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Slow oscillations of free intracellular calcium ion concentration in human fibroblasts responding to mechanical stretch
Author(s) -
Arora Pamela D.,
Bibby Kathryn J.,
McCulloch Christopher A. G.
Publication year - 1994
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041610202
Subject(s) - calcium , intracellular , biophysics , chemistry , ion , calcium in biology , microbiology and biotechnology , biochemistry , biology , organic chemistry
Abstract Calcium transients in single, human gingival fibroblasts were studied after mechanical stretching of flexible culture substrates. A model system was developed to reproducibly stretch and rapidly (< 1 sec) refocus cells in the same focal plane so that changes in the concentration of free intracellular calcium ions ([Ca 2+ ] i ) were monitored without delay. Attached cells were grown on flexible bottom Petriperm dishes, loaded with fura‐2/AM, and stretched by 1% or 2.8% of substrate area. The stretch caused no significant cell detachment or membrane lesions. A 1% stretch induced no calcium response, but a 2.8% stretch stimulated an initial calcium transient and the subsequent generation of [Ca 2+ ] i oscillations of up to 2,000 sec. At 1% stretch, there was no calcium response. Cell shape and plating time were important determinants in the calcium response to mechanical stimulation: the responder cells were small and round without long processes. Major calcium transients were inhibited completely by 5 mM EGTA or by 10 μM gadolinium ions, by 50 μM nifedipine, or 250 μM verapamil, suggesting an influx of calcium through stretch‐activated (SA) channels and L‐type calcium channels. Depolarization by high KCl (144 mM) in the extracellular medium enhanced the amplitude of calcium transients by 54%. Calcium oscillations were not inhibited by preincubation with thapsigargin, caffeine, cholera toxin, staurosporine or 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H‐7), indicating that IP 3 sensitive pools, IP 3 insensitive pools, G 5 α subunits, and protein kinase C, respectively, were not involved in the generation of calcium oscillations. Pretreatment with genistein, a specific tyrosine kinase inhibitor or cytochalasin D, an inhibitor of actin polymerization, or pertussis toxin, an inhibitor of G iα and G oα subunits, completely abolished calcium transients and oscillations. These results indicate that Ca 2+ flux due to mechanical stretching is likely mediated through SA ion channe s and is dependent on tyrosine kinases, pertussis toxin‐sensitive subunits of G‐proteins, and actin filaments. © 1994 Wiley‐Liss, Inc.