Effects of Ambient Air Pollution on Brain Cortical Thickness and Subcortical Volume: A Longitudinal Neuroimaging Study.

INTRODUCTION: Several cross-sectional studies have shown that long-term exposures to air pollutants are associated with smaller brain cortical volume or thickness. Here, we investigated longitudinal associations of long-term air pollution exposures with cortical thickness and subcortical volume. METHODS: In this longitudinal study, we included a prospective cohort of 361 adults residing in four cities in the Republic of Korea. Long-term concentrations of particulate matter with aerodynamic diameters of ≤10 µm (PM10) and ≤2.5 µm (PM2.5) and nitrogen dioxide (NO2) at residential addresses were estimated. Neuroimaging markers (cortical thickness and subcortical volume) were obtained from brain magnetic resonance images at baseline (August 2014 to March 2017) and at the 3-year follow-up (until September 2020). Linear mixed-effects models were used, adjusting for covariates. RESULTS: A 10-µg/m3 increase in PM10 was associated with reduced whole-brain mean (ß = -0.45, standard error [SE] = 0.10; p < 0.001), frontal (ß = -0.53, SE = 0.11; p < 0.001) and temporal thicknesses (ß = -0.37, SE = 0.12; p = 0.002). A 10-ppb increase in NO2 was associated with a decline in the whole-brain mean cortical thickness (ß = -0.23, SE = 0.05; p < 0.001), frontal (ß = -0.25, SE = 0.05; p < 0.001), parietal (ß = -0.12, SE = 0.05; p = 0.025), and temporal thicknesses (ß = -0.19, SE = 0.06; p = 0.001). Subcortical structures associated with air pollutants included the thalamus. CONCLUSIONS: Long-term exposures to PM10 and NO2 may lead to cortical thinning in adults.

Metabolic traits affecting the relationship between liver fat and intrapancreatic fat: a mediation analysis.

AIMS/HYPOTHESIS: The clinical importance of fat deposition in the liver and pancreas is increasingly recognised. However, to what extent deposition of fat in these two depots is affected by intermediate variables is unknown. The aim of this work was to conduct a mediation analysis with a view to uncovering the metabolic traits that underlie the relationship between liver fat and intrapancreatic fat deposition (IPFD) and quantifying their effect. METHODS: All participants underwent MRI/magnetic resonance spectroscopy on the same 3.0 T scanner to determine liver fat and IPFD. IPFD of all participants was quantified manually by two independent raters in duplicate. A total of 16 metabolic traits (representing markers of glucose metabolism, incretins, lipid panel, liver enzymes, pancreatic hormones and their derivatives) were measured in blood. Mediation analysis was conducted, taking into account age, sex, ethnicity and BMI. Significance of mediation was tested by computing bias-corrected bootstrap CIs with 5000 repetitions. RESULTS: A total of 353 individuals were studied. Plasma glucose, HDL-cholesterol and triacylglycerol mediated 6.8%, 17.9% and 24.3%, respectively, of the association between liver fat and IPFD. Total cholesterol, LDL-cholesterol, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyl transpeptidase, insulin, glucagon, amylin, C-peptide, HbA1c, glucagon-like peptide-1 and gastric inhibitory peptide did not mediate the association between liver fat and IPFD. CONCLUSIONS/INTERPRETATION: At least one-quarter of the association between liver fat and IPFD is mediated by specific blood biomarkers (triacylglycerol, HDL-cholesterol and glucose), after accounting for potential confounding by age, sex, ethnicity and BMI. This unveils the complexity of the association between the two fat depots and presents specific targets for intervention.

Remnant cholesterol, but not low-density lipoprotein cholesterol, is associated with intra-pancreatic fat deposition.

AIM: To investigate the associations of components of the lipid panel (and its derivatives) with intra-pancreatic fat deposition (IPFD). METHODS: All participants underwent abdominal magnetic resonance imaging on the same 3.0-Tesla scanner and IPFD was quantified. Blood samples were collected in the fasted state for analysis of lipid panel components. A series of linear regression analyses was conducted, adjusting for age, sex, ethnicity, body mass index, fasting plasma glucose, homeostatic model assessment of insulin resistance, and liver fat deposition. RESULTS: A total of 348 participants were included. Remnant cholesterol (P = 0.010) and triglyceride levels (P = 0.008) were positively, and high-density lipoprotein cholesterol level (P = 0.001) was negatively, associated with total IPFD in the most adjusted model. Low-density lipoprotein cholesterol and total cholesterol were not significantly associated with total IPFD. Of the lipid panel components investigated, remnant cholesterol explained the greatest proportion (9.9%) of the variance in total IPFD. CONCLUSION: Components of the lipid panel have different associations with IPFD. This may open up new opportunities for improving outcomes in people at high risk for cardiovascular diseases (who have normal low-density lipoprotein cholesterol) by reducing IPFD.

Negative self-regulation of transient receptor potential canonical 4 by the specific interaction with phospholipase C-δ1.

Transient receptor potential canonical (TRPC) channels are non-selective calcium-permeable cation channels. It is suggested that TRPC4ß is regulated by phospholipase C (PLC) signaling and is especially maintained by phosphatidylinositol 4,5-bisphosphate (PIP2). In this study, we present the regulation mechanism of the TRPC4 channel with PIP2 hydrolysis which is mediated by a channel-bound PLCδ1 but not by the GqPCR signaling pathway. Our electrophysiological recordings demonstrate that the Ca2+ via an open TRPC4 channel activates PLCδ1 in the physiological range, and it causes the decrease of current amplitude. The existence of PLCδ1 accelerated PIP2 depletion when the channel was activated by an agonist. Interestingly, PLCδ1 mutants which have lost the ability to regulate PIP2 level failed to reduce the TRPC4 current amplitude. Our results demonstrate that TRPC4 self-regulates its activity by allowing Ca2+ ions into the cell and promoting the PIP2 hydrolyzing activity of PLCδ1.

Intrapancreatic, Liver, and Skeletal Muscle Fat Depositions in First Attack of Acute Pancreatitis Versus Health.

INTRODUCTION: Increased intrapancreatic fat deposition (IPFD) has emerged as a harbinger of pancreatic cancer and chronic pancreatitis. Although it is well recognized that diseases of the exocrine pancreas often lie on a continuum (with acute pancreatitis preceding the development of chronic pancreatitis and/or pancreatic cancer), whether increased IPFD predisposes to acute pancreatitis is unknown. This study aimed to compare fat depositions in the pancreas (as well as the liver and skeletal muscle) between individuals who developed first attack of acute pancreatitis and healthy individuals. METHODS: This was a matched case-control study nested into population-based cohort. MRI on a single 3 T scanner was used to quantify intrapancreatic, liver, and skeletal muscle fat depositions using the same protocols in all study participants. Binary logistic regression with adjustment for body mass index and other possible confounders was performed. RESULTS: Fifty individuals with first attack of nonnecrotizing acute pancreatitis comprised the case group and 100 healthy individuals comprised the control group. A 1% increase in IPFD (but not the other fat depositions) was significantly associated with a more than 30% higher chance of developing first attack of acute pancreatitis, consistently in both the unadjusted ( P = 0.004) and all adjusted models. Furthermore, a 1% increase in IPFD (but not the other fat depositions) was significantly associated with up to a 27% higher chance of developing first attack of acute pancreatitis in individuals with normotriglyceridemia, consistently in both the unadjusted ( P = 0.030) and all adjusted models. DISCUSSION: Increased IPFD may predispose to the development of acute pancreatitis. This opens up opportunities for reducing the burden of acute pancreatitis by means of primary prevention.

Cytokine signature for predicting new-onset prediabetes after acute pancreatitis: A prospective longitudinal cohort study.

BACKGROUND/PURPOSE: Acute inflammation of the pancreas often leads to metabolic sequelae, the most common of which is new-onset prediabetes (and, ultimately, diabetes). However, there is a lack of studies on predictors of this sequela. The aim was to investigate whether cytokines/chemokines measured at baseline are predictive of new-onset prediabetes after acute pancreatitis (NOPAP). METHODS: This was a prospective longitudinal cohort study (as part of the LACERTA project) that included 68 individuals with non-necrotising acute pancreatitis who had no diabetes mellitus. Of them, 17 individuals had prediabetes at baseline and during follow-up, 37 individuals had normoglycaemia at baseline and during follow-up, and 14 individuals had normoglycaemia at baseline and developed NOPAP during follow-up. A commercially available human cytokine/chemokine multiplex kit was used to measure a total of 28 analytes at baseline. Multinomial regression analyses were conducted to investigate the associations between the cytokines/chemokines and the three study groups. RESULTS: Interleukin-1ß and interferon γ significantly predicted progression to NOPAP with an odds ratio (95% confidence interval) of 1.097 (1.002, 1.201) and 1.094 (1.003, 1.192), respectively (after accounting for age, sex, body mass index, and aetiology of acute pancreatitis). None of the studied cytokines/chemokines showed statistically significant associations with the antecedent prediabetes group (after accounting for the above covariates). CONCLUSION: Elevated levels of interleukin-1ß and interferon γ in acute pancreatitis individuals with normoglycaemia at baseline may predict progression to NOPAP during follow-up.

Higher expression of KCNK10 (TREK-2) K+ channels and their functional upregulation by lipopolysaccharide treatment in mouse peritoneal B1a cells.

Innate-like CD5+ B1a cells localized in serous cavities are activated by innate stimuli, such as lipopolysaccharide (LPS), leading to T cell-independent antibody responses. Although ion channels play crucial roles in the homeostasis and activation of immune cells, the electrophysiological properties of B1a cells have not been investigated to date. Previously, in the mouse B cell lymphoma cells, we found that the voltage-independent two-pore-domain potassium (K2P) channels generate a negative membrane potential and drive Ca2+ influx. Here, we newly compared the expression and activities of K2P channels in mouse splenic follicular B (FoB), marginal zone B (MZB), and peritoneal B1a cells. Next-generation sequencing analysis showed higher levels of transcripts for TREK-2 and TWIK-2 in B1a cells than those in FoB or MZB cells. Electrophysiological analysis, using patch clamp technique, revealed higher activity of TREK-2 with the characteristic large unitary conductance (~ 250 pS) in B1a than that in FoB or MZB cells. TREK-2 activity was further increased by LPS treatment (>2 h), which was more prominent in B1a than that in MZB or FoB cells. The cytosolic Ca2+ concentration of B cells was decreased by high-K+-induced depolarization (ΔRKCl (%)), suggesting the basal Ca2+ influx to be driven by negative membrane potential. The LPS treatment significantly increased the ΔRKCl (%) in B1a, though not in FoB and MZB cells. Our study was the first to compare the K2P channels in mouse primary B cell subsets, elucidating the functional upregulation of TREK-2 and augmentation of Ca2+ influx by the stimulation of Toll-like receptor 4 in B1a cells.

Acute Nutritional Ketosis and Its Implications for Plasma Glucose and Glucoregulatory Peptides in Adults with Prediabetes: A Crossover Placebo-Controlled Randomized Trial.

BACKGROUND: The potential of a ketone monoester (ß-hydroxybutyrate; KEßHB) supplement to rapidly mimic a state of nutritional ketosis offers a new therapeutic possibility for diabetes prevention and management. While KEßHB supplementation has a glucose-lowering effect in adults with obesity, its impact on glucose control in other insulin-resistant states is unknown. OBJECTIVES: The primary objective was to investigate the effect of KEßHB-supplemented drink on plasma glucose in adults with prediabetes. The secondary objective was to determine its impact on plasma glucoregulatory peptides. METHODS: This randomized controlled trial [called CETUS (Cross-over randomizEd Trial of ß-hydroxybUtyrate in prediabeteS)] included 18 adults [67% men, mean age = 55 y, mean BMI (kg/m2) = 28.4] with prediabetes (glycated hemoglobin between 5.7% and 6.4% and/or fasting plasma glucose between 100 and 125 mg/dL). Participants were randomly assigned to receive KEßHB-supplemented and placebo drinks in a crossover sequence (washout period of 7-10 d between the drinks). Blood samples were collected from 0 to 150 min, at intervals of 30 min. Paired-samples t tests were used to investigate the change in the outcome variables [ß-hydroxybutyrate (ßHB), glucose, and glucoregulatory peptides] after both drinks. Repeated measures analyses were conducted to determine the change in concentrations of the prespecified outcomes over time. RESULTS: Blood ßHB concentrations increased to 3.5 mmol/L within 30 minutes after KEßHB supplementation. Plasma glucose AUC was significantly lower after KEßHB supplementation than after the placebo [mean difference (95% CI): -59 (-85.3, -32.3) mmol/L × min]. Compared with the placebo, KEßHB supplementation led to significantly greater AUCs for plasma insulin [0.237 (0.044, 0.429) nmol/L × min], C-peptide [0.259 (0.114, 0.403) nmol/L × min], and glucose-dependent insulinotropic peptide [0.243 (0.085, 0.401) nmol/L × min], with no significant differences in the AUCs for amylin, glucagon, and glucagon-like peptide 1. CONCLUSIONS: Ingestion of the KEßHB-supplemented drink acutely increased the blood ßHB concentrations and lowered the plasma glucose concentrations in adults with prediabetes. Further research is needed to investigate the dynamics of repeated ingestions of a KEßHB supplement by individuals with prediabetes, with a view to preventing new-onset diabetes. This trial was registered at www.clinicaltrials.gov as NCT03889210.

Dietary carbohydrate intake and insulin traits in individuals after acute pancreatitis: Effect modification by intra-pancreatic fat deposition.

OBJECTIVES: Current knowledge of the link between dietary carbohydrate intake and insulin regulation in individuals after an attack of pancreatitis is limited. We aimed to investigate the associations between dietary carbohydrate intake and insulin traits in post-pancreatitis versus healthy individuals, taking into account intrapancreatic fat deposition (IPFD). METHODS: All participants underwent magnetic resonance imaging (using the same protocol and 3T scanner) to quantify IPFD. Dietary carbohydrate intake was assessed using a validated 131-item food frequency questionnaire. Insulin, HOMA-IR, HOMA-ß were determined in the fasted state. Linear regression and effect modification analyses were conducted in unadjusted and adjusted models (accounting for age, sex, body mass index, daily energy intake, use of anti-diabetic medications, and recurrence of acute pancreatitis). RESULTS: The study included 111 post-pancreatitis individuals (categorized into low IPFD (n = 33), moderate IPFD (n = 40), high IPFD (n = 38)) and 47 healthy controls. In the high IPFD group, starch intake was negatively associated with fasting insulin and HOMA-ß in both the unadjusted (p < 0.001 both) and fully adjusted models (p < 0.001 both); and with HOMA-IR in the fully adjusted model (p < 0.001) only. Total sugar intake was positively associated with fasting insulin (p = 0.015) and HOMA-ß (p = 0.007) in the fully adjusted model but not associated with HOMA-IR. None of the above associations was statistically significant in the low IPFD, moderate IPFD, and healthy controls groups. The studied associations were more pronounced in the high IPFD group but not in the moderate IPFD or low IPFD groups (when compared with the healthy controls group). CONCLUSIONS: Dietary carbohydrate intake is differentially associated with insulin traits in individuals after an attack of pancreatitis and the associations are modified by IPFD. These findings will be helpful for the development of dietary guidelines specifically for individuals after an attack of pancreatitis.

Relationship of pancreas volume to tobacco smoking and alcohol consumption following pancreatitis.

BACKGROUND: Tobacco smoking and alcohol consumption are established risk factors for diseases of the pancreas. With the recent advances in imaging modalities (such as magnetic resonance (MR) imaging), opportunities have arisen to study pancreas size, in both health and disease. Studies investigating the relationship between tobacco smoking, alcohol consumption, and total pancreas volume (TPV) - a holistic measure of pancreatic exocrine reserve - are lacking. The aim of the present study was to investigate the associations between MR-derived TPV and tobacco smoking/alcohol consumption. METHODS: This cross-sectional study recruited individuals with a history of pancreatitis and healthy controls. A validated questionnaire was used to ascertain current and lifetime tobacco smoking and alcohol consumption. TPV was quantified using MR images by two independent raters. Generalized additive models and linear regression analyses were conducted and adjusted for demographic, metabolic, and pancreatitis-related factors. RESULTS: A total of 107 individuals following pancreatitis and 38 healthy controls were included. There was no statistically significant difference in TPV between any of the tobacco smoking/alcohol consumption categories of individuals following pancreatitis and healthy controls, in both unadjusted and adjusted analyses. In individuals following pancreatitis, multivariate linear regression found no association between TPV and 7 smoking- and alcohol-related variables. Sensitivity analyses constrained to individuals who did not abstain from either smoking or drinking following their first attack of pancreatitis did not yield statistical significance with TPV. In post-hoc analysis, age was significantly inversely associated with TPV in the most adjusted model (p = 0.016). CONCLUSIONS: This is the first study to investigate the association between tobacco smoking, alcohol consumption, and MR-derived TPV following pancreatitis. It appears that age, but not tobacco smoking or alcohol consumption, is associated with a significantly reduced TPV.

Psoas muscle size as a magnetic resonance imaging biomarker of progression of pancreatitis.

OBJECTIVE: Pancreatitis often represents a continuous inflammatory process, from the first episode of acute pancreatitis (FAP) to recurrent acute pancreatitis (RAP) to chronic pancreatitis (CP). Psoas muscle size is a validated surrogate for global skeletal mass, changes in which are associated with inflammation. The objective was to investigate psoas muscle size in individuals following FAP, RAP, and CP, as well as its associations with pro-inflammatory cytokines. METHODS: Individuals following pancreatitis and healthy individuals were recruited. All participants underwent magnetic resonance imaging, from which psoas muscle volume was derived independently by two raters in a blinded fashion. Circulating levels of four major cytokines (interleukin-6, tumour necrosis factor-α, C-C motif chemokine ligand 2, and leptin) were measured. Five linear regression additive models were built to adjust for possible confounders (age, sex, body composition, physical activity, tobacco smoking, alcohol consumption, comorbidities, and endocrine and exocrine pancreatic functions). RESULTS: A total of 145 participants were enrolled. A significant downward trend in psoas muscle volume was observed between healthy controls and individuals following FAP, RAP, and CP in all adjusted models (p = 0.047, 0.005, 0.004, and < 0.001). Leptin was significantly associated with psoas muscle volume in all models (ß = - 0.16, p = 0.030 in the most adjusted model). The other studied cytokines were not significantly associated with psoas muscle volume. CONCLUSIONS: Psoas muscle size is significantly reduced along the continuum from FAP to RAP to CP. Leptin appears to be one of the factors implicated in this. Further studies are warranted to investigate the relationship between skeletal muscle and inflammation of the pancreas. KEY POINTS: • First acute pancreatitis, recurrent acute pancreatitis, and chronic pancreatitis were associated with progressively reduced psoas muscle size. • The findings were independent of age, sex, body fat composition, physical activity, tobacco smoking, alcohol consumption, comorbidities, and exocrine and endocrine functions of the pancreas. • The mechanism underlying the observed findings may involve hyperleptinaemia.

Analysis of interaction between intracellular spermine and transient receptor potential canonical 4 channel: multiple candidate sites of negatively charged amino acids for the inward rectification of transient receptor potential canonical 4.

Transient receptor potential canonical 4 (TRPC4) channel is a nonselective calcium-permeable cation channels. In intestinal smooth muscle cells, TRPC4 currents contribute more than 80% to muscarinic cationic current (mIcat). With its inward-rectifying current-voltage relationship and high calcium permeability, TRPC4 channels permit calcium influx once the channel is opened by muscarinic receptor stimulation. Polyamines are known to inhibit nonselective cation channels that mediate the generation of mIcat. Moreover, it is reported that TRPC4 channels are blocked by the intracellular spermine through electrostatic interaction with glutamate residues (E728, E729). Here, we investigated the correlation between the magnitude of channel inactivation by spermine and the magnitude of channel conductance. We also found additional spermine binding sites in TRPC4. We evaluated channel activity with electrophysiological recordings and revalidated structural significance based on Cryo-EM structure, which was resolved recently. We found that there is no correlation between magnitude of inhibitory action of spermine and magnitude of maximum current of the channel. In intracellular region, TRPC4 attracts spermine at channel periphery by reducing access resistance, and acidic residues contribute to blocking action of intracellular spermine; channel periphery, E649; cytosolic space, D629, D649, and E687.

The agonistic action of URO-K10 on Kv7.4 and 7.5 channels is attenuated by co-expression of KCNE4 ancillary subunit.

KCNQ family constitutes slowly-activating potassium channels among voltage-gated potassium channel superfamily. Recent studies suggested that KCNQ4 and 5 channels are abundantly expressed in smooth muscle cells, especially in lower urinary tract including corpus cavernosum and that both channels can exert membrane stabilizing effect in the tissues. In this article, we examined the electrophysiological characteristics of overexpressed KCNQ4, 5 channels in HEK293 cells with recently developed KCNQ-specific agonist. With submicromolar EC50, the drug not only increased the open probability of KCNQ4 channel but also increased slope conductance of the channel. The overall effect of the drug in whole-cell configuration was to increase maximal whole-cell conductance, to prolongate the activation process, and left-shift of the activation curve. The agonistic action of the drug, however, was highly attenuated by the co-expression of one of the ß ancillary subunits of KCNQ family, KCNE4. Strong in vitro interactions between KCNQ4, 5 and KCNE4 were found through Foster Resonance Energy Transfer and co-immunoprecipitation. Although the expression levels of both KCNQ4 and KCNE4 are high in mesenteric arterial smooth muscle cells, we found that 1 µM of the agonist was sufficient to almost completely relax phenylephrine-induced contraction of the muscle strip. Significant expression of KCNQ4 and KCNE4 in corpus cavernosum together with high tonic contractility of the tissue grants highly promising relaxational effect of the KCNQspecific agonist in the tissue.

TRPC1 as a negative regulator for TRPC4 and TRPC5 channels.

Transient receptor potential canonical (TRPC) channels are calcium permeable, non-selective cation channels with wide tissue-specific distribution. Among 7 TRPC channels, TRPC 1/4/5 and TRPC3/6/7 are subdivided based on amino acid sequence homology. TRPC4 and TRPC5 channels exhibit cationic current with homotetrameric form, but they also form heterotetrameric channel such as TRPC1/4 or TRPC1/5 once TRPC1 is incorporated. The expression of TRPC1 is ubiquitous whereas the expressions of TRPC4 and TRPC5 are rather focused in nervous system. With the help of conditional knock-out of TPRC1, 4 and/or 5 genes, TRPC channels made of these constituents are reported to be involved in various pathophysiological functions such as seizure, anxiety-like behaviour, fear, Huntington's disease, Parkinson's disease and many others. In heterologous expression system, many issues such as activation mechanism, stoichiometry and relative cation permeabilites of homomeric or heteromeric channels have been addressed. In this review, we discussed the role of TRPC1 channel per se in plasma membrane, role of TRPC1 in heterotetrameric conformation (TRPC1/4 or TRPC1/5) and relationship between TRPC1/4/5 channels, calcium influx and voltage-gated calcium channels.

Identification of phospholipase C ß downstream effect on transient receptor potential canonical 1/4, transient receptor potential canonical 1/5 channels.

Gαq-coupled receptor stimulation was implied in the activation process of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heterotetrameric channels. The inactivation occurs due to phosphatidylinositol 4,5-biphosphate (PI(4,5)P2) depletion. When PI(4,5)P2 depletion was induced by muscarinic stimulation or inositol polyphosphate 5-phosphatase (Inp54p), however, the inactivation by muscarinic stimulation was greater compared to that by Inp54p. The aim of this study was to investigate the complete inactivation mechanism of the heteromeric channels upon Gαq-phospholipase C ß (Gαq-PLCß) activation. We evaluated the activity of heteromeric channels with electrophysiological recording in HEK293 cells expressing TRPC channels. TRPC1/4 and TRPC1/5 heteromers undergo further inhibition in PLCß activation and calcium/protein kinase C (PKC) signaling. Nevertheless, the key factors differ. For TRPC1/4, the inactivation process was facilitated by Ca2+ release from the endoplasmic reticulum, and for TRPC1/5, activation of PKC was concerned mostly. We conclude that the subsequent increase in cytoplasmic Ca2+ due to Ca2+ release from the endoplasmic reticulum and activation of PKC resulted in a second phase of channel inhibition following PI(4,5)P2 depletion.

Englerin A-sensing charged residues for transient receptor potential canonical 5 channel activation.

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or GTPγS. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.

mTOR inhibition by rapamycin protects against deltamethrin-induced apoptosis in PC12 Cells.

The autophagy pathway can be induced and upregulated in response to intracellular reactive oxygen species (ROS). In this study, we explored a novel pharmacotherapeutic approach involving the regulation of autophagy to prevent deltamethrin (DLM) neurotoxicity. We found that DLM-induced apoptosis in PC12 cells, as demonstrated by the activation of caspase-3 and -9 and by nuclear condensation. DLM treatment significantly decreased dopamine (DA) levels in PC12 cells. In addition, we observed that cells treated with DLM underwent autophagic cell death, by monitoring the expression of LC3-II, p62, and Beclin-1. Exposure of PC12 cells to DLM led to the production of ROS. Treatment with N-acetyl cysteine (NAC) effectively blocked both apoptosis and autophagy. In addition, mitogen-activated protein kinase (MAPK) inhibitors attenuated apoptosis as well as autophagic cell death. We also investigated the modulation of DLM-induced apoptosis in response to autophagy regulation. Pretreatment with the autophagy inducer, rapamycin, significantly enhanced the viability of DLM-exposed cells, and this enhancement of cell viability was partially due to alleviation of DLM-induced apoptosis via a decrease in levels of cleaved caspase-3. However, pretreatment of cells with the autophagy inhibitor, 3-methyladenine (3MA), significantly increased DLM toxicity in these cells. Our results suggest that DLM-induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against DLM-induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 109-121, 2017.

Calcium permeability of transient receptor potential canonical (TRPC) 4 channels measured by TRPC4-GCaMP6s.

Conflicting evidence has been obtained regarding whether transient receptor potential cation channels (TRPC) are store-operated channels (SOCs) or receptor-operated channels (ROCs). Moreover, the Ca/Na permeability ratio differs depending on whether the current-voltage (I-V) curve has a doubly rectifying shape or inward rectifying shape. To investigate the calcium permeability of TRPC4 channels, we attached GCaMP6s to TRPC4 and simultaneously measured the current and calcium signals. A TRPC4 specific activator, (-)-englerin A, induced both current and calcium fluorescence with the similar time course. Muscarinic receptor stimulator, carbachol, also induced both current and calcium fluorescence with the similar time course. By forming heteromers with TRPC4, TRPC1 significantly reduced the inward current with outward rectifying I-V curve, which also caused the decrease of calcium fluorescence intensity. These results suggest that GCaMP6s attached to TRPC4 can detect slight calcium changes near TRPC4 channels. Consequently, TRPC4-GCaMP6s can be a useful tool for testing the calcium permeability of TRPC4 channels.

The interaction domains of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heteromultimeric channels.

Transient receptor potential canonical (TRPC) family contains a non-selective cation channel, and four TRPC subunits form a functional tetrameric channel. TRPC4/5 channels form not only the homotetrameric channel but also a heterotetrameric channel with TRPC1. We investigated the interaction domain required for TRPC1/4 or TRPC1/5 heteromultimeric channels using FRET and the patch-clamp technique. TRPC1 only localized at the plasma membrane (PM) when it was coexpressed with TRPC4 or TRPC5. The TRPC1/4 or TRPC1/5 heteromultimeric showed the typical outward rectifying I/V curve. When TRPC1 and TRPC4 form a heteromeric channel, the N-terminal coiled-coil domain (CCD) and C-terminal 725-745 region of TRPC1 interact with the N-terminal CCD and C-terminal 700-728 region of TRPC4. However, when TRPC1 and TRPC5 form a heteromeric channel, the N-terminal CCD and C-terminal 673-725 region of TRPC1 interact with the N-terminal CCD and C-terminal 707-735 region of TRPC5. In conclusion, the N-terminal CCD of TRPC channels is essential for the heteromultimeric structure of TRPC channels, whereas specific C-terminal regions are required for unique heteromerization between subgroups of TRPC channels.

Increased risk of developing cerebro-cardiovascular diseases in police officers: a nationwide retrospective cohort study.

BACKGROUND: Police officers face an increased risk of developing cerebro-cardiovascular diseases (CVD). However, current literature lacks population-based cohort studies specifically focusing on this association. This study aimed to investigate the association between police officers and the risk of developing CVD compared with education officers, while accounting for socioeconomic and demographic factors. METHODS: We used the Korean National Health Insurance Service data spanning from 2009 to 2020. In this population-based retrospective matched cohort study, we identified age, sex, and calendar years of job-enrollment-matched education officers for each police officer. This study evaluated the CVD occurrence, including acute myocardial infarction, ischemic stroke, and hemorrhagic stroke. Using multivariable Cox regression analysis, we determined the risk of developing CVD, expressed as a hazard ratio (HR) and 95% confidence interval (CI). RESULTS: Among 104,134 police officers and 104,134 education officers, 4,391(42.2%) cases and 3,631(34.9%) cases of CVD occurred, respectively. The mean ± standard deviation age was 38.4 ± 9.4 years in police officers and 38.6 ± 9.5 years in education officers. The proportion of men was 84.8 % in both groups. Police officers were significantly associated with a higher risk of developing CVD compared with education officers, with an adjusted HR of 1.15 (95% CI, 1.09-1.22). In addition, police officers had significantly higher risks for acute myocardial infarction (adjusted HR, 1.16; 95% CI, 1.06-1.26) and ischemic stroke (adjusted HR, 1.17; 95% CI, 1.09-1.25). CONCLUSIONS: The findings of our study highlight a significant increase in the risk of developing CVD among police officers, particularly among those aged 45 years and older and those with uncontrolled blood pressure compared to their education officer counterparts. Future cohort studies are required to confirm this association.