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Evidence for a functional adrenomedullin signaling pathway in the mouse retina.

Blom J, Giove TJ, Pong WW, Blute TA, Eldred WD - Mol. Vis. (2012)

Bottom Line: We found that calcitonin-receptor-like receptor and receptor activity modifying protein 2 had localization patterns similar to ADM, especially in somata in the inner nuclear and ganglion cell layers.These results are the first to show that ADM and functional ADM receptors are present in the retina.Since ADM is increased in eyes with ocular pathologies such as diabetic retinopathy, glaucoma, retinitis pigmentosa, and uveitis, the ADM signaling pathway may provide a new target for ameliorating these retinal pathologies.

View Article: PubMed Central - PubMed

Affiliation: Boston University, Laboratory of Visual Neurobiology, Department of Biology, Boston, MA 02215, USA.

ABSTRACT

Purpose: Adrenomedullin (ADM) is a small, secreted peptide often associated with vasodilation. However, ADM can also function as a neurotransmitter/neuromodulator, and studies suggest ADM is upregulated in the eye in several ocular diseases. However, no studies to date have described an ADM signaling pathway in the retina.

Methods: PCR, immunocytochemistry, nitric oxide imaging, western blots, and a nitrite assay were used to determine the localization of the components of the ADM signaling pathway in the mouse retina.

Results: We used reverse-transcriptase polymerase chain reaction to show that ADM and its primary receptor, calcitonin-receptor-like receptor, along with its associated receptor activity modifying proteins 2 and 3 are expressed in the retina. Using immunocytochemistry, we detected ADM staining throughout the retina in the photoreceptor outer segments, the outer nuclear layer, Müller and amacrine cell somata in the inner nuclear layer, and some somata in the ganglion cell layer. We found that calcitonin-receptor-like receptor and receptor activity modifying protein 2 had localization patterns similar to ADM, especially in somata in the inner nuclear and ganglion cell layers. Finally, we showed that the ADM receptor was functional in the retina. Stimulation of isolated retinas with ADM increased cyclic adenosine monophosphate- and cyclic guanosine monophosphate-like immunoreactivity, as well as nitric oxide production.

Conclusions: These results are the first to show that ADM and functional ADM receptors are present in the retina. Since ADM is increased in eyes with ocular pathologies such as diabetic retinopathy, glaucoma, retinitis pigmentosa, and uveitis, the ADM signaling pathway may provide a new target for ameliorating these retinal pathologies.

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Related in: MedlinePlus

Colocalization of adrenomedullin (ADM) with cell specific markers was done to accurately identify cell types with ADM-like immunoreactivity (LI). In all figures, ADM-LI was localized near the photoreceptor outer segments and in somata in the inner nuclear layer (INL) and the ganglion cell layer (GCL). A: Glutamine synthetase (GS, red) labeled Müller cells, and colocalized with ADM (green) in their somata in the INL (horizontal arrow). B: Protein kinase C α-like immunoreactivity (PKCα-LI; green) was present in rod bipolar cells and did not colocalize with ADM-LI (red). C: Calbindin-LI (green) was localized in horizontal cells in the INL and their process in the OPL, as well as in somata in the INL and GCL. ADM-LI (red) colocalized with calbindin-LI in somata in the lower tier of the INL that borders the IPL (vertical arrow) and in somata in the GCL (diagonal arrow). D: Calretinin-LI (green) was localized in amacrine cell somata in the INL, in processes in the inner plexiform layer (IPL), and within somata in the GCL. ADM-LI (red) did not colocalize with calretinin-LI. E: neuronal nitric oxide synthase (nNOS)-LI (green) was localized in select amacrine cell somata in the INL, in processes in the IPL, and in somata in the GCL. Some somata with nNOS-LI in the GCL colocalized with ADM-LI (red) (arrow). F: Glutamate-LI (red) was localized near the photoreceptor outer segments, in the OPL, in somata in the INL and the GCL, and as diffuse staining in the IPL. ADM-LI (green) colocalized with glutamate near the outer segments (arrow). G: Glycine-LI (red) was localized in the INL, in the OPL and the IPL, and in somata in the INL and the GCL. Glycine-LI was not colocalized with ADM-LI (green) in any somata, but there was some potential colocalization in puncta in the IPL (arrow). H: gamma-aminobutric acid (GABA)-LI (red) was localized to somata in the ONL, processes in the OPL, somata in the INL, diffusely in the IPL, and somata in the GCL. ADM-LI (green) did not colocalize with GABA-LI. Scale bars=20 µm.
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f3: Colocalization of adrenomedullin (ADM) with cell specific markers was done to accurately identify cell types with ADM-like immunoreactivity (LI). In all figures, ADM-LI was localized near the photoreceptor outer segments and in somata in the inner nuclear layer (INL) and the ganglion cell layer (GCL). A: Glutamine synthetase (GS, red) labeled Müller cells, and colocalized with ADM (green) in their somata in the INL (horizontal arrow). B: Protein kinase C α-like immunoreactivity (PKCα-LI; green) was present in rod bipolar cells and did not colocalize with ADM-LI (red). C: Calbindin-LI (green) was localized in horizontal cells in the INL and their process in the OPL, as well as in somata in the INL and GCL. ADM-LI (red) colocalized with calbindin-LI in somata in the lower tier of the INL that borders the IPL (vertical arrow) and in somata in the GCL (diagonal arrow). D: Calretinin-LI (green) was localized in amacrine cell somata in the INL, in processes in the inner plexiform layer (IPL), and within somata in the GCL. ADM-LI (red) did not colocalize with calretinin-LI. E: neuronal nitric oxide synthase (nNOS)-LI (green) was localized in select amacrine cell somata in the INL, in processes in the IPL, and in somata in the GCL. Some somata with nNOS-LI in the GCL colocalized with ADM-LI (red) (arrow). F: Glutamate-LI (red) was localized near the photoreceptor outer segments, in the OPL, in somata in the INL and the GCL, and as diffuse staining in the IPL. ADM-LI (green) colocalized with glutamate near the outer segments (arrow). G: Glycine-LI (red) was localized in the INL, in the OPL and the IPL, and in somata in the INL and the GCL. Glycine-LI was not colocalized with ADM-LI (green) in any somata, but there was some potential colocalization in puncta in the IPL (arrow). H: gamma-aminobutric acid (GABA)-LI (red) was localized to somata in the ONL, processes in the OPL, somata in the INL, diffusely in the IPL, and somata in the GCL. ADM-LI (green) did not colocalize with GABA-LI. Scale bars=20 µm.

Mentions: To accurately identify the specific cell types that were positive for ADM-LI, cross sections were double-labeled with ADM antiserum and cell specific markers. Colocalization of ADM-LI with the Müller cell marker glutamine synthetase (Figure 3A) demonstrated that the ADM-LI positive somata in the middle of the INL were Müller cells. The other cell somata in the middle of the INL were not rod bipolar cells as there was no colocalization of ADM-LI with the rod bipolar cell marker protein kinase Cα (Figure 3B). Additionally, ADM-LI was not localized in horizontal cells as demonstrated by the horizontal cell marker calbindin (Figure 3C). Although ADM-LI was not in horizontal cells, it was colocalized with calbindin-LI in somata in the INL on the border with the IPL, and within somata in the GCL, supporting that ADM-LI is in amacrine cells and either displaced amacrine or ganglion cells. ADM-LI positive cells did not colocalize with amacrine cells that were positive for calretinin-LI (Figure 3D) or neuronal nitric oxide synthase (nNOS)-LI (Figure 3E). However, some somata with nNOS-LI in the GCL did colocalize with ADM-LI. ADM-LI was colocalized with the excitatory neurotransmitter glutamate in the photoreceptor outer segments (Figure 3F), but not within the rest of the retina. ADM-LI did not colocalize with the inhibitory neurotransmitters glycine (Figure 3G) or gamma-aminobutyric acid (Figure 3H) in amacrine cell somata, but there was some potential colocalization of glycine in occasional puncta in the IPL.


Evidence for a functional adrenomedullin signaling pathway in the mouse retina.

Blom J, Giove TJ, Pong WW, Blute TA, Eldred WD - Mol. Vis. (2012)

Colocalization of adrenomedullin (ADM) with cell specific markers was done to accurately identify cell types with ADM-like immunoreactivity (LI). In all figures, ADM-LI was localized near the photoreceptor outer segments and in somata in the inner nuclear layer (INL) and the ganglion cell layer (GCL). A: Glutamine synthetase (GS, red) labeled Müller cells, and colocalized with ADM (green) in their somata in the INL (horizontal arrow). B: Protein kinase C α-like immunoreactivity (PKCα-LI; green) was present in rod bipolar cells and did not colocalize with ADM-LI (red). C: Calbindin-LI (green) was localized in horizontal cells in the INL and their process in the OPL, as well as in somata in the INL and GCL. ADM-LI (red) colocalized with calbindin-LI in somata in the lower tier of the INL that borders the IPL (vertical arrow) and in somata in the GCL (diagonal arrow). D: Calretinin-LI (green) was localized in amacrine cell somata in the INL, in processes in the inner plexiform layer (IPL), and within somata in the GCL. ADM-LI (red) did not colocalize with calretinin-LI. E: neuronal nitric oxide synthase (nNOS)-LI (green) was localized in select amacrine cell somata in the INL, in processes in the IPL, and in somata in the GCL. Some somata with nNOS-LI in the GCL colocalized with ADM-LI (red) (arrow). F: Glutamate-LI (red) was localized near the photoreceptor outer segments, in the OPL, in somata in the INL and the GCL, and as diffuse staining in the IPL. ADM-LI (green) colocalized with glutamate near the outer segments (arrow). G: Glycine-LI (red) was localized in the INL, in the OPL and the IPL, and in somata in the INL and the GCL. Glycine-LI was not colocalized with ADM-LI (green) in any somata, but there was some potential colocalization in puncta in the IPL (arrow). H: gamma-aminobutric acid (GABA)-LI (red) was localized to somata in the ONL, processes in the OPL, somata in the INL, diffusely in the IPL, and somata in the GCL. ADM-LI (green) did not colocalize with GABA-LI. Scale bars=20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f3: Colocalization of adrenomedullin (ADM) with cell specific markers was done to accurately identify cell types with ADM-like immunoreactivity (LI). In all figures, ADM-LI was localized near the photoreceptor outer segments and in somata in the inner nuclear layer (INL) and the ganglion cell layer (GCL). A: Glutamine synthetase (GS, red) labeled Müller cells, and colocalized with ADM (green) in their somata in the INL (horizontal arrow). B: Protein kinase C α-like immunoreactivity (PKCα-LI; green) was present in rod bipolar cells and did not colocalize with ADM-LI (red). C: Calbindin-LI (green) was localized in horizontal cells in the INL and their process in the OPL, as well as in somata in the INL and GCL. ADM-LI (red) colocalized with calbindin-LI in somata in the lower tier of the INL that borders the IPL (vertical arrow) and in somata in the GCL (diagonal arrow). D: Calretinin-LI (green) was localized in amacrine cell somata in the INL, in processes in the inner plexiform layer (IPL), and within somata in the GCL. ADM-LI (red) did not colocalize with calretinin-LI. E: neuronal nitric oxide synthase (nNOS)-LI (green) was localized in select amacrine cell somata in the INL, in processes in the IPL, and in somata in the GCL. Some somata with nNOS-LI in the GCL colocalized with ADM-LI (red) (arrow). F: Glutamate-LI (red) was localized near the photoreceptor outer segments, in the OPL, in somata in the INL and the GCL, and as diffuse staining in the IPL. ADM-LI (green) colocalized with glutamate near the outer segments (arrow). G: Glycine-LI (red) was localized in the INL, in the OPL and the IPL, and in somata in the INL and the GCL. Glycine-LI was not colocalized with ADM-LI (green) in any somata, but there was some potential colocalization in puncta in the IPL (arrow). H: gamma-aminobutric acid (GABA)-LI (red) was localized to somata in the ONL, processes in the OPL, somata in the INL, diffusely in the IPL, and somata in the GCL. ADM-LI (green) did not colocalize with GABA-LI. Scale bars=20 µm.
Mentions: To accurately identify the specific cell types that were positive for ADM-LI, cross sections were double-labeled with ADM antiserum and cell specific markers. Colocalization of ADM-LI with the Müller cell marker glutamine synthetase (Figure 3A) demonstrated that the ADM-LI positive somata in the middle of the INL were Müller cells. The other cell somata in the middle of the INL were not rod bipolar cells as there was no colocalization of ADM-LI with the rod bipolar cell marker protein kinase Cα (Figure 3B). Additionally, ADM-LI was not localized in horizontal cells as demonstrated by the horizontal cell marker calbindin (Figure 3C). Although ADM-LI was not in horizontal cells, it was colocalized with calbindin-LI in somata in the INL on the border with the IPL, and within somata in the GCL, supporting that ADM-LI is in amacrine cells and either displaced amacrine or ganglion cells. ADM-LI positive cells did not colocalize with amacrine cells that were positive for calretinin-LI (Figure 3D) or neuronal nitric oxide synthase (nNOS)-LI (Figure 3E). However, some somata with nNOS-LI in the GCL did colocalize with ADM-LI. ADM-LI was colocalized with the excitatory neurotransmitter glutamate in the photoreceptor outer segments (Figure 3F), but not within the rest of the retina. ADM-LI did not colocalize with the inhibitory neurotransmitters glycine (Figure 3G) or gamma-aminobutyric acid (Figure 3H) in amacrine cell somata, but there was some potential colocalization of glycine in occasional puncta in the IPL.

Bottom Line: We found that calcitonin-receptor-like receptor and receptor activity modifying protein 2 had localization patterns similar to ADM, especially in somata in the inner nuclear and ganglion cell layers.These results are the first to show that ADM and functional ADM receptors are present in the retina.Since ADM is increased in eyes with ocular pathologies such as diabetic retinopathy, glaucoma, retinitis pigmentosa, and uveitis, the ADM signaling pathway may provide a new target for ameliorating these retinal pathologies.

View Article: PubMed Central - PubMed

Affiliation: Boston University, Laboratory of Visual Neurobiology, Department of Biology, Boston, MA 02215, USA.

ABSTRACT

Purpose: Adrenomedullin (ADM) is a small, secreted peptide often associated with vasodilation. However, ADM can also function as a neurotransmitter/neuromodulator, and studies suggest ADM is upregulated in the eye in several ocular diseases. However, no studies to date have described an ADM signaling pathway in the retina.

Methods: PCR, immunocytochemistry, nitric oxide imaging, western blots, and a nitrite assay were used to determine the localization of the components of the ADM signaling pathway in the mouse retina.

Results: We used reverse-transcriptase polymerase chain reaction to show that ADM and its primary receptor, calcitonin-receptor-like receptor, along with its associated receptor activity modifying proteins 2 and 3 are expressed in the retina. Using immunocytochemistry, we detected ADM staining throughout the retina in the photoreceptor outer segments, the outer nuclear layer, Müller and amacrine cell somata in the inner nuclear layer, and some somata in the ganglion cell layer. We found that calcitonin-receptor-like receptor and receptor activity modifying protein 2 had localization patterns similar to ADM, especially in somata in the inner nuclear and ganglion cell layers. Finally, we showed that the ADM receptor was functional in the retina. Stimulation of isolated retinas with ADM increased cyclic adenosine monophosphate- and cyclic guanosine monophosphate-like immunoreactivity, as well as nitric oxide production.

Conclusions: These results are the first to show that ADM and functional ADM receptors are present in the retina. Since ADM is increased in eyes with ocular pathologies such as diabetic retinopathy, glaucoma, retinitis pigmentosa, and uveitis, the ADM signaling pathway may provide a new target for ameliorating these retinal pathologies.

Show MeSH
Related in: MedlinePlus