Method for measuring transporter activity, method for screening vesicular nucleotide transporter activity regulator, and vesicular nucleotide transporter activity regulator

Abstract

This method for measuring transporter activity comprises: exposing a membrane vesicle having a vacuolar ATPase and a vesicular nucleotide transporter to an anion which is labeled with a radionuclide and is different from adenosine triphosphate, in a reaction solution containing adenosine triphosphate and magnesium ions; and measuring the transport activity of the anion into the membrane vesicle on the basis of the radiation of the radionuclide.

Description

Method for measuring transporter activity, method for screening vesicular nucleotide transporter activity modifiers, and vesicular nucleotide transporter activity modifiers

[0001] The present invention relates to a method for measuring transporter activity, a method for screening vesicular nucleotide transporter activity modifiers, and vesicular nucleotide transporter activity modifiers.

[0002] Adenosine triphosphate (ATP), produced by glycolysis and oxidative phosphorylation, functions as the energy currency of cells. A significant portion of ATP is stored in secretory organelles such as secretory granules, synaptic vesicles, and secretory lysosomes. Upon stimulation, it is released extracellularly and, together with its metabolites adenosine diphosphate (ADP) and adenosine, functions as an intercellular signaling molecule, participating in various pathophysiological processes such as pain transmission and inflammation progression via purine receptors.

[0003] ATP secretion occurs when ATP is first packed into secretory vesicles, which then fuse with the cell membrane, releasing ATP into the extracellular space (exocytosis). The packing of ATP into vesicles involves vesicular nucleotide transporters (VNUT) and vacuolar ATPase (V-ATPase). V-ATPase hydrolyzes ATP, releasing protons (H). ＋ ) transports H into the vesicle, ＋ A driving force (membrane potential difference and pH gradient) is formed. Subsequently, VNUT is H ＋ The driving force is used as the driving force for transport to actively transport ATP into vesicles (Non-Patent Document 1).

[0004] VNUT is the ninth member of the SLC17 organic anion transporter family (SLC17A9). VNUT is present in ATP-filled granules of all animals. When VNUT expression is suppressed, vesicular ATP concentration decreases, and depending on the degree of suppression, the amount of ATP secreted from purinergic cells decreases or disappears.

[0005] SLC17A9 gene knockout mice appear to grow similarly to wild-type mice, but purine-mediated chemical transmission is consistently suppressed. As a result, the secretion of inflammatory cytokines such as IL-6 is reduced, insulin sensitivity is increased, and pain perception is alleviated. Furthermore, inflammatory cell infiltration and fibrosis in a high-fat diet-induced non-alcoholic steatohepatitis (NASH) model were significantly improved in SLC17A9 gene knockout mice. In contrast, overexpression of VNUT in neurons increases ATP secretion from neurons, leading to hyperalgesia and chronic inflammation due to excessive stimulation of purine receptors. A positive correlation has also been reported between SLC17A9 gene expression levels and the malignancy of several cancers, including liver cancer and lung cancer.

[0006] When VNUT inhibitors such as clodronate and Evans Blue are administered to mice, the concentration of ATP in vesicles decreases, leading to a reduction in ATP secretion. As a result, chronic neuropathic pain, complete Freund's adjuvant-induced inflammatory pain, carrageenan-induced inflammatory pain, and β-glucan-induced allodynia are alleviated. In a methionine / choline-deficient diet-induced NASH model, administration of clodronate dramatically improves NASH activity score, inflammatory cell infiltration, and hepatic fibrosis. Therefore, intravesicular ATP saturation is a new target for the development of treatments for intractable and metabolic diseases, and elucidating its regulatory mechanisms and developing VNUT inhibitors are medically important issues (Non-Patent Literature 2).

[0007] Studies on vesicular ATP filling have primarily used adrenal medulla chromaffin granules and synaptic vesicles of electric organs in electric rays. In the early stages of research, several research groups discovered that isolated chromaffin granules take up radioactive ATP, suggesting the existence of active ATP transporters. Non-patent documents 3 and 4 discuss mitochondrial ATP / ADP exchange inhibitor atractyroside, glycolysis intermediate phosphoenolpyruvate (PEP), and orthophosphate (H ３ PO ４ ２－ ; Pi), sulfates and thiocyanate ions (SCN －It has been reported that ) inhibits ATP uptake. Since chromaffin granules take up radioactive PEP, Pi, and sulfate in an ATP-inhibitory and membrane-voltage-dependent manner, it has been proposed that the ATP transporter is an anion transporter with low substrate specificity. On the other hand, Non-Patent Literature 5 concluded that membrane vesicles prepared by hypotonic treatment of chromaffin granules did not show these uptakes, and that the possibility of the existence of an ATP transporter was low. Subsequently, Non-Patent Literature 6 reported that membrane vesicles of bovine adrenal chromaffin granules take up ATP in an ATP-dependent manner, and that this uptake is sensitive to the V-ATPase inhibitor bafilomycin A1. This result suggests the existence of an ATP transporter energetically coupled with V-ATPase. However, research stalled at this stage. Therefore, although VNUT was identified as disclosed in Non-Patent Literature 1, the detailed mechanism of vesicle ATP filling in vivo and how vesicle ATP filling is controlled remain unclear.

[0008] International Publication No. 2008 / 126517

[0009] Yoshinori Moriya, et al., Vesicular nucleotide transporter (VNUT): appearance of an actress on the stage of purinergic signaling, 2017, Purinergic Signaling, 13:387 - 404 Nao Hasuzawa, et al., Physiopathological roles of vesicular nucleotide transporter (VNUT), an essential component for vesicular ATP release, 2020, BBA - Biomembranes, 1862, 183408 John H. Phillips and Yvonne P. Allison, Proton Translocation by the Bovine Chromaffin - Granule Membrane, 1978, Biochem. J., 170, 661 - 672 Andreas Weber, Edward W. Westhead and Hans Winkler, Specificity and properties of the nucleotide carrier in chromaffin granules from bovine adrenal medulla, 1983, Biochem. J., 210, 789 - 794 H. A. Gruninger, et al., Adenine nucleotide and phosphoenolpyruvate transport by bovine chromaffin granule “ghosts”, 1983, Neuroscience, 9(4):917 - 924 Laurie A. Bankston and Guido Guidotti, Characterization of ATP Transport into Chromaffin Granule Ghosts: SYNERGY OF ATP AND SEROTONIN ACCUMULATION IN CHROMAFFIN GRANULE GHOSTS, 1996, THE JOURNAL OF BIOLOGICAL CHEMISTRY, 271, 29,17132 - 17138Keisuke Sawada, 7 others, Identification of a vesicular nucleotide transporter, 2008, Proc Natl Acad Sci USA, 105(15):5683 - 5686Shohei Sakamoto, 12 others, Impairment of vesicular ATP release affects glucose metabolism and increases insulin sensitivity, 2014, Scientific Reports, 4, 6689Kaori Inoue, 6 others, Mechanism Underlying ATP Release in Human Epidermal Keratinocytes, 2014, J. Invest. Dermatol., 134, 1465 - 1468,

[0010] The activity of VNUT is defined by the rate at which ATP is transported into vesicles. In order for vesicles to be filled with ATP, ATP, which is a substrate of V - ATPase, must be hydrolyzed to transport protons into the vesicles. As a result, the concentration of ATP in the measurement solution, which is the transport substrate of VNUT, decreases, making it difficult to quantitatively analyze the transport of ATP. Therefore, currently, to proteoliposomes composed of purified VNUT and soybean phospholipids, K ＋ the potassium diffusion potential difference generated inside and outside the membrane by adding the potassium ionophore valinomycin to H ＋ is ＋By using VNUT as a substitute for a driving force and incorporating ATP electrophoretically, ATP transport by VNUT is measured without changing the ATP concentration in the measurement solution (Non-Patent Document 7 and Patent Document 1). This VNUT activity measurement system (proteoliposome system) consists of the minimum elements necessary for active ATP transport, namely VNUT and soybean phospholipids, and does not contain ATP-filled granular membrane components (V-ATPase, other membrane proteins and lipids). Therefore, various factors affecting ATP filling, such as the number and orientation of VNUTs, the composition of lipids and proteins in the membrane, the ion permeability and internal volume of membrane vesicles, and the properties of the driving force, differ from those of natural ATP-filled vesicles. Consequently, results obtained using proteoliposomes do not necessarily reflect intravesicular ATP filling under physiological conditions. In particular, little information can be obtained about the control of intravesicular ATP filling involving other biological components. Furthermore, methods using proteoliposomes involve many steps, such as the expression, solubilization, purification, and reconstitution of membrane proteins (transporters) into liposomes. These steps are complex, time-consuming, and result in high variability, making standardization difficult when handling a large number of samples. In fact, to date, there have been no reports from any research laboratory other than the one that published Non-Patent Document 7 mentioned above.

[0011] In other words, functional measurement systems for VNUT using proteoliposomes are not widely used, and functional evaluation of VNUT at the biological level, which should form the basis of research, is still in an extremely immature stage. In order to develop VNUT inhibitors, it is important to establish a highly sensitive and highly efficient in vitro ATP-filling measurement system. To that end, it is necessary to go beyond Non-Patent Document 6 mentioned above and elucidate in detail the properties of VNUT activity (ATP transport) coupled with V-ATPase. However, most of the results reported so far were performed before V-APase and VNUT were identified, and from the perspective of the present day, when V-APase and VNUT have been identified, most of them were measured under inappropriate conditions. For example, Non-Patent Document 4 discloses that sulfate is incorporated into chromaffin granules in a reaction solution containing sucrose. However, since this reaction solution does not contain magnesium ions, which are essential for V-ATPase activity, V-ATPase does not function under these conditions. In other words, it is difficult to say that Non-Patent Document 4 measures the VNUT activity that is energy-coupled with V-ATPase.

[0012] The present invention has been made in view of the above circumstances, and aims to provide a method for measuring transporter activity that can more easily and quantitatively measure VNUT activity energy-coupled with V-ATPase, and a method for screening vesicular nucleotide transporter activity modifiers. Furthermore, it aims to provide novel vesicular nucleotide transporter activity modifiers discovered by the screening method for vesicular nucleotide transporter activity modifiers.

[0013] The inventors of this invention have conducted extensive research on vesicular ATP packing and discovered that ATP-dependent ATP packing activity in membrane vesicles, i.e., VNUT activity energy-coupled with V-ATPase, can be replaced by the ATP-dependent uptake of anions other than ATP. They also found the optimal conditions for this, and while avoiding the complicated and specialized operation of proteoliposomes, they overcame the previous drawback of substrate ATP concentration changing during transport, thus completing the present invention.

[0014] A method for measuring transporter activity according to a first aspect of the present invention involves exposing a membrane vesicle having vacuolar ATPase and a vesicular nucleotide transporter to an anion different from adenosine triphosphate, labeled with a radionuclide, in a reaction solution containing adenosine triphosphate and magnesium ions, and measuring the transport activity of the anion into the membrane vesicle based on the radiation of the radionuclide.

[0015] A screening method for vesicular nucleotide transporter activity modifiers according to a second aspect of the present invention involves exposing membrane vesicles having vacuolar ATPase and vesicular nucleotide transporters to anions different from adenosine triphosphate, labeled with a test substance and a radionuclide, in a reaction solution containing adenosine triphosphate and magnesium ions; measuring the transport activity of the anions into the membrane vesicles based on the radiation of the radionuclide; comparing the transport activity with a reference value; and evaluating the effect of the test substance on the transport activity of the anions into the membrane vesicles.

[0016] The anion may be a sulfate ion or orthophosphate.

[0017] The reaction solution may further contain chloride ions or bromide ions.

[0018] The reaction solution may further contain chloride ions, and the concentration of chloride ions in the reaction solution may be 4 to 20 mM.

[0019] The anion may be a sulfate ion with a concentration of 0.1 mM in the reaction solution, and the concentration of the chloride ion in the reaction solution may be 15 mM.

[0020] The reaction solution may further contain bromide ions, and the concentration of bromide ions in the reaction solution may be 1 to 5 mM.

[0021] The anion may be a sulfate ion with a concentration of 0.1 mM in the reaction solution, and the concentration of the bromide ion in the reaction solution may be 2 mM.

[0022] A vesicular nucleotide transporter activity regulator according to a third aspect of the present invention comprises itaconic acid or an itaconic acid ester.

[0023] According to the present invention, the energy-coupled VNUT activity with V-ATPase can be measured more easily and quantitatively. Furthermore, the present invention provides a novel vesicular nucleotide transporter activity regulator.

[0024] This figure shows the time course of ATP uptake in the presence or absence of the ATP regeneration system. This figure shows the dependence of ATP uptake on chloride ion concentration. This figure shows the dependence of ATP uptake on bromide ion concentration. This figure shows the concentration dependence of inhibition of ATP uptake by sulfate ions in the presence or absence of chloride ions. This figure shows the concentration dependence of inhibition of ATP uptake by Pi. This figure shows the time course of sulfate ion uptake. This figure shows the uptake activity of ATP-dependent sulfate ions in the absence of chloride ions. This figure shows the uptake activity of ATP-dependent sulfate ions in the presence of chloride ions. This figure shows the dependence of ATP-dependent sulfate ion uptake on KCl concentration or KBr concentration. This figure shows the effect of Pi on ATP-dependent sulfate ion uptake. This figure shows the relationship between ATP-dependent sulfate ion uptake and sulfate ion concentration. This figure shows the effect of atractyroside on ATP-dependent sulfate ion uptake. This figure shows the effect of diisothiocyanostilbendisulfonic acid (DIDS) on ATP-dependent sulfate ion uptake. This figure shows the effect of PEP or pyruvate on ATP-dependent sulfate ion uptake. This figure shows the effect of sulfates or glucons on ATP-dependent Pi uptake. This figure shows the effect of PEP, atractyroside, or DIDS on ATP-dependent Pi uptake. This figure shows PEP-sensitive and ATP-dependent sulfate ion uptake in the adrenal membrane fraction of mice. This figure shows reserpine-sensitive and ATP-dependent dopamine uptake in the adrenal membrane fraction of mice. This figure shows the effect of itaconic acid on ATP-dependent sulfate ion uptake and Pi uptake. This figure shows the ATP secretion inhibitory effect of itaconic acid 4-octyl. This figure shows the intravesicular ATP filling inhibitory effect of itaconic acid 4-octyl.

[0025] Embodiments of the present invention will be described below with reference to the drawings. In each drawing, the same or equivalent parts are denoted by the same reference numerals. However, the present invention is not limited to the embodiments and drawings described below. In the embodiments described below, expressions such as “having,” “including,” or “containing” also include the meaning of “consisting of” or “composed of.”

[0026] (Embodiment 1) A method for measuring transporter activity according to this embodiment will be described. The measurement method includes an exposure step and a measurement step. In the exposure step, membrane vesicles having V-ATPase and VNUT are exposed to an anion different from ATP, which is labeled with a radionuclide, in a reaction solution containing ATP and magnesium ions.

[0027] The reaction solution is a biological buffer, preferably MOPS-Tris. The reaction solution contains ATP and magnesium ions. The reaction solution containing magnesium ions is obtained by adding a magnesium-donating salt to the reaction solution. The magnesium-donating salt is not particularly limited as long as the transporter activity can be measured, but magnesium acetate is preferred.

[0028] Preferably, the reaction solution contains sucrose and an ATP regeneration system. The ATP regeneration system consists of creatine phosphate and creatine kinase. To initiate substrate transport into membrane vesicles by VNUT, ATP hydrolysis by V-ATPase and subsequent H ＋ The formation of a driving force is necessary. By adding an ATP regeneration system to the reaction solution, the reduction of ATP as a substrate by V-ATPase and other ATP-consuming proteins is minimized, enabling more quantitative measurements.

[0029] A membrane vesicle is a sac-like structure enclosed in a membrane. Preferably, the membrane vesicle is a lipid bilayer, but is not limited to this. Membrane vesicles containing V-ATPase and VNUT can be prepared by known methods. Examples of membrane vesicles include platelet dense granulosa vesicles, lysosomes, pancreatic β-cell insulin granulosa vesicles, synaptic vesicles, and chromaffin granulosa vesicles. Membrane vesicles may also be prepared from cultured cells such as COS7 cells that express VNUT by introducing a VNUT expression plasmid. The VNUT expression plasmid may express a variant of VNUT.

[0030] The anion can be any anion other than ATP, and may be inorganic or organic. For example, inorganic anions include sulfate ions and orthophosphate. The anion is labeled with a radionuclide. The labeling may be done by substituting the constituent atoms of the anion with a radionuclide, or by adding a radionuclide to the anion. The radionuclide is not particularly limited, for example. ３５ S, ３２ P, ３ H is an example. When sulfate ions are used as the anion, the concentration of sulfate ions in the reaction solution is, for example, 0.05 to 10 mM, 0.08 to 5 mM, or 0.1 to 3 mM. Preferably, the concentration of sulfate ions in the reaction solution is 0.1 to 2 mM.

[0031] The reaction solution may further contain chloride ions or bromide ions. As shown in the examples below, in the transporter activity measurement method according to this embodiment, anion uptake by membrane vesicles is activated in the presence of chloride ions or bromide ions. When the reaction solution contains chloride ions, the concentration of chloride ions in the reaction solution is 4 to 20 mM, preferably 10 to 19 mM, more preferably 12 to 18 mM, and even more preferably 13 to 17 mM. The chloride salt added to the reaction solution as a chloride ion donor is not particularly limited, but examples include potassium chloride and sodium chloride.

[0032] Preferably, when sulfate ions with a concentration of 0.1 mM in the reaction solution are used as the above-mentioned anion, the concentration of chloride ions in the reaction solution is 15 mM.

[0033] If the reaction solution contains bromide ions, the concentration of bromide ions in the reaction solution is 1 to 5 mM, preferably 1.2 to 4.5 mM, more preferably 1.5 to 4 mM, and even more preferably 1.8 to 3 mM. The bromide salt added to the reaction solution as a bromide ion donor is not particularly limited, but examples include potassium bromide and sodium bromide. The reaction solution may also contain other salts such as potassium acetate, if necessary.

[0034] Preferably, when sulfate ions with a concentration of 0.1 mM in the reaction solution are used as the above-mentioned anion, the concentration of bromide ions in the reaction solution is 2 mM.

[0035] Exposure of membrane vesicles to anions in the reaction solution is sufficient if the membrane vesicles and anions come into contact, and the mode of contact is particularly arbitrary. The reaction conditions for the reaction solution containing the membrane vesicles and anions described above are, for example, a temperature of 25-35°C, 28-33°C, or 29-32°C, and a reaction time of, for example, 10-180 minutes, 10-120 minutes, 30-90 minutes, or 40-60 minutes.

[0036] In the measurement step, the transport activity of anions into membrane vesicles is measured based on the radiation of a radionuclide. Transport activity refers to the activity of transporting substances that cannot permeate the membrane forming the membrane vesicles (e.g., anions) across the membrane. Transport activity may also be the amount of anions transported into the membrane vesicles. In this measurement step, transport activity can be measured by any method, such as gel filtration, centrifugation, or transporter binding. Preferably, in the measurement step, transport activity is measured using the membrane filtration method, which is widely used in measuring transporter activity.

[0037] In the membrane filter method, the pore size is appropriately set according to the size of the membrane vesicles used, but for example, it is 0.2 to 0.7 μm, 0.3 to 0.6 μm, 0.4 to 0.5 μm, preferably 0.45 μm. In measurement using the membrane filter method, the reaction solution is filtered through a membrane filter, and the residue on the filter is washed with a washing solution. The washed residue is dissolved in a liquid scintillation reagent (cocktail) such as an emulsifying scintillator, and the radiation is measured with a liquid scintillation counter. The higher the radiation, the greater the amount of anion uptake by the membrane vesicles (transporter activity). In this case, the measured radiation value may be normalized by the amount of protein contained in the residue. Furthermore, since a higher radiation level in the filtrate indicates lower anion uptake activity by the membrane vesicles, the radiation level of the filtrate may be measured and compared in the membrane filter method.

[0038] The transporter activity measurement method according to this embodiment involves measuring the H accumulated as a result of ATP hydrolysis by V-ATPase. ＋This is based on the novel finding that anions different from ATP, taken into membrane vesicles using driving force, reflect ATP filling by VNUT. Because ATP is hydrolyzed by V-ATPase, it has been difficult to quantitatively analyze VNUT activity using conventional methods for measuring ATP uptake. However, the transporter activity measurement method according to this embodiment allows for simpler and more quantitative measurement of VNUT activity by measuring the uptake of anions other than ATP. Moreover, since this transporter activity measurement method includes the ATP hydrolysis process, it is possible to measure VNUT activity that is energy-coupled with V-ATPase.

[0039] This transporter activity measurement method can quantitatively measure VNUT activity in any membrane system containing V-ATPase and VNUT, and can be applied to membrane vesicles containing recombinant VNUT as VNUT.

[0040] On the other hand, the measurement methods using proteoliposomes described in Patent Document 1 and Non-Patent Document 7 above can only measure at an extremely low substrate (ATP) concentration of 0.1 mM, which is a fraction of the Km value, for a short period of time (up to about 10 minutes). Furthermore, because the liposome membrane has high ion permeability, H ＋ The driving force cannot be maintained for a long period of time, making it impossible to achieve high uptake levels, and the uptake level decreases rapidly over time. As a result, the measurement results are highly variable and the detection accuracy is low. Furthermore, the measurement method using proteoliposomes requires the separation of proteoliposomes and the external liquid by centrifugal column chromatography for each sample, and this measurement procedure takes more than ten minutes, making it unsuitable for high-throughput measurements.

[0041] In contrast, the method for measuring transporter activity allows measurement using any substrate in the presence of physiologically concentrated ATP (several mM) (see Figure 11), and H formed inside and outside the vesicle membrane. ＋The driving force is higher than that of proteoliposomes, and it remains stable for a long period (more than one hour). Furthermore, its large internal volume allows for highly sensitive measurement of uptake levels (see Figure 1). Additionally, by employing a membrane filter method, for example, in this transporter activity measurement method, high reproducibility can be achieved without significant dependence on the user's skill.

[0042] Furthermore, this transporter activity measurement method allows for the measurement of V-ATPase-energy-coupled VNUT activity in numerous samples using at least 25% less membrane vesicles compared to conventional methods. The measurement efficiency of this transporter activity measurement method is 10 to 100 times higher than that using proteoliposomes, enabling high-throughput testing.

[0043] Furthermore, in this transporter activity measurement method, highly sensitive activity measurement is possible by selecting anion labeled with a radionuclide having high specific radioactivity as the substrate.

[0044] From another perspective, a screening method for VNUT activity modifiers is provided. The differences between this screening method and the transporter activity measurement method described above are primarily explained below. Unless otherwise specified, the transporter activity measurement method described above can be used as the screening method for VNUT activity modifiers. Note that VNUT activity modifiers include VNUT activity inhibitors and VNUT activity enhancers.

[0045] The screening method includes an exposure step, a measurement step, and an evaluation step. The exposure step in the screening method is the same as the exposure step in the transporter activity measurement method described above, except that the reaction solution contains the test substance. That is, in the exposure step in the screening method, membrane vesicles having V-ATPase and VNUT are exposed to anions different from ATP, labeled with the test substance and a radionuclide, in a reaction solution containing ATP and magnesium ions. The test substance is not particularly limited and may include compounds, antibodies, proteins, peptides, DNA aptamers, and RNA aptamers.

[0046] In the measurement step, as described above, the transport activity of anions into membrane vesicles is measured based on the radiation of the radionuclide.

[0047] In the evaluation step, the transport activity is compared to a baseline value to assess the effect of the test substance on the anion transport activity into membrane vesicles. For example, the baseline value is the anion transport activity into membrane vesicles exposed to anions in a reaction solution that does not contain the test substance and has been measured in advance under the same reaction conditions as in the measurement step. Alternatively, the anion transport activity into membrane vesicles measured in a reaction solution containing a substance that inhibits ATP uptake or an ATP secretion inhibitor, such as clodronate, atractyroside, PEP, Pi, sulfate, or SCN, may be used as the baseline value. If the anion transport activity into membrane vesicles measured in the measurement step is lower than the baseline value determined by the positive control, the test substance is selected as a VNUT activity inhibitor. Alternatively, the anion transport activity into membrane vesicles measured in a reaction solution containing a substance that enhances ATP uptake may be used as the baseline value. In this case, if the transport activity of the anion into the membrane vesicle measured in the measurement step is higher than the threshold set by the substance that enhances ATP uptake, the test substance is selected as a VNUT activity enhancer.

[0048] Furthermore, since VNUT activity correlates with ATP secretion, the above screening method can also be used as a screening method for ATP secretion regulators.

[0049] (Embodiment 2) The VNUT activity regulator according to this embodiment comprises itaconic acid or an itaconic acid ester. Itaconic acid is synthesized in animal cells by aconitinate decarboxylase 1 using cis aconitinate, which constitutes the TCA cycle, as a raw material in the mitochondria of macrophages. The itaconic acid ester is not particularly limited as long as it is taken up into the cell and metabolized to produce itaconic acid, and examples include itaconic acid 4-octyl, ethyl itaconic acid, isopropyl itaconic acid, dimethyl itaconic acid, as well as esters of itaconic acid with trihydric alcohols such as glycerin.

[0050] Itaconic acid and itaconic acid esters may be commercially available or produced by known methods. Itaconic acid can be obtained by thermal decomposition of citric acid or aconitic acid, as well as by fermentation using itaconic acid-producing microorganisms.

[0051] The content of itaconic acid or itaconic acid ester in the VNUT activity regulator according to this embodiment can be adjusted as appropriate. For example, the VNUT activity regulator contains 0.000001 to 99.9% by mass, 0.00001 to 99.8% by mass, 0.0001 to 99.7% by mass, 0.001 to 99.6% by mass, 0.01 to 99.5% by mass, 0.1 to 99% by mass, 0.5 to 60% by mass, 1 to 50% by mass, or 1 to 2% by mass of itaconic acid or itaconic acid ester as an active ingredient.

[0052] The target recipients of the VNUT activity regulator according to this embodiment are cells, living tissues, organisms, animals, etc. The VNUT activity regulator as a pharmaceutical is administered to humans and non-human animals. Non-human animals are preferably mammals, more specifically dogs, cats, cattle, pigs, horses, sheep, deer, etc.

[0053] The route of administration of the VNUT activity regulator according to this embodiment to humans is not particularly limited. The VNUT activity regulator is preferably used as a topical preparation, an injection, or an oral preparation. The VNUT activity regulator may include, for example, itaconic acid or itaconic acid ester and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is various organic or inorganic carrier substances used as pharmaceutical materials. Examples of pharmaceutically acceptable carriers include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, etc. in solid preparations, or solvents, solubilizers, suspending agents, isotonic agents, buffers, flavoring agents, etc. in liquid preparations. In addition, additives such as preservatives, antioxidants, colorants, and sweeteners may be added as needed.

[0054] Excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, acacia gum, pullulan, soft anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminometasilicate, xylitol, sorbitol, and erythritol.

[0055] Examples of binders include, in addition to the above-mentioned excipients, pregelatinized starch, sucrose, gelatin, acacia gum, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and the like.

[0056] Examples of disintegrants include, in addition to the above-mentioned excipients, lactose, sucrose, starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethyl starch sodium, low-substituted hydroxypropylcellulose, soft anhydrous silicic acid, calcium carbonate, and the like.

[0057] Examples of lubricants include magnesium stearate, calcium stearate, talc, colloidal silica, and polyethylene glycol.

[0058] Examples of stabilizers include para-hydroxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; and benzalkonium chloride, acetic anhydride, and sorbic acid. Examples of flavoring and odor-modifying agents include sweeteners, acidulants, and fragrances.

[0059] Examples of solvents include sterile water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, and cottonseed oil. Examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, tris(hydroxymethyl)aminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate.

[0060] Examples of suspending agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; and polysorbates and polyoxyethylene hydrogenated castor oil.

[0061] Examples of isotonic agents include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, xylitol, and fructose. Examples of buffering agents include buffer solutions such as phosphates, acetates, carbonates, and citrates.

[0062] Examples of preservatives include para-hydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid. Examples of antioxidants include sulfites and ascorbic acid. Examples of coloring agents include water-soluble colored tar dyes, lake dyes, and natural pigments. Examples of sweeteners include sodium saccharin, dipotassium glycyrrhizinate, aspartame, and stevia.

[0063] The VNUT activity regulator according to this embodiment is manufactured by known methods and provided in the form of, for example, a liquid, cream, ointment, tablet, granule, fine granule, powder, capsule, etc.

[0064] The dosage of the VNUT activity regulator according to this embodiment is appropriately determined based on the age, weight, symptoms, etc., of the human or non-human animal being administered. The VNUT activity regulator is administered in an effective amount of itaconic acid or itaconic acid ester. The effective amount is the amount of itaconic acid or itaconic acid ester necessary to obtain the desired result, and is the amount necessary to delay, inhibit, prevent, reverse, or cure the progression of the condition being treated or managed.

[0065] The dosage of the VNUT activity regulator according to this embodiment is, for example, 0.000001 μg or more per day for adults, preferably 1 μg or more or 500 μg or more, and more preferably 1000 μg or more. The upper limit is, for example, 20000 μg per day, preferably 10000 μg. The VNUT activity regulator can be administered once or multiple times a day. Furthermore, the VNUT activity regulator may be administered at various frequencies such as every other day, once a week, every other week, or once a month. If necessary, amounts outside the above range may also be used.

[0066] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.

[0067] [Preparation of Chromaffin Granulosa] Bovine adrenal chromaffin granulosa (hereinafter simply referred to as "membrane vesicles") were prepared as follows: Approximately 50 adrenal medulla isolated from bovine adrenal glands were suspended in SME buffer (20 mM MOPS-Tris (pH 7.0), 0.3 M sucrose, 5 mM EDTA, 5 μg / ml pepstatin A, 5 μg / ml leupeptin) and homogenized in a Waring blender for approximately 5 seconds. The resulting mixture was filtered through a nylon filter (Sefar), and the obtained filtrate was centrifuged at 1000 × g for 15 minutes. Next, the supernatant was centrifuged at 10,000 × g for 20 minutes, and the pellet was gently suspended in approximately 30 ml of SME buffer. The suspended fractions (approximately 10 ml x 3) were layered on top of a sucrose gradient consisting of 12 ml of 1.8 M sucrose and 15 ml of 1.2 M sucrose, and centrifuged overnight at 20,000 rpm with an SW27 rotor at 4°C. After centrifugation, the fractions were aspirated and removed, and Chromaffin granules precipitated as a pellet were obtained at the bottom of the centrifuge tube. The obtained pellet was suspended in the minimum amount of SME buffer. After homogenizing the suspension with a glass Teflon® homogenizer, the suspension was diluted with approximately 300 ml of 5 mM MOPS-Tris (pH 7.0) containing 5 μg / ml pepstatin A and 5 μg / ml leupeptin. The resulting dilution was centrifuged at 3000 x g for 10 minutes, and the supernatant was centrifuged at 200,000 x g for 1 hour. Next, the obtained pellet was suspended in 5 ml of a solution containing SME buffer and 25% (w / v) glycerol to obtain the membrane vesicle fraction.

[0068] To prepare membrane vesicles with potassium chloride or potassium acetate trapped in the vesicle lumen, 10 mM potassium chloride or potassium acetate was added to the solution during hypotonic treatment of the membrane vesicles, and then 10 mM potassium chloride or potassium acetate was added to the final suspension of the Chromaffin granule membrane. The resulting suspension was frozen and stored at -80°C until use. The membrane vesicles prepared in this manner, with potassium chloride or potassium acetate trapped, maintained their activity even after being stored at -80°C for more than one year.

[0069] [Evaluation of ATP-dependent ATP uptake] ATP-dependent uptake by membrane vesicles [ ３The uptake of [H]ATP was evaluated as follows: 0.5 ml reaction solution (20 mM MOPS-Tris (pH 7.0), 5 mM potassium chloride, 0.3 M sucrose, 5 mM magnesium acetate, 1 mM [ ３ [H]ATP (15KBq / 1 assay, [2,8- ３ Approximately 20-40 μg of membrane vesicles were added to 0.5 mM creatine phosphate and 10 units of creatine kinase (Rabbit Muscle, 367 units / mg protein, Oriental Yeast Co., Ltd.) and incubated at 30°C. 200 μl was collected at regular intervals and filtered through an MF-Millipore™ 0.45 μm MCE membrane filter. The residue on the filter was washed with 10 ml of ice-cooled washing solution (20 mM MOPS-Tris (pH 7.0), 0.3 M sucrose, 5 mM potassium chloride, 5 mM magnesium acetate), dissolved in Clear-sol II (Nacalai Tesque), and the radiation was measured using a liquid scintillation counter.

[0070] [Evaluation of ATP-dependent sulfate ion, Pi, and dopamine uptake] ３５ S] SO ４ ２－ [ ３２ P]Pi and [ ３ [H] Dopamine uptake was evaluated as follows: ATP-dependent [ ３５ S] SO ４ ２－ For incorporation, use 0.5 ml of reaction solution (20 mM MOPS-Tris (pH 7.0), 0.1 M potassium acetate, 10 mM potassium chloride, 0.1 M sucrose, 5 mM magnesium acetate, 1 mM ATP, 0.1 mM [ ３５ S] SO ４ ２－ (37 kBq / 1 assay, [ ３５ S] Na ２ SO ４ ２－ (2 mCi / ml; 74 MBq, manufactured by New England Nuclear Corporation) or [ ３５ S] Na２ SO ４ ２－ (10 mCi / ml; 185 MBq, manufactured by American Radiolabed Chemicals), 0.5 mM creatine phosphate, 10 units creatine kinase) are used, and unless otherwise specified, approximately 10 μg of membrane vesicles are added to the reaction mixture, and the above [ ３ [H] ATP uptake was measured in the same manner.

[0071] [ ３２ For the uptake of P]Pi, 0.5 ml of reaction solution (20 mM MOPS-Tris (pH 7.0), 10 mM potassium chloride, 0.1 M potassium acetate, 0.1 M sucrose, 5 mM magnesium acetate, 1 mM ATP and 0.1 mM [ ３２ Using P]Pi (18.5 kBq / 1 assay, 2 mCi / ml; 74 MBq, New England Nuclear), unless otherwise specified, add approximately 10 μg of membrane vesicles to the reaction mixture, and proceed as described above. ３ [H] ATP uptake was measured in the same manner.

[0072] [ ３ In the case of dopamine uptake, 0.5 ml of reaction solution (20 mM MOPS-Tris (pH 7.0), 50 mM potassium chloride, 0.1 M sucrose, 5 mM magnesium acetate, 0.1 mM [ ３ H) Dopamine (15 KBq / 1 assay, 3, 4-[RING-2, 5, 6- ３ Using [H] dihydroxyphenylethylamine, 37.8 Ci / mmol, New England Nuclear), add approximately 10 μg of membrane vesicles to the reaction solution, and then [ ３ [H] ATP uptake was measured in the same manner.

[0073] The macrolide antibiotic conkanamycin B (conB), a V-ATPase inhibitor, was extracted from the mycelium of Streptomyces sp. AJ9467 with methanol. Further extraction was performed with ethyl acetate (pH 7.5), and purification was carried out by HPLC using silica gel with ethyl acetate and a 60-100% concentration gradient of acetonitrile / water using a YMC-Pack D-ODS-5 column.

[0074] Furthermore, Mg ２＋ In the presence of H, the uptake of dopamine driven by ATP addition is the result of dopamine being filled into vesicles by vesicular monoamine transporters (VMAT) that are present on chromaffin granulosa vesicles, similar to V-ATPase and VNUT (see Non-Patent Document 1 above). This transport is also carried out by H formed by V-ATPase. ＋ While both are active transport mechanisms driven by a motive force, VNUT utilizes the membrane potential difference as an energy source from the proton motive force, whereas VMAT primarily utilizes the pH gradient (ΔpH) as its driving force. Therefore, ATP-dependent dopamine transport (monoamine transport) provides an appropriate control experimental system for elucidating the properties of VNUT activity.

[0075] Protein concentrations were measured using the Bradford method with bovine serum albumin as the standard, according to the BIORAD protocol. Unless otherwise specified, all numerical values ​​in the results are expressed as mean ± standard error of the mean (SEM; n=3–6). Statistical significance was determined by Student's t-test.

[0076] The properties of VNUT activity under physiological conditions (i.e., ATP transport activity coupled with V-ATPase in vesicle membranes) were largely unknown. Therefore, we first clarified the properties of ATP-dependent ATP filling activity in membrane vesicles.

[0077] (Results) Figure 1 shows the time course of ATP uptake in the presence and absence of the ATP regeneration system. In the presence of the ATP regeneration system, time-dependent ATP uptake was observed. On the other hand, in the absence of the ATP regeneration system, ATP uptake hardly increased. When conB (1 μM), the macrolide antibiotic bafilomycin A1 (Baf, 1 μM, Fujifilm Wako Pure Chemical Industries), a V-ATPase inhibitor, or the uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP, 1 μM, Sigma-Aldrich), was added to the reaction mixture, ATP uptake was suppressed. Therefore, it was shown that ATP uptake, which is taken into vesicles by ATP addition and inhibited by conB or Baf, is energetically coupled by V-ATPase. Furthermore, this uptake amount was almost the same as the inhibition amount by CCCP, and H ＋ It was also confirmed that it is driven by a driving force.

[0078] One of the characteristics of the SLC17 transporter family to which VNUT belongs is that it is activated by low concentrations of chloride or bromide ions of about 5 mM (see Non-Patent Documents 1 and 7 above). The ATP-dependent ATP transport activity of Chromaffin granules has also been reported to be activated by chloride ions, but the concentration is very high, about 50 mM (see Non-Patent Document 6), and it was unclear whether the two represent the same activity. Therefore, in order to clarify this point, the chloride ion concentration dependence of ATP-dependent ATP uptake was investigated. [ ３ Figure 2 shows the dependence of ATP uptake on chloride ion concentration when the concentration of [H]ATP is 0.5 to 8 mM. While chloride ions are not necessarily essential for ATP uptake, their presence significantly enhances it. (Standard reaction solution: 1 mM ATP, 5 mM Mg) ＋In this study, the chloride ion concentration that showed maximum uptake was approximately 5 mM. Above this concentration, ATP uptake decreased significantly, and above 50 mM, ATP was hardly taken up at all. The concentration of the substrate, ATP, greatly influenced the activation and inhibitory effects of chloride ions on ATP transport. Specifically, as the ATP concentration increased, the optimal activation concentration increased, and the inhibitory effect of high concentrations of chloride ions decreased. These activation and inhibitory effects were considered to be allosteric effects mediated by chloride ions.

[0079] Next, the potassium chloride in the reaction solution was replaced with potassium bromide, and the concentration dependence of ATP-dependent ATP uptake on bromide ion levels was investigated. As shown in Figure 3, bromide ions also promoted ATP uptake at low concentrations and inhibited it at high concentrations. Bromide ions promoted ATP uptake at lower concentrations than chloride ions. The optimal concentration of bromide ions in the presence of 4 mM ATP was approximately 2 mM. No anion was found that activated ATP-dependent ATP uptake as strongly as chloride ions and bromide ions.

[0080] On the other hand, as shown in Figure 4, the concentration dependence of sulfate ions on ATP uptake in the presence or absence of chloride ions was investigated. Specifically, a predetermined concentration of SO was added to the reaction solution containing potassium chloride or to the reaction solution without potassium chloride. ４ ２－ 40 minutes later in [ ３ When ATP uptake was measured, as shown in Figure 4, sulfate ions strongly inhibited ATP uptake in the presence and absence of chloride ions. The 100% activity in relative activity corresponded to 13 nmol / mg protein in the presence of chloride ions and 4.1 nmol / mg protein in chloride-independent ATP uptake.

[0081] The inhibition of ATP transport by sulfate ions was observed regardless of the presence or absence of chloride ions, suggesting that this is clearly different from the allosteric activation effect of chloride ions and is directly related to the ATP transport mechanism mediated by VNUT.

[0082] Furthermore, the concentration dependence of Pi on ATP uptake in the presence of chloride ions was investigated. [ ３ When ATP uptake was measured, Pi strongly inhibited ATP uptake, as shown in Figure 5. The 100% activity in relative activity corresponded to 13 nmol / mg of protein.

[0083] Furthermore, the addition of potassium acetate, sodium acetate, potassium gluconate, and sodium gluconate up to 0.1 M did not affect ATP-dependent ATP uptake by the vesicle membrane.

[0084] The results of these measurements revealed that the anions can be broadly classified into three types: (1) anions that activate ATP-dependent ATP uptake by the vesicle membrane, (2) anions that inhibit ATP-dependent ATP uptake by the vesicle membrane, and (3) anions that do not affect ATP-dependent ATP uptake by the vesicle membrane. The ATP transport inhibitory effect of sulfate ions and Pi was specific, suggesting that sulfate ions and Pi may be transport substrates for VNUT.

[0085] Therefore, ATP-dependent [ ３５ When we examined sulfate ion uptake, as shown in Figure 6, membrane vesicles took up sulfate ions in an ATP and time-dependent manner (Complete). When 1 μM conB was added, ATP-dependent sulfate ion uptake was completely suppressed to the level of no ATP added. Furthermore, when 1 μM conB (at 35 min. + conB) or 1 μM CCCP (at 35 min. + CCCP) was added 30 minutes from the start of incubation, the amount of sulfate ions taken up by membrane vesicles in an ATP-dependent manner decreased, indicating that H ＋The disappearance of the driving force suggested that sulfate ions trapped within the membrane vesicles were released. The sulfate ion uptake (no ATP) when ATP was not added to the reaction mixture remained unchanged compared to when ATP was not added to the reaction mixture and conB (no ATP + conB) or CCCP (no ATP + CCCP), indicating that conB and CCCP do not affect ATP-independent sulfate ion uptake. Therefore, ATP-dependent sulfate ion uptake by membrane vesicles is linked to the formation of H by V-ATPase. ＋ We concluded that this is active transport using a gradient. The ATP-independent uptake of sulfate ions is thought to be due to the nonspecific binding of membrane vesicles to the membrane.

[0086] Using a reaction solution without added potassium chloride and membrane vesicles containing trapped potassium acetate, we investigated the ATP-dependent reaction in the absence of chloride ions. ３５ The uptake of sulfate ions was evaluated. As shown in Figure 7, in the absence of chloride ions, ATP-dependent sulfate [ ３５ S] The uptake of sulfate ions was minimal (ATP), and even with the addition of valinomycin (0.1 μg, Sigma-Aldrich) or CCCP (1 μM) it was minimal (+val. and +CCCP). Under these conditions, the solution was electrically neutral and H ＋ / K ＋ The addition of nigericin (0.1 μg, Sigma-Aldrich), an antiporter ionophore, introduces ATP-dependent H into the vesicle membrane. ＋ Of the driving force, only the membrane potential difference is formed, and [ ３５ S]Sulfate ion uptake was significantly enhanced (+nig.). This enhancement of sulfate ion uptake by nigericin was strongly suppressed by valinomycin, which eliminated the membrane potential (+nig. +val.). In the presence of 10 mM potassium chloride, sulfate ion uptake increased (+10 mM KCl). In the reaction solution with added potassium chloride, sulfate ion uptake increased in membrane vesicles trapped with potassium acetate, as shown in Figure 8 (ATP), sulfate ion uptake was significantly enhanced by nigericin, and suppressed by valinomycin or CCCP. These results suggest that ATP-dependent uptake in vesicles is present regardless of the presence or absence of chloride ions. ３５It was shown that the uptake of sulfate ions (S) is driven by the membrane potential formed by V-ATPase.

[0087] Next, ATP-dependent [ ３５ We investigated whether the uptake of sulfate ions (S) exhibits the same properties as ATP-dependent ATP uptake. ３５ Figure 9 shows the KCl or KBr concentration dependence of sulfate ion uptake. ATP dependence [ ３５ It was found that sulfate ion uptake, like ATP-dependent ATP uptake, is strongly activated by chloride ions and bromide ions. ATP-dependent sulfate ion uptake was maximized at 15 mM for chloride ions and decreased above 20 mM. For bromide ions, ATP-dependent sulfate ion uptake was maximized at 5 mM.

[0088] Furthermore, as shown in Figure 10, Pi inhibited ATP-dependent sulfate ion uptake, similar to ATP-dependent ATP uptake. Potassium acetate, sodium acetate, potassium gluconate, and sodium gluconate did not affect ATP-dependent sulfate ion uptake even when added up to 0.1 M. These results indicate that the effects of various anions on ATP-dependent sulfate ion uptake and ATP uptake are almost identical.

[0089] Figure 11 shows the relationship between the sulfate concentration in the reaction solution and the uptake of ATP-dependent sulfate ions. Sulfate ion uptake showed saturation with respect to the sulfate concentration in the reaction solution, and the Km value was ~1 mM.

[0090] Next, the effects of ATP-dependent ATP uptake inhibitors on ATP-dependent sulfate ion uptake were investigated. Atractyroside (Cayman Chemical), DIDS (Cayman Chemical, see Non-Patent Document 7 above), and PEP (Fujifilm Wako Pure Chemical Industries) are known to function as VNUT inhibitors. ATP-dependent sulfate ion uptake was evaluated in reaction solutions to which these VNUT inhibitors or pyruvate (Fujifilm Wako Pure Chemical Industries) were added at predetermined concentrations. As shown in Figure 12, atractyroside at 10 μM inhibited sulfate ion uptake by approximately 68%. As shown in Figures 13 and 14, DIDS and PEP strongly inhibited sulfate ion uptake, respectively. ５０ The concentrations were approximately 0.3 μM and 0.14 mM, respectively. On the other hand, pyruvate, which does not inhibit VNUT, was ineffective. These results indicate that ATP-dependent sulfate ion uptake by membrane vesicles has similar characteristics to ATP uptake. Note that 100% activity in the relative activity shown in Figures 12 to 14 corresponds to 0.6 nmol / mg of protein.

[0091] Add a predetermined concentration of potassium sulfate or potassium gluconate to the reaction solution, and ATP-dependent reaction by membrane vesicles [ ３２ P]Pi uptake was measured. As shown in Figure 15, sulfate ions inhibited ATP-dependent Pi uptake. On the other hand, potassium gluconate inhibited ATP-dependent [ ３２ P]Pi uptake was not inhibited. ATP-dependent [ ３２ P]Pi uptake was inhibited by PEP, atractyroside, and DIDS, as shown in Figure 16. From these results, ATP-dependent uptake by membrane vesicles [ ３２ P]Pi import is [ ３５ S] Sulfate ions and [ ３ It was found to exhibit anion sensitivity and drug sensitivity similar to that of [H]ATP uptake.

[0092] [SLC17A9 －／－ ATP-dependent SO2 in mice ４ ２－[Investigation of Uptake] To confirm that VNUT is involved in ATP-dependent sulfate ion uptake, we investigated wild-type and SLC17A9 －／－ ATP-dependent SO2 in the adrenal membrane fraction of mice ４ ２－ Uptake capacity was measured for wild-type and SLC17A9. －／－ Mouse adrenal membrane fractions were prepared as follows: wild type and SLC17A9 －／－ Fifty adrenal glands isolated from mice were suspended in SME buffer containing 10 μg / ml each of pepstatin A and leupeptin, homogenized in four strokes using a Downs homogenizer, and centrifuged at 1000 × g for 15 minutes. The supernatant was then centrifuged at 10,000 × g for 20 minutes. The pellet was gently suspended in approximately 1 ml of SME buffer containing 10 μg / ml each of pepstatin A and leupeptin. The suspension was then quickly poured into 40 ml of 5 mM MOPS-Tris (pH 7.0) containing 5 μg / ml pepstatin A and 5 μg / ml leupeptin, centrifuged at 3000 × g for 10 minutes, and the supernatant was centrifuged at 200,000 × g for 1 hour. The resulting palette was then homogenized with 5 ml of a solution containing SME and 25% (w / v) glycerol and used in the assay.

[0093] (Results) As shown in Figure 17, the adrenal membrane fraction of wild-type mice took up sulfate ions in an ATP-dependent manner. This uptake was suppressed in the presence of 1 mM PEP. The activity of ATP-dependent sulfate ion uptake in the adrenal membrane fraction of wild-type mice was found in SLC17A9. －／－ It disappeared in the adrenal membrane fraction of mice.

[0094] Furthermore, as a control experiment to confirm that this ATP-dependent sulfate ion uptake activity is caused by the gene deletion of SLC17A9 (VNUT), wild-type and SLC17A9 －／－The ATP-dependent and reserpine-sensitive dopamine uptake activity in the adrenal membrane fraction of mice was measured and compared with that of wild-type mice. Since this activity corresponds to the V-ATPase and energy-coupled vesicular monoamine transporter activity in the same chromaffin granule membrane, it is a good control experiment indicating that granule membrane function is retained (see Non-Patent Documents 1, 2, and 8).

[0095] As shown in Fig. 18, both adrenal membrane fractions showed ATP-dependent and reserpine-sensitive dopamine uptake. SLC17A9 －／－ The activity of dopamine uptake in the adrenal membrane fraction of mice was approximately 70% of that of wild-type mice. This result was in good agreement with the results of Non-Patent Document 8.

[0096] From the above results, it was concluded that VNUT, which is energetically coupled with V-ATPase in chromaffin granule membrane vesicles, transports sulfate ions and Pi.

[0097] 〔Inhibition of ATP-dependent sulfate ion and Pi uptake by itaconic acid〕 Preparation of chromaffin granule membrane vesicles and measurement of the uptake of ３５ [ ３２ S ] sulfate ions, [ ３ P ] Pi, and [ ＋ H ＋ ] dopamine were performed by the method described above. The membrane potential difference (V-ATPase membrane potential) among the H ＋ driving forces generated by the addition of ATP, and the ATP-dependent L-glutamate uptake in synaptic vesicles were measured based on Yoshinori Moriya, et al., Is the vesicular nucleotide transporter a molecular target of eicosapentaenoic acid?, 2022, Frontiers in Pharmacology, 1080189.

[0098] (Results) As shown in Fig. 19, the ATP-dependent ３５ [ ３２ S ] sulfate ion and [ ３２ ＋ P ] Pi uptake was inhibited by approximately 50% by 2 mM itaconic acid. On the other hand, ATP-dependent dopamine transport, H ＋The driving force (membrane potential) and ATP-dependent L-glutamate uptake by synaptic vesicles had little effect up to 4 mM. Furthermore, cis-aconitinate and succinate, TCA cycle intermediate metabolites near itaconic acid, did not affect VNUT function up to 5 mM.

[0099] [ATP secretion inhibitory effect of itaconic acid 4-octyl] We investigated whether VNUT inhibition by itaconic acid actually occurs within cells, suppresses ATP secretion, and is involved in anti-inflammatory effects. It is known that when itaconic acid 4-octyl is incubated with cells, it is taken up into the cells, and octyl alcohol and itaconic acid are released by cytoplasmic esterases. Itaconic acid 4-octyl is a reagent that supplies itaconic acid into cells and is widely used to investigate the effects of itaconic acid within cells.

[0100] Human epidermal keratinocytes (hereinafter referred to as keratinocytes) are the primary source of extracellular ATP in the skin. Similar to nerve and hormone-secreting cells, they accumulate ATP in secretory vesicles through the action of VNUT and V-ATPase, and secrete ATP through exocytosis induced by ultraviolet light, mechanical stimulation, or chemical stimulation. We investigated whether ATP secretion was suppressed when cultured keratinocytes were treated with 4-octyl eutaxone (4-OI).

[0101] [Culturing Keratinocytes] Keratinocytes (Cryopreserved HEK Lot 1758, Cell Applications) were cultured using the Epivita Keratinocyte Growth Medium Kit according to the instructions. The procedure was as follows: A complete medium was prepared by adding supplement medium to the hydrocortisone-containing basic medium from the culture medium preparation kit. The medium in the dish was aspirated and removed, then washed with 5 ml of balanced salt solution (BSS, Gibco) and aspirated again. 5-6 ml of trypsin / EDTA solution (Gibco) was added, and the culture was incubated at 37°C and 5% CO2. ２It was incubated for 2 minutes. 5 ml of trypsin neutralization solution was added, and the cell suspension was placed in two 15-ml Falcon tubes, centrifuged at 200×g for 5 minutes, and the supernatant was aspirated and removed. The pellet-like cell mass was suspended in 2 ml of medium, adjusted to 4.5×10 ５ cells / ml, part of it was cryopreserved, and part of it was cultured and used for measurement.

[0102] [ATP Secretion Measurement] Keratinocytes were placed in a 24-well plate at 37°C, 5% CO ４ to a density of 2.5×10 ２ cells / well and cultured for 4 days. The medium was Krebs ringer-HEPES supplemented with 0.2% BSA and 25 mM glucose (hereinafter also referred to as "Krebs / Iono(-)"). The medium was aspirated and removed, and Krebs / Iono(-) as a negative control, Krebs / Iono(-) containing a predetermined concentration of 4-octyl itaconate (4-IO), or Krebs / Iono(-) with 5 μM ionomycin added as a positive control (hereinafter also referred to as "Krebs / Iono(+)") were used to incubate at 37°C, 5% CO ２ for 2 hours. Ionomycin is a Ca ２＋ ionophore, and when added to cells, it promotes the influx of extracellular Ca ２＋ into the cells, increases the intracellular Ca ２＋ concentration, and as a result, is known to promote ATP secretion.

[0103] Subsequently, these solutions were aspirated and removed, the wells were replaced with 300 μl of control solution (Krebs / Iono(-) or Krebs / Iono(+)), and further incubated at 37°C, 5% CO ２ for 20 minutes, and 200 μl of the supernatant was collected (N = 5). The immediately collected supernatant was treated at 95°C for 10 minutes using a heat block to inactivate ectonucleotidase. The ATP in the supernatant was quantified using Dojindo ATP assay kit-Luminescence (manufactured by Dojindo).

[0104] (Results) As shown in Figure 20, ionomycin increased ATP secretion. On the other hand, keratinocytes treated with 4-octyl itaconic acid showed suppressed ATP secretion in a concentration-dependent manner with 4-octyl itaconic acid. Furthermore, the survival rate of keratinocytes treated with 4-octyl itaconic acid was no different from that of the control group. Clodronate (10 μM), known as a VNUT inhibitor, suppressed ionomycin-promoted ATP secretion to a similar extent as 4-octyl itaconic acid. These results indicate that itaconic acid suppresses ATP secretion within keratinocyte cells by inhibiting VNUT.

[0105] [Inhibition of intravesicular ATP filling by itaconic acid 4-octyl] The fluorescent ATP derivative 2' / 3'-O-(N-methyl-anthraniloyl)-adenosine-5'-triethylammonium phosphate (MANT-ATP, Invitrogen) is taken up into cells and accumulates in ATP-filled granules when incubated with keratinocytes. When VNUT expression is suppressed by RNAi, this accumulation ceases, suggesting that this accumulation is a result of VNUT recognizing MANT-ATP as a substrate and transporting it into the granules (Non-patent Literature 9). The fact that MANT-ATP is a transport substrate for VNUT has already been shown using bovine adrenal medulla chromaffin granulosa vesicles. MANT-ATP is thought to act as a competitive inhibitor of VNUT and inhibit ATP-dependent uptake of radioactive ATP.

[0106] To demonstrate that the target of the ATP secretion inhibitory effect of itaconic acid 4-octyl is ATP-filled vesicles, the effect of itaconic acid 4-octyl on the intragranular accumulation of MANT-ATP was investigated. Keratinocytes were cultured as described above. Keratinocytes were cultured in 4 × 10⁶ units. ４ Cells / ml are measured using a Multi-Well Glass Bottom Dish (D141410, 35mm type, well size: 9.5mm, manufactured by Matsunami Glass Industry Co., Ltd.) at 37°C and 5% CO2. ２The cells were cultured for two days, then the culture medium was replaced with one containing MANT-ATP (Invitrogen) to a final concentration of 0.3 mM, and the cells were incubated for 72 hours. Two hours or 18 hours before the end of incubation, 4-octyl itaconic acid was added to a concentration of 100 μM. Subsequently, the cells were washed twice with HBSS (Gibco) and observed using an inverted confocal laser microscope (Leica, STELLARIS 5). As a positive control, 10 μM clodronate was added 18 hours before the end of incubation, and the same procedure was followed before observation using an inverted confocal laser microscope.

[0107] (Results) As shown in Figure 21, in the control group, vesicles accumulating MANT-ATP were observed mainly around the nucleus, and MANT-ATP accumulated within the granules of keratinocytes, whereas this accumulation disappeared in cells treated with clodronate. This indicated that MANT-ATP was packed into ATP-filled granules in a VNUT-dependent manner. When itaconic acid 4-octyl was applied, the accumulation of MANT-ATP in vesicles was suppressed, similar to clodronate, and the accumulation of MANT-ATP in vesicles was more strongly suppressed by 18 hours of treatment than by 2 hours of treatment. These results indicate that itaconic acid suppresses ATP secretion within keratinocyte cells by inhibiting VNUT activity.

[0108] The embodiments described above are for illustrative purposes only and do not limit the scope of the present invention. That is, the scope of the present invention is defined not by the embodiments, but by the claims. Various modifications made within the scope of the claims and equivalent inventive meaning are considered to be within the scope of the present invention.

[0109] This application is based on Japanese Patent Application No. 2024-224658, filed on December 20, 2024. The entire specification, claims, and drawings of Japanese Patent Application No. 2024-224658 are incorporated herein by reference.

[0110] This invention is useful for screening pharmaceuticals.

Claims

1. A method for measuring transporter activity, comprising: exposing a membrane vesicle having vacuolar ATPase and a vesicular nucleotide transporter to an anion different from adenosine triphosphate, labeled with a radionuclide, in a reaction solution containing adenosine triphosphate and magnesium ions; and measuring the transport activity of the anion into the membrane vesicle based on the radiation of the radionuclide.

2. A screening method for vesicular nucleotide transporter activity modifiers, comprising: exposing membrane vesicles having vacuolar ATPase and vesicular nucleotide transporters to an anion different from adenosine triphosphate, labeled with a test substance and a radionuclide, in a reaction solution containing adenosine triphosphate and magnesium ions; measuring the transport activity of the anion into the membrane vesicles based on the radiation of the radionuclide; comparing the transport activity with a reference value; and evaluating the effect of the test substance on the transport activity of the anion into the membrane vesicles.

3. The method for measuring transporter activity according to claim 1, or the method for screening vesicular nucleotide transporter activity regulators according to claim 2, wherein the anion is a sulfate ion or orthophosphate.

4. The method for measuring transporter activity according to claim 1, or the method for screening vesicular nucleotide transporter activity regulators according to claim 2, wherein the reaction solution further comprises chloride ions or bromide ions.

5. The method for measuring transporter activity or screening a vesicular nucleotide transporter activity regulator according to claim 4, wherein the reaction solution further contains chloride ions, and the concentration of chloride ions in the reaction solution is 4 to 20 mM.

6. The method for measuring transporter activity or screening a vesicular nucleotide transporter activity regulator according to claim 5, wherein the anion is a sulfate ion at a concentration of 0.1 mM in the reaction solution, and the concentration of the chloride ion in the reaction solution is 15 mM.

7. The method for measuring transporter activity or screening a vesicular nucleotide transporter activity regulator according to claim 4, wherein the reaction solution further contains bromide ions, and the concentration of bromide ions in the reaction solution is 1 to 5 mM.

8. The method for measuring transporter activity or screening a vesicular nucleotide transporter activity regulator according to claim 7, wherein the anion is a sulfate ion at a concentration of 0.1 mM in the reaction solution, and the concentration of the bromide ion in the reaction solution is 2 mM.

9. A vesicular nucleotide transporter activity modulator containing itaconic acid or an itaconic acid ester.

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