Yuka Sudo

History of the G Protein–Coupled Receptor (GPCR) Assays From Traditional to a State-of-the-Art Biosensor Assay

The G protein-coupled receptors (GPCRs) form the largest and the most versatile superfamily that share a seven-transmembrane-spanning architecture. GPCR-signaling is involved in vision, taste, olfaction, sympathetic/parasympathetic nervous functions, metabolism, and immune regulation, indicating that GPCRs are extremely important therapeutic targets for various diseases. Cellular dielectric spectroscopy (CDS) is a novel technology that employs a label-free, real-time and cell-based assay approach for the comprehensive pharmacological evaluation of cells that exogenously or endogenously express GPCRs. Among the biosensors that use CDS technology, the CellKey™ system not only detects the activation of GPCRs but also distinguishes between signals through different subtypes of the Gα protein (Gs, Gi/o, and Gq). In this review, we discuss the traditional assays and then introduce the principles by which the CellKey™ system evaluates GPCR activation, followed by a perspective on the advantages and future prospects of this system.

A Review of Traditional Japanese Medicines and their Potential Mechanism of Action

Traditional Japanese herbal, or Kampo medicine was developed and modified from Chinese herbal medicine. After the Japanese government approved Kampo for clinical use, much attention has been paid to establishing scientific evidence for the effectiveness of these medicines. Recent progress has been made in elucidating the mechanisms of action of some types of Kampo medicine, including rikkunshito (RKT), daikenchuto, and yokukansan. In this review, we focused on identifying the target molecules and the active ingredients of RKT. Thus far, many target molecules have been implicated in the mechanism of action of Kampo medicines, such as ion channels, enzymes, and receptors. In particular, G protein-coupled receptors are attractive candidates for explaining herbal medicine activity. This is particularly true of RKT, which is composed of 8 independent, crude drug extracts. Recent reports have shown that RKT elicits its effects through dual action to the G protein-coupled receptors: inhibition of serotonergic 5-HT2C and 5-HT2B receptors and activation of ghrelin receptors via specific ingredients of RKT. In addition, we suggest that the identification of the effective ingredients from Kampo medicines could contribute to the discovery and development of new drugs by means of modern high-throughput drug screening technology.

New cancer cachexia rat model generated by implantation of a peritoneal dissemination-derived human stomach cancer cell line

Cancer cachexia (CC), a syndrome characterized by anorexia and body weight loss due to low fat-free mass levels, including reduced musculature, markedly worsens patient quality of life. Although stomach cancer patients have the highest incidence of cachexia, few experimental models for the study of stomach CC have been established. Herein, we developed stomach CC animal models using nude rats subcutaneously implanted with two novel cell lines, i.e., MKN45c185, established from the human stomach cancer cell line MKN-45, and 85As2, derived from peritoneal dissemination of orthotopically implanted MKN45c185 cells in mice. Both CC models showed marked weight loss, anorexia, reduced musculature and muscle strength, increased inflammatory markers, and low plasma albumin levels; however, CC developed earlier and was more severe in rats implanted with 85As2 than in those implanted with MKN45cl85. Moreover, human leukemia inhibitory factor (LIF), a known cachectic factor, and hypothalamic orexigenic peptide mRNA levels increased in the models, whereas hypothalamic anorexigenic peptide mRNA levels decreased. Surgical removal of the tumor not only abolished cachexia symptoms but also reduced plasma LIF levels to below detectable limits. Importantly, oral administration of rikkunshito, a traditional Japanese medicine, substantially ameliorated CC-related anorexia and body composition changes. In summary, our novel peritoneal dissemination-derived 85As2 rat model developed severe cachexia, possibly caused by LIF from cancer cells, that was ameliorated by rikkunshito. This model should provide a useful tool for further study into the mechanisms and treatment of stomach CC.

S(+)-ketamine suppresses desensitization of γ-aminobutyric acid type B receptor-mediated signaling by inhibition of the interaction of γ-aminobutyric acid type B receptors with G protein-coupled receptor kinase 4 or 5.

Background:Intrathecal baclofen therapy is an established treatment for severe spasticity. However, long-term management occasionally results in the development of tolerance. One of the mechanisms of tolerance is desensitization of &ggr;-aminobutyric acid type B receptor (GABABR) because of the complex formation of the GABAB2 subunit (GB2R) and G protein–coupled receptor kinase (GRK) 4 or 5. The current study focused on S(+)-ketamine, which reduces the development of morphine tolerance. This study was designed to investigate whether S(+)-ketamine affects the GABABR desensitization processes by baclofen. Methods:The G protein–activated inwardly rectifying K+ channel currents induced by baclofen were recorded using Xenopus oocytes coexpressing G protein–activated inwardly rectifying K+ channel 1/2, GABAB1a receptor subunit, GB2R, and GRK. Translocation of GRKs 4 and 5 and protein complex formation of GB2R with GRKs were analyzed by confocal microscopy and fluorescence resonance energy transfer analysis in baby hamster kidney cells coexpressing GABAB1a receptor subunit, fluorescent protein–tagged GB2R, and GRKs. The formation of protein complexes of GB2R with GRKs was also determined by coimmunoprecipitation and Western blot analysis. Results:Desensitization of GABABR-mediated signaling was suppressed by S(+)-ketamine in a concentration-dependent manner in the electrophysiologic assay. Confocal microscopy revealed that S(+)-ketamine inhibited translocation of GRKs 4 and 5 to the plasma membranes and protein complex formation of GB2R with the GRKs. Western blot analysis also showed that S(+)-ketamine inhibited the protein complex formation of GB2R with the GRKs. Conclusion:S(+)-Ketamine suppressed the desensitization of GABABR-mediated signaling at least in part through inhibition of formation of protein complexes of GB2R with GRK 4 or 5.

Hydroxy-α sanshool induces colonic motor activity in rat proximal colon: a possible involvement of KCNK9

Various colonic motor activities are thought to mediate propulsion and mixing/absorption of colonic content. The Japanese traditional medicine daikenchuto (TU-100), which is widely used for postoperative ileus in Japan, accelerates colonic emptying in healthy humans. Hydroxy-α sanshool (HAS), a readily absorbable active ingredient of TU-100 and a KCNK3/KCNK9/KCNK18 blocker as well as TRPV1/TRPA1 agonist, has been investigated for its effects on colonic motility. Motility was evaluated by intraluminal pressure and video imaging of rat proximal colons in an organ bath. Distribution of KCNKs was investigated by RT-PCR, in situ hybridization, and immunohistochemistry. Current and membrane potential were evaluated with use of recombinant KCNK3- or KCNK9-expressing Xenopus oocytes and Chinese hamster ovary cells. Defecation frequency in rats was measured. HAS dose dependently induced strong propulsive “squeezing” motility, presumably as long-distance contraction (LDC). TRPV1/TRPA1 agonists induced different motility patterns. The effect of HAS was unaltered by TRPV1/TRPA1 antagonists and desensitization. Lidocaine (a nonselective KCNK blocker) and hydroxy-β sanshool (a geometrical isomer of HAS and KCNK3 blocker) also induced colonic motility as a rhythmic propagating ripple (RPR) and a LDC-like motion, respectively. HAS-induced “LDC,” but not lidocaine-induced “RPR,” was abrogated by a neuroleptic agent tetrodotoxin. KCNK3 and KCNK9 were located mainly in longitudinal smooth muscle cells and in neural cells in the myenteric plexus, respectively. Administration of HAS or TU-100 increased defecation frequency in normal and laparotomy rats. HAS may evoke strong LDC possibly via blockage of the neural KCNK9 channel in the colonic myenteric plexus.

Involvement of lysosomal dysfunction in autophagosome accumulation and early pathologies in adipose tissue of obese mice

ABSTRACT Whether obesity accelerates or suppresses autophagy in adipose tissue is still debatable. To clarify dysregulation of autophagy and its role in pathologies of obese adipose tissue, we focused on lysosomal function, protease maturation and activity, both in vivo and in vitro. First, we showed that autophagosome formation was accelerated, but autophagic clearance was impaired in obese adipose tissue. We also found protein and activity levels of CTSL (cathepsin L) were suppressed in obese adipose tissue, while the activity of CTSB (cathepsin B) was significantly enhanced. Moreover, cellular senescence and inflammasomes were activated in obese adipose tissue. In 3T3L1 adipocytes, downregulation of CTSL deteriorated autophagic clearance, upregulated expression of CTSB, promoted cellular senescence and activated inflammasomes. Upregulation of CTSB promoted additional activation of inflammasomes. Therefore, we suggest lysosomal dysfunction observed in obese adipose tissue leads to lower autophagic clearance, resulting in autophagosome accumulation. Simultaneously, lysosomal abnormalities, including deteriorated CTSL function and compensatory activation of CTSB, caused cellular senescence and inflammasome activation. Our findings strongly suggest lysosomal dysfunction is involved in early pathologies of obese adipose tissue.

The endocannabinoid anandamide inhibits voltage-gated sodium channels Nav1.2, Nav1.6, Nav1.7, and Nav1.8 in Xenopus oocytes.

BACKGROUND:Anandamide is an endocannabinoid that regulates multiple physiological functions by pharmacological actions, in a manner similar to marijuana. Recently, much attention has been paid to the analgesic effect of endocannabinoids in terms of identifying new pharmacotherapies for refractory pain management, but the mechanisms of the analgesic effects of anandamide are still obscure. Voltage-gated sodium channels are believed to play important roles in inflammatory and neuropathic pain. We investigated the effects of anandamide on 4 neuronal sodium channel &agr; subunits, Nav1.2, Nav1.6, Nav1.7, and Nav1.8, to explore the mechanisms underlying the antinociceptive effects of anandamide. METHODS:We studied the effects of anandamide on Nav1.2, Nav1.6, Nav1.7, and Nav1.8 &agr; subunits with &bgr;1 subunits by using whole-cell, 2-electrode, voltage-clamp techniques in Xenopus oocytes. RESULTS:Anandamide inhibited sodium currents of all subunits at a holding potential causing half-maximal current (V1/2) in a concentration-dependent manner. The half-maximal inhibitory concentration values for Nav1.2, Nav1.6, Nav1.7, and Nav1.8 were 17, 12, 27, and 40 &mgr;mol/L, respectively, indicating an inhibitory effect on Nav1.6, which showed the highest potency. Anandamide raised the depolarizing shift of the activation curve as well as the hyperpolarizing shift of the inactivation curve in all &agr; subunits, suggesting that sodium current inhibition was due to decreased activation and increased inactivation. Moreover, anandamide showed a use-dependent block in Nav1.2, Nav1.6, and Nav1.7 but not Nav1.8. CONCLUSION:Anandamide inhibited the function of &agr; subunits in neuronal sodium channels Nav1.2, Nav1.6, Nav1.7, and Nav1.8. These results help clarify the mechanisms of the analgesic effects of anandamide.

Sterol regulatory element-binding protein-1c orchestrates metabolic remodeling of white adipose tissue by caloric restriction

Caloric restriction (CR) can delay onset of several age‐related pathophysiologies and extend lifespan in various species, including rodents. CR also induces metabolic remodeling involved in activation of lipid metabolism, enhancement of mitochondrial biogenesis, and reduction of oxidative stress in white adipose tissue (WAT). In studies using genetically modified mice with extended lifespans, WAT characteristics influenced mammalian lifespans. However, molecular mechanisms underlying CR‐associated metabolic remodeling of WAT remain unclear. Sterol regulatory element‐binding protein‐1c (Srebp‐1c), a master transcription factor of fatty acid (FA) biosynthesis, is responsible for the pathogenesis of fatty liver (steatosis). Our study showed that, under CR conditions, Srebp‐1c enhanced mitochondrial biogenesis via increased expression of peroxisome proliferator‐activated receptor gamma coactivator‐1α (Pgc‐1α) and upregulated expression of proteins involved in FA biosynthesis within WAT. However, via Srebp‐1c, most of these CR‐associated metabolic alterations were not observed in other tissues, including the liver. Moreover, our data indicated that Srebp‐1c may be an important factor both for CR‐associated suppression of oxidative stress, through increased synthesis of glutathione in WAT, and for the prolongevity action of CR. Our results strongly suggested that Srebp‐1c, the primary FA biosynthesis‐promoting transcriptional factor implicated in fatty liver disease, is also the food shortage‐responsive factor in WAT. This indicated that Srebp‐1c is a key regulator of metabolic remodeling leading to the beneficial effects of CR.

Ignavine: a novel allosteric modulator of the μ opioid receptor

Processed Aconiti tuber (PAT) is used to treat pain associated with various disorders. Although it has been demonstrated that the κ opioid receptor (KOR) signaling pathway is a mediator of the analgesic effect of PAT, active components affecting opioid signaling have not yet been identified. In this study, we explored candidate components of PAT by pharmacokinetic analysis and identified ignavine, which is a different structure from aconitine alkaloids. A receptor binding assay of opioid receptors showed that ignavine specifically binds the μ opioid receptor (MOR), not the KOR. Receptor internalization assay in MOR-expressing cell lines revealed that ignavine augmented the responses produced by D-Ala(2)-N-Me-Phe(4)-Gly-ol(5)-enkephalin (DAMGO), a representative MOR agonist, at a low concentration and inhibited it at a higher concentration. Ignavine also exerted positive modulatory activity for DAMGO, endomorphin-1 and morphine in cAMP assay. Additionally, ignavine alone showed an analgesic effect in vivo. In silico simulation analysis suggested that ignavine would induce a unique structural change distinguished from those induced by a representative MOR agonist and antagonist. These data collectively suggest the possibility that ignavine could be a novel allosteric modulator of the MOR. The present results may open the way for the development of a novel pain management strategy.

Neurosteroids allopregnanolone sulfate and pregnanolone sulfate have diverse effect on the α subunit of the neuronal voltage-gated sodium channels Nav1.2, Nav1.6, Nav1.7, and Nav1.8 expressed in xenopus oocytes.

Background:The neurosteroids allopregnanolone and pregnanolone are potent positive modulators of &ggr;-aminobutyric acid type A receptors. Antinociceptive effects of allopregnanolone have attracted much attention because recent reports have indicated the potential of allopregnanolone as a therapeutic agent for refractory pain. However, the analgesic mechanisms of allopregnanolone are still unclear. Voltage-gated sodium channels (Nav) are thought to play important roles in inflammatory and neuropathic pain, but there have been few investigations on the effects of allopregnanolone on sodium channels. Methods:Using voltage-clamp techniques, the effects of allopregnanolone sulfate (APAS) and pregnanolone sulfate (PAS) on sodium current were examined in Xenopus oocytes expressing Nav1.2, Nav1.6, Nav1.7, and Nav1.8 &agr; subunits. Results:APAS suppressed sodium currents of Nav1.2, Nav1.6, and Nav1.7 at a holding potential causing half-maximal current in a concentration-dependent manner, whereas it markedly enhanced sodium current of Nav1.8 at a holding potential causing maximal current. Half-maximal inhibitory concentration values for Nav1.2, Nav1.6, and Nav1.7 were 12 ± 4 (n = 6), 41 ± 2 (n = 7), and 131 ± 15 (n = 5) &mgr;mol/l (mean ± SEM), respectively. The effects of PAS were lower than those of APAS. From gating analysis, two compounds increased inactivation of all &agr; subunits, while they showed different actions on activation of each &agr; subunit. Moreover, two compounds showed a use-dependent block on Nav1.2, Nav1.6, and Nav1.7. Conclusion:APAS and PAS have diverse effects on sodium currents in oocytes expressing four &agr; subunits. APAS inhibited the sodium currents of Nav1.2 most strongly.