Reassessment of fura-2 and the ratio method for determination of intracellular Ca2+ concentrations

Uto, Akira; Arai, Hajime; Ogawa, Yasuo

doi:10.1016/0143-4160(91)90082-p

Cited by 116 publications

(71 citation statements)

References 12 publications

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“…A number of studies on many Ca 2+ indicators have shown that the indicator properties are likely to be altered in the cytoplasm, probably by binding to cellular macromolecules: fura-2 (Konishi et al, 1988;Uto, Arai, andOgawa, 1991), indo-1 (Hove-Madsen andBers, 1992;Westerblad and Allen, 1993;Baker et al, 1994), fluo-3 , fura red , and aequorin (Blatter and Blinks, 1991). The present results suggest that fura-2 conjugated to dextran can still bind to cellular proteins.…”

Section: Novel Calibration Methods Of the Intracellular Indicator Withmentioning

confidence: 56%

Resting cytoplasmic free Ca2+ concentration in frog skeletal muscle measured with fura-2 conjugated to high molecular weight dextran.

Konishi

Watanabe

1995

The Journal of General Physiology

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Intact frog skeletal muscle fibers were injected with the Ca 2+ indicator fura-2 conjugated to high molecular weight dextran (fura dextran, MW ,,d0,000; dissociation constant for Ca 2+, 0.52 p,M), and the fluorescence was measured from cytoplasm (17~ The fluorescence excitation spectrum of fura dextran measured in resting fibers was slighdy red-shifted compared with the spectrum of the Ca2+-free indicator in buffer solutions. A simple comparison of the spectra in the cytoplasm and the in vitro solutions indicates an apparently "negative" cytoplasmic [Ca2+], which probably reflects an alteration of the indicator properties in the cytoplasm. To calibrate the indicator's fluorescence signal in terms of cytoplasmic [Ca2+], we applied [3-escin to permeabilize the cell membrane of the fibers injected with fura dextran. After treatment with 5 ~M fl-escin for 30-35 min, the cell membrane was permeable to small molecules (e.g., Ca 2+, ATP), whereas the 10-kD fura dextran only slowly leaked out of the fiber. It was thus possible to estimate calibration parameters in the indicator fluorescence in the fibers by changing the bathing solution [Ca 2+] to various levels; the average values for the fraction of Ca2+-bound indicator in the resting fibers and the dissociation constant for Ca 2+ (KD) were, respectively, 0.052 and 1.0 I~M. For the comparison, the KD value was also estimated by a kinetic analysis of the indicator fluorescence change after an action potential stimulation in intact muscle fibers, and the average value was 2.5 wM. From these values estimated in the fibers, resting cytoplasmic [Ca 2+ ] in frog skeletal muscle fibers was calculated to be 0.06-0.14 ~M. The range lies between the high estimates from other tetracarboxylate indicators (0.1-0.3 ~M; Kurebayashi, N., A.

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Section: Novel Calibration Methods Of the Intracellular Indicator Withmentioning

confidence: 56%

Resting cytoplasmic free Ca2+ concentration in frog skeletal muscle measured with fura-2 conjugated to high molecular weight dextran.

Konishi

Watanabe

1995

The Journal of General Physiology

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“…Based on an increase in the f 340 /f 380 ratio, osmotic swelling was claimed to initially increase the Ca 2ϩ transient in the rat ventricle (4). Reduction in ionic strength decreases the dissociation constant for Ca 2ϩ (37,41), however, and a reduction of viscosity preferentially suppresses fluorescence at longer wavelengths (28,30). Both of these effects augment the f 340 /f 380 ratio, suggesting an increase in Ca 2ϩ when none has occurred.…”

Section: Discussionmentioning

confidence: 99%

Biphasic effects of cell volume on excitation-contraction coupling in rabbit ventricular myocytes

Zhang

Satin

et al. 2002

American Journal of Physiology-Heart and Circulatory Physiology

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.-We studied the effects of osmotic swelling on the components of excitation-contraction coupling in ventricular myocytes. Myocyte volume rapidly increased 30% in hyposmotic (0.6T) solution and was constant thereafter. Cell shortening transiently increased 31% after 4 min in 0.6T but then decreased to 68% of control after 20 min. In parallel, the L-type Ca 2ϩ current (ICa-L) transiently increased 10% and then declined to 70% of control. Similar biphasic effects on shortening were observed under current clamp. In contrast, action potential duration was unchanged at 4 min but decreased to 72% of control after 20 min. Ca 2ϩ transients were measured with fura 2-AM. The emission ratio with excitation at 340 and 380 nm (f 340/f380) decreased by 12% after 3 min in 0.6T, whereas shortening and I Ca-L increased at the same time. After 8 min, shortening, I Ca-L, and the f340/f380 ratio decreased 28, 25, and 59%, respectively. The results suggest that osmotic swelling causes biphasic changes in I Ca-L that contribute to its biphasic effects on contraction. In addition, swelling initially appears to reduce the Ca 2ϩ transient initiated by a given ICa-L, and later, both I Ca-L and the Ca 2ϩ transient are inhibited.osmolarity; action potential duration; calcium current; calcium transient; cell shortening SWELLING OF CARDIAC MYOCYTES is a prominent aspect of the response to ischemia-reperfusion and elective cardioplegia and also may arise in renal insufficiency and syndromes with inappropriate secretion of antidiuretic hormone. Altered myocyte hydration has important functional consequences. Osmotic perturbations modulate both electrical activity (for reviews, see 34,38,42) and the ability of cardiac muscle to contract (5,16,17). Although facets of the contractile response to osmotic swelling are known from studies on multicellular preparations, little mechanistic information is available at the single cell level to explain the implications of myocyte swelling for excitation-contraction (E-C) coupling. Moreover, the effect of swelling on L-type Ca 2ϩ current (I Ca-L ), a critical aspect of E-C coupling, remains controversial. Under ruptured patch voltage-clamp conditions, for example, swelling is reported to enhance I Ca-L in rabbit atrial and sinoatrial node cells (22), depress I Ca-L in rat ventricular cells (4), and to have no significant effect in guinea pig (13, 31) and canine (44) ventricular cells. A swelling-induced enhancement of I Ca-L also has been claimed based on fura 2 measurements of diltiazem-sensitive Ca 2ϩ influx (36). It is unclear whether these differences in the response of I Ca-L to myocyte swelling arise from species differences or from methodological issues, such as the composition of the patch pipette solution, the effectiveness of cell dialysis, or the variable extent of myocyte swelling under ruptured-patch conditions (6, 33). Several other species differences in E-C coupling, including the sensitivity of contraction to inhibition of the sarcoplasmic reticulum (SR), are well known (2).The present stu...

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“…Therefore, the PAF-induced increase in Finally, it should be mentioned that the [Ca 2+]i in resting human endothelial cells cali brated in the present study (34.5 ± 3.9 nM, n = 16) is less than half of the level reported by other workers (around 100 nM) in these cells loaded with fura-2 or quin-2 (25,(27)(28)(29). However, it should be noted that the value of [Ca 2+]i derived from the equation of Grynk iewicz et al (12) is not absolute, because it has been reported that the Kd value is variable according to the ionic strength, pH, magne sium concentration, and the concentration of cytosolic proteins (30,31). Moreover the vis cosity affects the fluorescent intensity (31,32).…”

Section: Discussionmentioning

confidence: 99%

Antagonism of Platelet-Activating Factor-Induced Increase in Cytosolic Free Calcium Concentration in Human Endothelial Cells.

Hirafuji

Satoh

1992

Jpn.J.Pharmacol

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ABSTRACT-The effects of platelet-activating factor (PAF) antagonists on the agonist-induced increase in cytosolic free calcium concentration, [Ca 2+]i, in human vascular endothelial cells grown in monolayer were investigated by a continuous su perfusion technique using a calcium fluorescent probe, fura-2. PAF caused a small but dose-dependent increase in [Ca 2+];. Seven structurally dissimilar PAF antagonists dose-dependently suppressed the peak response, among which WEB 2086 was the most potent, followed by WEB 2170 > FR 900452 ONO 6240 > BN 52021 kad surenone CV 3988. These antagonists except for CV 3988 were specific for PAF, since they had no effects on calcium mobilization induced by thrombin or histamine, while CV 3988 had a non-specific effect. PAF in the same range of concentration in creased prostacyclin release from human endothelial cells. WEB 2086 also inhibited the PAF-induced prostacyclin release, while it had no effect on the release induced by histamine and thrombin. These results demonstrate the specificity and dose-response characteristics of PAF antagonists in cultured human endothelial cells and suggest that a PAF antagonist could be a valuable therapeutic agent in certain human diseases where PAF activation of endothelial cells may have a critical role.

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Reassessment of fura-2 and the ratio method for determination of intracellular Ca2+ concentrations

Cited by 116 publications

References 12 publications

Resting cytoplasmic free Ca2+ concentration in frog skeletal muscle measured with fura-2 conjugated to high molecular weight dextran.

Resting cytoplasmic free Ca2+ concentration in frog skeletal muscle measured with fura-2 conjugated to high molecular weight dextran.

Biphasic effects of cell volume on excitation-contraction coupling in rabbit ventricular myocytes

Antagonism of Platelet-Activating Factor-Induced Increase in Cytosolic Free Calcium Concentration in Human Endothelial Cells.

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