Regulation of vascular smooth muscle cell bioenergetic function by protein glutathiolation

AbstractProtein thiolation by glutathione is a reversible and regulated post-translational modification that is increased in response to oxidants and nitric oxide. Because many mitochondrial enzymes contain critical thiol residues, it has been hypothesized that thiolation reactions regulate cell metabolism and survival. However, it has been difficult to differentiate the biological effects due to protein thiolation from other oxidative protein modifications. In this study, we used diamide to titrate protein glutathiolation and examined its impact on glycolysis, mitochondrial function, and cell death in rat aortic smooth muscle cells. Treatment of cells with diamide increased protein glutathiolation in a concentration-dependent manner and had comparably little effect on protein–protein disulfide formation. Diamide increased mitochondrial proton leak and decreased ATP-linked mitochondrial oxygen consumption and cellular bioenergetic reserve capacity. Concentrations of diamide above 200 μM promoted acute bioenergetic failure and caused cell death, whereas lower concentrations of diamide led to a prolonged increase in glycolytic flux and were not associated with loss of cell viability. Depletion of glutathione using buthionine sulfoximine had no effect on basal protein thiolation or cellular bioenergetics but decreased diamide-induced protein glutathiolation and sensitized the cells to bioenergetic dysfunction and death. The effects of diamide on cell metabolism and viability were fully reversible upon addition of dithiothreitol. These data suggest that protein thiolation modulates key metabolic processes in both the mitochondria and cytosol

Lipid Metabolites Enhance Secretion Acting on SNARE Microdomains and Altering the Extent and Kinetics of Single Release Events in Bovine Adrenal Chromaffin Cells

Lipid molecules such as arachidonic acid (AA) and sphingolipid metabolites have been implicated in modulation of neuronal and endocrine secretion. Here we compare the effects of these lipids on secretion from cultured bovine chromaffin cells. First, we demonstrate that exogenous sphingosine and AA interact with the secretory apparatus as confirmed by FRET experiments. Examination of plasma membrane SNARE microdomains and chromaffin granule dynamics using total internal reflection fluorescent microscopy (TIRFM) suggests that sphingosine production promotes granule tethering while arachidonic acid promotes full docking. Our analysis of single granule release kinetics by amperometry demonstrated that both sphingomyelinase and AA treatments enhanced drastically the amount of catecholamines released per individual event by either altering the onset phase of or by prolonging the off phase of single granule catecholamine release kinetics. Together these results demonstrate that the kinetics and extent of the exocytotic fusion pore formation can be modulated by specific signalling lipids through related functional mechanisms

Cell signalling by reactive lipid species: new concepts and molecular mechanisms

The process of lipid peroxidation is widespread in biology and is mediated through both enzymatic and non-enzymatic pathways. A significant proportion of the oxidized lipid products are electrophilic in nature, the RLS (reactive lipid species), and react with cellular nucleophiles such as the amino acids cysteine, lysine and histidine. Cell signalling by electrophiles appears to be limited to the modification of cysteine residues in proteins, whereas non-specific toxic effects involve modification of other nucleophiles. RLS have been found to participate in several physiological pathways including resolution of inflammation, cell death and induction of cellular antioxidants through the modification of specific signalling proteins. The covalent modification of proteins endows some unique features to this signalling mechanism which we have termed the ‘covalent advantage’. For example, covalent modification of signalling proteins allows for the accumulation of a signal over time. The activation of cell signalling pathways by electrophiles is hierarchical and depends on a complex interaction of factors such as the intrinsic chemical reactivity of the electrophile, the intracellular domain to which it is exposed and steric factors. This introduces the concept of electrophilic signalling domains in which the production of the lipid electrophile is in close proximity to the thiol-containing signalling protein. In addition, we propose that the role of glutathione and associated enzymes is to insulate the signalling domain from uncontrolled electrophilic stress. The persistence of the signal is in turn regulated by the proteasomal pathway which may itself be subject to redox regulation by RLS. Cell death mediated by RLS is associated with bioenergetic dysfunction, and the damaged proteins are probably removed by the lysosome-autophagy pathway

Signalling lipids dramatically enhance the amount of catecholamines released by event.

<p>Main amperometric parameters of the secretory spikes obtained from control (N=55 cells), as well as AA-treated cells (N=50) and SMase-treated cells (N=44). This analysis was performed by averaging the spikes obtained in every individual cell. A. Cumulative distribution of the Imax values averaged for individual cells. 20 pA bin width. B. The mean amplitude is enhanced by 2-fold in AA-treated cells and by 1.7-fold in SMase treated cells. C. Cumulative distribution of the averaged amount of catecholamine molecules released per event obtained for individual cells. 500.000 molecules/event bin width. D. Consequently, the mean value of released molecules per event increased by 2.1-fold in AA-treated cells and by 2-fold in cells incubated with SMase. E. Cumulative distribution of the t_1/2 parameter averaged for individual cells. 1 ms bin width. F. Mean t_1/2 parameter was slightly reduced with SMase-treatment (not significant at the statistical level. **P<0.01 and ***P<0.001comparing to control according to ANOVA with Dunnett’s test for multiple comparisons.</p

Granule motion is restricted after cell incubation with signalling lipids.

<p>Granule movement was studied in cells treated with AA and SMase as described in previous figures. Granules were labeled with acridine orange and motion was studied using TIRFM images acquired at 1 s intervals. Panels A, B, and C show images separated by 5 s intervals obtained in control (A), AA-treated cells (B), and SMase-treated cells (C). D. Averaged speed calculated for granules in control (n = 144), AA (n = 68), and SMase-treated cells (n = 73). <b>E</b>. Mean square displacement vs time for the granules indicate restriction in the mobility in SMase-treated cells and the high degree of immobilization found in the cells incubated with AA. <b>F</b>. Distribution of the Z distances corresponding to the granules in the indicated experimental conditions. 25 nm bin width. <b>G</b>. Calculation of the mean range of z displacement (ΔZ) obtained for granules in control, AA-treated cells, and SMase-treated cells. Bar represents 1 µm in A. * P<0.05 compared to control values using ANOVA with Dunnett’s post test.</p

Signalling lipids affect the kinetics of catecholamine release from individual granules.

<p>Analysis of the kinetic parameters of amperometric spikes for the same number of cells indicated in the previous figure. A. Cumulative distribution of the onset rise parameter (time corresponding to the amplitude rise between the 25 and 75% of the maximal amplitude) for the different experimental conditions showing a displacement to lower values in response to lipid metabolites. 0.2 ms bin width. B. The average values are reduced after both treatments in a similar proportion (20%). C and D. Analysis for the offset parameter corresponding to the decaying phase of amperometric events. AA treatment affects this decaying phase, prolonging catecholamine release. Bin width 1 ms. *P<0.05 comparing to control according to ANOVA with Dunnett’s test for multiple comparisons.</p

SNARE microdomain lateral motion is restricted in cells incubated with SMase and AA.

<p>The dynamics of the microdomains formed by expression of GFP-SNAP-25 were studied in time-lapse images taken at 1 s intervals in control chromaffin cells or cells treated with 100 μM AA or 1 U/ml SMase for 30 min. Shown are images separated by 5 s intervals of SNARE clusters in a control cell (A), a cell incubated with AA (B), and SMase (C). The microdomains display lateral mobility in the XY plane, which was studied by measuring the centroid coordinates, which were then used to calculate the average path speed (D) for the vesicles in control (N = 103), AA (N = 118), and SMase-treated cells (N = 105). E. Mean square displacement (MSD) vs time plot curves indicate a caged motion in the case of SMase-treated cells (downward curve) and almost immobile behaviour in AA-treated cells (flat curve, error bars smaller than symbols). The variations in the intensity of fluorescence indicate the different mobility in the Z plane for the same patches and were used to calculate Z distance distributions using 25 nm bin width (F), and increments (G). Bar represents 1 µm in A. *P<0.05 and **P<0.01 compared to controls using ANOVA with Dunnett’s multiple comparison test.</p

Effect of lipid metabolites on the amperometric granule release events.

<p>Depicted are amperometric recordings obtained from 59 mM KCl depolarizations in control (A), AA-treated cells (B), and SMase-treated cells. D. Amperometric fusion events were analyzed and pooled to generate the averaged amperometric spikes from control (N = 1262 events), AA (N = 828), and SMase-treated cells (N = 854). For kinetic studies the normalized spikes are compared to show an evident reduction in the t_1/2 parameter in SMase-treated cells (E). Evidently, secretory events exhibit higher amplitude in the cells treated with signalling lipids.</p