LGI1-ADAM22-MAGUK configures transsynaptic nanoalignment for synaptic transmission and epilepsy prevention.

Physiological functioning and homeostasis of the brain rely on finely tuned synaptic transmission, which involves nanoscale alignment between presynaptic neurotransmitter-release machinery and postsynaptic receptors. However, the molecular identity and physiological significance of transsynaptic nanoalignment remain incompletely understood. Here, we report that epilepsy gene products, a secreted protein LGI1 and its receptor ADAM22, govern transsynaptic nanoalignment to prevent epilepsy. We found that LGI1-ADAM22 instructs PSD-95 family membrane-associated guanylate kinases (MAGUKs) to organize transsynaptic protein networks, including NMDA/AMPA receptors, Kv1 channels, and LRRTM4-Neurexin adhesion molecules. Adam22ΔC5/ΔC5 knock-in mice devoid of the ADAM22-MAGUK interaction display lethal epilepsy of hippocampal origin, representing the mouse model for ADAM22-related epileptic encephalopathy. This model shows less-condensed PSD-95 nanodomains, disordered transsynaptic nanoalignment, and decreased excitatory synaptic transmission in the hippocampus. Strikingly, without ADAM22 binding, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Furthermore, forced coexpression of ADAM22 and PSD-95 reconstitutes nano-condensates in nonneuronal cells. Collectively, this study reveals LGI1-ADAM22-MAGUK as an essential component of transsynaptic nanoarchitecture for precise synaptic transmission and epilepsy prevention.

Biallelic ADAM22 pathogenic variants cause progressive encephalopathy and infantile-onset refractory epilepsy.

Pathogenic variants in A Disintegrin And Metalloproteinase (ADAM) 22, the postsynaptic cell membrane receptor for the glycoprotein leucine-rich repeat glioma-inactivated protein 1 (LGI1), have been recently associated with recessive developmental and epileptic encephalopathy. However, so far, only two affected individuals have been described and many features of this disorder are unknown. We refine the phenotype and report 19 additional individuals harbouring compound heterozygous or homozygous inactivating ADAM22 variants, of whom 18 had clinical data available. Additionally, we provide follow-up data from two previously reported cases. All affected individuals exhibited infantile-onset, treatment-resistant epilepsy. Additional clinical features included moderate to profound global developmental delay/intellectual disability (20/20), hypotonia (12/20) and delayed motor development (19/20). Brain MRI findings included cerebral atrophy (13/20), supported by post-mortem histological examination in patient-derived brain tissue, cerebellar vermis atrophy (5/20), and callosal hypoplasia (4/20). Functional studies in transfected cell lines confirmed the deleteriousness of all identified variants and indicated at least three distinct pathological mechanisms: (i) defective cell membrane expression; (ii) impaired LGI1-binding; and/or (iii) impaired interaction with the postsynaptic density protein PSD-95. We reveal novel clinical and molecular hallmarks of ADAM22 deficiency and provide knowledge that might inform clinical management and early diagnostics.

Venlafaxine ER Blocks the Norepinephrine Transporter in the Brain of Patients with Major Depressive Disorder: a PET Study Using [18F]FMeNER-D2.

BACKGROUND: The in vivo binding of clinical dose of venlafaxine on norepinephrine transporter has been questioned because venlafaxine has higher in vitro affinity to serotonin transporter than that to norepinephrine transporter. Although serotonin transporter occupancy of clinically relevant doses of venlafaxine has been reported, there has been no report of norepinephrine transporter occupancy in the human brain. METHODS: This was an open-label, single center, exploratory positron emission tomography study. Twelve major depressive disorder patients who had responded to venlafaxine extended-release and 9 control subjects were recruited. Each subject participated in one positron emission tomography measurement with [18F]FMeNER-D2. Binding potential in brain was quantified by the area under the curve ratio method with thalamus as target and white matter as reference regions. The difference of binding potential values between control and patient groups divided to 2 dose ranges were evaluated. Norepinephrine transporter occupancy (%) for all the major depressive disorder patients was calculated using mean binding potential of control subjects as baseline. The relationships between dose or plasma concentration of total active moiety and occupancies of norepinephrine transporter were also estimated. RESULTS: The binding potential of the patient group with 150 to 300 mg/d was significantly lower than that in the control subjects group (P = .0004 < .05/2). The norepinephrine transporter occupancy (8-61%) increased in a dose-dependent manner although a clear difference beyond 150 mg/d was not observed. CONCLUSIONS: This study demonstrates that clinically relevant doses of venlafaxine extended-release block the norepinephrine transporter of the major depressive disorder patient's brain. The data support the notion that the antidepressant effect of venlafaxine involves a combination of serotonin transporter and norepinephrine transporter blockades.

The Successful Removal of a Broken Needle as an Unusual Complication of Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA): A Case Report and Literature Review.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is recommended for the diagnosis of mediastinal lymph nodes by the American College of Chest Physicians guidelines; however, the guidelines state that this procedure should only be performed by a trained bronchoscopist. Complications related to needle malfunction during the EBUS-TBNA procedure have recently been reported. We herein describe a rare case involving the successful management of a needle breakage that occurred as an unusual complication of EBUS-TBNA. An 81-year-old male patient with a medical history of myocardial infarction was introduced to our institution to undergo an evaluation for mediastinal and right hilar lymphadenopathy on chest computed tomography (CT). We performed EBUS-TBNA in a 14×10 mm subcarinal lymph node station using a 22 G aspiration needle (NA-201SX-4022, Vizishot®, Olympus, Japan) for diagnosing and staging of the patient's lung cancer. After the second aspiration, we noticed that the needle tip was broken and that it was stuck in the right main bronchus. We immediately removed the broken needle tip from the right main bronchus by flexible bronchoscopy using an ID 8.5 mm tracheal tube without cuff inflation. The length of the needle tip was 13 mm and it was considerably bent. The EBUS scope did not suffer any apparent damage. The patient did not have any other procedure-related complications. Needle breakage during EBUS-TBNA is rare; however, inhaling or swallowing of a broken needle tip has the potential to cause serious complications. Bronchoscopists should therefore be aware of the possibility of needle breakage, which is an important complication during EBUS-TBNA.

[Evaluation of the Influence of the Experience and Training of EBUS-TBNA on Diagnostic Rate and Safety].

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has been widely used in Japan. The guidelines of the American College of Chest Physicians has recommended that EBUS-TBNA should be performed by well-trained operators who can perform highly accurate procedures, but the indicators of the degree of experience and training are unclear. In our department, physicians who do not have enough experience perform EBUS-TBNA under the supervision of bronchoscopic instructors who have EBUS-TBNA techniques (Board Certified Member of the Japan Society for Respiratory Endoscopy) after guidance and training in EBUS-TBNA using a simulator as an operator and helper. In order to evaluate the influence of the experience and training of EBUS-TBNA on diagnostic accuracy and safety, we retrospectively compared the diagnostic accuracy and safety of EBUS-TBNA performed by physicians within one year of experience of EBUS-TBNA and those performed by physicians with more than one year of experience. A total of 111 cases (148 lesions) who were eventually diagnosed as having primary lung cancer and underwent EBUS-TBNA in our department between April 2014 and January 2016 were divided into two groups. Group A (43 cases, 57 lesions) was examined by third-year doctors within one year of experience of EBUS-TBNA, and group B (68 cases, 91 lesions) was examined by doctors with four or more years of experience and with more than one year of experience of EBUS-TBNA. Diagnostic rate, examination time, and complications were evaluated. There were no significant differences between the two groups in the diagnostic rate (A, 89.5% vs. B, 90.1%, P = 1.0) or examination time (A, 27 min vs. B, 23 min, P = 0.149), and no complications were observed in either group. This study suggests that even less-experienced physicians may safely perform EBUS-TBNA as well as moderately-experienced physicians with more than 1 year experience of EBUS-TBNA with similar diagnostic rates when proper training and supervision are supplied.

A unique database for gathering data from a mobile app and medical prescription software: a useful data source to collect and analyse patient-reported outcomes of depression and anxiety symptoms.

A unique database named 'AN-SAPO' was developed by Iwato Corp. and Japan Brain Corp. in collaboration with the psychiatric clinics run by Himorogi Group in Japan. The AN-SAPO database includes patients' depression/anxiety score data from a mobile app named AN-SAPO and medical records from medical prescription software named 'ORCA'. On the mobile app, depression/anxiety severity can be evaluated by answering 20 brief questions and the scores are transferred to the AN-SAPO database together with the patients' medical records on ORCA. Currently, this database is used at the Himorogi Group's psychiatric clinics and has over 2000 patients' records accumulated since November 2013. Since the database covers patients' demographic data, prescribed drugs, and the efficacy and safety information, it could be a useful supporting tool for decision-making in clinical practice. We expect it to be utilised in wider areas of medical fields and for future pharmacovigilance and pharmacoepidemiological studies.

Systemic lupus erythematosus presenting with mixed-type fulminant autoimmune hemolytic anemia.

We report the case of a 9-year-old girl who presented with mixed-type fulminant autoimmune hemolytic anemia (AIHA) at the onset of systemic lupus erythematosus (SLE). On admission, laboratory investigations indicated very severe anemia (Hb, 2.7 g/dL) with reticulocytosis and positive direct/indirect Coombs tests. In addition, agglutinative reaction was clinically observed. Based on further examinations, the patient was diagnosed with AIHA complicated with SLE, and mixed-type AIHA was clinically identified. With oral prednisolone and methylprednisolone pulse therapy, the patient entered remission.

Post-renal Biopsy Retroperitoneal Haematoma Accompanied by Decreased Coagulation Factor XIII Levels in Immunoglobulin A Nephropathy.

Post-biopsy bleeding is the primary complication of renal biopsy. Retroperitoneal haematoma is a rare but severe bleeding complication; it commonly occurs among patients who have risk factors or vascular lesions. The bleeding risks in patients with immunoglobulin A (IgA) nephropathy (IgAN) have been discussed in the literature, but clinical data are lacking. Here, we report a case of a post-biopsy retroperitoneal haematoma accompanied by decreased coagulation factor XIII (FXIII) in a patient with IgAN. A 14-year-old male patient with haematuria and proteinuria but no bleeding or family history of bleeding underwent pre-renal biopsy evaluation that showed no coagulation abnormalities. He underwent percutaneous renal biopsy, and the histopathological diagnosis was IgAN. Five days after the biopsy, he presented with delayed bleeding from a retroperitoneal haematoma. During the workup for undiagnosed haemorrhagic diatheses, a mildly decreased FXIII level was discovered. This result suggested the possibility of bleeding complications associated with decreased FXIII. Some bleeding diatheses, including FXIII deficiency, cannot be evaluated in routine pre-biopsy coagulation tests. Mild FXIII deficiency can increase the risk of post-biopsy bleeding complications. Therefore, physicians should consider unevaluated haemorrhagic diatheses when a patient presents with major bleeding complications or delayed bleeding following renal biopsy without any known risk factors or vascular lesions.

Secukinumab-induced oral lichen planus in a psoriatic arthritis patient ameliorated after a switch to risankizumab.

Lichen planus is a chronic T-cell-mediated disorder in which lymphocytes, including Th17 cells, react toward the dermo-epidermal junction, which shows interface changes. Recently, IL-17-mediated changes in the oral mycobiome, including the proliferation of Candida and Aspergillus fungi, have been proposed as a possible pathomechanism of oral lichen planus (OLP). We treated a 54-year-old male who had been suffering from psoriatic arthritis. Secukinumab rapidly improved the skin and joint symptoms, but a painful erosion on the lip and thrush on the buccal mucosa appeared within 4 weeks. The erosion was histopathologically diagnosed as OLP. Although the candidiasis was successfully treated with topical miconazole nitrate, the labial OLP worsened during the secukinumab administration, despite the application of various topical agents. We finally switched from secukinumab to risankizumab, an anti-IL-23p19 agent, which dramatically improved the patient's OLP lesion in 4 weeks without candidiasis recurrence. Anti-IL-23p19 agents do not affect the oral mycobiome, and they are a potential therapeutic option for refractory OLP, including OLP induced by biologics.

Super-resolution imaging detects BP180 autoantigen in immunoglobulin M pemphigoid.

Bullous pemphigoid is generally caused by immunoglobulin (Ig)G autoantibodies against hemidesmosomal BP180 and/or BP230. Recently, the concept of IgM pemphigoid has been proposed. A 23-year-old Japanese woman presented with a 4-month history of severely itchy papules showing subepidermal separations with mild neutrophil infiltration. Direct immunofluorescence (DIF) revealed IgM deposits at the dermoepidermal junction, but neither IgG nor IgA deposits. Indirect immunofluorescence on 1 M NaCl-split skin demonstrated deposits on the epidermal side. The optical density (OD) value of a modified IgM enzyme-linked immunosorbent assay for full-length BP180, but not for BP180-NC16A, was increased. The patient was diagnosed with IgM pemphigoid and was treated with diphenyl sulfone at 50 mg/day without recurrence. To confirm the precise autoantigen, we tried to obtain super-resolution imaging. The deposition pattern of IgM autoantibodies seemed to be oriented parallel to that of BP180. The detailed images detect DIF deposits apart from BP180-NC16A staining, but are close to type VII collagen-NC1 staining. This result suggests that the IgM autoantibodies in the patient might target the C-terminus of BP180. IgM pemphigoid is still not a widely accepted concept, and the clinical course remains unknown. We will carefully follow-up the patient. Super-resolution images may help to detect precise autoantigens in autoimmune blistering diseases.

Trans-synaptic LGI1-ADAM22-MAGUK in AMPA and NMDA receptor regulation.

Exquisitely-regulated synaptic transmission and plasticity underlie higher brain functions such as learning and memory. PSD-95, a member of the MAGUK family, scaffolds an array of postsynaptic proteins including AMPA and NMDA receptors, and plays essential roles in excitatory synaptic transmission and postsynaptic organization. Epilepsy-related secreted protein LGI1 and its receptor ADAM22 represent major constituent elements of the PSD-95-containing synaptic protein complex in the brain. Recent studies begin to reveal a trans-synaptic configuration of the LGI1-ADAM22 complex and its pivotal role in AMPA and NMDA receptor-mediated synaptic transmission through regulating MAGUKs. Especially interesting is that without the association with LGI1-ADAM22, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Here, we review roles of LGI1-ADAM22 in synaptic function, and discuss its modes of action on the MAGUK regulation: as (i) a trans-synaptic hub, (ii) an extracellular scaffold, and (iii) an allosteric activator. We also highlight patho-physiological roles of the LGI1-ADAM22-MAGUK linkage in synaptic disorders such as epilepsy and autoimmune limbic encephalitis.

Risk of Extrapyramidal Syndromes Associated With Psychotropic Polypharmacy: A Study Based on Large-Scale Japanese Claims Data.

BACKGROUND: Psychotropic polypharmacy is common in clinical practice although supporting evidence is limited. Extrapyramidal syndromes (EPS) are adverse events associated with use of psychotropic drugs, especially antipsychotics. There are quite a few reports that suggest association between each psychotropic drug and EPS; however, the risk of EPS associated with their polypharmacy has not been fully evaluated. This study aimed at examining the influence of psychotropic polypharmacy on EPS occurrence by drug category (anxiolytics, hypnotics, antidepressants, and antipsychotics). METHODS: Sequence symmetry analyses were conducted using a large-scale Japanese health insurance claims database. This method assessed asymmetry in the distribution of EPS occurrence defined as both a diagnosis of EPS and a prescription for antiparkinsonian drugs before and after initiation of psychotropic drugs. The adjusted sequence ratio (ASR) and its 95% confidence interval (CI) were calculated. RESULTS: All categories of psychotropic drugs (anxiolytics, hypnotics, antidepressants, and antipsychotics) were significantly associated with EPS, and the tendency was stronger in polypharmacy associated with them. A clearer association between polypharmacy of BZs and EPS was indicated. In the analyses by subclasses of drugs, BZs, tetracyclic antidepressants, selective serotonin reuptake inhibitors, serotonin norepinephrine reuptake inhibitors, typical and atypical antipsychotics were significantly associated with EPS. The ASR of atypical antipsychotics (ASR 11.3, 95% CI 7.95-16.4) was higher than that of typical ones (ASR 2.96, 95% CI 2.06-4.33). CONCLUSIONS: The association between psychotropic polypharmacy and EPS was indicated. Further investigation is needed to confirm these findings because of the pharmacoepidemiologic nature of this study.

Characteristics of Patients with Hereditary Transthyretin Amyloidosis and an Evaluation of the Safety of Tafamidis Meglumine in Japan: An Interim Analysis of an All-case Postmarketing Surveillance.

PURPOSE: An all-case, single-arm, observational, postmarketing surveillance is underway to assess the safety of tafamidis in patients with hereditary transthyretin (ATTRv) amyloidosis with peripheral polyneuropathy, also called transthyretin-type familial amyloid polyneuropathy, in Japan. Results from an interim analysis (data cutoff date, May 15, 2018) are presented in this preliminary report. METHODS: Patients were registered and treated with tafamidis meglumine 20 mg/d in routine clinical practice (observation period, 156 weeks). Data on patient demographic and clinical characteristics and adverse drug reactions (ADRs) were captured using case-report forms. FINDINGS: Of 219 patients included (mean age, 59.7 years; patients with age at disease onset ≥50 years, 61.2%; mean treatment duration, 95.5 weeks), 143 (65.3%) were male, 126 (57.5%) had a family history of ATTRv amyloidosis, and 149 (68.0%) originated from nonendemic areas. The most common ADRs were diarrhea (1.4%) and hematuria (0.9%). Six serious ADRs (pneumonia, bacteremia, malignant melanoma, pancreatic carcinoma, hematuria, and hereditary neuropathic amyloidosis [primary disease exacerbation]) were reported; no ADRs leading to death were recorded. IMPLICATIONS: This interim analysis successfully provided comprehensive, nationwide epidemiologic data from 219 Japanese patients with ATTRv amyloidosis. The safety profile of tafamidis was largely consistent with that obtained from previous research. No new safety concerns were identified to date. Data presented in this interim analysis are subject to change following completion of the study, and we will continue to assess the safety and effectiveness of tafamidis throughout the study. ClinicalTrials.gov identifier: NCT02146378.

Changes in Prescription of Psychotropic Drugs After Introduction of Polypharmacy Reduction Policy in Japan Based on a Large-Scale Claims Database.

BACKGROUND AND OBJECTIVES: In Japan, polypharmacy reduction policy, which reduces the reimbursement of medical cost, was introduced to address unnecessary psychotropic polypharmacy. The rule was applied to the prescriptions of three or more anxiolytics or three or more hypnotics in the policy introduced in 2012. The prescriptions of four or more antidepressants or four or more antipsychotics were added to the rule in the policy revised in 2014. Furthermore, the prescriptions of three or more drugs of anxiolytics, hypnotics, antidepressants, or antipsychotics were subject to the reduction criteria of the policy revision in 2016. Benzodiazepine receptor agonists (BZs) are classified both into anxiolytics and hypnotics, and the reduction rule was not applied to the category of BZs before April 2018. This study aimed to examine the effect of the policy on the prescriptions of four drug categories as well as BZs from the point of view of the number of drugs and doses. METHODS: This was a retrospective observational study using a large-scale Japanese health insurance claims database. Patients who were prescribed at least one psychotropic drug (anxiolytic, hypnotic, antidepressant, or antipsychotic) during the study period (from April 2011 to March 2017) were selected. Segmented regression analysis was used to analyze the proportions of patients with three or more or four or more drugs as well as patients above clinically recommended doses, and the means of the average daily doses by drug category. RESULTS: A total of 312,167 patients were identified as a study population. The proportions of patients with three or more drugs in anxiolytics, hypnotics, antidepressants, and antipsychotics significantly decreased after the introduction or revisions of the policy, but not BZs. The proportions of patients with three or more drugs in March 2017 were 0.9%, 2.0%, 1.2%, 2.4%, and 8.9% in anxiolytics, hypnotics, antidepressants, antipsychotics, and BZs, respectively. The effect of the policy in reducing the proportions of patients above clinically recommended doses was identified in antipsychotics after the revision in 2016, but not identified in the sum of anxiolytics and hypnotics as well as BZs after the revision in 2014, and antidepressants after the revision in 2016. The proportions of monotherapy were increased from April 2011 to March 2017 only for antidepressants (76.9% → 80.8%) and antipsychotics (79.8% → 82.1%), and not changed or decreased for anxiolytics (85.2% → 85.7%), hypnotics (78.6% → 77.6%), sum of anxiolytics and hypnotics (68.1% → 65.7%), BZs (68.0% → 67.3%), and sum of psychotropic drugs (52.1% → 49.9%). CONCLUSIONS: The polypharmacy reduction policy reduced the proportions of patients with three or more drugs in four drug categories, but not BZs. Only limited effects were seen for reducing the proportions of patients above clinically recommended doses. The policy was revised in April 2018 again. Further investigation is needed to examine the effect of the revision in 2018.

Factors impacting the efficacy of venlafaxine extended release 75-225 mg/day in patients with major depressive disorder: exploratory post hoc subgroup analyses of a randomized, double-blind, placebo-controlled study in Japan.

PURPOSE: To explore the potential factors impacting the efficacy of venlafaxine extended release (ER) and treatment differences between 75 mg/day and 75-225 mg/day dose in patients with major depressive disorder (MDD). METHODS: We performed exploratory post hoc subgroup analyses of a randomized, double-blind, placebo-controlled study conducted in Japan. A total of 538 outpatients aged 20 years or older with a primary diagnosis of MDD who experienced single or recurrent episodes were randomized into three groups: fixed-dose, flexible-dose, or placebo. Venlafaxine ER was initiated at 37.5 mg/day and titrated to 75 mg/day for both fixed-dose and flexible-dose group, and to 225 mg/day for flexible-dose group (if well tolerated). Efficacy endpoints were changes from baseline at Week 8 using the Hamilton Rating Scale for Depression-17 items (HAM-D17) total score, Hamilton Rating Scale for Depression-6 items score, and Montgomery-Asberg Depression Rating Scale total score. The following factors were considered in the subgroup analyses: sex, age, HAM-D17 total score at baseline, duration of MDD, duration of current depressive episode, history of previous depressive episodes, history of previous medications for MDD, and CYP2D6 phenotype. For each subgroup, an analysis of covariance model was fitted and the adjusted mean of the treatment effect and corresponding 95% CI were computed. Due to the exploratory nature of the investigation, no statistical hypothesis testing was used. RESULTS: Venlafaxine ER improved symptoms of MDD compared with placebo in most subgroups. The subgroup with a long duration of MDD (>22 months) consistently showed greater treatment benefits in the flexible-dose group than in the fixed-dose group. CONCLUSION: These results suggest that a greater treatment response to venlafaxine ER (up to 225 mg/day) can be seen in patients with a longer duration of MDD. Further investigations are needed to identify additional factors impacting the efficacy of venlafaxine ER.

Possibility of Database Research as a Means of Pharmacovigilance in Japan Based on a Comparison with Sertraline Postmarketing Surveillance.

BACKGROUND: Many pharmacoepidemiologic studies using large-scale databases have recently been utilized to evaluate the safety and effectiveness of drugs in Western countries. In Japan, however, conventional methodology has been applied to postmarketing surveillance (PMS) to collect safety and effectiveness information on new drugs to meet regulatory requirements. Conventional PMS entails enormous costs and resources despite being an uncontrolled observational study method. This study is aimed at examining the possibility of database research as a more efficient pharmacovigilance approach by comparing a health care claims database and PMS with regard to the characteristics and safety profiles of sertraline-prescribed patients. METHODS: The characteristics of sertraline-prescribed patients recorded in a large-scale Japanese health insurance claims database developed by MinaCare Co. Ltd. were scanned and compared with the PMS results. We also explored the possibility of detecting signals indicative of adverse reactions based on the claims database by using sequence symmetry analysis. Diabetes mellitus, hyperlipidemia, and hyperthyroidism served as exploratory events, and their detection criteria for the claims database were reported by the Pharmaceuticals and Medical Devices Agency in Japan. RESULTS: Most of the characteristics of sertraline-prescribed patients in the claims database did not differ markedly from those in the PMS. There was no tendency for higher risks of the exploratory events after exposure to sertraline, and this was consistent with sertraline's known safety profile. CONCLUSIONS: Our results support the concept of using database research as a cost-effective pharmacovigilance tool that is free of selection bias . Further investigation using database research is required to confirm our preliminary observations.

Suppression of catechol-O-methyltransferase activity through blunting of alpha2-adrenoceptor can explain hypertension in Dahl salt-sensitive rats.

Catecholamines have been reported to be involved in the development of salt-sensitive hypertension. We investigated the relation between catechol-O-methyltransferase (COMT) and salt-sensitivity. In the first experiment, Dahl salt-sensitive (DS) rats were given a normal-salt (NS), high-salt (HS), or HS+hydralazine (80 mg/l water) diet for 4 or 13 weeks, and Dahl salt-resistant (DR) rats were given a NS or HS diet. COMT activities in both the kidneys and liver and urinary norepinephrine (NE) and dopamine (DA) excretion were measured. In the second experiment, HepG2 cells were used to investigate the role of NE in regulating COMT activity. In the third experiment, we investigated the reactivity of pre- and postsynaptic alpha(2)-adrenoceptor (AR) in DS rats. HS loading significantly suppressed the activities of membrane-bound COMT (MB-COMT) and, consistent with this finding, increased the urinary NE level in DS rats, but not in DR rats. Hydralazine did not restore the MB-COMT activities, which suggested that HS loading rather than elevated blood pressure suppressed the MB-COMT activities. The in vitro experiment using HepG2 cells revealed that NE increased the MB-COMT activity via the alpha(2)-AR. However, both the pre- and postsynaptic alpha(2)-AR reactivity was decreased by HS loading in DS rats. In conclusion, HS intake suppresses the MB-COMT activities in DS rats, presumably by blunting alpha(2)-AR signaling. The suppression of MB-COMT activities, consequent decrease in degradation of NE, and increase in NE release by blunting of alpha(2)-AR function may be involved in the development of salt-sensitive hypertension in DS rats, in whom DA-dependent natriuresis may be suppressed.

Rapid assay for catechol-O-methyltransferase activity by high-performance liquid chromatography-fluorescence detection.

A rapid assay for measuring the activities of catechol-O-methyltransferase (COMT) is described. The method is based on high-performance liquid chromatography (HPLC)-fluorescence detection, and includes on-line extraction of catecholamines with a precolumn, separation of norepinephrine (NE) and normetanephrine (NMN) on an ODS column, electrochemical oxidation, and post-column fluorogenic derivatization using ethylenediamine. The method took less than 25 min for one sample, which is half that of the previous method and the sensitivity was similar. The intra-day assay precisions were 0.52-1.6%, and the inter-day assay precisions were 3.6-5.8% for rat liver and cerebral cortex (n = 5). The method is suitable for the rapid measurement of COMT activities of many biological samples.