Effects of microtubules and microfilaments on [Ca2+](i) and contractility in a reconstituted fibroblast fiber

We used a reconstituted fiber formed when 3T3 fibroblasts are grown in coll agen to characterize nonmuscle contractility and Ca2+ signaling. Calf serum (CS) and thrombin elicited reversible contractures repeatable for >8 h. CS elicited dose-dependent increases in isometric force; 30% produced the lar gest forces of 106 +/- 12 mu N (n = 30), which is estimated to be 0.5 mN/mm (2) cell cross-sectional area. Half times for contraction and relaxation we re 4.7 +/- 0.3 and 3.1 +/- 0.3 min at 37 degrees C. With imposition of cons tant shortening velocities, force declined with time, yielding time-depende nt force-velocity relations. Forces at 5 s fit the hyperbolic Hill equation ; maximum velocity (V-max) was 0.035 +/- 0.002 L-o/s. Compliance averaged 0 .0076 +/- 0.0006 L-o/F-o. Disruption of microtubules with nocodazole in a C S-contracted fiber had no net effects on force, V-max, or stiffness; force increased in 8, but decreased in 13, fibers. Nocodazole did not affect base line intracellular Ca2+ concentration ([Ca2+](i)) but reduced (similar to 3 0%) the [Ca2+](i) response to CS. The force after nocodazole treatment was the primary determinant of stiffness and Vmax, suggesting that microtubules were not a major component of fiber internal mechanical resistance. Cytoch alasin D had major inhibitory effects on all contractile parameters measure d but little effect on [Ca2+](i).