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Ca 2+ increase and Ca 2+ ‐influx in human tracheal smooth muscle cells: role of Ca 2+ pools controlled by sarco‐endoplasmic reticulum Ca 2+ ‐ATPase 2 isoform
Author(s) -
Amrani Yassine,
Magnier Clarice,
Enouf Jocelyne,
Wuytack Frank,
Bronner Christian
Publication year - 1995
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1995.tb15026.x
Subject(s) - endoplasmic reticulum , calcium , chemistry , biophysics , biology , biochemistry , organic chemistry
1 The contribution of sarco‐endoplasmic reticulum Ca 2+ ‐ATPases (SERCA)‐regulated Ca 2+ stores to the increase in intracellular free calcium ([Ca 2+ ] 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 [Ca 2+ ]i in the presence of external Ca 2+ with an EC 50 value of 7.33±1.26 nM. In Ca 2+ ‐free conditions, the addition of Ca 2+ (1.25 mM) caused an increase in [Ca 2+ ] 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 Ca 2+ , BK induced a typical biphasic increase in [Ca 2+ ] 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 Ca 2+ . 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 Ca 2+ stores contribute significantly to the activation of human TSMC which suggests a role for these stores in the subsequent induction of Ca 2+ influx. These stores appear to be controlled by the Ca 2+ ‐ATPases (SERCA 2‐b isoform) which could also participate in the regulation of Ca 2+ influx through the plasma membrane.