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.