Amino-acid concentrations and NO production in murine jejunal tissue.

<p>(A) L-Citrulline supplementation (LPS-Cit) increased tissue citrulline concentration compared to the LPS-Ala, LPS-Arg and control groups (P<0.001) (B) Arginine concentrations were significantly reduced in the LPS-Ala group compared to the control group (P<0.05). Interestingly, this concentration was not increased by L-Arginine supplementation (LPS-Arg), whereas this concentration was significantly increased by L-Citrulline supplementation (LPS-Cit group; P<0.01). (C) Ornithine levels increased in the LPS-Ala treated group compared to the control group (P<0.05) and increased further upon L-Citrulline supplementation (P<0.0001 relative to the control group and P<0.05 relative to the LPS group). (D) In the jejunum, prolonged endotoxemia resulted in a significant decrease in NO production (measured as pmol mono-nitrosyl-iron complexes (MNIC)/mg wet tissue) compared to control (P<0.05). NO production was significantly improved in the LPS-Arg and LPS-Cit group compared to the LPS group (P<0.0001 and P<0.05 respectively).</p

Representative live images of the microcirculatory measurements in jejunal villi with SDF imaging.

<p>(A) Representative live image of the jejunal microcirculation in a control mouse. (B) Representative live image of the jejunal microcirculation in a LPS-Ala treated mouse which shows only perfusion of the larger vessels. (C) Representative live image of a LPS-Arg treated mouse which shows a comparable perfusion pattern as the LPS-Ala treated mouse. (D) Representative live image of a LPS-Cit treated mouse, which’s shows more small perfused vessels per villus.</p

Experimental set up of the prolonged endotoxemia model.

<p>Mice were fitted with a jugular vein cannula at t = 0 d. After 4 days (t = 4 d) an 18hours continuous infusion with lipopolysaccharides (LPS) was started, which was combined during the last 6 hours with an infusion of Citrulline (Cit), Arginine (Arg) or an isonitrogenous quantity of the placebo Alanine (Ala). After completing the treatment, sidestream dark-field (SDF) imaging was used to quantify the microcirculation in the jejunal villi or Nitric Oxide (NO) spin trapping with iron-diethyldithiocarbamate (DETC) complexes to measure NO production <i>in vivo</i>.</p

Phosphorylated eNOS and iNOS protein levels in murine jejunal tissue.

<p>(A) The degree of phosphorylation of eNOS protein (expressed in arbitrary units, AU) was higher in the LPS-Cit group than in the LPS-Ala and LPS-Arg group (P<0.05). (B) The iNOS protein concentration changed in an opposite detection, with significantly lower levels in the control (P<0.05) and LPS-Cit group (P<0.01) than in the LPS-Ala and LPS-Arg group. (C) Representative examples of expression of phosphorylated eNOS (Ser 1177), iNOS and beta-actin by Western blot analysis.</p

Amino-acid concentrations in plasma.

<p>(A) Citrulline plasma concentrations increased after L-Citrulline supplementation (LPS-Cit) compared to all other groups (P<0.0001). (B) Plasma arginine concentrations were significantly reduced in the LPS-Ala group compared to the control group (P<0.05), whereas this concentration was significantly increased by L-Arginine or L-Citrulline supplementation (LPS-Arg and LPS-Cit group) compared to both other groups (P<0.001). (C) Plasma ornithine levels increased in the LPS-Ala treated group compared to the control group (P<0.05) and increased further upon L-Arginine or L-Citrulline supplementation.</p

Detection of NO produced in explanted murine carotid arteries <i>ex</i><i>vivo</i> using Cu ₂FL2E (20 µM) after precontraction.

<p>(<b>a</b>) Detection of NO in response to NA (ECs and SMCs are not apparent), (<b>b</b>) Detection of NO in post NA and ACh stimulation (2.5min) (ECs and SMCs are apparent), (<b>c</b>) Syto 41 staining of nucleus of ECs and SMCs, (<b>d</b>) plot of fluorescence intensities of the ECs and SMCs (from carotid artery) measured with NA and ACh stimulation for 15min.</p

Detection of NO produced in explanted murine carotid arteries <i>ex</i><i>vivo</i> using Cu ₂FL2E (20 µM).

<p>(<b>a</b>) & (<b>b</b>) Magnified images of vessel showing basal NO signal detected after 5 min incubation of Cu ₂FL2E without any stimulus at medial and intimal focal planes, respectively. (<b>c</b>) NO signal detected in smooth muscle cells (SMCs) and (<b>d</b>) endothelial cells (ECs) of the tissue with 5 min incubation of Cu ₂FL2Eand, subsequently 45min incubation of H₂O₂ (150 µM). Scale bar is 50 µm, (<b>e</b>) & (<b>f</b>) Magnified images of vessel showing NO signal detected after 5 min incubation of Cu ₂FL2E and subsequently, 45 min incubation of H₂O₂ (150 µM) in SMCs at medial plane and in ECs at intimal plane respectively, (<b>g</b>) Quantification of spatial distribution of fluorescence intensity as measure of NO in cells of vessel wall stimulated with H₂O₂ (n = 5). (<b>h</b>) Quantification of spatial distribution of fluorescence intensity as measure of NO in cells of vessel wall stimulated with flow (flow rate= 2.1 Pa, time=45min), (n = 5).</p

Scheme of NO detection by Cu ₂FL2E in endothelial cells, showing the probe is trapped in the cell via hydrolysis of the pendant ester groups by intracellular esterases to give Cu ₂FL2A since the ester (E) is hydrolyzed to the acid (A).

<p>Scheme of NO detection by Cu ₂FL2E in endothelial cells, showing the probe is trapped in the cell via hydrolysis of the pendant ester groups by intracellular esterases to give Cu ₂FL2A since the ester (E) is hydrolyzed to the acid (A).</p

Detection of NO with Cu ₂FL2E produced by endothelial cells <i>in</i><i>vitro</i>.

<p>(<b>a</b>) NO detection in porcine aortic endothelial cells (PAECs); Left: 45 min incubation of Cu ₂FL2E (20 µM). Right: 45 min incubation of Cu ₂FL2E (20 µM) and H₂O₂ (150 µM). Top: bright-field images of cells. Bottom: fluorescence images of cells. Scale bar is50 µm. (<b>b</b>) Quantification of fluorescence intensity plotted against incubation time. (<b>c</b>) Detection of NO with Cu ₂FL2E in HCAECs cells, with or without NO-inhibitor (L-NAME). Shown are the fluorescence images after 45min co-incubation of the probe (Cu ₂FL2E =2 µM) with H₂O₂ (150 µM), L-NAME (100 µM), and/or ACh (10 µM) according to scheme. Scale bar is 75 µm. (<b>d</b>) Quantification of fluorescence intensity from (c) plotted against each condition mentioned in (c) (n = 5). Error bars indicate s.d.</p

Functional imaging of NO.

<p>(<b>a</b>) 3D reconstruction of vessels with Cu ₂FL2E (20 µM) without/ with stimulus (here ACh), (<b>b</b>) luminal diameter measured from arteries with conditions mentioned in (a), (<b>c</b>) normalized fluorescence intensities of the arteries with conditions mentioned in (a), (<b>d</b>) 3D reconstruction of vessels with Cu ₂FL2E without/ with stimulus (here ACh) and also in combination with L-NAME, (<b>e</b>) luminal diameter measured from arteries with conditions mentioned in (d), (<b>f</b>) normalized fluorescence intensities of the arteries with conditions mentioned in (c), error bars indicate s.d. (n=5).</p