Transient Impairment in Microglial Function Causes Sex-Specific Deficits in Synaptic and Hippocampal Function in Mice Exposed to Early Adversity.

Abnormal development and function of the hippocampus are two of the most consistent findings in humans and rodents exposed to early life adversity, with males often being more affected than females. Using the limited bedding (LB) paradigm as a rodent model of early life adversity, we found that male adolescent mice that had been exposed to LB exhibit significant deficits in contextual fear conditioning and synaptic connectivity in the hippocampus, which are not observed in females. This is linked to altered developmental refinement of connectivity, with LB severely impairing microglial-mediated synaptic pruning in the hippocampus of male and female pups on postnatal day 17 (P17), but not in adolescent P33 mice when levels of synaptic engulfment by microglia are substantially lower. Since the hippocampus undergoes intense synaptic pruning during the second and third weeks of life, we investigated whether microglia are required for the synaptic and behavioral aberrations observed in adolescent LB mice. Indeed, transient ablation of microglia from P13-21, in normally developing mice caused sex-specific behavioral and synaptic abnormalities similar to those observed in adolescent LB mice. Furthermore, chemogenetic activation of microglia during the same period reversed the microglial-mediated phagocytic deficits at P17 and restored normal contextual fear conditioning and synaptic connectivity in adolescent LB male mice. Our data support an additional contribution of astrocytes in the sex-specific effects of LB, with increased expression of the membrane receptor MEGF10 and enhanced synaptic engulfment in hippocampal astrocytes of 17-day-old LB females, but not in LB male littermates. This finding suggests a potential compensatory mechanism that may explain the relative resilience of LB females. Collectively, these studies highlight a novel role for glial cells in mediating sex-specific hippocampal deficits in a mouse model of early-life adversity.

Telehealth Availability for Cancer Care During the COVID-19 Pandemic: Cross-Sectional Study.

BACKGROUND: Telehealth was an important strategy for maintaining continuity of cancer care during the coronavirus pandemic and has continued to play a role in outpatient care; however, it is unknown whether services are equally available across cancer hospitals. OBJECTIVE: This study aimed to assess telehealth availability at cancer hospitals for new and established patients with common cancers to contextualize the impact of access barriers to technology on overall access to health care. METHODS: We conducted a national cross-sectional secret shopper study from June to November 2020 to assess telehealth availability at cancer hospitals for new and established patients with colorectal, breast, and skin (melanoma) cancer. We examined facility-level factors to determine predictors of telehealth availability. RESULTS: Of the 312 investigated facilities, 97.1% (n=303) provided telehealth services for at least 1 cancer site. Telehealth was less available to new compared to established patients (n=226, 72% vs n=301, 97.1%). The surveyed cancer hospitals more commonly offered telehealth visits for breast cancer care (n=266, 85%) and provided lower access to telehealth for skin (melanoma) cancer care (n=231, 74%). Most hospitals (n=163, 52%) offered telehealth for all 3 cancer types. Telehealth availability was weakly correlated across cancer types within a given facility for new (r=0.16, 95% CI 0.09-0.23) and established (r=0.14, 95% CI 0.08-0.21) patients. Telehealth was more commonly available for new patients at National Cancer Institute-designated facilities, medical school-affiliated facilities, and major teaching sites, with high total admissions and below-average timeliness of care. Telehealth availability for established patients was highest at Academic Comprehensive Cancer Programs, nongovernment and nonprofit facilities, medical school-affiliated facilities, Accountable Care Organizations, and facilities with a high number of total admissions. CONCLUSIONS: Despite an increase in telehealth services for patients with cancer during the COVID-19 pandemic, we identified differences in access across cancer hospitals, which may relate to measures of clinical volume, affiliation, and infrastructure.

Updates to the Autism Intervention Research Network on Physical Health (AIR-P) Research Agenda.

INTRODUCTION: Autistic individuals, now representing one in 36 individuals in the U.S., experience disproportionate physical health challenges relative to non-autistic individuals. The Health Resources and Services Administration's (HRSA) Autism Intervention Research Network on Physical Health (AIR-P) is an interdisciplinary, multi-center Research Network that aims to increase the health, well-being, and quality of life of autistic individuals. The current paper builds on the initial AIR-P Research Agenda (proposed in Year 1) and provides an updated vision for the Network. METHODS: Updates to the Research Agenda were made via the administration of a Qualtrics survey, and disseminated widely to all AIR-P entities, including the Research Node Leaders, Steering Committee, Autistic Researcher Review Board, and collaborating academic and non-academic entities. Network members were tasked with evaluating the Year 1 Research Agenda and proposing additional priorities. RESULTS: Within each Research Node, all Year 1 priorities were endorsed as continued priorities for research on autism and physical health. Specific topics, including co-occurring conditions and self-determination, advocacy, and decision-making, were particularly endorsed. Opportunities for exploratory studies and intervention research were identified across Research Nodes. Qualitative responses providing feedback on additional research priorities were collected. CONCLUSION: The updated AIR-P Research Agenda represents an important step toward enacting large-scale health promotion efforts for autistic individuals across the lifespan. This updated agenda builds on efforts to catalyze autism research in historically underrepresented topic areas while adopting a neurodiversity-oriented approach to health promotion.

Asymmetric dimethylation of AMPKα1 by PRMT6 contributes to the formation of phase-separated puncta.

AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase comprising α, ß, and γ subunits. AMPK is involved in intracellular energy metabolism and functions as a switch that turns various biological pathways in eukaryotes on and off. Several post-translational modifications regulating AMPK function have been demonstrated, including phosphorylation, acetylation, and ubiquitination; however, arginine methylation has not been reported in AMPKα1. We investigated whether arginine methylation occurs in AMPKα1. Screening experiments revealed arginine methylation of AMPKα1 mediated by protein arginine methyltransferase 6 (PRMT6). In vitro methylation and co-immunoprecipitation assays indicated that PRMT6 can directly interact with and methylate AMPKα1 without involvement of other intracellular components. In vitro methylation assays with truncated and point mutants of AMPKα1 revealed that Arg403 is the residue methylated by PRMT6. Immunocytochemical studies showed that the number of AMPKα1 puncta was enhanced in saponin-permeabilized cells when AMPKα1 was co-expressed with PRMT6, suggesting that PRMT6-mediated methylation of AMPKα1 at Arg403 alters the physiological characteristics of AMPKα1 and may lead to liquid-liquid phase separation.

Recent Development of Mechanical Stimuli Detectable Sensors, Their Future, and Challenges: A Review.

By virtue of their wide applications in transportation, healthcare, smart home, and security, development of sensors detecting mechanical stimuli, which are many force types (pressure, shear, bending, tensile, and flexure) is an attractive research direction for promoting the advancement of science and technology. Sensing capabilities of various force types based on structural design, which combine unique structure and materials, have emerged as a highly promising field due to their various industrial applications in wearable devices, artificial skin, and Internet of Things (IoT). In this review, we focus on various sensors detecting one or two mechanical stimuli and their structure, materials, and applications. In addition, for multiforce sensing, sensing mechanism are discussed regarding responses in external stimuli such as piezoresistive, piezoelectric, and capacitance phenomena. Lastly, the prospects and challenges of sensors for multiforce sensing are discussed and summarized, along with research that has emerged.

Corticosteroid Therapy in Immunoglobulin A Nephropathy: A Friend or Foe?

BACKGROUND: The administration of corticosteroids in addition to supportive care to delay progressive immunoglobulin A nephropathy (IgAN), the most common primary glomerulonephritis worldwide, remains controversial. This is partly due to the paucity of well-designed randomized controlled trials and well-known corticosteroid-related side effects. As a result, clinical equipoise in corticosteroid therapy exists depending on geographical regions and the clinician's preference. SUMMARY: Better understanding around the pathogenesis of IgAN has prompted several clinical trials exploring the effects of immunosuppressive agents including corticosteroids. Earlier studies of corticosteroids were limited by suboptimal study designs, inadequate implementation of standard of care, and inconsistent adverse event data collection. Two well-designed, adequately powered, multi-centre randomized controlled trials, the STOP-IgAN and TESTING studies, have reported contrasting kidney outcomes that have further fuelled the clinical conundrum regarding the efficacy of corticosteroids. Both studies independently reported greater adverse events with corticosteroids. A novel targeted release formulation of budesonide, which has been hypothesized to reduce the adverse events associated with systemic corticosteroids, has shown promising results in the Phase 3 NefigaRD trial. Studies of treatments targeting B cells and the complement cascade are currently underway, and early data appear encouraging. This review provides an overview of the current literature around the understanding of the pathomechanisms and benefits and harm of corticosteroid use in IgAN. KEY MESSAGES: Recent evidence suggests the use of corticosteroids in a selected cohort of people with IgAN at high risk of disease progression can improve kidney outcomes but comes with an associated risk of treatment-related adverse events, particularly with higher doses. Management decisions should therefore follow an informed patient-clinician discussion.

Practice patterns and perceptions of influenza testing amongst pediatric urgent care providers.

INTRODUCTION: Despite a sensitivity of 50% to 70% the rapid influenza diagnostic test (RIDT) continues to play an important role in clinical decision-making due to its quick turn-around time, high specificity, relative simplicity of use, and low cost. METHODS: A quantitative study using a web-based survey was distributed to 110 members of the Society of Pediatric Urgent Care aimed to assess RIDT use for diagnosis and management of influenza in outpatient pediatric patients. RESULTS: Responses from 61 providers were received. Forty-two percent (95% CI 29.5-54.5%) of respondents report higher confidence in their diagnosis of influenza with the aid of a positive RIDT. 28% of respondents (95% CI 16.6-39.4%) report a higher likelihood of prescribing antiviral medications to low-risk patients if an RIDT is positive than without laboratory confirmation. CONCLUSION: Most pediatric urgent care respondents reported higher confidence in their diagnosis and higher likelihood of prescribing antivirals with a positive RIDT rather than by clinical symptoms alone.

Acceptance of Simulated Adult Patients With Medicaid Insurance Seeking Care in a Cancer Hospital for a New Cancer Diagnosis.

Importance: Although there have been significant increases in the number of US residents insured through Medicaid, the ability of patients with Medicaid to access cancer care services is less well known. Objective: To assess facility-level acceptance of Medicaid insurance among patients diagnosed with common cancers. Design, Setting, and Participants: This national cross-sectional secret shopper study was conducted in 2020 in a random sample of Commission on Cancer-accredited facilities in the United States using a simulated cohort of Medicaid-insured adult patients with colorectal, breast, kidney, and melanoma skin cancer. Exposures: Telephone call requesting an appointment for a patient with Medicaid with a new cancer diagnosis. Main Outcomes and Measures: Acceptance of Medicaid insurance for cancer care. Descriptive statistics, χ2 tests, and multivariable logistic regression models were used to examine factors associated with Medicaid acceptance for colorectal, breast, kidney, and skin cancer. High access hospitals were defined as those offering care across all 4 cancer types surveyed. Explanatory measures included facility-level factors from the 2016 American Hospital Association Annual Survey and Centers for Medicare & Medicaid Services General Information database. Results: A nationally representative sample of 334 facilities was created, of which 226 (67.7%) provided high access to patients with Medicaid seeking cancer care. Medicaid acceptance differed by cancer site, with 319 facilities (95.5%) accepting Medicaid insurance for breast cancer care; 302 (90.4%), colorectal; 290 (86.8%), kidney; and 266 (79.6%), skin. Comprehensive community cancer programs (OR, 0.4; 95% CI, 0.2-0.7; P = .007) were significantly less likely to provide high access to care for patients with Medicaid. Facilities with nongovernment, nonprofit (vs for-profit: OR, 3.5; 95% CI, 1.1-10.8; P = .03) and government (vs for-profit: OR, 6.6; 95% CI, 1.6-27.2; P = .01) ownership, integrated salary models (OR, 2.6; 95% CI, 1.5-4.5; P = .001), and average (vs above-average: OR, 6.4; 95% CI, 1.4-29.6; P = .02) or below-average (vs above-average: OR, 8.4; 95% CI, 1.5-47.5; P = .02) effectiveness of care were associated with high access to Medicaid. State Medicaid expansion status was not significantly associated with high access. Conclusions and Relevance: This study identified access disparities for patients with Medicaid insurance at centers designated for high-quality care. These findings highlight gaps in cancer care for the expanding population of patients receiving Medicaid.

Access to telehealth services for colorectal cancer patients in the United States during the COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic yielded rapid telehealth deployment to improve healthcare access, including for surgical patients. METHODS: We conducted a secret shopper study to assess telehealth availability for new patient and follow-up colorectal cancer care visits in a random national sample of Commission on Cancer accredited hospitals and investigated predictive facility-level factors. RESULTS: Of 397 hospitals, 302 (76%) offered telehealth for colorectal cancer patients (75% for follow-up, 42% for new patients). For new patients, NCI-designated Cancer Programs offered telehealth more frequently than Integrated Network (OR: 0.20, p = 0.01), Academic Comprehensive (OR: 0.18, p = 0.001), Comprehensive Community (OR: 0.10, p < 0.001), and Community (OR: 0.11, p < 0.001) Cancer Programs. For follow-up, above average timeliness of care hospitals offered telehealth more frequently than average hospitals (OR: 2.87, p = 0.04). CONCLUSIONS: We identified access disparities and predictive factors for telehealth availability for colorectal cancer care during the COVID-19 pandemic. These factors should be considered when constructing telehealth policies.

Diagnostic yield of MR myelography in patients with newly diagnosed spontaneous intracranial hypotension: a systematic review and meta-analysis.

OBJECTIVES: To investigate the pooled diagnostic yield of MR myelography in patients with newly diagnosed spontaneous intracranial hypotension (SIH). METHODS: A literature search of the MEDLINE/PubMed and Embase databases was conducted until July 25, 2021, including studies with the following inclusion criteria: (a) population: patients with newly diagnosed SIH; (b) diagnostic modality: MR myelography or MR myelography with intrathecal gadolinium for evaluation of CSF leakage; (c) outcomes: diagnostic yield of MR myelography or MR myelography with intrathecal gadolinium. The risk of bias was evaluated using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. DerSimonian-Laird random-effects modeling was used to calculate the pooled estimates. Subgroup analysis regarding epidural fluid collection and meta-regression were additionally performed. RESULTS: Fifteen studies with 643 patients were included. Eight studies used MR myelography with intrathecal gadolinium, and 11 used MR myelography. The overall quality of the included studies was moderate. The pooled diagnostic yield of MR myelography was 86% (95% CI, 80-91%) and that of MR myelography with intrathecal gadolinium was 83% (95% CI, 51-96%). There was no significant difference in pooled diagnostic yield between MR myelography and MR myelography with intrathecal gadolinium (p = 0.512). In subgroup analysis, the pooled diagnostic yield of the epidural fluid collection was 91% (95% CI, 84-94%). In meta-regression, the diagnostic yield was unaffected regardless of consecutive enrollment, magnet strength, or 2D/3D. CONCLUSIONS: MR myelography had a high diagnostic yield in patients with SIH. MR myelography is non-invasive and not inferior to MR myelography with intrathecal gadolinium. KEY POINTS: • The pooled diagnostic yield of MR myelography was 86% (95% CI, 80-91%) in patients with spontaneous intracranial hypotension. • There was no significant difference in pooled diagnostic yield between MR myelography and MR myelography with intrathecal gadolinium. • MR myelography is non-invasive and not inferior to MR myelography with intrathecal gadolinium.

Colorimetric Sensor Based on Hydroxypropyl Cellulose for Wide Temperature Sensing Range.

Recently, temperature monitoring with practical colorimetric sensors has been highlighted because they can directly visualize the temperature of surfaces without any power sources or electrical transducing systems. Accordingly, several colorimetric sensors that convert the temperature change into visible color alteration through various physical and chemical mechanisms have been proposed. However, the colorimetric temperature sensors that can be used at subzero temperatures and detect a wide range of temperatures have not been sufficiently explored. Here, we present a colorimetric sensory system that can detect and visualize a wide range of temperatures, even at a temperature below 0 °C. This system was developed with easily affordable materials via a simple fabrication method. The sensory system is mainly fabricated using hydroxypropyl cellulose (HPC) and ethylene glycol as the coolant. In this system, HPC can self-assemble into a temperature-responsive cholesteric liquid crystalline mesophase, and ethylene glycol can prevent the mesophase from freezing at low temperatures. The colorimetric sensory system can quantitatively visualize the temperature and show repeatability in the temperature change from -20 to 25 °C. This simple and reliable sensory system has great potential as a temperature-monitoring system for structures exposed to real environments.

Patient and Visit Characteristics of Pediatric Patients With High-frequency Low-acuity Emergency Department Visits.

INTRODUCTION: Pediatric patients account for a disproportionate number of low-acuity emergency department (ED) visits. The aim of this study is to describe pediatric patient and visit characteristics for high-frequency users for low-acuity visits. METHODS: This was a retrospective cohort study of children presenting to a tertiary care pediatric ED and an affiliated community ED, over a 2-year period, with at least 10 low-acuity visits. Twenty patients with the highest number of visits were classified as "superusers." We analyzed patient data from the larger sample of high-frequency users and visit specific data from superuser visits. IBM SPSS Statistics 25 (SPSS Inc., Chicago, IL) was used to perform descriptive statistics and to summarize demographic and visit specific variables. RESULTS: We identified 181 high-frequency users with a mean number of visits of 14.3 ± 4.3 and a subpopulation of 20 superusers accounting for 434 visits. The majority of high-frequency users (89%) identified as African American and had public insurance (96.1%). Many patients received primary care affiliated with the home institution. In the first year of the study, 50.3% of high-frequency users were infants younger than 1 year at the index visit and 47.4% of superusers were infants at the index visit.Superuser visits were evenly distributed among seasons and the majority of visits occurred during the weekdays (70.7%). The majority of visits were for medical complaints (86.6%) and almost half (47.6%) resulted in some testing (24.9%) or treatment (30.6%); however, only 1.4% resulted in hospital admission. CONCLUSIONS: In our sample, most high-frequency low-acuity ED patients were infants, African American and have public insurance. Many are seen during clinic hours and are paneled at affiliated clinics. Among superusers, the majority of the visits did not require any testing, intervention, or treatment.

Songwriting intervention for cognitively-impaired children with life-limiting conditions: Study protocol.

Investigations evaluating the effectiveness of music-based interventions as a complementary approach to symptom management and health promotion for cognitively-impaired children with life-limiting conditions and their families are needed to minimize the physical and psychological burdens on individuals and their caregivers. Songwriting is one music-based strategy for a remote family-centered complementary approach to improve outcomes for cognitively-impaired children with life-limiting conditions, their parents, and families. The overall purpose of this study is to test the feasibility and preliminary efficacy of a songwriting intervention for cognitively-impaired children (ages 5-17 years) who are receiving palliative or complex care and their parents. Specific aims are to: (1) determine the feasibility of songwriting for cognitively-impaired children with life-limiting conditions and their parents, and (2) examine preliminary efficacy of songwriting for child psychological distress and physical symptoms, parent psychological distress, and family environment. The protocol for this 1-group pre-and post-intervention clinical trial (N = 25) is described.

Epidemiology and burden of chronic kidney disease-associated pruritus.

Chronic kidney disease-associated pruritus (CKD-aP) is a common, yet underrecognized condition in patients with CKD and end-stage kidney disease (ESKD). Real-world observational studies indicate that CKD-aP affects up to 80% of ESKD patients undergoing haemodialysis (HD), with ∼40% experiencing moderate to severe itch. CKD-aP can negatively impact patients' mental and physical health-related quality of life (HRQoL) and is also associated with sleep disturbance and depression. Several studies have found that CKD-aP is a predictor of adverse medical outcomes, including an increased risk of hospitalizations and mortality. In this article we review the literature relating to the epidemiology of CKD-aP to describe its prevalence across the treatment spectrum of CKD (non-dialysis, HD, peritoneal dialysis and transplant recipients) and to summarize potential risk factors associated with its development. We also review key data from studies that have evaluated the impact of CKD-aP on HRQoL and medical outcomes.

Factors Influencing Pain and Anxiety Before Endodontic Treatment: A Cross-Sectional Study Amongst American Individuals.

OBJECTIVE: The present study aimed to assess preoperative levels of patient anxiety and pain before root canal treatment, and to explore variables that may affect these levels. METHODS: Ninety-five patients presenting for an endodontic visit were recruited for the study. A questionnaire was administered. Visual analog scales were used to record levels of pain and anxiety. Data was tabulated, and analysis was performed using the Pearson Chi-Squire test with continuity correction, and the level of significance was set at 0.05 (P=0.05). RESULTS: Anxiety was detected more frequently in females (60%) than in males (33%) (P=0.016). Sixty-two percent of patients who were waiting for a new treatment were anxious, compared to 39% of those who were returning to continue treatment (P=0.049). Sixty-nine percent of patients in pain reported being anxious (P=0.015). Patients aged 18-30 years reported more pain than those older than 30 years (P=0.023). Forty-three percent of new patients reported being in pain, whereas only 20% of patients returning for a treatment reported pain (P=0.027). CONCLUSION: Anxiety associated with root canal treatment is prevalent, and it was reported primarily by young females who were presenting for a new treatment. Pain and anxiety are highly inter-related, and they are usually reduced after the first endodontic session.

Increased expression of nucleophosmin is associated with the pathophysiology of chronic rhinosinusitis with nasal polyposis.

OBJECTIVE: Nucleophosmin (NPM1) has been suggested to be involved in the pathophysiologic mechanism of inflammatory disorders. We measured the expression level of NPM1 in nasal polyp (NP) tissues of patients with chronic rhinosinusitis with nasal polyposis (CRSwNP). We also assessed the correlation between NPM1 expression and other parameters such as eosinophilic infiltration, inflammatory cytokines, and clinical indicators such as Lund-Mackay computed tomography (CT) score. METHODS: Thirty patients with CRSwNP were included. We performed pre-operative CT scan to determine Lund-Mackay CT scores. During endoscopic sinus surgery, we harvested NP tissues from patients with CRSwNP. We performed Sirius red staining to evaluate eosinophilia and conducted immunohistochemical staining for NPM1 and real-time PCR for cytokines including interleukin (IL)-5, IL-17A, and IL-32. RESULTS: The mRNA expression of NPM1 was significantly up-regulated in eosinophilic NP tissues (RQ 0.58 ± 0.06), compared to non-eosinophilic NP tissues (RQ 0.38 ± 0.08, p < 0.05). In the epithelium of NP tissue, a significant positive correlation was observed between eosinophilic infiltration and NPM1 expression. The expression of NPM1 was significantly correlated with that of IL-5 (r = 0.6229, p = 0.0004), IL-17A (r = 0.5971, p = 0.001), and IL-32 (r = -0.5985, p = 0.0068). There was no significant correlation between the mRNA expression of NPM1 and the Lund-Mackay CT score (Spearman r = -0.2563, p = 0.1879). CONCLUSION: Expression of NPM1 was significantly increased in eosinophilic NP tissues from patients with CRSwNP. We observed an association between NPM1 expression and various pro-inflammatory cytokines such as IL-5, IL-17, and IL-32 and eosinophilic infiltration, which is thought to contribute to the pathophysiology of NP.

Site-specific phosphorylation of Fbxw7 by Cdk5/p25 and its resulting decreased stability are linked to glutamate-induced excitotoxicity.

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine protein kinase that regulates brain development and neurodegeneration. Cdk5 is activated by p25 that is generated from calpain-dependent cleavage of p35. The generation of p25 is responsible for the aberrant hyper-activation of Cdk5, which causes neurodegeneration. Using in vitro assays, we discovered that F-box/WD repeat-containing protein 7 (Fbxw7) is a new substrate of Cdk5. Additionally, Cdk5-dependent phosphorylation of Fbxw7 was detected in the presence of p25, and two amino acid residues (S349 and S372) were determined to be major phosphorylation sites. This phosphorylation was eventually linked to decreased stability of Fbxw7. Using a culture model of cortical neurons challenged with glutamate, we confirmed that decreased stability of Fbxw7 was indeed Cdk5-dependent. Furthermore, diminished levels of Fbxw7 led to increased levels of transcription factor AP-1 (c-Jun), a known substrate of Fbxw7. Given that previous reports demonstrate that c-Jun plays a role in accelerating neuronal apoptosis in these pathological models, our data support the concepts of a molecular cascade in which Cdk5-mediated phosphorylation of Fbxw7 negatively regulates Fbxw7 expression, thereby contributing to neuronal cell death following glutamate-mediated excitotoxicity.

Bupivacaine-induced contraction is attenuated by endothelial nitric oxide release modulated by activation of both stimulatory and inhibitory phosphorylation (Ser1177 and Thr495) of endothelial nitric oxide synthase.

This study examined the mechanism associated with the endothelium-dependent attenuation of vasoconstriction induced by bupivacaine (BPV), with a particular focus on the upstream cellular signaling pathway of endothelial nitric oxide synthase (eNOS) phosphorylation induced by BPV in human umbilical vein endothelial cells (HUVECs). BPV concentration-response curves were investigated in the isolated rat aorta. The effects of Nω-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), methylene blue, calmidazolium, the Src kinase inhibitor 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and the combination of L-arginine and L-NAME on BPV-induced contraction in endothelium-intact aorta preparations were examined. The effects of BPV alone and in combination with PP2 on the phosphorylation of eNOS (at Ser1177 or Thr495), caveolin-1 and Src kinase were examined in HUVECs. BPV-induced contraction was lower in endothelium-intact aortae than in endothelium-denuded aortae. L-NAME, ODQ, methylene blue and calmidazolium increased BPV-induced contraction in endothelium-intact aortae, whereas PP2 alone and combined treatment with L-arginine and L-NAME inhibited BPV-induced contraction. Low-concentration BPV (30 µM) induced both stimulatory (Ser1177) and inhibitory (Thr495) phosphorylation of eNOS in HUVECs. However, high-concentration BPV (150 µM) induced only stimulatory (Ser1177) eNOS phosphorylation. Additionally, phosphorylation of Src kinase, caveolin-1 and inhibitory eNOS (Thr495) induced by low-concentration BPV was inhibited by PP2. These results suggest that contraction induced by low-concentration BPV is attenuated by endothelial nitric oxide release, which is modulated both stimulatory (Ser1177) and inhibitory eNOS phosphorylation (Thr495). BPV-induced phosphorylation of eNOS (Thr495) is indirectly mediated by an upstream cellular signaling pathway involving Src kinase (Tyr416) and caveolin-1 (Tyr14).