Alterations in metabolites in the anterior cingulate cortex and thalamus and their associations with pain and empathy in patients with chronic mild pain: a preliminary study.

Proton magnetic resonance spectroscopy (1H-MRS) has shown inconsistent alterations in the brain metabolites of individuals with chronic pain. We used 3T 1H-MRS to investigate the brain metabolites in the anterior cingulate cortex and thalamus of 22 patients with chronic mild pain and no gait disturbance and 22 healthy controls. The chronic-pain group included patients with chronic low back pain and/or osteoarthritis but none suffering from hypersensitivity. There were no significant between group-differences in glutamate, glutamate plus glutamine (Glx), N-acetylaspartate, glycerophosphorylcholine (GPC), glutamine, creatine plus phosphocreatine, or myo-inositol in the anterior cingulate cortex, but the patients showed a significant decrease in GPC, but not other metabolites, in the thalamus compared to the controls. The GPC values in the patients' thalamus were significantly correlated with pain components on the Short-Form McGill Pain Questionnaire (SF-MPQ-2) and affective empathy components on the Questionnaire of Cognitive and Affective Empathy (QCAE). The GPC in the patients' anterior cingulate cortex showed significant correlations with cognitive empathy components on the QCAE. Myo-inositol in the controls' anterior cingulate cortex and Glx in the patients' thalamus each showed significant relationships with peripheral responsivity on the QCAE. These significances were not significant after Bonferroni corrections. These preliminary findings indicate important roles of GPC, myo-inositol, and Glx in the brain of patients with chronic mild pain.

The Inhibition of TREK-1 K+ Channels via Multiple Compounds Contained in the Six Kamikihito Components, Potentially Stimulating Oxytocin Neuron Pathways.

Oxytocin, a significant pleiotropic neuropeptide, regulates psychological stress adaptation and social communication, as well as peripheral actions, such as uterine contraction and milk ejection. Recently, a Japanese Kampo medicine called Kamikihito (KKT) has been reported to stimulate oxytocin neurons to induce oxytocin secretion. Two-pore-domain potassium channels (K2P) regulate the resting potential of excitable cells, and their inhibition results in accelerated depolarization that elicits neuronal and endocrine cell activation. We assessed the effects of KKT and 14 of its components on a specific K2P, the potassium channel subfamily K member 2 (TREK-1), which is predominantly expressed in oxytocin neurons in the central nervous system (CNS). KKT inhibited the activity of TREK-1 induced via the channel activator ML335. Six of the 14 components of KKT inhibited TREK-1 activity. Additionally, we identified that 22 of the 41 compounds in the six components exhibited TREK-1 inhibitory effects. In summary, several compounds included in KKT partially activated oxytocin neurons by inhibiting TREK-1. The pharmacological effects of KKT, including antistress effects, may be partially mediated through the oxytocin pathway.

Therapeutic Potential of Orally Administered Rubiscolin-6.

Rubiscolins are naturally occurring opioid peptides derived from the enzymatic digestion of the ribulose bisphosphate carboxylase/oxygenase protein in spinach leaves. They are classified into two subtypes based on amino acid sequence, namely rubiscolin-5 and rubiscolin-6. In vitro studies have determined rubiscolins as G protein-biased delta-opioid receptor agonists, and in vivo studies have demonstrated that they exert several beneficial effects via the central nervous system. The most unique and attractive advantage of rubiscolin-6 over other oligopeptides is its oral availability. Therefore, it can be considered a promising candidate for the development of a novel and safe drug. In this review, we show the therapeutic potential of rubiscolin-6, mainly focusing on its effects when orally administered based on available evidence. Additionally, we present a hypothesis for the pharmacokinetics of rubiscolin-6, focusing on its absorption in the intestinal tract and ability to cross the blood-brain barrier.

Distinct Profiles of Desensitization of µ-Opioid Receptors Caused by Remifentanil or Fentanyl: In Vitro Assay with Cells and Three-Dimensional Structural Analyses.

Remifentanil (REM) and fentanyl (FEN) are commonly used analgesics that act by activating a µ-opioid receptor (MOR). Although optimal concentrations of REM can be easily maintained during surgery, it is sometimes switched to FEN for optimal pain regulation. However, standards for this switching protocol remain unclear. Opioid anesthetic efficacy is decided in part by MOR desensitization; thus, in this study, we investigated the desensitization profiles of REM and FEN to MOR. The efficacy and potency during the 1st administration of REM or FEN in activating the MOR were almost equal. Similarly, in ß arrestin recruitment, which determines desensitization processes, they showed no significant differences. In contrast, the 2nd administration of FEN resulted in a stronger MOR desensitization potency than that of REM, whereas REM showed a higher internalization potency than FEN. These results suggest that different ß arrestin-mediated signaling caused by FEN or REM led to their distinct desensitization and internalization processes. Our three-dimensional analysis, with in silico binding of REM and FEN to MOR models, highlighted that REM and FEN bound to similar but distinct sites of MOR and led to distinct ß arrestin-mediated profiles, suggesting that distinct binding profiles to MOR may alter ß arrestin activity, which accounts for MOR desensitization and internalization.

Discovery of 7-Azanorbornane-Based Dual Agonists for the Delta and Kappa Opioid Receptors through an In Situ Screening Protocol.

In medicinal chemistry, the copper-catalyzed click reaction is used to prepare ligand candidates. This reaction is so clean that the bioactivities of the products can be determined without purification. Despite the advantages of this in situ screening protocol, the applicability of this method for transmembrane proteins has not been validated due to the incompatibility with copper catalysts. To address this point, we performed ligand screening for the µ, δ, and κ opioid receptors using this protocol. As we had previously reported the 7-azanorbornane skeleton as a privileged scaffold for the G protein-coupled receptors, we performed the click reactions between various 7-substituted 2-ethynyl-7-azanorbornanes and azides. Screening assays were performed without purification using the CellKeyTM system, and the putative hit compounds were re-synthesized and re-evaluated. Although the "hit" compounds for the µ and the δ receptors were totally inactive after purifications, three of the four "hits" for the κ receptor were true agonists for this receptor and also showed activities for the δ receptor. Although false positive/negative results exist as in other screening projects for soluble proteins, this in situ method is effective in identifying novel ligands for transmembrane proteins.

Possible mechanism for improving the endogenous immune system through the blockade of peripheral µ-opioid receptors by treatment with naldemedine.

BACKGROUND: It has been considered that activation of peripheral µ-opioid receptors (MORs) induces side effects of opioids. In this study, we investigated the possible improvement of the immune system in tumour-bearing mice by systemic administration of the peripheral MOR antagonist naldemedine. METHODS: The inhibitory effect of naldemedine on MOR-mediated signalling was tested by cAMP inhibition and ß-arrestin recruitment assays using cultured cells. We assessed possible changes in tumour progression and the number of splenic lymphocytes in tumour-bearing mice under the repeated oral administration of naldemedine. RESULTS: Treatment with naldemedine produced a dose-dependent inhibition of both the decrease in the cAMP level and the increase in ß-arrestin recruitment induced by the MOR agonists. Repeated treatment with naldemedine at a dose that reversed the morphine-induced inhibition of gastrointestinal transport, but not antinociception, significantly decreased tumour volume and prolonged survival in tumour-transplanted mice. Naldemedine administration significantly decreased the increased expression of immune checkpoint-related genes and recovered the decreased level of toll-like receptor 4 in splenic lymphocytes in tumour-bearing mice. CONCLUSIONS: The blockade of peripheral MOR may induce an anti-tumour effect through the recovery of T-cell exhaustion and promotion of the tumour-killing system.

Effects of an oral mucosa protective formulation on chemotherapy- and/or radiotherapy-induced oral mucositis: a prospective study.

BACKGROUND: Oral mucositis (OM) associated with cancer treatment not only impairs patients' quality of life but also causes treatment delays or changes. This prospective exploratory study was conducted to evaluate the efficacy of Episil® oral liquid, which is an approved protective formulation for the oral mucosa in patients with OM. The extent of the pain-relieving effect, feeling during use, and adverse events or problems were evaluated. METHODS: In total, 10 Japanese cancer patients with OM receiving chemotherapy, pretreatment therapy for hematopoietic stem cell transplantation, or radiation therapy for head and neck cancer were enrolled. RESULTS: A numerical rating scale (NRS) was used to assess oral pain intensity due to OM. Compared to baseline, the mean NRS began to decrease at 5 min after using Episil® (7.1 ± 1.4 to 4.6 ± 2.87; p = 0.264). A significant decrease was observed in the pain score after using Episil® compared with that before using Episil®, and this effect lasted up to 120 min. The protective effects of Episil® were observed 3-5 min after application. Some patients felt slight soreness or discomfort when applying Episil®. However, this discomfort due to Episil®'s stimulation was within the allowable range and transient. No adverse events were observed in any of the cases. CONCLUSIONS: The results of this prospective study showed that Episil® could be an effective treatment to relieve oral pain in Japanese patients with moderate to severe OM, and this newly approved product might adequately support patients' oral intake. TRIAL REGISTRATION: University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR) ( UMIN000031921 ).

Novel Opioid Analgesics for the Development of Transdermal Opioid Patches That Possess Morphine-Like Pharmacological Profiles Rather Than Fentanyl: Possible Opioid Switching Alternatives Among Patch Formula.

BACKGROUND: Transdermal fentanyl is widely used in the treatment of severe pain because of convenience, safety, and stable blood concentrations. Nevertheless, patients often develop tolerance to fentanyl, necessitating the use of other opioids; transdermal buprenorphine patch is widely used as an analgesic agent, though available formulation does not provide comparable analgesic effect as transdermal fentanyl patch. Opioids bind to the opioid receptor (OR) to activate both G protein-mediated and ß-arrestin-mediated pathways. We synthesized morphine-related compounds with high transdermal absorbability (N1 and N2) and evaluated their OR activities pharmacologically in comparison with fentanyl and morphine. METHODS: In cells stably expressing µ-opioid receptor (MOR), δ-opioid receptor (DOR), and κ-opioid receptor (KOR), G protein-mediated pathways were assessed using the CellKey and an intracellular cyclic adenosine monophosphate (cAMP) assay, while ß-arrestin-mediated pathways were analyzed with ß-arrestin recruitment and receptor internalization assays. Furthermore, analgesic effects were evaluated using a tail-flick test in mice, and the analgesic effect on fentanyl-tolerant mice was evaluated. RESULTS: In the CellKey and cAMP assays, both N1 and N2 showed the highest affinity for MOR and acted as full agonists as well as partial agonists for DOR and KOR. In the ß-arrestin and internalization assays, only fentanyl acted as a full agonist; N1 and N2 acted as partial agonists of MOR. In the mouse tail-flick test, N1 and N2 showed analgesic effects equivalent to those of fentanyl and morphine. In fentanyl-tolerant mice, fentanyl showed a diminished analgesic effect, whereas N1 and N2 as well as morphine retained their analgesic effects. CONCLUSIONS: While N1 and N2 have higher transdermal absorbability than fentanyl, they also have analgesic effects comparable to those of morphine, suggesting that they may be attractive compounds for the development of novel opioid patches for transitioning from fentanyl patches.

Evaluation of the Intracellular Signaling Activities of κ-Opioid Receptor Agonists, Nalfurafine Analogs; Focusing on the Selectivity of G-Protein- and ß-Arrestin-Mediated Pathways.

Opioid receptors (ORs) are classified into three types (µ, δ, and κ), and opioid analgesics are mainly mediated by µOR activation; however, their use is sometimes restricted by unfavorable effects. The selective κOR agonist nalfurafine was initially developed as an analgesic, but its indication was changed because of the narrow safety margin. The activation of ORs mainly induces two intracellular signaling pathways: a G-protein-mediated pathway and a ß-arrestin-mediated pathway. Recently, the expectations for κOR analgesics that selectively activate these pathways have increased; however, the structural properties required for the selectivity of nalfurafine are still unknown. Therefore, we evaluated the partial structures of nalfurafine that are necessary for the selectivity of these two pathways. We assayed the properties of nalfurafine and six nalfurafine analogs (SYKs) using cells stably expressing κORs. The SYKs activated κORs in a concentration-dependent manner with higher EC50 values than nalfurafine. Upon bias factor assessment, only SYK-309 (possessing the 3S-hydroxy group) showed higher selectivity of G-protein-mediated signaling activities than nalfurafine, suggesting the direction of the 3S-hydroxy group may affect the ß-arrestin-mediated pathway. In conclusion, nalfurafine analogs having a 3S-hydroxy group, such as SYK-309, could be considered G-protein-biased κOR agonists.

Further investigation of the rapid-onset and short-duration action of the G protein-biased µ-ligand oliceridine.

TRV130 (oliceridine), a G protein-biased ligand for µ-opioid receptor, has recently been synthesized. It is considered to have strong antinociceptive effects and only minor adverse effects. However, whether or not oliceridine actually exhibits an ideal pharmacological profile as an analgesic has not yet been fully clarified in animal studies. This study examined the pharmacological profile of oliceridine in cells and animals. Oliceridine (10 µM) did not produce any µ-opioid receptor internalization in cells even though it increased impedance, which reflects the activation of Gi protein using the CellKey™ system, and inhibited the formation of cAMP. In mice, oliceridine (0.3-10 mg/kg) produced a dose-dependent antinociceptive effect with a rapid-onset and short-duration action in the hot-plate test, as well as antihyperalgesia after sciatic nerve ligation without the development of antinociceptive tolerance using the thermal hyperalgesia test. On the other hand, oliceridine inhibited gastrointestinal transit. Furthermore, oliceridine produced rapid-onset hyperlocomotion at antinociceptive doses; sensitization developed in mice and an emetic effect was observed in ferrets. These results indicate that, although oliceridine may produce dopamine-related behaviors even through selective stimulation of the G-protein-biased µ-opioid receptor pathway, it still offers advantages for breakthrough pain without antinociceptive tolerance with adequate doses.

In Vitro Analyses of Spinach-Derived Opioid Peptides, Rubiscolins: Receptor Selectivity and Intracellular Activities through G Protein- and ß-Arrestin-Mediated Pathways.

Activated opioid receptors transmit internal signals through two major pathways: the G-protein-mediated pathway, which exerts analgesia, and the ß-arrestin-mediated pathway, which leads to unfavorable side effects. Hence, G-protein-biased opioid agonists are preferable as opioid analgesics. Rubiscolins, the spinach-derived naturally occurring opioid peptides, are selective δ opioid receptor agonists, and their p.o. administration exhibits antinociceptive effects. Although the potency and effect of rubiscolins as G-protein-biased molecules are partially confirmed, their in vitro profiles remain unclear. We, therefore, evaluated the properties of rubiscolins, in detail, through several analyses, including the CellKeyTM assay, cADDis® cAMP assay, and PathHunter® ß-arrestin recruitment assay, using cells stably expressing µ, δ, κ, or µ/δ heteromer opioid receptors. In the CellKeyTM assay, rubiscolins showed selective agonistic effects for δ opioid receptor and little agonistic or antagonistic effects for µ and κ opioid receptors. Furthermore, rubiscolins were found to be G-protein-biased δ opioid receptor agonists based on the results obtained in cADDis® cAMP and PathHunter® ß-arrestin recruitment assays. Finally, we found, for the first time, that they are also partially agonistic for the µ/δ dimers. In conclusion, rubiscolins could serve as attractive seeds, as δ opioid receptor-specific agonists, for the development of novel opioid analgesics with reduced side effects.

A novel method for evaluating activity of transient receptor potential channels using a cellular dielectric spectroscopy.

Cellular dielectric spectroscopy (CDS) is a novel technology enabling pharmacological evaluation of multiple receptor types with a label-free cell-based assay. We evaluated activities of a family of ligand-gated channels, transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) channels by an electrical impedance-based biosensor (CellKey™ system) using CDS. Measures of both potency (EC50) and efficacy (Emax) of these agonists with CellKey™ were almost identical to those made using the traditional Ca2+ influx assay in TRPV1- or TRPA1-expressing cells, suggesting that CellKey™ is a simpler and easier means of evaluating TRP activities.

A novel strategy for treatment of cancer cachexia targeting xanthine oxidase in the brain.

Cancer cachexia is a systemic wasting syndrome characterized by anorexia and loss of body weight. The xanthine oxidase (XO) inhibitor febuxostat is one of the promising candidates for cancer cachexia treatment. However, cachexic symptoms were not alleviated by oral administration of febuxostat in our cancer cachexia model. Metabolomic analysis with brains of our cachexic model showed that purine metabolism was activated and XO activity was increased, and thus suggested that febuxostat would not reach the brain. Accordingly, targeting XO in the brain, which controls appetite, may be an effective strategy for treatment of cancer cachexia.

Possible biased analgesic of hydromorphone through the G protein-over ß-arrestin-mediated pathway: cAMP, CellKey™, and receptor internalization analyses.

Morphine, fentanyl, and oxycodone are widely used as analgesics, and recently hydromorphone has been approved in Japan. Although all of these are selective for µ-opioid receptors (MORs) and have similar structures, their analgesic potencies and adverse effects (AEs) are diverse. Recent molecular analyses of MOR signaling revealed that the G protein-mediated signaling pathway causes analgesic effects and the ß-arrestin-mediated signaling pathway is responsible for AEs. We used several cell-based analyses that selectively measure cellular responses activated by either G protein- or ß-arrestin-mediated pathways. GloSensor™ cAMP, CellKey™, and receptor internalization assays were performed with four different types of cells stably expressing differentially labelled MOR. EC50 values measured by cAMP and CellKey™ assays had potencies in the order fentanyl ≤ hydromorphone < morphine ≤ oxycodone, all also exhibiting full agonist responses. However, in the internalization assay, only fentanyl elicited a full agonist response. Hydromorphone had the strongest potency next to fentanyl; however, contribution of the ß-arrestin-mediated pathway was small, suggesting that its effect could be biased toward the G protein-mediated pathway. Based on these properties, hydromorphone could be chosen as an effective analgesic.

Carboplatin Enhances the Activity of Human Transient Receptor Potential Ankyrin 1 through the Cyclic AMP-Protein Kinase A-A-Kinase Anchoring Protein (AKAP) Pathways.

Carboplatin, an anticancer drug, often causes chemotherapy-induced peripheral neuropathy (PN). Transient receptor potential ankyrin 1 (TRPA1), a non-selective cation channel, is a polymodal nociceptor expressed in sensory neurons. TRPA1 is not only involved in pain transmission, but also in allodynia or hyperalgesia development. However, the effects of TRPA1 on carboplatin-induced PN is unclear. We revealed that carboplatin induced mechanical allodynia and cold hyperalgesia, and the pains observed in carboplatin-induced PN models were significantly suppressed by the TRPA1 antagonist HC-030031 without a change in the level of TRPA1 protein. In cells expressing human TRPA, carboplatin had no effects on changes in intracellular Ca2+ concentration ([Ca2+]i); however, carboplatin pretreatment enhanced the increase in [Ca2+]i induced by the TRPA1 agonist, allyl isothiocyanate (AITC). These effects were suppressed by an inhibitor of protein kinase A (PKA). The PKA activator forskolin enhanced AITC-induced increase in [Ca2+]i and carboplatin itself increased intracellular cyclic adenosine monophosphate (cAMP) levels. Moreover, inhibition of A-kinase anchoring protein (AKAP) significantly decreased the carboplatin-induced enhancement of [Ca2+]i induced by AITC and improved carboplatin-induced mechanical allodynia and cold hyperalgesia. These results suggested that carboplatin induced mechanical allodynia and cold hyperalgesia by increasing sensitivity to TRPA1 via the cAMP-PKA-AKAP pathway.

Relief from neuropathic pain by blocking of the platelet-activating factor-pain loop.

Neuropathic pain resulting from peripheral neuronal damage is largely resistant to treatment with currently available analgesic drugs. Recently, ATP, lysophosphatidic acid, and platelet-activating factor (PAF) have been reported to play important inductive roles in neuropathic pain. In the present study, we found that pain-like behaviors resulting from partial sciatic nerve ligation (PSL) were largely attenuated by deficiency of lysophosphatidylcholine acyltransferase (LPCAT)2, which is one of the PAF biosynthetic enzymes. By contrast, deficiency of the other PAF biosynthetic enzyme, LPCAT1, did not ameliorate neuropathic pain. With regard to the mechanism of the observed effects, LPCAT2 was detected in wild-type spinal cord microglia, and the absence of LPCAT2 expression precluded spinal PAF expression in LPCAT2-knockout mice. Furthermore, ATP-stimulated PAF biosynthesis in macrophages was decreased by pretreatment with the PAF receptor antagonist ABT-491, indicating the existence of a positive feedback loop of PAF biosynthesis, which we designated the PAF-pain loop. In conclusion, LPCAT2 is a novel therapeutic target for newly categorized analgesic drugs; in addition, our data call for the re-evaluation of the clinical utility of PAF receptor antagonists.-Shindou, H., Shiraishi, S., Tokuoka, S. M., Takahashi Y., Harayama, T., Abe, T., Bando, K., Miyano, K., Kita, Y., Uezono, Y., Shimizu, T. Relief from neuropathic pain by blocking of the platelet-activating factor-pain loop.

Neuropeptide oxytocin enhances µ opioid receptor signaling as a positive allosteric modulator.

Oxytocin (OT) is a 9-amine neuropeptide that plays an essential role in mammalian labor, lactation, maternal bonding, and social affiliation. OT has been reported to exert an analgesic effect in both humans and animals, and the results of certain animal experiments have shown that the analgesic effect of OT is partially blocked by opioid receptor antagonists. To investigate the relationship between OT and µ opioid receptor (MOR), we evaluated how OT affects MOR in vitro by performing an electrical impedance-based receptor biosensor assay (CellKey™ assay), an intracellular cAMP assay, and a competitive receptor-binding analysis by using cells stably expressing human MOR and OT receptor. In both the CellKey™ assay and the intracellular cAMP assay, OT alone exerted no direct agonistic effect on human MOR, but treatment with 10-6 M OT markedly enhanced the MOR signaling induced by 10-6 M endomorphin-1, ß-endorphin, morphine, fentanyl, and DAMGO. Moreover, in the competitive receptor-binding assay, 10-6 M OT did not alter the affinity of endomorphin-1 or morphine for MOR. These results suggest that OT could function as a positive allosteric modulator that regulates the efficacy of MOR signaling, and thus OT might represent a previously unrecognized candidate analgesic agent.

A Novel Method for Determination of Methadone in the Serum by High-Performance Liquid Chromatography with Electrochemical Detection.

In March 2013, the clinical use of oral methadone tablets was initiated in Japan. There are many factors responsible for the change in blood concentrations of methadone, and its pharmacokinetics is very complex. Therefore, a simple and accurate measurement method for methadone blood concentrations was developed using HPLC/electrochemical detector (ECD). An eluent of 10 mM Na2HPO4/CH3CN/CH3OH (20 : 19 : 3) was used as the mobile phase. The column was used the XTerra® RP18, and the voltage of the ECD was set at 400 to 800 mV. As a result, the calibration curve was linear in the ranges of 10 to 100 ng/mL (y=5012.7x+1041.1, r=0.999). The intra- and inter-day coefficients of variation were <5.2 and <5.8%, respectively. Therefore, this method was considered to be useful for the measurement of methadone blood levels in cancer patients. Also, using this method, blood methadone concentration was measured over time in a patient with cancer-associated pain who was treated with methadone. The estimated clearance (CL/F) and distribution volume (Vd/F) of methadone were 2.84 L/h and 502.8 L, respectively, and took about two weeks to reach steady state.

Differential Metabolic Responses to Adipose Atrophy Associated with Cancer Cachexia and Caloric Restriction in Rats and the Effect of Rikkunshito in Cancer Cachexia.

Despite the similar phenotypes, including weight loss, reduction of food intake, and lower adiposity, associated with caloric restriction (CR) and cancer cachexia (CC), CC is a progressive wasting syndrome, while mild CR improves whole body metabolism. In the present study, we compared adipose metabolic changes in a novel rat model of CC, mild CR (70% of the food intake of control rats, which is similar to the food consumption of CC rats), and severe CR (30% of the food intake of controls). We show that CC and severe CR are associated with much smaller adipocytes with significantly lower mitochondrial DNA content; but, that mild CR is not. CC and both mild and severe CR similarly upregulated proteins involved in lipolysis. CC also downregulated proteins involved in fatty acid biosynthesis, but mild CR upregulated these. These findings suggest that CC might impair de novo fatty acid biosynthesis and reduce mitochondrial biogenesis, similar to severe CR. We also found that rikkunshito, a traditional Japanese herbal medicine, does not ameliorate the enhanced lipolysis and mitochondrial impairment, but rather, rescues de novo fatty acid biosynthesis, suggesting that rikkunshito administration might have partially similar effects to mild CR.

Identification of Lysophosphatidic Acid Receptor 1 in Astroglial Cells as a Target for Glial Cell Line-derived Neurotrophic Factor Expression Induced by Antidepressants.

Preclinical and clinical evidence suggests that glial cell line-derived neurotrophic factor (GDNF) is important in the therapeutic effect of antidepressants. A previous study demonstrated that the tricyclic antidepressant amitriptyline induces Gαi/o activation, which leads to GDNF expression in astrocytes. However, the specific target expressed in astrocytes that mediates antidepressant-evoked Gαi/o activation has yet to be identified. Thus, the current study explored the possibility that antidepressant-induced Gαi/o activation depends on lysophosphatidic acid receptor 1 (LPAR1), a Gαi/o-coupled receptor. GDNF mRNA expression was examined using real-time PCR and Gαi/o activation was examined using the cell-based receptor assay system CellKeyTM in rat C6 astroglial cells and rat primary cultured astrocytes. LPAR1 antagonists blocked GDNF mRNA expression and Gαi/o activation evoked by various classes of antidepressants (amitriptyline, nortriptyline, mianserin, and fluoxetine). In addition, deletion of LPAR1 by RNAi suppressed amitriptyline-evoked GDNF mRNA expression. Treatment of astroglial cells with the endogenous LPAR agonist LPA increased GDNF mRNA expression through LPAR1, whereas treatment of primary cultured neurons with LPA failed to affect GDNF mRNA expression. Astrocytic GDNF expression evoked by either amitriptyline or LPA utilized, in part, transactivation of fibroblast growth factor receptor and a subsequent ERK cascade. The current results suggest that LPAR1 is a novel, specific target of antidepressants that leads to GDNF expression in astrocytes.