Calcium and Sodium Transport in Smooth Muscle

描述（由申请人提供）：该提案侧重于Na+运输对血管平滑肌细胞（VSMC）中Ca2+调节至关重要的假设。这是由于关键的Na+和Ca2+转运体在质膜（PM）微域和邻近的（亚PM）“连接”肌浆网（JSR）中的定位，包括PM Na+泵a2/ct3异构体、Na/Ca交换器（NCX）、储存操作通道（soc）和一些SR Ca2+泵（SERCA）。为了验证这一假设，我们提出了三个具体的目标：1)确定培养VSMC中SR Ca2+储存和PM-SR连接的组织，以及它们如何影响Ca2+信号传导。Na+泵a2/a3亚基、l型Ca2+通道（LVGCs）、SOCs和NCX的抑制如何影响静息和激动剂刺激的原代培养大鼠VSMC的亚pm （SPM）和“散装”细胞质Ca2+浓度（[Ca2+] SPM和[Ca2+] cyt） ？高分辨率成像的Ca2+染料，Fura 2， Furaptra和FFP-18（近膜指示剂），将分别用于测量全局，SR和Spm [Ca2+]，并阐明激动剂诱发的Ca2+信号的起源位置和传播机制。VSMC中表达的特异性转运体亚型将被鉴定（PCR，免疫印迹），并通过免疫细胞化学在用于研究Ca2+的相同细胞中定位，以将信号起始位点与转运体位置联系起来。转运蛋白水平降低的细胞，从反义（AS-）寡核苷酸处理或小鼠零突变也将被检查。这些研究将测试a2/a3 Na+泵和NCX通过间接调节PM和jSR之间空间的局部[ca2+]（即[ca2+]spm）以及ca2+的储存和释放来调节Ca+信号的想法。2)确定SR Ca2+储存是如何组织的，以及在完整的约250pm od动脉中，哪些转运蛋白参与了VSM中的Ca2+调节。3)确定Na+和Ca2+转运蛋白如何促进这些动脉的肌生成和激动剂诱发的收缩。直径将监测和细胞质和SR Ca2+将测量共聚焦显微镜使用高和低亲和力的Ca2+染料，Fluo-4和Fluo-5N，在孤立的，加压的动脉。NCX, lvgc, soc， IP3和ry敏感Ca2+存储的存在和性质将被确定。这些转运蛋白如何影响单个细胞中的Ca2+储存和信号，以及它们如何促进肌原性张力和激动剂诱发的动脉收缩，将被研究。将检查正常动脉和转运蛋白水平降低的动脉，这些转运蛋白水平来自AS-oligo治疗或零突变小鼠。Ca2+信号将与收缩相关，以阐明Na+泵，Ca2+瞬态和血管张力之间的关系，以获得对控制血流和压力机制的新见解。

DESCRIPTION (provided by the applicant): This proposal focuses on the hypothesis that Na+ transport is crucial for Ca2+ regulation in vascular smooth muscle cells (VSMC). This is due to localization of key Na+ and Ca2+ transporters in plasma membrane (PM) micro-domains and adjacent (sub-PM) "junctional" sarcoplasmic reticulum (JSR), including PM Na+ pump a2/ct3 isoforms, Na/Ca exchangers (NCX), store-operated channels (SOCs), and some SR Ca2+ pumps (SERCA). Three specific aims are proposed to test the hypothesis: 1) To determine the organization of SR Ca2+ stores and PM-SR junctions in cultured VSMC, and how this influences Ca2+ signaling. How do inhibition of Na+ pump a2/a3 subunits, L-type Ca2+ channels (LVGCs), SOCs, and NCX, affect sub-PM (SPM) and "bulk" cytosolic Ca2+ concentrations ([Ca2+] spm and [Ca2+] cyt) in resting and agonist-stimulated, primary cultured rat VSMC? High resolution, imaging with Ca2+ dyes, Fura 2, Furaptra and FFP-18 (near-membrane indicator), will be used to measure, respectively, global, SR, and Spm [Ca2+], and to elucidate the sites of origin and mechanism(s) of propagation of agonist-evoked Ca2+ signals. Specific transporter isoforms expressed in VSMC will be identified (PCR, immunoblot) and localized by immunocytochemistry in the same cells used to study Ca2+, to relate signal initiation sites to transporter location. Cells with reduced transporter levels, from antisense (AS-) oligo treatment or mice with null mutations will also be examined. These studies will test the idea that a2/a3 Na+ pumps and NCX modulate Ca+ signaling by regulating indirectly local [ca2+] in the space between the PM and jSR (i.e., [ca2+]spm), and Ca2+ storage and release. 2) To determine how SR Ca2+ stores are organized, and which transporters contribute to Ca2+ regulation in VSM within intact about 250pm O.D. arteries. And 3) To determine how Na+ and Ca2+ transporters contribute to myogenic and agonist-evoked constriction in these arteries. Diameter will be monitored and cytosolic and SR Ca2+ will be measured with confocal microscopy using high-and low-affinity Ca2+ dyes, Fluo-4 and Fluo-5N, in isolated, pressurized arteries. The presence and properties of the NCX, LVGCs, SOCs, and IP3- and RY-sensitive Ca2+ stores will be determined. How these transporters influence Ca2+ stores and signaling in individual cells, and how they contribute to myogenic tone and agonist-evoked arterial constriction, will be investigated. Normal arteries and those with reduced transporter levels, from AS-oligo treatment or mice with null mutations, will be examined. Ca2+ signals will be correlated with contraction to elucidate relationships among Na+ pumps, Ca2+ transients and vascular tone, to obtain novel insight into mechanisms that control blood flow and pressure.