Crossbridge attachment, resistance to stretch, and viscoelasticity in resting mammalian smooth muscle.

There exist a calcium-dependent resistance to stretch in resting mammalian smooth muscle that is not caused by depolarization of the cell membrane or release of calcium from intracellulr sites. The similarity of the resistance to stretch in the resting state to that in rigor suggests that most, if not all, crossbridges are attached and thus able to resist stretch in noncontracting smooth muscles. When the muscle is stretched the breaking and subsequent reformation of links in nonstrained positions accounts for most of the so-called viscoelasticity, except at extreme lengths.

Myosin light chain phosphorylation does not modulate cross-bridge cycling rate in mouse skeletal muscle.

An attempt was made to determine whether phosphorylation of the myosin light chain represents a thick filament-associated mechanism for modulating the rate of cross-bridge cycling in mouse skeletal muscle. When the degree of light chain phosphorylation was varied independently of tetanus duration, there was no correlation of phosphorylation with cross-bridge turnover rate, as measured by the shortening velocity of the muscle. It is concluded that in intact skeletal muscle phosphorylation of the myosin light chain does not in itself modulate cross-bridge cycling rate and that previously reported changes in cycling rate were due to other factors that may vary with tetanus duration.

Chemical energetics of force development, force maintenance, and relaxation in mammalian smooth muscle.

High-energy phosphate utilization (delta approximately P) associated with force development, force maintenance, and relaxation has been determined during single isometric tetani in the rabbit taenia coli. ATP resynthesis from glycolysis and respiration was stopped without deleterious effects on the muscle. At 18 degrees C and a muscle length of 95% l0, the resting rate of energy utilization is 1.8 +/- 0.2 nmol/g . s-1, or 0.85 +/- 0.2 mmol approximately P/mol of total creatine (Ct) . s-1, where Ct = 2.7 mumol/g wet wt. During the initial 25 s of stimulation when force is developed, the average rate of delta approximately P was -8.2 +/- 0.8 mmol/mol Ct . s-1, some four times greater than during the subsequent 35 s of force maintenance, when the rate was -2.0 +/- 0.6 mmol approximately P/mol Ct . s-1. The energy cost of force redevelopment (0 to 95% P0) after a quick release from the peak of a tetanus is very low compared with the initial force development. Therefore, the high rate of energy utilization during force development is not due only to internal work done against the series elasticity nor to any high rate of cross-bridge cycling inherently associated with force development. The high economy of force maintenance compared with other muscle types is undoubtedly due to a slower cross-bridge cycle time. The energy utilization during 45 s of relaxation was not statistically significant, and integral of Pdt/delta approximately P was higher during relaxation than during force maintenance in the stimulated muscle.

Chemical energy usage and myosin light chain phosphorylation in mammalian smooth muscle.

Experiments have been done to determine the relationships among active force output, average rate of high-energy phosphate utilization, and the degree of phosphorylation of the 20,000-dalton myosin light chain in the rabbit tenia coli at 18 C. During an isometric tetanus at l0 the degree of light chain phosphorylation increases to a maximum of 30-40% before maximum force is developed, and then phosphorylation slowly decreases while active force is maintained. During the period when there is a small decrease in degree of phosphorylation, the average rate of chemical energy usage falls by fourfold. In contrast, when the calcium concentration of the bathing medium is lowered from 1.9 to 1.0 mM a very large decrease in degree of phosphorylation is associated with only a small decrease in both energy usage and active force. At lower calcium levels both force and chemical energy usage decrease proportionately with little further decrease in degree of phosphorylation. We conclude that under isometric conditions there is no consistent relationship between degree of myosin light chain phosphorylation and the rate of cross-bridge cycling as measured by the rate of high-energy phosphate usage in this mammalian smooth muscle.

Chemical Energetics of contraction in mammalian smooth muscle.

Measurements of high-energy phosphate utilization during isometric tetanic contractions were made on a rabbit taenia coli preparation that had been treated so that respiration and glycolysis were blocked without altering the mechanical response to the muscle. At 18 C the first sign of high-energy phosphate usage after electrical stimulation was a net breakdown of ATP with a subsequent net resynthesis when the rate of phosphocreatine breakdown was high. The average rate of chemical energy usage was more than four times as high during the period of initial force development compared to that during maximum force maintenance. This initial high rate of energy usage was not due only to the internal work done against the series elasticity during force development. Comparison of energetics and mechanics data for the rabbit taenia coli and frog sartorius showed that smooth muscle was 100-fold more economical in maintaining isometric force and probably had a cross-bridge cycle time that was about 150-fold slower. The rate of energy usage during relaxation from an isometric tetanus in smooth muscle was very low, and force was exerted with a lower expenditure of energy during relaxation than during maximum force maintenance. Phosphorylation of the 20,000-dalton light chain of myosin occurred to the extent of 32% during a maximally activated isometric tetanus and was not proportional to the rate of energy usage during different stages of the tetanus. During isometric relaxation, the degree of phosphorylation, rate of energy usage, and active state all decreased more quickly than did active force output.

Control of cross-bridge cycling by myosin light chain phosphorylation in mammalian smooth muscle.

This review focuses on experiments in which the single turnover of myosin-bound ADP is used to characterize the regulation of the cross-bridge cycle by myosin light chain phosphorylation in mammalian smooth muscle. Under isometric conditions, at rest, when the myosin light chain is not phosphorylated, myosin cycles very slowly (about 0.004 s-1), while phosphorylation of the light chain results in a 50-fold increase in cycling rate of 0.2 s-1. Experiments consistently show that some myosin does not increase its cycling rate although its light chain is phosphorylated. Studies at low levels of myosin light chain phosphorylation show that phosphorylation also induces an increase in the cycling rate of unphosphorylated myosin. The fast cycling phosphorylated myosin is the main determinant of suprabasal myosin ATPase activity, while the cycling rate of cooperatively activated unphosphorylated myosin is slow and appears to depend on the extent of phosphorylation of the entire thick filament. Single turnover experiments measuring the rate of phosphorylation and dephosphorylation of myosin light chain show that the turnover of light chain phosphate can be very rapid (0.3-0.4 s-1) at suprabasal calcium concentrations. The expected effect of such a rapid turnover of light chain phosphorylation on the turnover of myosin-bound ADP is not observed. The effects of low levels of myosin light chain phosphorylation on the single turnover of myosin suggest that the same small pool of myosin remains phosphorylated for relatively long periods of time rather than the entire pool of myosin spending a small fraction of its cycle time in the phosphorylated state.

Metabolic characteristics of alpha-toxin-permeabilized smooth muscle.

Rabbit portal veins were permeabilized using Staphylococcus aureus alpha-toxin, and adenosinetriphosphatase (ATPase) was measured as the formation of [3H]ADP, [3H]AMP, and [3H]adenosine from [3H]ATP in the solution bathing the muscle. The resting ATPase (1.96 +/- 0.15 mM/min, n = 13) is approximately 5-10 times higher than that measured in Triton X-100-permeabilized muscles (0.28 +/- 0.01 mM/min, n = 4), with nucleotide accumulating as ADP, AMP, and adenosine. The ATPase activity is also seen when the intact muscle is incubated in a Krebs solution containing 1 mM MgATP (2.76 +/- 0.10 mM/min, n = 73). This suggests that it is due primarily to an ecto-ATPase. The ectoenzyme is capable of hydrolyzing both ATP and ADP, and in both cases there is a higher rate at 3 than at 1 mM nucleotide. The high resting ATPase compromises the control of nucleotide concentrations within the permeabilized tissue even in the presence of an ATP-regenerating system consisting of phosphocreatine (PCr, 35mM) and creatine kinase (1 mg/ml). Treatment of the intact muscle with the ectonucleotidase inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) followed by alpha-toxin permeabilization and inclusion of sodium azide in subsequent solutions reduces the ecto-ATPase by approximately 70%. Addition of PCr and creatine kinase then results in the maintenance of high [ATP] and low [ADP] in the muscle, and importantly, there are no significant changes in [ATP], [ADP], [adenosine/AMP], or the ADP-to-ATP ratio upon activation of the muscle in pCa 4.5. In general, the force output in high Ca2+ increased as the metabolic profile of the muscle improved. When ATPase was measured as the appearance of [32P]Pi from [32P]PCr and [gamma-32P]ATP, the alpha-toxin-permeabilized muscle subjected to the above treatment showed only approximately 30% higher total ATPase under activated conditions compared with the freeze-glycerinated Triton-treated portal vein. The suprabasal ATPase is similar in both preparations. We conclude that the reduction of the basal ATPase by the DIDS-azide treatment permits both rigorous control of nucleotide contents and accurate measurement of ATPase activity in alpha-toxin-permeabilized smooth muscle.

Slowing of cross-bridge cycling in smooth muscle without evidence of an internal load.

We have tested whether the slowing in velocity of shortening that occurs in mammalian smooth muscle under conditions of high force output and dephosphorylated myosin light chains is due to an internal load presented by non- or slowly cycling cross bridges. The experimental design included stimulation of the rabbit taenia coli for 25 s under isometric conditions followed by 30 s of relaxation prior to the test stimulus. There was a large decrease in maximum velocity of shortening and chemical energy usage as well as a significantly decreased myosin light-chain phosphorylation response during the test stimulus even though active force developed to 90 +/- 4% of the original. The chemical energy cost of active external work production was not significantly different from that of the initial stimulation. There is, therefore, no energetic evidence for the dissipation of work against an internal load under conditions during which most of the force would be expected to be generated by latch bridges. We conclude that force maintenance with a low energy input and a slow velocity of shortening in mammalian smooth muscle represents a general slowing of the cross-bridge cycling rate rather than the appearance of a class of latch bridges that provide an internal load against which other cross bridges must operate.

Phosphatase inhibition with okadaic acid does not alter the relationship between force and myosin light chain phosphorylation in permeabilized smooth muscle.

The phosphatase inhibitor, okadaic acid, has been used to test the hypothesis that myosin light chain phosphatase activity plays a central role in latchbridge formation in smooth muscle. In the permeabilized rabbit portal vein there is a non-linear relationship between myosin light chain phosphorylation and force production such that maximum force output occurs with about 50% phosphorylation. Treatment of the muscle with okadaic acid does not change this relationship even though there is a profound inhibition of phosphatase activity. The data suggest that dephosphorylation of the myosin light chain while the myosin is in the force producing state does not account for the high force output with low levels of light chain phosphorylation in smooth muscle.

ADP release from myosin in permeabilized smooth muscle.

The purpose of this study was to determine the nucleotide bound to myosin and its rate of release under relaxed and activated conditions in permeabilized rabbit portal veins. Incubation of the muscles in a relaxing solution containing [3H]-ATP resulted in the formation of 60-70 microM radiolabeled ADP in the muscle whether or not the myosin light chains had been thiophosphorylated. This value was similar to the estimate of the concentration of myosin subfragment 1. Upon transfer of the muscles to a chase solution containing no labeled ATP, there was a very slow loss of labeled ADP when the light chains were unphosphorylated, but a much faster release occurred when the light chains were thiophosphorylated. The results suggest that smooth muscle myosin exists primarily in a complex with ADP under both relaxed and phosphorylated conditions and that phosphorylation of all of the light chains results in a large increase in the rate of release of the products of ATP splitting from all of the myosin. Interestingly, the exponential release of ADP in relaxed muscle shows two components, one of which contains about two-thirds of the total ADP and is 5- to 10-fold faster than the other. If the difference in rates of ADP release observed in relaxed muscle persists when the myosin is phosphorylated, then it is possible that there is a 5- to 10-fold difference in rates of cycling for different phosphorylated cross bridges in smooth muscle.

Rigor and resistance to stretch in vertebrate smooth muscle.

Depletion of the APT content of the rabbit taenia coli muscle to 0.07 mumol/g caused an increase in force equal to 11% of maximum active tension (Po) when the muscle was bathed in a calcium-containing Krebs solution at 21 degrees C, but no increase in force in a calcium-free solution. Stimulation of the muscle in calcium-containing medium during the loss of ATP increased the force maintained and permitted demonstration of an increased stiffness by shortening of the muscle. There was no increase in resistance to stretch in the ATP-depleted state compared with the resting state. There was, however, a calcium-dependent resistance to stretch with properties that suggested the presence of attached crossbridges in the resting state. Bathing of the resting muscle in a calcium-free medium decreased the resting resistance to stretch and permitted demonstration of an increased resistance to stretch in the rigor state. It is concluded that the rigor state exists in vertebrate smooth muscle.

Chemical energy usage during stimulation and stretch of mammalian smooth muscle.

High energy phosphate usage was measured in the rabbit taenia coli subjected to stimulation and stretch (0.12 Lo/min) and was compared to that observed previously under isometric conditions. When the muscle was bathed in a medium containing 1.9 mM Ca2+, stretch during the period of initial force development substantially decreased the rate of chemical energy usage compared to that under isometric conditions. When crossbridge cycling rate under isometric conditions was increased by incubation of the muscle in a medium containing 4.5 mM Ca2+, there was a greater decrease in rate of high energy phosphate usage during stretch compared to isometric conditions. The low energy usage during stretch occurs even though average active force output was approximately 40% higher than that under isometric conditions. During the period of subsequent force maintenance when both energy usage and crossbridge cycling rate under isometric conditions were low, there was no significant effect of stretch on the average rate of energy usage at either Ca2+ level. These results are consistent with the hypothesis that during stimulation and stretch in smooth muscle, crossbridge attachment and force production can occur even though the actin-activated myosin ATPase activity normally associated with isometric force development is greatly suppressed.

Myosin-product complex in the resting state and during relaxation of smooth muscle.

Previous findings suggested that in resting smooth muscle ADP is bound to myosin and that phosphorylation of the myosin, and its subsequent interaction with actin, increases the rate of ADP release. We have now extended these studies to include measurements of bound Pi as well as bound ADP in permeabilized rabbit portal vein. We report that in resting smooth muscle that has been exposed to [3H]ATP and [gamma-32P]ATP, followed by a chase in an unlabeled relaxing solution, the ratio of bound [3H]ADP to bound [32P]Pi is close to unity, and both are released at approximately the same rate. This suggests that myosin exists predominantly with both ADP and Pi bound under resting conditions and that the release of one is quickly followed by the release of the other. In contrast, there is a significant 30% excess of bound Pi over ADP in a muscle during relaxation from an isometric contraction. Under these conditions, while force output is slowly decreasing, both light chain phosphorylation and adenosinetriphosphatase (ATPase) activity have decreased to near-resting values. The time course of relaxation is similar to the time course of Pi release from both the resting and relaxing muscle. We propose that during relaxation the dephosphorylated cross bridges which are bearing force have Pi but not ADP bound and that detachment of the cross bridge (and thus force decay) is limited by Pi release from myosin which occurs at the same rate as in the resting muscle.

Altered isoactin gene expression in the affected bowel segments of the lethal spotted mouse.

BACKGROUND: Actin is a key contractile protein associated with the normal differentiation and function of gastrointestinal smooth muscle cells. Distinct changes in gastrointestinal smooth muscle cell morphology and function have been reported for the aganglionic rectum and megacolon of the adult lethal spotted mouse. This study examines what effect these changes in smooth muscle cell morphology and function have on the expression of the actin multigene family in both the aganglionic rectum and megacolon of the lethal spotted mouse. METHODS: Expression of the smooth muscle and cytoplasmic isoactins was examined by Northern blot analysis of the aganglionic rectum and megacolon of the homozygotic lethal spotted mouse and the equivalent bowel segments of control animals. RESULTS: The megacolon of the lethal spotted mouse showed a significant increase in gamma-smooth muscle isoactin expression. The aganglionic rectum of the lethal spotted mouse displayed a complex pattern of altered isoactin gene expression that included changes in both gamma-smooth muscle and beta-cytoplasmic isoactin expression. Strain-specific differences in the quantitative levels of isoactin gene expression were observed for the various bowel segments examined in this study. CONCLUSIONS: These results show that the changes in smooth muscle cell morphology and function observed in the lethal spotted mutant mouse are accompanied by significant alterations in isoactin gene expression.

Rapid turnover of myosin light chain phosphate during cross-bridge cycling in smooth muscle.

The rate of phosphatase-mediated dephosphorylation of the regulatory light chain of smooth muscle myosin was determined under nearly steady-state conditions in permeabilized muscles, from the time course of incorporation of 33P-labeled phosphate into the light chain after the photolytic release of [gamma-33P]ATP from high specific activity caged [gamma-33P]ATP. The extent of myosin light chain phosphorylation is unchanged, and, if the kinase and phosphatase reactions are irreversible, the rate constant for the exponential increase in 33P in the light chain is equal to the rate constant for the phosphatase reaction. Under activated conditions (pCa 4.5) at 20 degrees C, the incorporation of 33P into approximately 80% of the phosphorylated light chain is fit by a single exponential with a rate constant of 0.37 s-1. ATP usage due to phosphorylation and dephosphorylation of the light chain is about one-third of the suprabasal energy requirement. The high phosphatase rate constant suggests that dephosphorylation of the light chain is rapid enough to interact with and potentially modify the completion of the cross-bridge cycle.

Thiophosphorylation of the 130-kDa subunit is associated with a decreased activity of myosin light chain phosphatase in alpha-toxin-permeabilized smooth muscle.

Pretreatment of alpha-toxin-permeabilized smooth muscle with ATP gamma S (adenosine 5'-O-(thiotriphosphate)) under conditions resulting in minimal (< 1%) thiophosphorylation of the myosin light chain increases the subsequent calcium sensitivity of force output and myosin light chain phosphorylation. The change in calcium sensitivity results at least in part from a 5-fold decrease in myosin light chain phosphatase activity. One of the few proteins thiophosphorylated under these conditions is the 130-kDa subunit of myosin light chain phosphatase. These results suggest that thiophosphorylation of this subunit leads to a decrease in the activity of the phosphatase, and that phosphorylation and dephosphorylation of the subunit may play a role in regulating myosin light chain phosphatase activity.

Regulation of catch muscle by twitchin phosphorylation: effects on force, ATPase, and shortening.

Recent experiments on permeabilized anterior byssus retractor muscle (ABRM) of Mytilus edulis have shown that phosphorylation of twitchin releases catch force at pCa > 8 and decreases force at suprabasal but submaximum [Ca2+]. Twitchin phosphorylation decreases force with no detectable change in ATPase activity, and thus increases the energy cost of force maintenance at subsaturating [Ca2+]. Similarly, twitchin phosphorylation causes no change in unloaded shortening velocity (Vo) at any [Ca2+], but when compared at equal submaximum forces, there is a higher Vo when twitchin is phosphorylated. During calcium activation, the force-maintaining structure controlled by twitchin phosphorylation adjusts to a 30% Lo release to maintain force at the shorter length. The data suggest that during both catch and calcium-mediated submaximum contractions, twitchin phosphorylation removes a structure that maintains force with a very low ATPase, but which can slowly cycle during submaximum calcium activation. A quantitative cross-bridge model of catch is presented that is based on modifications of the Hai and Murphy (1988. Am. J. Physiol. 254:C99-C106) latch bridge model for regulation of mammalian smooth muscle.

Calcium-dependent resistance to stretch and stress relaxation in resting smooth muscles.

Mechanical responses to stretch and length-tension relations were examined in rabbit taenia coli, mesenteric vein, aorta, and myometrium and in guinea pig taenia coli made atonic by incubation in Krebs-bicarbonate solution at 20-22 degrees C. When stretched 10% of the length at which maximum active tension is observed (Lo) in 0.5 s, the muscles showed a transient large force (resistance to stretch) that decayed to a new constant level within minutes (stress relaxation). The resistance to stretch decreased markedly in Ca2+-free [disodium ethylene glycolbis-(beta-aminoethylether)-N,N-tetraacetic acid (EGTA)] Krebs but was restored in normal Krebs solution. Calcium removal did not affect the passive length-tension curve. The absence of Ca2+ did not change the steady-state force maintained by the muscle; thus stretch resistance was not due to tone. Blockade of Ca2+ influx associated with electrical activity with 5-[3,4-dimethoxyphenethyl)methylamino]-2-(3,4,5-trimethoxyphenyl-2-isopropylvaleronitrile (D-600) and of Ca2+ release from intracellular sites with thymol (1 mM) completely blocked contraction but did not alter the responses to stretch, thus dissociating the responses to stretch from these processes and tension development. The Ca2+-dependent stress relaxation showed a dependence on muscle length similar to that for active tension development. Except at long muscle lengths, where connective tissue markedly affects length-tension relations, most of the "viscoelasticity" of these smooth muscles is dependent on calcium and may be largely due to the straining of crossbridges that are attached, but not generating a net force, in the resting state.

Chemical energetics of single isometric tetani in mammalian smooth muscle.

A rabbit taenia coli preparation has been used to study the chemical energetics of smooth muscle contraction. Under the experimental conditions, the muscle had no spontaneous mechanical activity, but could be fully activated with the use of electrical field stimulation. ATP resynthesis from glycolysis and respiration was stopped with a procedure that involved treatment with metabolic inhibitors at 5 degrees C followed by rewarming to 18 degrees C. This procedure did not alter the high-energy phosphate contents of mechanical responses of the muscle. Use of this preparation to determine ATP and phosphorylcreatine changes during isometric tetani at 18 degrees C and a length of 86% LO showed that there was net ATP breakdown initially with no significant phosphorylcreatine splitting. This was followed by an increase in the rate of phosphorylcreatine splitting with net ATP synthesis. The average rate of total high-energy phosphate utilization was about 0.01 mumol/g.s for up to 60 s or about 150 times less than that of frog sartorius at the same temperature.