Limits...
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.

Show MeSH

Related in: MedlinePlus

Endogenous adenosine signaling is required to prevent the osmotic swelling of Müller cells. Data were obtained in freshly isolated retinal slices (A) and Müller cells (B) from wild-type mice. Perfusion of the slices or cells with a hypoosmolar solution containing 1 mM barium chloride, 100 nM DPCPX, a A1 receptor blocker, 100 µM AOPCP, an ecto-5′-nucleotidase inhibitor, 10 µM clofilium, a potassium channel blocker, or 100 µM NPPB, a chloride channel blocker, resulted in a swelling of Müller cell somata. C: Original records of a dye-filled isolated Müller cell before (left) and during (right) perfusion with the barium-containing hypoosmolar solution. The arrowhead indicates the cell soma, and the arrow refers to the cell endfoot. The scale bar equals 5 µm. Data are mean (±SEM) soma areas (n=7–14 cells per bar) which were measured after a 4 min perfusion of the hypoosmolar solution. Data are expressed in percent of the soma size measured before hypotonic challenge (100%). Significant differences versus control (the asterisk indicates a p<0.05, the double asterisk indicates a p<0.01, and the triple asterisk indicates a p<0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2743807&req=5

f3: Endogenous adenosine signaling is required to prevent the osmotic swelling of Müller cells. Data were obtained in freshly isolated retinal slices (A) and Müller cells (B) from wild-type mice. Perfusion of the slices or cells with a hypoosmolar solution containing 1 mM barium chloride, 100 nM DPCPX, a A1 receptor blocker, 100 µM AOPCP, an ecto-5′-nucleotidase inhibitor, 10 µM clofilium, a potassium channel blocker, or 100 µM NPPB, a chloride channel blocker, resulted in a swelling of Müller cell somata. C: Original records of a dye-filled isolated Müller cell before (left) and during (right) perfusion with the barium-containing hypoosmolar solution. The arrowhead indicates the cell soma, and the arrow refers to the cell endfoot. The scale bar equals 5 µm. Data are mean (±SEM) soma areas (n=7–14 cells per bar) which were measured after a 4 min perfusion of the hypoosmolar solution. Data are expressed in percent of the soma size measured before hypotonic challenge (100%). Significant differences versus control (the asterisk indicates a p<0.05, the double asterisk indicates a p<0.01, and the triple asterisk indicates a p<0.001).

Mentions: We found that Müller cells in freshly isolated retinal slices from wild-type mice did not significantly swell within 4 min of hypotonic exposure (Figure 1B). To reveal whether endogenous adenosine signaling is required for this volume homeostasis of Müller cells, we recorded the osmotic swelling of Müller cells in retinal slices from wild-type animals in the presence of different pharmacological antagonists. As shown in Figure 3A, the presence of a selective inhibitor of A1 receptors resulted in a swelling of the Müller cell bodies under hypoosmotic conditions which was not observed in the absence of the blocking agent. In addition, pharmacological inhibition of the ecto-5′-nucleotidase (CD73) resulted in osmotic swelling of Müller cells (Figure 3A). CD73 is known to hydrolyze nucleoside monophosphates such as AMP to the respective nucleosides [34]; Müller cells are known to express CD73 [35,36]. The amplitude of soma swelling in the presence of the blocking agents was smaller than the amplitude of the barium-evoked swelling (Figure 3A). The data may suggest that endogenous adenosine signaling (involving extracellular generation of adenosine and activation of A1 receptors) is required to prevent osmotic swelling of Müller cells under hypoosmotic conditions. The swelling-inducing effects of the potassium and chloride channel blockers, respectively, clofilium and NPPB (Figure 3A), suggest that hypoosmotic swelling of Müller cells is normally prevented by opening of ion channels in the plasma membrane.


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)

Endogenous adenosine signaling is required to prevent the osmotic swelling of Müller cells. Data were obtained in freshly isolated retinal slices (A) and Müller cells (B) from wild-type mice. Perfusion of the slices or cells with a hypoosmolar solution containing 1 mM barium chloride, 100 nM DPCPX, a A1 receptor blocker, 100 µM AOPCP, an ecto-5′-nucleotidase inhibitor, 10 µM clofilium, a potassium channel blocker, or 100 µM NPPB, a chloride channel blocker, resulted in a swelling of Müller cell somata. C: Original records of a dye-filled isolated Müller cell before (left) and during (right) perfusion with the barium-containing hypoosmolar solution. The arrowhead indicates the cell soma, and the arrow refers to the cell endfoot. The scale bar equals 5 µm. Data are mean (±SEM) soma areas (n=7–14 cells per bar) which were measured after a 4 min perfusion of the hypoosmolar solution. Data are expressed in percent of the soma size measured before hypotonic challenge (100%). Significant differences versus control (the asterisk indicates a p<0.05, the double asterisk indicates a p<0.01, and the triple asterisk indicates a p<0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Endogenous adenosine signaling is required to prevent the osmotic swelling of Müller cells. Data were obtained in freshly isolated retinal slices (A) and Müller cells (B) from wild-type mice. Perfusion of the slices or cells with a hypoosmolar solution containing 1 mM barium chloride, 100 nM DPCPX, a A1 receptor blocker, 100 µM AOPCP, an ecto-5′-nucleotidase inhibitor, 10 µM clofilium, a potassium channel blocker, or 100 µM NPPB, a chloride channel blocker, resulted in a swelling of Müller cell somata. C: Original records of a dye-filled isolated Müller cell before (left) and during (right) perfusion with the barium-containing hypoosmolar solution. The arrowhead indicates the cell soma, and the arrow refers to the cell endfoot. The scale bar equals 5 µm. Data are mean (±SEM) soma areas (n=7–14 cells per bar) which were measured after a 4 min perfusion of the hypoosmolar solution. Data are expressed in percent of the soma size measured before hypotonic challenge (100%). Significant differences versus control (the asterisk indicates a p<0.05, the double asterisk indicates a p<0.01, and the triple asterisk indicates a p<0.001).
Mentions: We found that Müller cells in freshly isolated retinal slices from wild-type mice did not significantly swell within 4 min of hypotonic exposure (Figure 1B). To reveal whether endogenous adenosine signaling is required for this volume homeostasis of Müller cells, we recorded the osmotic swelling of Müller cells in retinal slices from wild-type animals in the presence of different pharmacological antagonists. As shown in Figure 3A, the presence of a selective inhibitor of A1 receptors resulted in a swelling of the Müller cell bodies under hypoosmotic conditions which was not observed in the absence of the blocking agent. In addition, pharmacological inhibition of the ecto-5′-nucleotidase (CD73) resulted in osmotic swelling of Müller cells (Figure 3A). CD73 is known to hydrolyze nucleoside monophosphates such as AMP to the respective nucleosides [34]; Müller cells are known to express CD73 [35,36]. The amplitude of soma swelling in the presence of the blocking agents was smaller than the amplitude of the barium-evoked swelling (Figure 3A). The data may suggest that endogenous adenosine signaling (involving extracellular generation of adenosine and activation of A1 receptors) is required to prevent osmotic swelling of Müller cells under hypoosmotic conditions. The swelling-inducing effects of the potassium and chloride channel blockers, respectively, clofilium and NPPB (Figure 3A), suggest that hypoosmotic swelling of Müller cells is normally prevented by opening of ion channels in the plasma membrane.

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