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At least 19 recordsLinked to original sources
PubMed1992

On the significance of different aequorin loading techniques on intracellular aequorin discharge, baseline calcium, platelet aggregation and aequorin-indicated Ca(2+)-transients.

The study compares the decay of intracellular luminescence activity (Lmax), the levels of basal [Ca2+]i in resting platelets, and agonist-induced peak [Ca2+]i-signals in platelets loaded with aequorin using the EGTA-, DMSO- and hypoosmotic shock treatment (HOST)-techniques. The highest load of intracellular aequorin with almost unchanged luminescence activity during 4 h was achieved with HOST. Lmax decreased linearly in EGTA- and HOST-platelets, but the decay rate and the levels of basal [Ca2+]i were significantly lower in HOST-platelets. Platelet aggregation and aequorin-indicated [Ca2+]i-rise induced by thrombin and collagen were similar in EGTA- and HOST-platelets. In HOST-platelets, ADP-induced platelet aggregation was always accompanied by aequorin-signals, while at a similar time point, aequorin-signals were absent in 3 of 5 cases in EGTA-platelets. The initial aequorin loading was highest in DMSO-platelets, but Lmax described an exponential decay, which was most pronounced when DMSO-platelets were maintained in Ca(2+)-free buffer (R2 = 0.86). Agonist-induced platelet aggregation was significantly reduced in DMSO-platelets: thrombin-stimulation was accompanied by a significantly lower and delayed [Ca2+]i-rise and no aequorin-signal was obtained in response to ADP in 3 of 5 cases. The study shows that in addition of being a rapid loading-technique, the criteria of high intracellular aequorin load with low luminescence consumption, low basal [Ca2+]i and completely preserved platelet functions are most convincingly met by the HOST-method.

Aequorin↗
PubMed1990

Recombinant aequorin and recombinant semi-synthetic aequorins. Cellular Ca2+ ion indicators.

Properties of a recombinant aequorin were investigated in comparison with those of natural aequorin. In chromatographic behaviour the recombinant aequorin did not match any of ten isoaequorins tested, although it was very similar to aequorin J. Its sensitivity to Ca2+ was found to be higher than that of any isoaequorin except aequorin D. The recombinant aequorin exhibited no toxicity when tested in various kinds of cells, even where samples of natural aequorin had been found to be toxic. Properties of four recombinant semi-synthetic aequorins (fch-, hcp-, e- and n-types), prepared from the recombinant apo-aequorin and synthetic analogues of coelenterazine, were approximately parallel with those of corresponding semi-synthetic aequorins prepared from natural apo-aequorin. Both recombinant e-aequorin and natural e-aequorin J luminesced with high values of the luminescence intensity ratio I400/I465, although the ratios were not pCa-dependent. The recombinant aequorin and recombinant semi-synthetic aequorins are highly suited for monitoring cellular Ca2+.

Aequorin↗
PubMed1989

Time dependence of aequorin-indicated calcium levels in stimulated and unstimulated platelets: evidence for multiple aequorin environments in platelets.

Aequorin-induced calcium signals were examined in human unstimulated platelets and platelets stimulated with various agonists as a function of time. The total aequorin response in unstimulated platelets, obtained by Triton x-100 lysis in the presence of 1 mM Ca, decreased in a distinctly non-linear manner over 20-60 min. This decrease was slightly, but significantly, greater in platelets maintained in the continuous presence of 1 mM Ca than in platelets maintained without external Ca, and could not be accounted for completely by leakage of aequorin from the cells. Basal Ca levels in unstimulated platelets also decreased in a non-linear manner, with a similar sensitivity to the continuous presence or absence of external Ca. These observed changes in aequorin response thus appear to be at least partially due to an intracellular discharge of aequorin, and are therefore consistent with the view that aequorin in platelets is heterogeneously distributed among localized environments differing in Ca concentration. The aequorin signals observed initially in platelets stimulated by ADP or epinephrine were lost completely over a period of 30-60 min in almost all cases studied, while initial rates of aggregation were either unchanged (epinephrine) or only partially decreased (ADP) over this same time period. In contrast, thrombin- and A23187-induced aequorin signals were virtually unchanged over periods up to 90 min. Minimal changes with time also occurred in the aequorin signals induced by phorbol ester or by collagen in the presence of indomethacin. These differences in time dependence suggest that the signals generated by ADP and epinephrine may derive from different sources of aequorin than those associated with the signals induced by other agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Aequorin↗
PubMed1993

Light-emitting properties of recombinant semi-synthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium.

15 kinds of recombinant semi-synthetic aequorins and a recombinant fluorescein-conjugated aequorin were prepared and their properties in Ca(2+)-triggered luminescence were studied. The semi-synthetic aequorins showed a wide range of Ca(2+)-sensitivity. The luminescence intensity of a high-sensitivity type (hcp-aequorin) was greater than 10(4)-times that of a low-sensitivity type (n-aequorin) at pCa 6.0-6.5. The fluorescein-conjugated aequorin exhibited fluorescence in addition to the Ca(2+)-triggered luminescence, thus it can be used to visualize the diffusion and distribution of aequorin in cells. The data obtained, particularly the Ca(2+)-sensitivity curves, are useful in selecting a suitable semi-synthetic aequorin for an experiment.

Aequorin↗
PubMed1993

A significant portion of the aequorin luminescent signal from stimulated human and rabbit platelets is due to exposure of the aequorin to calcium in the suspending medium.

We have examined in unstimulated and thrombin-stimulated human and rabbit platelets the localization and behavior of aequorin loaded by a variety of published methods. When platelets were suspended at 37 degrees C in Tyrode-albumin medium containing 2 mM Ca2+ and apyrase, we found with all preparations that total aequorin revealed by addition of Triton X-100 decreased by more than 50% over one hour. Incubation in the presence of 5 mM EGTA followed by addition of Ca2+ to restore the concentration to 2 mM showed that some aequorin had entered the medium; subsequent addition of Triton X-100 showed that the increase in aequorin in the medium matched the decrease in aequorin in the platelets, such that total aequorin remained unchanged. However, comparison of aequorin in platelets incubated in media with and without Ca2+ showed a larger decrease in platelets incubated in the presence of Ca2+; this finding may indicate the presence of an intracellular pool of Ca2+ which is more dependent on external Ca2+. Stimulation of platelets with thrombin in the presence of EGTA resulted in a smaller luminescent signal than in the presence of Ca2+. Subsequent addition of Ca2+ to 2 mM in the platelet suspension that originally contained EGTA or to its supernate (after centrifugation of the platelet suspension), resulted in a larger luminescent signal compared with controls, indicating that stimulation of the platelets had increased loss of the aequorin into the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Aequorin↗
PubMed1991

Simultaneous measurement of Ca2+ in muscle with Ca electrodes and aequorin. Diffusible cytoplasmic constituent reduces Ca(2+)-independent luminescence of aequorin.

Estimates of cytoplasmic Ca2+ concentration ([Ca2+]i) were made essentially simultaneously in the same intact frog skeletal muscle fibers with aequorin and with Ca-selective microelectrodes. In healthy fibers under truly resting conditions [Ca2+]i was too low to be measured reliably with either technique. The calibration curves for both indicators were essentially flat in this range of [Ca2+], and the aequorin light signal was uniformly below the level to be expected in the total absence of Ca2+. When [Ca2+]i had been raised to a stable level below the threshold for contracture by increasing [K+]o to 12.5 mM, [Ca2+]i was 38 nM according to aequorin and 59 nM according to the Ca-selective microelectrodes. These values are not significantly different. Our estimates of [Ca2+]i are lower than most others obtained with microelectrodes, probably because the presence of aequorin in the cells allowed us to detect damaging microelectrode impalements that otherwise we would have had no reason to reject. The observation that the light emission from aequorin-injected fibers in normal Ringer solution was below the level expected from the Ca(2+)-independent luminescence of aequorin in vitro was investigated further, with the conclusion that the myoplasm contains a diffusible macromolecule (between 10 and 30 kD) that interacts with aequorin to reduce light emission in the absence of Ca2+.

Aequorin↗
PubMed1997

Interaction of immobilized avidin with an aequorin-biotin conjugate: an aequorin-linked assay for biotin.

Biotinylated recombinant aequorin was used in the development of a heterogeneous bioluminescence binding assay for biotin. This assay is based on a competition between a biotinylated aequorin conjugate and biotin for the binding sites of avidin immobilized on solid particles. Dose-response curves were obtained that relate solid-phase aequorin activity to the concentration of biotin. Under certain experimental conditions these curves were biphasic; i.e., as the biotin concentration increased, the solid-phase aequorin activity first increased reaching a maximum and then decreased at higher biotin concentrations. This "hook" effect was observed with four different types of immobilization supports. The effect was more pronounced when low concentrations of aequorin-biotin conjugate were used, and diminished at a high conjugate concentration. This behavior indicates a possible positive cooperativity in the interaction between the immobilized avidin and biotin. Scatchard plot analysis was also consistent with a positive cooperativity mechanism. By using the ascending portion of the dose-response curve, the detection limit of the assay for biotin was 1 x 10(-15) M (100 zmol of biotin in the sample).

Aequorin↗
PubMed1999

The in situ regeneration and extraction of recombinant aequorin from Escherichia coli cells and the purification of extracted aequorin.

Recombinant apoaequorin expressed in the periplasmic space of Escherichia coli cells was regenerated into aequorin and extracted from the cells, simultaneously, using a buffer that contained coelenterazine. Due to the mild extraction conditions, the impurities in the extract were minimal. Thus, the purification of extracted aequorin could be accomplished in only two steps, anion-exchange chromatography and hydrophobic interaction chromatography, simply by adsorption and elution in both steps. The purified recombinant aequorin was pure, based on various data, including HPLC analysis and light-emitting activity. The yield of purified aequorin was 25-35 mg from 600 ml of culture, which was over 75% of the total amount of apoaequorin expressed in E. coli cells.

Aequorin↗
PubMed1997

Expression of apo-aequorin during embryonic development; how much is needed for calcium imaging?

Aequorin is a bioluminescent calcium indicator consisting of a 21 kDa protein (apo-aequorin) that is covalently linked to a lipophilic cofactor (coelenterazine). The aequorin gene can be expressed in a variety of cell lines and tissues, allowing non-invasive calcium imaging of specific cell types. In the present paper, we describe the possibilities and limitations of calcium imaging with genetically introduced apo-aequorin during embryonic development. By injecting aequorin into sea urchin, Drosophila and zebrafish eggs, we found that higher aequorin concentrations are needed in smaller eggs. Our results suggest that for measuring resting levels of free cytosolic calcium, one needs aequorin concentrations of at least 40 microM in sea urchin eggs, 2 microM in Drosophila eggs, and only 0.11 microM in zebrafish eggs. A simple assay was used to determine the absolute concentrations of expressed apo-aequorin and the percentage of aequorin formation in vivo. The use of this assay is illustrated by expression of the aequorin gene in Drosophila oocytes. These oocytes form up to 1 microM apo-aequorin. In our hands, only 0.3% of this apo-aequorin combined with coelenterazine entering from the medium to form aequorin, which was not enough for calcium imaging of the oocytes, but did allow in vivo imaging of the ovaries. From these studies, we conclude that coelenterazine entry into the cell is the rate limiting step in aequorin formation. Based on the rate of coelenterazine uptake in Drosophila, we estimate that complete conversion of 1 microM apo-aequorin would take 50 days in zebrafish eggs, 2 days [corrected] in Drosophila eggs, 7 days in sea urchin eggs or 18 h in a 10 microm tissue culture cell. Our results suggest that work based on genetically introduced apo-aequorin will be most successful when large amounts of small cells can be incubated in coelenterazine. During embryonic development this would involve introducing coelenterazine into the circulatory system of late stage embryos. Calcium imaging in early stage embryos may be best done by injecting aequorin, which circumvents the slow process of coelenterazine entry.

Aequorin↗
PubMed1986

Isolation and properties of various molecular forms of aequorin.

The photoprotein aequorin emits light by an intramolecular reaction when a trace of Ca2+ is added. The samples of aequorin that were purified by the conventional methods of column chromatography were separated by high-performance liquid chromatography into eight molecular forms (isoaequorins), which were designated aequorins A-H. Aequorins A, C and F were obtained in crystalline states. A wide range of properties were studied with aequorins A-F, which were essentially pure. These six isoaequorins showed relatively small differences in their spectroscopic properties, but their values of A0.1%/1 cm, 280 were found to be close to 3.0, about 10% more than the previously reported value of 2.70-2.71 that was obtained with the samples of conventionally purified aequorin. The Mr values ranged from 20,100 (aequorin F) to 22,800 (aequorin A), the luminescence activities ranged from 4.35 X 10(15) photons/mg (aequorin A) to 5.16 X 10(15) photons/mg (aequorin F), and the first-order reaction rate constants of luminescence ranged from 0.95 s-1 (aequorin A) to 1.33 s-1 (aequorin F). As regards sensitivity to Ca2+, aequorin D was the most sensitive, having a sensitivity about 0.4-0.5 pCa unit above that of the least sensitive kind (aequorin A).

Aequorin↗
PubMed1995

Calcium compartments in vascular smooth muscle cells as detected by aequorin signal.

1. To examine whether cytosolic Ca2+ in smooth muscle cells distributes evenly, cytosolic Ca2+ levels were measured with two different Ca2+ indicators in the ferret isolated portal vein; a fluorescent indicator, fura-PE3, that shows the average Ca2+ level, and a photoprotein, aequorin, that preferentially shows a high Ca2+ compartment. 2. A noradrenaline (10 microM)-induced sustained contraction was associated with a sustained increase in the fura-PE3 signal, or a transient increase followed by small sustained increase in the aequorin signal. A high K(+)-induced contraction was associated with a sustained increase in both the fura-PE3 and aequorin signals. 3. A second application of noradrenaline or high K+ induced reproducible contractions and fura-PE3 signals. In contrast, the aequorin signal resulting from a second application of noradrenaline or high K+ was much smaller than the first signal. 4. Following a 13 h but not a 3 h resting period, the aequorin signal stimulated by noradrenaline or high K+ recovered, without any change in the contractile response. 5. In Ca(2+)-free solution, high K+ was ineffective, whereas noradrenaline induced only a small aequorin signal and contraction compared to those obtained in the presence of external Ca2+. After the addition of Ca2+, the first application of noradrenaline induced a large aequorin signal and a large contraction, although a second application induced a much smaller aequorin signal accompanied by a large contraction. 6. These results suggest that high K+ and noradrenaline increase Ca2+ in at least two cytosolic compartments; a compartment that is coupled to the contractile mechanism ('contractile' Ca2+ compartment; major portion of cytoplasm containing contractile elements) and a compartment that is not coupled to contractile mechanisms ('non-contractile' Ca2+ compartment; small sub-membrane area that does not contain contractile elements). On stimulation, the Ca2+ level in the 'contractile' compartment may increase to a level high enough to stimulate myosin light chain kinase but not so high as to consume aequorin rapidly. In contrast, the Ca2+ level in the 'non-contractile' compartment may increase so greatly that aequorin in this compartment is rapidly consumed. These two compartments may be separated by a diffusion barrier and, during a resting period, aequorin may slowly diffuse from the 'contractile' compartment to the 'non-contractile' compartment and thus restore the full aequorin signal. An increase in Ca2+ in the 'non-contractile' compartment seems to be dependent mainly on Ca2+ influx and partly on Ca2+ release.

Aequorin↗
PubMed1978

Calcium transients in isolated amphibian skeletal muscle fibres: detection with aequorin.

1. Single twitch muscle fibres isolated from frogs and toads were microinjected with the Ca2+-sensitive bioluminescent protein aequorin. The fibres contracted normally and emitted flashes of light (aequorin responses) in response to stimulation for many hours thereafter. 2. No luminescence was detected from healthy fibres at rest. 3. The aequorin diffused from the site of injection at a rate consistent with a diffusion coefficient of 5 x 10(-8) cm2/sec. 4. During trains of isometric contractions there was a progressive reduction in both the amplitude and the rate of decline of the aequorin response, an observation consistent with the theory that Ca is redistributed from sites of release to sites of sequestration under such circumstances. 5. In isometric tetani light emission continued to rise long after the plateau of force had been achieved. This and the fact that the amplitude of the tetanic aequorin response increased steeply with increasing stimulus frequency suggest that in tetani the sarcoplasmic [Ca2+] may normally be above the level required to saturate the contractile apparatus. 6. Both in twitches and in tetani the amplitude of the aequorin response increased slightly and then decreased substantially as the fibre was stretched progressively beyond slack length. 7. In potassium contractures the luminescent and mechanical responses first became detectable at about the same [K+], but for equivalent force luminescence was less intense than in twitches. The aequorin response was biphasic in solutions of high [K+]. 8. Exposure of the fibre to Ca2+-free solutions had no influence on either the mechanical or the luminescent responses in twitches. In Ca2+-free solutions tetanic aequorin responses tended not to be maintained as well as normally, suggesting that intracellular Ca stores do become somewhat depleted. 9. In twitches the amplitude of the aequorin response probably reflects the amount of Ca2+ liberated into the cytoplasm rather than a [Ca2+] in equilibrium with the myofilaments. Changes in the rate of decay of the aequorin response may reflect changes in the rate of Ca sequestration by the sarcoplasmic reticulum. 10. In K+-contractures and during the plateaus of tetani the aequorin signal changes slowly enough so that it seems unlikely that substantial gradients of [Ca2+] exist at the sarcomere level. Under such circumstances the amplitude of the aequorin response probably does reflect the [Ca2+] in equilibrium with the myofilaments.

Aequorin↗
PubMed1994

The effects of theophylline on aequorin light transients and force in the isolated dog right ventricular myocardium.

Experiments were carried out to investigate the changes in intracellular Ca2+ transients associated with biphasic contractions that were elicited during interaction of theophylline with isoproterenol in the dog ventricular myocardium. For this purpose, effects of theophylline and isoproterenol on aequorin light transients and isometric contractions were assessed in the isolated canine ventricular trabeculae, superficial cells of which had been microinjected with the Ca2+ sensitive bioluminescent protein aequorin. The positive inotropic effect of theophylline (0.1-0.3 mM) was consistently associated with an increase in the amplitude of aequorin light transients. Theophylline at concentrations of 0.6 mM and higher decreased the amplitude of aequorin light transients, but the force of contraction increased further in association with a prominent prolongation of time to peak force. Theophylline (0.3 mM) enhanced the forskolin-induced increase in aequorin light transients and force. Theophylline (2 mM) inhibited the isoproterenol-induced increase in aequorin light transients associated with early phase of contraction in a reversible manner. A late phase of aequorin light transients was induced in association with late phase of contraction in the presence of both isoproterenol and theophylline. Thus, both the early and late phase of contraction were accompanied by corresponding phases of aequorin light transients. The relation between the amplitude of force and Ca2+ transients was markedly different and the late phase of contraction was associated with much lower aequorin light transients. The late phase of aequorin light transients induced by theophylline at a high concentration (10 mM) was enhanced by isoproterenol. These results indicate that theophylline (0.1-0.3 mM) increases the amplitude of Ca2+ transients through an accumulation of cyclic AMP by inhibition of the cyclic AMP phosphodiesterase activity. In concentrations of 0.6 mM and higher theophylline decreases the amplitude of the early phase aequorin light transients probably by inhibition of release of Ca2+ from the sarcoplasmic reticulum and induces simultaneously the late phase of contraction that may be associated with an increase in responsiveness to Ca2+ of myofibrils.

Aequorin↗
PubMed1974

Changes in intracellular free calcium concentration during illumination of invertebrate photoreceptors. Detection with aequorin.

Aequorin, which luminesces in the presence of calcium, was injected into photoreceptor cells of Limulus ventral eye. A bright light stimulus elicited a large increase in aequorin luminescence, the aequorin response, indicating a rise of intracellular calcium ion concentration, Ca(i). The aequorin response reached a maximum after the peak of the electrical response of the photoreceptor, decayed during a prolonged stimulus, and returned to an undetectable level in the dark. Reduction of Ca(o) reduced the amplitude of the aequorin response by a factor no greater than 3. Raising Ca(o) increased the amplitude of the aequorin response. The aequorin response became smaller when membrane voltage was clamped to successively more positive values. These results indicate that the stimulus-induced rise of Ca(i) may be due in part to a light-induced influx of Ca and in part to release of Ca from an intracellular store. Our findings are consistent with the hypothesis that a rise in Ca(i) is a step in the sequence of events underlying light-adaptation in Limulus ventral photoreceptors. Aequorin was also injected into photoreceptors of Balanus. The aequorin responses were similar to those recorded from Limulus cells in all but two ways: (a) A large sustained aequorin luminescence was measured during a prolonged stimulus, and (b) removal of extracellular calcium reduced the aequorin response to an undetectable level.

Acetates↗
PubMed1977

The effect of physiologically occurring cations upon aequorin light emission. Determination of the binding constants.

1. The effect of K+, Na+, Mg2+ and pH upon the rate of aequorin utilization has been investigated in the presence of Ca2+. 2. The aequorin light emission in a medium simulating the in vivo cationic conditions for barnacle muscle fibres indicates that two Ca2+ are apparently involved in this process for free calcium concentrations higher than approx. 10(-5) M. However, for free calcium concentrations lower than 10(-6) M, the intensity of light emitted by aequorin shows a steeper dependency upon [Ca2+] than the square low relationship, indicating that a third Ca2+ should be involved in the process of aequorin light emission, as it has been previously predicted (Moisescu, D.G., Ashley, C.C. and Campbell, A.K. (1975) Biochim. Biophys. Acta. 396, 133-140). 3. The inhibitory effect of physiologically occurring cations upon the aequorin light emission can be explained by the cooperative action of two cations, competing with Ca2+ for the reactive sites on aequorin. 4. At a given concentration, Na2+ was found to have a stronger inhibitory effect upon the aequoring light emission than K+. 5. The experiments indicate a strong interaction between Na+ and K+ in this inhibitory process, since for a given total concentration of monovalent cations, a mixture containing both Na+ and K+ has a larger inhibitory effect on the aequorin light response than solutions containing either Na+ or K+ alone. 6. All other interactions between K+, Na+, H+ and Mg2+ appear to be weak. 7. The reaction schemes used for the explanation of these and other published results on aequorin (Moisescu, D.G., Ashley, C.C. and Campbell, A.K. (1975) Biochim. Biophys, Acta 396, 133-140 and Blinks, J.R. (1973) Eur. J. Cardiol. 1, 135-142) are described, and the 'absolute' binding constants of all physiologically occurring cations for aequorin have been determined. 8. Based on these parameters one can make accurate quantitative predictions for the aequoring light response under a variety of ionic conditions, and this suggests that it is possible to determine absolute free calcium concentrations providing that the ionic composition of the solutions is known, and that the relative rate of aequorin utilization is higher than 0.005.

Aequorin↗
PubMed1999

Targeted recombinant aequorins: tools for monitoring [Ca2+] in the various compartments of a living cell.

In the last decade, the study of Ca2+ homeostasis within organelles in living cells has been greatly enhanced by the utilisation of a recombinant Ca(2+)-sensitive photoprotein, aequorin. Aequorin is a Ca2+ sensitive photoprotein of a coelenterate that, in the past, was widely employed to measure Ca2+ concentration in living cells. In fact, the purified protein was widely used to monitor cytoplasmic [Ca2+] changes in invertebrate muscle cells after microinjection. However, due to the time-consuming and traumatic procedure of microinjection, the role of aequorin in the study of Ca2+ homeostasis remained confined to a limited number of cells (giant cells) susceptible to microinjection. Thus, in most instances, it was replaced by the fluorescent indicators developed by Roger Tsien and coworkers. The cloning of aequorin cDNA [Inouye et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:3154-3158] and the explosive development of molecular biology offered new possibilities in the use of aequorin, as microinjection has been replaced by the simpler technique of cDNA transfection. As a polypeptide, aequorin allows the endogenous production of the photoprotein in cell systems as diverse as bacteria, yeast, slime molds, plants, and mammalian cells. Moreover, it is possible to specifically localise it within the cell by including defined targeting signals in the amino acid sequence. Targeted recombinant aequorins represent to date the most specific means of monitoring [Ca2+] in subcellular organelles. In this review, we will not discuss the procedure of aequorin microinjection and its use as purified protein but we will present the new advances provided by recombinant aequorin in the study of intracellular Ca2+ homeostasis, discussing in greater detail the advantages and disadvantages in the use of this probe.

Aequorin↗
PubMed1985

Modification by dantrolene, procaine and suxamethonium of caffeine-induced changes in aequorin luminescence transients and twitch tensions of directly-stimulated diaphragm muscle of mouse.

A convenient method is described for measuring simultaneously Ca2+-related aequorin luminescence and twitch tension in the isolated diaphragm muscle of the mouse. Forty to fifty fibres were injected intracellularly with aequorin solution and the mechanical and luminescence responses to direct stimulation were recorded. The replacement of Na+ by K+ (to obtain 59 or 143.4 mM K+) in the nutrient solution decreased both aequorin luminescence and twitch tensions, but after a time lag, it produced a contracture. Caffeine (5 or 10 mM) increased both aequorin luminescence and twitch tensions, and after a time lag, it also produced a contracture. Dantrolene (1 and 30 microM) and procaine (10 microM, 300 microM and 1 mM) decreased aequorin luminescence transients and twitch tension. In addition procaine inhibited the caffeine-induced increase of aequorin luminescence, but dantrolene did not have this effect. At concentrations causing neuromuscular block, suxamethonium (130 microM) decreased aequorin luminescence transients and twitch tension. By contrast, (+)-tubocurarine (6.5 microM) did not affect the aequorin luminescence in directly stimulated muscles. These results suggest that Ca+-related aequorin luminescence transients accompanied by twitch tensions reflect the intracellular fast mobilization of compartmentalized Ca2+ from plasma membrane or sarcoplasmic reticulum, and that the increase in resting luminescence caused by a K+- or caffeine-induced contracture may be produced by the slow mobilization of Ca2+ from sarcoplasmic reticulum.

Aequorin↗
PubMed1996

Generation of cell transfectants expressing cardiac calcium ion channel and calcium indicator protein aequorin.

Chinese hamster ovary (CHO) cells stably coexpressing cardiac calcium ion channel [L-type calcium channel or ryanodine receptor (RyR)] and the calcium-sensitive bioluminescent protein aequorin were generated by transfecting aequorin cDNA. In a selected clone, C1-17, carrying the L-type calcium channel, depolarization induced by high concentration of K+ produces aequorin luminescence. In another clone, R3-7, carrying RyR, caffeine produces aequorin luminescence. In the presence of selective calcium ion channel blockers, the aequorin luminescence was inhibited in a dose-dependent manner. These results indicate that functionally expressed calcium ion channels in these transformants can be monitored through the activation of endogenous aequorin luminescence following a physiological signal similar to that of native calcium channel. Moreover, the aequorin system compared very well with Fura-2 measurements. Thus, the recombinant cell models, which expressed cloned calcium channel and aequorin, will contribute to the elucidation of Ca2+ movement through the cell surface and intracellular calcium ion channels.

Aequorin↗