The muscle sound properties of different muscle fiber types during voluntary and electrically induced contractions.

Soundmyogram (SMG) and electromyogram signals were recorded simultaneously from the relatively fast medial gastrocnemius (MG) and slow soleus (SOL) during voluntary and electrically induced contractions. Using a spike-triggered averaging technique, the averaged elementary sound and corresponding MU spikes were also obtained from about 35 different MUs identified. The rms-SMG of MG increased as a function of force (P < 0.01). On the contrary, these values for SOL increased up to 60% MVC (P < 0.01), but decreased at 80% MVC. The relationship between the peak to peak amplitude of SMG and MU spike indicated significant positive correlations (r = 0.631 to approximately 0.657, P < 0.01). During electrical stimulation at 5 Hz, the SMG power spectral peak frequency (PF) was matched with stimulation frequency in both muscles. At higher stimulation frequencies, e.g., > 15 Hz, only in the MG was SMG-PF synchronized with stimulation frequency; the slow SOL did not show such synchronization. Our data suggest that the SMG frequency components might reflect active motor unit firing rates, and that the SMG amplitude depends upon mechanical properties of contraction, muscle fiber composition, and firing rate during voluntary and electrically induced contractions.     

Calcium transients and calcium binding to troponin at the contraction threshold in skeletal muscle.

Antipyrylazo III calcium transients from voltage-clamped, cut skeletal muscle fibers of the frog were recorded, and the calcium binding to the regulatory sites of troponin C was calculated. The strength-duration curve for the contraction threshold was determined. It was found that the increase in myoplasmic calcium concentration necessary to produce the same level of contractile activation, i.e., the just visible movement, was approximately 60% higher at more positive membrane potentials resulting from short depolarizing pulses than at rheobase. However, using biochemical data for the kON and kOFF rate coefficients of the binding sites, the calculated maximums of the calcium binding curves were about the same at different voltages, and the time to maximum saturation was roughly equal to the latency of the contractions. To characterize the calcium binding in intact fibers more accurately, those values of the kON and kOFF rate coefficients that gave equal peak saturations during threshold movement at different membrane potentials were determined.     

Calcium and contractions of isolated smooth muscle cells from rat myometrium

Smooth muscle cells were isolated from estrogenized rat myometrium by collagenase digestion. Electron microscopic examination and measurement of cell lengths by image-splitting micrometry were carried out after fixation with acrolein. Mean lengths of cells before and after isolation were 81.7 and 66.9 micron, respectively. Responses of cells were compared with contractions of isolated strips recorded isometrically. Effects of carbachol and KCl were examined in 2 mM Ca, 2 mM Ca + 4 mM EGTA, and 2 mM Ca + 10(-8) M nitrendipine solution. Carbachol and KCl produced concentration-dependent shortening of isolated cells maximal at 30 s after addition. The concentrations of carbachol required to produce shortenings were about 100-fold less than those required to produce isometric contractions; but no major difference was observed in the concentration dependence of cell shortening and isometric contraction produced by potassium-induced depolarization. In 2 mM Ca solution, there was a phasic response, followed by a tonic response such that more than 50% of maximum cell shortening was maintained for 10 min. However, in 2 mM Ca + 4 mM EGTA or 10(-8) M nitrendipine, the tonic contraction was abolished and cells rapidly relaxed after 30 s. If carbachol was added to cells after varying times in the EGTA-containing solution, the ability to initiate a contraction declined exponentially with a half-time of 160 s. Effects of depolarization by KCl were examined in 2 mM Ca plus nitrendipine and 2 mM Ca + 4 mM EGTA solution. Shortening occurred in 2 mM Ca solution by depolarization but not if nitrendipine was added. Though shortening was not observed in 2 mM Ca + 4 mM EGTA solution by KCl, subsequent addition of carbachol induced shortening. These results suggested that there was an intracellular Ca store site from which Ca was released by carbachol and which was not affected by depolarization in the absence of external Ca. No evidence was obtained that the contraction persists in Ca2+-free medium in isolated cells, which is in agreement with previous findings in small muscle strips in which only a similar transient response was obtained.     

Changes in voluntary and electrically induced contractions during strength training and detraining

To elucidate the changes in neuro-muscular function during strength training and detraining, five male subjects underwent progressive isotonic strength training of their calf muscles three times a week for 8 weeks with additional detraining for the same periods. Electrically evoked twitch contractions were induced in the triceps surae muscles of each subject every 4 weeks during the training and detraining periods. At the same time, maximal voluntary isometric contractions (MVC) and the maximal girth of the calf (MGC) were measured. During the training period, MVC increased significantly from 98.4 to 129.6 Nm (31.7%, P less than 0.01) for the first 4 weeks of training but MGC showed little increase. Neither of the changes correlated with each other. Twitch contraction parameters, i.e. maximal twitch torque (Pt), maximal rate of torque development (max dT/dt) and rate of relaxation (relax dT/dt) showed no statistical change. During detraining, on the contrary, a large and significant increase (22.5%, P less than 0.01) was observed in max dT/dt without any changes in Pt and relax dT/dt. The MVC/Pt showed both significant increases during training and decreases during detraining. Our data suggest that short term strength training as employed in the present study does not induce changes in the contractile properties of the muscle during training, but may significantly affect the rate of force development during the subsequent detraining period, indicating the possible existence of complex post-training muscle adaptation.     

A realistic model of biphasic calcium transients in electrically nonexcitable cells.

In many electrically nonexcitable cells, the release of calcium from internal stores is followed by a much slower phase in which the intracellular calcium concentration decreases gradually to a sustained value higher than the concentration before stimulation. This elevated calcium plateau has been shown to be the result of calcium influx. The model presented in this work describes a system consisting of a cytoplasmic calcium store and a plasma membrane calcium channel, both excitable by a membrane receptor; a fast cytoplasmic calcium buffer; and calcium pumps in both the calcium store and cellular membranes. Inherent difficulties in the numerical evaluation of the model, caused by very large calcium fluxes across the store membrane, were overcome by analytically separating the fast processes of calcium release from the slower processes of calcium cycling across the plasma membrane. This enabled the simulation of realistic biphasic calcium transients similar to those observed experimentally. The model predicted 1) a strong correlation between the rate of calcium cycling across the plasma membrane and the rate of calcium decay; and 2) a dependence on the level of cell excitation of the maximum rise in cytoplasmic calcium concentration, the level of the elevated calcium plateau, and the rate of calcium decay. Using the model, we simulated the washout of agonist from the bathing solution and the depletion of the calcium store by a pharmacological agent (such as thapsigargin) under several experimental conditions.     

Charge-coupled device imaging of rapid calcium transients in cultured arterial smooth muscle cells

Transient changes in the concentration of intracellular free calcium are associated with the transduction of primary signals and the subsequent employment of Ca2+ as a second messenger in a multitude of cell types. These transients, typically monitored with the calcium-sensitive fluorescent dye Fura-2, are known to occur with a time course in the order of seconds. In order to accurately monitor such rapid changes in intracellular free calcium concentration in both single cells and simultaneously in several cells in a single field, we have developed a digital fluorescence imaging system based on a charge-coupled device (CCD) camera. We report here on the detailed kinetics of calcium increases in cultured arterial swine smooth muscle cells in response to the agonist ATP.     

Calcium regulation by and buffer capacity of molluscan neurons during calcium transients

The properties of Ca-regulation and -buffering of physiological levels of Ca-transients were examined in the soma of Archidoris monteryensis neurons. The rate of recovery from a Ca-transient was examined with two experimental protocols; in one the pulse duration was kept constant and its amplitude was varied, and in the other the duration was varied while the amplitude was kept constant. These experiments revealed that the recovery from a Ca-transient was approximately a first order process and the apparent first order rate constant was dependent on the duration of Ca-influx. The calcium buffer capacity of the cytoplasm was determined by an indirect method which utilised measured amounts of intracellular EGTA to reduce transient changes in free calcium. An equation for the cytoplasmic buffer capacity was derived on the assumption that the capacities of exogenous and endogenous Ca buffers summate linearly. The resting cytoplasmic Ca buffer capacity was 45.2 microM/delta pCa, when it was assumed that the incoming Ca diffuses a distance of 10 microns into the cytoplasm. For a diffusion distance of 5 microns it was 34.5 microM/delta pCa. In both cases, the buffer capacity increased with an increase in the size of Ca transient.     

Calcium transients and calcium signalling during early neurogenesis in the amphibian embryo Xenopus laevis

Development of the vertebrate embryonic nervous system is characterized by a cascade of signalling events. In Xenopus, the initial step in this cascade results from signals emanating from the dorsal mesoderm that divert the fate of the ectoderm from an epidermal to a neural lineage. These signals include extracellular antagonists of the bone morphogenetic protein (BMP). Experiments performed with isolated ectoderm suggest that epidermis is induced by BMP, whereas neural fates arise by default following BMP inhibition; however, we show that this mechanism is not sufficient for neural determination. Ca2+ imaging of intact Xenopus embryos reveals patterns of Ca2+ transients in the dorsal ectoderm but not in the ventral ectoderm. These increases in intracellular calcium concentration ([Ca2+](i)), which occur via the activation of dihydropyridine (DHP)-sensitive Ca2+ channels, are necessary and sufficient to orientate the ectodermal cells toward a neural fate. On the one hand, the treatments that antagonize the increase in [Ca2+](i), inhibit neuralization, while on the other hand, an artificial increase in [Ca2+](i), whatever its origin, neuralizes the ectoderm. Using these properties, we have constructed a subtractive cDNA library between untreated ectoderm and caffeine-treated ectoderm. The caffeine stimulates an increase in [Ca2+](i) and thus orientates the cells towards the neural pathway. We have identified early Ca2+ target genes expressed in neural territories. One of these genes, an arginine methyl transferase, controls the expression of the early proneural gene, Zic3. Here, we discuss an alternative model where Ca2+ plays a central regulatory role in early neurogenesis. This model integrates the activation of a Ca2+ -dependent signalling pathway due to an influx of Ca2+ through DHP-Ca2+ channels. While Ca2+ is required for neural determination, epidermal determination occurs when Ca2+ -dependent signalling pathways are inactive.     

Imaging of calcium transients in skeletal muscle fibers.

Epifluorescence images of Ca2+ transients elicited by electrical stimulation of single skeletal muscle fibers were studied with fast imaging techniques that take advantage of the large fluorescence signals emitted at relatively long wavelengths by the dyes fluo-3 and rhod-2 in response to binding of Ca2+ ions, and of the suitable features of a commercially available CCD video camera. The localized release of Ca2+ in response to microinjection of InsP3 was also monitored to demonstrate the adequate space and time resolutions of the imaging system. The time resolution of the imager system, although limited to the standard video frequency response, still proved to be adequate to investigate the fast Ca2+ release process in skeletal muscle fibers at low temperatures.     

Mechanical transients of single toad stomach smooth muscle cells. Effects of lowering temperature and extracellular calcium

Smooth muscle's slow, economical contractions may relate to the kinetics of the crossbridge cycle. We characterized the crossbridge cycle in smooth muscle by studying tension recovery in response to a small, rapid length change (i.e., tension transients) in single smooth muscle cells from the toad stomach (Bufo marinus). To confirm that these tension transients reflect crossbridge kinetics, we examined the effect of lowering cell temperature on the tension transient time course. Once this was confirmed, cells were exposed to low extracellular calcium [( Ca2+]o) to determine whether modulation of the cell's shortening velocity by changes in [Ca2+]o reflected the calcium sensitivity of one or more steps in the crossbridge cycle. Single smooth muscle cells were tied between an ultrasensitive force transducer and length displacement device after equilibration in temperature-controlled physiological saline having either a low (0.18 mM) or normal (1.8 mM) calcium concentration. At the peak of isometric force, after electrical stimulation, small, rapid (less than or equal to 1.8% cell length in 3.6 ms) step stretches and releases were imposed. At room temperature (20 degrees C) in normal [Ca2+]o, tension recovery after the length step was described by the sum of two exponentials with rates of 40-90 s-1 for the fast phase and 2-4 s-1 for the slow phase. In normal [Ca2+]o but at low temperature (10 degrees C), the fast tension recovery phase slowed (apparent Q10 = 1.9) for both stretches and releases whereas the slow tension recovery phase for a release was only moderately affected (apparent Q10 = 1.4) while unaffected for a stretch. Dynamic stiffness was determined throughout the time course of the tension transient to help correlate the tension transient phases with specific step(s) in the crossbridge cycle. The dissociation of tension and stiffness, during the fast tension recovery phase after a release, was interpreted as evidence that this recovery phase resulted from both the transition of crossbridges from a low- to high-force producing state as well as a transient detachment of crossbridges. From the temperature studies and dynamic stiffness measurements, the slow tension recovery phase most likely reflects the overall rate of crossbridge cycling. From the tension transient studies, it appears that crossbridges cycle slower and have a longer duty cycle in smooth muscle. In low [Ca2+]o at 20 degrees C, little effect was observed on the form or time course of the tension transients.(ABSTRACT TRUNCATED AT 400 WORDS)     

A viscoelastic model of mechanically induced and spontaneous contractions of vascular smooth muscle

A new viscoelastic model was developed for the mathematical characterisation of mechanically induced and intrinsic contractional responses of the vascular smooth muscle. To this end, the elastic and viscous analogue elements were supplemented with a new active element generating stress proportional to its momentary elongation. The four-element model consisting of an active element, a parallel viscous element and both series and parallel elastic elements predicted biphasic or damped oscillatory stress relaxation and creep responses which were similar to that found experimentally earlier. Above a certain exciting frequency the model exhibited dissipative and below energy producing behaviour, as indicated by the sign change of the hysteresis loop area. In the case of sinusoidal modulation of the stress generation parameter the model showed parametric resonance, which was regarded as a simulation of intrinsic oscillation of the smooth muscle.     

Interleukin-1beta induces calcium transients and enhances basal and store operated calcium entry in human myometrial smooth muscle

We have previously reported increased protein expression of sarcoplasmic reticulum calcium ATPase (SERCA) 2b in myometrium from women in labor at term, but the stimulus for this change is unknown. Proinflammatory cytokines have been implicated in the cascade of events leading to preterm and term labor, and we hypothesize that interleukin (IL)-1beta may induce changes in key calcium homeostatic mechanisms and, in turn, augment myometrial contractility before labor. The aim of the present study was to investigate the long-term effects of IL-1beta on SERCA 2b protein expression, calcium mobilization from intracellular stores, and store-operated calcium entry. Myometrial biopsies were obtained, with consent, from women undergoing elective cesarean section at term. Primary-cultured human myometrial smooth muscle (HMSM) cells were exposed to IL-1beta (10 ng/ml) for 24 h or to culture medium alone (control). Cells were subsequently used in Western blot studies or loaded with fura-2 to assess calcium dynamics using fluorescent digital imaging. The present study clearly demonstrated that IL-1beta significantly increased SERCA 2b protein expression in HMSM cells. Cyclopiazonic acid-induced calcium transients were also augmented, predominantly by activation of lanthanum-sensitive, store-operated calcium entry. HMSM cell excitability was enhanced, as evidenced by increased basal calcium entry and the initiation of spontaneous calcium transients in 37% of IL-1beta-treated cells. IL-1beta modulation of calcium mobilization may be an important mechanism in the cascade of events preparing the pregnant uterus for labor.     

Sodium and calcium interactions in vascular smooth muscle cells of the rabbit ear artery

The effects of Na-free and of K-free solutions on the membrane potential, on tension development, and on 45Ca exchange have been investigated in rabbit ear artery. The contraction induced by Na-free solutions and the tension which develops in K-free solutions after a delay of about 1 h are both submaximal. Exposure for 4 h to K-free solutions does not affect the membrane potential, whereas Na-free solutions depolarize the cells by 10-20 mV, depending on the Na-substitute. Neither the amplitude nor the rate constant of the slowly exchanging 45Ca-fraction is affected by these experimental procedures. Substituting external Na by choline or TMA induces a transient increase of the 45Ca-efflux rate which does not occur in a Ca-free efflux medium, and which can be blocked with La. K readmission to Na-enriched tissues hyperpolarizes the cells up to -100 mV and induces a relaxation, without exerting any effect on the 45Ca efflux rate. The release of Ca from intracellular stores, induced by histamine and FCCP, and its subsequent extrusion through the plasma membrane produce a transient stimulation of the 45Ca efflux, which is not affected by the reduction of the Na gradient. The transient contraction induced by histamine in Ca-free solutions is affected in a different way by different Na substitutes. The results do not fit the Na-Ca exchange hypothesis but are consistent with an effect of the Na gradient on the passive Ca influx.     

Effects of capacitative calcium entry on agonist-induced calcium transients in A7r5 vascular smooth muscle cells

OBJECTIVE: The purpose of this study was to evaluate the contribution of capacitative calcium influx to intracellular calcium levels during agonist-induced stimulation of vascular smooth muscle cells. METHODS: Aortic vascular smooth muscle cells (A7r5) were loaded with Indo-1 and intracellular calcium transients were measured. Cells were challenged with either arginine vasopressin (0. 5 microM) or thapsigargin (1 microM). Lanthanum (1 mM) was used to block capacitative calcium influx through store-operated channels. Calcium traces were analyzed for basal, peak and plateau responses. Recordings were derivatized and integrated to gain additional information. Nonlinear regression provided a time constant that describes restoration of ionic equilibrium involving both sequestration and extrusion pathways. RESULTS: Stimulation of cells with thapsigargin produced a non-L-type calcium influx that was attenuated by lanthanum. Cells excited with vasopressin exhibited a rapid calcium increase followed by a gradual decrease to a plateau level. Lanthanum pretreatment prior to stimulation caused no significant change in baseline, peak or plateau calcium levels as compared to control. Lanthanum caused no significant change in maximal calcium release rate, calcium integrals or time constant as compared to control. CONCLUSIONS: Capacitative calcium entry can occur in vascular smooth muscle cells, but does not appear to contribute significantly to the vasopressin response.     

Delay of intracellular calcium transients using a calcium chelator: application to high-throughput screening of the capsaicin receptor ion channel and G-protein-coupled receptors.

Whole-cell functional assays are often used for high-throughput screening (HTS) of molecular targets such as ion channels and G-protein-coupled receptors. A common method for assaying the activity of these membrane proteins is to measure the change in intracellular calcium concentration upon receptor stimulation. These changes in calcium concentration are typically transient and therefore not readily adapted to high-density plate formats used in HTS instruments. We have demonstrated that an intracellular calcium chelator, BAPTA, was able to delay by 5- to 20-fold and extend for several minutes the observed calcium signals initiated by extracellular calcium influx or release of calcium from intracellular stores. As examples, we used cells expressing a calcium-permeable ion channel, vanilloid receptor type 1 (the capsaicin receptor), and two G-protein-coupled receptors. These receptor-mediated increases in intracellular calcium concentration were measured by both fluorescence-based and luminescence-based detection methods. The use of an intracellular calcium chelator to delay calcium signaling should have wide application since it allows the measurement of the functional activity of any cellular receptor that signals through calcium. With this procedure, calcium fluorescence and luminescence whole-cell functional assays may be performed with standard laboratory pipetting and detection systems.     

External cadmium and internal calcium block of single calcium channels in smooth muscle cells from rabbit mesenteric artery.

The patch clamp technique was used to record unitary currents through single calcium channels from smooth muscle cells of rabbit mesenteric arteries. The effects of external cadmium and cobalt and internal calcium, barium, cadmium, and magnesium on single channel currents were investigated with 80 mM barium as the charge carrier and Bay K 8644 to prolong openings. External cadmium shortened the mean open time of single Ca channels. Cadmium blocking and unblocking rate constants of 16.5 mM-1 ms-1 and 0.6 ms-1, respectively, were determined, corresponding to dissociation constant Kd of 36 microM at -20 mV. These results are very similar to those reported for cardiac muscle Ca channels (Lansman, J. B., P. Hess, and R. W. Tsien. 1986. J. Gen. Physiol. 88:321-347). In contrast, Cd2+ (01-10 mM), when applied to the internal surface of Ca channels in inside-out patches, did not affect the mean open time, mean unitary current, or the variance of the open channel current. Internal calcium induced a flickery block, with a Kd of 5.8 mM. Mean blocking and unblocking rate constants for calcium of 0.56 mM-1 ms-1 and 3.22 ms-1, respectively, were determined. Internal barium (8 mM) reduced the mean unitary current by 36%. We conclude that under our experimental conditions, the Ca channel is not symmetrical with respect to inorganic ion block and that intracellular calcium can modulate Ca channel currents via a low-affinity binding site.     

Strain-dependent modulation of phosphate transients in rabbit skeletal muscle fibers.

When inorganic phosphate (Pi) is photogenerated from caged Pi during isometric contractions of glycerinated rabbit psoas muscle fibers, the released Pi binds to cross-bridges and reverses the working stroke of cross-bridges. The consequent force decline, the Pi-transient, is exponential and probes the kinetics of the power-stroke and Pi release. During muscle shortening, the fraction of attached cross-bridges and the average strain on them decreases (Ford, L. E., A.F. Huxley, and R.M. Simmons, 1977. Tension responses to sudden length change in stimulated frog muscle fibers near slack length. J. Physiol. (Lond.). 269:441-515; Ford, L. E., A. F. Huxley, and R.M. Simmons, 1985. Tension transients during steady state shortening of frog muscle fibers. J. Physiol. (Lond.). 361:131-150. To learn to what extent the Pi transient is strain dependent, muscle fibers were activated and shortened or lengthened at a fixed velocity during the photogeneration of Pi. The Pi transients observed during changes in muscle length showed three primary characteristics: 1) during shortening the Pi transient rate, Kpi, increased and its amplitude decreased with shortening velocity; Kpi increased linearly with velocity to > 110 s-1 at 0.3 muscle lengths per second (ML/s). 2) At a specific shortening velocity, increases in [Pi] produce increases in Kpi that are nonlinear with [Pi] and approach an asymptote. 3) During forced lengthening Kpi and the amplitude of the Pi transient are little different from the isometric contractions. These data can be approximated by a strain-dependent three-state cross-bridge model. The results show that the power stroke's rate is strain-dependent, and are consistent with biochemical studies indicating that the rate-limiting step at low strains is a transition from a weakly to a strongly bound cross-bridge state.