4 research outputs found

    Novel Nongenomic Signaling by Glucocorticoid May Involve Changes to Liver Membrane Order in Rainbow Trout

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    <div><p>Stress-induced glucocorticoid elevation is a highly conserved response among vertebrates. This facilitates stress adaptation and the mode of action involves activation of the intracellular glucocorticoid receptor leading to the modulation of target gene expression. However, this genomic effect is slow acting and, therefore, a role for glucocorticoid in the rapid response to stress is unclear. Here we show that stress levels of cortisol, the primary glucocorticoid in teleosts, rapidly fluidizes rainbow trout (<em>Oncorhynchus mykiss</em>) liver plasma membranes <em>in vitro</em>. This involved incorporation of the steroid into the lipid domains, as cortisol coupled to a membrane impermeable peptide moiety, did not affect membrane order. Studies confirmed that cortisol, but not sex steroids, increases liver plasma membrane fluidity. Atomic force microscopy revealed cortisol-mediated changes to membrane surface topography and viscoelasticity confirming changes to membrane order. Treating trout hepatocytes with stress levels of cortisol led to the modulation of cell signaling pathways, including the phosphorylation status of putative PKA, PKC and AKT substrate proteins within 10 minutes. The phosphorylation by protein kinases in the presence of cortisol was consistent with that seen with benzyl alcohol, a known membrane fluidizer. Our results suggest that biophysical changes to plasma membrane properties, triggered by stressor-induced glucocorticoid elevation, act as a nonspecific stress response and may rapidly modulate acute stress-signaling pathways.</p> </div

    Cortisol effect on rapid cell signaling in trout hepatocytes.

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    <p>Rainbow trout hepatocytes were incubated either with cortisol (0, 100 or 1000 ng/mL) or benzyl alcohol (BA; 25 mM) for 10 min. Cell homogenates (40 µg protein) were probed with polyclonal rabbit antibody (Cell Signaling Technology, Beverly, MA) to either phospho-(Ser) PKC substrate (A), phospho-PKA Substrate (RRXS/T) (B) or phospho-Akt substrate (RXXS/T) (C). Equal loading was confirmed with β-actin (monoclonal mouse antibody; Sigma, St. Louis, MO). A representative immunoblot for each is shown; values are plotted as % control and shown as mean ± S.E.M (n = 3 independent fish); bars with different letters are significantly different (repeated measures ANOVA, p<0.05). *significantly different from control (Paired Student’s t-test; p<0.05).</p

    Cortisol effect on plasma membrane order <i>in vitro.</i>

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    <p>A) 1,6-Diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy of enriched hepatic plasma membranes isolated from rainbow trout treated with or without benzyl alcohol for 30 min prior to anisotropy measurement at various temperatures. Data represents mean ± S.E.M (n = 7 independent fish). Different upper case letters indicate significant temperature effects and inset indicates significant treatment effects (two-way repeated measures ANOVA, p<0.05). B) DPH fluorescence anisotropy of enriched hepatic plasma membrane fractions treated with cortisol (0, 10, 100, 500, and 1000 ng/ml) for 30 min at various temperatures. Data represents mean ± S.E.M (n = 6 independent fish). Different upper case letters indicate significant temperature effects and inset indicates significant treatment effects (two-way repeated measures ANOVA, p<0.05). C) DPH fluorescence anisotropy of isolated hepatic plasma membrane fractions treated with the peptide conjugate (PEP, equivalent to 1000 ng/ml, 2.75 µM), or cortisol-conjugated peptide (cortisol-PEP, 0.275 µM and 2.75 µM) for 30 min prior to anisotropy measurement at various temperatures. Data represents mean ± S.E.M (n = 5 independent fish). Different upper case letters indicate significant temperature effects (one-way repeated measures ANOVA, p<0.05). D) DPH fluorescence anisotropy of enriched trout hepatic plasma membrane fractions treated with 17β-estradiol (E; 1 µM) or testosterone (T; 1 µM) for 30 min. Data shown as mean ± S.E.M (n = 6 independent fish).</p

    Cortisol effect on liver plasma membrane topography and surface adhesion.

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    <p>A) Representative atomic force microscopy (AFM) images of supported hepatic plasma membrane topography (membrane height). Images were taken prior to (a) and 30 min after cortisol (100 ng/mL) treatment (e) in liquid cell at room temperature. A zoomed in scan is also shown for the control (c) and cortisol treated (g) membranes that was scanned for 60 min. The approximate scan region of the zoomed in image is indicated by the dashed red box in the control image (a) and solid red box in the cortisol-treated image (e). Two distinct domains, which differ in height, are visible in both control and cortisol-treated membranes. A representative higher domain is indicated by the dotted arrow, while the lower domain is indicated by the solid arrow (c). Short-term cortisol treatment altered the topography of the plasma membrane. The cross-section graph featured below each image was calculated from points along the white horizontal line. The y-axis represents vertical height (nm), whereas the x-axis represents the horizontal distance (nm). B) Representative AFM images of supported hepatic plasma membrane phase (surface adhesion properties). Images were taken prior to (a) and 30 min after cortisol (100 ng/mL) treatment (e) in liquid cell at room temperature. A zoomed in scan is also shown of the control (c) and cortisol treated (g) membranes that was scanned for 60 min. The approximate scan region of the zoomed in image is indicated by the dashed red box in the control image (a) and solid red box in the cortisol-treated image (e). Two distinct domains, which differ in their viscoelastic (surface adhesion) are visible in both control and cortisol-treated membranes. Acute cortisol treatment altered the viscoelastic properties of the plasma membrane within 30 min of treatment. The cross-section graph featured below each image was calculated from points along the white horizontal line. The y-axis represents degree of deflection (degrees), whereas the x-axis represents the horizontal distance (nm). C) A schematic representation of cortisol’s effect on plasma membrane properties. Short-term incubation with cortisol (b) increased surface roughness (height difference between higher and lower domains) compared to control membrane (a).</p
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