Hiroaki Murata

Glutaredoxin Exerts an Antiapoptotic Effect by Regulating the Redox State of Akt

Glutaredoxin (GRX) is a small dithiol protein involved in various cellular functions, including the redox regulation of certain enzyme activities. GRX functions via a disulfide exchange reaction by utilizing the active site Cys-Pro-Tyr-Cys. Here we demonstrated that overexpression of GRX protected cells from hydrogen peroxide (H2O2)-induced apoptosis by regulating the redox state of Akt. Akt was transiently phosphorylated, dephosphorylated, and then degraded in cardiac H9c2 cells undergoing H2O2-induced apoptosis. Under stress, Akt underwent disulfide bond formation between Cys-297 and Cys-311 and dephosphorylation in accordance with an increased association with protein phosphatase 2A. Overexpression of GRX protected Akt from H2O2-induced oxidation and suppressed recruitment of protein phosphatase 2A to Akt, resulting in a sustained phosphorylation of Akt and inhibition of apoptosis. This effect was reversed by cadmium, an inhibitor of GRX. Furthermore an in vitro assay revealed that GRX reduced oxidized Akt in concert with glutathione, NADPH, and glutathione-disulfide reductase. Thus, GRX plays an important role in protecting cells from apoptosis by regulating the redox state of Akt.

17β-Estradiol Protects against Oxidative Stress-induced Cell Death through the Glutathione/Glutaredoxin-dependent Redox Regulation of Akt in Myocardiac H9c2 Cells

The GSH/glutaredoxin (GRX) system is involved in the redox regulation of certain enzyme activities, and this system protects cells from H2O2-induced apoptosis by regulating the redox state of Akt (Murata, H., Ihara, Y., Nakamura, H., Yodoi, J., Sumikawa, K., and Kondo, T. (2003) J. Biol. Chem. 278, 50226–50233). Estrogens, such as 17β-estradiol (E2), play an important role in development, growth, and differentiation and appear to have protective effects on oxidative stress mediated by estrogen receptor α (ERα). However, the role of the ERβ-mediated pathway in this cytoprotection and the involvement of E2 in the redox regulation are not well understood. In the present study, we demonstrated that E2 protected cardiac H9c2 cells, expressing ERβ from H2O2-induced apoptosis concomitant with an increase in the activity of Akt. E2 induced the expression of glutaredoxin (GRX) as well as γ-glutamylcysteine synthetase, a rate-limiting enzyme for the synthesis of GSH. Inhibitors for both γ-glutamylcysteine synthetase and GRX and ICI182,780, a specific inhibitor of ERs, abolished the protective effect of E2 on cell survival as well as the activity of Akt, suggesting that ERβ is involved in the cytoprotection and redox regulation by E2. Transcription of the GRX gene was enhanced by E2. The promoter activity of GRX was up-regulated by an ERβ-dependent element. These results suggest that the GRX/GSH system is involved in the cytoprotective and genomic effects of E2 on the redox state of Akt, a pathway that is mediated, at least in part, by ERβ. This mechanism may also play an antiapoptotic role in cancer cells during carcinogenesis or chemotherapy.

Glutaredoxin Modulates Platelet-derived Growth Factor-dependent Cell Signaling by Regulating the Redox Status of Low Molecular Weight Protein-tyrosine Phosphatase

Glutaredoxin (GRX) is a glutathione-disulfide oxidoreductase involved in various cellular functions, including the redox-dependent regulation of certain integral proteins. Here we demonstrated that overexpression of GRX suppressed the proliferation of myocardiac H9c2 cells treated with platelet-derived growth factor (PDGF)-BB. After stimulation with PDGF-BB, the phosphorylation of PDGF receptor (PDGFR) β was suppressed in GRX gene-transfected cells, compared with controls. Conversely, the phosphorylation was enhanced by depletion of GRX by RNA interference. In this study we focused on the role of low molecular weight protein-tyrosine phosphatase (LMW-PTP) in the dephosphorylation of PDGFRβ via a redox-dependent mechanism. We found that depletion of LMW-PTP using RNA interference enhanced the PDGF-BB-induced phosphorylation of PDGFRβ, indicating that LMW-PTP works for PDGFRβ. The enhancement of the phosphorylation of PDGFRβ was well correlated with inactivation of LMW-PTP by cellular peroxide generated in the cells stimulated with PDGF-BB. In vitro, with hydrogen peroxide treatment, LMW-PTP showed decreased activity with the concomitant formation of dithiothreitol-reducible oligomers. GRX protected LMW-PTP from hydrogen peroxide-induced oxidation and inactivation in concert with glutathione, NADPH, and glutathione disulfide reductase. This strongly suggests that retention of activity of LMW-PTP by enhanced GRX expression suppresses the proliferation of cells treated with PDGF-BB via enhanced dephosphorylation of PDGFRβ. Thus, GRX plays an important role in PDGF-BB-dependent cell proliferation by regulating the redox state of LMW-PTP.

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.

Anesthetic management of peroral endoscopic myotomy for esophageal achalasia: a retrospective case series

Peroral endoscopic myotomy (POEM) is a newly developed, less invasive treatment for esophageal achalasia that requires general anesthesia under positive pressure ventilation. In this retrospective case series, we describe the anesthetic management of 28 consecutive patients who underwent POEM for esophageal achalasia. Anesthesia was maintained with sevoflurane and remifentanil under positive pressure ventilation through a tracheal tube. Retained contents in the esophagus were evacuated just before anesthesia induction to prevent regurgitation into the trachea. The POEM procedure was performed using an orally inserted flexible fiberscope. Elevation of end-tidal carbon dioxide after initiating esophageal carbon dioxide insufflation was observed in all patients and was treated by minute adjustments to the ventilation volume. Scopolamine butylbromide-induced tachycardia in one patient was treated with landiolol hydrochloride, which is a short-acting beta 1-selective blocker. Minor subcutaneous emphysema around the neck was observed in one patient. POEM was successfully completed, and tracheas were extubated immediately after the procedure in all patients. Our findings suggest that prevention of aspiration pneumonia during anesthesia induction, preparation for carbon dioxide insufflation-related complications, and treatment of scopolamine butylbromide-induced tachycardia play important roles in safe anesthesia management of POEM for esophageal achalasia.

Ultrasound-guided Continuous Thoracic Paravertebral Block for Outpatient Acute Pain Management of Multilevel Unilateral Rib Fractures

A 61-year-old man with multiple unilateral rib fractures (T3-T8) gained the ability to breathe deeply and to ambulate after ultrasound-guided continuous thoracic paravertebral block and was discharged home after being observed for 15 hours after the block. The ultrasound guidance was helpful in determining the site of rib fractures and the optimal level for catheter placement. This report also discusses the management of analgesia using continuous paravertebral block in an outpatient with trauma.

Overexpression of glutaredoxin protects cardiomyocytes against nitric oxide-induced apoptosis with suppressing the S-nitrosylation of proteins and nuclear translocation of GAPDH

There is increasing evidence demonstrating that glutaredoxin 1 (GRX1), a cytosolic enzyme responsible for the catalysis of protein deglutathionylation, plays distinct roles in inflammation and apoptosis by inducing changes in the cellular redox system. In this study, we investigated whether and how the overexpression of GRX1 protects cardiomyocytes against nitric oxide (NO)-induced apoptosis. Cardiomyocytes (H9c2 cells) were transfected with the expression vector for mouse GRX1 cDNA, and mock-transfected cells were used as a control. Compared with the mock-transfected cells, the GRX1-transfected cells were more resistant to NO-induced apoptosis. Stimulation with NO significantly increased the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a pro-apoptotic protein, in the mock-transfected cells, but did not change GAPDH localization in the GRX1-transfected cells. Furthermore, we found that NO stimulation clearly induced the oxidative modification of GAPDH in the mock-transfected cells, whereas less modification of GAPDH was observed in the GRX1-transfected cells. These data suggest that the overexpression of GRX1 could protect cardiomyocytes against NO-induced apoptosis, likely through the inhibition of the oxidative modification and the nuclear translocation of GAPDH.

The presence of transverse cervical and dorsal scapular arteries at three ultrasound probe positions commonly used in supraclavicular brachial plexus blockade.

BACKGROUND:Ultrasound-guided supraclavicular brachial plexus block carries a risk for puncture of vascular structures. In this study, we determined the frequency with which the transverse cervical artery (TCA) and the dorsal scapular artery (DSA) are detected by ultrasound evaluation at 3 probe positions during supraclavicular block. METHODS:Ultrasound examinations of the supraclavicular region were performed in 53 healthy adult volunteers. Ultrasound images of the supraclavicular region were acquired at 3 probe positions: position A (the brachial plexus and the subclavian artery both lying on the first rib); position B (the brachial plexus on the first rib; the artery on the pleura); and position C (the brachial plexus between the anterior and middle scalene muscles). The primary outcome variables were the frequencies with which TCA and DSA were detected by 2-dimensional and color Doppler imaging at 3 specified probe positions. RESULTS:One hundred six supraclavicular regions were examined in 53 subjects. The subclavian artery was detected in all subjects. TCA was more often detected than DSA, 94 (88.7%, 95% confidence interval [CI] 80.7%–93.8%) and 36 (34%, 95% CI 25.3%–43.9%) of 106 scans, respectively (McNemar P value <0.001). TCA was detected in 2 (1.9%, 95% CI 0.3%–7.3%), 31 (29.2%, 95% CI 20.9%–38.9%), and 61 (57.5%, 95% CI 47.5%–66.9%) of scans at probe positions A, B, and C, respectively, whereas DSA was detected in 3 (2.8%, 95% CI 0.7%–8.6%), 23 (21.7%, 95% CI 14.5%–30.9%), and 10 (9.4%, 95% CI 4.8%–17.0%) of scans at probe positions A, B, and C, respectively. Thus, the TCA and DSA were less likely to be present with probe position A (all P < 0.001). CONCLUSION:TCA was more often detected than DSA in the vicinity of the brachial plexus in the supraclavicular region. Both TCA and DSA were least likely to be present in probe position A. Color Doppler, particularly for probe position A, may help to reduce the risk for inadvertent vascular puncture during ultrasound-guided supraclavicular block.

Extent of sympathectomy affects postoperative compensatory sweating and satisfaction in patients with palmar hyperhidrosis

PurposeEndoscopic thoracic sympathectomy (ETS) for the treatment of palmar hyperhidrosis is generally performed at one or two levels ranging between T2 and T4; however, compensatory sweating (CS) is an occasional bothersome side effect. The aim of our study was to evaluate the association between the extent of ETS and the degree of postoperative CS and palmar sweating, as well as patient satisfaction.MethodsThe participants represented a consecutive series of 76 patients who underwent bilateral ETS for palmar hyperhidrosis at level T2 and/or T3. Patients were interviewed by postal questionnaires to assess their self-reported degree of postoperative palmar sweating and CS and their outcome satisfaction. Of the 53 patients who replied to the postal questionnaire, 25 underwent bilateral ETS at one level (group A), and 27 underwent bilateral ETS at two levels (group B). One patient who underwent asymmetrical sympathectomy was excluded.ResultsThe degree of postoperative palmar sweating was significantly lower in group B than in group A. The severity of CS was significantly higher in group B than in group A. The severity of CS was significantly inversely correlated with the degree of patient satisfaction. However, the degree of postoperative palmar sweating was not correlated with the degree of patient satisfaction.ConclusionsCompared to ETS at two levels, single-level ETS of T2 or T3 reduces postoperative palmar sweating to a milder degree, and causes CS to a less severe degree. The severity of CS is inversely correlated with the degree of patient satisfaction.

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.