Mg2+- or Ca2+-activated ATPase activities of plasma membranes isolated from vascular smooth muscle

Studies of ATP hydrolysis by various subcellular fractions isolated from rat mesenteric arteries and veins indicate that an apparent ATPase activity, which can be activated by Mg2+ or Ca2+, is primarily associated with the plasma membranes. Although both Mg2+-activated and Ca2+-activated ATPase activities under the optimal condition are substantially lower in venous than in arterial plasma membrane fraction, their dependence on the concentration of Mg2+ and Ca2+ are quite similar in arterial as well as venous plasma membrane fractions. No synergistic effect on ATP hydrolysis was observed in the presence of both Mg2+ and Ca2+. In addition, Mg2+-activated and Ca2+-activated ATPase activities show similar pH dependence, inhibition by deoxycholate, stability toward heat inactivation and substrate specificity. Furthermore, Mg2+-activated and Ca2+-activated ATPase activities were similarly reduced in vascular smooth muscles of spontaneously hypertensive rats. These results suggest that the activation of ATP hydrolysis by Mg2+ or Ca2+ may represent a single enzyme moiety in the plasma membrane of vascular smooth muscle. The possible involvement of such ATPase in the Ca2+ transport function of vascular smooth muscle is discussed.     

Functional transformation of fast posterior latissimus dorsi muscle by "slow" nerve implanted in newly hatched chickens.

1. Contraction properties and the activity of Ca2+ - ATPase were investigated 2 and 5 to 6 1/2 months after transposition of the fast posterior latissimus dorsi muscle (PLD) to the other side in newly hatched chickens. At the same time the muscle was cross-innervated by the nerve originally supplying the slow anterior latissimus dorsi muscle (ALD). 2. The mean isometric twitch contraction time of these transposed, cross-innervated PLD muscles in the 2-month-old and 5 to 6 1/2-month-old groups was 61.6 +/- 4.2 msec and 72.5 +/- 10.8 msec respectively. When compared with values obtained in control PLD and ALD muscles (21.9 +/- 0.6 msec and 107.7 +/- 5.6 msec), contraction time was significantly prolonged by this procedure. 3. Ca2+ - ATPase activity was also found to change towards the slow muscle (activity in control PLD was 0.600 micronmoles Pi/mg myosin/min, in the transposed, cross-innervated PLD 0.462 and in control ALD muscle 0.156 respectively). 4. Foreign innervation may thus induce changes in functional and biochemical properties even in muscles considerably different in structure and function, if transformation is allowed to take place at a sufficiently early stage of development. The muscle transposition itself, by introducing the element of muscle dedifferentiation and regeneration, probably assists the transformation process by making the muscle more plastic to the neural influences.     

In vitro differentiation in the absence of nerve of avian myoblasts derived from slow and fast muscle rudiments

Slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) muscles of 9-day-old quail embryos were cultured in vitro without neurons for 1 to 12 weeks. Several differences could be observed between ALD- and PLD-derived cells. PLD cultures proliferated less rapidly than ALD cultures. ALD-derived muscle fibres exhibited wide Z lines, numerous mitochondria, and a poorly developed sarcotubular system, while PLD-derived muscle fibres exhibited narrow Z lines, few mitochondria, and an abundant sarcotubular system. Staining for myofibrillar ATPase revealed that all well-differentiated ALD-derived muscle fibres were of the beta' type, while PLD-derived fibres were of beta and beta R types. These results show that myoblasts from slow and fast muscle rudiments can express in vitro some of the characteristic features of slow and fast muscle fibres, independently of motor innervation.     

Electrophoretic analysis of multiple forms of myosin in fast-twitch and slow-twitch muscles of the chick.

1. A method is described for the electrophoretic analysis of intact myosin in polyacrylamide gel in a buffer system containing 0.02 M-pyrophosphate and 10% (v/v) glycerol, pH 8.8. 2. In this system chicken skeletal-muscle myosins reveal five distinct electrophoretic components, three components from the fast-twitch posterior latissimus dorsi muscle and two slower-migrating components from the slow-twitch anterior latissimus dorsi muscle. 3. The Ca2+-activated ATPase (adenosine triphosphatase) activity of myosin components was measured by densitometric scanning of the gel for the Ca3(PO4)2 precipitate formed during the ATPase reaction and subsequently for stained protein. Each component from the same muscle appears to have identical ATPase activity, but components from the fast-twitch muscle had an activity 2.2 times higher than those from the slow-twitch muscle. 4. On re-electrophoresis in the same buffer system, individual fractions of fast-twitch myosin did not reproduce the three-band pattern of the original myosin, but migrated at rates consistent with their original mobility. 5. Analysis of the mobility of the three fast-twitch myosin components in gels of different concentrations suggests that they are not stable oligomers of each other. 6. It is suggested that these components of fast-twitch myosin and slow-twitch myosin are isoenzymes of myosin.     

Elevated levels of a calcium-activated muscle protease in rapidly atrophying muscles from vitamin E-deficient rabbits.

A Ca2+-activated proteolytic enzyme that partially degrades myofibrils was isolated from hind limb muscles of normal rabbits and rabbits undergoing rapid muscle atrophy as a result of vitamin E deficiency. Extractable Ca2+-activated protease activity was 3.6 times higher in muscle tissue from vitamin E-deficient rabbits than from muscle tissue of control rabbits. Ultrastructural studies of muscle from vitamin E-deficient rabbits showed that the Z disk was the first myofibrillar structure to show degradative changes in atrophying muscle. Myofibrils prepared from muscles from vitamin E-deficient rabbits showed partial or complete loss of Z-disk density. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the amount of troponin-T (37 000 daltons) and alpha-actinin (96 000 daltons) was reduced in myofibrils from atrophying muscle as compared to myofibrils prepared from control muscle. In vitro treatment of purified myofibrils with purified Ca2+-activated proteolytic enzyme produced alterations in myofibrillar ultrastructure that were identical to the initial alterations occurring in myofibrils from atrophying muscle (i.e. weakening and subsequent removal of Z disks). Additonally the electrophoretic banding pattern of Ca2+-activated proteolytic enzyme-treated myofibrils is very similar to that of myofibrils prepared from muscles atrophying as a result of nutritional vitamin E deficiency. The possible role of Ca2+-activated proteolytic enzyme in disassembly and degradation of the myofibril is discussed.     

Effects of denervation and direct electrical stimulation upon the post-hatching differentiation of posterior latissimus dorsi muscle in chicken

The influences of denervation and of direct electrical stimulation of denervated muscle upon the post-hatching differentiation of fibre types in the fast avian muscle posterior latissimus dorsi have been investigated. Denervation inhibits the normal decrease in number of muscle fibres exhibiting acid-stable myofibrillar ATPase activity and leads to weak oxidative activity in all the fibres. Direct stimulation at a low rhythm of denervated muscle induces the normal decrease of fibres exhibiting acid-stable myofibrillar ATPase but does not allow the occurrence of normal oxidative activity pattern. The results emphasize the role of muscular activity upon the differentiation of fibre types in a developing muscle.     

Elevated levels of a calcium-activated muscle protease in rapidly atrophying muscles from vitamin E-deficient rabbits

A Ca²⁺-activated proteolytic enzyme ¹ that partially degrades myofibrials was isolated from hind limb muscles of normal rabbits and rabbits undergoing rapid muscle atrophy as a result of vitamin E deficiency. Extractable Ca²⁺-activated protease activity was 3.6 times higher in muscle tissue from vitamin E-deficient rabbits than from muscle tissue of control rabbits. Ultrastructural studies of muscle from vitamin E-deficient rabbits showed that the Z disk was the first myofibrillar structure to show degradative changes in atrophying muscle. Myofibris prepared from muscles vitamin E-deficient rabbits showed partial or complete loss of Z-disk density. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the amount of troponin-T (37 000 daltons) and α-actinin (96 000 daltons) was reduced in myofibrils from atrophying muscle as compared to myofibrils prepared from control muscle. In vitro treatment of purified myofibrils with purified Ca²⁺-activated proteolytic enzyme produced alterations in myofibrillar ultrastructure that were identical to the initial alterations occuring in myofibrils from atrophying muscle (i.e. weakening and subsequent removal of Z disks). Additionally the electrophoretic banding pattern of Ca²⁺-activated proteolytic enzyme-treated myofibrils is very similar to that of myofibrils prepared from muscles atrophying as a result of nutritional vitamin E deficiency. The possible role of Ca²⁺-activated proteolytic enzyme in disassembly and degradation of the myofibril is discussed.     

Inhibition of actomyosin ATPase by high concentrations of 5-hydroxytryptamine. Possible basis of lesion in 5HT-induced experimental myopathy.

The effect of 5-hydroxytryptamine (5HT) on the ATPase activity and sulphydryl group reactivity of mammalian skeletal muscle actomyosin has been studied. 5HT inhibited the Mg2+-activated but not the Ca2+-activated ATPase activity of actomyosin. It slightly activated myosin ATPase. The sulphydryl groups of actomyosin reacting with 5,5'-dithiobis-(2-nitrobenzoic acid) were blocked by concentrations of 5HT which inhibited the Mg2+-activated ATPase. The significance of the results are discussed in relation to the muscle lesions in the experimental myopathy induced by 5HT and imipramine.     

Effect of ovarian steroids on membrane ATPase activities in microsomes (microsomal fractions) from rat myometrium. Inhibition of a component of the Mg2+-activated ATPase by Ca2+-calmodulin and by oxytocin.

The activities of Mg2+-ATPase (Mg2+-activated ATPase), (Ca2+ + Mg2+)-activated ATPase and (Na+ + K+)-activated ATPase have been determined in microsomes (microsomal fractions) obtained from rat myometrium under different hormonal conditions. Animals were either ovariectomized and treated for a prolonged period of time with 17 beta-oestradiol or progesterone, or myometria were obtained at day 21 of pregnancy. In each case the endometrium was carefully removed. The Mg2+-ATPase consists of two components: an inactivating labile component and a second constant component. The rate of ATP hydrolysis by the labile component of the Mg2+-ATPase declines exponentially as a function of time after adding the membranes to the assay medium; this inactivation is caused by the presence of ATP in the medium. This ATPase activity inhibited by ATP is catalysed by a labile enzyme and hence it gradually diminishes within a few hours, even when the microsomes are kept on ice. This labile component has the highest activity in microsomes from pregnant rats, a lower activity in progesterone-treated rats, and the lowest in 17 beta-oestradiol-treated rats. This component of the Mg2+-ATPase is not affected by 90 nM-oxytocin. The constant component of the Mg2+-ATPase must be ascribed to a different enzyme, which, in contrast with the labile component, is very stable and not affected by the hormonal status of the animal. This constant component of the Mg2+-ATPase is inhibited both by Ca2+-calmodulin, and by oxytocin in microsomes from pregnant and from progesterone-treated animals, whereas such inhibition does not occur in microsomes from 17 beta-oestradiol-treated animals. The activity of the (Na+ + K+)-activated ATPase is not dependent on the hormonal status of the animal. Myometrial microsomes present an ATP-dependent Ca2+ transport, irrespective of the hormonal condition, but only in microsomes obtained from rats treated with 17 beta-oestradiol, can a (Ca2+ + Mg2+)-activated ATPase activity be demonstrated. This activity can be stimulated by calmodulin.     

Characterization of the C-protein from posterior latissimus dorsi muscle of the adult chicken: heterogeneity within a single sarcomere

Specific isoforms of myofibrillar proteins are expressed in different muscles and in various fiber types within a single muscle. We have isolated and characterized monoclonal antibodies against C-proteins from slow tonic (anterior latissimus dorsi, ALD) and fast twitch (pectoralis major) muscles of the chicken. Although the antibody against "fast" C-protein (MF-1) did not bind to the "slow" isoform and the antibody to the "slow" C-protein (ALD-66) did not bind to the "fast" isoform, we observed that both antibodies bound C-protein from the posterior latissimus dorsi (PLD) muscle. Here we demonstrate that in the PLD muscle the binding sites of these two antibodies reside in two different C-protein isoforms which have different molecular weights and can be separated by hydroxylapatite column chromatography. Since we have shown previously that both these antibodies stain all myofibers and myofibrils derived from PLD muscle, we conclude that all myofibers in this muscle contain both isoforms with all sarcomeres.     

Non-correlation of phosphorylation of the P-light chain and the actin activation of the ATPase of chicken gizzard myosin.

1. The enzymic properties of myosin isolated from chicken gizzard by three different methods have been compared. 2. Although the specific Ca2+-stimulated ATPases of all preparations were similar and high, there were significant differences in the specific activities of the Mg2+-stimulated actomyosin ATPases. 3. There was no direct correlation between the Mg2+-stimulated actomyosin ATPase activity and the extent of P-light-chain phosphorylation in any of the three myosin preparations. 4. A fraction that activates the Mg2+-stimulated actomyosin ATPase of gizzard muscle has been isolated from a gizzard muscle filament preparation. 5. The activator was specific for the Mg2+-activated actomyosin ATPase of smooth muscle. 6. The activator required the addition of calmodulin for full effect.     

Turnover of muscle protein in the fowl (Gallus domesticus). Rates of protein synthesis in fast and slow skeletal, cardiac and smooth muscle of the adult fowl.

Rates of protein synthesis in skeletal, cardiac and smooth muscle of fully grown fowl (Gallus domesticus) were determined in vivo by means of the constant infusion method using [14C]proline. In the anterior latissimus dorsi muscle, containing predominantly slow fibres, the average synthesis rate of non-collagen muscle proteins was 17.0 +/- 3.1% per day, a value higher than that obtained for cardiac muscle (13.8 +/- 1.3% per day) and for smooth muscle of the gizzard (12.0 +/- 1.9% per day). In the posterior latissimus dorsi muscle, containing predominantly fast fibres, synthesis rates were much lower (6.9 +/- 1.8% per day). In each case these average rates for the non-collagen protein were similar to the average rate for the sarcoplasmic and myofibrillar protein fractions. The RNA concentration of these four muscles showed that relative rates of protein synthesis were determined mainly by the relative RNA concentrations. The rate of protein synthesis per unit of DNA (the DNA activity) was similar in the two skeletal muscles, but somewhat lower in cardiac muscle and gizzard, possibly reflecting the larger proportion of less active cell types in these two muscles. These quantitative aspects of protein turnover in the two skeletal muscles are discussed in terms of the determination of ultimate size of the DNA unit, and in relation to muscle ultrastructure.     

Differentiation of slow and fast muscles in chickens

1. The development of the characteristic histochemical appearance of the slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) was studied in chickens during embryonic development as well as during regeneration of minced muscle. 2. During embryonic development the activity of the oxidative enzyme succinic dehydrogenase (SDH) is higher in the slow ALD muscle already at 16 days of incubation. At this time the fast PLD has a higher activity of the glycolytic enzyme, phosphorylase. Although the histochemical appearance of the two types of muscle is already different at 16 days, their contractile speeds are still similar. No difference in myosin ATP-ase was found in the two muscles in young embryos but in 20-day old embryos the two muscles became distinctly different when stained for this enzyme. 3. When PLD muscles in hatched chickens redeveloped during regeneration in place of ALD the histochemical characteristics of the regenerated muscle resembled ALD, and when ALD regenerated in place of PLD it resembled PLD. 4. It is concluded that the histochemical characteristics of slow and fast muscles become determined during early development, even before any difference in contractile properties can be detected and that they are determined by the nerve.     

Histochemical differentiation of extrafusal muscle fibres of the anterior latissimus dorsi in the chick

Histochemical differentiation of the chick anterior latissimus dorsi (ALD) muscle was studied during embryonic development and after hatching. The two types of adult ALD tonic fibres (alpha' and beta') differentiate from a pool of acid and alkali-stable myofibrillar ATPase fibres. Intermediate stages of the transformation from beta' to alpha' were observed. At all developmental stages studied, a low percentage of formalin-resistant, alkali-stable and acid-labile ATPase fibres were observed. Such fibres have the histochemical properties of the alpha R or fast oxidative-glycolytic fibres and are assumed to be focally innervated.     

Myofibrillar and membrane-bound enzymes in skeletal muscle from myodystrophic mice.

1. Experiments were carried out to examine the biochemical changes, such as contractile protein biochemistry and membrane bound enzyme alterations associated with skeletal muscles of myd/myd. 2. Our studies demonstrate that there was a progressive decline in myofibrillar ATPase activity, and this decrease is greatest in 30 weeks old animals of myd/myd as compared to controls. 3. The proteolytic activity of myofibrils isolated from myd/myd was significantly higher than controls. 4. There was no significant difference in Ca2+ ATPase activity of myosin and actin-activated myosin ATPase activity of myd/myd and their controls. 5. Mg2+ ATPase and Na(+)+K(+)-ATPase of myodystrophic SL showed significant increase compared to controls. 6. Isoproterenol stimulated adenylate cyclase activity was significantly lower in the SL of dystrophic mice compared to controls. 7. GTP+isoproterenol stimulate adenylate cyclase was significantly higher in control SL and SR when compared to SL and SR isolated from myd/myd. 8. Guanylate cyclase activity was greater in myodystrophic mice both in the absence and presence of Triton X-100. cGMP and cAMP phosphodiesterase activities were greater in dystrophic mice as compared to controls. 9. These observations suggest that there are significant changes in myofibrillar ATPase, myofibrillar protease and membrane bound enzymes of myd/myd compared to control.     

ATPase Activity of Myosin Correlated with Speed of Muscle Shortening

Myosin was isolated from 14 different muscles (mammals, lower vertebrates, and invertebrates) of known maximal speed of shortening. These myosin preparations were homogeneous in the analytical ultracentrifuge or, in a few cases, showed, in addition to the main myosin peak, part of the myosin in aggregated form. Actin- and Ca⁺⁺-activated ATPase activities of the myosins were generally proportional to the speed of shortening of their respective muscles; i.e. the greater the intrinsic speed, the higher the ATPase activity. This relation was found when the speed of shortening ranged from 0.1 to 24 muscle lengths/sec. The temperature coefficient of the Ca⁺⁺-activated myosin ATPase was the same as that of the speed of shortening, Q₁₀ about 2. Higher Q₁₀ values were found for the actin-activated myosin ATPase, especially below 10°C. By using myofibrils instead of reconstituted actomyosin, Q₁₀ values close to 2 could be obtained for the Mg⁺⁺-activated myofibrillar ATPase at ionic strength of 0.014. In another series of experiments, myosin was isolated from 11 different muscles of known isometric twitch contraction time. The ATPase activity of these myosins was inversely proportional to the contraction time of the muscles. These results suggest a role for the ATPase activity of myosin in determining the speed of muscle contraction. In contrast to the ATPase activity of myosin, which varied according to the speed of contraction, the F-actin-binding ability of myosin from various muscles was rather constant.     

Differences in calcium transport mechanism of the sarcoplasmic reticulum of the slow-twitch and fast-twitch muscle

The Ca2+ uptake mechanism of sarcoplasmic reticulum (SR) was comparatively examined in fast-twitch and slow-twitch muscles. The competition of Mg2+ and Ca2+ at the binding sites is important in the function of the Mg2+-activated Ca2+-ATPase of the SR. The best ratio of divalent cations for Ca2+ uptake is not the same in the two kinds of muscle. The formation of the phosphorylated intermediate in more dependent on changes in the concentrations of the two divalent cations in the SR membrane of the fast-twitch than in that of the slow-twitch muscle. The requirement for Mg2+ to an efficient function of the transport ATPase and Ca2+ uptake of SR is greater in the latter than in the former.     

Biochemical transformation of canine skeletal muscle for use in cardiac-assist devices

Skeletal muscle has an inherent biochemical phenotypic plasticity that provides the possibility for it to be remodeled into a "heart-like" muscle for use in cardiac-assist devices. The purpose of this study was to chronically stimulate skeletal muscle electrically to transform the biochemical capacities of the three major subcellular systems (i.e., metabolic, calcium regulating, and contractile) to resemble those of heart muscle. The latissimus dorsi muscle (LDM) of mongrel dogs weighing 22-27 kg was stimulated via the thoracodorsal nerve at 2 Hz for 6-8 wk. This stimulation protocol reduced the phosphorylase (glycogenolytic) and phosphofructokinase (glycolytic) activities by 70%. The aerobic (citrate synthase activity) and fatty acid oxidative (3-hydroxyacyl-CoA dehydrogenase activity) capacities were not significantly increased by chronic stimulation and remained at about one-fourth those in the canine heart. The calcium-dependent sarcoplasmic reticulum adenosinetriphosphatase (ATPase) activity in the microsomal fraction, which was sixfold greater in the nonstimulated LDM than in the heart, was reduced by electrical stimulation to a level similar to that of the dog heart. The contractile capacity was evaluated by determining the percentage of types I and II fibers, the myofibrillar ATPase activity, and the proportion of myosin isoforms. The transformed muscle was comprised of 93 +/- 2% type I fibers, a myofibrillar ATPase activity similar to that in heart with primarily a slow-twitch muscle myosin isoform. In conclusion, electrical stimulation of canine LDM at 2 Hz for 6-8 wk resulted in two of the three biochemical systems, which confer physiological expression and fatigue resistance to muscle being transformed to resemble those of the myocardium.     

The effects of ATP, inorganic phosphate, protons, and lactate on isolated myofibrillar ATPase activity.

The purpose of this study was to examine the effects of lactate, protons, inorganic phosphate, and ATP on myofibrillar ATPase activity. Myofibrils were isolated from carp (Cyprinius carpio L.) fast-twitch white muscle, and myofibrillar ATPase activities were assessed under maximal activating calcium levels (pCa 4.0) at 10 degrees C in reaction media containing metabolic profiles similar to those seen in fatiguing muscles. The Ca(2+)-activated ATPase activity was assessed by an ATP regenerating assay that coupled the myofibrillar ATPase to pyruvate kinase and lactate dehydrogenase. This assay allowed the effects of ATP, inorganic phosphate, protons, and lactate on myofibrillar ATPase activity to be assessed. The coupled assay was found to give similar myofibrillar ATPase kinetics, with the exception of higher maximal activities, to those seen with a standard end-point assay. Myofibrillar ATPase activity was depressed by 35% when ATP concentrations were lowered to 2.5 mM. Lowering ATP levels to 0.5 mM reduced the myofibrillar ATPase activities by 85%. Lactate had no effect on myofibrillar ATPase activities. Inorganic phosphate levels up to about 20 mM significantly decreased the myofibrillar ATPase activities, after which further increases in inorganic phosphate content had minimal effects. The changes in ATPase activities were related to total inorganic phosphate, not to the content of diprotonated inorganic phosphate. Myofibrillar ATPase activity was highest at pH 7.5 and lowest at pH 6.0. The interactive effects of low ATP, decreased pH, and high inorganic phosphate levels were not additive, giving similar decreases in activity to those produced by increased inorganic phosphate levels alone.(ABSTRACT TRUNCATED AT 250 WORDS)