Aequorin measurements of free calcium in single heart cells

Cobbold, P H; Bourne, P.K.

doi:10.1038/312444a0

Cited by 144 publications

(72 citation statements)

References 17 publications

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“…Because the speed of aequorin consumption is dependent on [Ca 2ϩ ], [Ca 2ϩ ] i can be estimated by a rate constant that is represented as a ratio of luminescence intensity above the background (photon count/s) to the product of the amount of remaining chromophores multiplied by the quantum yield (it should be equal to the total photon count after subtraction of background counts) (55,67,68,429).…”

Section: Aequorinmentioning

confidence: 99%

Measurement of Intracellular Calcium

Takahashi

Camacho

Lechleiter

et al. 1999

Physiological Reviews

674

511

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To a certain extent, all cellular, physiological, and pathological phenomena that occur in cells are accompanied by ionic changes. The development of techniques allowing the measurement of such ion activities has contributed substantially to our understanding of normal and abnormal cellular function. Digital video microscopy, confocal laser scanning microscopy, and more recently multiphoton microscopy have allowed the precise spatial analysis of intracellular ion activity at the subcellular level in addition to measurement of its concentration. It is well known that Ca2+ regulates numerous physiological cellular phenomena as a second messenger as well as triggering pathological events such as cell injury and death. A number of methods have been developed to measure intracellular Ca2+. In this review, we summarize the advantages and pitfalls of a variety of Ca2+ indicators used in both optical and nonoptical techniques employed for measuring intracellular Ca2+ concentration.

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Section: Aequorinmentioning

confidence: 99%

Measurement of Intracellular Calcium

Takahashi

Camacho

Lechleiter

et al. 1999

Physiological Reviews

674

511

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“…Similar studies on the mammalian heart, however, show that ICa contributes at best between 5 and 30o to the activation of contraction, depending on experimental conditions (8). The release of Ca24 from the intracellular compartments has been measured in intact cardiac muscle (9,10), in isolated single cells (11)(12)(13)(14), and in skinned cells (15)(16)(17) with aequorine, Ca24-sensitive dyes, and intrinsic birefringence signal. The similarity in the voltage dependence of tension and ICa suggest that ICa may gate the release of intracellular pools by a Ca2+-induced Ca2+ release mechanism.…”

mentioning

confidence: 91%

Epinephrine enhances Ca2+ current-regulated Ca2+ release and Ca2+ reuptake in rat ventricular myocytes.

Callewaert

Cleemann

Morad

1988

Proc. Natl. Acad. Sci. U.S.A.

125

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The voltage dependence of the intracellular Ca2 + transients was measured in single rat ventricular myocytes with the fluorescent Ca24 indicator dye fura-2. The whole-cell voltage damp technique was used to measure the membrane current, and 0.9 mM fura-2 was loaded into the cell by including it in the dialyzing solution of the patch electrode. A mechanical light chopper operating at 1200 Hz was used to obtain simultaneous measurements of the intracellular Ca2+ activity with fluorescence excitation on either side of the isosbestic point (330 am and 410 nm). The symmetry of the two optical Ca24 signals was used as a criterion to guard against artifacts resulting, for instance, from motion. The voltage dependence of peak Ca2+ current and the Ca24 transient measured 25 ms after depolarizing clamps from a holding potential of -40 mV were bell-shaped and virtually identical. The Ca2+ entry estimated from the integral of the Ca2+ current (0 mV, 25 ms) corresponds to a 5-10 pM increase in the total intracellular Ca24 concentration, whereas the optical signal indicated a 100 FM increase in total intracellular Ca24. Repolarization of clamp pulses from highly positive potentials were accompanied by a second Ca2+ transient, the magnitude of which, when summed with that measured during depolarization, was nearly constant. Ryanodine (10 #M) had little or no effect on the peak Ca2+ current but reduced the magnitude of the early Ca2+ transients by 70-90%. Epinephrine (1 FM) increased the Ca2+ current and the Ca2+ transients, accelerated the rate of decline of the Ca2+ transients at potentials between -30 and + 70 mV, and reduced the intracellular [Ca2+] below baseline at potentials positive to + 80 or negative to -40 mV, where clamp pulses did not elicit any Ca2+ release. Elevation of intracellular cAMP mimicked the relaxant effect of epinephrine at depolarizing potentials, whereas elevation of extracellular [Ca2+] did not. These results suggest that most of the activator Ca2+ in rat ventricular cells is released from the sarcoplasmic reticulum as a graded response to sarcolemmal Ca2+ influx. Consistent with a graded Ca2+-induced Ca2+ release we find that epinephrine increases the internal Ca2+ release by increasing the Ca2+ current. Epinephrine may also increase the Ca2+ content of the sarcoplasmic reticulum that may, in turn,

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“…anoxia, inhibition of oxidative phosphorylation and metabolic blockade (prevention of both oxidative phosphorylation and anaerobic glycolysis) (Dahl & Isenberg, 1980;Allen & Orchard, 1983b;Cobbold & Bourne, 1984). However, in these experimental models perfusate flow continues so that the consequences of accumulation of ions and products of metabolism, such as K+, CO2 and lactate, are not usually observed.…”

Section: Introductionmentioning

confidence: 99%

The consequences of simulated ischaemia on intracellular Ca2+ and tension in isolated ferret ventricular muscle.

Allen

Lee

Smith

1989

The Journal of Physiology

104

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SUMMARY1. In order to study cellular events occurring in ischaemia, we have developed a method for simulating ischaemia in an isolated papillary muscle. Muscles were suspended in a chamber and changed from conventional superfusion with Tyrode solution to gas perfusion with 95% N2/5 % CO2 (N2 gas perfusion), thus simultaneously stopping oxygenation and flow. Surface cells of the preparation were injected with the photoprotein aequorin in order to monitor intracellular free calcium2. Gas perfusion with 95% 02/5% CO2 (02 gas perfusion) had little effect on the tension or Ca2+ transients. Superfusion with Tyrode solution equilibrated with 95% N2/5% CO2 (N2 Tyrode) caused tension to decline to 30-40% of control, but had little effect on the amplitude of the Ca2+ transients. N2 gas perfusion caused tension to fall more rapidly and to a lower level than superfusion with N2 Tyrode. Ca2+ transients showed a small initial decline followed by a slowly developing increase in magnitude and duration.3. Long exposures to N2 gas perfusion caused tension to decline to very low levels and Ca2+ transients to increase to a maximum. After a variable length of time, resting tension began to increase. At approximately the same time, Ca2+ transients began to decrease and eventually disappeared. Resting Ca2+ increased during N2 gas perfusion and remained elevated when the Ca2+ transients had declined. These changes could be reversed by restarting superfusion with standard Tyrode or by perfusion with 02 gas.4. N2 gas perfusion caused a depolarization of the resting potential and an abbreviation of the action potential. In a long exposure the action potential eventually failed. These changes could be reversed by restarting superfusion with standard Tyrode or by perfusion with 02 gas.5. Many of the effects of N2 gas perfusion could be mimicked by the addition of 20 mM-lactic acid to the superfusing solution, which caused a profound reduction of tension and also an increase in the amplitude and duration of the Ca21 transients. Calculation of the changes in intracellular pH caused by the addition of lactic acid suggest that the fall in intracellular pH produced by lactic acid was similar to that occurring in ischaemia. D. G. ALLEN, J. A. LEE AND G. L. SMITH6. Repeated exposures to N2 gas perfusion caused tension to fall more rapidly and an increased resting tension to develop more rapidly. The slowly developing rise in Ca2+ transients was abolished and a rise in resting Ca2+ occurred more quickly.7. When muscles were quiescent, exposure to N2 gas perfusion caused an increase in resting light. Low-frequency, spontaneous oscillations of [Ca2+]i appeared which increased in magnitude and frequency with increasing duration of exposure. Such oscillations were suppressed by stimulation during exposure to N2 gas perfusion but appeared when stimulation was stopped. 8. Addition of 20 mM-lactic acid to the solution superfusing a quiescent muscle caused an increased resting Ca2+. Large, low-frequency oscillations of [Ca2+]i appeared, which increased in ...

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Aequorin measurements of free calcium in single heart cells

Cited by 144 publications

References 17 publications

Measurement of Intracellular Calcium

Measurement of Intracellular Calcium

Epinephrine enhances Ca2+ current-regulated Ca2+ release and Ca2+ reuptake in rat ventricular myocytes.

The consequences of simulated ischaemia on intracellular Ca2+ and tension in isolated ferret ventricular muscle.

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