A key unanswered question in smooth muscle biology is whether phosphorylation of the myosin regulatory light chain (RLC) is sufficient for regulation of contraction, or if thin-filament-based regulatory systems also contribute to this process. To address this issue, the endogenous RLC was extracted from single smooth muscle cells and replaced with either a thiophosphorylated RLC or a mutant RLC (T18A/S19A) that cannot be phosphorylated by myosin light chain kinase. The actin-binding protein calponin was also extracted. Following photolysis of caged ATP, cells without calponin that contained a nonphosphorylatable RLC shortened at 30% of the velocity and produced 65% of the isometric force of cells reconstituted with the thiophosphorylated RLC. The contraction of cells reconstituted with nonphosphorylatable RLC was, however, specifically suppressed in cells that contained calponin. These results indicate that calponin is required to maintain cells in a relaxed state, and that in the absence of this inhibition, dephosphorylated cross-bridges can slowly cycle and generate force. These findings thus provide a possible framework for understanding the development of latch contraction, a widely studied but poorly understood feature of smooth muscle.

The role of Ca2+ in regulating smooth muscle contraction was investigated by measuring isometric force and [Ca2+] simultaneously in individual single smooth-muscle cells. [Ca2+] was measured with fura-2 and a high time-resolution dual-wavelength digital microfluorimeter, and force was measured with an ultrasensitive force transducer attached to a probe around which was tied one end of the cell. Both [Ca2+] and force increase after maximal electrical stimulus, with [Ca2+] increasing considerably before the first detectable increase in force. Force development exhibited maximal sensitivity to [Ca2+] between 150 and 500 nM Ca2+. This Ca2+ sensitivity can account for the fact that many physiological stimuli produce full contraction even though such stimuli only increase Ca2+ to 600-800 nM. When Ca2+ was induced to increase rapidly, the relation between [Ca2+] and force exhibited hysteresis. During the onset of contraction, force at a given [Ca2+] was lower than during the muscle's return to rest, thus suggesting the existence of a slow step(s) linking Ca2+ and force development in smooth muscle. The direction of this hysteresis reversed during contractions in which Ca2+ increased slowly, suggesting that the contractile process becomes desensitized to [Ca2+] with time. These relations between calcium and force in intact single smooth-muscle cells differ in many respects from the relation found previously in chemically permeabilized multicellular preparations of smooth muscle.