Fibroblast fiber contraction: role of C and Rho kinase in activation by thromboxane A(2)

We investigated the mechanisms underlying regulation of contraction with measurements of isometric force and intracellular Ca2+ concentration ([Ca2+](i)) in NIH 3T3 fibroblast reconstituted into fibers with the use of a collagen matrix. Treatment with the major phospholipids, neurotransmitters, and growth factors had little effect on baseline isometric force. However, U-46619, a thromboxane A(2) (TxA(2)) analog, increased force and [Ca2+](i); EC50 values were 11.0 and 10.0 nM, respectively. The time courses were similar to those induced by calf serum ( CS), and the maximal force was 65% of a CS-mediated contraction. The selective TxA(2) receptor antagonist SQ-29548 abolished the U-46619-induced responses. CS-induced contractions are dependent on an intracellular Ca2+ store function; however, the U-46619 response depended not only on intracellular Ca2+ stores, but also on Ca2+ influx from the extracellular medium. Inhibition of Rho kinase suppressed U-46619- and CS-induced responses; in contrast, inhibition of C kinase (PKC) reduced only the U-46619 response. Moreover, addition of U-46619 to a CS contracture enhanced force and [Ca2+](i) responses. These results indicate that U-46619- induced responses involve PKC and Rho kinase pathways, in contrast to activation by CS. Thus TxA(2) may have a role in not only the initial step of wound repair as an activator of blood coagulation, but also in fibroblast contractility in later stages.