JP5250542B2 - Overactive bladder prevention, screening method for ameliorating agent - Google Patents

Description

  The present invention relates to a method for screening an agent for preventing or improving overactive bladder.

  Overactive bladder (OAB) is a disease that presents with dysuria such as urgency, frequent urination, and urge incontinence. Symptoms of overactive bladder interfere with daily life and reduce the quality of life, especially in elderly people, and it is estimated that the number of affected people over the age of 40 is over 8 million. Attention has been gathered with the changing society. In healthy people, the amount of urine stored in the bladder and urinary urgency are correlated, but in patients with overactive bladder, it is thought that the urinary urgency is caused because the bladder contracts regardless of urine accumulation. However, there are still many unclear points in the onset mechanism.

  In recent years, increased excitability of the bladder sensory nervous system (bladder afferent nervous system) has been pointed out as one of the factors that cause overactive bladder. This sensory nervous system excitement is caused by various mediators released from bladder epithelial cells upon stimulation of bladder expansion by storing urine (adenosine triphosphate (ATP), acetylcholine, etc.) It is thought to be caused by. It has been reported that the amount of ATP released from the bladder epithelium increases with age in humans, and that the amount of ATP released during bladder extension increases in patients with overactive bladder (Non-patent Document 1). There is also a report that administration of ATP to the rat bladder induces detrusor overactivity and shortens the urination interval (Non-patent Document 2).

  At present, anticholinergic drugs that suppress the action of acetylcholine, which promotes bladder contraction, are mainly used to treat overactive bladder with drugs. However, it is known that anticholinergic drugs cause side effects such as dry mouth, constipation, difficulty in urination, etc. associated with taking them. From such a situation, it is desired to search for a more effective therapeutic agent and method for overactive bladder.

Journal of the Japanese Society for Spinal Cord Disorders, 2005, Vol. 18, p.18-19 Journal of Urology, 2002, 168, p.1230-1234

  The present invention is capable of efficiently evaluating a test substance, preventing and / or improving urination disorders such as overactive bladder or urinary urgency, frequent urination, urge urinary incontinence with a simple method with high accuracy and high sensitivity. It is an object to provide a method for screening a possible substance. In addition, the present invention provides a method for screening a substance that can efficiently evaluate a test substance and can relieve and / or suppress the activation of bladder afferent nerve with high accuracy and high sensitivity by a simple technique. This is the issue.

  The present inventors sought a search method for searching for a new material that can be used as an agent for preventing / ameliorating overactive bladder. Among various factors that cause overactive bladder, focusing on the excitability of the bladder afferent nerve described above, a substance that suppresses ATP release from the bladder epithelium causing the excitement is excessive. It was considered useful as a preventive / ameliorating agent for active bladder. Based on this idea, we have intensively studied the construction of a system that can reproduce ATP release accompanying bladder extension stimulation in vivo. Using a bladder epithelial cell line that can be subcultured and obtain a large amount of uniform cells, It was found that by applying osmotic pressure stimulation to a cell line, it is possible to construct a system that can achieve both screening accuracy and efficiency. The present invention has been completed based on these findings.

That is, the present invention suppresses ATP release by measuring a bladder epithelial cell line brought into contact with a test substance by an osmotic pressure change and measuring the amount of ATP released from the cell line after the osmotic pressure change. The present invention relates to a method for screening an agent for preventing and / or improving overactive bladder comprising a step of evaluating or selecting a test substance.
The present invention also includes a step of extending a bladder epithelial cell line brought into contact with a test substance by a change in osmotic pressure, and measuring the amount of ATP released from the cell line after the change in osmotic pressure to suppress ATP release. The present invention relates to a method for screening for an agent for preventing and / or improving urination disorder, comprising a step of evaluating or selecting a test substance.
Furthermore, the present invention suppresses ATP release by measuring a bladder epithelial cell line brought into contact with a test substance by an osmotic pressure change and measuring the amount of ATP released from the cell line after the osmotic pressure change. The present invention relates to a method for screening an agent for alleviating and / or suppressing bladder afferent nerve activation, comprising a step of evaluating or selecting a test substance.

  According to the method of the present invention, a test substance can be efficiently evaluated, and a urinary disorder such as overactive bladder or urinary urgency, frequent urination, urge urinary incontinence can be prevented with a simple method with high accuracy and high sensitivity. And / or substances that can be improved can be screened. In addition, according to the method of the present invention, a large number of test substances can be efficiently evaluated, and a substance capable of alleviating and / or suppressing bladder afferent nerve activation with high accuracy and high sensitivity can be screened with a simple technique. can do.

(A) is a figure which shows the relationship between the osmotic pressure change and ATP density | concentration in HT-1376 strain. (B) is a figure which shows the relationship between the osmotic pressure change and ATP density | concentration in RT-4 stock | strain. In addition, the bar in a figure represents a standard deviation. It is a figure which shows the relationship between the addition of capsazepine and ATP concentration in HT-1376 strain. In addition, the bar in a figure represents a standard deviation. It is a figure which shows the relationship between the incubation time (osmotic pressure stimulation time) after addition of a low osmotic pressure liquid, and ATP density | concentration in HT-1376 strain. In addition, the bar in a figure represents a standard deviation. It is a figure which shows the relationship between the capsazepine density | concentration and ATP density | concentration in HT-1376 strain. In addition, the bar in a figure represents a standard deviation. It is a figure which shows the relationship between the low osmotic pressure addition amount and ATP density | concentration in HT-1376 strain. In addition, the bar in a figure represents a standard deviation.

The screening method of the present invention comprises a step of extending a bladder epithelial cell line brought into contact with a test substance by an osmotic pressure change, and measuring the amount of ATP released from the cell line after the osmotic pressure change, thereby suppressing ATP release. A test substance selected by the screening method has an action of alleviating and / or suppressing bladder afferent nerve activation, and comprises an overactive bladder Or it is useful as a preventive and / or ameliorating agent for dysuria. The present invention focuses on the onset mechanism of overactive bladder in which ATP is released from bladder epithelial cells and acts on the afferent nervous system of the bladder in response to bladder extension stimulation by storing urine, and is used to prevent and improve overactive bladder. Search for useful new materials. This has been achieved by approaching the onset mechanism of overactive bladder from a different aspect from the mechanism of action of conventional therapies and therapeutic agents.
Hereinafter, the screening method of the present invention will be described in detail.

  The screening method of the present invention includes a step of using a bladder epithelial cell line as a bladder epithelial cell and extending the cell line by osmotic pressure stimulation. The bladder epithelial cell line used in the present invention has the ability to respond to ATP release in response to stretching stimulation caused by changes in osmotic pressure. Furthermore, since the bladder epithelial cell line used in the present invention is an established cell line, it can be passaged over a long period of time, and it is possible to continuously obtain a large amount of homogeneous cells without fear of contamination with other types of cells. Is possible. For example, there are concerns that the number of cells that can be collected from one individual is low, such as primary cultured bladder epithelial cells, contamination of other types of cells is unavoidable, and it is difficult to obtain a homogeneous and large number of cells that are easily altered in character. There is no. Therefore, the screening method of the present invention using a bladder epithelial cell line is excellent in efficiency, accuracy, operational simplicity, and the like.

To date, reports on extension stimulation in an in vitro system include the release of ATP by fixing the bladder epithelial tissue to a tissue ring and applying water pressure to the extension (The Journal of Clinical Investigation, 2005, 115, p.2412-2422), primary cultured bladder epithelial cells by culturing the primary cultured bladder epithelial cells on a stretchable stretch plate and stretching the plate or refluxing the low osmotic pressure solution. (Journal of Urology, 2001, 166, p.1951-1956 and American journal of physiology Renal physiology, 2003, 285, p.F423-F429) Yes. However, in a system using a bladder epithelial cell line that can be subcultured, the ATP release response when an extension stimulus is applied by osmotic pressure change such as a low osmotic pressure solution has not been known so far. This is a finding obtained by the inventors.
Furthermore, the extension method described in the above document requires a special device, and the number of samples that can be collected from one individual is low for bladder epithelial tissue and primary cultured cells. In addition, when primary cultured cells are used as described above, it is inevitable that other types of cells are mixed in due to their properties. Therefore, when these extension methods and cells are applied to a screening system, it is sufficient from the viewpoint of simplicity of operation, the efficiency of experiments that can evaluate a large number of samples at once, and the accuracy of the experimental system. I can't say that.
In the present invention, use of a bladder epithelial cell line that can be passaged and can obtain a large amount of uniform cells, and osmotic stimulation is given to the cell line can improve convenience and evaluation efficiency required for screening. In addition, an experimental system closer to the in vivo environment of an overactive bladder patient can be established, and the usefulness and reliability of the screened substance can be improved. Examples of bladder epithelial cell lines that can be subcultured and obtain uniform cells in large quantities include UM-UC-3, SW780, 1A6 (RTA-556), Hs195. T, J82, SCaBER, T24, TCCSUP, HT-1197, HT-1376, RT-4, NBT-II, TBN-54 and the like are known, and these cell lines can be used in the present invention.

  As described above, ATP is released from bladder epithelial cells in response to bladder extension stimulation by storing urine, and this ATP release involves a receptor (mechanosensor) that senses mechanical stimulation of bladder epithelial extension. It is considered. It is known that there are mechanosensors belonging to ENaC (Epithelial Na channel) family and TRP (transient receptor potential) family in human bladder epithelium. In bladder epithelial cells of overactive bladder and urinary tract obstruction patients, There are reports that these mechanosensors are highly expressed compared to healthy individuals (Urologia Internationals, 2006, Vol.76, p.289-295, Urology View2, Vol.5, 2007), p.31. -36).

The bladder epithelial cell line used in the present invention is preferably a cell line in which ENaC, which is a mechanosensor belonging to the above-mentioned ENaC family, and / or TRPV1, which is a mechanosensor belonging to the TRP family, is expressed. In overactive bladder patients, these two mechanosensors are highly expressed compared to healthy individuals. Furthermore, it has been reported that the micturition reflex is suppressed by inhibiting its function (Journal of Urology, 2009, Vol.181, p.379-386, Urology, 2007, Vol.69, p.590). -595), particularly important among the factors associated with ATP release from bladder epithelial cells. Therefore, by using a cell line expressing these two mechanosensors, the reproducibility of the system can be improved and the accuracy can be increased.
In particular, it has been pointed out that ENaC has a significantly different expression pattern between healthy and affected individuals. ENaC has known α, β, γ, and δ subunits, and is considered to function in the αβγ type or the δβγ type. In healthy individuals, the expression of αa, δ and γ subunits of ENaC is very weak and the expression level of β subunits is also low. In contrast, overactive bladder shows a high expression level for any subunit (Urology View 2, 2007, Vol. 5, p. 31-36). Therefore, in the present invention, bladder epithelial cell lines expressing ENaC α subunit, β subunit and γ subunit, or bladder epithelial cell lines expressing δ subunit, β subunit and γ subunit Is preferably used.

In addition, from the viewpoint of usefulness and reliability of the screened substance, the cell line is preferably derived from a human. This takes into consideration the case where the structure of the molecule, the signal transduction pathway, the expression pattern of the mechanosensor, and the like related to ATP release by the above-described extension stimulation differ between humans and non-human animals.
Examples of bladder epithelial cell lines preferably used in the present invention include HT-1376 and RT-4. All of these cell lines are of human origin, and the HT-1376 strain has all αa, β, γ and δ subunits of ENaC and TRPV1, and the RT-4 strain has ENaC α, β and γ subunits and TRPV1. Are highly expressed. HT-1376 and RT-4 are both available from the American Type Culture Collection (ATCC) and the European Collection of Cell Cultures (ECACC).
Furthermore, in the present invention, it is also a preferred embodiment that a combination of a plurality of these bladder epithelial cell lines, for example, a combination of HT-1376 and RT-4 is used for screening. By using a combination of cells with different expression patterns, a screening system showing an expression pattern closer to the ecological environment can be constructed.

The bladder epithelial cell line can be cultured in a medium and culture conditions suitable for each type and used in the present invention. For example, using MEM medium containing 10% FBS, DMEM medium, McCoy's 5a medium, RPMI 1640, etc. as a medium, culturing at 37 ° C. in a 5% CO ₂ environment for about 15 to about 80 hours. For example, it is preferable to perform culture at 0.1 to 0.2 mL / well on a 96 well plate.

  In the screening method of the present invention, after contacting the aforementioned bladder epithelial cell line and the test substance, the bladder epithelial cell line is extended by osmotic pressure change. The bladder epithelial cell line that has been brought into contact with the test substance may be any substance that can directly or indirectly contact the test substance and the bladder epithelial cell line. For example, the test substance is added to the culture medium in advance. Thereafter, the culture solution is added to the culture solution of the bladder epithelial cell line, the culture solution of the bladder epithelial cell line is replaced with the culture solution, or the culture solution in which the bladder epithelial cell line is seeded is tested. This can be done by adding a substance. The extension of the bladder epithelial cell line may be performed in contact with a test substance or in a non-contact state.

  Extension of the bladder epithelial cell line is performed by osmotic pressure change. In addition to the method of changing the osmotic pressure, the cells can be expanded by fixing the cells and applying water pressure to expand the cells, culturing the cells on a stretchable stretch plate, and stretching the plate in a certain cycle. Compared with the method of extending these cells by mechanical stimulation, the method of expanding cells by changing osmotic pressure can simultaneously apply the same extension stimulation to many cells. is there. Therefore, according to the method of the present invention using osmotic pressure change, a large amount of test substance can be evaluated at a time under the same conditions, and rapid and efficient screening becomes possible.

In the present invention, any method for extending cells by changing osmotic pressure may be used as long as the cell environment can be changed to low osmotic pressure conditions and low osmotic pressure stimulation can be given to the cells. By being placed under a low osmotic pressure condition, the cells swell and the structure of the cell membrane changes, and an extension stimulus is applied to the cell membrane. As a method of giving such a low osmotic pressure stimulus to a cell, first, a bladder epithelial cell line is brought into contact with an isotonic solution (isotonic solution), and then brought into contact with a hypotonic solution (hypotonic solution) to give the cell. It is preferable to apply a low osmotic pressure stimulus. Contact with a low osmotic pressure solution after stabilization by contacting with an osmotic pressure solution in advance, thereby reducing physical or chemical irritation and more accurately confirming the ATP release response specific to the low osmotic pressure solution. can do.
In the present invention, contacting cells with an isotonic solution or a hypotonic solution is not limited as long as the cells can be brought into contact with the isotonic solution or the hypotonic solution via the cell membrane surface. In the present invention, firstly, the bladder epithelial cell line is brought into contact with an isotonic solution, and then a hypotonic solution is added to the isotonic solution, thereby creating a low osmotic pressure condition and bringing the bladder epithelial cell line into contact with the hypotonic solution. Is particularly preferred. By adding a hypotonic solution to the isotonic solution, the osmotic pressure gradually decreases, so that rapid changes in the extracellular environment can be prevented, and mechanical extension in vivo due to the gradual decrease in osmotic pressure. Since a stimulus closer to the stimulus can be obtained, the accuracy of screening can be increased.

The isotonic solution that can be used in the present invention may be a solution having the same or similar osmotic pressure as that of the cells used for screening, and a solution having an osmotic pressure of 280 to 350 mOsm can be used. Specifically, as an isotonic solution, a pH 7.4 solution of 105 mM NaCl, 6 mM CsCl, 1 mM MgCl ₂ , 5 mM CaCl ₂ , 10 mM HEPES, 90 mM D-Mannitol, 10 mM glucose, 4.8 mM KCl, 120 mM NaCl, 1. 1 mM MgCl ₂ , 2 mM CaCl ₂ , 11 mM glucose, 10 mM Hepes solution, PBS, physiological saline and the like can be used.
The hypotonic solution may be a solution having an osmotic pressure lower than that of the cells used for screening, and a solution having an osmotic pressure of 0 to 300 mOsm can be used, preferably an osmotic pressure of 0 to 250 mOsm. Has an osmotic pressure of 50 to 200 mOsm. Specifically, as the hypotonic solution, a pH 7.4 solution composed of 5 mM CaCl ₂ , 1 mM MgCl ₂ , 10 mM Hepes, diluted physiological saline, PBS, Krebs solution, distilled water, or the like can be used.
These isotonic and hypotonic solutions may be used by appropriately adding other components within a range not impairing the function. The osmotic pressure of the isotonic solution and the hypotonic solution can be calculated from the molar concentration of the solvent in the solution, and can be measured using, for example, an osmometer by a vapor pressure method or a freezing point depression method (freezing point depression method).
The degree of osmotic pressure change before and after the low osmotic pressure stimulation is not particularly limited as long as it can cause the cell to exert a stretching stimulus, but is preferably in the range of 10 to 80% with respect to the osmotic pressure of the isotonic pressure solution. More preferably, it is in the range of 40 to 70% of the isotonic solution. It is preferable to change the osmotic pressure within this range because cells can be stretch-loaded and sufficient ATP release is observed.
The contact time between the isotonic solution and the bladder epithelial cell line is not particularly limited, but is preferably from 5 minutes to 120 minutes, more preferably from 30 minutes to 90 minutes. The contact time between the hypotonic solution and the bladder epithelial cell line is not particularly limited, but is preferably 5 minutes or longer and 120 minutes or shorter, and more preferably 5 minutes or longer and 60 minutes or shorter. When the isotonic solution is brought into contact within this range, the background ATP release amount (the amount of ATP released in a non-osmotic solution stimulating non-specific manner) can be reduced. Moreover, it is preferable to contact the low osmotic pressure liquid in this range because a reproducible result can be obtained.
Moreover, the temperature at the time of osmotic pressure stimulation is not particularly limited, but is preferably 35 ° C to 38 ° C.
The total amount of the isotonic solution and the low osmotic solution added to the cells is only required to be able to cover the cell surface. For example, if it is 96 holes, 75 to 200 μL / well is preferable, and 100 to 175 μL / well. Is more preferable. If the amount of liquid is within this range, the isotonic solution and the low osmotic solution are uniformly mixed in the well, so that the reproducibility of screening can be ensured.

After extending the bladder epithelial cell line by the osmotic pressure change, the amount of ATP released from the bladder epithelial cell line by the osmotic pressure change is measured. Specifically, as described above, the bladder epithelial cell line is contacted with the hypotonic solution for a certain period of time, and then the solution is recovered and the ATP amount contained therein is measured by any one of the following ATP amount measuring methods. Can be mentioned.
The method for measuring the amount of ATP in the present invention is not particularly limited as long as it has sensitivity that can be used for comparison and evaluation of the amount of ATP released, and a commonly used ATP measurement method can be applied. For example, bioluminescence method, electrochemical measurement method, enzyme method and the like can be mentioned. In the present invention, from the viewpoint of detection sensitivity and rapidity, it is preferable to use an ATP measurement method (luciferase assay) based on a luciferin luciferase bioluminescence reaction. The ATP measurement method by the luciferase assay is well known to those skilled in the art, but the light generated based on biochemiluminescence is measured with a luminescence detector by allowing a biochemiluminescence reaction reagent using luciferase and luciferin to act on an ATP-containing sample. The ATP is measured using the fact that the amount of light generated is proportional to the amount of ATP.

  Based on the measured ATP amount, a test substance that suppresses ATP release is evaluated or selected, and a test substance that has an action of suppressing ATP release is determined as a prophylactic / ameliorating agent for overactive bladder. Specifically, in the bladder epithelial cell line contacted with the test substance and the bladder epithelial cell line not contacted, the amount of ATP released by the osmotic pressure change was compared, and the bladder epithelium contacted with the test substance When the amount of ATP released from the cell line is substantially less than the amount of ATP released from the bladder epithelial cell line not in contact with the test substance, the test substance is used as a prophylactic / ameliorating agent for overactive bladder Can be determined. In addition, when comparing the measured ATP amount, it can be determined whether or not the ATP amount measurement method used is substantially changed from the literature describing the used ATP amount measurement method or common knowledge of those skilled in the art. In the present invention, since the test substance is evaluated and determined based on the measurement result of the amount of ATP, highly sensitive and quantitative evaluation is possible.

The test substance used in the screening method of the present invention is not particularly limited, and for example, compounds, plant extracts, microbial metabolites, derivatives thereof, and the like can be used. Usually one kind of substance can be used as the test substance, but by using two or more substances, the characteristics as a prophylactic / ameliorating agent for overactive bladder can be determined for the combination of two or more substances. Is also possible.
As described above, one of the causes of overactive bladder is thought to be that ATP released by bladder extension stimulation excites and activates afferent nerves of the bladder and causes overactivity of the bladder. As a result of bladder overactivation, the patient presents with dysuria. Since the substance obtained by the screening method of the present invention can suppress ATP release by extension stimulation, it suppresses and / or alleviates excitement / activation of bladder afferent nerve, and prevents and / or improves overactive bladder And as a preventive and / or ameliorating agent for dysuria.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.

In the following test examples and examples, cell culture was performed under the following conditions unless otherwise specified.
(Cell culture)
HT-1376 (purchased from ATCC) and RT-4 (purchased from ATCC) were used as bladder epithelial cell lines, and each cell was cultured in a medium having the following composition under conditions of 37 ° C. and 5% CO ₂ .

10% FCS (fetal calf serum), sodium pyruvate (0.055 g / 500 mL), L-glutamine (0.146 g / 500 mL) were added to MEM Earle's (manufactured by Invitrogen) for HT-1376
10% FCS (fetal calf serum) and L-glutamine (0.146 g / 500 mL) were added to the medium for RT-4 McCoys 5A Medium (manufactured by Invitrogen)

Test Example 1 Gene Expression Analysis of ENaC Subunits and TRPV1 in Bladder Epithelial Cell Line Bladder epithelial cell line HT-1376 or RT-4 (both purchased from ATCC) to be 1 × 10 ⁵ cells / well in a 24-well plate After seeding and culturing for 24 hours under the above culture conditions, total RNA was extracted using RNeasy Mini Kit (manufactured by QIAGEN). The obtained RNA was subjected to a reverse transcription reaction with a high capacity cDNA reverse transcription kit (Applied Biosystems), and the gene expression levels of ENaC subunits α, β, γ, and δ and TRPV1 were expressed by TaqMan Fast Universal PCR Master Mix ( (Reagent: TaqMan Fast Universal PCR Master Mix, apparatus: 7500 Fast Real-Time PCR System). The expression level of each gene was corrected using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as an internal standard, and was set as a relative value with respect to the expression level of GAPDH. The primers and probes purchased from Applied Biosystems were used (product numbers: GAPDH Hs99999905_m1, αENaC Hs00168906z_m1, βENaC Hs00165722_m1, γENaC Hs00168918_m1, δENaC 00161595_m1, TRPV1 Hs00218912_).
The results are shown in Table 1. Table 1 shows the expression level of HT-1376 strain as a reference.

As is clear from Table 1, all four subunits of α subunit, β subunit, γ subunit, and δ subunit were expressed in bladder epithelial cell line HT-1376. In the bladder epithelial cell line RT-4, α subunit, β subunit, and γ subunit were expressed. As is clear from Table 1, both the HT-1376 strain and the RT-4 strain expressed the TRPV1 gene.
As described above, ENaC expresses only a part of subunits in healthy human bladder epithelial cells, but expresses all subunits of ENaC in bladder epithelial cells of overactive bladder patients. In addition, TRPV1 has been reported to be expressed in human bladder epithelial cells, and TRPV1 expression has been reported to be elevated in overactive bladder patients (Urologia Internationals, 2006, Vol. 76, p.289-295). Therefore, in bladder epithelial cell lines HT-1376 and RT-4, the expression pattern of each ENaC subunit is different from that of non-overactive bladder patients and is similar to the expression pattern of overactive bladder patients. It was also found that TRPV1 expressed in overactive bladder patients was expressed.

Test Example 2 Verification of relationship between osmotic pressure change and ATP release amount Bladder epithelial cell line HT-1376 or RT-4 was seeded in a 96-well plate at 4.1 × 10 ⁴ cells / well, and It was cultured for 24 hours under the culture conditions. The medium was removed by suction and washed with the isotonic solution described in Table 2 below, 75 μL / well of isotonic solution was added, and the mixture was incubated at 37 ° C. for 1 hour. Thereafter, 75 μL / well of the low osmotic pressure solution shown in Table 2 or 75 μL / well of the isotonic solution shown in Table 2 was further added and incubated at 37 ° C. for 5 minutes to collect the solution. The ATP concentration in the collected sample was measured by luciferin luciferase assay using ATP Bioluminescent Assay Kit (manufactured by SIGMA).
The results are shown in FIG. 1A shows the ATP concentration when HT-1376 is used, and FIG. 1B shows the ATP concentration when RT-4 is used.

  As is clear from FIG. 1, the ATP concentration was significantly increased in the system to which the low osmotic pressure solution was added as compared with the system to which the isotonic pressure solution was added. That is, in the system in which the bladder epithelial cells were stimulated and expanded by changing the osmotic pressure from isotonic pressure to low osmotic pressure, the amount of ATP released was significantly increased compared to the system in which the osmotic pressure was not changed. . From this, it was confirmed that a system in which the amount of ATP released from bladder epithelial cells was increased could be constructed by changing the osmotic pressure.

Example 1
The bladder epithelial cell line HT-1376 was seeded in a 96-well plate so as to be 4.1 × 10 ⁴ cells / well, and cultured under the above culture conditions for 24 hours. The medium was removed by suction, and the cell line was washed with the isotonic solution described in Table 2. After dissolving capsazepine (manufactured by SIGMA) to a concentration of 50 mM with ethanol, add 75 μL / well of an isotonic solution that was diluted 1/1000 with an isotonic solution to a final concentration of 50 μM and added at 37 ° C. Incubated for hours. Thereafter, 75 μL / well of a solution in which capsazepine was added to the low osmotic pressure solution shown in Table 2 to a final concentration of 50 μM was further added and incubated at 37 ° C. for 1 hour to collect the solution. The ATP concentration in the collected sample was measured by luciferin luciferase assay using ATP Bioluminescent Assay Kit (manufactured by SIGMA). As a control, instead of adding capsazepine, a sample to which ethanol was added to a final concentration of 0.1% was prepared, and the ATP concentration was measured in the same manner. The results are shown in FIG.
Capsazepine is a known substance as a competitive inhibitor of TRPV1. It has been reported that ATP release is suppressed by inhibition or deficiency of TRPV1 in both physical extension stimulation and hypoosmotic stimulation of bladder epithelial cells (Nature Neuroscience, Vol. 5, 2002, p.856). -860). In this example, capsazepine was used as a test substance in order to confirm whether ATP release could be detected by the same route as that of the previously reported stretching stimulus.

  As is clear from FIG. 2, the ATP concentration was greatly increased in the system in which the osmotic pressure was changed without adding capsazepine. In contrast, in the system in which capsazepine was added to change the osmotic pressure, the increase in ATP concentration was suppressed. These features were consistent with the results reported as features of ATP release from bladder epithelial cells caused by overactive bladder. From this result, the screening system of the present invention can reproduce ATP release from cell epithelial cells by extension stimulation in vivo, and the test substance is released by cell extension using the screening method of the present invention. To determine whether or not a test substance that suppresses ATP release can be determined or selected as a prophylactic / ameliorating agent for overactive bladder or dysuria, or a mitigating / suppressing agent for bladder afferent nerve activation Was confirmed to be possible.

Example 2
An experiment was conducted in the same manner as in Example 1 except that the incubation time (osmotic pressure stimulation time) after addition of the low osmotic pressure solution was changed as follows, and the relationship between the osmotic pressure stimulation time and the ATP concentration was compared and evaluated. Went.
For osmotic stimulation, first, 75 μL / well of an isotonic solution (capsazepine final concentration 50 μM) was added and incubated at 37 ° C. for 1 hour. Thereafter, a low osmotic pressure solution (final concentration of capsazepine 50 μM) was further added at 75 μL / well, and incubated at 37 ° C. for 5 minutes, 20 minutes, and 60 minutes, respectively. The results are shown in FIG.

  As is clear from FIG. 3, the osmotic pressure stimulation times of 5 minutes, 20 minutes, and 60 minutes all increased the amount of ATP released due to osmotic pressure change from isosmotic pressure to low osmotic pressure. The ATP release response to osmotic pressure stimulation could be confirmed. Further, in the system to which capsazepine was added, the ATP concentration was suppressed lower than that in the control, and the ATP release inhibitory action of the test substance could be confirmed. In particular, the system with an osmotic pressure stimulation time of 60 minutes had the smallest variation between the wells and good reproducibility.

Example 3
An experiment was carried out in the same manner as in Example 1 except that the addition concentration of capsazepine was changed as follows, and the relationship between the change in the concentration of the test substance and the ATP concentration was compared and evaluated.
Regarding the capsazepine concentration, first, capsazepine (manufactured by SIGMA) dissolved in ethanol to a concentration of 5 mM, 10 mM, and 50 mM was diluted to 1/1000 with an isotonic solution, and the final concentrations of capsazepine were 5 μM, 10 μM, and 50 μM, respectively. Then, 75 μL / well of an isotonic solution prepared so as to be obtained was added and incubated at 37 ° C. for 1 hour. Then, 75 μL / well of a solution in which capsazepine was added to the low osmotic pressure solution to final concentrations of 5 μM, 10 μM, and 50 μM, respectively, was added and incubated at 37 ° C. for 1 hour. The results are shown in FIG.

  As apparent from FIG. 4, the higher the concentration of added capsazepine, the lower the ATP concentration. That is, it was found that the screening method of the present invention can evaluate the ATP release inhibitory action according to the concentration change of the test substance.

Example 4
An experiment was conducted in the same manner as in Example 1 except that the addition amount of the low osmotic pressure solution was changed as follows, and the relationship between the change in osmotic pressure and the ATP concentration was compared and evaluated.
To change the osmotic pressure, first, 75 μL / well of isotonic solution (capsazepine final concentration 50 μM) was added and incubated at 37 ° C. for 1 hour. Thereafter, a low osmotic pressure solution (capsazepine final concentration 50 μM) was further added to 75 μL / well or 150 μL / well and incubated at 37 ° C. for 1 hour. The results are shown in FIG.

  As is clear from FIG. 5, in each system to which low osmotic pressure solution was added at 75 μL / well and 150 μL / well, the ATP release amount increased due to the osmotic pressure change from isosmotic pressure to low osmotic pressure. The ATP release response to pressure stimulation could be confirmed. Further, in the system to which capsazepine was added, the ATP concentration was suppressed lower than that in the control, and the ATP release inhibitory action of the test substance could be confirmed.

Claims (8)

  A step of extending the bladder epithelial cell line brought into contact with the test substance by osmotic pressure change, and measuring the amount of ATP released from the cell line after the osmotic pressure change, and evaluating the test substance suppressing ATP release or A method for screening an agent for preventing and / or improving overactive bladder comprising a step of selecting.   A step of extending the bladder epithelial cell line brought into contact with the test substance by osmotic pressure change, and measuring the amount of ATP released from the cell line after the osmotic pressure change, and evaluating the test substance suppressing ATP release or A method for screening a preventive and / or ameliorating agent for dysuria, comprising a step of selecting.   A step of extending the bladder epithelial cell line brought into contact with the test substance by osmotic pressure change, and measuring the amount of ATP released from the cell line after the osmotic pressure change, and evaluating the test substance suppressing ATP release or A method of screening for an agent for alleviating and / or suppressing bladder afferent nerve activation, comprising a step of selecting.   The screening method according to any one of claims 1 to 3, wherein the bladder epithelial cell line is derived from a human.   The screening method according to any one of claims 1 to 4, wherein the bladder epithelial cell line is HT-1376 or RT-4.   The screening method according to claim 5, wherein the bladder epithelial cell lines HT-1376 and RT-4 are used in combination. The screening method according to any one of claims 1 to 6, wherein the step of extending the bladder epithelial cell line by osmotic pressure change includes the following steps (a) and (b).
(A) contacting the bladder epithelial cell line with an isotonic solution (b) contacting the bladder epithelial cell line with a hypotonic solution The screening method according to claim 7, wherein the step (b) is the following step (b ′).
(B ′) a step of bringing a bladder epithelial cell line into contact with a hypotonic solution by adding a hypotonic solution to the isotonic solution of (a)

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