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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

Immunocytochemical localization of the adrenomedullin (ADM) receptor calcitonin receptor like receptor (CRLR) and receptor activity modifying protein (RAMP2). A: The ADM receptor CRLR-like immunoreactivity (LI) was localized near the outer segments of photoreceptors (diagonal arrow), in faint somata in the outer nuclear layer (ONL; horizontal arrow), in puncta in the outer plexiform layer (OPL; asterisk), in select cell somata in the inner nuclear layer (INL; arrowhead), in delicate puncta in the inner plexiform layer (IPL), and in numerous somata in the ganglion cell layer (GCL; vertical arrow). B: RAMP2-LI was localized near the photoreceptor outer segments (diagonal arrow), in somata in the ONL (horizontal arrow), and in somata in the INL (arrowhead) and GCL (vertical arrow). Scale bars=20 µm. C: western blot of mouse retinal homogenate probed with the same CRLR and RAMP2 antisera used for immunocytochemistry. Both antisera recognized single proteins with the correct molecular weights.
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f4: Immunocytochemical localization of the adrenomedullin (ADM) receptor calcitonin receptor like receptor (CRLR) and receptor activity modifying protein (RAMP2). A: The ADM receptor CRLR-like immunoreactivity (LI) was localized near the outer segments of photoreceptors (diagonal arrow), in faint somata in the outer nuclear layer (ONL; horizontal arrow), in puncta in the outer plexiform layer (OPL; asterisk), in select cell somata in the inner nuclear layer (INL; arrowhead), in delicate puncta in the inner plexiform layer (IPL), and in numerous somata in the ganglion cell layer (GCL; vertical arrow). B: RAMP2-LI was localized near the photoreceptor outer segments (diagonal arrow), in somata in the ONL (horizontal arrow), and in somata in the INL (arrowhead) and GCL (vertical arrow). Scale bars=20 µm. C: western blot of mouse retinal homogenate probed with the same CRLR and RAMP2 antisera used for immunocytochemistry. Both antisera recognized single proteins with the correct molecular weights.

Mentions: We used RT–PCR to determine whether the primary ADM receptor, CRLR, and its associated receptor activity modifying proteins were expressed in the retina. We detected the expression of CRLR, RAMP2, and RAMP3 (Figure 1B). We then used immunocytochemistry to localize the ADM receptors in the retina. Because we were unable to find a reliable antiserum against RAMP3, we focused on CRLR and RAMP2. CRLR-LI was detected primarily in large somata in the GCL, in faint puncta in the OPL, in photoreceptor outer segments or the RPE, in photoreceptor inner segments, and in the cytoplasm of numerous faint somata in the ONL and the INL (Figures 4A and Figure 5A). We found RAMP2-LI was strongly localized in many somata in the INL and the GCL, in faint somata in the outer ONL, and in photoreceptor outer segments (Figure 4B). The RAMP2-LI in the INL was present in numerous somata that resembled amacrine cells by location and morphology. The specificity of the CRLR and RAMP2 antisera was confirmed using western blots that recognized proteins of the correct molecular weight (Figure 4C). Labeling in CRLR or RAMP2 knockout mice as a control for specificity was not possible as CRLR (CRLR−/−) and RAMP2 (RAMP2−/−) knockout mice are embryonic lethal due to vascular complications [26,27].


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

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

Immunocytochemical localization of the adrenomedullin (ADM) receptor calcitonin receptor like receptor (CRLR) and receptor activity modifying protein (RAMP2). A: The ADM receptor CRLR-like immunoreactivity (LI) was localized near the outer segments of photoreceptors (diagonal arrow), in faint somata in the outer nuclear layer (ONL; horizontal arrow), in puncta in the outer plexiform layer (OPL; asterisk), in select cell somata in the inner nuclear layer (INL; arrowhead), in delicate puncta in the inner plexiform layer (IPL), and in numerous somata in the ganglion cell layer (GCL; vertical arrow). B: RAMP2-LI was localized near the photoreceptor outer segments (diagonal arrow), in somata in the ONL (horizontal arrow), and in somata in the INL (arrowhead) and GCL (vertical arrow). Scale bars=20 µm. C: western blot of mouse retinal homogenate probed with the same CRLR and RAMP2 antisera used for immunocytochemistry. Both antisera recognized single proteins with the correct molecular weights.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369892&req=5

f4: Immunocytochemical localization of the adrenomedullin (ADM) receptor calcitonin receptor like receptor (CRLR) and receptor activity modifying protein (RAMP2). A: The ADM receptor CRLR-like immunoreactivity (LI) was localized near the outer segments of photoreceptors (diagonal arrow), in faint somata in the outer nuclear layer (ONL; horizontal arrow), in puncta in the outer plexiform layer (OPL; asterisk), in select cell somata in the inner nuclear layer (INL; arrowhead), in delicate puncta in the inner plexiform layer (IPL), and in numerous somata in the ganglion cell layer (GCL; vertical arrow). B: RAMP2-LI was localized near the photoreceptor outer segments (diagonal arrow), in somata in the ONL (horizontal arrow), and in somata in the INL (arrowhead) and GCL (vertical arrow). Scale bars=20 µm. C: western blot of mouse retinal homogenate probed with the same CRLR and RAMP2 antisera used for immunocytochemistry. Both antisera recognized single proteins with the correct molecular weights.
Mentions: We used RT–PCR to determine whether the primary ADM receptor, CRLR, and its associated receptor activity modifying proteins were expressed in the retina. We detected the expression of CRLR, RAMP2, and RAMP3 (Figure 1B). We then used immunocytochemistry to localize the ADM receptors in the retina. Because we were unable to find a reliable antiserum against RAMP3, we focused on CRLR and RAMP2. CRLR-LI was detected primarily in large somata in the GCL, in faint puncta in the OPL, in photoreceptor outer segments or the RPE, in photoreceptor inner segments, and in the cytoplasm of numerous faint somata in the ONL and the INL (Figures 4A and Figure 5A). We found RAMP2-LI was strongly localized in many somata in the INL and the GCL, in faint somata in the outer ONL, and in photoreceptor outer segments (Figure 4B). The RAMP2-LI in the INL was present in numerous somata that resembled amacrine cells by location and morphology. The specificity of the CRLR and RAMP2 antisera was confirmed using western blots that recognized proteins of the correct molecular weight (Figure 4C). Labeling in CRLR or RAMP2 knockout mice as a control for specificity was not possible as CRLR (CRLR−/−) and RAMP2 (RAMP2−/−) knockout mice are embryonic lethal due to vascular complications [26,27].

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