Simultaneous measurement of cytosolic free calcium concentration and cell circumference during contraction, both in a single rat cardiomuscular cell, by digital imaging microscopy with indo-1

We have developed a novel system using digital imaging microscopy with indo-1 to measure the cytosolic free calcium concentration [( Ca2+]i). The method is particularly suitable for measuring the rapid change in [Ca2+]i in relation to the cell motion. With this system, we made the first successful simultaneous measurement of [Ca2+]i and cell circumference during contraction in an electrically stimulated single rat ventricular myocyte. It was found that the level of [Ca2+]i was elevated during contraction, and that the onset and peak time of the calcium transient preceded that of the decrease in circumference.     

Phytochrome and Calcium Regulated Protein Phosphorylation in Sorghum bicolor

Irradiation with red light of Sorghum bicolor seedlings stimulated in vitro phosphorylation of 55 kD and several other soluble polypeptides in a development-dependent manner. The red light stimulated phosphorylation of 55 kD polypeptide was more in 6-day-old etiolated plants as compared to 5-day-old plants. The in vitro phosphorylation of 55 kD polypeptide was enhanced further when calcium was added to the extracts obtained from red light irradiated tissues of 6-day-old seedlings. This effect was inhibited in the presence of calmodulin inhibitors. There was no significant stimulation in the phosphorylation of this polypeptide by calcium in 5-day-old and 7-day-old etiolated plants. Besides 55 kD, the phosphorylation of several other polypeptides was either stimulated or inhibited by light, calcium and calmodulin inhibitors suggesting involvement of both kinases and phosphatases in light-mediated phosphorylation.     

Characterization of an Acute Muscle Contraction Model Using Cultured C2C12 Myotubes

A cultured C2C12 myotube contraction system was examined for application as a model for acute contraction-induced phenotypes of skeletal muscle. C2C12 myotubes seeded into 4-well rectangular plates were placed in a contraction system equipped with a carbon electrode at each end. The myotubes were stimulated with electric pulses of 50 V at 1 Hz for 3 ms at 997-ms intervals. Approximately 80% of the myotubes were observed to contract microscopically, and the contractions lasted for at least 3 h with electrical stimulation. Calcium ion (Ca²⁺) transient evoked by the electric pulses was detected fluorescently with Fluo-8. Phosphorylation of protein kinase B/Akt (Akt), 5′ AMP-activated protein kinase (AMPK), p38 mitogen-activated protein kinase (p38), and c-Jun NH2-terminal kinase (JNK)1/2, which are intracellular signaling proteins typically activated in exercised/contracted skeletal muscle, was observed in the electrically stimulated C2C12 myotubes. The contractions induced by the electric pulses increased glucose uptake and depleted glycogen in the C2C12 myotubes. C2C12 myotubes that differentiated after exogenous gene transfection by a lipofection or an electroporation method retained their normal contractile ability by electrical stimulation. These findings show that our C2C12 cell contraction system reproduces the muscle phenotypes that arise in vivo (exercise), in situ (hindlimb muscles in an anesthetized animal), and in vitro (dissected muscle tissues in incubation buffer) by acute muscle contraction, demonstrating that the system is applicable for the analysis of intracellular events evoked by acute muscle contraction.     

Detection of a calcium-activated protein kinase in mougeotia by using synthetic Peptide substrates

By using a synthetic peptide, KM-14, a protein kinase was detected and partially purified from Mougeotia sp. The peptide contains the sequence of the regulatory light chain of smooth muscle myosin that is phosphorylated by calcium-calmodulin-dependent myosin light chain kinase (MLCK). The Mougeotia kinase was stimulated 40-fold by calcium with half-maximal stimulation occurring at 1.5 micromolar. The enzyme was fractionated from calmodulin and was depleted of calmodulin based on enzyme activator analysis. The calmodulin-depleted enzyme was fully active and calcium dependent, and was not stimulated further by exogenous calmodulin nor by the calcium effectors phosphatidylserine and diacylglycerol. The enzyme phosphorylated intact chicken gizzard myosin light chain as well as the KM-14 substrate. KM-13, a peptide analog of KM-14 with a deletion of a glutamine at position 5, was a poor substrate with a V_max/K_m ratio 200-fold lower than KM-14. Thus, similarly to vertebrate MLCK, the Mougeotia enzyme is very sensitive to changes in sequence surrounding the phosphorylation site. Calcium-dependent KM-14 kinase activity also was detected in two other algae, Mesotaenium caldariorum and Spirogyra sp., as well as in pea seedlings. The data suggest that plant and algal tissues possess an enzyme with a substrate specificity similar to MLCK, but unlike MLCK, does not appear to require calmodulin for activity.     

Glucose transport and metabolism in the perfused hindquarters of lean and obese-hyperglecemic (db/db) mice effects of insulin and electrical stimulation

Changes in glucose transport and metabolism in skeletal muscles of the obese-diabetic mice (db/db) was characterized using the perfused mouse hindquarter preparation. Metabolism of [5-³H]glucose, uptake of 3-O-[methyl-³H]glucose (methylglucose) and [2-¹⁴C]deoxyglucose (deoxyglucose) was studied under resting, electrically stimulated contracting, and insulin-stimulated conditions. Basal rate of methylglucose uptake was 255 ± 18 and 180 ± 9 μl/15 min per ml intracellular fluid space for lean and db/db mice, respectively. The V_? of methylglucose transport was decreased with no change in K_m in the db/db mice. Both electrical stimulation and insulin (1/mU/ml) increased methylglucose uptake rate 2-fold in both lean and obese mice. We observed no significant change in insulin sensitivity in the db/db mice in stimulating methylglucose uptake which was subnormal under all conditions. Similar results were obtained using deoxyglucose. Likewise, uptake of glucose and ³H₂O production from [5-³H]glucose were significantly reduced, both at rest and during electrically stimulated contraction in the db/db mouse. However, lactate production in the electrically stimulated db/db mouse preparations was not significantly different from that in the lean mice. These data suggest a major contribution from an impaired glucose transport activity to the reduction in glucose metabolism in the db/db mouse skeletal muscle.     

Contraction of protoplasm. I. Cinematographic analysis of the anodally stimulated contraction of Spirostomum ambiguum

Electrically stimulated contraction of Spirostomum ambiguum was investigated by high speed cinematography (up to 6,000 pps). Contraction is completed in about 4 msec following a latent period of up to 30 msec. Reduction in length during contraction followed a sigmoid curve, and final length was about 50% of the original length. Contraction always started at the end of the animal directed towards the anode. When the length of each half was measured separately, it was found that the cathodal end lagged about 1 msec in all cases observed. Rate of contraction was increased when the external calcium contraction was increased, and was decreased in Ca-free and K-free solutions, but was unchanged in K-rich solutions. These results are interpreted in terms of contraction being associated with a relative increase of calcium bound to the contractile protein. The differential migration of potassium and calcium ions in an electric current would result in a temporary lowering of K⁺ at the anodal end of the animal, hence a relative rise would take place in the Ca⁺⁺ available for binding. The results of experiments using changed calcium and potassium concentrations can be explained by this hypothesis which is in general agreement with modern work on muscle contraction and relaxation.     

Fibroblast contraction of collagen gels requires activation of protein kinase C

Fibroblasts stimulated to contract collagen gels with serum were completely inhibited by staurosporine, a broad spectrum kinase inhibitor. Further analysis demonstrated that staurosporine is potent (IC₅₀ 17 nM), rapid in onset, and completely reversible. Complete inhibition of gel contraction was also observed with calphostin C (IC₅₀ 48 nM), an inhibitor specific for protein kinase C (PKC). Similar effects were not observed with KT5926 or KT5720, inhibitors for myosin light chain kinase and cAMP-dependent kinase, respectively. These data suggested that serum-stimulated fibroblast contraction is dependent upon activation of PKC. This was also observed with fibroblast contraction stimulated with endothelin-1, platelet-derived growth factor, and transforming growth factor beta. PKC activated directly with low concentrations of phorbol ester was observed to stimulate fibroblast contraction of collagen gels, in some cases within 30 minutes of exposure. © 1993 Wiley-Liss, Inc.     

Photosensitivity of the Frog Iris

The absorption of quanta by rhodopsin leads to the contraction of frog iris muscle. The contractions reach a maximum after about 8 sec. in the light. When the light is turned off the irises relax exponentially with a half-time of about 6 sec. Membrane polarization is not necessary for the response but calcium movement and membrane permeability changes probably are. The response is not mediated by acetylcholine or epinephrine. The curves of log It vs. log t for constant response amplitude bend progressively upward away from a unit slope line at short times as larger response criteria are used because (a) light influences tension development over longer times and (b) the higher intensity, shorter duration flashes are less effective.     

Central inhibition of the aortic baroreceptors-heart rate reflex at the onset of spontaneous muscle contraction.

Animals decerebrated at the precollicular-premammillary body level exhibit spontaneous locomotion without any artificial stimulation. Our laboratory reported that the cardiovascular and autonomic responses at the onset of spontaneous locomotor events are evoked by central command, generated from the caudal diencephalon and the brain stem (Matsukawa K, Murata J, and Wada T. Am J Physiol Heart Circ Physiol 275: H1115-H1121, 1998). In this study, we examined whether central command and/or a reflex resulting from muscle afferents modulates arterial baroreflex function using a decerebrate cat model. The baroreflex was evoked by stimulating the aortic depressor nerve (ADN) at the onset of spontaneous muscle contraction (to test the possible influence of central command) and during electrically evoked contraction or passive stretch (to test the possible influence of the muscle reflex). When the ADN was stimulated at rest, heart rate and arterial blood pressure decreased by 40 +/- 2 beats/min and 11 +/- 1 mmHg, respectively. The baroreflex bradycardia was attenuated to 55 +/- 4% at the onset of spontaneous contraction. The attenuating effect on the baroreflex bradycardia was not observed at the onset and middle of electrically evoked contraction or passive stretch. The depressor response to ADN stimulation was identical among resting and any muscle interventions. The inhibition of the baroreflex bradycardia during spontaneous contraction was seen after beta-adrenergic blockade but abolished by muscarinic blockade, suggesting that the bradycardia is mainly evoked through cardiac vagal outflow. We conclude that central command, produced within the caudal diencephalon and the brain stem, selectively inhibits the cardiac component, but not the vasomotor component, of the aortic baroreflex at the onset of spontaneous exercise.     

Inhibition of stem elongation in cucumis seedlings by blue light requires calcium

The effects of blue light and calcium on elongation of hypocotyl segments of Cucumber (Cucumis sativa L. cv Burpee's Pickler) were studied. Cucumber seedlings grown in dim red light showed a rapid decline in the rate of hypocotyl elongation when irradiated with high intensity (100 micromoles per square meter per second) blue light. In intact, 4-day-old seedlings the inhibition began within 2 minutes after the onset of blue-light irradiation and reached a maximum of approximately 55% within 4 minutes. Hypocotyl segments cut from 4-day-old seedlings also showed an inhibition of elongation in response to blue light when segments were floated on aqueous buffer and exposed to blue light for 3 hours. In the presence of 2 micromolar indole-3-acetic acid, blue light caused a 50% inhibition of elongation. Buffering free calcium in the incubation medium with 0.1 millimolar ethylene glycol bis(-aminoethyl ether)- N,N,N′,N′-tetraacetic acid eliminated the blue-light inhibition of segment elongation. Several experiments confirmed a specific requirement for calcium for the blue-light-induced inhibition of segment elongation. Treating segments with 0.2 micromolar fusicoccin abolished the inhibition of elongation by blue light as did buffering the medium at pH 4. Adding 1 millimolar ascorbate to incubation medium also eliminated the inhibition of segment elongation caused by blue light. Several compounds implicated in cell-wall redox reactions alter the magnitude of the blue-light-induced inhibition. The activity of peroxidase isolated from the cell-wall free space of cucumber hypocotyls was inhibited by ascorbate and low pH. The results are consistent with the hypothesis that blue light inhibits elongation by inducing an increase in cell-wall peroxidase activity and implicate calcium ions in the response to blue light.     

Unloaded shortening velocity of voluntarily and electrically activated human dorsiflexor muscles in vivo

We have previously shown that unloaded shortening velocity (V ₀) of human plantar flexors can be determined in vivo, by applying the “slack test” to submaximal voluntary contractions (J Physiol 567:1047–1056, 2005). In the present study, to investigate the effect of motor unit recruitment pattern on V ₀ of human muscle, we modified the slack test and applied this method to both voluntary and electrically elicited contractions of dorsiflexors. A series of quick releases (i.e., rapid ankle joint rotation driven by an electrical dynamometer) was applied to voluntarily activated dorsiflexor muscles at three different contraction intensities (15, 50, and 85% of maximal voluntary contraction; MVC). The quick-release trials were also performed on electrically activated dorsiflexor muscles, in which three stimulus conditions were used: submaximal (equal to 15%MVC) 50-Hz stimulation, supramaximal 50-Hz stimulation, and supramaximal 20-Hz stimulation. Modification of the slack test in vivo resulted in good reproducibility of V ₀, with an intraclass correlation coefficient of 0.87 (95% confidence interval: 0.68–0.95). Regression analysis showed that V ₀ of voluntarily activated dorsiflexor muscles significantly increased with increasing contraction intensity (R ² = 0.52, P<0.001). By contrast, V ₀ of electrically activated dorsiflexor muscles remained unchanged (R ²<0.001, P = 0.98) among three different stimulus conditions showing a large variation of tetanic torque. These results suggest that the recruitment pattern of motor units, which is quite different between voluntary and electrically elicited contractions, plays an important role in determining shortening velocity of human skeletal muscle in vivo.     

Light-induced generation of a protonmotive force and Ca2+-transport in membrane vesicles of Streptococcus cremoris fused with bacteriorhodopsin proteoliposomes

The light-driven primary proton pump bacteriorhodopsin has been incorporated in the cytoplasmic membrane of Streptococcus cremoris, in order to generate a protonmotive force across these membranes. This has been achieved by fusion of S. cremoris membrane vesicles with bacteriorhodopsin proteoliposomes. This fusion occurred when both preparations were mixed at low pH (less than 6.0), as shown by sucrose density gradient centrifugation and by dilution of fluorescent phospholipids incorporated into the bacteriorhodopsin proteoliposomes. Fusion was strongly enhanced by the presence of negatively charged phospholipids in the liposomal bilayer. When proteoliposomes were used that showed light-dependent proton uptake, the orientation of bacteriorhodopsin in the fused membranes was inside-out with respect to the in vivo orientation in Halobacterium halobium. Consequently, in the light a ΔΨ, interior positive and a ΔpH, interior acid were generated. This protonmotive force could drive calcium uptake in the fused membranes. The uptake increased hyperbolically with increasing light intensity and was abolished by bleaching of bacteriorhodopsin. Addition of the ionophore valinomycin stimulated calcium uptake and led to an increase of the ΔpH. Calcium uptake was strongly decreased in the dark and in the light in the presence of uncouplers, nigericin or both valinomycin and nigericin.     

Effect of calcium ions on ecdysteroid secretion by testes of Heliothis virescens

• 1.1. Testes of Heliothis virescens synthesized ecdysteroid in media containing low titers of calcium; the optimum calcium titer for testis sheaths stimulated to synthesize ecdysteroid in vivo was ca 1 mM, while the optimum of testes stimulated in vitro with the peptide testis ecdysiotropin was ca 0.3 mM calcium.
• 2.2. Verapamil at concentrations lower than 10⁻³ M induced increases in ecdysteroid synthesis, indicating more efficient synthesis when calcium influx was inhibited.
• 3.3. Hemolymph of H. virescens was 7 mM in calcium, while whole testes were maintained at 1–2 μM calcium.

     

Volume contraction of isolated pea chloroplasts promoted by bivalent cations

1. Chloroplasts isolated from pea seedlings were incubated in sucrose–tris medium reinforced with salts of calcium, magnesium, manganese or iron, at concentrations up to 10mm. 2. Measurements of chloroplast-pellet volume and water content showed that the bivalent cations brought about a contraction in chloroplast volume and a loss of chloroplast water. This was further substantiated by density-gradient centrifugations. 3. Measurements of the light-scattering and apparent fluorescence of chloroplast suspensions confirmed this conclusion and eliminated the possibility of contraction being caused by centrifugal forces. 4. The uptake of ⁴⁵Ca²⁺ was measured and shown to be competitive with diluent Ca²⁺, Mg²⁺ or Mn²⁺ ions, indicating a mechanism of low specificity. 5. The chloroplast contraction was insensitive to light but could be made sensitive by the addition of ferric EDTA. This light-sensitivity was inhibited by added 3-(p-chlorophenyl)-1,1-dimethylurea and so probably involves the Hill reaction. 6. On the basis of these observations it is suggested that the process of contraction does not consume much energy, but that in light-activated contraction a previous step occurs that is conducive to contraction and that is energy-transducing. It is postulated that this step results in a local increase in concentration of bivalent ions, which promotes contraction.     

High frequency vibration conditioning stimulation centrally reduces myoelectrical manifestation of fatigue in healthy subjects

ObjectiveVibration conditioning has been adopted as a tool to improve muscle force and reduce fatigue onset in various rehabilitation settings. This study was designed to asses if high frequency vibration can induce some conditioning effects detectable in surface EMG (sEMG) signal; and whether these effects are central or peripheral in origin.Design300 Hz vibration was applied for 30 min during 5 consecutive days, to the right biceps brachii muscle of 10 healthy males aged from 25 to 50 years. sEMG was recorded with a 16 electrode linear array placed on the skin overlying the vibrated muscle. The test protocol consisted of 30% and 60% maximal voluntary contraction (MVC) as well as involuntary (electrically elicited) contractions before and after treatment.ResultsNo statistically significant differences were found between PRE and POST vibration conditioning when involuntary stimulus-evoked contraction and 30% MVC were used. Significant differences in the initial values and rates of change of muscle fibre conduction velocity were found only at 60% MVC.Conclusions300 Hz vibration did not induce any peripheral changes as demonstrated by the lack of differences when fatigue was electrically induced. Differences were found only when the muscle was voluntarily fatigued at 60% MVC suggesting a modification in the centrally driven motor unit recruitment order, and interpreted as an adaptive response to the reiteration of the vibratory conditioning.     

Atrial myocardium is the predominant inotropic target of adrenomedullin in the human heart

Adrenomedullin (ADM) is an endogenous peptide with favorable hemodynamic effects in vivo. In this study, we characterized the direct functional effects of ADM in isolated preparations from human atria and ventricles. In electrically stimulated human nonfailing right atrial trabeculae, ADM (0.0001-1 micromol/l) increased force of contraction in a concentration-dependent manner, with a maximal increase by 35 +/- 8% (at 1 micromol/l; P < 0.05). The positive inotropic effect was accompanied by a disproportionate increase in calcium transients assessed by aequorin light emission [by 76 +/- 20%; force/light ratio (DeltaF/DeltaL) 0.58 +/- 0.15]. In contrast, elevation of extracellular calcium (from 2.5 to 3.2 mmol/l) proportionally increased force and aequorin light emission (DeltaF/DeltaL 1.0 +/- 0.1; P < 0.05 vs. ADM). Consistent with a cAMP-dependent mechanism, ADM (1 micromol/l) increased atrial cAMP levels by 90 +/- 12%, and its inotropic effects could be blocked by the protein kinase A (PKA) inhibitor H-89. ADM also exerted positive inotropic effects in failing atrial myocardium and in nonfailing and failing ventricular myocardium. The inotropic response was significantly weaker in ventricular vs. atrial myocardium and in failing vs. nonfailing myocardium. In conclusion, ADM exerts Ca(2+)-dependent positive inotropic effects in human atrial and less-pronounced effects in ventricular myocardium. The inotropic effects are related to increased cAMP levels and stimulation of PKA. In heart failure, the responsiveness to ADM is reduced in atria and ventricles.     

Ionic Strength and the Contraction Kinetics of Skinned Muscle Fibers

The influence of KCl concentration on the contraction kinetics of skinned frog muscle fibers at 5–7°C was studied at various calcium levels. The magnitude of the calcium-activated force decreased continuously as the KCl concentration of the bathing solution was increased from 0 to 280 mM. The shortening velocity at a given relative load was unaffected by the level of calcium activation at 140 mM KCl, as has been previously reported by Podolsky and Teichholz (1970. J. Physiol. [Lond.]. 211: 19), and was independent of ionic strength when the KCl concentration was increased from 140 to 280 mM. In contrast, the shortening velocity decreased as the KCl concentration was reduced below 140 mM; the decrease in velocity was enhanced when the fibers were only partially activated. In the low KCl range, the resting tension of the fibers increased after the first contraction cycle. The results suggest that in fibers activated at low ionic strength some of the cross bridges that are formed are abnormal in the sense that they retard shortening and persist in relaxing solution.     

Propagation of bioluminescence in Euphysa japonica hydromedusae, (Tubulariidae)

The jellyfish Euphysa emits light when touched, stimulated electrically or shaken in the water. Light is emitted over the whole subumbrellar surface. The source of light is the subumbrellar endodermal epithelium. The response accompanies muscular involution (crumpling) and is spread by all-or-none action potentials propagated in the epithelium itself. Light emission is calcium dependent, but propagation of the triggering events is not. Octanol blocks propagation but light is still emitted in the immediate vicinity of the stimulating electrode. Light emission consists of discrete flashes which sum and facilitate with repeated stimulation. Although the triggering impulses propagate over the entire epithelium, the threshold for light emission varies regionally. The response more closely resembles that described for the siphonophore Hippopodius than those of leptomedusae such as Aequorea. Light emission is best viewed as a startle response.     

The Interval-Strength Relationship in Mammalian Atrium: A Calcium Exchange Model: I. Theory

A model is developed for predicting the interval-strength relationship in mammalian atrium. The postulates underlying the model relate the intracellular and transmembrane calcium fluxes to changes in contractility. The predictions of the model agree qualitatively with the behavior of atrium for the following patterns of stimulation: (a) constant interval between stimuli, (b) a rest, or period with no stimuli, after the attainment of a steady-state force level, (c) a sudden change in the interval between stimuli, and (d) paired pulse stimulation. The effects of varying several parameters of the model on both the contraction staircases, after a rested-state contraction, and the steady-state interval-strength relationship are examined. Additional considerations are made: (a) estimates are made of the tissue calcium content available for contraction; (b) the physical meaning of the rested-state contraction is discussed; and (c) estimates are made of the proportionality constant between the maximum value of the contractile tension and the amount of calcium released before a contraction.     

Preliminary measurements of intracellular calcium in an insect salivary gland using a calcium-sensitive microelectrode

A calcium-sensitive microeletrode was used to measure free intracellular calcium in salivary gland cells of Calliphora during stimulation with 5-hydroxytryptamine (5-HT). The resting level of calcium was approximately 10^?7M or less but increased in a dose-dependent way sometimes to levels in excess of 10^?6M. The onset of the calcium signal was closely related to changes in membrane and transepithelial potential. This calcium response was greatly reduced when the extracellular calcium concentration was reduced from 10^?3 to 10^?4M. This dependence on external calcium is consistent with previous observations that 5-HT acts to increase the permeability of the basal plasma membrane to calcium. These observations indicate that an increase in the intracellular level of calcium is an early event associated with the onset of fluid secretion in this insect salivary gland.