Stimulation by chemotactic factor of actin association with the cytoskeleton in rabbit neutrophils. Effects of calcium and cytochalasin B.

The amounts of actin and myosin in rabbit neutrophils expressed as micrograms/10(6) cells are 5.6 +/- 0.75 and 0.56 +/- 0.08, respectively. The average value of the total actin in rabbit neutrophils under unstimulated conditions is distributed between Triton X-100 soluble fraction (74 +/- 7%) and Triton X-100 insoluble fraction (26 +/- 3%). The Triton X-100 soluble and insoluble fractions will be referred to as the cytoplasmic and the cytoskeletal components. When the cells are stimulated by the chemotactic factor formyl-Met-Leu-Phe the amount of actin associated with the cytoskeleton increases to 73.7 +/- 6% of the total cell actin. This increase is rapid, dose-dependent and mediated through fMet-Leu-Phe receptors. Neither the time course of the response nor the dose-response curve is affected by the removal of calcium from the suspending medium. Calcium ions at concentrations greater than 10(-7) M added after Triton X-100 extraction dissociate actin from the cytoskeleton. Calcium at 1.9 microM added after Triton X-100 extraction reduces the amount of cytoskeletal actin under control and stimulated conditions to 10.3 +/- 0.9 and 33 +/- 1.5% of the total cell actin, respectively. The average value of the total myosin in rabbit neutrophils under unstimulated conditions is distributed between the cytosol (32 +/- 10%) and the cytoskeleton (68 +/- 18%). When neutrophils are stimulated with the chemotactic factor fMet-Leu-Phe the amount of myosin associated with the cytoskeleton does not increase significantly. Cytochalasin B decreases cytoskeletal actin and myosin and causes a shift in the amount of actin and myosin from the cytoskeleton to the cytoplasm both under fMet-Leu-Phe-stimulated and control conditions. In the presence of 1.6 mM extracellular Ca2+ and cytochalasin B (5 micrograms/ml) the amount of actin associated with the cytoskeleton under control and stimulated conditions is reduced to 13 +/- 2.2 and 10.2 +/- 3.5% of total cell actin, and that of myosin is reduced to 50.2 +/- 14 and 2.3 +/- 0.8% of the total cell myosin. The effect of cytochalasin B on actin does not depend on the time of its addition relative to that of fMet-Leu-Phe and is more pronounced in the presence of Ca2+. These results are discussed in terms of the roles of cytochalasin B and calcium in the overall mechanism of neutrophil degranulation induced by chemotactic factors.