Inhibition of force and shortening in smooth muscle by vanadate

Abstract

We have investigated the effects of vanadate (Vi) on force generation by, and shortening of, chemically skinned smooth muscle preparations from guinea-pig taenia coli at 22 degrees C. A method, using phosphatase inhibitors, was introduced to obtain stable, long-lasting contractions in thiophosphorylated preparations. Vi (10-1000 microM) dose-dependently inhibited active force, to about 20% of its maximum level. At a higher temperature (30 degrees C), the rate of inhibition was faster but the extent of inhibition was less. The rate of contraction following photolytic release of ATP to fibres in rigor was not affected by Vi (30 microM). The maximal shortening velocity (Vmax) was inhibited in a similar manner as active force by Vi (30 microM). In conclusion, the results suggest that Vi interacts with a force-generating actomyosin-ADP (AMADP) state reached after phosphate release. The rate of inhibition of smooth muscle contraction was markedly lower than in skeletal muscle, suggesting differences either in properties of the Vi-bound states or, more likely, in the concentration of AMADP states capable of binding Vi. This suggests that the long duty cycle in smooth muscle is not associated with a higher relative population of AMADP states reached immediately after Pi release, but rather by an increase in the population of subsequent force-generating cross-bridge states. The Vi-bound cross-bridges introduce an internal load to shortening, possibly acting in a similar manner as cross-bridge states introduced at low levels of activation.

Effects of vanadate, phosphate and 2,3-butanedione monoxime (BDM) on skinned molluscan catch muscle

“…As in mammalian striated and smooth muscle [5,9,22,23,26,35,36,55], V i , P i and BDM depressed the maximal Ca 2+ -activated force in ABRM fibres. It is thought that V i and P i induce isomerisation to a stable non-or low-force-generating complex after binding to the AM.ADP state [9].…”

“…The exact [V i ] was determined by absorption measurement at 265 nm, assuming an extinction coefficient of 2925 M À1 cm À1 [26]. A stock solution of 300 mM V i (pH 10) was prepared in double-distilled water and boiled until colourless [26]. After adding, the pH of skinned fibre bath solutions was readjusted to 6.7 with HCl.…”

“…When required, orthovanadate (0.01-10 mM, Na 3 VO 4 ) or phosphate (10 mM, mixture of Na 2 HPO 4 and NaH 2 PO 4 ) was added to these solutions and removing KCl to keep the ionic strength constant. The exact [V i ] was determined by absorption measurement at 265 nm, assuming an extinction coefficient of 2925 M À1 cm À1 [26]. A stock solution of 300 mM V i (pH 10) was prepared in double-distilled water and boiled until colourless [26].…”

“…Orthovanadate (V i ), a well-known phosphate analogue, inhibits force and ATPase activity of skinned muscle preparations of skeletal, cardiac and smooth muscle [5,9,19,23,26]. The inhibition is thought to result from V i binding to the AM.ADP state, which induces isomerisation to a stable non-or low-force-generating complex resulting in depression of force and ATPase activity of skinned muscle fibres.…”

The catch state of mollusc catch muscle is established during activation:experiments on skinned fibre preparations of the anterior byssus retractor muscle of Mytilus edulis L. using the myosin inhibitors orthovanadate and blebbistatin

“…Therefore, in the present study we substantially extended the approach of Butler et al (Butler et al, 2006) by using maximally Ca 2+ -activated skinned ABRM preparations and by including rapid changes of the fibre length: as in the work of Butler et al (Butler et al, 2006), we inhibited the active cycling cross-bridges by specific myosin inhibitors during Ca 2+ activation. Orthovanadate (Herzig et al, 1981;Dantzig and Goldman, 1985;Chase et al, 1993;Jaworowski et al, 1999;Galler et al, 2005) and blebbistatin (Kovacs et al, 2004;Butler et al, 2006) were chosen as myosin inhibitors. The force remaining after myosin inhibition was investigated by addressing the following questions.…”