Activation of skinned muscle fibers by calcium and strontium ions

Intact and mechanically skinned skeletal muscle fibers of the crab Carcinus maenas have been used. The aim of the experiments was to determine the origin of the mechanical activity recorded in intact crab muscle fibers exhibiting an inward strontium current in strontium solution without calcium. To do so, the effect of strontium ions in inducing activation of contractile proteins and calcium release from the sarcoplasmic reticulum has been studied. The properties of the sarcoplasmic reticulum membrane towards strontium ions, i.e., the efficiency of the calcium ATPase towards strontium ions and the capability to release strontium ions have been investigated. Results show that the contractile proteins have a lower affinity for strontium than for calcium ions. However, the maximum bound strontium is identical to the maximum bound calcium. As for the sarcoplasmic reticulum, strontium ions can induce a calcium release and also can be taken up by the calcium ATPase and be released. We concluded that the mechanical activity in intact fibers bathed in a strontium medium has two origins: first, a direct and partial activation of the contractile proteins by strontium ions flowing through the calcium channel; second, a contractile proteins activation of calcium ions released by the sarcoplasmic reticulum by a "strontium-induced calcium release" mechanism.     

Contracture of Slow Striated Muscle during Calcium Deprivation

When deprived of calcium the slow striated muscle fibers of the frog develop reversible contractures in either hypertonic or isotonic solutions. While calcium deprivation continues because of a flowing calcium-free solution the muscles relax slowly and completely. Restoration of calcium during contracture relaxes the muscle promptly to initial tension. When relaxed during calcium lack the return of calcium does not change tension and the muscle stays relaxed. When contractures are induced by solutions containing small amounts of calcium relaxation does not occur or requires several hours. The rate of tension development depends upon the rate at which calcium moves outward since the contractures develop slower in low concentrations of calcium and are absent or greatly slowed in a stagnant calcium-free solution. Withdrawal of calcium prevents the contractile responses to ACh, KCl, or electrical stimulation through the nerve. Muscles return to their original excitability after calcium is restored. Origin of the contractures is unrelated to nerve activity since they are maximal during transmission failure from calcium lack, occur in denervated muscles, and are not blocked by high concentrations of d-tubocurarine, procaine, or atropine. The experiments also indicate that the contractures do not originate from repetitive activity of muscle membranes. The findings are most simply explained by relating the outward movement of calcium as a link for initiating contraction in slow type striated muscle.     

Effect of halothane on the Ca2+-transport system of surface membranes isolated from normal and malignant hyperthermia pig skeletal muscle

Trapezius muscle from normal and malignant hyperthermia (MH) pigs was used to investigate the effects of halothane on contractile properties and on the calcium transport system of isolated surface membranes. We observed that (i) halothane, diluted in dimethyl sulfoxide, induced a higher isometric contracture response in MH muscle than in normal muscle, (ii) halothane had a more pronounced inhibitory effect on the sarcolemmal Ca2+-ATPase activity in MH membrane, and (iii) the actively accumulated calcium was released in higher amounts in MH muscle than in normal muscle. These results suggest that halothane might induce, in vivo, an important influx of extracellular calcium ions through the MH sarcolemmal membranes and this pool of intracellular calcium may constitute the trigger for the defective sarcoplasmic reticulum "calcium-induced calcium-release" system.     

Cholinergic regulation of body-wall muscle contraction of the ascidian Styela rustica (Linnaeus, 1767)

The regulation of body-wall muscle contraction in the ascidian Styela rustica was studied. Acetylcholine (ACh, 1?C10 ??M) induced a significant contraction of isolated muscle strips. The ACh-induced contractile response was potentiated and prolonged in the presence of proserine (15 ??M), which confirms acetylcholinesterase activity in the S. rustica body-wall muscle. Atropine (1?C100 ??M, M-cholinoreceptor blocker) did not prevent the ACh-induced contractile response, while d-tubocurarine (1?C100 ??M, N-cholinoreceptor blocker) progressively reduced muscle contraction induced by 10 ??M ACh. Thus, neuromuscular transmission in the S. rustica body-wall muscle is mediated by nicotinic-like ACh-receptors. Procaine reduced ACh-induced (10 ??M) muscle contraction. As well, our experiments showed spontaneous rhythmic contractile activity in isolated muscle strips of S. rustica. Atropine, d-tubocurarine, procaine, and proserine did not alter rhythmic activity. Myogenic automaticity is suggested as a possible cause of the rhythmic contraction of the ascidian body-wall muscle.     

Effect of papaverine on the tonus and contraction of depolarized taenia coli musculature in guinea pigs

Experiments were performed on the isolated strips of guinea pig taenia coli. The smooth muscle was depolarized in a solution with high potassium concentration (120 mM KCl). The effect of papaverine (in concentration of from 10(-5) to 3.10(-5) g/ml) on the tonus and the contractile off-response originating after the ending of longlasting strong polarizing current was investigated. It was found that: 1) papaverine abolished the concentrations induced by drugs (histamine, acetylcholine, bradykinin); 2) papaverine reduced the tonus of depolarized muscle and eliminated its increase under the effect of a rise of the external calcium concentration; 3) papaverine had no effect on the amplitude and the ascending phase of the contractile off-response; 4) papaverine accelerated the discending phase of the contractile off-response. The data obtained suggest: 1) there are chemoexcitable calcium channels in the cellular membrane which are blocked by papaverin; 2) there are calcium "leakage" channels in the cellular membrane responsible for the tone maintenance which are blocked by papaverine; 3) papaverine has a negligible effect on the electroexcitable calcium channels.     

Ionic Control of the Reversal Response of Cilia in Paramecium caudatum : A calcium hypothesis

The duration of ciliary reversal of Paramecium caudatum in response to changes in external ionic factors was determined with various ionic compositions of both equilibration and stimulation media. The reversal response was found to occur when calcium ions bound by an inferred cellular cation exchange system were liberated in exchange for externally applied cations other than calcium. Factors which affect the duration of the response were (a) initial amount of calcium bound by the cation exchange system, (b) final amount of calcium bound by the system after equilibration with the stimulation medium, and (c) concentration of calcium ions in the stimulation medium. An empirical equation is presented which relates the duration of the response to these three factors. On the basis of these and previously published data, the following hypothesis is proposed for the mechanism underlying ciliary reversal in response to cationic stimulation: Ca⁺⁺ liberated from the cellular cation exchange system activates a contractile system which is energized by ATP. Contraction of this component results in the reversal of effective beat direction of cilia by a mechanism not yet understood. The duration of reversal in live paramecia is related to the time course of bound calcium release.     

The effect of ions upon the response of smooth muscle to cooling

The slow tonic responses of the anterior byssus retractor of Mytilus edulis to rapid cooling were investigated by simultaneously recording tension and resting potential changes after soaking the muscle in banthine, a powerful neuromuscular blocking agent. The quantitative relations between the amount of cooling and the amount of associated depolarization necessary for contraction at various concentrations of potentiating potassium can be expressed in a family of curves. The plateaus of the curves for sea water and for potassium-free sea water were beneath the depolarization value necessary for contraction, so that it is clear that no amount of cooling with sea water alone or with potassium-free sea water would ever be effective. When the muscle is treated with subthreshold amounts of potassium and rapidly cooled in various concentrations of sodium ion and calcium ion, respectively, the sodium and calcium do not affect the amount of depolarization. Acetylcholine, in subthreshold amounts, has a potentiating effect, but, unlike potassium and cooling, acts through the nervous apparatus. Mytilus muscle will respond to cooling with tonic contraction whenever a critical threshold amount of depolarization is achieved. Cooling alone cannot trigger the contraction since it cannot bring about sufficient depolarization. Cooling can result in contraction, however, if used in conjunction with some other subthreshold depolarizing agent. Cooling affects the contractile mechanism by first causing membrane breakdown and depolarization.     

The role of proctolin and glutamate in the excitation-contraction coupling of insect visceral muscle

Proctolin (1 X 10(-10) to 1 X 10(-9) M) had a minimal effect on the spontaneous and evoked electrical events of the hindgut of the cockroach Leucophea maderae. Spontaneous action potentials and contractile activity stopped when the hindgut was exposed to 2 mM Mn2+. Eighty per cent of the response of the hindgut to glutamate was blocked by manganese, but only 35% of the response to proctolin. Hindguts were responsive to proctolin in a calcium-free medium, but not to glutamate. Moreover, proctolin appeared to facilitate the reentry of calcium after depleted preparations were returned to normal levels of external calcium. The results offer evidence for two calcium transmembrane channels in insect visceral muscle.     

Effects of nifedipine on the mechanical properties of sarcolemma and modulation of calcium accumulation dynamics in fibers of the rat soleus muscle under short-term hypogravity conditions

It has been shown that the modulation of the mechanical properties of sarcolemma by nifedipine may be related to the dynamics of accumulation of calcium ions under short-term rat hindlimb suspension. The basal calcium level was measured by the fluorescence probe Fluo-4AM, the transversal stiffness of different parts of the contractile apparatus and sarcolemma was estimated by atomic force microscopy, and the content of desmin was determined by gel electrophoresis with immunoblotting. It has been found that nifedipine has a protecting effect on muscle fibers under hypogravity by decreasing the degradation of desmin and proteins that determine the transversal stiffness of sarcolemma and the contractile apparatus, and the intensity of the increase in the basal calcium level. It was shown that the selective blocking of L-channels leads to an increase in the basal calcium level in intact soleus fibers. At the same time, the transversal stiffness of sarcolemma and the contractile apparatus increased. The mechanism of this increase is still unclear; however, it is assumed that it mediates the protecting action of nifedipine.     

Actin dynamics mediates the changes of calcium level during the pulvinus movement of Mimosa pudica

The bending movement of the pulvinus of Mimosa pudica is caused by a rapid change in volume of the abaxial motor cells, in response to various environmental stimuli. We investigated the relationship between the actin cytoskeleton and changes in the level of calcium during rapid contractile movement of the motor cells that was induced by electrical stimulation. The bending of the pulvinus was retarded by treatments with actin-affecting reagents and calcium channel inhibitors. The actin filaments in the motor cells were fragmented in response to electrical stimulation. Further investigations were performed using protoplasts from the motor cells of M. pudica pulvini. Calcium-channel inhibitors and EGTA had an inhibitory effect on contractile movement of the protoplasts. The level of calcium increased and became concentrated in the tannin vacuole after electrical stimulation. Ruthenium Red inhibited the increase in the level of calcium in the tannin vacuole and the contractile movement of the protoplasts. However, treatment with latrunculin A abolished the inhibitory effect of Ruthenium Red. Phalloidin inhibited the contractile movement and the increase in the level of calcium in the protoplasts. Our study demonstrates that depolymerization of the actin cytoskeleton in pulvinus motor cells in response to electrical signals results in increased levels of calcium.Key words: actin, calcium, pulvinus movement, the tannin vacuole, Mimosa pudica     

Contractile effects of 16-methyl analogues of PGE2 on the circular and longitudinal muscles of the guinea-pig isolated colon.

The mechanical effects of 16-methyl analogues of PGE2, mainly 16,16-dimethyl PGE2, on circular and longitudinal muscles of the guinea-pig isolated proximal colon were investigated. In circular muscle strips, PGE2 100 nM produced an initial contraction followed by relaxation, while 16(R)-methyl PGE2 and 16,16-dimethyl PGE2 (1 nM - 1 microM) produced sustained contractions. In longitudinal muscle strips, PGE2 and 16-methyl analogues of PGE2 produced only contractions. The contractile responses of both muscle strips to 16,16-dimethyl PGE2 were not influenced by atropine or tetrodotoxin, indicating that these analogues act directly on the muscles, but were eliminated by the omission of extracellular Ca ions or in the presence of 1 mM lanthanum ions. However, verapamil, a Ca channel blocker, did not block the contractile response to the methyl analogues in circular muscle strips, although it completely inhibited the contractile response of longitudinal muscle strips. These results suggest that the contractile effect of 16-methyl analogues of PGE2 on the circular muscle may be due to an increased influx of Ca ions mainly via receptor-sensitive and partly voltage-sensitive Ca channels, while the contractile effect of the analogues on the longitudinal muscle may be due to an increase in influx of Ca ions via voltage-sensitive Ca channels.     

Increasing O-GlcNAcylation Level on Organ Culture of Soleus Modulates the Calcium Activation Parameters of Muscle Fibers

O-N-acetylglucosaminylation is a reversible post-translational modification which presents a dynamic and highly regulated interplay with phosphorylation. New insights suggest that O-GlcNAcylation might be involved in striated muscle physiology, in particular in contractile properties such as the calcium activation parameters. By the inhibition of O-GlcNAcase, we investigated the effect of the increase of soleus O-GlcNAcylation level on the contractile properties by establishing T/pCa relationships. We increased the O-GlcNAcylation level on soleus biopsies performing an organ culture of soleus treated or not with PUGNAc or Thiamet-G, two O-GlcNAcase inhibitors. The enhancement of O-GlcNAcylation pattern was associated with an increase of calcium affinity on slow soleus skinned fibers. Analysis of the glycoproteins pattern showed that this effect is solely due to O-GlcNAcylation of proteins extracted from skinned biopsies. We also characterized the O-GlcNAcylated contractile proteins using a proteomic approach, and identified among others troponin T and I as being O-GlcNAc modified. We quantified the variation of O-GlcNAc level on all these identified proteins, and showed that several regulatory contractile proteins, predominantly fast isoforms, presented a drastic increase in their O-GlcNAc level. Since the only slow isoform of contractile protein presenting an increase of O-GlcNAc level was MLC2, the effect of enhanced O-GlcNAcylation pattern on calcium activation parameters could involve the O-GlcNAcylation of sMLC2, without excluding that an unidentified O-GlcNAc proteins, such as TnC, could be potentially involved in this mechanism. All these data strongly linked O-GlcNAcylation to the modulation of contractile activity of skeletal muscle.     

Kinetic analysis of contraction-relaxation process in smooth muscles

A method is proposed for analysing kinetic curves of the smooth muscle contraction--relaxation approved on smooth muscle preparations of ureter and taenia coli. A notion of the velocity coefficient is introduced. It is the parameter which gives a quantitative characteristics of changes in the dynamics of muscle contraction and relaxation under the effect of factors modifying the contractile response. In order to illustrate the application of this parameter studies were carried out of the effect of sodium-free medium, monensin, and of temperature decrease on the contractile activity of the smooth muscle tissue of the ureter. The method can be useful while investigating the regularities of electro- and pharmacomechanical conjunction in the smooth muscles, as well as during pharmacological screening of compounds--regulators of calcium homeostasis in myocytes and their contractile activity.     

Effects of inhibitors of protein kinase C and phosphodiesterase on the contractile effects of endothelin in dog femoral artery

Endothelin-induced contraction in dog femoral artery strips was relaxed by a protein kinase C inhibitor (MDL 27,032), cyclic nucleotide phosphodiesterase (PDE) inhibitors (enoximone, piroximone, rolipram, 3-isobutyl-1-methylxanthine), an adenylcyclase activator (forskolin), a guanylcyclase activator (nitroprusside), but only slightly relaxed by a calcium channel blocker (nifedipine).

Endothelin-induced contractions were effectively relaxed by MDL 27,032, but were only slightly antagonized by nifedipine. Endothelin lost its contractile effect in the absence of external calcium whereas phorbol 12, 13-dibutyrate produced a contractile effect in the presence or absence of external calcium. Thus, endothelin-induced contraction requires external calcium ions which may enter vascular smooth muscle cells through nifedipine resistant channels. Endothelin-induced contraction also appears to involve activation of protein kinase C. PDE inhibitors, forskolin and nitroprusside all antagonized the contractile effect of endothelin in femoral arteries.

These results indicate that protein kinase C inhibitors and compounds which increase cyclic nucleotide levels can be used to antagonize the vasoconstriction produced by endothelin.     

Regulation of intracellular pH in the myocardium; relevance to pathology

Intracellular pH affects the contractile function of the heart, metabolic reactions, ion exchange and calcium homoeostasis. Numerous studies have concluded that a fall of extracellular pH, by whatever mechanism, causes a fall of contractility by alteration of intracellular pH. Measurement of cytosolic intracellular pH using microelectrodes has confirmed that earlier deduction. Acidosis reduces the slow calcium current and the release of calcium from the sarcoplasmic reticumul but, because the cytosolic calcium does not fall, the major site of action of hydrogen ions appears to be on the calcium sensitivity of the contractile proteins. In man acidosis can be detected 15 s after the occlusion of a coronary artery and is a major mechanism for the simultaneous loss of contractility in ischaemia. A transient alkalosis is not detected in man but has been reported in isolated heart preparations where ATP consumption is low.An imposed mild respiratory acidosis during hypoxia increases the subsequent recovery of mechanical function on reoxygenation whereas a severe acidosis can be harmful. Acidosis in ischaemic may be advantageous due to a cardioplegic effect, inhibition of transsarcolemmal calcium fluxes or a reduction of mitochondrial calcium overload. Calcium uptake on reperfusion or reoxygenation has been linked to an inward movement of sodium in exchange for hydrogen ions on reperfusion and subsequent sodium-calcium exchange. Such a mechanism in its simplest form cannot account for the similar uptake of calcium on reoxygenation and reperfusion. Acidosis is a cause of early contractile failure in ischaemia but the role of acidosis in causing cell necrosis is not established.     

Actomyosin isolated from bovine tracheal smooth muscle.

A contractile protein (actomyosin) was isolated from bovine tracheal smooth muscle by the use of "classical" procedures. The protein was considered to be actomyosin because it demonstrated: ATPase activity; superprecipitation upon the addition of ATP, and the solubility and extraction characteristics of actomyosin. The ATPase and superprecipitation reactions were not inhibited by EGTA, and did not require calcium. Lack of an effect of either calcium or EGTA could not be reversed by the addition of active bovine skeletal muscle troponin and tropomyosin. No troponin-tropomyosin like activities could be demonstrated in various tracheal muscle fractions.     

Mechanisms of regulation of Ca2+ levels in myometrium cells

The ways and mechanisms of the Ca2+ concentration regulation in myometrium cells are analyzed. The plasma membrane is thoroughly studied for its role in the calcium control provision for the contractile activity of the uterus. The systems of Mg2+-ATP-dependent transport of Ca2+, sodium-calcium metabolism as well as regularities of the Ca2+ passive transfer in the sarcolemma vesicles are considered. The systems of the Mg2+-ATP- and N+-dependent transport of calcium are discussed for their contribution into regulation of calcium concentration in the myoplasm. Oxytocin and ions of bivalent metals (stimulators of the contractile activity of the uterus) are studied for their effect on the activity of the sarcolemma calcium pump.     

Effects of nifedipine on the mechanical properties of sarcolemma and modulation of dynamics of the increase in the basal calcium level in fibers of rat soleus muscle under short-term hypogravity conditions

It has been shown that the modulation of the mechanical properties of sarcolemma mediated by nifedipine may be related to the dynamics of accumulation of calcium ions under short-term rat hindlimb suspension. The basal calcium level was measured with a fluorescent probe Fluo-4AM, the transverse stiffness of different parts of the contractile apparatus and sarcolemma was estimated by atomic force microscopy, and the content of desmin was determined by gel electrophoresis with immunoblotting. It has been found that nifedipine has a protective effect on muscle fibers under hypogravity by decreasing the degradation of desmin and proteins that determine the transverse stiffness of sarcolemma and the contractile apparatus, and the intensity of the increase in the basal calcium level. It was shown that selective blocking of L-channels leads to an increase in the basal calcium level in intact soleus fibers. At the same time, the transverse stiffness of sarcolemma and the contractile apparatus increases. The mechanism of this increase is still unclear, but it is thought to mediate the protective action of nifedipine.     

Distribution of calcium during contraction and relaxation of crayfish skeletal muscle fibre.

A model of activation of muscle contraction has been applied to the crayfish isolated skeletal muscle fibre. The model is based on calcium diffusion and binding to specific regulatory sites in a sarcomere. Calcium ions activate interactions of contractile proteins and thus the generation of force. The model quantifies the relation between calcium released from intracellular stores and force elicited. Experimental tension records from isolated crayfish skeletal muscle fibres under voltage clamp conditions are analyzed. Model parameters were determined either via approximation of the onset of tension by the model solution or from the model based relations between the tension maximum, and depolarizing pulse length and amplitude. This allowed to determine time changes of free and bound calcium distribution in the sarcomere and the calcium release from terminal cisternae. The steady state calcium concentration at terminal cisternae showed S-shaped voltage dependence with saturation below approx. 10 mumol/l at positive membrane potentials.     

Influence of the sodium gradient on contractile activity in pregnant rat myometrium

The effects of varying the sodium gradient-either by lowering [Na+]o or by increasing [Na+]i on the electromechanical properties of pregnant rat uterine smooth muscle were studied. In normal tissues, complete removal of external sodium ions (choline, Tris or sucrose as substitutes) induced a strong and maintained contraction which was dependent on the presence of extracellular calcium ions, and was sensitive to Ca2+-antagonist drugs (Nifedipine; D 600, Mn2+). Electrical recordings showed that the membrane was transiently hyperpolarized (-10 +/- 2.4 mV, n = 20); after 1 minute depolarization accompanied by a spontaneous spike discharge occurred. Partial withdrawal of external sodium ions resulted in following changes in twitch contractions evoked by electrical stimulation: a linear relationship was found between the time constant of twitch relaxation and the external Na-concentration. In Na-rich tissues, where the Na/K pump was blocked, or in the presence of monensin, Na-free solutions (whatever the substitute, even K+ ions) again triggered strong contractions entirely dependent on external calcium but rather insensitive to Ca-antagonists. The Na-free (K+) induced contraction was larger than the Na-free (choline or Tris)-induced contraction. It was concluded that the sodium gradient was an important factor for the regulation of contractile activity of uterine smooth muscle. Na-Ca exchange appeared to mediate twitch relaxation in normal tissues and was responsible for Ca-influx in Na-rich tissues.