Cocaine binding to σ₁ receptor modulates the ERK 1/2 signaling in transfected cells.

<p>CHO cells were transfected with D₂ receptor cDNA (1 µg, black bars) or cotransfected (white bars) with D₂ receptor cDNA and σ₁ receptor siRNA (6.25 µg of oligonucleotides). Cells were incubated for 30 min (a) or 10 min (b) with medium (basal) or with 30 µM cocaine (a) or 1 µM quinpirole (b) in the absence or in the presence of 10 µM raclopride or 100 nM PD144418. In (<b>c</b>) cells were treated with medium (basal), 30 µM cocaine for 30 min, 1 µM quinpirole for 10 min or 30 µM cocaine for 30 min and, during the last 10 min, with 1 µM quinpirole. In all cases, ERK 1/2 phosphorylation is represented as percentage over basal levels (100%). Results are mean ± SEM of six to eight independent experiments performed in duplicate. Bifactorial ANOVA showed a significant (**p<0.01 and ***P<0.005) effect over basal.</p

ADA enhances CD83, CD80 and CD86 expression on iDCs.

<p>iDCs, obtained as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051287#s2" target="_blank">Materials and Methods</a>, were cultured for 48 h in medium in the absence (−ADA) or in the presence (+ADA) of 2 µM ADA or in the presence of maturating cocktail (mDCs). Expression of CD83 (A and B), CD80 (C and D) or CD86 (E and F) in the DCs gate was assessed by flow cytometry. In A, C and E, histogram overlays and the percentage of positive cells (A and B) or geometric mean (C) for a representative healthy donor and HIV-infected subject are shown. In B, D and F, values obtained from 16 to 19 healthy donors (circles) or 12 to 16 HIV-infected individuals (triangles) in the absence (open symbols) or in the presence (filled symbols) of 2 µM ADA are plotted. Each pair of linked symbols represents results from a particular individual. *<i>P</i><0.05; ***<i>P</i><0.001.</p

Functionality of dopamine D₄ receptors in pineal gland and pinealocytes.

<p>Pineal glands extracted at 9:00 h were treated for 10 min with increasing amounts of dopamine or with 1 µM of RO 10-5824 (RO). The immunoreactive bands, corresponding to ERK 1/2 (Thr¹⁸³-Tyr¹⁸⁵) phosphorylation (A) and Akt (Ser⁴⁷³) phosphorylation (B), of two separate experiments performed in duplicate were quantified and values represent the mean ± S.D. of the fold increase relative to basal levels found in untreated cells. Significant differences with respect to basal levels were determined by one-way ANOVA followed by a Dunnett's multiple comparison post hoc test (*<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001). A representative Western blot is shown at the top (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>). (C) Pinealocytes were isolated from pineal glands extracted at 9:00 h and were treated with medium (Control), 1 µM of RO 10-5824 (RO), 1 µM phenylephrine (Phenyl), or 1 µM isoproterenol (Iso) for 10 min before labeling with anti-S-arrestin (green) and anti-phospho-ERK1/2 (red), as indicated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>. Cell nuclei were stained with DAPI (blue). Scale bar, 5 µm.</p

Cross-antagonism between D₄ and α_1B or β₁ receptors in transfected cells and in pineal gland.

<p>In (A to D) CHO cells were transiently co-transfected with 2 µg of plasmid coding for D₄ receptors and with 3 µg of plasmid coding for α_1B receptors (A and B) or β₁ receptors (C and D). In (E and F) rat pineal glands were extracted at 9:00 h and processed as indicated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>. Cells were treated for 7 min and pineal glands were treated for 10 min with 500 nM of RO 10-5824 (RO), phenylephrine (Phenyl), or isoproterenol (Iso) or with 1 µM of L-745,870 (L-745), REC 15/2615 (REC), or CGP 20712 (CGP), alone or in combination. The immunoreactive bands, corresponding to ERK 1/2 (Thr¹⁸³-Tyr¹⁸⁵) phosphorylation (A, C, and E) and Akt (Ser⁴⁷³) phosphorylation (B, D, and F) of four experiments were quantified and values represent the mean ± S.E.M. of the fold increase with respect to basal levels found in untreated cells. Significant differences were calculated by a one-way ANOVA followed by post hoc Bonferroni's tests (***<i>p</i><0.001, as compared to the basal level; ^#<i>p</i><0.001, as compared to the sample treated with RO 10-5824; ^$<i>p</i><0.001, as compared to the sample treated with phenylephrine; ^&<i>p</i><0.001, as compared to the sample treated with isoproterenol). A representative Western blot is shown at the top of each panel.</p

Effect of ADA on the iDCs viability.

<p>iDCs, obtained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051287#s2" target="_blank">Materials and Methods</a>, from healthy or HIV-infected donors were cultured during 48 h in the absence (−ADA) or in the presence (+ADA) of 2 µM ADA. Cell viability was assessed through DIOC₆ and propidium iodide (PI) staining and measured by flow cytometry. In (A), contour plots showing the percentage of viable (bright DIOC₆ and negative propidium iodide staining), apoptotic (low DIOC₆ and negative propidium iodide staining) and necrotic (low DIOC₆ and positive propidium iodide staining) populations from a representative healthy or HIV-infected donor are shown. Percentage of viable (B), apoptotic (C) and necrotic (D) DCs from 7 different healthy and 6 HIV-infected donors are shown.*<i>P</i><0.05.</p

Negative cross-talk between cocaine and the D₂ receptor agonist quinpirole on ERK 1/2 phosphorylation in mice striatum.

<p>In (<b>a</b>) WT (black bars) and σ₁ receptor KO (white bars) mouse striatal slices were treated with 1 µM quinpirole for 10 min, with 150 µM cocaine for 30 min or with cocaine for 30 min and, during the last 10 min, with quinpirole. Immunoreactive bands from six slices obtained from five WT or five KO animals were quantified for each condition. Values represent mean ± SEM of percentage of phosphorylation relative to basal levels found in untreated slices. No significant differences were obtained between the basal levels of the WT and the σ₁ receptor KO mice. Bifactorial ANOVA showed a significant (*p<0.05, **p<0.01, ***p<0.005) effect over basal. One-way ANOVA followed by Bonferroni post hoc tests showed a significant cocaine-mediated counteraction of quinpirole (^&p<0.05, ^&&p<0.01). In (<b>b</b>) a representative scheme summarizing the overall results is shown. Top images represent D₂ and D₁ receptors signaling in the indirect and direct striatal pathway neurons after dopamine binding. Bottom images represent the effect of cocaine increasing the dopamine by inhibiting dopamine transporters (DAT) and interacting with σ₁ receptors within σ₁-D₂ and σ₁-D₁ receptor heteromers, changing the dopamine receptor signaling.</p

Molecular interaction between σ₁ receptors and D₂ receptors in living cells.

<p>BRET saturation experiments were performed with HEK-293T cells co-transfected with: (<b>a</b>) D₂-RLuc cDNA (0.4 µg, squares) or adenosine A_2A-RLuc cDNA as negative control (0.2 µg, triangles) and increasing amounts of σ₁-YFP cDNA (0.1 to 1 µg cDNA), (<b>b</b>) D₃-RLuc cDNA (0.5 µg, squares) or D₄-RLuc cDNA (0.5 µg, triangles) and increasing amounts of σ₁-YFP cDNA (0.1 to 1 µg cDNA). The relative amount of BRET acceptor is given as the ratio between the fluorescence of the acceptor minus the fluorescence detected in cells only expressing the donor, and the luciferase activity of the donor (YFP/Rluc). BRET data are expressed as means ± S.D. of five to six different experiments grouped as a function of the amount of BRET acceptor. In (<b>c</b>) confocal microscopy images of HEK-293T cells transfected with D₂-YFP or σ₁-RLuc (top panels) or co-transfected with D₂-YFP and σ₁-RLuc (bottom panels), treated (right images) or not (left images) with 30 µM cocaine for 30 min. σ₁ receptors (red) were identified by immunocytochemistry and D₂ receptors (green) were identified by its own fluorescence. Co-localization is shown in yellow. Scale bar:10 µm.</p

Clinical information of patients with HIV-1.

*<p>VL is expressed as log viral copies/ml.</p><p>NC: Non controller.</p><p>VC: Viremic Controller.</p><p>EC: Elite Controller.</p

ADA increases cytokines and chemokines secretion.

<p>iDCs, obtained as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051287#s2" target="_blank">Materials and Methods</a>, from 9 healthy (circles) and 8 HIV-infected (triangles) donors were cultured during 48 h in medium in the absence (iDCs) or in the presence of 2 µM ADA and the indicated cytokines and chemokines were determined in the supernatant as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051287#s2" target="_blank">Materials and Methods</a>. Values are expressed as the ratio (in-fold) of cytokine or chemokine levels obtained in the presence of ADA versus levels obtained in the absence of ADA (iDCs, the reference value of 1 is represented by a dotted line). For each group, the median is indicated by a thick line. *<i>P</i><0.05; **<i>P</i><0.01 with respect to iDCs.</p

Enzymatic and non-enzymatic activities are implicated on ADA-mediated effects.

<p>iDCs, obtained as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051287#s2" target="_blank">Materials and Methods</a>, from 4 to 8 healthy donors were cultured for 48 h in medium in the absence (iDC) or in the presence of 2 µM ADA (+ADA) or 2 µM HgCl₂ inactivated ADA (ADA-Hg) or iDCs were pre-incubated with the mAb anti-CD26 TA5.9 and incubated with 2 µM ADA (ADA+TA5.9). In (A) and (B), the expression of CD83 and CD80 was assessed in the DCs gate by flow cytometry. In C) the indicated cytokines and chemokines were determined in the supernatants after 48 h of cell culture. Each pair of linked symbols represents results from a particular individual. Results are expressed as the ratio (in-fold) of the values obtained in the presence of ADA, ADA-Hg or ADA+TA5.9 versus untreated cells (iDCs, the reference value of 1 is represented by a dotted line). *<i>P</i><0.05; **<i>P</i><0.01 with respect to iDCs.</p