title: Thapsigargin decreases the Na(+)- Ca(2+) exchanger mediated Ca(2+) entry in pig coronary artery smooth muscle.
abstract: Na(+)- Ca(2+) exchanger (NCX) has been proposed to play a role in refilling the sarco/endoplasmic reticulum (SER) Ca(2+) pool along with the SER Ca(2+) pump (SERCA). Here, SERCA inhibitor thapsigargin was used to determine the effects of SER Ca(2+) depletion on NCX-SERCA interactions in smooth muscle cells cultured from pig coronary artery. The cells were Na(+)-loaded and then placed in either a Na(+)-containing or in a Na(+)-substituted solution. Subsequently, the difference in Ca(2+) entry between the two groups was examined and defined as the NCX mediated Ca(2+) entry. The NCX mediated Ca(2+) entry in the smooth muscle cells was monitored using two methods: Ca(2+)sensitive fluorescence dye Fluo-4 and radioactive Ca(2+). Ca(2+)-entry was greater in the Na(+)-substituted cells than in the Na(+)-containing cells when measured by either method. This difference was established to be NCX-mediated as it was sensitive to the NCX inhibitors. Thapsigargin diminished the NCX mediated Ca(2+) entry as determined by either method. Immunofluorescence confocal microscopy was used to determine the co-localization of NCX1 and subsarcolemmal SERCA2 in the cells incubated in the Na(+)-substituted solution with or without thapsigargin. SER Ca(2+) depletion with thapsigargin increased the co-localization between NCX1 and the subsarcolemmal SERCA2. Thus, inhibition of SERCA2 leads to blockade of constant Ca(2+) entry through NCX1 and also increases proximity between NCX1 and SERCA2. This blockade of Ca(2+) entry may protect the cells against Ca(2+)-overload during ischemia-reperfusion when SERCA2 is known to be damaged.
authors.affiliation: Department of Biology, McMaster University, Hamilton, ON, Canada.
authors.forename: Gauri
authors.initials: G
authors.lastname: Akolkar
authors.forename: Jyoti
authors.initials: J
authors.lastname: Pande
authors.forename: Sue E
authors.initials: SE
authors.lastname: Samson
authors.forename: Ashok K
authors.initials: AK
authors.lastname: Grover
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003331:Coronary Vessels / Q000378:metabolism* / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D017136:Ion Transport / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000473:pathology; D015427:Reperfusion Injury / Q000378:metabolism / Q000473:pathology; D012508:Sarcolemma / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000378:metabolism; D013552:Swine; D019284:Thapsigargin / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D019284:Thapsigargin; D012964:Sodium; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium