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Activation of Neuropeptide Y Receptors Modulates Retinal Ganglion Cell Physiology and Exerts Neuroprotective Actions In Vitro.

Martins J, Elvas F, Brudzewsky D, Martins T, Kolomiets B, Tralhão P, Gøtzsche CR, Cavadas C, Castelo-Branco M, Woldbye DP, Picaud S, Santiago AR, Ambrósio AF - ASN Neuro (2015)

Bottom Line: We found that NPY attenuated the increase in the [Ca2+]i triggered by glutamate mainly via Y1 receptor activation.Using in vitro cultures of rat retinal explants exposed to NMDA, we found that NPY pretreatment prevented NMDA-induced cell death.In conclusion, we found modulatory effects of NPY application that for the first time were detected at the level of RGCs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3004-548 Coimbra, Portugal CNC.IBILI, University of Coimbra, 3004-548 Coimbra, Portugal.

No MeSH data available.


Related in: MedlinePlus

NPY stimulates [35S]GTPγS functional binding in retinal sections. (a) Examples of retinal sections from autoradiography films using [35S]GTPγS-binding assay. (b) Quantification of [35S]GTPγS binding in inner retinal layers. Incubation with 10 µM NPY increased [35S]GTPγS binding in inner retinal layers (ab, black arrow) compared with basal binding (aa). (c) [35S]GTPγS binding signal (green) merged with hematoxylin-eosin staining (red). The green represents the [35S]GTPγS binding signal as in Figure 2ab, where the black was converted to green in order to be better visualized in the merged picture with hematoxylin-eosin staining which is in red. Note the increased signal in IPL (black arrow). An intense signal was found in the photoreceptor layer (white arrows) both under basal and NPY-stimulated binding conditions that may represent the high amount of G proteins in photoreceptor outer segments, mainly transducin. The blockade of Y1, Y2, and Y5 receptors prevented the increased [35S]GTPγS binding induced by NPY (ac). NSB refers to nonspecific binding, a competitive control with nonradioactive GTPγS (ad). Antagonists used: Y1, BIBO 3304 (10 µM); Y2, BIIE 0246 (10 µM); Y5, L-152,804 (100 µM). Bar: 2 mm. Data are presented as mean ± SEM of n = 2 to 10 independent experiments. *p < .05, compared with basal; ##p < .01, compared with 10 µM NPY. One-way analysis of variance followed by Bonferroni’s test.
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fig2-1759091415598292: NPY stimulates [35S]GTPγS functional binding in retinal sections. (a) Examples of retinal sections from autoradiography films using [35S]GTPγS-binding assay. (b) Quantification of [35S]GTPγS binding in inner retinal layers. Incubation with 10 µM NPY increased [35S]GTPγS binding in inner retinal layers (ab, black arrow) compared with basal binding (aa). (c) [35S]GTPγS binding signal (green) merged with hematoxylin-eosin staining (red). The green represents the [35S]GTPγS binding signal as in Figure 2ab, where the black was converted to green in order to be better visualized in the merged picture with hematoxylin-eosin staining which is in red. Note the increased signal in IPL (black arrow). An intense signal was found in the photoreceptor layer (white arrows) both under basal and NPY-stimulated binding conditions that may represent the high amount of G proteins in photoreceptor outer segments, mainly transducin. The blockade of Y1, Y2, and Y5 receptors prevented the increased [35S]GTPγS binding induced by NPY (ac). NSB refers to nonspecific binding, a competitive control with nonradioactive GTPγS (ad). Antagonists used: Y1, BIBO 3304 (10 µM); Y2, BIIE 0246 (10 µM); Y5, L-152,804 (100 µM). Bar: 2 mm. Data are presented as mean ± SEM of n = 2 to 10 independent experiments. *p < .05, compared with basal; ##p < .01, compared with 10 µM NPY. One-way analysis of variance followed by Bonferroni’s test.

Mentions: We performed [35S]GTPγS-binding assay (Christensen et al., 2006) to assess the functional activity of NPY receptors in retinal slices from adult Wistar rats (Figure 2). [35S]GTPγS-binding assay is able to evaluate the activation of GPCRs taking advantage of a radiolabeled nonhydrolyzable GTP analog—[35S]GTPγS. Upon activation of the GPCR by a receptor agonist, Gα binds [35S]GTPγS allowing the measurement of the amount of radiolabeled GTP bound to the cell membrane using an autoradiography film. After incubation with 10 µM NPY for 60 min (n = 10 animals), we detected an increase in [35S]GTPγS binding in inner retinal layers comparing to basal conditions (n = 10)—nonstimulated retinal slices; Figure 2(a) and (b). The small size of the rat retinal tissue and the resolution of autoradiography films made it difficult to identify the exact part of the inner retinal layers responsible for the increased NPY-stimulated [35S]GTPγS functional binding. However, when comparing the hematoxylin-eosin staining of native retinal slices with autoradiography film pictures of the same retinal slices, we found that the retinal layers displaying increased [35S]GTPγS binding upon NPY stimulation corresponded to the GCL and IPL (Figure 2(c)). It is also of note that an intense binding signal corresponding to the photoreceptor layer was detected both under basal and NPY-stimulated conditions (Figure 2(aa) and (ab), white arrows). This likely represents the high amount of G proteins in the photoreceptor outer segments, mainly transducin (Arshavsky et al., 2002). Moreover, NPY did not induce a statistically significant increase in binding signal in the photoreceptor layer (data not shown). Since 1 µM NPY (n = 2, not used for statistical tests) was not sufficient to increase [35S]GTPγS binding in retinal sections, we speculated that low levels of NPY receptors were functionally active in frozen retinal sections, thus requiring increased concentrations of NPY. A cocktail of NPY receptor antagonists (10 µM BIBO 3304—Y1 receptor antagonist; 10 µM BIIE 0246 for Y2; 100 µM L-152,804 for Y5) was used to evaluate the selectivity of NPY-stimulated [35S]GTPγS binding (Figure 2(ac) and (b)). The blockade of Y1, Y2, and Y5 receptors (n = 8) prevented the increased [35S]GTPγS binding induced by NPY, confirming the selectivity of the signal corresponding to the GCL and IPL. To assess the nonspecific binding, a competitive control with nonradioactive GTPγS was used, which exhibit no clear binding signal in the autoradiography film (Figure 2(ad)).Figure 2.


Activation of Neuropeptide Y Receptors Modulates Retinal Ganglion Cell Physiology and Exerts Neuroprotective Actions In Vitro.

Martins J, Elvas F, Brudzewsky D, Martins T, Kolomiets B, Tralhão P, Gøtzsche CR, Cavadas C, Castelo-Branco M, Woldbye DP, Picaud S, Santiago AR, Ambrósio AF - ASN Neuro (2015)

NPY stimulates [35S]GTPγS functional binding in retinal sections. (a) Examples of retinal sections from autoradiography films using [35S]GTPγS-binding assay. (b) Quantification of [35S]GTPγS binding in inner retinal layers. Incubation with 10 µM NPY increased [35S]GTPγS binding in inner retinal layers (ab, black arrow) compared with basal binding (aa). (c) [35S]GTPγS binding signal (green) merged with hematoxylin-eosin staining (red). The green represents the [35S]GTPγS binding signal as in Figure 2ab, where the black was converted to green in order to be better visualized in the merged picture with hematoxylin-eosin staining which is in red. Note the increased signal in IPL (black arrow). An intense signal was found in the photoreceptor layer (white arrows) both under basal and NPY-stimulated binding conditions that may represent the high amount of G proteins in photoreceptor outer segments, mainly transducin. The blockade of Y1, Y2, and Y5 receptors prevented the increased [35S]GTPγS binding induced by NPY (ac). NSB refers to nonspecific binding, a competitive control with nonradioactive GTPγS (ad). Antagonists used: Y1, BIBO 3304 (10 µM); Y2, BIIE 0246 (10 µM); Y5, L-152,804 (100 µM). Bar: 2 mm. Data are presented as mean ± SEM of n = 2 to 10 independent experiments. *p < .05, compared with basal; ##p < .01, compared with 10 µM NPY. One-way analysis of variance followed by Bonferroni’s test.
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fig2-1759091415598292: NPY stimulates [35S]GTPγS functional binding in retinal sections. (a) Examples of retinal sections from autoradiography films using [35S]GTPγS-binding assay. (b) Quantification of [35S]GTPγS binding in inner retinal layers. Incubation with 10 µM NPY increased [35S]GTPγS binding in inner retinal layers (ab, black arrow) compared with basal binding (aa). (c) [35S]GTPγS binding signal (green) merged with hematoxylin-eosin staining (red). The green represents the [35S]GTPγS binding signal as in Figure 2ab, where the black was converted to green in order to be better visualized in the merged picture with hematoxylin-eosin staining which is in red. Note the increased signal in IPL (black arrow). An intense signal was found in the photoreceptor layer (white arrows) both under basal and NPY-stimulated binding conditions that may represent the high amount of G proteins in photoreceptor outer segments, mainly transducin. The blockade of Y1, Y2, and Y5 receptors prevented the increased [35S]GTPγS binding induced by NPY (ac). NSB refers to nonspecific binding, a competitive control with nonradioactive GTPγS (ad). Antagonists used: Y1, BIBO 3304 (10 µM); Y2, BIIE 0246 (10 µM); Y5, L-152,804 (100 µM). Bar: 2 mm. Data are presented as mean ± SEM of n = 2 to 10 independent experiments. *p < .05, compared with basal; ##p < .01, compared with 10 µM NPY. One-way analysis of variance followed by Bonferroni’s test.
Mentions: We performed [35S]GTPγS-binding assay (Christensen et al., 2006) to assess the functional activity of NPY receptors in retinal slices from adult Wistar rats (Figure 2). [35S]GTPγS-binding assay is able to evaluate the activation of GPCRs taking advantage of a radiolabeled nonhydrolyzable GTP analog—[35S]GTPγS. Upon activation of the GPCR by a receptor agonist, Gα binds [35S]GTPγS allowing the measurement of the amount of radiolabeled GTP bound to the cell membrane using an autoradiography film. After incubation with 10 µM NPY for 60 min (n = 10 animals), we detected an increase in [35S]GTPγS binding in inner retinal layers comparing to basal conditions (n = 10)—nonstimulated retinal slices; Figure 2(a) and (b). The small size of the rat retinal tissue and the resolution of autoradiography films made it difficult to identify the exact part of the inner retinal layers responsible for the increased NPY-stimulated [35S]GTPγS functional binding. However, when comparing the hematoxylin-eosin staining of native retinal slices with autoradiography film pictures of the same retinal slices, we found that the retinal layers displaying increased [35S]GTPγS binding upon NPY stimulation corresponded to the GCL and IPL (Figure 2(c)). It is also of note that an intense binding signal corresponding to the photoreceptor layer was detected both under basal and NPY-stimulated conditions (Figure 2(aa) and (ab), white arrows). This likely represents the high amount of G proteins in the photoreceptor outer segments, mainly transducin (Arshavsky et al., 2002). Moreover, NPY did not induce a statistically significant increase in binding signal in the photoreceptor layer (data not shown). Since 1 µM NPY (n = 2, not used for statistical tests) was not sufficient to increase [35S]GTPγS binding in retinal sections, we speculated that low levels of NPY receptors were functionally active in frozen retinal sections, thus requiring increased concentrations of NPY. A cocktail of NPY receptor antagonists (10 µM BIBO 3304—Y1 receptor antagonist; 10 µM BIIE 0246 for Y2; 100 µM L-152,804 for Y5) was used to evaluate the selectivity of NPY-stimulated [35S]GTPγS binding (Figure 2(ac) and (b)). The blockade of Y1, Y2, and Y5 receptors (n = 8) prevented the increased [35S]GTPγS binding induced by NPY, confirming the selectivity of the signal corresponding to the GCL and IPL. To assess the nonspecific binding, a competitive control with nonradioactive GTPγS was used, which exhibit no clear binding signal in the autoradiography film (Figure 2(ad)).Figure 2.

Bottom Line: We found that NPY attenuated the increase in the [Ca2+]i triggered by glutamate mainly via Y1 receptor activation.Using in vitro cultures of rat retinal explants exposed to NMDA, we found that NPY pretreatment prevented NMDA-induced cell death.In conclusion, we found modulatory effects of NPY application that for the first time were detected at the level of RGCs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3004-548 Coimbra, Portugal CNC.IBILI, University of Coimbra, 3004-548 Coimbra, Portugal.

No MeSH data available.


Related in: MedlinePlus