Fumiyo Kasuya

Involvement of the long-chain fatty acid receptor GPR40 as a novel pain regulatory system.

G-protein receptor (GPR) 40 is known to be activated by docosahexaenoic acid (DHA). However, reports studying the role and functions (including pain regulation) of GPR40 in the brain are lacking. We investigated the involvement of GPR40 in the brain on DHA-induced antinociceptive effects. Expression of GPR40 protein was observed in the olfactory bulb, striatum, hippocampus, midbrain, hypothalamus, medulla oblongata, cerebellum and cerebral cortex in the brain as well as the spinal cord, whereas GPR120 protein expression in these areas was not observed. Intracerebroventricular (i.c.v.), but not intrathecal (i.t.) injection of DHA (25 and 50μg/mouse) and GW9508 (a GPR40- and GPR120-selective agonist; 0.1 and 1.0μg/mouse) significantly reduced formalin-induced pain behavior. These effects were inhibited by pretreatment with the μ opioid receptor antagonist β-funaltrexamine (β-FNA), naltrindole (δ opioid receptor antagonist) and anti-β-endorphin antiserum. The κ opioid receptor antagonist norbinaltorphimine (nor-BNI) did not affect the antinociception of DHA or GW9508. Furthermore, the immunoreactivity of β-endorphin in the hypothalamus increased at 10 and 20min after i.c.v. injection of DHA and GW9508. These findings suggest that DHA-induced antinociception via β-endorphin release may be mediated (at least in part) through GPR40 signaling in the supraspinal area, and may provide valuable information on a novel therapeutic approach for pain control.

Hypothalamic GPR40 Signaling Activated by Free Long Chain Fatty Acids Suppresses CFA-Induced Inflammatory Chronic Pain

GPR40 has been reported to be activated by long-chain fatty acids, such as docosahexaenoic acid (DHA). However, reports studying functional role of GPR40 in the brain are lacking. The present study focused on the relationship between pain regulation and GPR40, investigating the functional roles of hypothalamic GPR40 during chronic pain caused using a complete Freunds adjuvant (CFA)-induced inflammatory chronic pain mouse model. GPR40 protein expression in the hypothalamus was transiently increased at day 7, but not at days 1, 3 and 14, after CFA injection. GPR40 was co-localized with NeuN, a neuron marker, but not with glial fibrillary acidic protein (GFAP), an astrocyte marker. At day 1 after CFA injection, GFAP protein expression was markedly increased in the hypothalamus. These increases were significantly inhibited by the intracerebroventricular injection of flavopiridol (15 nmol), a cyclin-dependent kinase inhibitor, depending on the decreases in both the increment of GPR40 protein expression and the induction of mechanical allodynia and thermal hyperalgesia at day 7 after CFA injection. Furthermore, the level of DHA in the hypothalamus tissue was significantly increased in a flavopiridol reversible manner at day 1, but not at day 7, after CFA injection. The intracerebroventricular injection of DHA (50 µg) and GW9508 (1.0 µg), a GPR40-selective agonist, significantly reduced mechanical allodynia and thermal hyperalgesia at day 7, but not at day 1, after CFA injection. These effects were inhibited by intracerebroventricular pretreatment with GW1100 (10 µg), a GPR40 antagonist. The protein expression of GPR40 was colocalized with that of β-endorphin and proopiomelanocortin, and a single intracerebroventricular injection of GW9508 (1.0 µg) significantly increased the number of neurons double-stained for c-Fos and proopiomelanocortin in the arcuate nucleus of the hypothalamus. Our findings suggest that hypothalamic GPR40 activated by free long chain fatty acids might have an important role in this pain control system.

Participation of a medium chain acyl-CoA synthetase in glycine conjugation of the benzoic acid derivatives with the electron-donating groups

Glycine conjugation of a series of benzoic acid derivatives was investigated in bovine liver mitochondria. Benzoic acids with chlorine, methyl, methoxy or ethoxy substituents in the para-or meta-positions of the benzene ring showed a high degree of glycine conjugation. In contrast, the acids with cyano, nitro, amino, or acetylamino groups were conjugated to a small extent with glycine. A medium chain acyl-CoA synthetase that activates carboxylic acids was purified from bovine liver mitochondria. The purified medium chain acyl-CoA synthetase accepted not only medium chain fatty acids but also aromatic and arylacetic acids as substrates. There was a good correlation between the activity of the purified medium chain acyl-CoA synthetase and glycine conjugation of ten benzoic acids with electron-donating substituents. These findings indicate that the purified medium chain acyl-CoA synthetase is a major enzyme for glycine conjugation of benzoic acids with electron-donating groups in bovine live mitochondria.

The analysis of cocaine and its metabolites by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS)

The method for simultaneous determination of cocaine and its four metabolites (benzoylecgonine, ecgonine methyl ester, ecgonine and norcocaine) in urine by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS) was studied. The mass spectra showed the quasi-molecular ions, [M+H]+ as the base peak. LC/APCI-MS analysis was performed by focusing the characteristic ions at m/ = 186, 290, 200, 304 and 290 for ecgonine, benzoylecgonine, ecgonine methyl ester, cocaine and norcocaine, respectively. Cocaine and its four metabolites were well separated by high performance liquid chromatography (HPLC). The recoveries of cocaine and its metabolites from the spiked urine were 40.3-94.7% by solid-phase extraction with two type cartridges (Bond Elut Certify and Bond Elut SCX).

Determination of cabergoline and l-dopa in human plasma using liquid chromatography–tandem mass spectrometry

We determined cabergoline and L-dopa in human plasma using liquid chromatography-mass spectrometry with tandem mass spectrometry (LC-MS-MS). The deproteinized plasma samples with organic solvent or acid were analyzed directly by reversed-phase liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 381 of m/z 452 for cabergoline and m/z 152 of m/z 198 for L-dopa) on LC-MS-MS with electrospray ionization (ESI), cabergoline and L-dopa in human plasma were determined. Calibration curves of the method showed a good linearity in the range 5-250 pg/ml for cabergoline and 1-200 ng/ml for L-dopa, respectively. The limit of determination was estimated to be approximately 2 pg/ml for cabergoline and approximately 0.1 ng/ml for L-dopa, respectively. The method was applied to the analysis of cabergoline and L-dopa in plasma samples from patients treated with these drugs. The precision of analysis showed coefficients of variation ranging from 3.8% to 10.5% at cabergoline concentration of 13.8-26.2 pg/ml and from 2.9% to 8.9% at an L-dopa concentration of 302.5-522.1 ng/ml in patient plasma. As a result, the procedure proved to be very suitable for routine analysis.

Possible involvement of β-endorphin in docosahexaenoic acid-induced antinociception.

We have previously demonstrated that the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) has an antinociceptive effect on various pain stimuli in a naloxone-reversible manner. In the present study, the role of the endogenous opioid peptide β-endorphin in DHA-induced antinociception was examined. DHA-induced antinociception was abolished when mice were pretreated with the μ-opioid receptor antagonist β-funaltrexamine (β-FNA) and the δ-opioid receptor antagonist naltrindole, but not by the κ-opioid receptor antagonist nor-binaltorphimine (nor-BNI) in the acetic acid-induced writhing test. In the radioligand binding assay, DHA itself did not have affinity for μ- , δ- or κ-opioid receptors. On the other hand, the pretreatment of anti-β-endorphin antiserum inhibited DHA-induced antinociception. Furthermore, the intracerebroventricular injection of DHA dose-dependently reduced writhing behavior, and this effect was inhibited by d-Phe-Cys-Tyr-Orn-Thr-Pen-Thr-NH(2) (CTOP) and naltrindole, but not nor-BNI. β-endorphin-induced antinociception was inhibited by the pretreatment of β-FNA, but not naltrindole or nor-BNI, and its levels in plasma were increased by DHA treatment. These findings suggest that the induction of antinociception by DHA may partially involve the μ-opioid receptor via the release of β-endorphin.

Differentiation of regioisomeric fluoroamphetamine analogs by gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry

In recent years, a large number of clandestinely produced controlled-substance analogs (designer drugs) of amphetamine with high structural variety have been detected in forensic samples. Analytical differentiation of regioisomers is a significant issue in forensic drug analysis because, in most cases, legal controls are placed only on one or two of the three isomers. In this study, we used gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the differentiation of regioisomers of fluoroamphetamine analogs (fluoroamphetamines and fluoromethamphetamines), which were synthesized in our laboratories. Free bases and their acylated and silylated derivatives were subjected to GC–MS analysis using DB-1ms, DB-5ms, and DB-17ms capillary columns. The separation of free bases was incomplete on all columns. Trifluoroacetyl derivatives of 3- and 4-positional isomers showed slight separation on DB-1ms and DB-5ms. On the other hand, trimethylsilyl derivatization enabled baseline separation of six fluoroamphetamine analogs on DB-1ms and DB-5ms columns, which was sufficient for unequivocal identification. For LC–MS/MS, a pentafluorophenyl column was able to separate six regioisomeric fluoroamphetamine analogs but a conventional C18 column could not achieve separation between 3- and 4-positional isomers. These results show that a suitable choice of derivatization and analytical columns allows the differentiation of regioisomeric fluoroamphetamine analogs.

Difference of the liver and kidney in glycine conjugation of ortho-substituted benzoic acids

The relative importance of the liver and kidney for glycine conjugation of ortho-substituted benzoic acids was investigated. Glycine conjugation of ortho-substituted benzoic acids was investigated in mouse liver and kidney mitochondria. The extent of glycine conjugation of benzoic acids with the halogen group decreased in the order F > Cl > Br > I. The conjugation of salicylic acid with glycine took place in only the kidney. 2-Methoxybenzoic acid exhibited no activity in the liver and kidney. The difference in glycine conjugation of ortho-substituted benzoic acids was observed between liver and kidney. The kidney was more active in glycine conjugation of ortho-substituted acids than the liver. In addition, the relationship between glycine conjugation and the chemical structure of ortho-substituted acids was examined in the liver and kidney. The size of the substituent had a far greater influence over glycine conjugation in the liver and kidney. Glycine conjugation was also dependent on the substituent electronegativity. It may be important that the substrates undergoing glycine conjugation contain a flat region coplanar to the carboxylate group.

Release of vesicular Zn2+ in a rat transient middle cerebral artery occlusion model

In the brain, Zn(2+) is stored in synaptic vesicles of a subgroup of glutamatergic nerve terminals. Although it has been reported that this Zn(2+) is released upon the excitation of nerves in vitro, there has been little study of the release of Zn(2+) during ischemia in vivo. Here, using brain microdialysis, the release of vesicular Zn(2+) was investigated in vivo. When the vesicular Zn(2+) was released into the synaptic cleft by a depolarizing stimulation achieved by perfusion with Ringers solution containing high K(+) (100mM KCl), a significant increase in the extracellular concentration of Zn(2+) could be detected by microdialysis. Then, we investigated the release of vesicular Zn(2+) in a rat transient middle cerebral artery occlusion model using microdialysis. Consequently, the extracellular Zn(2+) level in the cortex increased within 15 min of the start of occlusion and reached a peak at 30 min, which was about twice the basal level. After 30 min, it declined with time returning to the basal level 15 min after reperfusion, which was performed after 60 min of occlusion. The results suggest that vesicular Zn(2+) would be released into the synaptic cleft during brain ischemia in vivo.

The activation of supraspinal GPR40/FFA1 receptor signalling regulates the descending pain control system

The ω‐3 polyunsaturated fatty acids exert antinociceptive effects in inflammatory and neuropathic pain; however, the underlying mechanisms remain unclear. Docosahexaenoic acid‐induced antinociception may be mediated by the orphan GPR40, now identified as the free fatty acid receptor 1 (FFA1 receptor). Here, we examined the involvement of supraspinal FFA1 receptor signalling in the regulation of inhibitory pain control systems consisting of serotonergic and noradrenergic neurons.