Ca2+ increase and Ca(2+)-influx in human tracheal smooth muscle cells: role of Ca2+ pools controlled by sarco-endoplasmic reticulum Ca(2+)-ATPase 2 isoform.
1. The contribution of sarco-endoplasmic reticulum Ca(2+)-ATPases (SERCA)-regulated Ca2+ stores to the increase in intracellular free calcium ([Ca2+]i) induced by bradykinin (BK) was investigated in fura-2 loaded human tracheal smooth muscle cells (TSMC). For this purpose, we used thapsigargin, a selective inhibitor of Ca(2+)-ATPases of intracellular organelles. 2. Thapsigargin (10(-9) to 10(-6) M) induced a dose-dependent increase in [Ca2+]i in the presence of external Ca2+ with an EC50 value of 7.33 +/- 1.26 nM. In Ca(2+)-free conditions, the addition of Ca2+ (1.25 mM) caused an increase in [Ca2+]i which was directly proportional to the pre-incubation time of the cells with thapsigargin. Net increases of 60 +/- 9, 150 +/- 22 and 210 +/- 27 nM were obtained after 1, 3 and 5 min, respectively. 3. In the presence of extracellular Ca2+, BK induced a typical biphasic increase in [Ca2+]i with a fast transient phase and a sustained phase. The sustained component was reversed by addition of a bradykinin B2-receptor antagonist (Hoe 140, 10(-6) M) to the buffer as well as by deprivation of Ca2+. The transient phase induced by BK, histamine and carbachol was inhibited in a time-dependent way by preincubation of the cells with thapsigargin. 4. Comparative western blotting of human TSMC membranes using anti-SERCA2 isoform-specific antibodies clearly showed the greater expression of the 100-kDa SERCA2-b isoform compared with the SERCA2-a isoform. 5. Our data show that thapsigargin-sensitive Ca2+ stores contribute significantly to the activation of human TSMC which suggests a role for these stores in the subsequent induction of Ca2+ influx. These stores appear to be controlled by the Ca2+-ATPases (SERCA2-b isoform) which could also participate in the regulation of Ca2+ influx through the plasma membrane.
Amrani
Y
Y
INSERM U 425, Université Louis Pasteur Strasbourg I, Illkirch, France.
Magnier
C
C
Enouf
J
J
Wuytack
F
F
Bronner
C
C
D001692:Biological Transport; D001920:Bradykinin / Q000031:analogs & derivatives / Q000494:pharmacology; D065168:Bradykinin Receptor Antagonists; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D043782:Receptor, Bradykinin B2; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014132:Trachea / Q000166:cytology / Q000378:metabolism*
D065168:Bradykinin Receptor Antagonists; D004791:Enzyme Inhibitors; D043782:Receptor, Bradykinin B2; D013729:Terpenes; D019284:Thapsigargin; C065679:icatibant; D006632:Histamine; D002217:Carbachol; D000252:Calcium-Transporting ATPases; D001920:Bradykinin; D002118:Calcium