Effects of microtubules and microfilaments on [Ca(2+)](i) and contractility in a reconstituted fibroblast fiber.
We used a reconstituted fiber formed when 3T3 fibroblasts are grown in collagen to characterize nonmuscle contractility and Ca(2+) signaling. Calf serum (CS) and thrombin elicited reversible contractures repeatable for >8 h. CS elicited dose-dependent increases in isometric force; 30% produced the largest forces of 106 +/- 12 microN (n = 30), which is estimated to be 0.5 mN/mm(2) cell cross-sectional area. Half times for contraction and relaxation were 4.7 +/- 0.3 and 3.1 +/- 0.3 min at 37 degrees C. With imposition of constant shortening velocities, force declined with time, yielding time-dependent 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 CS-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 baseline intracellular Ca(2+) concentration ([Ca(2+)](i)) but reduced ( approximately 30%) the [Ca(2+)](i) response to CS. The force after nocodazole treatment was the primary determinant of stiffness and V(max), suggesting that microtubules were not a major component of fiber internal mechanical resistance. Cytochalasin D had major inhibitory effects on all contractile parameters measured but little effect on [Ca(2+)](i).
Obara
K
K
Departments of Physiology and Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA.
Nobe
K
K
Nobe
H
H
Kolodney
M S
MS
De Lanerolle
P
P
Paul
R J
RJ
D016475:3T3 Cells; D008841:Actin Cytoskeleton / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003187:Compliance; D015638:Cytochalasin D / Q000494:pharmacology; D004548:Elasticity; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000378:metabolism*; D051379:Mice; D008870:Microtubules / Q000187:drug effects / Q000502:physiology*; D015739:Nocodazole / Q000494:pharmacology; D009994:Osmolar Concentration; D013997:Time Factors
D015638:Cytochalasin D; D015739:Nocodazole; D002118:Calcium