The role of Ca24 in regulating smooth muscle contraction was investigated by measuring isometric force and [Ca2I] simultaneously in individual single smooth-muscle cells. [Ca24] was measured with fura-2 and a high timeresolution 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+1 and force increase after maximal electrical stimulus, with [Ca24] Calcium is considered the key regulator of contraction of smooth muscle (1, 2), although details of this regulation remain somewhat unclear. The relationship between force and [Ca2 '] has been studied in skinned smooth-muscle strips (3-11) and in smooth muscles that were loaded with aequorin by temporarily permeabilizing the tissue (12). The sensitivity of force to [Ca24] found in chemically permeabilized (skinned) preparations by various investigators varies by almost two orders of magnitude, and several studies showed that the sensitivity can be altered by calmodulin addition (5,6,13) per se, accounts for the several hundred-msec delay between stimulation and onset of active-force production characteristic of smooth muscle. Finally, our results show that the contractile process exhibits decreasing sensitivity to calcium during slow contraction-relaxation cycles. METHODSSmooth muscle cells were isolated from the stomach of the toad Bufo marinus as described (14). Briefly, the procedure involves incubating thin slices of stomach wall first in a dilute solution of collagenase and trypsin and then in a solution of collagenase alone. Cells released from the tissue in this latter solution are collected by mechanically separating the tissue from the cell suspension. Soybean trypsin inhibitor was added to all cell suspensions to suppress any remaining trypsin activity.Cells were loaded with the calcium-sensitive dye fura-2 (15) by incubating them in a solution of the acetoxymethyl ester form ofthe dye, fura-2/AM (Molecular Probes, Eugene, OR). Cells were incubated in this solution at 31°C for 60 min and then returned to room temperature. The amount of fura-2/AM added to the cell suspension was adjusted according to cell density at the ratio of 1.25 x 10-9 mol of fura-2/AM per 106 cells. These loading conditions were found to produce intracellular fura-2 concentrations of 30-100 ,M as estimated by comparing total fluorescence to that from droplets of fura-2 solutions in oil.A dilute suspension of cells that had been loaded with fura-2 was placed on a cover-slip chamber on the stage of an inverted microscope, and the cells were allowed to settle. A single cell was then attached at both ends to two special glass microprobes, each of which had a single anionic-exchangeresin particle cemented to its tip. After attachment of cell to probe, a small section of the muscle cell was wrapped at each end by micromanipulation around the attached probe. The procedure was basically that described by Warshaw and Fay (16). One probe was attached to a micr...

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 (T18A͞ 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.

Changes in skeletal muscle of a patient with malignant hyperthermia (MH) were examined in skinned fibers. In the patient's muscle, the Ca-induced Ca release mechanism showed a significantly higher sensitivity to Ca than that in normal muscles and the maximum rate of Ca release at a sufficiently high concentration of Ca was also significantly higher. Halothane accelerated Ca-induced Ca release to a similar extent both in the patient's and normal muscles. No difference was observed in the properties of Ca uptake by the sarcoplasmic reticulum (SR) and of the contractile protein system between the patient's and normal muscles. The changes observed in the Ca-induced Ca release mechanism were considered to be sufficient to explain the disease.

Ca-induced Ca release (CICR) first found in the sarcoplasmic reticulum of skeletal muscle1 '2 is an interesting biological phenomenon, but it does not appear to play a major role in normal excitation-contraction coupling in the amphibian skeletal muscle.3>>4> However, it might play an important physiological role in other kinds of cells such as medaka eggs, as suggested by propagation of `calcium wave' upon their fertilization.5> If a response of some cells is mediated by the CICR mechanism, it should be inhibited by inhibitors of CICR. Therefore, to examine whether or not inhibitors of CICR inhibit the response constitutes a good screening test for the possibility of involvement of the CICR mechanism in evoking the response in question. Indeed, the absence of inhibition of normal excitation-contraction coupling of amphibian skeletal muscle by inhibitors of CICR°>,7) furnished strong evidence against physiological role of the mechanism in this tissue. In the present article, the effect of procaine and adenine, two different inhibitors of CICR,6) 8 ° was examined on contractions of smooth muscles evoked by transmitters and on shape changes of platelets. Thin bundles of smooth muscle fibers were dissected from taenia caeci or portal vein from the guinea pig. They were suspended in a trough in which solutions could be exchanged rapidly. Isometric tension was monitored by a strain-gauge transducer (UL-2, Shinkoh, Tokyo) and displayed on a pen recorder. Contractions were evoked by applying caffeine or neurotransmitters, acetylcholine or carbachol in taenia caeci and noradrenaline in portal vein. To see the result of Ca release from an intracellular store without participation of changes in membrane potential and of Ca entry from the extracellular fluid, experiments were done in a medium in which all Na ions were replaced by K ions and 4 mM EGTA (ethyleneglycolbis-(,Q-aminoethylether)-N-N'-tetraacetic acid) was present. Platelets were obtained from arterial blood drawn through a plastic cannula inserted into the femoral artery of albino rabbits under ether anesthesia. Blood was immediately mixed with 1/9 volume of sodium citrate solution (final concentration about 11 mM) and platelet-rich plasma obtained as the supernatant by centrifuging the citrated blood at 230 X g for 7 min. One volume of the platelet-rich plasma was mixed with 4 volume of a buffer (Na2SO4 or K2SO4 t)