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Involvement of A(1) adenosine receptors in osmotic volume regulation of retinal glial cells in mice.

Wurm A, Lipp S, Pannicke T, Linnertz R, Färber K, Wiedemann P, Reichenbach A, Bringmann A - Mol. Vis. (2009)

Bottom Line: The size of Müller cell somata was recorded before and during perfusion of retinal sections and isolated Müller cells with a hypoosmolar solution.The data suggest that autocrine activation of A(1) receptors by extracellularly generated adenosine mediates the volume homeostasis of Müller cells in the murine retina.The swelling-inhibitory effect of triamcinolone is mediated by enhancement of endogenous adenosine signaling.

View Article: PubMed Central - PubMed

Affiliation: Paul Flechsig Institute of Brain Research, University of Leipzig, D-04103 Leipzig, Germany.

ABSTRACT

Purpose: Osmotic swelling of Müller glial cells has been suggested to contribute to retinal edema. We determined the role of adenosine signaling in the inhibition of Müller cell swelling in the murine retina.

Methods: The size of Müller cell somata was recorded before and during perfusion of retinal sections and isolated Müller cells with a hypoosmolar solution. Retinal tissues were freshly isolated from wild-type mice and mice deficient in A(1) adenosine receptors (A(1)AR(-/-)), or cultured as whole-mounts for three days. The potassium conductance of Müller cells was recorded in isolated cells, and retinal slices were immunostained against Kir4.1.

Results: Hypotonic exposure for 4 min induced a swelling of Müller cell bodies in retinal slices from A(1)AR(-/-) mice but not wild-type mice. Pharmacological inhibition of A(1) receptors or of the ecto-5'-nucleotidase induced hypoosmotic swelling of Müller cells from wild-type mice. Exogenous adenosine prevented the swelling of Müller cells from wild-type but not A(1)AR(-/-) mice. The antiinflammatory corticosteroid, triamcinolone acetonide, inhibited the swelling of Müller cells from wild-type mice; this effect was blocked by an antagonist of A(1) receptors. The potassium conductance of Müller cells and the Kir4.1 immunolabeling of retinal slices were not different between A(1)AR(-/-) and wild-type mice, both in freshly isolated tissues and retinal organ cultures.

Conclusions: The data suggest that autocrine activation of A(1) receptors by extracellularly generated adenosine mediates the volume homeostasis of Müller cells in the murine retina. The swelling-inhibitory effect of triamcinolone is mediated by enhancement of endogenous adenosine signaling.

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Organ culturing alters the potassium conductance of Müller glial cells and the retinal immunolabeling of Kir4.1 protein. Retinal tissues from wild-type (Wt) and A1AR−/− mice were used. The tissues were freshly isolated or derived from retinal organ cultures. A: Shown are examples of original records of whole-cell potassium currents, which were obtained in isolated Müller cells. Outward (upwardly depicted) and inward (downwardly depicted) currents were evoked by 20 mV incremental voltage steps up to +20 and −180 mV from a holding potential of −80 mV. The lines at left of each trace indicate zero current levels. B: Retinal organ culturing results in a decrease of the mean amplitude of the Kir currents of Müller cells. C: Retinal organ culturing results in a slight decrease in the mean resting membrane potential of Müller cells from A1AR−/− mice. D: Retinal organ culturing results in a decrease in the intensity of the Kir4.1 immunoreactivity. The arrows indicate perivascular staining, and the arrowheads point to the limiting membranes of the retina. Abbreviations: ganglion cell layer (GCL); inner nuclear layer (INL); inner plexiform layer (IPL); outer nuclear layer (ONL); outer plexiform layer (OPL); photoreceptor segments (PRS). Scale bars equal to 20 µm. The bar diagrams display mean (±SD) values obtained in 6–22 cells. Significant differences versus the respective control obtained in freshly isolated cells (the double closed circles indicate a p<0.01 and the triple closed circles indicate a p<0.001).
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f5: Organ culturing alters the potassium conductance of Müller glial cells and the retinal immunolabeling of Kir4.1 protein. Retinal tissues from wild-type (Wt) and A1AR−/− mice were used. The tissues were freshly isolated or derived from retinal organ cultures. A: Shown are examples of original records of whole-cell potassium currents, which were obtained in isolated Müller cells. Outward (upwardly depicted) and inward (downwardly depicted) currents were evoked by 20 mV incremental voltage steps up to +20 and −180 mV from a holding potential of −80 mV. The lines at left of each trace indicate zero current levels. B: Retinal organ culturing results in a decrease of the mean amplitude of the Kir currents of Müller cells. C: Retinal organ culturing results in a slight decrease in the mean resting membrane potential of Müller cells from A1AR−/− mice. D: Retinal organ culturing results in a decrease in the intensity of the Kir4.1 immunoreactivity. The arrows indicate perivascular staining, and the arrowheads point to the limiting membranes of the retina. Abbreviations: ganglion cell layer (GCL); inner nuclear layer (INL); inner plexiform layer (IPL); outer nuclear layer (ONL); outer plexiform layer (OPL); photoreceptor segments (PRS). Scale bars equal to 20 µm. The bar diagrams display mean (±SD) values obtained in 6–22 cells. Significant differences versus the respective control obtained in freshly isolated cells (the double closed circles indicate a p<0.01 and the triple closed circles indicate a p<0.001).

Mentions: We found that Kir channel-blocking barium ions induced a swelling of Müller cell bodies in freshly isolated retinal slices of wild-type mice (Figures 1A,B). Because Müller cells in slices from A1AR−/− mice displayed an alteration in the osmotic swelling characteristics when compared to cells from wild-type mice, we determined whether this alteration was associated with a decrease in the Kir currents of the cells. As shown in Figure 5A, freshly isolated Müller cells of both wild-type and A1AR−/− mice displayed large potassium currents around the resting membrane potential (at the zero current level) and upon membrane hyperpolarization. The mean amplitude of the Kir currents (Figure 5B) and the resting membrane potential (Figure 5C) were not different between freshly isolated cells from both strains. Furthermore, the mean membrane capacitance which is proportional to the plasma membrane area of the cells did not differ between freshly isolated cells from both mouse strains (data not shown), disclosing the possibility of cellular hypertrophy of cells from the knockout mice.


Involvement of A(1) adenosine receptors in osmotic volume regulation of retinal glial cells in mice.

Wurm A, Lipp S, Pannicke T, Linnertz R, Färber K, Wiedemann P, Reichenbach A, Bringmann A - Mol. Vis. (2009)

Organ culturing alters the potassium conductance of Müller glial cells and the retinal immunolabeling of Kir4.1 protein. Retinal tissues from wild-type (Wt) and A1AR−/− mice were used. The tissues were freshly isolated or derived from retinal organ cultures. A: Shown are examples of original records of whole-cell potassium currents, which were obtained in isolated Müller cells. Outward (upwardly depicted) and inward (downwardly depicted) currents were evoked by 20 mV incremental voltage steps up to +20 and −180 mV from a holding potential of −80 mV. The lines at left of each trace indicate zero current levels. B: Retinal organ culturing results in a decrease of the mean amplitude of the Kir currents of Müller cells. C: Retinal organ culturing results in a slight decrease in the mean resting membrane potential of Müller cells from A1AR−/− mice. D: Retinal organ culturing results in a decrease in the intensity of the Kir4.1 immunoreactivity. The arrows indicate perivascular staining, and the arrowheads point to the limiting membranes of the retina. Abbreviations: ganglion cell layer (GCL); inner nuclear layer (INL); inner plexiform layer (IPL); outer nuclear layer (ONL); outer plexiform layer (OPL); photoreceptor segments (PRS). Scale bars equal to 20 µm. The bar diagrams display mean (±SD) values obtained in 6–22 cells. Significant differences versus the respective control obtained in freshly isolated cells (the double closed circles indicate a p<0.01 and the triple closed circles indicate a p<0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2743807&req=5

f5: Organ culturing alters the potassium conductance of Müller glial cells and the retinal immunolabeling of Kir4.1 protein. Retinal tissues from wild-type (Wt) and A1AR−/− mice were used. The tissues were freshly isolated or derived from retinal organ cultures. A: Shown are examples of original records of whole-cell potassium currents, which were obtained in isolated Müller cells. Outward (upwardly depicted) and inward (downwardly depicted) currents were evoked by 20 mV incremental voltage steps up to +20 and −180 mV from a holding potential of −80 mV. The lines at left of each trace indicate zero current levels. B: Retinal organ culturing results in a decrease of the mean amplitude of the Kir currents of Müller cells. C: Retinal organ culturing results in a slight decrease in the mean resting membrane potential of Müller cells from A1AR−/− mice. D: Retinal organ culturing results in a decrease in the intensity of the Kir4.1 immunoreactivity. The arrows indicate perivascular staining, and the arrowheads point to the limiting membranes of the retina. Abbreviations: ganglion cell layer (GCL); inner nuclear layer (INL); inner plexiform layer (IPL); outer nuclear layer (ONL); outer plexiform layer (OPL); photoreceptor segments (PRS). Scale bars equal to 20 µm. The bar diagrams display mean (±SD) values obtained in 6–22 cells. Significant differences versus the respective control obtained in freshly isolated cells (the double closed circles indicate a p<0.01 and the triple closed circles indicate a p<0.001).
Mentions: We found that Kir channel-blocking barium ions induced a swelling of Müller cell bodies in freshly isolated retinal slices of wild-type mice (Figures 1A,B). Because Müller cells in slices from A1AR−/− mice displayed an alteration in the osmotic swelling characteristics when compared to cells from wild-type mice, we determined whether this alteration was associated with a decrease in the Kir currents of the cells. As shown in Figure 5A, freshly isolated Müller cells of both wild-type and A1AR−/− mice displayed large potassium currents around the resting membrane potential (at the zero current level) and upon membrane hyperpolarization. The mean amplitude of the Kir currents (Figure 5B) and the resting membrane potential (Figure 5C) were not different between freshly isolated cells from both strains. Furthermore, the mean membrane capacitance which is proportional to the plasma membrane area of the cells did not differ between freshly isolated cells from both mouse strains (data not shown), disclosing the possibility of cellular hypertrophy of cells from the knockout mice.

Bottom Line: The size of Müller cell somata was recorded before and during perfusion of retinal sections and isolated Müller cells with a hypoosmolar solution.The data suggest that autocrine activation of A(1) receptors by extracellularly generated adenosine mediates the volume homeostasis of Müller cells in the murine retina.The swelling-inhibitory effect of triamcinolone is mediated by enhancement of endogenous adenosine signaling.

View Article: PubMed Central - PubMed

Affiliation: Paul Flechsig Institute of Brain Research, University of Leipzig, D-04103 Leipzig, Germany.

ABSTRACT

Purpose: Osmotic swelling of Müller glial cells has been suggested to contribute to retinal edema. We determined the role of adenosine signaling in the inhibition of Müller cell swelling in the murine retina.

Methods: The size of Müller cell somata was recorded before and during perfusion of retinal sections and isolated Müller cells with a hypoosmolar solution. Retinal tissues were freshly isolated from wild-type mice and mice deficient in A(1) adenosine receptors (A(1)AR(-/-)), or cultured as whole-mounts for three days. The potassium conductance of Müller cells was recorded in isolated cells, and retinal slices were immunostained against Kir4.1.

Results: Hypotonic exposure for 4 min induced a swelling of Müller cell bodies in retinal slices from A(1)AR(-/-) mice but not wild-type mice. Pharmacological inhibition of A(1) receptors or of the ecto-5'-nucleotidase induced hypoosmotic swelling of Müller cells from wild-type mice. Exogenous adenosine prevented the swelling of Müller cells from wild-type but not A(1)AR(-/-) mice. The antiinflammatory corticosteroid, triamcinolone acetonide, inhibited the swelling of Müller cells from wild-type mice; this effect was blocked by an antagonist of A(1) receptors. The potassium conductance of Müller cells and the Kir4.1 immunolabeling of retinal slices were not different between A(1)AR(-/-) and wild-type mice, both in freshly isolated tissues and retinal organ cultures.

Conclusions: The data suggest that autocrine activation of A(1) receptors by extracellularly generated adenosine mediates the volume homeostasis of Müller cells in the murine retina. The swelling-inhibitory effect of triamcinolone is mediated by enhancement of endogenous adenosine signaling.

Show MeSH
Related in: MedlinePlus