Minoru Hojo

Desensitization of GABAB receptor signaling by formation of protein complexes of GABAB2 subunit with GRK4 or GRK5

We investigated the role of G protein coupled‐receptor kinases (GRKs) in the desensitization of GABAB receptor‐mediated signaling using Xenopus oocytes and baby hamster kidney (BHK) cells. Baclofen elicited inward K+ currents in oocytes coexpressing heterodimeric GABAB receptor, GABAB1a subunit (GB1aR) and GABAB2 subunit (GB2R), together with G protein‐activated inwardly rectifying K+ channels (GIRKs), in a concentration‐dependent manner. Repetitive application of baclofen to oocytes coexpressing GABABR and GIRKs did not change peak K+ currents in the first and second responses, but the latter responses were significantly attenuated by coexpression of either GRK4 or GRK5 with attenuation efficacy of GRK4 > GRK5. Coexpression of other GRKs including GRK2, GRK3, and GRK6 had no effect on GABAB receptor‐mediated desensitization processes. In BHK cells coexpressing GRK4 fused to Venus (brighter variant of yellow fluorescent protein, GRK4‐Venus) with GB1aR and GB2R, GRK4‐Venus was expressed in the cytosol but was translocated to the plasma membranes by GABABR activation. In BHK cells coexpressing GRK4 fused to Cerulean (brighter variant of cyan fluorescent protein, GRK4‐Cerulean) with GB1aR and GB2R‐Venus, fluorescence resonance energy transfer (FRET) analysis demonstrated that GRK4‐Cerulean formed a protein complex with GB2R‐Venus. Immunoprecipitation and Western blot analysis confirmed GB2R‐GRK4 complex formation. GRK5 also formed a complex with GB2R on the plasma membranes as determined by FRET and Western blotting but not GRK2, GRK3, and GRK6. Our results indicate that GRK4 and GRK5 desensitize GABAB receptor‐mediated responses by forming protein complexes with GB2R subunit of GABABR at the plasma membranes. J. Cell. Physiol. 210: 237–245, 2007.

Adhesiolysis and targeted steroid/local anesthetic injection during epiduroscopy alleviates pain and reduces sensory nerve dysfunction in patients with chronic sciatica

PurposeThe aim of this study was to evaluate the effect of adhesiolysis followed by the injection of steroid and local anesthetic during epiduroscopy on sensory nerve function, pain, and functional disability in patients with chronic sciatica.MethodsEpidural adhesiolysis, using epiduroscopy, followed by the injection of steroid and local anesthetic, was scheduled in 19 patients with chronic sciatica refractory to lumbar epidural block. Sensory nerve function in the legs was tested with a series of 2000-Hz (Aβ-fiber), 250-Hz (Aδ-fiber), and 5-Hz (C-fiber) stimuli, using the current perception threshold (CPT), and CPT values and intensity of pain and Roland Morris Disability Questionnaire (RMDQ) scores were assessed before and 1 and 3 months after the epiduroscopy.ResultsAt all frequencies, the CPT values in the affected legs of patients before the epiduroscopy were significantly higher than those in the unaffected legs. Epidural adhesiolysis was successfully performed in 16 of the 19 patients. In these patients, the CPT values at 2000 and 250 Hz, and the pain and RMDQ scores 1 and 3 months after the epiduroscopy were significantly lower than those before the epiduroscopy, while the CPT value at 5 Hz did change.ConclusionEpidural adhesiolysis followed by the injection of steroid and local anesthetic during epiduroscopy alleviated pain, and functional disability, and reduced dysfunction of Aβ and Aδ fibers in patients with chronic sciatica.

Possible involvement of prolonging spinal µ-opioid receptor desensitization in the development of antihyperalgesic tolerance to µ-opioids under a neuropathic pain-like state.

In the present study, we investigated the possible development of tolerance to the antihyperalgesic effect of µ‐opioid receptor (MOR) agonists under a neuropathic pain‐like state. Repeated treatment with fentanyl, but not morphine or oxycodone, produced a rapid development of tolerance to its antihyperalgesic effect in mice with sciatic nerve ligation. Like the behavioral study, G‐protein activation induced by fentanyl was significantly reduced in membranes obtained from the spinal cord of nerve‐ligated mice with in vivo repeated injection of fentanyl. In β‐endorphin‐knockout mice with nerve ligation, developed tolerance to the antihyperalgesic effect of fentanyl was abolished, and reduced G‐protein activation by fentanyl after nerve ligation with fentanyl was reversed to the normal level. The present findings indicate that released β‐endorphin within the spinal cord may be implicated in the rapid development of tolerance to fentanyl under a neuropathic pain‐like state.

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.

GABAB receptors do not internalize after baclofen treatment, possibly due to a lack of β-arrestin association: Study with a real-time visualizing assay

The mechanism of agonist‐induced GABAB receptor (GABABR) internalization is not well understood. To investigate this process, we focused on the interaction of GABABR with β‐arrestins, which are key proteins in the internalization of most of the G protein‐coupled receptors, and the agonist‐induced GABABR internalization and the interaction of GABABR with β‐arrestin1 and β‐arrestin2 were investigated in real time using GABABR and β‐arrestins both of which were fluorescent protein‐tagged. We then compared these profiles with those of μ‐opioid receptors (μOR), well‐studied receptors that associate and cointernalize with β‐arrestins. When stimulated by the specific GABABR agonist baclofen, GABABR composed of GABAB1aR (GB1aR) and fluorescent protein‐tagged GABAB2R‐Venus (GB2R‐V) formed functional GABABR; they elicited G protein‐activated inwardly rectifying potassium channels as well as nontagged GABABR. In cells coexpressing GB1aR, GB2R‐V, and β‐arrestin1‐Cerulean (βarr1‐C) or β‐arrestin2‐Cerulean (βarr2‐C), real‐time imaging studies showed that baclofen treatment neither internalized GB2R‐V nor mobilized βarr1‐C or βarr2‐C to the cell surface. This happened regardless of the presence of G protein‐coupled receptor kinase 4 (GRK4), which forms a complex with GABABR and causes GABABR desensitization. On the other hand, in cells coexpressing μOR‐Venus, GRK2, and βarr1‐C or βarr2‐C, the μOR molecule formed μOR/βarr1 or μOR/βarr2 complexes on the cell surface, which were then internalized into the cytoplasm in a time‐dependent manner. Fluorescence resonance energy transfer assay also indicated scarce association of GB2R‐V and β‐arrestins‐C with or without the stimulation of baclofen, while robust association of μOR‐V with β‐arrestins‐C was detected after μOR activation. These findings suggest that GABABRs failure to undergo agonist‐induced internalization results in part from its failure to interact with β‐arrestins. Synapse 66:759–769, 2012.© 2012 Wiley Periodicals, Inc.

Effect of ONO-1101, a novel short-acting β-blocker on hemodynamic responses to isoflurane inhalation and tracheal intubation

PurposeWe investigated the effect of a new ultrashort-acting β-blocker, ONO-1101, on hemodynamic responses to isoflurane inhalation and tracheal intubation.MethodsFifty-four ASA PS 1 or 2 patients were randomly allocated to receive either ONO-1101, 0.04 mg·kg−1·min−1, or saline prior to tracheal intubation. Anesthesia was induced with thiamylal, 4 mg·kg−1, and vecuronium, 0.15 mg·kg−1. Tracheal intubation was carried out after 3 min controlled mask ventilation with 66% N2O and 3% inspired isoflurane in oxygen. Heart rate and blood pressure were continuously recorded from the start of induction until 5 min after intubation. Plasma concentrations of catecholamines were measured before induction, 3 min after initiating inhalation of isoflurane, and 1 min after tracheal intubation.ResultsSignificant increases in heart rate occurred in both groups in response to isoflurane inhalation and tracheal intubation, but the magnitude of the increase was significantly less in the ONO-1101 group. Blood pressure increased after tracheal intubation in the saline group but remained unchanged in the ONO-1101 group. Plasma concentrations of norepinephrine increased after induction and intubation in both groups, with no significant difference between the groups.ConclusionONO-1101 infusion is effective for the attenuation of hemodynamic responses to isoflurane inhalation and tracheal intubation.

Possible involvement of β-endorphin in a loss of the coordinated balance of μ-opioid receptors trafficking processes by fentanyl.

SATOSHI IMAI, YUKA SUDO, ATSUSHI NAKAMURA, AYUMI OZEKI, MEGUMI ASATO, MINORU HOJO, LAKSHMI A. DEVI, NAOKO KUZUMAKI, TSUTOMU SUZUKI, YASUHITO UEZONO, AND MINORU NARITA* Department of Toxicology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan Cancer Pathophysiology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan Department of Anesthesiology, Nagasaki University Graduate School of Biomedical Sciences,1-12-4 Sakamoto, Nagasaki-shi, Nagasaki 852-8523, Japan Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, 1468 Madison Avenue, New York 10029 Department of Cellular and Molecular Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki-shi, Nagasaki 852-8523, Japan

Efficacy and Safety of Oxycodone Injection for Relieving Cancer Pain: A Study in Japan Consisting of Two Open Trials for Intravenous and Subcutaneous Administration

Pure oxycodone injection became increasingly necessary after oral oxycodone was launched in Japan in 2003. However, trials clarifying the efficacy and safety of injection are rare. Therefore, a multicenter open study on injection was designed and carried out in 2010, resulting in the launch of injection therapy in 2012. As published domestic case reports on efficacy already show widespread prescription, this study aimed to provide useful information for cancer pain relief in Japan and other countries. Our oxycodone injection study consisted of two trials, one of intravenous (S#9131) and the other of subcutaneous (S#9132) administration. The minimum required number of enrolled patients suffering cancer pain was determined to be 70 in S#9131 and 20 in S#9132. These studies had the same dose-titration protocol as the main endpoint, i.e., pain relief rate (PRR) defined as the rate of achieving adequate pain control (APC), as in prior oral oxycodone trials in Japan. In S#9131, PRR was 81.4% (95% confidence interval: 70.3-89.7%), therefore, the null hypothesis of PRR<70% was rejected using the binominal one-sided test (p=0.0217). In S#9132, PRR was 73.7% also surpassing 70%. Safety was also assessed in the same way as in prior trials. The majority of adverse effects were moderate or mild and recovered with no sequelae. As shown above, the injection was considered to be effective and safe in cancer pain treatment. The details of these trials, particularly the dose-titration protocol for achieving APC and route switching information, are expected to enhance injection convenience for prescribers.