Calcium and Sodium Transport in Smooth Muscle

描述（由申请方提供）：该提案侧重于Na+转运对血管平滑肌细胞（VSMC）中Ca 2+调节至关重要的假设。这是由于质膜（PM）微域和相邻（亚PM）“交界”肌浆网（JSR）中关键Na+和Ca 2+转运蛋白的定位，包括PM Na+泵α 2/α 3亚型、Na/Ca交换剂（NCX）、储存操纵通道（SOC）和一些SR Ca 2+泵（SERCA）。我们提出了三个具体的目标来验证这一假设：1）确定培养的VSMC中SR钙库和PM-SR连接的组织结构，以及这如何影响钙信号。抑制Na+泵a2/a3亚单位、L型钙通道（LVGCs）、SOC和NCX如何影响静息和激动剂刺激的原代培养大鼠VSMC中亚PM（SPM）和“本体”胞浆Ca 2+浓度（[Ca 2 +] spm和[Ca 2 +] cyt）？ 将使用高分辨率的Ca 2+染料Fura 2、Furaptra和FFP-18（近膜指示剂）成像，分别测量全局、SR和Spm [Ca 2 +]，并阐明激动剂诱发的Ca 2+信号的起源部位和传播机制。将鉴定（PCR，免疫印迹）VSMC中表达的特异性转运蛋白亚型，并通过免疫细胞化学在用于研究Ca 2+的相同细胞中进行定位，以将信号起始位点与转运蛋白位置联系起来。还将检查来自反义（AS-）寡核苷酸处理的转运蛋白水平降低的细胞或具有无效突变的小鼠。这些研究将测试a2/a3 Na+泵和NCX通过间接调节PM和jSR之间空间中的局部[ca 2 +]来调节Ca+信号传导的想法（即，[ca2+]spm），以及Ca ~（2+）的储存和释放。2)目的：研究在250 pm O.D.左右，VSM中SR Ca ~（2+）库是如何组织的，以及哪些转运蛋白参与了VSM的Ca ~（2+）调节。动脉（3）探讨Na+和Ca ~（2+）转运体在肌源性和激动剂诱发的收缩中的作用。在分离的加压动脉中，将监测直径，并使用高亲和力和低亲和力Ca 2+染料Fluo-4和Fluo-5 N，通过共聚焦显微镜测量细胞溶质和SR Ca 2+。将确定NCX、LVGC、SOC以及IP 3和RY敏感性Ca 2+储存的存在和性质。这些转运蛋白如何影响Ca 2+储存和信号在个别细胞，以及它们如何有助于肌张力和激动剂诱发的动脉收缩，将进行调查。将检查正常动脉和来自AS-oligo治疗或具有无效突变的小鼠的转运蛋白水平降低的动脉。Ca 2+信号将与收缩相关，以阐明Na+泵，Ca 2+瞬变和血管张力之间的关系，以获得控制血流和压力的机制的新见解。

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.