Regulation of isometric force and isotonic shortening velocity by phosphorylation of the 20,000 dalton myosin light chain of rat uterine smooth muscle

Abstract

The regulation of isometric force maintenance and isotonic shortening velocity by phosphorylation of the 20,000 dalton light chain of myosin has been examined for potassium-depolarized rat uterine smooth muscle. Following a transient peak in myosin light chain (LC20) phosphorylation at 20 s of contraction (0.46 mol PO4/mol LC20), phosphorylation declined to a steady-state by 2 min (0.28 mol PO4/mol LC20) with no significant change from 2-90 min of contraction. Isometric force developed more slowly, reaching a maximum at 2 min with no further change out to 90 min. Lightly-loaded (0.1 F0) shortening velocity, like LC20 phosphorylation, increased initially to a peak of 0.034 L0/s at 20 s of contraction and then declined to 0.023 L0/s by 2 min. However, unlike LC20 phosphorylation and isometric force, shortening velocity decreased approximately 4-fold from 0.023 L0/s at 2 min to 0.006 L0/s at 90 min of contraction. Graded activation with reduced extracellular calcium was associated with proportional changes in steady-state isometric force and LC20 phosphorylation. Shortening velocity was also decreased with reduced calcium, however, unlike LC20 phosphorylation, the greatest changes in velocity occurred at low levels of developed force. Moreover, in contrast to the large reductions in shortening velocity observed during 90 min contractions where force and LC20 phosphorylation were unchanged, similar reductions in shortening velocity did not occur with graded activation in spite of significant (greater than 3-fold) decreases in both force and LC20 phosphorylation. These results suggest that factors other than light chain phosphorylation are involved in the regulation of isotonic shortening velocity during extended isometric contractions of uterine smooth muscle.

Correlations between myosin heavy chain isoforms and mechanical parameters in rat myometrium.

“…In addition, the relationship of light chain phosphorylation and shortening velocity is a tenuous one. Haeberle et al (1985b) could not demonstrate a consistent correlation between these two parameters in uterine smooth muscle. We conclude from these data that the difference in velocity between the two groups 362 SMOOTH MUSCLE MYOSIN ISOFORMS AND FUNCTION cannot be wholly attributable to differences in phosphorylation which might result from either preferential induction or inhibition of myosin light chain kinase or phosphatase activity by oestrogen.…”

“…Only fibres from ovariectomized and oestrogen-treated rats that were paired by weight, and run on the same gel, were compared. The relative proportions of the myosin heavy chain isoforms in tissue extracts or fibres were estimated by comparing peak heights (mm) from densitometric gel scans, as per Rovner et al (1986 Haeberle, Hott & Hathaway (1985b) as adapted by Obara & de Lanerolle (1989) using affinity-purified anti-myosin light chain antibodies (anti MLC) to quantitate the myosin light chains. Gels were composed of 7 5% acrylamide,0-67 % bis-acrylamide,6-00 M urea, 10% glycerol, 2% ampholytes (Pharmalyte,, and were polymerized with 0-01 % temed and 0-1 mg ml-' ammonium persulphate.…”

Rapid myosin phosphorylation transients in phasic contractions in chicken gizzard smooth muscle

“…Muscle strips of approximately 15 mm length and 0.4-0.5 mm diameter were cut out of the superficial circumferential At selected times the muscle strips were frozen within 60 ms by clamping them between two brass-bolts precooled to -100°C with liquid nitrogen. The frozen strips were stored in a 10% (w/v) TCA/acetone solution at -80°C for at least 48 h. They were allowed to warm to room temperature in this solution over a period of 1 h [4], and were further processed for ZD-gel electrophoresis, as described previously [5]. The relative amounts of phosphorylated and nonphosphorylated myosin light chains were determined by densitometry scans of the Coomassie stained gels.…”

Contractile elements and myosin light chain phosphorylation in myometrial tissue from nonpregnant and pregnant women.

“…Contraction and relaxation of myometrium (and other smooth muscles) are regulated by phosphorylation and dephosphorylation of the 20-kD light chain of myosin (4)(5)(6)(7)(8)(9)(10) This study was conducted as part of an investigation to identify differences in contractile properties (mechanical and biochemical) of myometrium from pregnant and nonpregnant women. In the rat, the stress-generating capacity of the myometrium increases threefold during pregnancy ( 11).…”