MECHANISMS OF CA2+-INDEPENDENT CONTRACTION IN SINGLE PERMEABILIZED FERRET AORTA CELLS

The mechanisms by which prostaglandin F2alpha (PGF2alpha) can cause contractions at constant intracellular Ca2+ were investigated by the direct measurement of force from single saponin-permeabilized smooth muscle cells from the ferret aorta. The size of PGF2alpha contractions did not change between pCa 9.0) and pCa 6.6. The remainder of the experiments were carried out at pCa 7.0. At pCa 7.0, PGF2alpha (0.1-100 muM) induced sustained force in a dose-dependent manner, reaching a maximum (2.61+/-0.20 muN, n=14) in 10 minutes. Both protein kinase C pseudosubstrate inhibitor (3 muM) and staurosporine (1 muM) significantly inhibited PGF2alpha (100 muM)-induced contractions, but staurosporine was more effective. Staurosporine caused 88.8+/-13.3% inhibition, whereas protein kinase C pseudosubstrate inhibitor inhibited 62.3+/-9.6% of the PGF2alpha-induced contraction. An inhibitor of type-1 and type-2A protein phosphatases, microcystin-LR, at a concentration of 1 muM induced a gradual and sustained contraction (1.53+/-0.21 muN). A lower concentration of microcystin-LR (100 nM) also induced a small but significant contraction (0.36+/-0.26 muN). Pretreatment with both 1 muM and 100 nM microcystin-LR caused significant inhibition of the PGF2alpha-induced contraction from 2.61+/-0.20 muN (n = 14) to 0.32+/-0.20 muN (n=6) (p<0.01) and 1.52+/-0.21 muN (n=6) (p<0.01), respectively. These results indicate that the part of the PGF2alpha-induced contraction that occurs at a constant, low intracellular Ca2+ is the combined result of activation of protein kinase C and phosphatase inhibition.