CA2+ INCREASE AND CA2+-INFLUX IN HUMAN TRACHEAL SMOOTH-MUSCLE CELLS - ROLE OF CA2+ POOLS CONTROLLED BY SARCO-ENDOPLASMIC RETICULUM CA2+-ATPASE 2 ISOFORM

1 The contribution of sarco-endoplasmic reticulum Ca2+-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 Ca2+-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 EC(50) value of 7.33+/-1.26 nM. In Ca2+-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 B-2-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-SERCA(2) isoform-specific antibodies clearly showed the greater expression of the 100-kDa SERCA(2-b) isoform compared with the SERCA(2-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+ flux. These stores appear to be controlled by the Ca2+-ATPases (SERCA(2-b) isoform) which could also participate in the regulation of Ca2+ influx through the plasma membrane.