Calcium and Sodium Transport in Smooth Muscle

描述(由申请人提供)：本提案侧重于Na+转运对血管平滑肌细胞(VSMC)的钙调节至关重要的假设。这是由于关键的Na+和Ca2+转运体位于质膜(PM)微域和相邻的(亚PM)“连接”肌质网(JSR)中，包括PM的Na+泵A2/CT3亚型、Na/Ca交换器(NCX)、存储操作通道(SOCs)和一些SR钙泵(SERCA)。我们提出了三个具体的目标来检验这一假说：1)确定培养的VSMC中SR钙离子储存库和PM-SR连接的组织，以及这是如何影响钙信号转导的。抑制Na+泵A2/A3亚单位，L型钙通道(LVGCs)、SOCs和NCx，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“大量”胞浆钙浓度([Ca2+]Spm和[Ca2+]Cyt)？高分辨率的钙离子染料Fura-2、Furaptra和FFP-18(近膜指示剂)将被用来分别测量GLOBAL、SR和SPM[Ca~(2+)]，并阐明激动剂诱导的Ca~(2+)信号的起源和传播机制(S)。在VSMC中表达的特定转运蛋白亚型将被鉴定(聚合酶链式反应，免疫印迹)，并在用于研究钙离子的同一细胞中通过免疫细胞化学进行定位，以将信号起始位置与转运蛋白位置联系起来。转运蛋白水平降低的细胞、反义(AS-)寡核苷酸治疗的细胞或零突变的小鼠也将被检查。这些研究将验证A2/A3Na+泵和NCX通过间接调节PM和JSR之间的局部[Ca~(2+)](即[Ca~(2+)]SPM)以及Ca~(2+)的储存和释放来调制Ca~+信号的想法。2)确定肌浆网钙离子的存储是如何组织的，以及哪些转运体参与了大约250 pm的完整动脉内钙离子的调节。3)确定Na+和Ca~(2+)转运体在这些动脉的肌源性收缩和激动剂诱发收缩中的作用。将使用高亲和力和低亲和力的钙染料Fluo-4和Fluo-5N在分离的加压动脉中监测直径，并使用共聚焦显微镜测量胞浆和肌浆钙离子。将确定NCX、LVGCs、SOCs以及IP3和RY敏感的钙离子存储的存在和性质。这些转运体如何影响单个细胞中的钙储存和信号传递，以及它们如何参与肌源性张力和激动剂引起的动脉收缩，将被研究。将检查正常动脉和那些转运蛋白水平降低的动脉，这些动脉来自反义寡核苷酸治疗或零突变小鼠。钙信号将与收缩相关，以阐明钠泵、钙瞬变和血管紧张性之间的关系，以获得对血流和血压控制机制的新见解。

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.