Biphasic Changes Research Articles

6-Monoacetylmorphine (6-MAM), Not Morphine, Is Responsible for the Rapid Neural Effects Induced by Intravenous Heroin.

Heroin rapidly enters the CNS but is quickly metabolized into 6-monoacetylmorphine (6-MAM) and then morphine. Although morphine is often thought to mediate heroin's neural effects, pharmacokinetic data question this view. To further understand the effects of heroin and its metabolites, oxygen sensors were used to examine changes in nucleus accumbens (NAc) oxygen levels. Heroin, 6-MAM, and morphine were all administered intravenously at twohuman-relevant doses (0.25 μmol/kg and 0.98 μmol/kg) in freely moving rats. Intravenous heroin induced a biphasic change in NAc oxygen, with a decrease resulting from respiratory depression and an increase resulting from cerebral vasodilation. 6-MAM caused similar but more rapid and slightly weaker effects than heroin. The stronger response to heroin can be primarily attributed to heroin's permeability and metabolism resulting in more 6-MAM in the brain. Morphine only induced weak increases in NAc oxygen. Therefore, it appears that 6-MAM is the major contributor to acute neural effects induced by iv heroin.

Biphasic Changes in β-Cell Mass Around Parturition Accompanied by Increased Serotonin Production

Pancreatic β-cell mass is known to be considerably altered during pregnancy and after parturition in rodents and humans. Although βcell mass is increased during pregnancy and starts to return toward its original level shortly after parturition, the cellular mechanisms by which β-cell mass during this period is regulated remains unclear. To address this issue in mice, we quantified β-cell mass and investigated the mechanisms underlying its regulation throughout the perinatal and postpartum period. The increased β-cell size and proliferation during pregnancy were significantly reduced shortly after parturition, whereas there was no evidence of β-cell reprogramming or increased apoptosis. Direct RNA sequencing of islets from pregnant and postpartum mice demonstrated dynamic changes in gene expression patterns, showing robust down-regulation of cell cycle-related genes 1 day after parturition, and the biphasic up-regulation of serotonin metabolism-related genes during pregnancy and at postpartum day 7. Serotonin synthesis was activated during lactation in human as well as in mice. Taken together, these findings demonstrate that β-cell mass is decreased shortly after parturition owing to reduced β-cell size and proliferation, and is subsequently increased, in association with lactation and serotonin biosynthesis. Funding Statement: This work was supported by research grant for Cross-disciplinary Collaboration, Juntendo University 2017 (29-35). Declaration of Interests: The authors stated: There is no conflict of interest on this study. Ethics Approval Statement: The study design was approved by the ethics committee of the Hirosaki University School of Medicine (approval number 2014-269), and the study conforms to the provision of the Declaration of Helsinki.

SAT-080 Dexamethasone Administration Stimulates Acute Increases in Natriuretic Peptides in Humans: A Potential Diagnostic Test for "Natriuretic Peptide Hormone Deficiency"?

Background: Animal models and human genetic investigations suggest that lower levels of natriuretic peptides (NP), which are cardiac-derived hormones, are associated with the development of obesity, insulin resistance, type 2 diabetes, and hypertension. However, whether lower resting NP levels in humans reflect a true hormone deficiency (analogous to other hormone deficiencies like adrenal insufficiency) is unknown, in part, because a diagnostic test for NP deficiency is currently lacking. One strategy for developing a diagnostic test for NP deficiency is to identify a stimulus that provokes an increase in NP concentrations. Dexamethasone is a potent stimulus for NP production in animals, but its effect on NP levels in humans is not well defined. Objective: We sought to define the effects of dexamethasone on circulating NPs over time in healthy humans. We hypothesized that dexamethasone would stimulate increases in NP levels, and that we could characterize the magnitude and timing of the changes as a primary step toward developing a diagnostic test for NP deficiency. Methods: 10 healthy, lean women (mean age 28 + 5 years, mean BMI 22.5 + 1.6 kg/m2) received a single 4 mg IV dose of dexamethasone after an overnight fast. Plasma levels of N-terminal pro-atrial natriuretic peptide (NT-proANP) and N-terminal pro- b-type natriuretic peptide (NT-proBNP) were serially measured during 2 time periods following dexamethasone: 1) “Acute Phase”: 0-8 hours, and 2) “Extended Phase”: at 24, 48, and 72 hours. Changes in NPs were analyzed using a piecewise random effects model for the 2 time periods. Results: During the Acute Phase after dexamethasone, a biphasic change in NT-proANP was observed (nonlinear, p<0.001). Median NT-proANP initially increased, with a peak increase of 24% occurring at 2.5 hours, and thereafter decreased until 8 hours (to 21% below baseline). During the “Extended Phase,” NT-proANP levels rebounded (p<0.001) and returned to near baseline by 72 hours. Next, median NT-proBNP increased during the Acute Phase (p= 0.016), with a peak increase of 27% occurring at 8 hours, and subsequently declined during the Extended Phase (p= 0.04) to a median value 24% lower than baseline at 72 hours. Discussion: Dexamethasone appears to acutely stimulate NP production in humans, as evidenced by an initial increase, followed by a subsequent decline, in circulating NP levels. The time course differs between NT-proANP and NT-proBNP, with NT-proANP peaking earlier than NT-proBNP. These pilot data may inform larger studies for establishing population norms for the NP response to glucocorticoids and evaluating whether a NP stimulation test with glucocorticoids may be a useful test for diagnosing NP deficiency.

Time–temperature superposition principle for the kinetic analysis of destabilization of pharmaceutical emulsions

The time–temperature superposition principle (TTSP) was applied to the destabilization kinetics of a pharmaceutical emulsion. The final goal of this study is to predict precisely the emulsion stability after long-term storage from the short-period accelerated test using TTSP. As the model emulsion, a cream preparation that is clinically used for the treatment of pruritus associated with chronic kidney disease was tested. After storage at high temperatures ranging from 30 to 45 °C for designated periods, the emulsion state was monitored using magnetic resonance imaging, and then the phase separation behaviors observed were analyzed according to the Arrhenius approach applying TTSP. The Arrhenius plot showed a biphasic change around 35 °C, indicating that the separation behaviors of the sample were substantially changed between the lower (30–35 °C) and higher (35–45 °C) temperature ranges. This study also monitored the coalescence behavior using a backscattered light measurement. The experiment verified that the destabilization was initiated by coalescence of oil droplets and then it eventually led to obvious phase separation via creaming. Furthermore, we note the coalescence kinetics agreed well with the phase separation kinetics. Therefore, in the case of the sample emulsion, the coalescence behavior has a dominant influence on the destabilization process. This study offers a profound insight into the destabilization process of pharmaceutical emulsions and demonstrates the promising applicability of TTSP to pharmaceutical research.

K+ influx triggers slow K+/H+ exchange detected by biphasic changes in matrix pH in Guinea pig cardiomyocyte mitochondria

Recent findings in cell models supports the importance of cation homeostasis by a K+/H+ exchanger (KHE) encoded by the gene LETM1, although LETM1 may also encode Ca2+/H+ exchange and or Na+/H+ exchanger (mNCE). In mitochondria, K+ influx occurs through K+ channels sensitive to Ca2+, Na+, low ATP, and other ligands. K+ influx promotes mitochondrial (m) swelling and contributes to regulation of mitochondrial respiration, membrane potential (mMP) and redox signaling. The influx of mK+ requires a mK+ efflux mechanism (mK+ cycle) paired with H+ influx, which in turn would affect mitochondrial bioenergetics, e.g. respiration, ADP phosphorylation, and mMP due effectively to a “proton leak”. We have previously reported that increased cytosolic and mCa2+ stimulate small and large Ca2+‐sensitive mK+ channels to permit mK+ influx. Cytosolic and mCa2+ increase during ischemia or hypoxia in association with an increase in mitochondrial volume so a high mK+ concentration may be a parallel effect of mCa2+ loading. Experimental conditions that modulate mKHE activity in mammalian cardiac tissue and its consequences have not been examined. Our aim was to provide evidence of mKHE activity in mitochondria isolated from Guinea pig cardiac cell mitochondria and to better understand the circumstances and kinetics of mKHE activity. We proposed that the subsequent efflux of mK+ via mKHE would re‐establish mK+ homeostasis but also provide a “H+ leak” thereby affecting bioenergetics activity. We reasoned that mKHE becomes active once K+ has accumulated in the matrix and that K+ efflux is linked to H+ influx as a marker of mKHE activity. mKHE might be affected by extra‐matrix pH (pHm) as it can change the trans‐membrane pH gradient. To test these ideas, we first added CGP37157 to the mitochondrial buffer to inhibit mNCE. Then we added 25 nM valinomycin, a K+ ionophore, and measured the time‐dependent changes in matrix pHm, using the fluorescent probe BCECF‐AM. We found that across the three extra‐matrix pHs of 6.9, 7.15, and 7.6, matrix pHm transiently increased over approximately 100 s and then transiently decreased over the next 100 s with a slower decline over 20 min. The initial increase in pHm may reflect enhanced proton pumping by respiratory complexes and the secondary decrease in pHm may reflect H+ influx in exchange for K+ efflux, via mKHE. When 500 μM quinine was given before valinomycin to attenuate mKHE, we found that the peak increase in pHm (first phase) occurred later and the subsequent decrease in pHm (second phase) also occurred later by 100 s and was slower to fall over 20 min. The changes to pHm in the presence of quinine provide evidence that the delay in pHm changes were due to block of mKHE by quinine. Interestingly the biphasic effects of valinomycin and quinine on pHm did not appear to be dependent on the extra‐matrix pH values of 6.9, 7.15, 7.6. Additional studies will determine concomitant effects of these protocols on respiration, ADP phosphorylation, mMP, and mitochondrial volume.Support or Funding InformationVA Merit BX‐002539‐01 and NIH T35 HL072483This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Concurrent recording of neurometabolic changes and local field potential in the central nervous system of awake-behaving rodent models of epilepsy

Introduction: Temporal lobe epilepsy (TLE) is a form of acquired epilepsy characterized as an electro-clinical syndrome in which seizures emanate from the limbic system [1,2]. TLE is particularly disabling due to the unpredictable and recurrent nature of seizures and incidence of antiepileptic drug resistance [3] many times an indication for resection surgery [4,5]. Pre-surgical identification of the seizure onset zone relies on multimodal methods based on different pathophysiological principles. The golden standard technique is long-term intracranial electroencephalogram (EEG) [6] combined neuroimaging techniques, which explore brain hemodynamics as a surrogate signal for brain activity [7,8]. To date, no single sensor approach allows simultaneous and seamless recording of electrical and hemodynamic responses. Materials and methods: We used multisite ceramic-based Pt microelectrode arrays (MEAs) to perform high-frequency amperometric recording of pO2 and local field potential (LFP)-related currents in the CNS of chronically implanted freely-moving rats during pilocarpine-evoked status epilepticus. Using fast Fourier transform (FFT) filtering of the raw electrochemical signal we separated into the low frequency component (<1 Hz), corresponding to the electrochemical reduction of O2 and high (>1 Hz) frequency component, corresponding to the LFP currents. Results: We determined the mean resting level of pO2 in two subregions of the hippocampus as well as in the striatum and the cortex of freely moving rats. The hippocampal DG showed lowest (p < 0.01) resting pO2 level (6.6 ± 0.7 μM) when compared to CA1 (22.1 ± 3.4 μM), striatum (17.2 ± 1.7 μM), and cortex (22.1 ± 4.9 μM). Evoked changes in interstitial pO2 in vivo we performed a 5 min tail pinch stress paradigm test, which induced a transient increase pO2 in the hippocampus of 15 ± 4% from baseline, corresponding to an average Δ[O2] of 3.7 ± 0.6 μM. Induction of status epilepticus induced biphasic changes in pO2 in the hippocampus. The initial dip at seizure onset (−4.5 ± 0.7 µM) was followed by a prolonged hyperoxygenation phase (+10.4 ± 2.9 µM). Analysis of high-frequency component of the amperometric signal allowed the simultaneous monitoring LFP at the site of pO2 recording. This has high potential for translation into the clinical setting supported on intracranial grid or strip electrodes. Discussion and conclusions: This strategy revealed that a single sensor can simultaneously report chemical (pO2) and electrophysiological (LFP currents) information, allowing concurrent monitoring of electrical and neurovascular/neurometabolic activities.

Involvement of the Na+,K+‐ATPase isoforms in control of cerebral perfusion

What is the topic of this review? In this review, we consider the role of the Na+ ,K+ -ATPase in cerebrovascular function and how it might be changed in familial hemiplegic migraine type2 (FHM2). The primary focus is involvement of the Na+ ,K+ -ATPase isoforms in regulation of cerebrovascular tone. What advances does it highlight? In this review, we discuss three overall distinct mechanisms whereby the Na+ ,K+ -ATPase might be capable of regulating cerebrovascular tone. Furthermore, we discuss how changes in the Na+ ,K+ -ATPase in cerebral arteries might affect brain perfusion and thereby be involved in the pathology of FHM2. Familial hemiplegic migraine type2 (FHM2) has been characterized by biphasic changes in cerebral blood flow during a migraine attack, with initial hypoperfusion followed by abnormal hyperperfusion of the affected hemisphere. We suggested that FHM2-associated loss-of-function mutation(s) in the Na+ ,K+ -ATPase α2 isoform might be responsible for these biphasic changes in several ways. We found that reduced expression of the α2 isoform leads to sensitization of the contractile machinery to [Ca2+ ]i via Src kinase-dependent signal transduction. This change in sensitivity might be the underlying mechanism for both abnormally potentiated vasoconstriction and exaggerated vasorelaxation. Moreover, the functional significance of the Na+ ,K+ -ATPase α2 isoform in astrocytes provides for the possibility of elevated extracellular potassium signalling from astrocytic endfeet to the vascular wall in neurovascular coupling.

Biphasic changes in spontaneous cardiovagal baroreflex sensitivity during passive hyperthermia

Successful adaptation to passive hyperthermia requires continual adjustment of circulation, which is mediated mainly by the autonomic nervous system. The goal of this study was to explore the alterations in spontaneous cardiovagal baroreflex sensitivity (BRS) during exposure to a hot environment. To continuously follow changes in core body temperature (Tc), haemodynamics, and BRS, male Wistar-Kyoto rats were implanted with telemetric transmitters. BRS at an ambient temperature of 23 °C was not steady but oscillated with a maximum power in the range of 0.02–0.2 Hz. Exposure to hot air immediately shifted the distribution of BRS to higher values, although Tc remained unchanged (37.2 (0.3) °C), and the average BRS changed from 1.3 (0.3) to 3 (1.4) ms.mmHg−1, p < 0.0001. The degree of initial cardiovagal baroreflex sensitization explained 57% of the variability in the time to the onset of arterial pressure decline (p = 0.0114). With an increasing Tc (>38.8 (0.6) °C), BRS non-linearly declined, but haemodynamic parameters remained stable even above a Tc of 42 °C when the cardiovagal baroreflex was virtually non-operative. Abrupt full desensitization of the cardiovagal baroreflex with a muscarinic blocker did not induce arterial pressure decline. Our data indicate that a progressive decrease in BRS during passive hyperthermia does not induce haemodynamic instability. The positive association between initial cardiovagal baroreflex sensitization and the time to the onset of arterial pressure decline may reflect the potential protective role of parasympathetic activation during exposure to a hot environment.

The role of DNA methyltransferase activity in cocaine treatment and withdrawal in the nucleus accumbens of mice.

An increasing number of reports have provided crucial evidence that epigenetic modifications, such as DNA methylation, may be involved in initiating and establishing psychostimulant-induced stable changes at the cellular level by coordinating the expression of gene networks, which then manifests as long-term behavioral changes. In this study, we evaluated the enzyme activity of DNA methyltransferases (DNMTs) after cocaine treatment and during withdrawal. Furthermore, we studied how genetic or pharmacological inhibition of DNMTs in mouse nucleus accumbens (NAc) affects the induction and expression of cocaine-induced behavioral sensitization. Our results showed that after silencing Dnmt3a in the NAc during the induction phase of cocaine-induced sensitization, overall DNMT activity decreases, correlating negatively with behavioral sensitization. Reduced Dnmt3a mRNA during this phase was the largest contributing factor for decreased DNMT activity. Cocaine withdrawal and a challenge dose increased DNMT activity in the NAc, which was associated with the expression of behavioral sensitization. Long-term selective Dnmt3a transcription silencing in the NAc did not alter DNMT activity or the expression of cocaine-induced behavioral sensitization. However, bilateral intra-NAc injection of a non-specific inhibitor of DNMT (RG108) during withdrawal from cocaine decreased DNMT activity in the NAc and had a small effect on the expression of cocaine-induced behavioral sensitization. Thus, cocaine treatment and withdrawal is associated with biphasic changes in DNMT activity in the NAc, and the expression of behavioral sensitization decreases with non-selective inhibition of DNMT but not with selective silencing of Dnmt3a.

Sympathoexcitation in response to cardiac and pulmonary afferent stimulation of TRPA1 channels is attenuated in rats with chronic heart failure.

Excessive sympathoexcitation characterizes the chronic heart failure (CHF) state. An exaggerated cardiac sympathetic afferent reflex (CSAR) contributes to this sympathoexcitation. Prior studies have demonstrated that the CSAR to capsaicin [transient receptor potential (TRP) vanilloid 1 agonist] is exaggerated in CHF animal models. We recently discovered that capsaicin application to the lung visceral pleura in anesthetized, vagotomized, open-chested rats increases mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA). We named this response the pulmonary spinal afferent reflex (PSAR). Due to the similarities between TRP vanilloid 1 and TRP ankyrin 1 (TRPA1) channels as well as the excessive sympathoexcitation of CHF, we hypothesized that stimulation of the CSAR and PSAR with a specific TRPA1 agonist would result in an augmented response in CHF rats (coronary ligation model) compared with sham control rats. In response to a TRPA1 agonist, both CSAR and PSAR in sham rats resulted in biphasic changes in MAP and increases in HR and RSNA 10-12 wk postmyocardial infarction (post-MI). These effects were blunted in CHF rats. Assessment of TRPA1 expression levels in cardiopulmonary spinal afferents by immunofluorescence, quantitative RT-PCR, and Western blot analysis 10-12 wk post-MI all indicates reduced expression in CHF rats but no reduction at earlier time points. TRPA1 protein was reduced in a dorsal root ganglia cell culture model of inflammation and simulated tissue ischemia, raising the possibility that the in vivo reduction of TRPA1 expression was, in part, caused by CHF-related tissue ischemia and inflammation. These data provide evidence that reflex responses to cardiopulmonary spinal afferent TRPA1 stimulation may be attenuated in CHF rather than enhanced. NEW & NOTEWORTHY Excessive sympathoexcitation characterizes chronic heart failure (CHF). The contribution of transient receptor potential ankyrin 1 (TRPA1) channel-mediated reflexes to this sympathoexcitation is unknown. We found that application of TRPA1 agonist to the heart and lung surface resulted in increased heart rate and sympathetic output and a biphasic change in mean arterial pressure in control rats. These effects were attenuated in CHF rats, decreasing the likelihood that TRPA1 channels contribute to cardiopulmonary afferent sensitization in CHF.

Successful treatment for disseminated intravascular coagulation (DIC) corresponding to phenotype changes in a heat stroke patient

BackgroundHeat stroke induces coagulofibrinolytic activation, which leads to life-threatening disseminated intravascular coagulation (DIC). However, treatment strategies for DIC in heat stroke have not yet been established, and also, the time course changes in coagulofibrinolytic markers have not been thoroughly evaluated. We report a severe heat stroke case with DIC who was eventually saved by anti-DIC treatments in accordance with changes in coagulofibrinolytic markers.Case presentationA 45-year-old man was found unconscious outside, and his body temperature was elevated to 41.9 °C. For heat stroke, we performed an immediate tracheal intubation under the general anesthesia along with cooling by iced gastric lavage, cold fluid administration, and an intravascular cooling using Thermogard™. About 4 h after admission, his core temperature fell to 37 °C. We assessed coagulofibrinolytic biomarkers and treated in accordance with changes in these parameters. This case exhibited a biphasic change varying from an enhanced to a suppressed fibrinolytic type of DIC depending on the relative balance between fibrinolytic activation and the level of plasminogen activator inhibitor-1 (PAI-1). In the early phase with consumption coagulopathy and enhanced fibrinolysis, we transfused a large amount of fresh frozen plasma (FFP) and platelets with tranexamic acid, an antifibrinolytic agent, possibly providing relief for the bleeding tendency. Anticoagulant therapy using recombinant human thrombomodulin-α (rh-TM-α) and antithrombin III (ATIII) concentrate was especially effective for DIC with a suppressed fibrinolytic phenotype in the later phase, after which organ failure that included severe hepatic failure was remarkably improved.ConclusionThe present case may indicate the clinical significance of monitoring coagulifibrinolytic changes and the potential benefits of anticoagulants for heat stroke-induced DIC.

Evaluation of leptin concentration in Gingival Crevicular Fluid (GCF) during orthodontic tooth movement and its correlation to the rate of tooth movement.

OBJECTIVES:Leptin, a polypeptide which is related to body fat regulation, is also found to have a role in the inflammatory reaction. The aim of this study is to assess the concentration of leptin in Gingival Crevicular Fluid (GCF) during orthodontic force application and to correlate its concentration to rate of tooth movement.METHODS:Twenty orthodontic patients (10 males and 10 females) were selected for the study. Leptin concentration was measured at T0, before force application; T1, one hour after force application; T2, one day after force application; T3, one week after force application; T4, one month after force application. GCF was collected using filter paper strips from the distal aspect of gingival sulcus of the right maxillary canine distalized by an active lace-backs of tooth movement was measured on dental casts, before and one month after force application. One-way ANOVA with Bonferroni correction and Pearson's correlation test were used to analyze the data.RESULTS:The mean GCF leptin concentration increased from T0 to T1, rose to a peak at T2, then declined to a minimum value at T3 and then increased to a value at T4, closer to the base line value (T0), and it was statistically significant (P < 0.05). There was positive correlation of the overall mean leptin concentration to rate of tooth movement (correlation coefficient = 0.634).CONCLUSION:There was a biphasic change in GCF leptin concentration during one cycle of orthodontic force application. There was a positive correlation between the GCF leptin concentration and rate of tooth movement.

Neuroendocrine Insights into Burnout Syndrome

Debates flow in the medical and psychological field about burnout symptoms: from considering it as a distinct illness, a separate entity or correlated with physiological changes and/or job-related reaction. Seen as a form of depression, the researches are investigating the correlations between various changes in the normal human body functioning, environmental and job lever / implications. The following pages refer to the recent studies of neuroendocrine indicators involved in burnout. It is known that the endocrine system is highly interrelated with the immune and neural systems, the neuro-immuno-endocrine axis is subject to clear biphasic changes in the acute and chronic phases of a critical illness, most likely reflecting a beneficial adaptation.

Systemic Morphine Produces Dose-dependent Nociceptor-mediated Biphasic Changes in Nociceptive Threshold and Neuroplasticity

We investigated the dose dependence of the role of nociceptors in opioid-induced side-effects, hyperalgesia and pain chronification, in the rat. Systemic morphine produced a dose-dependent biphasic change in mechanical nociceptive threshold. At lower doses (0.003–0.03 mg/kg, s.c.) morphine induced mechanical hyperalgesia, while higher doses (1–10 mg/kg, s.c.) induced analgesia. Intrathecal (i.t.) oligodeoxynucleotide (ODN) antisense to mu-opioid receptor (MOR) mRNA, attenuated both hyperalgesia and analgesia. 5 days after systemic morphine (0.03–10 mg/kg s.c.), mechanical hyperalgesia produced by intradermal (i.d.) prostaglandin E2 (PGE2) was prolonged, indicating hyperalgesic priming at the peripheral terminal of the nociceptor. The hyperalgesia induced by i.t. PGE2 (400 ng/10 µl), in groups that received 0.03 (that induced hyperalgesia) or 3 mg/kg (that induced analgesia) morphine, was also prolonged, indicating priming at the central terminal of the nociceptor. The prolongation of the hyperalgesia induced by i.d. or i.t. PGE2, in rats previously treated with either a hyperalgesic (0.03 mg/kg, s.c.) or analgesic (3 mg/kg, s.c.) dose, was reversed by i.d. or i.t. injection of the protein translation inhibitor cordycepin (1 µg), indicative of Type I priming at both terminals. Although pretreatment with MOR antisense had no effect on priming induced by 0.03 mg/kg morphine, it completely prevented priming by 3 mg/kg morphine, in both terminals. Thus, the induction of hyperalgesia, but not priming, by low-dose morphine, is MOR-dependent. In contrast, induction of both hyperalgesia and priming by high-dose morphine is MOR-dependent. The receptor at which low-dose morphine acts to produce priming remains to be established.

Biphasic changes in airway epithelial cell EGF receptor binding and phosphorylation induced by components of hogbarn dust

Purpose of the study: Workers in enclosed hogbarns experience an increased incidence of airway inflammation and obstructive lung disease, and an aqueous hogbarn dust extract (HDE) induces multiple inflammation-related responses in cultured airway epithelial cells. Epidermal growth factor receptor (EGFR) phosphorylation and activation has been identified as one important mediator of inflammatory cytokine release from these cells. The studies here investigated both early and late phase adaptive changes in EGFR binding properties and subcellular localization induced by exposure of cells to HDE. Materials and Methods: Cell surface EGFRs were quantified as binding to intact cells on ice. EGFR phosphorylation, expression, and localization were assessed with anti-EGFR antibodies and either blotting or confocal microscopy. Results: In BEAS-2B and primary human bronchial epithelial cells, HDE induced decreases in cell surface EGFR binding following both 15-min and 18-h exposures. In contrast, H292 cells exhibited only the 15-min decrease, with binding near the control level at 18 hr. Confocal microscopy showed that the 15-min decrease in binding is due to EGFR endocytosis. Although total EGFR immunoreactivity decreased markedly at 18 hr in confocal microscopy with BEAS-2B cells, immunoblots showed no loss of EGFR protein. HDE stimulated EGFR phosphorylation at both 15 min and 18 hr in BEAS-2B cells and primary cells, but only at 15 min in H292 cells, indicating that the different EGFR binding changes among these cell types is likely related to their different time-dependent changes in phosphorylation. Conclusions: These studies extend the evidence for EGFRs as important cellular targets for components of HDE and they reveal novel patterns of EGFR phosphorylation and binding changes that vary among airway epithelial cell types. The results provide both impetus and convenient assays for identifying the EGFR-activating components and pathways that likely contribute to hogbarn dust-induced lung disease in agricultural workers.

Gramicidin Lateral Distribution in Phospholipid Membranes: Fluorescence Phasor Plots and Statistical Mechanical Model.

When using small mole fraction increments to study gramicidins in phospholipid membranes, we found that the phasor dots of intrinsic fluorescence of gramicidin D and gramicidin A in dimyristoyl-sn-glycero-3-phosphocholine (DMPC) unilamellar and multilamellar vesicles exhibit a biphasic change with peptide content at 0.143 gramicidin mole fraction. To understand this phenomenon, we developed a statistical mechanical model of gramicidin/DMPC mixtures. Our model assumes a sludge-like mixture of fluid phase and aggregates of rigid clusters. In the fluid phase, gramicidin monomers are randomly distributed. A rigid cluster is formed by a gramicidin dimer and DMPC molecules that are condensed to the dimer, following particular stoichiometries (critical gramicidin mole fractions, Xcr including 0.143). Rigid clusters form aggregates in which gramicidin dimers are regularly distributed, in some cases, even to superlattices. At Xcr, the size of cluster aggregates and regular distributions reach a local maximum. Before a similar model was developed for cholesterol/DMPC mixtures (Sugar and Chong (2012) J. Am. Chem. Soc. 134, 1164–1171) and here the similarities and differences are discussed between these two models.

Rapid peroxynitrite reduction by human peroxiredoxin 3: Implications for the fate of oxidants in mitochondria

Mitochondria are main sites of peroxynitrite formation. While at low concentrations mitochondrial peroxynitrite has been associated with redox signaling actions, increased levels can disrupt mitochondrial homeostasis and lead to pathology. Peroxiredoxin 3 is exclusively located in mitochondria, where it has been previously shown to play a major role in hydrogen peroxide reduction. In turn, reduction of peroxynitrite by peroxiredoxin 3 has been inferred from its protective actions against tyrosine nitration and neurotoxicity in animal models, but was not experimentally addressed so far. Herein, we demonstrate the human peroxiredoxin 3 reduces peroxynitrite with a rate constant of 1 × 107 M−1 s−1 at pH 7.8 and 25 °C. Reaction with hydroperoxides caused biphasic changes in the intrinsic fluorescence of peroxiredoxin 3: the first phase corresponded to the peroxidatic cysteine oxidation to sulfenic acid. Peroxynitrite in excess led to peroxiredoxin 3 hyperoxidation and tyrosine nitration, oxidative post-translational modifications that had been previously identified in vivo. A significant fraction of the oxidant is expected to react with CO2 and generate secondary radicals, which participate in further oxidation and nitration reactions, particularly under metabolic conditions of active oxidative decarboxylations or increased hydroperoxide formation. Our results indicate that both peroxiredoxin 3 and 5 should be regarded as main targets for peroxynitrite in mitochondria.

A high-throughput real-time in vitro assay using mitochondrial targeted roGFP for screening of drugs targeting mitochondria

Most toxic compounds including cancer drugs target mitochondria culminating in its permeabilization. Cancer drug-screening and toxicological testing of compounds require cost-effective and sensitive high-throughput methods to detect mitochondrial damage. Real-time methods for detection of mitochondrial damage are less toxic, allow kinetic measurements with good spatial resolution and are preferred over end-stage assays.Cancer cell lines stably expressing genetically encoded mitochondrial-targeted redox-GFP2 (mt-roGFP) were developed and validated for its suitability as a mitochondrial damage sensor. Diverse imaging platforms and flow-cytometry were utilized for ratiometric analysis of redox changes with known toxic and cancer drugs. Key events of cell death and mitochondrial damage were studied at single-cell level coupled with mt-roGFP. Cells stably expressing mt-roGFP and H2B-mCherry were developed for high-throughput screening (HTS) application.Most cancer drugs while inducing mitochondrial permeabilization trigger mitochondrial-oxidation that can be detected at single-cell level with mt-roGFP. The image-based assay using mt-roGFP outperformed other quantitative methods of apoptosis in ease of screening. Incorporation of H2B-mCherry ensures accurate and complete automated segmentation with excellent Z value. The results substantiate that most cancer drugs and known plant-derived antioxidants trigger cell-death through mitochondrial redox alterations with pronounced ratio change in the mt-roGFP probe.Real-time analysis of mitochondrial oxidation and mitochondrial permeabilization reveal a biphasic ratio change in dying cells, with an initial redox surge before mitochondrial permeabilization followed by a drastic increase in ratio after complete mitochondrial permeabilization. Overall, the results prove that mitochondrial oxidation is a reliable indicator of mitochondrial damage, which can be readily determined in live cells using mt-roGFP employing diverse imaging techniques. The assay described is highly sensitive, easy to adapt to HTS platforms and is a valuable resource for identifying cytotoxic agents that target mitochondria and also for dissecting cell signaling events relevant to redox biology.

Discovery and Structure-Based Optimization of Benzimidazole-Derived Activators of SOS1-Mediated Nucleotide Exchange on RAS.

Son of sevenless homologue 1 (SOS1) is a guanine nucleotide exchange factor that catalyzes the exchange of GDP for GTP on RAS. In its active form, GTP-bound RAS is responsible for numerous critical cellular processes. Aberrant RAS activity is involved in ∼30% of all human cancers; hence, SOS1 is an attractive therapeutic target for its role in modulating RAS activation. Here, we describe a new series of benzimidazole-derived SOS1 agonists. Using structure-guided design, we discovered small molecules that increase nucleotide exchange on RAS in vitro at submicromolar concentrations, bind to SOS1 with low double-digit nanomolar affinity, rapidly enhance cellular RAS-GTP levels, and invoke biphasic signaling changes in phosphorylation of ERK 1/2. These compounds represent the most potent series of SOS1 agonists reported to date.

Promoter Usage and Dynamics in Vascular Smooth Muscle Cells Exposed to Fibroblast Growth Factor-2 or Interleukin-1\u03b2

Smooth muscle cells (SMC) in blood vessels are normally growth quiescent and transcriptionally inactive. Our objective was to understand promoter usage and dynamics in SMC acutely exposed to a prototypic growth factor or pro-inflammatory cytokine. Using cap analysis gene expression (FANTOM5 project) we report differences in promoter dynamics for immediate-early genes (IEG) and other genes when SMC are exposed to fibroblast growth factor-2 or interleukin-1β. Of the 1871 promoters responding to FGF2 or IL-1β considerably more responded to FGF2 (68.4%) than IL-1β (18.5%) and 13.2% responded to both. Expression clustering reveals sets of genes induced, repressed or unchanged. Among IEG responding rapidly to FGF2 or IL-1β were FOS, FOSB and EGR-1, which mediates human SMC migration. Motif activity response analysis (MARA) indicates most transcription factor binding motifs in response to FGF2 were associated with a sharp induction at 1 h, whereas in response to IL-1β, most motifs were associated with a biphasic change peaking generally later. MARA revealed motifs for FOS_FOS{B,L1}_JUN{B,D} and EGR-1..3 in the cluster peaking 1 h after FGF2 exposure whereas these motifs were in clusters peaking 1 h or later in response to IL-1β. Our findings interrogating CAGE data demonstrate important differences in promoter usage and dynamics in SMC exposed to FGF2 or IL-1β.