Ca2+ increase and Ca2+‐influx in human tracheal smooth muscle cells: role of Ca2+ pools controlled by sarco‐endoplasmic reticulum Ca2+‐ATPase 2 isoform

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Abstract
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 EC50 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 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.

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