Kinetic analysis of calcium desaturation curves from isolated kidney cells

Uchikawa, T.; Borle, A. B.

doi:10.1152/ajpregu.1978.234.1.r29

Cited by 12 publications

(19 citation statements)

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“…Previous studies of 45Ca2+ ef flux rates in other cells have shown that the first Ca2+ compartment represents Ca2+ bound to the outer plasma membrane [25][26][27]30] which in the case of Caco-2 cells would also include the glycocalyx of the api cal microvilli. The second and third Ca2+ compartments usually represent cytoplasmic and endoplasmic reticulum/mitochondrial stores, respectively [25][26][27]30]. The addi tion of higher amounts of ionomycin cou pled with the loss of the second and third exponential Ca2+ compartments suggests that ionomycin was having a progressive perturbation on intracellular Ca2+ stores.…”

Section: Discussionmentioning

confidence: 99%

“…All calculations of 45Ca2t efflux were taken from the mathematical formulations of Uchikawa and Borle [25] and Borle [26]. The total initial radioactiv ity (45Ca'*) was obtained by adding the sum of all the radioactivity at each time point plus the radioactivity left in the cells at the end of the experiment.…”

Section: Caeffluxmentioning

confidence: 99%

“…The parameters B|, B:, B3, p i, P2, and p3 were determined by means of an iterative Marquardt-Levenberg algorithm [25][26][27] run on an IBM PS/2-60 computer using the nonlinear least-squares method for exponential components [25,26]. The criteria for accepting the appropriate curve-fitting equation were: (1) if addition of an extra exponential did not significantly decrease the mean square error, and (2) the plotted equation should fit most of the experimental points.…”

Section: Caeffluxmentioning

confidence: 99%

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Physiological and Cytotoxic Effects of Ca2+ Ionophores on Caco-2 Paracellular Permeability: Relationship of 45Ca2+ Efflux to 51Cr Release

Rutten¹,

Cogburn²,

Schasteen³

et al. 1991

Pharmacology

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The human intestinal cell line, Caco-2, and the Ca²⁺ ionophores, A23187 and ionomycin, were used to determine the interrelationships of ⁴⁵Ca²⁺ efflux, transepithelial electrical resistance (R_t), and [³H]-mannitol flux to changes in ⁵¹Cr release and lactate dehydrogenase (LDH) activity. Treatment of Caco-2 monolayers with ionomycin at concentrations of between 0.25 and 2.50 μmol/l showed similar ⁴⁵Ca²⁺ efflux rate constants and coefficients. Analysis of the control and ionomycin-induced ⁴⁵Ca²⁺ efflux values showed the data to best fit a three Ca²⁺ compartmental model. All changes in Caco-2 Rt and [³H]-mannitol flux were reversible with no significant increases in ⁵¹Cr release with ionomycin concentrations of ≤2.5μmol/l. Caco-2 monolayers treated with ionomycin at concentrations of between 5.0 and 50.0 μmol/l showed rapid non-exponential ⁴⁵Ca²⁺ effluxes with irreversible changes in R_t, [³H]-mannitol flux, and significant increases in ⁵¹Cr release. There was no change in media LDH activity using either ionomycin or A23187 at concentrations of up to 50 μmol/l for 60 min. The results of our study show that: (1) disruption of Ca²⁺ homeostasis in Caco-2 cells will occur with the addition of Ca²⁺ ionophores at concentrations of >2.50 μmol/l; (2) high concentrations (>2.5 μmol/l) of ionomycin will cause non-exponential ⁴⁵Ca²⁺ efflux rates with irreversible changes in transcellular R_t and ¹⁴C-mannitol flux, and (3) early signs of Ca²⁺ ionophore-induced damage can be detected by 51Cr release from Caco-2 cells into the media and not by changes in LDH media activity.

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Section: Discussionmentioning

confidence: 99%

Section: Caeffluxmentioning

confidence: 99%

Section: Caeffluxmentioning

confidence: 99%

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Physiological and Cytotoxic Effects of Ca2+ Ionophores on Caco-2 Paracellular Permeability: Relationship of 45Ca2+ Efflux to 51Cr Release

Rutten¹,

Cogburn²,

Schasteen³

et al. 1991

Pharmacology

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“…The calculation of the rate constants (kij) and the pool sizes (&J were done according to the equations derived by Uchikawa and Borle (1978). The amount of Ca++ in each pool was calculated from the specific activity of the medium, the number of viable cells in the plate, the value of C,, and the fraction of total cell radioactivity calculated to be located in the specific pool (S(,-3J.…”

Section: Calculationsmentioning

confidence: 99%

“…assumptions and equations of Uchikawa and Borle (1978). The cell Ca++ system was represented by the following scheme:…”

Section: Efflux Studiesmentioning

confidence: 99%

Calcium transport and cellular distribution in quiescent and serum‐stimulated primary cultures of bone cells and skin fibroblasts

Eilam

Szydel

1981

Journal Cellular Physiology

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Primary cultures of bone cells and skin fibroblasts were examined for their Ca++ content, intracellular distribution and Ca++ fluxes. Kinetic analysis of 45Ca++ efflux curves indicated the presence of three exchangeable Ca++ compartments which turned over at different rates: a "very fast turnover" (S1), a "fast turnover" (S2, and a "slow turnover" Ca++ pool (S3). S1 was taken to represent extracellular membrane-bound Ca++, S2 represented cytosolic Ca++, and S3 was taken to represent Ca++ sequestered in some intracellular organelles, probably the mitochondria. Bone cells contained about twice the amount of Ca++ as compared with cultured fibroblasts. Most of this extra Ca++ was localized in the "slow turnover" intracellular Ca++ pool (S3). Serum activation caused the following changes in the amount, distribution, and fluxes of Ca++: (1) In both types of cells serum caused an increase in the amount of Ca++ in the "very fast turnover" Ca++ pool, and an increase in the rate constant of 45Ca++ efflux from this pool, indicating a decrease in the strength of Ca++ binding to ligands on cell membranes. (2) In fibroblasts, serum activation also caused a marked decrease in the content of Ca++ in the "slow turnover" Ca++ pool (S3), an increase in the rates of Ca++ efflux from the cells to the medium, and from S3 to S2, as well as a decrease in the rate of influx into S3. (3) In bone cells the amount of Ca++ in S3 remained high in "serum activated" cells, the rate of efflux from S3 to S2 increased, and the rate of influx into S3 also increased. The rate of efflux from the cells to the medium did not change. The results suggest specific properties of bone cells with regard to cell Ca++ presumably connected with their differentiation. Following serum activation we investigated the time course of changes in the amount of exchangeable Ca++ in bone cells and fibroblasts, in parallel with measurements of 3H-thymidine incorporation and cell numbers. Serum activation caused a rapid decrease in the content of cell Ca++ which was followed by a biphasic increase lasting until cell division.

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Efflux of ⁴⁵CA²⁺ from human fibroblasts in response to serum or growth factors

Owen

Villereal

1983

Journal Cellular Physiology

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Previous studies have indicated that intracellular Ca2+ is involved in fetal bovine serum (FBS)- or growth factor (GF)-stimulated Na+ influx in human foreskin fibroblasts (HSWP). In the present study, 45Ca2+ efflux from serum-deprived HSWP cells was measured in response to 10% FBS or GF [lys-bradykinin, vasopressin, epidermal growth factor, and insulin]. Efflux data were analyzed using a computer program and the best fit indicated the presence of three Ca2+ compartments: a compartment (C1) with a very fast turnover rate, one (C2) with a fast turnover rate, and one (C3) with a slow turnover rate. When serum-deprived cells were treated with 10% FBS, efflux from C2 and C3 increased significantly (p less than 0.05). Similar effects on efflux were observed when serum-deprived cells were treated with individual GFs. Combination of the four GFs produced a higher stimulation than any single factor and a response that was equal to that of FBS. On the other hand, when cells were serum-treated in the presence of the intracellular Ca2+ antagonist, B-(N-N,diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), 45Ca2+ efflux from C2 was substantially reduced. Finally, when cells were treated with the Na+ transport inhibitor amiloride, there was no significant effect on serum-stimulated Ca2+ efflux. These results are consistent with a FBS- or GF-induced mobilization of Ca2+ that can be blocked by intracellular Ca2+ antagonists, and support the hypothesis that the action of these agents on Na+ influx may be via their effects on intracellular Ca2+.

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Kinetic analysis of calcium desaturation curves from isolated kidney cells

Cited by 12 publications

References 0 publications

Physiological and Cytotoxic Effects of Ca<sup>2+</sup> Ionophores on Caco-2 Paracellular Permeability: Relationship of 45Ca<sup>2+</sup> Efflux to <sup>51</sup>Cr Release

Physiological and Cytotoxic Effects of Ca<sup>2+</sup> Ionophores on Caco-2 Paracellular Permeability: Relationship of 45Ca<sup>2+</sup> Efflux to <sup>51</sup>Cr Release

Calcium transport and cellular distribution in quiescent and serum‐stimulated primary cultures of bone cells and skin fibroblasts

Efflux of ⁴⁵CA²⁺ from human fibroblasts in response to serum or growth factors

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Kinetic analysis of calcium desaturation curves from isolated kidney cells

Cited by 12 publications

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Physiological and Cytotoxic Effects of Ca<sup>2+</sup> Ionophores on Caco-2 Paracellular Permeability: Relationship of 45Ca<sup>2+</sup> Efflux to <sup>51</sup>Cr Release

Physiological and Cytotoxic Effects of Ca<sup>2+</sup> Ionophores on Caco-2 Paracellular Permeability: Relationship of 45Ca<sup>2+</sup> Efflux to <sup>51</sup>Cr Release

Calcium transport and cellular distribution in quiescent and serum‐stimulated primary cultures of bone cells and skin fibroblasts

Efflux of 45CA2+ from human fibroblasts in response to serum or growth factors

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Efflux of ⁴⁵CA²⁺ from human fibroblasts in response to serum or growth factors