Sex-related differences in single nucleotide polymorphisms associated with dyslipidemia in a Korean population.

BACKGROUND: The prevalence of dyslipidemia has increased steadily in Korea, and the incidence of dyslipidemia differs by sex. In this study, we identified single nucleotide polymorphisms (SNPs) related to dyslipidemia in Korean cohorts through genome-wide association study (GWAS) analysis. METHODS: Genotyping was conducted to determine the genotypes of 72,298 participants and investigate genotypes for 7,079,946 SNPs. Sex, age, and BMI were set as covariates for GWAS, and significant SNPs were identified in the discovery and replication stages using logistic regression. RESULTS: GWAS of the entire cohort revealed a total of five significant SNPs: rs117026536 (LPL), rs651821 (APOA5), rs9804646 (APOA5), rs9926440 (CETP), and rs429358 (APOE). GWAS of the male subjects revealed a total of four significant SNPs. While rs9804646 (APOA5) and rs429358 (APOE) were significant for all the subjects, rs662799 (APOA5) and rs56156922 (CETP) were significant only for the male subjects. GWAS of the female subjects revealed two significant SNPs, rs651821 (APOA5) and rs9804646 (APOA5), both of which were significant in all the subjects. CONCLUSION: This is the first study to identify sex-related differences in genetic polymorphisms in Korean populations with dyslipidemia. Further studies considering environmental variables will be needed to elucidate these sex-related genetic differences in dyslipidemia.

Smartphone-based home workout program for shift-work nurses working during the COVID-19 pandemic.

This study aimed to develop a smartphone-based home workout program for shift-work nurses to increase their levels of exercise and examine its effects on health (sleep disturbance, fatigue, musculoskeletal problems, and resilience) and nursing performance. For this quasiexperimental study with a nonequivalent control group, 54 shift-work nurses were recruited from two general wards at a hospital in Korea and assigned to the intervention and control groups. Nurses in the intervention group were encouraged to exercise regularly using the home workout application for 18 weeks. For the first 12 weeks, text-message counseling and environmental improvement were carried out; only environmental improvement was implemented in the remaining 12-18 weeks. The control group did not receive any intervention. After excluding dropouts, the data of the final 25 participants in the intervention group and 24 participants in the control group were analyzed. Compared with the control group, the intervention group showed statistically significant improvements in physical and psychological health. Hospital organizations could adopt a smartphone-based home workout program to overcome obstacles to exercise, which could lead to positive health outcomes for shift-work nurses.

Oxygen-dependent regulation of ion channels: acute responses, post-translational modification, and response to chronic hypoxia.

Oxygen is a vital element for the survival of cells in multicellular aerobic organisms such as mammals. Lack of O2 availability caused by environmental or pathological conditions leads to hypoxia. Active oxygen distribution systems (pulmonary and circulatory) and their neural control mechanisms ensure that cells and tissues remain oxygenated. However, O2-carrying blood cells as well as immune and various parenchymal cells experience wide variations in partial pressure of oxygen (PO2) in vivo. Hence, the reactive modulation of the functions of the oxygen distribution systems and their ability to sense PO2 are critical. Elucidating the physiological responses of cells to variations in PO2 and determining the PO2-sensing mechanisms at the biomolecular level have attracted considerable research interest in the field of physiology. Herein, we review the current knowledge regarding ion channel-dependent oxygen sensing and associated signalling pathways in mammals. First, we present the recent findings on O2-sensing ion channels in representative chemoreceptor cells as well as in other types of cells such as immune cells. Furthermore, we highlight the transcriptional regulation of ion channels under chronic hypoxia and its physiological implications and summarize the findings of studies on the post-translational modification of ion channels under hypoxic or ischemic conditions.

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.

Increased inward rectifier K+ current of coronary artery smooth muscle cells in spontaneously hypertensive rats; partial compensation of the attenuated endothelium-dependent relaxation via Ca2+ -activated K+ channels.

Endothelium-dependent vasorelaxation is partly mediated by small-conductance (SK3) and intermediate-conductance Ca2+ -activated K+ channels (SK4) in the endothelium that results in endothelium-dependent hyperpolarization (EDH). Apart from the electrical propagation through myoendothelial gap junctions, the K+ released from the endothelium facilitates EDH by increasing inward rectifier K+ channel (Kir) conductance in smooth muscle cells. The EDH-dependent relaxation of coronary artery (CA) and Kir current in smooth muscle cells (CASMCs) of hypertensive animals are poorly understood despite the critical role of coronary flow in the hypertrophic heart. In spontaneously hypertensive (SHR) and control (WKY) rats, we found attenuation of the CA relaxation by activators of SK3 and SK4 (NS309 and 1-EBIO) in SHR. In isolated CASMCs, whole-cell patch-clamp study revealed larger IKir in SHR than WKY, whereas the myocytes of skeletal and cerebral arteries showed smaller IKir in SHR than WKY. While the treatment with IKir inhibitor (0.1 mmol/L Ba2+ ) alone did not affect the WKY-CA, the SHR-CA showed significant contractile response, suggesting relaxing influence of the higher IKir in the CASMCs of SHR. Furthermore, the attenuation of NS309-induced relaxation of CA by the combined treatment with 0.1 mmol/L Ba2+ was more prominent in SHR than WKY. Our study firstly shows a distinct increase of IKir in the CASMCs of SHR, which could partly compensate for the attenuated relaxation via endothelial SK3 and SK4.

Teaching cardiac excitation-contraction coupling using a mathematical computer simulation model of human ventricular myocytes.

To understand the excitation-contraction (E-C) coupling of cardiomyocytes, including the electrophysiological mechanism of their characteristically long action potential duration, is one of the major learning goals in medical physiology. However, the integrative interpretation of the responses occurring during the contraction-relaxation cycle is challenging due to the dynamic interaction of underlying factors. Starting in 2017, we adopted the mathematical computer simulation model of human ventricular myocyte (Cardiac E-C_Sim), hypothesizing that this educational technology may facilitate students' learning of cardiac physiology. Here, we describe the overall process for the educational application of Cardiac E-C_Sim in the human physiology practicum of Seoul National University College of Medicine. We also report the results from questionnaires covering detailed assessment of the practicum class. The analysis of results and feedback opinions enabled us to understand how the students had approached the problem-solving process. As a whole, the students could better accomplish the learning goals using Cardiac E-C_Sim, followed by constructive discussions on the complex and dynamic mechanisms of cardiac E-C coupling. We suggest that the combined approach of lecture-based teaching and computer simulations guided by a manual containing clinical context would be broadly applicable in physiology education.

Anti-Inflammatory Effects of Alnus Sibirica Extract on In Vitro and In Vivo Models.

Alnus sibirica extracts (ASex) have long been used in Oriental medicine to treat various conditions. To provide a scientific basis for this application and the underlying mechanism, we investigated the anti-inflammatory effects of ASex in vitro and in vivo. The in vitro model was established using human dermal fibroblasts (HDFs) treated with inflammatory stimulants (lipopolysaccharide, tumor necrosis factor-alpha, interferon-gamma). Lactate dehydrogenase and reverse transcription-polymerase chain reaction showed that ASex inhibited the increased expression of acute-phase inflammatory cytokines. The in vivo model was established by inducing skin inflammation in NC/Nga mice via the repeated application of house dust mite (HDM) ointment to the ears and back of the mice for eight weeks. HDM application increased the severity of skin lesions, eosinophil/mast cell infiltration, and serum immunoglobulin E levels, which were all significantly decreased by ASex treatment, demonstrating the same degree of protection as hydrocortisone. Overall, ASex showed excellent anti-inflammatory effects both in vitro and in vivo, suggesting its potential as an excellent candidate drug to reduce skin inflammation.

Decreased inward rectifier and voltage-gated K+ currents of the right septal coronary artery smooth muscle cells in pulmonary arterial hypertensive rats.

In vascular smooth muscle, K+ channels, such as voltage-gated K+ channels (Kv), inward-rectifier K+ channels (Kir), and big-conductance Ca2+-activated K+ channels (BKCa), establish a hyperpolarized membrane potential and counterbalance the depolarizing vasoactive stimuli. Additionally, Kir mediates endothelium-dependent hyperpolarization and the active hyperemia response in various vessels, including the coronary artery. Pulmonary arterial hypertension (PAH) induces right ventricular hypertrophy (RVH), thereby elevating the risk of ischemia and right heart failure. Here, using the whole-cell patch-clamp technique, we compared Kv and Kir current densities (IKv and IKir) in the left (LCSMCs), right (RCSMCs), and septal branches of coronary smooth muscle cells (SCSMCs) from control and monocrotaline (MCT)-induced PAH rats exhibiting RVH. In control rats, (1) IKv was larger in RCSMCs than that in SCSMCs and LCSMCs, (2) IKv inactivation occurred at more negative voltages in SCSMCs than those in RCSMCs and LCSMCs, (3) IKir was smaller in SCSMCs than that in RCSMCs and LCSMCs, and (4) IBKCa did not differ between branches. Moreover, in PAH rats, IKir and IKv decreased in SCSMCs, but not in RCSMCs or LCSMCs, and IBKCa did not change in any of the branches. These results demonstrated that SCSMC-specific decreases in IKv and IKir occur in an MCT-induced PAH model, thereby offering insights into the potential pathophysiological implications of coronary blood flow regulation in right heart disease. Furthermore, the relatively smaller IKir in SCSMCs suggested a less effective vasodilatory response in the septal region to the moderate increase in extracellular K+ concentration under increased activity of the myocardium.

Fast relaxation and desensitization of angiotensin II contraction in the pulmonary artery via AT1R and Akt-mediated phosphorylation of muscular eNOS.

Angiotensin II (AngII) triggers a transient contraction of pulmonary arteries (PAs) followed by protracted desensitization. Based on the unconventional eNOS expression in PA smooth muscle cells (PASMCs), we hypothesized that activation of smooth muscle eNOS by AngII might be responsible for fast relaxation and tachyphylaxis. Using dual-wire myograph, mechanically endothelium-denuded rat PA [E(-)PA] showed AngII concentration-dependent transient contractions (ΔTAngII, 95% decay within 1 min), which were abolished by losartan (AT1R antagonist). Neither PD123319 (AT2R antagonist) nor A779 (MasR antagonist) affected ΔTAngII. When the vessels were pretreated with L-NAME (NOS inhibitor), ODQ (guanylate cyclase inhibitor), or KT5823 (PKG inhibitor), ΔTAngII of E(-)PA became larger and sustained, whereas nNOS or iNOS inhibitors had no such effect. Immunoblotting of human PASMCs (hPASMCs) also showed eNOS expression, and AngII treatment induced activating phosphorylations of Ser1177 in eNOS and of Ser473 in Akt (Ser/Thr protein kinase B), an upstream signal of eNOS phosphorylation. In addition, L-NAME co-treatment promoted AngII-induced Ser19 phosphorylation of myosin light chain. In hPASMCs, AngII abolished plasma membrane expression of AT1R, and recovery by washout took more than 1 h. Consistent with the data from hPASMCs, the second application of AngII to E(-)PA did not induce contraction, and significant recovery of ΔTAngII required prolonged washout (> 2 h) in the myography study. L-NAME treatment before the second application facilitated recovery of ΔTAngII. Muscular eNOS plays an auto-inhibitory role in ΔTAngII of PAs. The molecular changes investigated in hPASMCs revealed eNOS phosphorylation and internalization of AT1R by AngII. We propose that the rat PA smooth muscle eNOS-induced lusitropy and slow recovery of AT1R from tachyphylaxis might counterbalance the excessive contractile response to AngII, contributing to the distinctive low-pressure pulmonary circulation.

The novel high-frequency variant of TRPV3 p.A628T in East Asians showing faster sensitization in response to chemical agonists.

TRPV3, a member of the thermosensitive Ca2+-permeable TRPV channel subfamily expressed in skin and sensory nerves, is also activated by chemical agonists such as 2-aminoethyl diphenylborinate (2-APB). Repetitive stimuli induce sensitization of TRPV3 activation, characterized by the cumulative increase in current amplitude and linearization of current-voltage relation (I/V curve). Through genomic analysis of various populations, we found non-rare TRPV3 mutation (p.A628T) in East Asian people with an allele frequency of 0.249 while 0.007 in Caucasian. Slope conductance of unitary channel was not different between WT and p.A628T. Whole-cell patch clamp study of wildtype TRPV3 (WT) and p.A628T overexpressed in HEK293T cells showed similar sensitization by the repetitive increase in temperature from 23 to 37 °C, while slightly higher sensitization to 43 °C in p.A628T. In contrast, the repetitive application of 2-APB (10 µM) or carvacrol (100 µM) induced faster sensitization in p.A628T than WT. However, 1 µM farnesyl pyrophosphate, an intrinsic lipid metabolite agonist, induced similar level of slow activations in WT and p.A628T. In Fura-2 microspectrofluorimetry, the 2-APB pulses induced a faster increase of [Ca2+]c in p.A628T than WT. In terms of ionic selectivity of channels, WT and p.A628T showed similar Ca2+ permeability (PCa/PNa) calculated from the reversal potential of I/V curves. Taken together, p.A628T shows faster sensitization to chemical agonists that are reflected as higher [Ca2+]c signaling. Based on the intriguing pharmacological sensitivity, the physiological implications of p.A628T in the East Asian population require further investigation.

Parental Occupational Status and Suicidal Ideation in Adolescent: Cross-Sectional Secondary Data Analysis.

PURPOSE: This study aimed to identify the effect of parental occupational status on adolescents' suicidal ideation from a representative sample of the Korean population. DESIGN AND METHODS: A cross-sectional analysis was performed. Participants were 3201 adolescents aged 12-18 years from the Korean National Health and Nutrition Examination Survey. RESULTS: Adolescents experiencing suicidal ideation were found to be more likely to be female, have a history of smoking or alcohol use, a shorter sleep duration, greater perception of stress, and greater experience of depression when compared to those who did not report suicidal ideation. There was a statistically significant difference in perception of stress, experience of depression, and experience of suicidal ideation according to parental occupational status. Adolescents' suicidal ideation was associated with parents' employment status, work status, work schedule patterns, and working hours per week. CONCLUSIONS: Parental occupational status was associated with adolescents' suicidal ideation, but fathers' and mothers' occupational status affected children's suicidal ideation in different ways. Adolescents' suicidal ideation seems to have been affected by economic difficulty and stress, resulting from the father's occupational status, amount of time spent interacting with the mother, and the mother's employment status. Policies are needed to improve the quality of employment to reduce the financial difficulties and stresses of the father. A strategy for work-family compatibility is needed so that the mother can have enough time with her children while she has a job. PRACTICE IMPLICATIONS: It is necessary to consider parents' occupational status when assessing adolescents' suicidal ideation and providing interventions.

Differential effects of saturated and unsaturated fatty acids on vascular reactivity in isolated mesenteric and femoral arteries of rats.

Free fatty acid (FFA) intake regulates blood pressure and vascular reactivity but its direct effect on contractility of systemic arteries is not well understood. We investigated the effects of saturated fatty acid (SFA, palmitic acid), polyunsaturated fatty acid (PUFA, linoleic acid), and monounsaturated fatty acid (MUFA, oleic acid) on the contractility of isolated mesenteric (MA) and deep femoral arteries (DFA) of Sprague-Dawley rats. Isolated MA and DFA were mounted on a dual wire myograph and phenylephrine (PhE, 1-10 µM) concentration-dependent contraction was obtained with or without FFAs. Incubation with 100 µM of palmitic acid significantly increased PhE-induced contraction in both arteries. In MA, treatment with 100 µM of linoleic acid decreased 1 µM PhE-induced contraction while increasing the response to higher PhE concentrations. In DFA, linoleic acid slightly decreased PhE-induced contraction while 200 µM oleic acid significantly decreased it. In MA, oleic acid reduced contraction at low PhE concentration (1 and 2 µM) while increasing it at 10 µM PhE. Perplexingly, depolarization by 40 mM KCl-induced contraction of MA was commonly enhanced by the three fatty acids. The 40 mM KCl-contraction of DFA was also augmented by linoleic and oleic acids while not affected by palmitic acid. SFA persistently increased alpha-adrenergic contraction of systemic arteries whereas PUFA and MUFA attenuated PhE-induced contraction of skeletal arteries. PUFA and MUFA concentration-dependent dual effects on MA suggest differential mechanisms depending on the types of arteries. Further studies are needed to elucidate underlying mechanisms of the various effects of FFA on systemic arteries.

Enhancing undergraduate nursing students' global health competencies in South Korea.

OBJECTIVES: As the need for greater global health competency increases for health care professionals in South Korea, educational efforts for nursing students have begun. This study examined the effectiveness of two educational courses for freshmen and sophomores that were designed to improve students' global health competencies. DESIGN AND SAMPLE: A trend study was conducted for all undergraduate nursing students enrolled in a 4-year undergraduate nursing program in 2013 and 2014. MEASURES: We assessed students' global health competencies (1-knowledge and interests in global health and health equity, 2-global health skills, and 3-learning needs) in 2013 and 2014 and analyzed variance between mean scores by year and by course exposure, using 95% confidence intervals. RESULTS: Students who took both global health courses (sophomores in both years) reported higher global health-related knowledge and interests than did freshmen (p < .01); these scores were not sustained a year later. CONCLUSIONS: The two courses may have improved students' global health competencies. Reinforcement of knowledge in later courses may be needed to build on the global competencies.

Effects of high glucose with or without other metabolic substrates on alpha-adrenergic contractions in rat mesenteric and femoral arteries.

Hyperglycemia is associated with an increased risk of cardiovascular diseases. It has been demonstrated that chronic exposure to high glucose impaired endothelial functions. However, specific effects of short-term exposure to high glucose on vascular reactivity are controversial. Moreover, the combined effects of other metabolic substrates such as free fatty acids (FFA) on vascular reactivity remain poorly understood. Here we investigate the effects of short-term exposure to high glucose with or without other metabolic substrates including FFAs termed "nutrition full" (NF) solution, on mesenteric (MA) and deep femoral arteries (DFA) of rats. Arterial ring segments were mounted in a double-wire myograph. Contraction in response to phenylephrine (PhE) was determined in control (5 mM) and high glucose (23 mM, HG) environments over a 30 min period. In both arteries, PhE-inducedvasocontraction was enhanced by pre-incubation of HG solution. A combined incubation with HG and palmitic acid (100 µM) induced similar sensitization of PhE-contractions in both arteries. In contrast, high K+-induced contractions were not affected by HG. Interestingly, pre-incubation with NF solution decreased PhE-induced contraction in MA but increased the contraction in DFA. In NF solution, the HG-induced facilitation of PhE-contraction was not observed in MA. Furthermore, the PhE-induced contraction of DFA was attenuated by HG in NF solution. Our results demonstrate that the sensitization of PhE-induced arterial contraction by HG is differentially affected by other metabolic substrates. The conversation of skeletal arterial contractility by HG in NF solution requires careful interpretation of the previous in vitro studies where only glucose is included in physiological salt solutions. Further studies are required to elucidate the mechanism underlying the inconsistent effect of NF solution on MA and DFA.

Biphasic augmentation of alpha-adrenergic contraction by plumbagin in rat systemic arteries.

Plumbagin, a hydroxy 1,4-naphthoquinone compound from plant metabolites, exhibits anticancer, antibacterial, and antifungal activities via modulating various signaling molecules. However, its effects on vascular functions are rarely studied except in pulmonary and coronary arteries where NADPH oxidase (NOX) inhibition was suggested as a mechanism. Here we investigate the effects of plumbagin on the contractility of skeletal artery (deep femoral artery, DFA), mesenteric artery (MA) and renal artery (RA) in rats. Although plumbagin alone had no effect on the isometric tone of DFA, 1 µM phenylephrine (PhE)-induced partial contraction was largely augmented by plumbagin (ΔTPlum, 125% of 80 mM KCl-induced contraction at 1 µM). With relatively higher concentrations (>5 µM), plumbagin induced a transient contraction followed by tonic relaxation of DFA. Similar biphasic augmentation of the PhE-induced contraction was observed in MA and RA. VAS2870 and GKT137831, specific NOX4 inhibitors, neither mimicked nor inhibited ΔTPlum in DFA. Also, pretreatment with tiron or catalase did not affect ΔTPlum of DFA. Under the inhibition of PhE-contraction with L-type Ca2+ channel blocker (nifedipine, 1 µM), plumbagin still induced tonic contraction, suggesting Ca2+-sensitization mechanism of smooth muscle. Although ΔTPlum was consistently observed under pretreatment with Rho A-kinase inhibitor (Y27632, 1 µM), a PKC inhibitor (GF 109203X, 10 µM) largely suppressed ΔTPlum. Taken together, it is suggested that plumbagin facilitates the PKC activation in the presence of vasoactive agonists in skeletal arteries. The biphasic contractile effects on the systemic arteries should be considered in the pharmacological studies of plumbagin and 1,4-naphthoquinones.

Role of muscular eNOS in skeletal arteries: Endothelium-independent hypoxic vasoconstriction of the femoral artery is impaired in eNOS-deficient mice.

We previously reported that hypoxia augments α-adrenergic contraction (hypoxic vasoconstriction, HVC) of skeletal arteries in rats. The underlying mechanism may involve hypoxic inhibition of endothelial nitric oxide synthase (eNOS) expressed in skeletal arterial myocytes (16). To further explore the novel role of muscular eNOS in the skeletal artery, we compared HVC in femoral arteries (FAs) from eNOS knockout (KO) mice with that from wild-type (WT) and heterozygous (HZ) mice. Immunohistochemical assays revealed that, in addition to endothelia, eNOS is also expressed in the medial layer of FAs, albeit at a much lower level. However, the medial eNOS signal was not evident in HZ FAs, despite strong expression in the endothelium; similar observations were made in WT carotid arteries (CAs). The amplitude of contraction induced by 1 µM phenylephrine (PhE) was greater in HZ than in WT FAs. Hypoxia (3% Po2) significantly augmented PhE-induced contraction in WT FAs but not in HZ or KO FAs. No HVC was observed in PhE-pretreated WT CAs. The NOS inhibitor nitro-l-arginine methyl ester (0.1 mM) also augmented PhE contraction in endothelium-denuded WT FAs but not in WT CAs. Inhibitors specific to neuronal NOS and inducible NOS did not augment PhE-induced contraction of WT FAs. NADPH oxidase 4 (NOX4) inhibitor (GKT137831, 5 µM), but not NOX2 inhibitor (apocynin, 100 µM), suppressed HVC. Consistent with the role of reactive oxygen species (ROS), HVC was also inhibited by pretreatment with tiron or polyethylene glycol-catalase. Taken together, these data suggest that the eNOS expressed in smooth muscle cells in FAs attenuates α-adrenergic vasoconstriction; this suppression is alleviated under hypoxia, which potentiates vasoconstriction in a NOX4/ROS-dependent mechanism.

Inhibition of TREK-2 K(+) channels by PI(4,5)P2: an intrinsic mode of regulation by intracellular ATP via phosphatidylinositol kinase.

TWIK-related two-pore domain K(+) channels 1 and 2 (TREKs) are activated under various physicochemical conditions. However, the directions in which they are regulated by PI(4,5)P2 and intracellular ATP are not clearly presented yet. In this study, we investigated the effects of ATP and PI(4,5)P2 on overexpressed TREKs (HEK293T and COS-7) and endogenously expressed TREK-2 (mouse astrocytes and WEHI-231 B cells). In all of these cells, both TREK-1 and TREK-2 currents were spontaneously increased by dialysis with ATP-free pipette solution for whole-cell recording (ITREK-1,w-c and ITREK-2w-c) or by membrane excision for inside-out patch clamping without ATP (ITREK-1,i-o and ITREK-2,i-o). Steady state ITREK-2,i-o was reversibly decreased by 3 mM ATP applied to the cytoplasmic side, and this reduction was prevented by wortmannin, a PI-kinase inhibitor. An exogenous application of PI(4,5)P2 inhibited the spontaneously increased ITREKs,i-o, suggesting that intrinsic PI(4,5)P2 maintained by intracellular ATP and PI kinase may set the basal activity of TREKs in the intact cells. The inhibition of intrinsic TREK-2 by ATP was more prominent in WEHI-231 cells than astrocytes. Interestingly, unspecific screening of negative charges by poly-L-lysine also inhibited ITREK-2,i-o. Application of PI(4,5)P2 after the poly-L-lysine treatment showed dose-dependent dual effects, initial activation and subsequent inhibition of ITREK-2,i-o at low and high concentrations, respectively. In HEK293T cells coexpressing TREK-2 and a voltage-sensitive PI(4,5)P2 phosphatase, sustained depolarization increased ITREK-2,w-c initially (<5 s) but then decreased the current below the control level. In HEK293T cells coexpressing TREK-2 and type 3 muscarinic receptor, application of carbachol induced transient activation and sustained suppression of ITREK-2,w-c and cell-attached ITREK-2. The inhibition of TREK-2 by unspecific electrostatic quenching, extensive dephosphorylation, or sustained hydrolysis of PI(4,5)P2 suggests the existence of dual regulatory modes that depend on PI(4,5)P2 concentration.

Wall stretch and thromboxane A2 activate NO synthase (eNOS) in pulmonary arterial smooth muscle cells via H2O2 and Akt-dependent phosphorylation.

Pulmonary arteries (PAs) have high compliance, buffering the wide ranges of blood flow. Here, we addressed a hypothesis that PA smooth muscle cells (PASMCs) express nitric oxide synthases (NOS) that might be activated by mechanical stress and vasoactive agonists. In the myograph study of endothelium-denuded rat PAs, NOS inhibition (L-NAME) induced strong contraction (96 % of 80 mM KCl-induced contraction (80K)) in the presence of 5 nM U46619 (thromboxane A2 (TXA2) analogue) with relatively high basal stretch (2.94 mN, S(+)). With lower basal stretch (0.98 mN, S(-)), however, L-NAME application following U46619 (TXA2/L-NAME) induced weak contraction (27 % of 80K). Inhibitors of nNOS and iNOS had no such effect in S(+) PAs. In endothelium-denuded S(+) mesenteric and renal arteries, TXA2/L-NAME-induced contraction was only 18 and 21 % of 80K, respectively. Expression of endothelial-type NOS (eNOS) in rat PASMCs was confirmed by RT-PCR and immunohistochemistry. Even in S(-) PAs, pretreatment with H2O2 (0.1-10 µM) effectively increased the sensitivity to TXA2/L-NAME (105 % of 80K). Vice versa, NADPH oxidase inhibitors, reactive oxygen species scavengers, or an Akt inhibitor (SC-66) suppressed TXA2/L-NAME-induced contraction in S(+) PAs. In a human PASMC line, immunoblot analysis showed the following: (1) eNOS expression, (2) Ser(1177) phosphorylation by U46619 and H2O2, and (3) Akt activation (Ser(473) phosphorylation) by U46619. In the cell-attached patch clamp study, H2O2 facilitated membrane stretch-activated cation channels in rat PASMCs. Taken together, the muscular eNOS in PAs can be activated by TXA2 and mechanical stress via H2O2 and Akt-mediated signaling, which may counterbalance the contractile signals from TXA2 and mechanical stimuli.

Hypoxic pulmonary vasoconstriction and vascular contractility in monocrotaline-induced pulmonary arterial hypertensive rats.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vascular remodeling of pulmonary arteries (PAs) and increased vascular resistance in the lung. Monocrotaline (MCT), a toxic alkaloid, is widely used for developing rat models of PAH caused by injury to pulmonary endothelial cells; however, characteristics of vascular functions in MCT-induced PAH vary and are not fully understood. Here, we investigated hypoxic pulmonary vasoconstriction (HPV) responses and effects of various vasoconstrictors with isolated/perfused lungs of MCT-induced PAH (PAH-MCT) rats. Using hematoxylin and eosin staining, we confirmed vascular remodeling (i.e., medial thickening of PA) and right ventricle hypertrophy in PAH-MCT rats. The basal pulmonary arterial pressure (PAP) and PAP increase by a raised flow rate (40 mL/min) were higher in the PAH-MCT than in the control rats. In addition, both high K+ (40 mM KCl)- and angiotensin II-induced PAP increases were higher in the PAH-MCT than in the control rats. Surprisingly, application of a nitric oxide synthase inhibitor, L-NG-Nitroarginine methyl ester (L-NAME), induced a marked PAP increase in the PAH-MCT rats, suggesting that endothelial functions were recovered in the three-week PAH-MCT rats. In addition, the medial thickening of the PA was similar to that in chronic hypoxia-induced PAH (PAH-CH) rats. However, the HPV response (i.e., PAP increased by acute hypoxia) was not affected in the MCT rats, whereas HPV disappeared in the PAH-CH rats. These results showed that vascular contractility and HPV remain robust in the MCT-induced PAH rat model with vascular remodeling.