Mechanical characteristics of skinned and intact muscle fibres from the giant barnacle,Balanus nubilus

Intact muscle fibres fromBalanus nubilus develop tensions of up to 600 kN sd m⁻² during electrical stimulation. The rise of tension occurs with a half-time (177 ms at 12° C) about fivefold longer than that of tetanised frog muscle at the same temperature. The response of myofibrillar bundles to a rapid stretch resembles that of frog muscle but has a y_o value (i.e. the size of an instantaneous release necessary to just discharge tension) which is ca. 2.5 times smaller, and phase 2 of the tension transient (the “quick phase”) occurs at a rate comparable to that of frog muscle. In contrast, the ATPase activity (0.018 mmoles · kg wet weight⁻¹ · s⁻¹) of this preparation and its maximum shortening velocity (0.15–0.16 muscle lengths · s⁻¹) are both at least fivefold slower than frog muscle. These findings can be accounted for by a cross-bridge cycle in barnacle muscle in which events prior and subsequent to the tension generating step(s) occur at a rate at least fivefold slower than comparable steps in frog muscle, but the step(s) associated with tension development occur at similar rates in the two preparations. Since the rate of mechanical relaxation in barnacle muscle is modified in the presence of intracellular calcium buffers and by depolarisation-induced elevation of the free calcium during the relaxation phase, it is proposed that the time course of relaxation is not determined exclusively by the kinetics of the cross-bridge cycle, but is also dependent on the free calcium concentration during relaxation.     

The effects of repeated exposure to anoxia on intracellular calcium,glycogen and lactate in isolated ferret heart muscle

Isolated cardiac tissue from the ferret was repeatedly exposed to anoxia while perfused with glucose-containing Tyrode solution. In one series of experiments, papillary muscles were injected with aequorin to measure intracellular Ca²⁺. On the first exposure to anoxia, the Ca²⁺ transients often increased, but on subsequent exposures this increase disappeared and eventually the Ca²⁺ transients declined on exposure to anoxia. This decline in the Ca²⁺ transients could be converted back to an increase by a 1 h exposure to an elevated (×5) glucose concentration. Exposure of aerobic muscles to 10 mM lactic acid caused a similar increase in the Ca²⁺ transients to that seen in early exposures to anoxia. In a second series of experiments, performed on Langendorff-perfused hearts, measurements were made of glycogen concentration preceding, and lactate production during, exposures to anoxia. At a constant level of glucose, glycogen concentration and lactate production were found to decline on repeated exposures to anoxia, and both were increased after a period of elevated glucose and reduced stimulation frequency. These results suggest that the response of the Ca²⁺ transients to anoxia is dependent on the metabolic status of the muscle. The increase in the Ca²⁺ transients during an early eposure to anoxia may be a consequence of lactic acid production due to accelerated glycolysis. Repeated exposures to anoxia reduce glycogen concentration and lactate production and this reduces the rise in the Ca²⁺ transients.     

Effects of β-adrenoceptor stimulation on intracellular Ca transients and tension in rat ventricular muscle

Effects of -adrenoceptor stimulation on intracellular Ca²⁺ transients and tension were explored in rat ventricular muscles injected with aequorin. Adrenaline (0.05–5.0 M) and isoproterenol (0.05–1.0 M) increased the peak of twitch tension and accelerated relaxation. The former effect depended on Ca²⁺ concentration in Tyrode's solution ([Ca²⁺]_o) and the stage of the experiment. Low concentrations of these drugs added to normal Tyrode's solution containing 2 mM [Ca²⁺]_o did not potentiate twitch tension in the early stage of the experiments. These drugs increased the peak of the aequorin light signal and slightly accelerated the falling phase of the light especially the tail. Effects of dibutyryl-cyclic AMP (DB-cAMP) (0.1–5.0 mM) and 3-isobutyl-1-methylxanthine (IBMX) (0.01–0.5 mM) were qualitatively similar to those of adrenaline and isoproterenol.Isoproterenol applied at the peak of Na-deficient contracture decreased tension without significantly changing the light signal; similar results were obtained in the presence of ryanodine (1 M).The results were interpreted as follows: The increase of intracellular cAMP induced by -adrenoceptor stimulation facilitated Ca²⁺ uptake by sarcoplasmic reticulum (SR) and decreased Ca²⁺ sensitivity of contractile elements. Faster relaxation induced by cAMP was considered to be due to the decrease of Ca²⁺ sensitivity of contractile elements and faster Ca²⁺ uptake by SR. The slightly faster falling phase of light transient might be due to the faster Ca²⁺ uptake by SR, which predominates over the slower fall of [Ca²⁺]_i induced by the decreased Ca²⁺ sensitivity of the contractile element.     

Differential effects of reoxygenation on intracellular calcium and isometric tension

We used the bioluminescent Ca²⁺ indicator, aequorin to record intracellular calcium transients during reoxygenation of hypoxic ferret ventricular muscle in order to determine whether alterations in the amplitude and time course of isometric contraction are emdiated by changes in [Ca²⁺]_i. Papillary muscles 1 mm in diameter were removed from the hearts of male ferrets and perfused with a bicarbonate-buffered physiologic salt solution at 30°C. Muscles were stimulated to contract isometrically at 0.33 Hz and were loaded with aequorin by a chemical procedure. Hypoxia was induced by changing the gas mixture bubbling the perfusate to 95% N₂, 5% CO₂; reoxygenation was accomplished by switching the gas mixture to 95% O₂, 5% CO₂. Hypoxia produced a decrease in peak Ca²⁺ and tension that was reversed by reoxygenation. However, the effects on tension of changes in oxygenation were greater than expected from the degree of change in [Ca²⁺]_i. The time courses of the Ca²⁺ transient and isometric twitch moved in opposite directions and were respectively prolonged/abbreviated by hypoxia and abbreviated/prolonged by reoxygenation. These results indicate that changes in the amplitude and time course of the isometric twitch induced by hypoxia and reoxygenation cannot be attributed alone to changes in intracellular Ca²⁺ availability and are caused in part by a significant decrease in the calcium sensitivity of the contractile apparatus.     

The effects of temperature on relaxation in frog skeletal muscle: the role of parvalbumin

Isolated muscle fibers fromRana temporaria tibialis anterior muscles were microinjected with aequorin. The force responses and the Ca²⁺ transients associated with twitch and tetanic contractions were studied at several temperatures. The declines of the Ca²⁺ transients were well described by single exponential equations and the effects of temperature were complex (multi-exponential). To determine if these temperature effects on the Ca²⁺ transients were influenced by the Ca²⁺ indicator itself, samples of the injected aequorin were studied in vitro using a Gibson stopped-flow apparatus. The quenching of aequorin luminescence with either EGTA or de-calcifiedRana temporaria parvalbumin were mono-exponential. These overall quenching reactions had single exponential temperature dependencies. The effects of temperature on the declines of the single fiber Ca²⁺ transients did not appear to be influenced by the kinetics of the aequorin reaction. The disparity in the effects of temperature on the single fiber Ca²⁺ transients versus the in vitro quenching of aequorin luminescence with parvalbumin, were interpreted to indicate that in twitch and tetanic contractions of these fibers, it was unlikely that soluble Ca²⁺ binding proteins played a major role in the regulation of myoplasmic Ca²⁺.     

Effects of enflurane on excitation-contraction coupling in frog skeletal muscle fibers

The effect of enflurane on the excitation-contraction (E-C) coupling of frog skeletal muscle fiber was studied.Low (19.13±0.70 mg%) and high (108.52±4.52 mg%) concentrations of enflurane did not cause substantial changes of the resting membrane potential but a high concentration of enflurane reduced the peak of action potentials and prolonged the duration measured at 50% peak amplitude.During the twitch response, enflurane reduced the peak response of light signal measured with calcium-sensitive photoprotein, aequorin, butpeak tension was not diminished by a low concentration of enflurane. A high concentration of enflurane remarkably inhibited both light signal and tension.During tetanus, a low concentration of enflurane partially abolished light signal and tension. At the high concentration, this inhibitory effect was pronuunced and action potentials were only observed in the initial phase of tetanic stimulation.Enflurane enhanced the increase of light signal observed in rapid cooling contracture.In glycerinated muscle fiber, enflurane shifted the pCa-tension relation to the left in low calcium and suppressed maximally activated tension at high calcium concentration.Inhibitory effect of enflurane on light signal in twitch response without abolition of action potential suggests that enflurane might inhibit E-C coupling in frog skeletal muscle fiber (but it enhances direct calcium release from SR induced by caffeine) and increase calcium sensitivity of contractile element.     

Concurrent measurement of platelet ionized calcium concentration and aggregation: studies with the lumiaggregometer

Platelet aggregation and aequorin-indicated cytoplasmic ionized calcium concentration ([Cai2+]) were measured simultaneously using a modification of a commercially available instrument, the Chronolog Whole Blood Lumiaggregometer. The [Cai2+] rise in response to arachidonic acid, the Ca2+-ionophore A23187, collagen, and epinephrine occurred either before or simultaneously with platelet shape change or aggregation. PGD2 caused concentration-dependent inhibition of both the [Cai2+] rise and aggregation. However, suppression of the [Cai2+] rise alone did not fully account for the inhibition of aggregation due to the elevated cAMP produced by PGD2. Agonist-induced elevation of [Cai2+] did not depend on platelet aggregation, but was enhanced by close platelet-to-platelet contact. Concurrent measurement of platelet aggregation and [Cai2+] may further clarify pathways to platelet activation.     

The effects of digitalis on intracellular calcium transients in mammalian working myocardium as detected with aequorin

The effects of positive inotropic agents on the amplitude and time course of the light signal and corresponding tension response were studied in cat and human working myocardium microinjected with the bioluminescent Ca2+ indicator aequorin. Distinctive patterns of light and tension responses were identified that are consistent with known actions of the various agents on the release of Ca2+ from intracellular stores, rate of uptake of Ca2+ by the sarcoplasmic reticulum and sensitivity of the myofilaments to Ca2+. In common with most other inotropic drugs, the cardiotonic steroid, acetylstrophanthidin, in doses of 4 X 10(-7) to 2 X 10(-6)M increases the amount of Ca2+ available for excitation-contraction coupling in the heart. However, in contrast to most other agents, acetylstrophanthidin does not affect the time course of the calcium transient. In common with changes in [Ca2+]o, acetylstrophanthidin does not alter the relationship between the amplitude of the aequorin light signal and developed tension, which, in contrast to caffeine and isoproterenol, indicates that the increase in tension is fully accounted for by the increase in systolic free calcium. These findings suggest that the cardiotonic steroids increase loading of intracellular calcium stores without affecting the kinetics of subcellular handling of Ca2+. In doses of 8 X 10(-7) to 2 X 10(-6)M, acetylstrophanthidin produces a calcium-overload state characterized by 'after-contractions' and 'after-glimmers' that are associated with the development of automatic and triggerable dysrhythmias. These studies provide direct evidence that the inotropic and toxic effects of digitalis on animal and human working myocardium are produced by changes in intracellular Ca2+.     

Luminometry: a novel bioluminescent immunoassay enhances the quantitation of mucosal and systemic antibody responses

We have directly compared enzyme-linked immunoassays (ELISAs) with bioluminescent immunoassays employing derivatives of the bioluminescent molecule aequorin, and have shown that detection of mucosal and serum antibodies is considerably more sensitive when detected by luminometry. Luminometry is based upon counting photons of light via phototubes and is generally similar to scintillation spectrometry. Current commercial luminometric technology employs a phototube which is most efficient for light emission in the 400–420 nm wavelength range. For this reason, we have chosen the bioluminescent molecule, aequorin, which upon the addition of Ca²⁺ undergoes a conformational change resulting in the emission of blue light at 469 nm. The high quantum yield is reflected by the fact that addition of Ca²⁺ to 1 ng of recombinant streptaequorin, a covalent conjugate of streptavidin and aequorin, resulted in the production of 7 × 10⁸ relative light units. In this study, we show the superior sensitivity of biotin-streptaequorin when directly compared with biotin-streptavidin linked horseradish peroxidase commonly used for ELISA. For example, mice orally immunized once with cholera toxin (CT) did not exhibit detectable fecal IgA antibodies as determined by ELISA, whereas use of streptaequorin and the bioluminescent immunoassay revealed fecal IgA anti-CT-B subunit antibody titers of 1 : 24 500. In addition, no detectable anti-CT-B antibodies were noted in saliva samples by ELISA 7 days following oral immunization with CT, while IgA endpoint titers could be extrapolated to 1 : 393 000. The 21 day fecal IgA anti-CT-B titers were 1 : 512 by ELISA, whereas titers determined by luminometry reached 1 : 10⁷ when Neutralite avidin and biotinylated aequorin were employed. In general, the bioluminescent immunoassay was > 10⁴-fold more sensitive when compared with ELISA for detection of mucosal and serum antigen- and isotype-specific antibody responses. Thus, the bioluminescent immunoassay is a more sensitive assay for detection of antibodies in dilute external secretions.     

NMDA-evoked consumption and recovery of mitochondrially targeted aequorin suggests increased Ca2+ uptake by a subset of mitochondria in hippocampal neurons

Activation of NMDA receptors produces large increases in cytosolic Ca(2+) that are taken up into mitochondria. We used recombinant aequorin targeted to mitochondria to report changes in matrix Ca(2+) in rat hippocampal neurons in culture. Upon binding Ca(2+), aequorin emits a photon in a one-shot reaction that consumes the indicator. Here we show that stimulation with NMDA produced a mitochondrial Ca(2+) response that rapidly inactivated. However, following a 30-min recovery period the response was restored, suggesting the presence of a pool of indicator that was not exposed to high Ca(2+) during the initial stimulus. We speculate that aequorin distant from the Ca(2+) source was protected from microdomains of high Ca(2+) near the plasmalemma and that this aequorin moved, either by movement of individual mitochondria or via the mitochondrial tubular network, to replenish consumed indicator during the recovery time. A large Ca(2+) increase in a subset of mitochondria could produce local changes in energy metabolism, regional Ca(2+) buffering, and foci that initiate neurotoxic processes.