JP3348039B2 - Method for measuring intracellular calcium ion concentration - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring intracellular calcium ion concentration, and more particularly, to a method for measuring a change in calcium ion concentration in neutrophil cells.
The present invention also relates to a method for screening a neutrophil cell superoxide production inhibitor.

[0002]

2. Description of the Related Art It is known that superoxide produced by human neutrophils plays an important role in biological defense by killing bacteria, viruses and the like that have invaded the human body (JM McCord & R.S.
Roy Can. J. Physiol. P
Pharmacol. vol. 60: 1346-1
352 (1982) pathophysiolo
gy of superoxide: role i
n inflammation and ischem
ia, E.I. L. Thomas, R.A. I. Le
hrer & R. F. Rest. Rev ..
Infect. Dis. vol. 10: S4
50-S456 (1988) neurophil
antimicrobial activity).
Moreover, the O ₂ ^- in the production mechanism, the concentration of calcium ions in the neutrophil cells are also known to play an important role (T. Pozzan, D. P. Le
w, C.I. B. Wollheim & R.A. Y.
Tsien Science vol. 221:
1413-1415 (1983) cytosol
ic ionized calcium regula
ting neutrophil activatio
n, P. H. Kazanjian & J.M. E. FIG.
Pennington J. et al. Infect. Di
s. vol. 151: 15-22 (198
5) of drugs that block ca
lcium channels on the mic
robotical function of hum
an neutrophils). For example, neutrophils in patients with chronic granulomatosis that lack superoxide-producing function
Formylmethionylleucylphenylalanine (for
myl-methionyl-leucyl-phen
ylalanine, hereinafter referred to as fMLP. A neutrophil chemotactic peptide that specifically binds to the fMLP receptor on the surface of neutrophil cells and transmits a stimulus that increases intracellular calcium concentration. From this calcium response further stimuli are transmitted intracellularly towards the expression of several cellular mechanisms, one of which is superoxide production. ), No depolarization of the cell membrane was observed, so calcium ions in the extracellular fluid could not be mobilized into the cells, and it was thought that a sufficient intracellular calcium ion concentration for superoxide production could not be obtained. (Cohen, H .;
J. , Newburger, P .; E. FIG. , Chov
aniec, M .; E. FIG. , Whitin, J. et al. C.
& Simons, E.A. R. Blood vol.
58, No. 5, 975-982 (198
1) zymosan-stimulated gran
ulocytes-activation and ac
activity of the superoxide-
generating system and mem
brane potentialchanges, L
azzari, K .; G. FIG. Proto, P .; &
Simons, E.C. R. J. Biol. Che
m. vol. 265, No. 19, 10959-
10967 (1990) hyperpolariza
Tion in response to a che
motopeptide, Ahlin,
A. , Gyllenhammar, H .; , Ring
ertz, B .; & Palmblad, J. et al. .
Lab. Clin. Med. vol. 125, N
o. 3, 392-401 (1995) membr
an potentialal changes and
homotypic aggregation kin
etics are pH-dependent: S
tumors of chronic granulo
matose disease).

[0003] While there are a plurality of pathways that cause such a change in intracellular calcium ion concentration, and it is known that they constitute a change in intracellular calcium ion concentration in the whole cell, the role of each pathway in the expression of cell functions has been clarified. Therefore, there is a problem that the method of decomposing the change in intracellular calcium ion concentration and identifying individual pathways is not sufficient.

[0004]

SUMMARY OF THE INVENTION The present invention provides a method for identifying three pathways that cause a change in calcium ion concentration in neutrophil cells, and for individually measuring the calcium ion concentration change in each pathway. The purpose is to do. It is another object of the present invention to provide a method for screening for an inhibitor that inhibits neutrophil cell superoxide production using such a method.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, a novel method for measuring intracellular calcium ion concentration, which causes a change in intracellular calcium ion concentration in cell function expression. The present inventors have found a method capable of identifying a plurality of pathways and quantitatively measuring a change in intracellular calcium ion concentration in each pathway, and completed the present invention.

More specifically, the method for measuring the intracellular calcium ion concentration according to the present invention is a method for measuring the intracellular calcium ion concentration, wherein neutrophil cells treated with an indicator for detecting calcium and neutrophil cells are detected. To prepare a mixed solution, and (1) a calcium ion remover is present in the mixed solution, a neutrophil chemotactic peptide is added to the mixed solution, and calcium ions in the cells are removed. Measuring the concentration and the concentration of the superoxide to obtain the concentration of calcium ions from the intracellular calcium store; (2) allowing the intracellular calcium ion channel inhibitor to be present in the mixed solution; Neutrophil-migratory peptide was added, and the concentration of calcium ions in the cells and the concentration of the superoxide were measured. And (3) adding calcium ions to the mixed solution, adding a calcium concentration increasing stimulant to the mixed solution to obtain a concentration of calcium ions in the cells, The intracellular superoxide concentration is measured, and the calcium ion concentration from the intracellular calcium store obtained in (1) above and (2)
And reducing the concentration of calcium ions from the extracellular calcium obtained in step (a) to obtain the concentration of extracellular calcium ions by SOCI.

Further, in the method for measuring the intracellular calcium ion concentration according to the present invention, the indicator for detecting calcium is a fluorescent indicator, and the indicator for detecting superoxide is a chemiluminescent indicator. .

[0008] Further, in the method for measuring the intracellular calcium ion concentration according to the present invention, the indicator for detecting calcium preferably comprises:
1- [2-amino-5- (2,7-dichloro-6-hydroxy-3-oxy-9-xantenyl) phenoxy]-
2- (2-amino-5-methylphenoxy) ethane-
N, N, N ', N'-tetraacetic acid (1- [2-Amino-
5- (2,7-dichloro-6-hydroxy)
-3-oxy-9-xanthenyl) phenox
y] -2- (2-amino-5-methylphenyl
noxy) ethane-N, N, N ', N'-tet
raaceticacid, hereinafter referred to as fluo-3)
And the indicator for superoxide detection,
2-methyl-6-phenyl-3,7-dihydroimidazo [1,2-a] pyrazin-3-one (2-methyl
-6-phenyl-3,7-dihydroimid
azo [1,2-a] pyrazin-3-one, hereinafter referred to as CLA).

Further, in the method for measuring the intracellular calcium ion concentration according to the present invention, the calcium concentration-increasing stimulant is formylmethionyl leucylphenylalanine (fo).
rmyl-methionyl-leucyl-phe
Nylalanine, hereinafter referred to as fMLP)
The intracellular calcium ion channel inhibitor comprises 8-
(Diethylamino) octyl 3,4,5-trimethoxybenzoate hydrochloride (8-Diethyl
lamino) octyl 3,4,5-trimet
hoxybenzoate hydrochrolli
de, hereinafter referred to as TMB-8).

Further, the method for screening a neutrophil cell superoxide production inhibitor according to the present invention is directed to a method for inhibiting neutrophil cell superoxide production against a change in calcium ion concentration in neutrophil cells by a calcium ion concentration increasing stimulant. A method for screening said inhibitor based on the inhibitory activity of an agent, wherein the calcium ion concentration change is prepared by mixing a neutrophil cell treated with a calcium detection indicator and a superoxide detection indicator. (1) A calcium ion remover is present in the mixture, and a calcium concentration increasing stimulant is added to the mixture to measure the calcium ion concentration in the cells and the superoxide concentration. A change in the concentration of calcium ions from the intracellular calcium store, The presence of an intracellular calcium ion channel inhibitor in the mixture, the addition of a neutrophil chemotactic peptide to the mixture, and the measurement of the concentration of calcium ions in the cells and the concentration of the superoxide. Or (3) the presence of calcium ions in the mixture, the addition of a neutrophil chemotactic peptide to the mixture, and the intracellular The concentration of calcium ions and the concentration of the superoxide are measured, and the concentration of calcium ions from the intracellular calcium store obtained in (1),
The change in the concentration of calcium ions from the outside due to the volume-dependent calcium influx obtained by reducing the concentration of calcium ions from the extracellular calcium obtained in the above (2). And

[0011]

Further, in the method for screening a neutrophil cell superoxide production inhibitor according to the present invention, the calcium detection indicator is a fluorescent indicator, and the superoxide detection indicator is a chemiluminescent indicator. It is characterized by the following.

The method for screening a neutrophil cell superoxide production inhibitor according to the present invention is characterized in that the calcium detection indicator is fluo-3 and the superoxide detection indicator is CLA. Features.

Further, in the method for screening a neutrophil cell superoxide production inhibitor according to the present invention, the calcium concentration-increasing stimulant is fMLP and the intracellular calcium ion channel inhibitor is TMB-8. It is characterized by.

The method for screening for a neutrophil cell superoxide production inhibitor according to the present invention is characterized in that the neutrophil cells are human neutrophil-like differentiated THP-1 cells. .

Furthermore, the present invention relates to a method for converting the rate of superoxide production from a chemiluminescence intensity value to a molar concentration, comprising: (1) a reaction between XOD (xanthine oxidase) having a known enzyme activity value and hypoxanthine. (2) Superoxide generated under the same enzyme activity conditions as in (1) above, by deriving a relational expression between the measured intensity value and the XOD activity. The reduced cytochrome c produced by the reduction is quantified, and based on the molar concentration of the generated superoxide, a relational expression between the molar concentration and the XOD activity is derived, and (3) obtained by the above (1) and (2). From the obtained relational expression, a relational expression between the chemiluminescence intensity value and the generation rate of the superoxide molar concentration is derived,
(4) Based on the molar concentration conversion formula of superoxide obtained in the above (3), the XOD activity is plotted on the horizontal axis, the chemiluminescence intensity value and the superoxide molar concentration generation rate are plotted on the vertical axis, Another object of the present invention is to provide a method for preparing a calibration curve for estimating the superoxide molar concentration generation rate from an emission intensity value.

Hereinafter, the present invention will be described in detail with reference to embodiments of the present invention.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION (Identification of a Plurality of Pathways Which Cause Changes in Intracellular Calcium Ion Concentration) The method for measuring a change in intracellular calcium ion concentration according to the present invention employs a plurality of pathways which cause a change in intracellular calcium ion concentration. , And a change in calcium ion concentration in each of the plurality of pathways is measured.

As schematically shown in FIG. 28, the change in intracellular calcium ion concentration due to stimulation is the sum of changes due to a plurality of pathways by which cells mobilize calcium ions into their cytoplasm (introduce them into the cytoplasm). It is. The pathways for influx of a plurality of calcium ions include (1) a pathway for taking in calcium ions from the extracellular fluid via cell surface molecules called extracellular calcium ion channels, and (2) an intracellular calcium ion channel called intracellular calcium ion channels It is roughly classified into a pathway in which the surface molecules of the store function to send calcium ion ions to the cytoplasm.

Furthermore, in neutrophil cells, in addition to the above two pathways, (3) SOCI (storeoperated)
There is a third path, called calcium influx. This pathway plays a role in sensing the decrease in calcium ion concentration in the intracellular calcium ion store and sending calcium ions from the extracellular fluid.
This pathway consists of a single calcium ion channel located at the cell surface (Kiselyov, et a
l. Nature, 396, 478, 199
8).

In order to identify each of the calcium ion concentration changes based on these at least three kinds of pathways, it becomes possible by blocking specific pathways.

The pathway (1) is blocked by adding a calcium ion chelating agent such as EGTA to the extracellular fluid to form a complex with calcium ions. In addition, the pathway (2) is a compound that specifically inhibits the function of an intracellular calcium ion channel, for example, TM
Blocking by treating cells with B-8.

Further, the route of (3) subtracts the change of calcium ion concentration based on the route from the intracellular store of (1) and the extracellular fluid of (2) from the change of calcium ion concentration of the whole cell. Will be obtained by

In the present invention, the three types of routes are
By selectively using one calcium ion mobilization pathway by selectively using EGTA and TMB-8, a pathway that causes a change in calcium ion concentration in the whole cell is identified, and the change in calcium ion concentration in each pathway is individually determined. Enables measurement and quantification.

(Method of measuring intracellular calcium ion concentration)
In the present invention, the method for measuring the calcium ion concentration in the cytoplasm is not particularly limited, and a known method is preferably used. Specifically the method according to fluorescent indicator, a method may be mentioned by a radioisotope (e.g., ⁴⁵ Ca). Further, the fluorescent indicator (i) is preferable, and specific examples thereof include fra-2, quin-2, and indo-1. Particularly, use of fluo-3 is preferred. When a fluorescent indicator is used, the measuring device is not particularly limited, and a generally known fluorescent measuring device (including a fluorescence microscope) can be used. If necessary, it is also preferable to use an appropriate image analysis device together.

(Method for Measuring Superoxide Production Amount) In the present invention, the method for measuring the amount of superoxide production is not particularly limited, and a known method is preferably used. Specifically, cytotome c reduction method, chemiluminescence method,
Examples thereof include a nitro blue tetrazolium method (NBT method) and an ESR spectrum method. Of these, the use of a chemiluminescent method is preferred, and the use of a chemiluminescent method such as luminol or Cypridina luciferin derivatives is particularly preferred. When the chemiluminescence method is used, the luminescence measuring device is not particularly limited, and a generally known photon counter can be used.

(Screening method for neutrophil cell superoxide production inhibitor)
The method for measuring the change in calcium ion concentration in neutrophil cells based on stimulation has been described. Further, the change in intracellular calcium ion concentration is measured in the presence of an appropriate test substance (inhibitor) together with the stimulator. This makes it possible to measure the superoxide production inhibitory activity of the test substance, and to screen for a neutrophil cell superoxide production inhibitor.

For measuring changes in intracellular calcium ion concentration
Screening by (a) The change in intracellular calcium ion concentration when cells not treated with the inhibitor are stimulated with the stimulant fMLP is measured, and the measurement result is defined as (i). Similarly, a change in intracellular calcium ion concentration when cells treated with the inhibitor were stimulated with fMLP is measured, and the measurement result is defined as (ii). (B) Changes in intracellular calcium ion concentration when cells treated with TMB-8 which inhibits intracellular calcium ion channel function were stimulated with stimulant fMLP without treatment with an inhibitor were measured. (Iii). Similarly, the change in intracellular calcium ion concentration when the cells are treated with the inhibitor and stimulated with the stimulant fMLP is measured, and the measurement result is defined as (iv). (C) Based on the obtained measurement results, a pathway that causes a change in intracellular calcium ion concentration blocked by the inhibitor is identified as follows. Calcium ion C blocked by the inhibitor
a Change in concentration (ΔCa1) is determined. ΔCa1 = (i) − (ii) The obtained ΔCa1 is calculated as the result (iii), (iv), or calculated value ((i) − ((iii) + (iv), SOC)
I). Preferably, the start time, peak time, and end time of the concentration change are compared. Particularly preferably, the change over time from the stimulation time (0 hour) to the start of change to the peak is compared. A pathway that causes a change in Ca concentration corresponding to ΔCa1 is identified.

For measuring the change in superoxide production rate
Screening (a) Superoxide production rate when cells not treated with the inhibitor are stimulated with the stimulant fMLP is measured, and the measurement result is defined as (i ′). Similarly, the rate of superoxide production when the cells treated with the inhibitor were stimulated with fMLP is measured, and the measurement result is defined as (ii ′). (B) The rate of superoxide production when cells treated with TMB-8 which inhibits intracellular calcium ion channel function were stimulated with the stimulant fMLP without treatment with the inhibitor was measured, and the measurement results were shown as (iii) ').
Similarly, the superoxide production rate when treated with the inhibitor and stimulated with the stimulant fMLP is measured, and the measurement result is defined as (iv ′). (C) Based on the obtained measurement results, a pathway that causes a change in superoxide production rate by the inhibitor is identified as follows. Changes in superoxide production rate by the inhibitor (ΔO ₂ ^- 1) obtained. ^{_{ΔO 2 - 1 = (i '}} ) - (ii') ΔCa1 obtained results (iii '), (i
v ′) or a calculated value ((i ′) − ((iii ′)) +
(Iv ′), change in superoxide production rate by SOCI). Preferably, the start time, peak time, and end time of the change in superoxide production rate are compared. Particularly preferably, the change over time from the stimulation time (0 hour) to the start of change to the peak is compared. Delta O.D. ₂ ^- identifying a path leading to superoxide production rate change corresponding to 1.

The change in the intracellular calcium ion concentration and the measurement of the superoxide production rate as a cell function based on the change described above may be measured individually, substantially simultaneously or simultaneously. . Less than,
The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

[0031]

EXAMPLES (Example 1) Method and Results Human monocyte cell line THP-1 was replaced with 0.5 mM Bt ₂ c.
10 containing AMP (dibutyryl cyclic adenosine phosphate)
Cell density is 3X1 with RPMI medium containing 5% serum
0 ⁵ suit cells / ml, 3 days 37 ° C., were cultured in ₂ the presence 5% CO, were differentiated into neutrophil-like. This cell (100
ml) at 37 ° C., 5% CO 2
₂ Incorporated for 30 minutes in the presence. 20 ml of these cells
RH buffer (154 mM NaCl, 5.6 mM
KCl, 10 mM Hepes (2- [4- (2-H
hydroxyethyl) -1-piperaziny
l] ethanesulfonic Acid) (pH
Washed twice in 7.4)) and suspended in 3 ml of the same buffer. This cell suspension (the number of cells is 8.3 × 10 ⁵ ) is placed in a quartz cell, 1 mM CaCl ₂ and 4 μM CLA are added, and the cell suspension is placed in a cell holder for a measurement sample of a simultaneous chemiluminescence and fluorescence measurement apparatus. And stirred at 37 ° C. To the cell suspension, 0 or 750 μM of TMB-8 was further added and incubated at 37 ° C. for 10 minutes. 500 msec for the same cell in the cell holder
Irradiation to the cell suspension was started using argon laser light (wavelength 488 nm) as excitation light while operating the chopper at intervals. 50 seconds later, 0 or 5 mM E
GTA was added to the cell suspension using a sample dispenser, and after 50 seconds, changes in the fluorescence intensity of fluo-3 and the chemiluminescence of CLA when 1 μM fMLP was added using the same dispenser were detected and monitored almost simultaneously.

(1) Waveform analysis of a change in calcium ion concentration obtained as a stimulus response of cells treated with EGTA, and the rise time, peak time, and end time of the pathway for feeding calcium ions from the intracellular calcium ion store to the cytoplasm I asked. To further characterize this path, the rise was enlarged and the slope was expressed as a function of time (curve in FIG. 2).

(2) For the change in calcium ion concentration of the cells treated with TMB-8, the same waveform analysis as in (1) was performed to characterize the pathway for sending calcium ions from the extracellular fluid to the cytoplasm (FIG. 3). Curve).

(3) The sum of the changes in the calcium ion concentration obtained from the above (1) and (2) was obtained, and the obtained waveform (curve in FIG. 4) was used to calculate the change in the total calcium ion concentration in the untreated cells (FIG. 4). 1 curve) to obtain a change in concentration due to SOCI (curve in FIG. 5).

(4) The waveform analysis of SOCI is (1)
The SOCI was characterized as in (2) (curve in FIG. 5). The following (5) is associated with the waveform analysis of the intracellular calcium ion concentration change in the above (1) to (4).
In (7), waveform analysis on superoxide production was performed.

(5) Waveform analysis of superoxide production based on calcium ion mobilization from intracellular calcium ion stores obtained as a stimulus response of cells treated with EFTA, and the rise time, peak time, and end time of the production response I asked. The slope of the rising portion of the response was represented as a function of time (curve in FIG. 7).

(6) With respect to O ₂ ^- production of cells treated with TMB-8, the same waveform analysis as in (5) was carried out to characterize superoxide production based on calcium ion mobilization from extracellular fluid (FIG. 8 curve).

(7) The waveform (curve in FIG. 9) obtained by calculating the sum of the change values of the superoxide production rate from the results of (5) and (6) is plotted as the change value of the total superoxide production rate of the untreated cells. When subtracted from (curve in FIG. 6), the difference is found to be substantially 0 (curve in FIG. 10), and it is clear that the sum of the speed change values well matches the total speed change value. became.

(8) From the results obtained from the above (1) to (7), the behavior of the mobilization pathway of each calcium ion causing a change in the intracellular calcium ion concentration of neutrophils when the stimulation time is 0 second Is shown over time. Furthermore, the behavior of superoxide production based on each pathway was shown.

[0040] Capacities from intracellular calcium stores to the cytoplasm
Pathway for feeding lucium ion Changes in intracellular calcium ion concentration Rise (3.3
1 sec) → peak (9.84 sec) → end (5
6.02 sec) Superoxide production Start up (4.
82 sec) → peak (28.41 sec) → end (212.14 sec)

Calcium ion from extracellular fluid to cytoplasm
The feed path intracellular calcium ion concentration changes rising (2.8
1 sec) → peak (12.85 sec) → end (5
6.52 sc) Superoxide production Start up (6.5.
82 sec) → peak (18.87 sec) → end (79.11 sec)

Calcium ion deficiency in calcium store
Pathway for sensing calcium and sending calcium ions from external solution
(SOCI) Intracellular calcium ion concentration change Rise (19.
88sec) → peak (55.52) → end (unknown)

(Example 2) A model cell for chronic granulomatosis was prepared, and a simulation of calcium ion mobilization pathway using the cell was performed.

The human neutrophil-like differentiated THP-1 cells obtained by the simultaneous chemiluminescence and fluorescence measurement system were
From data on superoxide production / intracellular calcium ion concentration change when stimulated with MLP, calcium ion influx from extracellular fluid responsible for rapid and transient superoxide production was found, and this phenomenon was analyzed. f
Since it is known that the depolarization of the plasma membrane of neutrophils is caused by the stimulation of MLP, it is considered that the calcium ion channel related to the influx of calcium ions may be a membrane voltage-dependent calcium ion channel. On the other hand, it is considered that neutrophils of a chronic granulomatosis patient (hereinafter, referred to as CGD) whose superoxide production function has failed cannot increase intracellular calcium ion concentration due to fMLP stimulation. As described above, by examining the effects of selective inhibition of the function of the membrane potential-dependent calcium channel on the production of superoxide, it is possible to obtain not only important clues for investigating the disease mechanism of CGD, but also the calcium channel. Neutrophils whose function is selectively inhibited are expected to be a good model for CGD neutrophils.

Therefore, superoxide production / intracellular calcium ion concentration by a membrane potential-dependent calcium ion channel inhibitor called verapamil, which is known to inhibit superoxide production of human neutrophils upon fMLP stimulation, The effect on the change was examined using a simultaneous fluorescence and chemiluminescence measurement device.

Method and Results The human monocyte cell line THP-1 was isolated from 0.5 mM Bt2c.
10 containing AMP (dibutyryl cyclic adenosine phosphate)
Cell density is 3X1 with RPMI medium containing 5% serum
0 ⁵ suit cells / ml, 3 days 37 ° C., were cultured in ₂ the presence 5% CO, were differentiated into neutrophil-like. This cell (50m
1) 3 μM fluo3-AM at 37 ° C., 5% CO ₂
Incorporated for 1 hour in presence. The cells are added to 20 ml of R
H buffer (154 mM NaCl, 5.6 mM
Washed twice with KCl, 10 mM Hepes (pH 7.4)) and suspended in 3 ml of the same buffer. This cell suspension ( ^4.5 × 10 ⁵ cells) was placed in a quartz cell, and
mM CaCl ₂ and 4 μM CLA were added, and the cell suspension was placed in the cell holder for the measurement sample of the simultaneous chemiluminescence and fluorescence measurement apparatus, stirred at 37 ° C., and stirred at 37 ° C. with 0, 1, or 10 μM verapamil.
Minutes.

The same cell in the cell holder has 500 ms
Irradiation to the cell suspension was started using argon laser light (wavelength 488 nm) as excitation light while operating the chopper at ec intervals. 100 seconds later, 1 μM fML
P was added to the cell suspension using a sample dispenser, and changes in the fluorescence intensity of fluo-3 and the chemiluminescence of CLA caused by the stimulation of fMLP were monitored almost simultaneously.

To calculate the intracellular calcium concentration (nM), the cell suspension was placed in a quartz cell, 1 mM CaCl ₂ was added to the cell, and the chopper was operated at 500 msec intervals while using an argon laser beam. (Wavelength 4
After irradiating the cell suspension with excitation light of 88%), 0.1% (final concentration) Triton X-100 was used.
(Surfactant) was added to the cell suspension by a sample dispenser, and fluo-3 was added to the cells upon lysis.
Increase in the fluorescence intensity was detected. 250 seconds later, 10
mM (final concentration) EGTA was added by the dispenser, and the change in the fluorescence intensity when the calcium ion was chelated was detected and monitored.

First, the intracellular calcium concentration (nM) was determined from the change in the obtained fluorescence intensity value. As a specific example, the peak value (nM) of intracellular calcium concentration was calculated from the maximum value of the fluorescence intensity when untreated cells were stimulated by the following method (the details of the method were Example 5). Triton
The value of the maximum fluorescence intensity obtained by the addition of X-100 was defined as the Fmax value in the above equation, and the fluorescence intensity when quenched by adding EGTA was defined as the Fmin value. Obtained Fmax
The Fmin value was as follows. Fmax = 14,438, Fmin = 136

The above Fmax and Fmin are set to K of fluo3.
It was substituted into the formula for the calcium ion concentration [nM] together with the d value. Ca ²⁺ [nM] = (F-136) / (14,438−
F) x390

Since the maximum fluorescence intensity produced by the change in intracellular calcium concentration following stimulation is 8,100, Ca ²⁺ [nM] = (8,100-136) / (14,4)
38−8,100) × 390 = 490.1 [nM] On the other hand, since the fluorescence intensity without stimulation is 1,800, Ca ²⁺ [nM] = (1,800-136) / (14,4)
38-1,800) × 390 = 51.3 [nM]

Accordingly, the maximum intracellular Ca concentration following stimulation is 490.1-51.3 = 438.8 [nM].

Second, from the change in the obtained chemiluminescence intensity value, the superoxide production rate (pmol / sec /
ml). As a specific example, the peak value of the superoxide production rate (pmol / sec / ml) was calculated from the maximum value of the chemiluminescence intensity when untreated cells were stimulated by the following method (the details of the method are Example 4).

The maximum value of chemiluminescence produced by superoxide production upon stimulation was 17,500 (cou).
nts / 0.2 sec), and the baseline chemiluminescence value without stimulation is 1,250 (counts /
0.2 sec), the maximum change in superoxide production rate due to stimulation is 27,000-1,
180 = 16,320 (counts / 0.2 sec)
It became. Above measured values (counts / 0.2se
Since c) was obtained for 0.5 ml of the sample solution, the luminescence intensity I (k
cps (kcounts per second) / m
l). 16,320 (counts / 0.2
sec), 163.2 (kcps / ml) is obtained. This value is calculated using the formula (superoxide (pmol /
sec / ml) = 3.1 × I (kcps / ml) +1.
Substituted in 62), O ₂ ^- was determined (nmol / min / ml). O ₂ ⁻ (pmol / sec / ml) = 507.5

[0055] were obtained from more than 0 ₂ ^- production rate change (pm
ol / sec / ml) or changes in intracellular calcium concentration (nM) are plotted on the vertical axis and the stimulation time of fMLP is plotted on the horizontal axis (FIGS. 11, 12, 13, 16, 17, 17, 1).
8, 19, 22). Verapa with the stimulation
The following remarkable changes were observed in the mil-treated cells. (A) Superoxide production: Vera added
Depending on the concentration of pamil, the rapid and transient kinetic components seen immediately after the stimulation were removed, leaving low and sustained kinetic components (FIGS. 17, 18, and 19). (B) Change in intracellular calcium concentration: added ver
The transient increase in calcium ion concentration seen immediately after stimulation depending on the concentration of apamil was cut off and the SOCI
A response component centering on a continuous increase in concentration due to γ was left (FIGS. 11, 12, and 13).

Further, in order to confirm that the kinetic component removed in (a) was similar to the pattern obtained with the TMB-8-treated cells, 750 μM of TMB-
The cells were treated at 37 ° C. for 10 minutes at 8, and stimulated under exactly the same conditions as described above to perform simultaneous measurements.

As a result, a result was reproduced in which the reaction rate component for rapid and transient superoxide production was removed and the other components were removed (FIG. 22).

Finally, for the purpose of simulating the reaction rate component of superoxide production impaired by the action of verapamil, each measured value constituting the waveform (FIG. 17) of superoxide production of untreated cells of verapamil (control cells) From, verapam
The corresponding measurements of the il-treated cells were subtracted, and their aging (FIGS. 20 and 21) was reconstituted and compared to the TMB-8-treated cell waveforms measured as described above, resulting in the subtraction. The resulting waveform is the ve added to the cells.
It was revealed that the pattern and level were very similar to those of the TMP-8-treated cells (FIG. 22) depending on the dose of rapamil (FIG. 22).

The above-mentioned simulation was carried out for the change in intracellular calcium concentration, and the same tendency was shown.
In order to know the temporal relationship between intracellular calcium ion concentration change and superoxide production based on the above results, the time schedule when the stimulation start time was set to 0 second for all analyzed waveforms was shown below. . (I) Cells treated with 0 μM verapamil Intracellular calcium ion change Rise (2.51
sec)> peak (9.04)> end (121.48s)
ec) Superoxide production rise (5.52se)
c)> Peak (18.07)> End (333.83se)
c) (ii) Cells treated with 1 μM verapamil Intracellular calcium ion change Rising (2.51
sec)> peak (9.04)> end (121.48s)
ec) Superoxide production rise (5.52se)
c)> Peak (44.18)> End (332.83se)
c) (iii) Cells treated with 10 μM verapamil Intracellular calcium ion change Rising (2.51
sec)> peak (9.04)> end (121.48s)
ec) Superoxide production rise (5.52se)
c)> Peak (18.07)> End (322.79se)
c) Simulation (Response component removed by verapamil treatment) (i)-(ii) 1 μM verapamil Intracellular calcium ion change Rise (2.51)
sec)> peak (19.54)> end (76.81s)
ec) Superoxide production (5.52 sec)> peak (18.07)> end (130.52 sec) (i)-(iii) 10 μM verapamil Intracellular calcium ion change Rise (2.51)
sec)> peak (19.54)> end (76.81s)
ec) Superoxide production (5.52 sec)> peak (18.07)> end (113.95 sec) (iv) Cells treated with 750 μM TMB-8 Intracellular calcium ion change Rise (2.51)
sec)> peak (19.54)> end (76.81s)
ec) Superoxide production Rise (5.52 sec)
c)> Peak (18.07)> End (112.95se)
c)

Since the above (i)-(iii) and (iv) have almost the same temporal pattern, ver.
It was concluded that apamil treatment impaired the rapid and transient superoxide production function, which was due to the inability to recruit calcium ions from the extracellular fluid into the cytoplasm.

Although the detailed mechanism is unknown, the effect of a calcium channel inhibitor on superoxide production is examined by confirming or comparing changes in intracellular Ca concentration of the same cell by a simultaneous chemiluminescence and fluorescence measurement system. It has become possible for the first time to identify the nature of superoxide production that is affected by the inactivity of voltage-gated calcium channels.

The neutrophil-like cells treated with verapamil seemed to well mimic neutrophils in patients with chronic granulomatosis in which no depolarization of the cell membrane was observed, but the findings obtained this time indicate that A voltage-dependent calcium ion channel inhibitor inhibits the production of superoxide, which has an adverse effect on the human body during blood, as a (therapeutic agent) (JJZZ)
Immerman et al., Biochem. Pharmac
ol. 38, 3601-3610 (1989);
Levy et al., Eur. J. Clin. Invest. 2
6: 376-381 (1996)).

Example 3 An attempt was made to calculate the amount of increase in intracellular calcium Ca ion and the amount of superoxide release to the extracellular fluid per neutrophil cell.

Methods and Results The human monocyte cell line THP-1 was isolated from 0.5 mM Bt ₂ c
10 containing AMP (dibutyryl cyclic adenosine phosphate)
Cell density is 3X1 with RPMI medium containing 5% serum
0 ⁵ suit cells / ml, 3 days 37 ° C., were cultured in ₂ the presence 5% CO, were differentiated into neutrophil-like.

3 μM flu was added to the cells (100 ml).
o3-AM was incorporated at 37 ° C. in the presence of 5% CO ₂ for 1 hour.

The cells were added to 20 ml of RH buffer (1
54 mM NaCl, 5.6 mM KCl, 10 mM H
epes (pH 7.4)) twice, and suspended in 3 ml of the same buffer. This cell suspension (the number of cells is 1.6 ×
10 ⁶ ) in a quartz cell, and 1 mM CaCl ₂ and 4 μm
M CLA was added, and the cell suspension was placed in a cell holder for a measurement sample of a simultaneous chemiluminescence and fluorescence measurement apparatus, and was heated at 37 ° C
While stirring.

The cell in the cell holder is placed for 500 ms.
Irradiation to the cell suspension was started using argon laser light (wavelength 488 nm) as excitation light while operating the chopper at ec intervals. 100 seconds later, 1 μM fML
P was added to the cell suspension using a sample dispenser, and changes in the fluorescence intensity of fluo-3 and the chemiluminescence of CLA caused by the stimulation of fMLP were monitored almost simultaneously.

In order to calculate the intracellular calcium concentration (nM), this cell suspension was placed in a quartz cell, and 1 mM
CaCl ₂ was added, and the cell suspension was placed in a cell holder for a measurement sample and stirred at 37 ° C. Argon laser light (wavelength 488 n) was operated on the same cells in the cell holder at 500 msec intervals while operating the chopper.
After irradiation of the cell suspension with m) as the excitation light was started,
1% (final concentration) Triton X-100 (detergent) was added to the cell suspension using a sample dispenser, and an increase in the fluorescence intensity of fluo-3 due to cell lysis was detected. 250 seconds later, 10 mM (final concentration) EGTA was added by the dispenser, and Ca was added.
The change in the fluorescence intensity when the ions were chelated was detected and monitored.

First, the change in calcium concentration due to the stimulation of cells was converted into a molar concentration as described below (the details of the conversion method are described in Example 5). Triton X-10
The value of the maximum fluorescence intensity obtained by the addition of
The fluorescence intensity when quenched by adding EGTA to the max value was defined as the Fmin value. The obtained Fmax and F
The min values were as follows. Fmax = 38,502, Fmin = 163

The above Fmax and Fmin are changed to K of fluo3.
Together with the d value, it was substituted into the above-mentioned calculation formula for the calcium ion concentration [nM]. Ca ²⁺ [nM] = (F-163) / (38,502−)
F) x390

Since the maximum fluorescence (intensity) value generated by the change in intracellular Ca concentration due to the stimulation is 25,500, Ca ²⁺ [nM] = (25,500-163) / (38,
502-25, 500) x 390 = 759.9 [n
M].

On the other hand, the baseline fluorescence value without stimulation was 13,100, so Ca ²⁺ [nM] = (13,100-163) / (38,
502-13, 100) × 390 = 198.6 [nM]

Thus, the change in intracellular calcium concentration due to stimulation is 760.0-198.6 = 561.4.
[NM].

(1) Calculation of cell volume: Measurement of the radius of neutrophil-like differentiated THP-1 cells by phase-contrast microscopy (assuming the sphere is the average + -SE value using the ARGUS area analysis menu) 13.34 + -1.31μ.
m / cell. The volume is 1274 + -357f as a sphere
1 / cell (2) Calculation of the maximum increase in cytoplasmic calcium upon fMLP stimulation: It was obtained using the maximum cytoplasmic calcium ion concentration (561.4 nM) and the cell volume obtained in (i). 561.4x (1274 + -357) = 715.2 +-
200.4 pmol (3) Calculation of maximum superoxide production (rate) following fMLP stimulation:

The following formula was used to convert the chemiluminescence intensity value (kcps / ml) into a molar concentration (pmol / sec / ml) (the details of how to derive the formula were described in Example 4). O ₂ ⁻ (pmol / sec / ml) = 3.1 × I
_{(Kcps / ml) +1.62 O 2} - and (pmol / sec / ml) O 2 - (nmol /
When revised to ^{_{min / ml), O 2 -}} (nmol / min / ml) = 0.186 ×
I (kcps / ml) + 0.0972

The peak value of chemiluminescence produced by the production of superoxide following stimulation was 27,000 (co
counts / 0.2 sec), and the baseline chemiluminescence value without stimulation is 1,250 (counts
/0.2 sec), the maximum change in superoxide production rate following stimulation is 27,000-
1,250 = 25,750 (counts / 0.2se
c). Above measured values (counts / 0.2s
Since ec) was obtained for a 0.5 ml sample solution, the luminescence intensity I per unit time and per unit volume was obtained.
(Kcps (kcounts per second)
/ Ml). 25,750 (counts /
From 0.2 sec) to 257.5 (kcps / ml).

[0077] substituting this value to the above equation, O ₂ ^- (n
mol / min / ml). O ₂ ⁻ (nmol / min / ml) = 0.186 × 25
7.5 (kcps / ml) + 0.0972 = 48.0

The cell density (the number of cells per unit volume) of the sample was determined as follows. 1.6 × 10 ⁶ /0.5 (volume of sample (ml)) =
3.2 × 10 ⁶ / ml

From the above, based on the maximum superoxide production rate (48 nmol / min / ml) and the cell density of the sample (3.2 × 10 ⁶ cells / ml), the amount of superoxide produced by one cell per minute (mol ) Was calculated. 48 / 3.2 × 10 ⁶ = 15 fmol / min / cel
l or = 250 amol / sec / ce
ll

From the above calculation results, one cell stimulated with fMLP began to recruit calcium ions into the cytoplasm 2.5 seconds after stimulation, and at 9.5 seconds, 715.2 + −
200.4 pmol of calcium ions are retained in the cell compartment. This increase in calcium ions is a factor that causes various cell functions such as superoxide production. At 18.5 seconds, the superoxide production capacity of the cells becomes the most active, and 15 fmol per minute (or 250 amol per second). It can be concluded that the superoxide of (1) can be released into the external liquid.

Example 4 Establishment of Method for Converting Superoxide Production Rate from Chemiluminescence Intensity Value to Molar Concentration The Conversion Method Established: Data on superoxide production / intracellular calcium concentration change of human neutrophils have been obtained. ,
Although it was expressed as the chemiluminescence intensity of CLA / the fluorescence intensity of fluo3, it is necessary to revise the data of both the above as the stoichiometry to make it easy to handle biochemically and clinically when diagnosing cell function of human neutrophils. It was thought that there was. In both cases, the conversion of intracellular calcium concentration into molar concentration was carried out according to a conventional method.

On the other hand, there is no method for converting the amount (or rate) of superoxide production obtained as the intensity of chemiluminescence into a molar concentration, and it is necessary to establish a new method.

XOD (xanthine oxidase) is an enzyme that generates superoxide using hypoxanthine as a substrate. This enzymatic reaction can be conveniently performed in a test tube, and the resulting amount of reaction product (superoxide) is very well reproduced between multiple experiments. On the other hand, superoxide produced as a biological response (eg, in neutrophils)
The reproducibility of the results is always questioned.

As described above, XOD having a known enzyme activity
Based on the chemiluminescence intensity resulting from the oxidation of the chemiluminescent reagent CLA by the superoxide generated by the method described above, the chemiluminescence intensity (increase) associated with the production of superoxide by human neutrophils is defined as the XOD activity unit (unit). The method of expressing has already been reported (Minoru Nakano,
Edited by Toshiichi Yoshikawa, active oxygen and luminescence, 107-112).

However, the method of estimating the unit value of a commercially available XOD purified sample varies depending on the manufacturer, and cannot be used as an index of the amount (or rate) of superoxide production widely used.

Thus, the superoxide produced by an in vitro enzymatic reaction using XOD (xanthine oxidase) whose enzyme activity is known, as the molar concentration of cytochrome c by the reduction method, and as CLA
We obtained the intensity of chemiluminescence and indirectly derived the correspondence between them.

Specifically, the CLA chemiluminescence intensity of superoxide was converted into a molar concentration through the following steps. (1) The intensity (maximum luminescence intensity) of CLA chemiluminescence generated by superoxide generated by XOD (xanthine oxidase) having a known enzyme activity acting on hypoxanthine in a test tube is determined. A relational expression between this intensity value and XOD activity (mU / ml) is derived. (2) Quantifying reduced cytochrome c generated by superoxide generated under the same enzyme activity conditions as in (1). Since one molecule of cytochrome c is reduced by one molecule of superoxide, from the molar extinction coefficient (21.0 / mM · cm) of reduced cytochrome c,
A molar concentration of superoxide is obtained. A relational expression between this molar concentration and XOD activity (unit) is derived. (3) The relational expressions obtained in (1) and (2) are integrated to derive a relational expression between the chemiluminescence intensity value and the generation rate of superoxide (molar concentration). (4) Based on the formula for converting the molar concentration of superoxide obtained in (3), the XOD activity (mU / ml) was plotted on the horizontal axis, and both the chemiluminescence intensity value and the superoxide (molar concentration) generation rate were plotted vertically. On the axis, a calibration curve is prepared for estimating the superoxide (molar concentration) generation rate at a glance from the chemiluminescence intensity value. The details are as follows in “Methods and results”.

Method and Results (1) Measurement of CLA Chemiluminescence Intensity Using Superoxide Generated by XOD Enzyme Reaction RH buffer (154 mM NaCl, 5.63 containing 1.5 mM hypoxanthine and 4 μM CLA)
mM, 10 mM Hepes (pH 7.4)) in a cell holder for a measurement sample in a simultaneous chemiluminescence and fluorescence measurement apparatus, and stirred at 37 ° C. for 5 minutes (pre-incubation). 5.0, 10.
0, 25.0, 50.0, 75.0, 100 mU / ml
(Final concentration) of XOD was added by a sample dispenser, and CLA emanating from the reaction solution with the addition.
The change in chemiluminescence intensity (counts / 0.2 sec) was monitored over time.

The above measurement was carried out at 500 m in order to match the experimental conditions with the simultaneous measurement of intracellular calcium concentration change and superoxide production accompanying cell stimulation.
While operating the chopper at intervals of sec, the irradiation was performed under the condition of irradiating the sample solution with argon laser light (wavelength 488 nm) as excitation light.

As a result, under all the XOD concentration conditions, the maximum luminescence intensity was obtained within 10 seconds after the addition of XOD (FIG. 23 shows data for one measurement). The maximum luminescence intensity at each XOD concentration was as follows (data is 3
Mean ± standard error measured twice).

The measured value of the maximum emission intensity (kcounts /
Since 0.2 sec) was obtained for a 0.5 ml sample solution, the luminescence intensity I (kcps (kcounts per sec.) Per unit time and unit volume) was obtained.
nd) / ml) and arranged as follows. 2mU / ml XOD; 3.541 ± 0.47 (kco
unts / 0.2 sec) or 35.41 ± 4.7
(Kcps / ml) 5 mU / ml XOD; 11.299 ± 2.76 (kc
counts / 0.2 sec) or 112.99 ± 2
7.6 (kcps / ml) 10 mU / ml XOD; 21.605 ± 0.785
(Kcounts / 0.2 sec) or 216.05
± 7.85 (kcps / ml) 25 mU / ml XOD; 49.750 ± 6.477
(Kcounts / 0.2 sec) or 97.50 ±
64.77 (kcps / ml) 50 mU / ml XOD; 70.975 ± 12.125
(Kcounts / 0.2 sec) or 709.75
± 121.25 (kcps / ml) 75 mU / ml XOD; 93.185 ± 5.04 (k
counts / 0.2 sec) or 931.85 ± 5
0.04 (kcps / ml) 100 mU / ml XOD; 100.510 ± 7.90
(Kcounts / 0.2 sec) or 1005.1
0 ± 79.0 (kcps / ml)

Next, XO is plotted on the vertical axis of I (kcps / ml).
When D (mU / ml) was plotted on the horizontal axis, XOD
It was shown that both had a linear correlation up to = 25 mU / ml (FIG. 24).

The relationship between the two was obtained from each point on the coordinates by the least squares method, and could be expressed as the following linear equation. I (kcps / ml) = 19.92 x XOD (mU / m
l) + 5.03- (i) ( 2) chemiluminescent intensity measured at O produced in the same enzymatic reaction conditions with ₂ ^- Determination by cytochrome c reduction method

XOD of each concentration (mU / ml) was 1.5 m
RH containing M hypoxanthine, 80 μM cytochrome c
buffer (154 mM NaCl, 5.63 mM
KCl, 10 mM Hepes (pH 7.4)) and the absorbance of the reaction solution over time (measured wavelength = 550 n)
m and 540 nm) and the difference between the absorbance values at 550 nm and 540 nm at each XOD concentration (OD550).
-540) was determined as follows.

[0095] Next, since the cytochrome c of 1 molecule by superoxide one molecule is reduced, O ₂ is the number of moles equal consumption of cytochrome c to be reduced ^- relationship moles of is established.

[0096] Depending, cell path width if 1 cm, since the molar extinction coefficient = 21.0 / mM of reduced cytochrome c, O ₂ as follows: ^- the molar concentration is obtained. ^{_{O 2 - (mM) = OD}} 550-540 / 21.0 O 2 - by 1,000 times the value of ^{_{(mM) O 2 - (nmol}} /
ml), and the O per reaction time (min)
₂ ^- The (nmol / min / ml) was determined and summarized in the table below.

[0097]

[Table 1]

From the results in the above table, the relationship between the XOD concentration and the superoxide generation reaction rate is summarized below. XOD concentration 2 mU / ml; 1.01 ± 0.02 (nm
ol / min / ml) XOD concentration 5 mU / ml; 2.36 ± 0.41 XOD concentration 10 mU / ml; 3.85 ± 0.13 XOD concentration 25 mU / ml; 7.42 ± 0.45 XOD concentration 18.30 ± 0.96 XOD concentration 75 mU / ml; 27.09 ± 5.18 XOD concentration 100 mU / ml; 40.35 ± 0.58

Referring to these values, XOD (mU / m
l) is plotted on the abscissa and the reaction rate of superoxide (O ₂ ⁻ ) production (nmol / min / ml) is plotted on the ordinate, and there is a linear correlation between them up to XOD = 50 mU / ml. As shown (FIG. 25). This relationship is
It could be expressed as a linear expression of a straight line obtained by the least square method from each point on the coordinates. O ₂ ⁻ (nmol / min / ml) = 0.3666 ×
XOD (mU / ml) + 0.1896

Since the superoxide generation rate I (kcps / ml) determined by chemiluminescence in (1) is per second, (nmol / min / ml) is converted to (pmol / min).
/ Sec / ml). O ₂ ⁻ (pmol / sec / ml) = 6.11 × XOD
(MU / ml) + 3.16− (ii)

(3) Conversion of molar concentration of superoxide in extracellular solution (1) From (2), (i) is replaced by XOD in the formula (ii).
(MU / ml), and the following equation relating to emission intensity was used. O ₂ ⁻ (pmol / sec / ml) = 3.1 × I (kcp
s / ml) + 1.62- (iii)

When the CLA chemiluminescence intensity value (kcps / ml) is known from the above equation (iii), the superoxide molar concentration (pmol / sec / ml) can be obtained.

(4) Preparation of Calibration Curve for Conversion of Superoxide Molar Concentration Apart from the equation obtained in (3), it is possible to roughly calculate the intensity of chemiluminescence emitted with the production of superoxide by neutrophil cells. Since it was considered very convenient to be able to grasp the superoxide (molar) concentration at a glance, the following calibration curve was created.

The XOD concentration is plotted on the abscissa, and the superoxide formation rate is plotted on the ordinate both for the rate (pmol / sec / ml) determined by the cytochrome c reduction method and the maximum chemiluminescence intensity (kcps / ml) of CLA. i) and (i
The calibration curve was obtained by superimposing the straight lines of the equation (i) (FIG. 2).
6).

(Example 5) Method (Principle) and Result of Establishment of Method for Converting Change in Intracellular Calcium Concentration from Fluorescence Intensity Value to Molar Concentration

Generally, the dissociation constant Kd for calcium ions
Is determined from the fluorescence intensity F of the one-wavelength excitation / one-wavelength fluorescence type Ca indicator with a clear calcium ion concentration (Ca ²⁺ [n
M]) can be easily obtained by the following formula. Ca ²⁺ [nM] = (F−Fmin) / (Fma
x-F) xKd

Since the Kd value of fluo3 is 390 nM (Handbook of Fluorescence)
nt Probes and Research Ch
chemicals, Sixth Edition, 1
996), knowing the Fmin and Fmax values, the above equation for calculating the intracellular calcium concentration is satisfied.

A human monocyte cell line THP-1 was
The cell density was adjusted to 3 × 10 ⁵ cells / ml with RPMI medium containing 10% serum containing M Bt ₂ cAMP (dibutyryl cyclic adenosine phosphate) for 37 days for 3 days.
The cells were cultured at 5 ° C. in the presence of 5% CO ₂ to differentiate into neutrophils. 3 μM fluo3-AM was added to the cells (50 ml).
At 37 ° C. in the presence of 5% CO ₂ for 1 hour. The cells were added to 20 ml of RH buffer (154 mM Na).
Cl, 5.6 mM KCl, 10 mM Hepes
(PH 7.4)) and suspended in 3 ml of the same buffer. This cell suspension (the number of cells was 6.0 × 10 6
⁵ ) were placed in a quartz cell, 1 mM CaCl ₂ was added, and the cell suspension was placed in a measurement sample cell holder of a simultaneous chemiluminescence and fluorescence measurement apparatus and stirred at 37 ° C.

[0109] The same cell in the cell holder
After irradiating the cell suspension with argon laser light (wavelength 488 nm) as excitation light while operating the chopper at ec intervals, 0.1% (final concentration) Triton
The time during which X-100 (surfactant) was added to the cell suspension using a sample dispenser was set to 0 second, and an increase in the fluorescence intensity of fluo-3 due to cell lysis was detected. 250 seconds later, 10 mM (final concentration) EGTA
Was added by the dispenser, and the change in the fluorescence intensity when the calcium ion was chelated was detected and monitored almost simultaneously (FIG. 27).

The value of the maximum fluorescence intensity obtained by the addition of Triton X-100 was defined as the Fmax value of the above equation,
The fluorescence intensity when GTA was added and quenched was defined as the Fmin value. The obtained Fmax and Fmin values were as follows. Fmax = 11, 289, Fmin = 106

[0111] The above Fmax and Fmin are set to K of fluo3.
Together with the d value, it was substituted into the above-mentioned calculation formula for the calcium ion concentration [nM]. Ca ²⁺ [nM] = (F−106) / (11,289−)
F) x390

For example, if the fluorescence intensity F generated upon stimulation is 6,300, Ca ²⁺ [nM] = (6,30
0-106) / (11,289-6,300) × 390
= 484.2 [nM]

If the fluorescence intensity F without stimulation is 1,900, Ca ²⁺ [nM] = (19,00−
106) / (11,289-1,900) × 390 = 7
4.5 [nM]

Therefore, the change in intracellular Ca concentration due to stimulation is 484.2-74.5 = 409.7 [nM].

[0115]

According to the present invention, it is possible to identify a plurality of pathways that cause a change in the calcium ion concentration in a cell, especially in a neutrophil cell, and to individually quantify the concentration change due to the pathway. Furthermore, it becomes possible to screen for a neutrophil cell superoxide production inhibitor based on the inhibitory activity on each calcium ion concentration change pathway in neutrophil cells.

Therefore, the calcium ion concentration in neutrophil cells and the screening method for neutrophil cell superoxide production inhibitor according to the present invention can be used to increase the intracellular calcium concentration responsible for superoxide production. It is possible to contribute to the elucidation and to establish a new method for diagnosing cell function which leads to treatment of patients with chronic granulomatosis. In addition, by applying the present invention to screening of a drug whose mechanism of action is unknown, it becomes possible to identify a pathway that leads to a change in intracellular calcium concentration that is responsible for an expected drug effect or an unexpected side effect.

This is extremely important for research and development of a therapeutic agent or a therapeutic method for a disease relating to intracellular calcium ions (eg, an ischemic disease). Furthermore, for a system for analyzing cells mimicking neutrophils (simulated patient neutrophils) of a patient with chronic granulomatosis, for example, bacteria bactericidal function and the like are separately measured and compared with normal cells. It can also be used to evaluate (quantify) the biological defense ability of the neutrophil.

[Brief description of the drawings]

FIG. 1. Intracellular calcium ion (Ca ²⁺ ) following stimulation
It is a figure which shows the density change (sum total).

FIG. 2 is a graph showing a change in intracellular Ca ²⁺ concentration when calcium ions (Ca ²⁺ ) are mobilized from an intracellular calcium store.

FIG. 3 is a graph showing changes in intracellular Ca ²⁺ concentration when calcium ions (Ca ²⁺ ) are mobilized from extracellular fluid.

FIG. 4. Intracellular Ca when mobilizing calcium ions (Ca ²⁺ ) from extracellular fluid and intracellular calcium store.
It is a figure which shows the density change of ²⁺ .

FIG. 5: SOCI (store operated c)
It is a figure which shows the simulation of (alkium influx).

FIG. 6 is a graph showing a change (total) of superoxide production rates following stimulation.

FIG. 7 shows changes in superoxide production rate based on mobilization from intracellular calcium stores.

FIG. 8 is a graph showing changes in superoxide production rate based on mobilization of calcium ions (Ca ²⁺ ) from extracellular fluid.

9 is a diagram showing a change in superoxide production rate obtained by integrating the change values of the superoxide production rate shown in FIGS. 7 and 8. FIG.

10 is a diagram showing a change in superoxide production rate when the change value shown in FIG. 9 is subtracted from a change value in superoxide production rate shown in FIG. 6;

FIG. 11 is a graph showing changes in intracellular calcium ion (Ca ²⁺ ) concentration following stimulation of untreated cells.

FIG. 12 shows changes in intracellular calcium concentration following stimulation of cells treated with 1 μM verapamil.

FIG. 13 shows changes in intracellular calcium concentration following stimulation of cells treated with 10 μM verapamil.

14 is a diagram showing a change in intracellular calcium ion concentration obtained by subtracting the change value shown in FIG. 12 from the value of change in intracellular Ca ²⁺ concentration shown in FIG. 11;

FIG. 15 is a diagram showing a change in intracellular calcium ion concentration obtained by subtracting the change value shown in FIG. 13 from the change value in intracellular Ca ²⁺ concentration shown in FIG. 11;

FIG. 16 shows changes in intracellular calcium ion concentration treated with 750 μM TMB-8.

FIG. 17 is a diagram showing a change in superoxide production rate accompanying stimulation of untreated cells.

FIG. 18 is a graph showing changes in superoxide production rate following stimulation of cells treated with 1 μM verapamil.

FIG. 19 is a graph showing changes in superoxide production rate following stimulation of cells treated with 10 μM verapamil.

20 is a diagram showing a change in superoxide production rate obtained by subtracting the change value shown in FIG. 18 from the value of the change in superoxide production rate shown in FIG. 17;

21 is a diagram showing a change in superoxide production rate obtained by subtracting the change value shown in FIG. 19 from the value of change in superoxide production rate shown in FIG. 17;

FIG. 22 shows changes in the superoxide production rate of cells treated with 750 μM TMB-8.

FIG. 23 is a diagram showing the maximum chemiluminescence intensity due to superoxide generated by XOD at each concentration.

FIG. 24 is a diagram showing the maximum chemiluminescence intensity due to superoxide generated by XOD at each concentration.

FIG. 25 is a diagram showing the generation rate of superoxide by XOD at each concentration.

FIG. 26 is a diagram showing the generation rate of superoxide by XOD at each concentration.

FIG. 27 is a diagram showing the results of an experiment for conversion of the intracellular calcium ion (Ca ²⁺ ) molar concentration.

FIG. 28 shows that cells have calcium ions (C
FIG. 2 is a diagram showing a plurality of pathways for recruiting (a ²⁺ ) recruitment (inflow into the cytoplasm).

──────────────────────────────────────────────────続 き Continued on front page (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 33/48-33/98 A61K 31/34

Claims (4)

(57) [Claims] 1. A mixed solution is prepared by mixing a neutrophil cell treated with an indicator for detecting calcium and an indicator for detecting superoxide, (1) causing a calcium ion removing agent to be present in the mixed solution, Adding a neutrophil-migratory peptide to the mixture, measuring the concentration of calcium ions in the cells and the concentration of superoxide, and calculating the concentration of calcium ions from the calcium store in the cells; The presence of an intracellular calcium ion channel inhibitor in the mixture, the addition of a neutrophil chemotactic peptide to the mixture, and the measurement of the concentration of calcium ions in the cells and the concentration of the superoxide. The concentration of calcium ions from the extracellular calcium ions, and (3) the presence of calcium ions in the mixture; A calcium concentration increasing stimulant is added to the mixture to measure the concentration of calcium ions in the cells and the concentration of superoxide, and the calcium ions from the intracellular calcium store obtained in (1) are obtained. And the concentration of calcium ions from the extracellular calcium obtained in the above (2) is reduced to obtain the concentration of calcium ions from the extracellular due to the volume-dependent calcium influx, Method for measuring calcium ion concentration in neutrophil cells. 2. The method according to claim 1, wherein the indicator for detecting calcium is a fluorescent indicator, and the indicator for detecting superoxide is a chemiluminescent indicator. 3. A method for screening an inhibitor of neutrophil cell superoxide production based on the inhibitory activity of a neutrophil cell superoxide production inhibitor on a change in calcium ion concentration in neutrophil cells by a calcium ion concentration increasing stimulant, The ion concentration change is as follows: a neutrophil cell treated with a calcium detection indicator and a superoxide detection indicator are mixed to prepare a mixed solution; A calcium concentration increase stimulant is added to the mixture, and the concentration of calcium ions in the cells and the concentration of the superoxide are obtained, and the concentration change of calcium ions from the calcium store in the cells is obtained. (2) An intracellular calcium ion channel inhibitor is present in the mixture, A change in the concentration of calcium ions from the extracellular calcium ions, which is obtained by measuring the concentration of intracellular calcium ions and the concentration of superoxide by adding a neutrophil chemotactic peptide; or Calcium ions are present in the mixture, a neutrophil chemotactic peptide is added to the mixture, and the concentration of calcium ions in the cells and the concentration of the superoxide are measured. The extracellular calcium influx obtained by reducing the concentration of calcium ions from the intracellular calcium store obtained in step (2) and the concentration of calcium ions from the extracellular calcium obtained in step (2). Neutrophil cell superoxide production characterized by any one of calcium ion concentration changes from Inhibitor screening method. 4. The method according to claim 1, wherein the calcium concentration increasing stimulant is formylmethionyl leucylphenylalanine, and the intracellular calcium ion channel inhibitor is 8-
The method according to claim 1, wherein the method is (diethylamino) octyl 3,4,5-trimethoxybenzoate hydrochloride.