title: Clearance of store-released Ca2+ by the Na+-Ca2+ exchanger is diminished in aortic smooth muscle from Na+-K+-ATPase alpha 2-isoform gene-ablated mice.
abstract: Two alpha-isoforms of the Na+-K+-ATPase are expressed in vascular smooth muscle cells (VSMCs). The alpha 1-isoform is proposed to serve a cytosolic housekeeping role, whereas the alpha 2-isoform modulates Ca2+ storage via coupling to the Na+-Ca2+ exchanger (NCX) in a subsarcolemmal compartment. To evaluate the ramifications of this proposed interaction, Ca2+-store load and the contributions of the primary Ca2+ transporters to Ca2+ clearance were studied in aortic VSMCs from embryonic wild-type (WT) and Na+-K+-ATPase alpha 2-isoform gene-ablated, homozygous null knockout (alpha 2-KO) mice. Ca2+ stores were unloaded by inhibiting the sarco(endo)plasmic reticulum Ca2+-ATPase with cyclopiazonic acid (CPA) in Ca2+-free media to limit Ca2+ influx. Ca2+ clearance by the plasma membrane Ca2+-ATPase (PMCA), NCX, or mitochondria was selectively inhibited. In WT VSMCs, NCX accounted for 90% of the Ca2+ efflux. In alpha 2-KO VSMCs, preferential clearance of store-released Ca2+ by NCX was lost, whereas PMCA activity was increased. Selective inhibition of the alpha 2-isoform (0.5 microM ouabain for 20 min), before treatment with CPA enhanced the store load in VSMCs from WT, but not alpha 2-KO mice. A subsequent analysis of capacitative Ca2+ entry (CCE) indicated that the magnitude of Ca2+ influx was significantly greater in alpha 2-KO cells. Our findings support the concept of a subsarcolemmal space where the alpha 2-isoform coupled with NCX modulates Ca2+-store function and, thereby, CCE.
authors.affiliation: Department of Physiology, University of Arizona, Arizona Health Sciences Ctr., Tucson, AZ 85718, USA. rlynch@u.arizona.edu
authors.forename: Ronald M
authors.initials: RM
authors.lastname: Lynch
authors.forename: Craig S
authors.initials: CS
authors.lastname: Weber
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Nullmeyer
authors.forename: Edwin D W
authors.initials: ED
authors.lastname: Moore
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Paul
mesh_terms: D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002469:Cell Separation; D003600:Cytosol / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D012430:Ruthenium Red; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D007093:Imidazoles; D007527:Isoenzymes; D019831:Sodium-Calcium Exchanger; D012430:Ruthenium Red; C401838:Atp1a2 protein, mouse; D000254:Sodium-Potassium-Exchanging ATPase; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium