Inhibition of tension development and actomyosin ATPase activity in barnacle muscle by the Ca2+-indicator dye antipyrylazo III.

We have investigated the effects of the Ca2+-indicator dye antipyrylazo III on: (1) tension development in myofibrillar preparations from barnacle depressor muscle; and (2) actomyosin ATPase activity in myofibrils/native actomyosin isolated from barnacle muscle and in actomyosin hybrids prepared from pure (unregulated) rabbit F-actin and purified barnacle myosin. In all solutions, pCa was either heavily buffered with suitable [CaEGTA]/[EGTA] and/or measured with a calcium-electrode so as to offset the appreciable Ca2+-buffering effects of the dyes. Antipyrylazo III produced a rapid, reversible and concentration-dependent inhibition of: (1) tension development by isolated barnacle myofibrils; (2) calcium-regulated ATPase activity in barnacle myofibrils and native actomyosin; and (3) calcium-independent actin-activated ATPase activity in hybrid actomyosins prepared from purified barnacle myosin and rabbit actin. This latter observation indicated that the inhibitory effect of the dye on calcium-regulated tension and ATPase in intact myofibrils is due to specific repression of active crossbridge formation rather than modification of calcium-regulatory mechanisms. The hypothesis that antipyrylazo III specifically represses active crossbridge formation was supported by the observation that the dye had no effect on rigor tension development. Specific and saturable binding of the dye to these myofibrils was characterized by a maximum capacity of 1.3 mumol dye g-1 myofibrillar protein, consistent with a calculated 1 dye: 1 myosin stoichiometry. These various biological effects were observed with both commercially available antipyrylazo III and highly purified dye preparations. Preliminary studies using myofibrillar preparations from rabbit psoas muscle, guinea-pig portal vein smooth muscle, and scallop adductor muscle have indicated that contractile function in these muscle types does not appear to be inhibited by antipyrylazo III.