLC delta1 show a dramatic deficiency in their ability to respond appropriately to water deprivation.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.