Effects of calcium and substrate on force-velocity relation and energy turnover in skinned smooth muscle of the guinea-pig.

Mechanical properties and rate of ATP breakdown (JATP) have been determined in the chemically skinned guinea-pig taenia coli at 22 degrees C. The influence of varied [Ca2+], [Mg ATP] and muscle length were investigated. The shortening response after a step decrease in force (isotonic quick release) was highly curvilinear in the first 100-200 ms. This effect was shown to be a time-dependent response to the force step and not primarily caused by the shift along the length-force relation associated with shortening. Maximal shortening velocity (Vmax) decreased gradually following the release. At pCa (= -log [Ca2+]) 4.5, Vmax at 20 and 1000 ms after release was 0.49 +/- 0.07 and 0.041 +/- 0.004 (mean +/- S.E. of mean, n = 5) lengths s-1 respectively. Unloaded shortening velocity obtained from length steps of different magnitude (slack test) also showed a gradual decrease after the release, consistent with the isotonic release results. Increasing [Ca2+] from the relaxed state at pCa 9 (1 microM-calmodulin present) gave increased isometric force to a maximum at pCa 4.5. Half-maximal response was obtained at pCa 6.1. JATP at maximal force at pCa 4.5 was about 3 times the basal rate at pCa 9. The relation between JATP and force was highly non-linear, with a marked increase in JATP with little alteration in force at the highest [Ca2+]. When force was reduced to zero at pCa 4.5 by shortening the muscle to 0.3 L0 (L0 being the length giving maximal active force), JATP decreased by about 30%. At two levels of [Ca2+] giving similar force (pCa 5.75 and 4.5) the energetic tension cost obtained by length variations was lower at the low [Ca2+]. At pCa 6.0, Vmax and force were decreased to the same extent relative to their values at pCa 4.5. At pCa 5.75, where there was no reduction in force but a 25% decrease in isometric JATP, Vmax was unchanged relative to pCa 4.5. Force, Vmax and JATP were all dependent on [Mg ATP]. Half-maximal response was obtained at 0.1 mM for force and Vmax, and at 0.5 mM for JATP. The results are discussed in relation to a possible influence of both Ca2+ and Mg ATP on kinetic properties of the cross-bridge cycle

Cross-bridge behaviour in skinned smooth muscle of the guinea-pig taenia coli at altered ionic strength.

1. The effects of varied levels (25-300 mM) of ionic strength on mechanical properties and ATP hydrolysis rate of chemically skinned guinea-pig taenia coli fibres were investigated. 2. The tension development following activation by calcium (pCa 4.8), and relaxation following removal of calcium (pCa 9), were slower in 25 mM compared to 150 mM ionic strength. In fibres activated by thiophosphorylation of myosin light chains, by exposure to ATP-gamma-S, the tension development was rapid and independent of ionic strength. 3. The maximal shortening velocity (Vmax) was obtained from force-velocity relations determined by the quick-release method. The rate of ATP hydrolysis (JATP) was determined by measurement of pyruvate released from phosphoenolpyruvate (PEP). In order to obtain maximal Vmax and JATP at a Mg-ATP concentration of 1 mM, an ATP regenerating system was required. In thiophosphorylated fibres 2 mM-phosphocreatine (PCr) or 3.2 mM-PEP were adequate for maximal Vmax and JATP respectively. In calcium-activated fibres 5 mM-PCr was required for maximal Vmax. 4. The isometric force of thiophosphorylated fibres showed a biphasic dependence on ionic strength with a maximum at 90 mM. Vmax was essentially unchanged between 50 and 200 mM ionic strength. At 25 mM ionic strength, isometric force and Vmax were decreased by, respectively, about 15 and 25%. At 250 mM ionic strength, isometric force and Vmax were decreased by, respectively, 47 and 33%. 5. Vmax decreased with decreasing [Mg-ATP]. At [Mg-ATP] less than 0.1 mM there was no difference in Vmax between 35 and 150 mM ionic strength. At 250 mM ionic strength Vmax was lower than that at 150 mM at all [Mg-ATP]. 6. JATP during contraction in thiophosphorylated fibres at 35, 150 and 250 mM ionic strength was respectively, 0.62, 0.98 and 0.93 mumol g-1 min-1. The energetic tension cost (JATP/force) increased with ionic strength. 7. The force response to a quick stretch was investigated in the relaxed, contracted and rigor states at 25, 150 and 250 mM ionic strength. Stiffness in the relaxed state increased with speed of stretch and was higher the lower the ionic strength. In the contracted and rigor states, stiffness was also affected by ionic strength, but the relative effect in the contracted state was small.(ABSTRACT TRUNCATED AT 400 WORDS

Myosin composition and functional properties of smooth muscle from the uterus of pregnant and non-pregnant rats

The myosin heavy chain stoichiometry and the force-velocity relation have been determined in the myometrium of the non-pregnant and pregnant rat. The relative proportions of the slower migrating heavy chain (MHC1) greatly exceeded that of the faster migrating heavy chain (MHC2) as shown by electrophoresis on SDS 4%-polyacrylamide gels. The ratios of MHC1/MHC2 were 2.2/1 in the non-pregnant rats, 2.6/1 in the pregnant rat, and contrasted with 0.8/1 in the rat portal vein. This stoichiometry was unchanged by extracting the myosin from the smooth muscle as native myosin in a salt extract, as dissociated myosin using sodium dodecyl sulphate (SDS) or by isolating the native myosin first by a non-dissociating (pyrophosphate) electrophoresis step and subsequently analysing the protein bands on the SDS 4%-polyacrylamide gel. Although the unequal proportions of the heavy chains suggested the possibility that the native myosin molecule may be arranged as homodimeric heavy chains, no evidence for or against the existence of native myosin isoforms could be obtained by electrophoresing native myosin extracts on pyrophosphate-polyacrylamide gels. The force-velocity relations of the intact electrically stimulated myometrium from the non-pregnant and pregnant rats gave isometric force of 45 and 135 mN/mm2 and Vmax of 0.71 and 0.52 lengths/s (37 degrees C) when measured at 95% of optimal length, whereas in chemically skinned uterine strips at 22 degrees C Vmax was 0.09 and 0.13 lengths/s, respectively. The length-force relationship was of similar shape in the non-gravid and gravid skinned tissues. The energetic tension cost (ATP-turnover/active stress) in skinned fibres was also similar. The mechanical and metabolic characteristics of the gravid and non-gravid uterus found in the present study do not suggest an obvious difference in the intrinsic properties of the myosin, although significant functional alterations in the tissue appear during pregnancy. This corresponds to the lack of a difference in the pattern of the heavy chains