Relaxant effect of phalloidin on Triton-skinned microvascular and other smooth muscle preparations

Guinea pig mesenteric microarteries (diameter 60-100 microns), the main branch of the mesenteric artery and taenia coli were skinned with 1% Triton X-100 for 4 h at 4 degrees C. Microarteries, mounted for circumferential force measurement, developed maximal active force in response to elevation of the free Ca2+ (pCa = 4.52, in EGTA buffer) in the presence of ATP (7.5 mM) and calmodulin (0.1-0.3 microM). In these preparations, addition of phalloidin (1-100 microM) slowly (greater than 1 h) relaxed submaximal contractions (pCa greater than 4.52) in a dose-dependent manner. Relaxation was irreversible as, after phalloidin wash-out, subsequent active force to pCa = 4.52 was also reduced. By contrast, phalloidin preincubation and wash-out under relaxed conditions (pCa greater than 8) only reduced subsequent force to pCa = 4.52 on prolonged stimulation. The extent of phalloidin-induced relaxation was not dependent on free Ca2+ between pCa 6.40 and 4.52. Phalloidin-induced relaxation did not occur during rigor contractions (i.e. absence of ATP and Ca2+). These mechanical effects of phalloidin were accompanied by a decreased leak of actin out of the skinned preparations and by the prevention of guanidine extraction of actin from these preparations. Phalloidin did not inhibit the myosin light chain kinase or phosphatase activity isolated from these preparations. In addition, the relaxant effects were also noted in taenia coli and the main branch of the mesenteric artery but not in skinned porcine ventricular heart muscle. These experiments suggest the possible participation of actin filament dynamics on the maintenance of active force in Triton-skinned smooth muscle.