Characterization of the effects of Mg2+ on Ca2+- and Sr2+-activated tension generation of skinned skeletal muscle fibers

Changes in [Mg2+] in a millimolar range have a significant inverse effect on the Ca2+- (or Sr2+)activated tension generation of skeletal muscle fibers. Single frog (Rana pipiens) semitendinosus muscle fibers were "skinned" (sarcolemma removed) and contracted isometrically in bathing solutions of varying [Ca2+] or [Sr2+] and [Mg2+] but a constant pH, [MgATP2-], [K+], [CP2-], [CPK], and ionic strength. Ca2+- (or Sr2+- )activated steady-state tensions were recorded for three [Mg2+]'s: 5 X 10(-5)M, 1 X 10(-3) M, and 2 X 10(-3) M; and these tensions were expressed as the percentages of maximum tension generation of the fibers for the same [Mg2+]. Maximum tension was not affected by [Mg2+] within Ca2+-activating or Sr2+-activating sets of solutions; however, the submaximum Ca2+-(or Sr2+)activated tension is strongly affected in an inverse fashion by increasing [Mg2+]. Mg2+ behaves as a competitive inhibitor of Ca2+ and also affects the degree of cooperativity in the system. At [Mg2+] = 5 X 10(-5)M the shape of tension versus [Ca2+] (or [Sr2+]) curve showed evidence of cooperativity of Ca2+ (or Sr2+) binding or activation of the contractile system. As [Mg2+] increased, the apparent affinity for Ca2+ or Sr2+ and cooperativity of the contractile system declined. The effect on cooperativity suggests that as [Mg2+] decreases a threshold for Ca2+ activation appears.     

The regulation of tension in a chemically skinned molluscan smooth muscle: effect of Mg2+ on the Ca2+-activated tension generation

Chemically skinned anterior byssus retractor muscle (ABRM) preparations were prepared by treatment with the nonionic detergents saponin and Triton X-100. Both maximum peak tension and rate of contraction were found to be greater in saponin-treated ABRM than in ABRM treated with Triton X-100. Active tension was initiated at a concentration of free Ca2+ above 0.1 microM, and maximum tension development was found at a [Ca2+] = approximately 32 microM. During exposure of the muscle preparation to optimal Ca2+ concentration, a high and almost constant tension level was sustained. The force recovery was high after a quick release during this period indicating the presence of an "active" state rather than a "catch" state. Actually, a state equivalent to the catch state in the living ABRM could not be induced, if the Ca2+ concentration was above 0.1 microM. Variations in the ionic strength in the range of 0.07--0.28 M had no influence on active state and only slightly affected the maximum tension developed. The influence of Mg2+ on the Ca2+-activated tension was examined by studying the tension-pCa relation at two concentrations of free Mg2+ (0.43 and 4.0 mM). The tension-pCa relation was found to be S-shaped with tension increasing steeply over approximately 1 pCa unit, indicating the existence of cooperativity between Ca2+ sites. Increasing the free concentration of Mg2+ shifted the tension-pCa relation to lower pCa as in striated muscles, demonstrating a decreasing Ca2+ sensitivity with increasing Mg2+. At [Mg2+] = 4.0 mM the half-maximum tension was found at [Ca2+] = 0.43 microM, decreasing to 0.20 microM at [Mg2+] = 0.43 mM. At both Mg2+ concentrations studied, plots of log Prel/(1--Prel) vs. log [Ca2+] were nonlinear with a shape indicating a rather complicated model for cooperativity, probably involving four sites for Ca2+. These Ca2+--Mg2+ interactions are most probably taking place at the myosin head itself because troponin is absent in this myosin-regulated muscle.     

Characteristics of Ca2+- and Mg2+-induced tension development in chemically skinned smooth muscle fibers

Chemically skinned fibers from guinea pig taenia caecum were prepared by saponin treatment to study the smooth muscle contractile system in a state as close to the living state as posible. The skinned fibers showed tension development with an increase of Ca2+ in the solution, the threshold tension occurring as 5 X 10(-7) M Ca2+. The maximal tension induced with 10(-4) M Ca2+ was as large and rapid as the potassium-induced contracture in the intact fibers. The slope of the pCa tension curve was less steep than that of skeletal muscle fibers and shifted in the direction of lower pCa with an increase of MgATP. The presence of greater than 1 mM Mg2+ was required for Ca2+-induced contraction in the skinned fibers as well as for the activation of ATPase and superprecipitation in smooth muscle myosin B. Mg2+ above 2 mM caused a slow tension development by itself in the absence of Ca2+. Such a Mg2+-induced tension showed a linear relation to concentrations up to 8 mM in the presence of MgATP. Increase of MgATP concentration revealed a monophasic response without inhibition of Ca2+-induced tension development, unlike the biphasic response in striated muscle. When MgATP was removed from the relaxing solution, the tension developed slowly and slightly, even though the Mg2+ concentrations was fixed at 2 mM. These results suggest a substantial difference in the mode of actin-myosin interaction between smooth and skeletal muscle.     

Sarcomere length effects on the Sr2+- and Ca2+-activation curves in skinned frog muscle fibres.

1. A procedure recently described to produce rapid changes in [Ca2+] and [Sr2+] within the whole cross-section of skinned muscle preparations (Moisescu, D.G. (1976) Nature 262, 610--613, and Moisescu, D.G. and Thieleczek, R. (1978) J. Physiol. 275, 241--262) has enabled us to obtain whole Ca2+- or Sr2+-activation curves at different sacromere lengths with the same preparation. 2. The maximal isometric force response was found to be very similar in Ca2+-and Sr2+-buffered solutions for otherwise identical conditions. 3. The change in sarcomere length between approx. 2.2 and 2.6 micron reversibly shifted both the Ca2+- and the Sr2+-activation curves by approx. 0.1 log units towards lower concentrations of the activator, without affecting their shape. However, the change in sarcomere length in the range above 2.6 micron did not have an effect upon the relative isometric force response-pCa (and -pSr) relationship. 4. All the Ca2+- and Sr2+-activation curves present a similar steepness and indicate that the relative isometric force increases from approx. 10 to 90% if the concentration of the activator is increased 3-fold. 5. The half time for force development in these experiments did not appear to be influenced by the length of the sarcomeres. 6. A potentiometric method for determining the apparent affinity constants of Ca2+, Mg2+ and Sr2+ to EGTA and ATP under various conditions is described.     

Alterations in Ca2+ sensitive tension due to partial extraction of C- protein from rat skinned cardiac myocytes and rabbit skeletal muscle fibers

C-protein, a substantial component of muscle thick filaments, has been postulated to have various functions, based mainly on results from biochemical studies. In the present study, effects on Ca(2+)-activated tension due to partial removal of C-protein were investigated in skinned single myocytes from rat ventricle and rabbit psoas muscle. Isometric tension was measured at pCa values of 7.0 to 4.5: (a) in untreated myocytes, (b) in the same myocytes after partial extraction of C-protein, and (c) in some myocytes, after readdition of C-protein. The solution for extracting C-protein contained 10 mM EDTA, 31 mM Na2HPO2, 124 mM NaH2PO4, pH 5.9 (Offer et al., 1973; Hartzell and Glass, 1984). In addition, the extracting solution contained 0.2 mg/ml troponin and, for skeletal muscle, 0.2 mg/ml myosin light chain-2 in order to minimize loss of these proteins during the extraction procedure. Between 60 and 70% of endogenous C-protein was extracted from cardiac myocytes by a 1-h soak in extracting solution at 20-23 degrees C; a similar amount was extracted from psoas fibers during a 3-h soak at 25 degrees C. For both cardiac myocytes and skeletal muscle fibers, partial extraction of C-protein resulted in increased active tension at submaximal concentrations of Ca2+, but had little effect upon maximum tension. C-protein extraction also reduced the slope of the tension-pCa relationships, suggesting that the cooperativity of Ca2+ activation of tension was decreased. Readdition of C-protein to previously extracted myocytes resulted in recovery of both tension and slope to near their control values. The effects on tension did not appear to be due to disruption of cooperative activation of the thin filament, since C-protein extraction from cardiac myocytes that were 40-60% troponin-C (TnC) deficient produced effects similar to those observed in cells that were TnC replete. Measurements of the tension-pCa relationship in skeletal muscle fibers were also made at a sarcomere length of 3.5 microns which, because of the distribution of C-protein on the thick filament, should eliminate any interaction between C-protein and actin. The effects of C-protein extraction were similar at long and short sarcomere lengths. These data are consistent with a model in which C-protein modulates the range of movement of myosin, such that the probability of myosin binding to actin is increased after its extraction.     

Ca2+ dependence of tension and ADP production in segments of chemically skinned muscle fibers.

Both ADP production and tension have been measured in segments of chemically skinned fibers contracting at different Ca2+ concentrations. Full mechanical activation occurred between pCa 7.00 and pCa 6.50. The total ATPase was due to both actomyosin and non-actomyosin ATPase. Actomyosin ATPase was observed at pCa 7.09 without accompanying tension. The Ca2+ dependence of tension was steeper than actomyosin ATPase. This finding implies some rate constants of the mechano-chemical cycle are Ca2+ dependent. Non-actomyosin ATPase was measured in fibers stretched beyond overlap of the thick and thin filaments. Sarcoplasmic reticulum was isolated and sarcoplasmic reticulum activity was measured in vitro under the same conditions as the single-fiber experiments. Non-actomyosin ATPase in the single fibers was found to be small compared to maximally activated actomyosin ATPase but larger than the ATPase that could be attributed to sarcoplasmic reticulum activity.     

Ca(2+) measurements in skinned cardiac fibers: effects of Mg(2+) on Ca(2+) activation of force and fiber ATPase.

In contrast to previous studies, a new fluorescent method was used to accurately determine the Ca(2+) concentration in test solutions used to activate skinned rat cardiac cells. This method used the calcium green-2 fluorescent indicator, which is shown to change its fluorescence over the Ca(2+) range responsible for Ca(2+) activation of force and ATPase. The dissociation constant (K(d)) of calcium green-2 for Ca(2+) was determined for three different Mg(2+) concentrations in solutions similar to those used in the experiment. Increasing Mg(2+) concentration from 1.0 to 8.0 mM had no significant effect on the Ca(2+) sensitivity of either force or actomyosin ATPase activity, in contrast to previous reported studies on force. The ATPase activity was activated at lower Ca(2+) concentration than the force. The ratio (ATPase/force) is proportional to the dissociation rate of force-generating myosin cross bridges and decreased during Ca(2+) activation. These findings are consistent with the hypothesis that cardiac muscle contraction is activated by a single Ca(2+)-specific binding site on troponin C.     

Restoration of Ca2+-activated tension of CDTA-treated single skeletal muscle fibers by troponin C

Single fibers from glycerinated rabbit psoas muscle were treated with a solution containing CDTA, a strong chelator of metal ions. The CDTA-treated fibers lost all of the troponin C and showed no Ca2+-activated tension development. The addition of troponin C restored the Ca2+-activated tension of CDTA-treated fibers. The tension-pCa relationship in the case of the CDTA-treated fibers reconstituted with troponin C was almost the same as that in the case of the same fibers before the CDTA treatment. These results are consistent with those of the previous study on the Ca2+-activated ATPase of CDTA-treated rabbit skeletal myofibrils.     

The effects of Mg2+ on rat liver microsomal Ca2+ sequestration

The effects of Mg2+ on the hepatic microsomal Ca2(+)-sequestering system was tested. Ca2(+)-ATPase activity and Ca2+ uptake were both dependent on the concentration of free Mg2+, reaching maximum levels at 2 mM. The effects of Mg-ATP were also influenced by the concentration of free Mg2+, being maximally effective at a ratio of 1:1. The results suggest that Mg2+ influences Ca2+ sequestration at various steps, namely in addition to forming the substrate of the Ca2(+)-ATPase reaction, Mg-ATP, Mg2+ stimulates the reaction at an additional step, as indicated by its stimulatory effect on the Ca2(+)-ATPase reaction and on Ca2+ uptake, even at optimal Mg-ATP levels. The stimulatory effect of Mg2+ was evident at various pH levels tested, and it was nucleotide specific. The stimulatory effect of Mg2+ might be exerted at the dephosphorylation step of the enzymatic reaction or at an other, yet undefined, site. The results demonstrate a plural effect of Mg2+ on the hepatic microsomal sequestration system. This indicates that, depending on its magnitude, changes in Mg2+ distribution might influence cytosolic Ca2+ levels.     

Evidence that Mg2+- or Ca2+-activated adenosine triphosphatase in rat pancreas is a plasma-membrane ecto-enzyme.

Preparations of enzymically dispersed rat pancreatic cells hydrolyse externally added nucleoside triphosphates and diphosphates at high rates in the presence of Mg2+ or Ca2+. The lack of response to specific inhibitors and activators differentiates this hydrolytic activity from that of other well-characterized ion-transporting ATPases. Studies based on inactivation of this hydrolytic activity by the covalently reacting, slowly permeating probe diazotized sulphanilic acid indicated that this nucleoside tri- and di-phosphatase is primarily a plasma-membrane ecto-enzyme. It is the major ATPase activity associated with intact cells, homogenates and isolated plasma-membrane fractions. Concanavalin A stimulates this ATPase activity of intact cells and isolated plasma-membrane fractions. The insensitivity of this ATPase activity to univalent ions and inhibitors of pancreatic electrolyte secretion, taken together with the evidence that the active site is externally located, suggests that this enzyme is not directly involved in HCO3- secretion in the pancreas. Its actual function remains unknown.     

Ca2+ dependence of stimulated 45Ca efflux in skinned muscle fibers

Stimulation of sarcoplasmic reticulum Ca release by Mg reduction of caffeine was studied in situ, to characterize further the Ca2+ dependence observed previously with stimulation by Cl ion. 45Ca efflux and isometric force were measured simultaneously at 19 degrees C in frog skeletal muscle fibers skinned by microdissection; EGTA was added to chelate myofilament space Ca either before or after the stimulus. Both Mg2+ reduction (20 or 110 microM to 4 microM) and caffeine (5 mM) induced large force responses and 45Ca release, which were inhibited by pretreatment with 5 mM EGTA. In the case of Mg reduction, residual efflux stimulation was undetectable, and 45Ca efflux in EGTA at 4 microM Mg2+ was not significantly increased. Residual caffeine stimulation at 20 microM Mg2+ was substantial and was reduced further in increased EGTA (10 mM); at 600 microM Mg2+, residual stimulation in 5 mM EGTA was undetectable. Caffeine appears to initiate a small Ca2+-insensitive efflux that produces a large Ca2+-dependent efflux. Additional experiments suggested that caffeine also inhibited influx. The results suggest that stimulated efflux is mediated mainly or entirely by a channel controlled by an intrinsic Ca2+ receptor, which responds to local [Ca2+] in or near the channel. Receptor affinity for Ca2+ probably is influenced by Mg2+, but inhibition is weak unless local [Ca2+] is very low.     

Ca2+sensitivity and caffeine-induced changes in skinned cardiac muscle fibers of the carp,Cyprinus carpio

 Ca²⁺ sensitivity and caffeine-induced sensitivity changes in skinned carp heart fibers were compared with those of guinea pig and rat heart. The Ca²⁺ concentration-response curves of saponin-treated left atrial skinned fibers obtained from guinea pig and rat were almost identical. Doses of 5 and 20 mmol ⋅ l^-1 caffeine shifted this curve to the left. However, when a relatively high concentration (50 mmol ⋅ l^-1) of caffeine was used, the left-ward shift was reduced. Caffeine reduced the peak of the Ca²⁺ concentration-response curve. The Ca²⁺ concentration-response curve of carp atrial skinned fiber is almost identical to that of guinea pig and rat. However, a further increase in Ca²⁺ sensitivity was observed even when 50 mmol ⋅ l^-1 caffeine was added. Similarly, a decrease in the response curve peak was also observed. Ca²⁺ sensitivity in ventricular skinned fibers obtained from carp was almost the same as that observed for the atrial, but the increase in Ca²⁺ sensitivity due to caffeine was larger. In addition, a further increase was also observed when 50 mmol ⋅ l^-1 caffeine was added. These results indicate that the Ca²⁺ sensitivity of contractile proteins in atrial muscles from carp heart is the same as that of guinea pig and rat. It is, however, assumed that there are some differences in properties in the contractile proteins. It is also assumed that there are some differences between the atrial and ventricular muscles of carp heart. Accepted: 17 May 1996     

Conformational transitions in the Ca2+ + Mg2+-activated ATPase and the binding of Ca2+ ions.

We have studied the fluorescence of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum labelled with fluorescein isothiocyanate. The change in intensity of fluorescein fluorescence caused by addition of Ca2+ to the labelled ATPase can be interpreted in terms of a two-conformation model for the ATPase, one conformation (E1) having a high affinity for Ca2+, the other (E2) a low affinity. Effects of Ca2+ as a function of pH allow an estimate of the effect of pH on the E1/E2 ratio, consistent with kinetic studies. A model is presented for binding of Ca2+ to the ATPase as a function of pH that is consistent both with the data on the E1/E2 equilibrium and with literature data on Ca2+ binding.     

Competitive Mg2+ block of a large-conductance, Ca(2+)-activated K+ channel in rat skeletal muscle. Ca2+, Sr2+, and Ni2+ also block

The patch-clamp technique was used to investigate the effect of intracellular Mg2+ (Mgi2+) on the conductance of the large-conductance, Ca(2+)-activated K+ channel in cultured rat skeletal muscle. Measurements of single-channel current amplitudes indicated that Mgi2+ decreased the K+ currents in a concentration-dependent manner. Increasing Mgi2+ from 0 to 5, 10, 20, and 50 mM decreased channel currents by 34%, 44%, 56%, and 73%, respectively, at +50 mV. The magnitude of the Mgi2+ block increased with depolarization. For membrane potentials of -50, +50, and +90 mV, 20 mM Mgi2+ reduced the currents 22%, 56%, and 70%, respectively. Mgi2+ did not change the reversal potential, indicating that Mg2+ does not permeate the channel. The magnitude of the Mgi2+ block decreased as the concentration of K+ was increased. At a membrane potential of +50 mv, 20 mM Mgi2+ reduced the currents 71%, 56%, and 25% for Ki+ of 75, 150, and 500 mM. These effects of Mgi2+, voltage, and K+ were totally reversible. Although the Woodhull blocking model could approximate the voltage and concentration effects of the Mgi2+ block (Kd approximately 30 mM with 150 mM symmetrical K+; electrical distance approximately 0.22 from the inner surface), the Woodhull model could not account for the effects of K+. Double reciprocal plots of 1/single channel current vs. 1/[K+] in the presence and absence of Mgi2+, indicated that the Mgi2+ block is consistent with apparent competitive inhibition between Mgi2+ and Ki+. Cai2+, Nii2+, and Sri2+ were found to have concentration- and voltage-dependent blocking effects similar, but not identical, to those of Mgi2+. These observations suggest the blocking by Mgi2+ of the large-conductance, Ca(2+)-activated K+ channel is mainly nonspecific, competitive with K+, and at least partially electrostatic in nature.     

Effects of Mg2+, anions and cations on the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum.

In a previous paper [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227] we presented a kinetic model for the activity of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum. Here we extend the model to account for the effects on ATPase activity of Mg2+, cations and anions. We find that Mg2+ concentrations in the millimolar range inhibit ATPase activity, which we attribute to competition between Mg2+ and MgATP for binding to the nucleotide-binding site on the E1 and E2 conformations of the ATPase and on the phosphorylated forms of the ATPase. Competition is also suggested between Mg2+ and MgADP for binding to the phosphorylated form of the ATPase. ATPase activity is increased by low concentrations of K+, Na+ and NH4+, but inhibited by higher concentrations. It is proposed that these effects follow from an increase in the rate of dephosphorylation but a decrease in the rate of the conformational transition E1'PCa2-E2'PCa2 with increasing cation concentration. Li+ and choline+ decrease ATPase activity. Anions also decrease ATPase activity, the effects of I- and SCN- being more marked than that of Cl-. These effects are attributed to binding at the nucleotide-binding site, with a decrease in binding affinity and an increase in 'off' rate constant for the nucleotide.     

Altered Ca2+ dependence of tension development in skinned skeletal muscle fibers following modification of troponin by partial substitution with cardiac troponin C

Binding of Ca2+ to the troponin C (TnC) subunit of troponin is necessary for tension development in skeletal and cardiac muscles. Tension was measured in skinned fibers from rabbit skeletal muscle at various [Ca2+] before and after partial substitution of skeletal TnC with cardiac TnC. Following substitution, the tension-pCa relationship was altered in a manner consistent with the differences in the number of low-affinity Ca2+-binding sites on the two types of TnC and their affinities for Ca2+. The alterations in the tension-pCa relationship were for the most part reversed by reextraction of cardiac TnC and readdition of skeletal TnC into the fiber segments. These findings indicate that the type of TnC present plays an important role in determining the Ca2+ dependence of tension development in striated muscle.