Perturbation of monoamine metabolism and enhanced fear responses in mice defective in the regeneration of tetrahydrobiopterin.

Increasing evidence suggests the involvement of peripheral amino acid metabolism in the pathophysiology of neuropsychiatric disorders, whereas the molecular mechanisms are largely unknown. Tetrahydrobiopterin (BH4) is a cofactor for enzymes that catalyze phenylalanine metabolism, monoamine synthesis, nitric oxide production, and lipid metabolism. BH4 is synthesized from guanosine triphosphate and regenerated by quinonoid dihydropteridine reductase (QDPR), which catalyzes the reduction of quinonoid dihydrobiopterin. We analyzed Qdpr-/- mice to elucidate the physiological significance of the regeneration of BH4. We found that the Qdpr-/- mice exhibited mild hyperphenylalaninemia and monoamine deficiency in the brain, despite the presence of substantial amounts of BH4 in the liver and brain. Hyperphenylalaninemia was ameliorated by exogenously administered BH4, and dietary phenylalanine restriction was effective for restoring the decreased monoamine contents in the brain of the Qdpr-/- mice, suggesting that monoamine deficiency was caused by the secondary effect of hyperphenylalaninemia. Immunohistochemical analysis showed that QDPR was primarily distributed in oligodendrocytes but hardly detectable in monoaminergic neurons in the brain. Finally, we performed a behavioral assessment using a test battery. The Qdpr-/- mice exhibited enhanced fear responses after electrical foot shock. Taken together, our data suggest that the perturbation of BH4 metabolism should affect brain monoamine levels through alterations in peripheral amino acid metabolism, and might contribute to the development of anxiety-related psychiatric disorders. Cover Image for this issue: https://doi.org/10.1111/jnc.15398.

Tokishakuyakusan ameliorates lowered body temperature after immersion in cold water through the early recovery of blood flow in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: 'Cold feeling' is a subjective feeling of unusual coldness that aggravates fatigue, stiffness, and other symptoms, thereby reducing quality of life. Tokishakuyakusan (TSS) is a Kampo medicine reported to improve cold feeling and is used to treat symptoms aggravated by cold feeling. However, the mechanism of action of TSS is unclear. Cold feeling may involve reduced blood flow and subsequent inhibition of heat transport. Therefore, elucidating the effects of TSS on blood flow is one of the most important research topics for clarifying the mechanism of action of TSS. AIM OF THE STUDY: We aimed to evaluate the effect of TSS on recovery from lowered body temperature by the immersion of rats in cold water and to clarify the involvement of blood flow in the action of TSS. MATERIALS AND METHODS: After female Wistar rats underwent 9 days of low room temperature stress loading (i.e. room temperature of 18 °C), they were subjected to immersion in cold water (15 °C) for 15 min. Body surface temperature, rectal temperature, and plantar temperature were measured before and after immersion in cold water. Blood flow was measured before and after immersion in cold water without low room temperature stress loading. TSS (0.5 g/kg or 1 g/kg) or the vehicle (i.e. distilled water) was orally administered once daily for 10 days for the measurement of body temperature or once 30 min before immersion in cold water for the measurement of blood flow. In addition, we examined the effect of TSS on calcitonin gene-related peptide (CGRP) release from dorsal root ganglion (DRG) cells, the effect of TSS ingredients on transient receptor potential (TRP) channels, and the effect of TSS ingredients on the membrane potential of vascular smooth muscle cells and evaluated the mechanism of the effects of TSS on blood flow. RESULTS: Body temperature and blood flow decreased after immersion in cold water and then recovered over time. A comparison of body temperature at each timepoint or area under the curve showed that TSS (1 g/kg) accelerated the recovery of body surface temperature, rectal temperature, and blood flow. TSS significantly increased CGRP release from DRG cells, which disappeared after pretreatment with HC-030031 (a transient receptor potential ankyrin 1 [TRPA1] antagonist). The effects of seven TSS ingredients on TRP channels were examined. The agonistic effect on TRPA1 was observed for atractylodin, atractylodin carboxylic acid and levistolide A. Among the TSS ingredients, atractylodin carboxylic acid had significant hyperpolarising effects. CONCLUSIONS: The mechanism by which TSS accelerates the recovery of lowered body temperature in rats after immersion in cold water may involve the acceleration of the recovery of lowered blood flow. Increased CGRP release from DRG cells by TSS, TRPA1 activation by TSS ingredients, and membrane potential changes in vascular smooth muscle cells caused by TSS ingredients are part of the mechanism of action of TSS. These findings may partly contribute to the interpretation of the beneficial effects of TSS on cold feeling.

Pharmacokinetic study of traditional Japanese Kampo medicine shimotsuto used to treat gynecological diseases in rats.

Shimotsuto is a traditional Japanese Kampo medicine used to treat gynecological diseases, such as irregular menstruation, in addition to oversensitivity to cold and chilblains. Part of the pharmacological actions of shimotsuto is traditionally considered to be exerted by an improvement effect of the blood and the circulatory system. Multiple ingredients (e.g., catalpol and paeoniflorin) contained in shimotsuto have been reported to have pharmacological activities on the blood and circulatory system, and thus been considered to contribute to the pharmacological actions of shimotsuto. However, it remains unclear whether the ingredients can be absorbed into the body following oral administration of shimotsuto. The aim in the present study was to specify shimotsuto ingredient absorbed into the systemic circulation in rats. Seven candidate active ingredients (catalpol, paeoniflorin, albiflorin, ligustilide, senkyunolide A, butylphthalide, and ferulic acid) in plasma after oral administration of shimotsuto were quantified by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This study also performed nontargeted LC-MS/MS analysis of plasma following administration of constituent crude drugs of shimotsuto to find extensively blood-absorbed ingredients of shimotsuto. Among detected peaks in the nontargeted analysis, two peaks could be identified as bergapten and 8-debenzoylpaeoniflorin, subsequently their concentrations in shimotsuto-treated rat plasma were quantified. These pharmacokinetic studies indicated that catalpol showed the highest plasma concentration following administration of shimotsuto, followed by 8-debenzoylpaeoniflorin. This study suggests that all nine ingredients are absorbed into the blood following oral administration of shimotsuto and possibly contribute to its pharmacological action.

Identification of herbal components as TRPA1 agonists and TRPM8 antagonists.

Transient receptor potential (TRP) channels are non-selective cation channels that are implicated in analgesia, bowel motility, wound healing, thermoregulation, vasodilation and voiding dysfunction. Many natural products have been reported to affect the activity of TRP channels. We hypothesize that numerous traditional herbal medicines (THMs) might exert their pharmacological activity through modulating the activity of TRP channels. The present study aimed to evaluate the effects of flavonoid aglycones and their glycosides, which are the main components of many THMs, on the TRP channel subtypes. A Ca2+ influx assay was performed using recombinant human TRPA1, TRPV1, TRPV4 and TRPM8 cell lines. Our findings showed that flavonoid aglycones and glycycoumarin activated TRPA1. In particular, isoflavone and chalcone compounds displayed potent TRPA1 agonistic activity. Furthermore, flavone aglycones showed concomitant potent TRPM8 inhibiting activity. Indeed, flavone, isoflavone aglycones, non-prenylated chalcones and glycycoumarin were found to be TRPM8 inhibitors. Hence, flavonoid aglycones metabolized by lactase-phlorizin hydrolase and ß-glucosidase in the small intestine or gut microbiota of the large intestine could generate TRPA1 agonists and TRPM8 antagonists.

Disturbed biopterin and folate metabolism in the Qdpr-deficient mouse.

Quinonoid dihydropteridine reductase (QDPR) catalyzes the regeneration of tetrahydrobiopterin (BH4), a cofactor for monoamine synthesis, phenylalanine hydroxylation and nitric oxide production. Here, we produced and analyzed a transgenic Qdpr(-/-) mouse model. Unexpectedly, the BH4 contents in the Qdpr(-/-) mice were not decreased and even increased in some tissues, whereas those of the oxidized form dihydrobiopterin (BH2) were significantly increased. We demonstrated that unlike the wild-type mice, dihydrofolate reductase regenerated BH4 from BH2 in the mutants. Furthermore, we revealed wide alterations in folate-associated metabolism in the Qdpr(-/-) mice, which suggests an interconnection between folate and biopterin metabolism in the transgenic mouse model.

Recovery of neurogenic amines in phenylketonuria mice after liver-targeted gene therapy.

Phenylketonuria (PKU) is a common genetic disorder arising from a deficiency of phenylalanine hydroxylase. If left untreated, the accumulation of phenylalanine leads to brain damage and neuropsychological dysfunction. One of the abnormalities found in hyperphenylalaninemic patients and a mouse model of PKU is an aminergic deficit in the brain. We previously showed correction of hyperphenylalaninemia and concomitant behavioral recovery in PKU mice after liver-targeted gene transfer with a viral vector. Here, we addressed whether such a functional recovery was substantiated by an improved amine metabolism in the brain. After gene transfer, brain dopamine, norepinephrine, and serotonin levels in the PKU mice were significantly elevated to normal or near-normal levels, along with systemic improvement of phenylalanine catabolism. The results of biochemical analyses validated the efficacy of PKU gene therapy in the central nervous system.