Preferential role of intracellular Ca2+ stores in regulation of isometric force in NIH 3T3 fibroblast fibres

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Abstract
1 Fibroblast contraction plays a major role in wound repair, but the regulatory mechanisms are not well known. We investigated the relations between isometric force and intracellular calcium concentration ([Ca2+]i) in fibroblast fibres. These fibres were made with mouse NIH 3T3 fibroblasts cultured with native collagen in a three-dimensional matrix. 2 Calf serum (CS; 30 %) elicited a monotonic increase in force that attained a maximum within 15 min and could be sustained indefinitely. In contrast, [Ca2+]i increased to a peak at 3 min after CS stimulation, then returned to baseline levels by 10 min. Pretreatment with Ca2+-free medium or the Ca2+-channel antagonist nicardipine (10 μM) blocked the CS-induced [Ca2+]i increase, but force was not affected. 3 KCl (50 mM) stimulation on the other hand, elicited a prolonged increase in [Ca2+]i but did not increase force. 4 Inhibition of the endoplasmic reticulum Ca2+ release with Ca2+-ATPase inhibitors cyclopiazonic acid (5 μM) or thapsigargin (5 μM) nearly abolished (< 20 % control) the increase in [Ca2+]i and force response to CS. Treatment with ryanodine (10 μM) and caffeine (20 mM) had a similar effect. The phospholipase C inhibitor U73122 (3 μM) reduced the CS-induced increases in [Ca2+]i and force by 70 and 40 %, respectively. 5 We conclude that fibroblast isometric force is not coupled to Ca2+ arising from transmembrane influx but is correlated with the transient [Ca2+]i increase due to release from intracellular stores. Store-released Ca2+ may initiate activation pathways for fibroblast force development, but is not required for force maintenance.