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Osmotic pressure-adaptive responses in the eye tissues of rainbow smelt (Osmerus mordax).

Gendron RL, Armstrong E, Paradis H, Haines L, Desjardins M, Short CE, Clow KA, Driedzic WR - Mol. Vis. (2011)

Bottom Line: The effects that such massive changes in osmolarity have on both its visual system and its highly evolved and specialized circulation are not known.We propose a hypothesis that in a state of cold-induced hyperosmolarity, changes in ZO-1 expression are associated with the passage of small solutes from the plasma space to ocular fluid, while changes in Tbdn expression regulate the passage of proteins between the ocular fluid and plasma space.This work also provides fundamental insight into the mechanisms underlying the adaptation of the blood-retinal barrier to metabolically relevant compounds such as glycerol.

View Article: PubMed Central - PubMed

Affiliation: Division of BioMedical Sciences, Faculty of Medicine, Memorial University, St. John’s, NL, A1B 3V6, Canada. rgendron@mun.ca

ABSTRACT

Purpose: The rainbow smelt (Osmerus mordax), is a teleost fish, which avoids freezing by becoming virtually isosmotic with seawater. The effects that such massive changes in osmolarity have on both its visual system and its highly evolved and specialized circulation are not known. New knowledge about the osmotic adaptation of the rainbow smelt eye is highly relevant to the adaptation and survival of this species and to its ability to feed as a visual predator in the face of environmental pressures. Moreover, the molecular physiologic response of the smelt to osmotic stress might provide valuable insights into understanding and managing mammalian pathological hyperosmolarity conditions, such as diabetes. We undertook the present study to provide an initial assessment of gene expression in ocular vasculature during osmotic adaptation in rainbow smelt.

Methods: Immunohistochemistry with species cross reactive antibodies was used to assess blood vessel protein expression in paraffin sections. Western blotting was used to further verify antibody specificity for orthologs of mammalian blood vessel proteins in rainbow smelt. Thermal hysteresis and the analysis of glycerol concentrations in vitreous fluid were used to assess the physiologic adaptive properties of cold stressed eyes.

Results: Glycerol levels and osmotic pressure were significantly increased in the vitreal fluid of smelt maintained at <0.5 °C versus those maintained at 8-10 °C. Compared to the 8-10 °C adapted specimens, the rete mirabile blood vessels and connecting regions of the endothelial linings of the choroidal vessels of the <0.5 °C adapted specimens showed a higher expression level of Tubedown (Tbdn) protein, a marker of the endothelial transcellular permeability pathway. Expression of the zonula occludens protein ZO-1, a marker of the endothelial paracellular permeability pathway showed a reciprocal expression pattern and was downregulated in rete mirabile blood vessels and connecting regions in the endothelial linings of choroidal vessels in <0.5 °C adapted specimens. Smelt orthologs of the mammalian Tbdn and zoluna occludens protein 1 (ZO-1) proteins were also detected by western blotting using anti-mammalian antibodies raised against the same epitopes as those used for immunohistochemistry.

Conclusions: This work provides the first evidence that molecules known to play a role in ocular vascular homeostasis are expressed and may be differentially regulated during anti-freezing cold adaptation in smelt eyes. We propose a hypothesis that in a state of cold-induced hyperosmolarity, changes in ZO-1 expression are associated with the passage of small solutes from the plasma space to ocular fluid, while changes in Tbdn expression regulate the passage of proteins between the ocular fluid and plasma space. This work also provides fundamental insight into the mechanisms underlying the adaptation of the blood-retinal barrier to metabolically relevant compounds such as glycerol.

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Western blot analyses of smelt orthologs of mammalian Tbdn and ZO-1 proteins. A: A Tbdn western blot analysis showing a major band of approximately 103 kDa in smelt and 100 kDa in primate cells reacting with affinity purified rabbit anti-mouse Tbdn C10–20 antibody, which are completely competed out by the addition of the C10–20 Tbdn peptide, but not by a control peptide, as indicated. The bottom panel shows Tubulin as a protein loading and integrity control. B: A ZO-1 western blot analysis showing a doublet in primate and smelt cells reacting with the anti-ZO-1 antibody, as indicated. The bottom panel shows Tubulin as a protein loading and integrity control.
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f5: Western blot analyses of smelt orthologs of mammalian Tbdn and ZO-1 proteins. A: A Tbdn western blot analysis showing a major band of approximately 103 kDa in smelt and 100 kDa in primate cells reacting with affinity purified rabbit anti-mouse Tbdn C10–20 antibody, which are completely competed out by the addition of the C10–20 Tbdn peptide, but not by a control peptide, as indicated. The bottom panel shows Tubulin as a protein loading and integrity control. B: A ZO-1 western blot analysis showing a doublet in primate and smelt cells reacting with the anti-ZO-1 antibody, as indicated. The bottom panel shows Tubulin as a protein loading and integrity control.

Mentions: A teleost ortholog of the mouse Tbdn gene, the protein product of which represents a marker of the endothelial transcellular permeability pathway, exists [National Center for Biotechnology Information resources: Expressed sequence tags (EST): GE781036.1, EG915740.1, DY704791.1, DY734706.1, CX355128.1, and CX066490.1]. In addition, the mouse Tbdn peptide epitope (C10–20) against which we raised a Tbdn specific monoclonal antibody reagent (OE5) displays approximately 90% homology with salmonid and osmerus mordax expressed sequence tags (EST: CX355128.1, CX066490.1, EL547336 and EL536341.1). Moreover, our anti-Tbdn monoclonal antibody (OE5) stained retinal choriocapillaris and rete mirabile blood vessels in smelt eyes, and these have similar anatomic structures to those in which we observed Tbdn immunostaining in mammals [9–12] (Figure 4). To further confirm the specificity of mouse Tbdn peptide epitope C10–20 and the OE5 monoclonal antibody for a putative smelt Tbdn protein, western blot analyses on smelt retinal tissues were performed. Since our Tbdn monoclonal antibody OE5 is not useful for western blotting applications, our affinity purified rabbit anti-Tbdn antibody C10–20, which is raised against the same epitope as our OE5 antibody, was used. A western blot analysis of smelt retinal tissue using the anti-Tbdn antibody C10–20 revealed a major band of ~103 kDa (Figure 5). Moreover, the reactivity of the C10–20 Tbdn antibody with the 103 kDa protein present in smelt retinal tissues was competed away by the presence of the competing peptide Tbdn C10–20 as compared to a control peptide (Figure 5). Similar results were obtained with primate retinal endothelial cell line protein extracts (Figure 5). This data strongly suggests that smelt harbor a Tbdn ortholog. The slight difference in molecular weight between the smelt (103 kDa) and primate (100 kDa) Tbdn proteins might reflect their evolutionary divergence. Additional studies are required to clarify these differences. However, this is the first report of a putative smelt ortholog for the mammalian Tbdn protein.


Osmotic pressure-adaptive responses in the eye tissues of rainbow smelt (Osmerus mordax).

Gendron RL, Armstrong E, Paradis H, Haines L, Desjardins M, Short CE, Clow KA, Driedzic WR - Mol. Vis. (2011)

Western blot analyses of smelt orthologs of mammalian Tbdn and ZO-1 proteins. A: A Tbdn western blot analysis showing a major band of approximately 103 kDa in smelt and 100 kDa in primate cells reacting with affinity purified rabbit anti-mouse Tbdn C10–20 antibody, which are completely competed out by the addition of the C10–20 Tbdn peptide, but not by a control peptide, as indicated. The bottom panel shows Tubulin as a protein loading and integrity control. B: A ZO-1 western blot analysis showing a doublet in primate and smelt cells reacting with the anti-ZO-1 antibody, as indicated. The bottom panel shows Tubulin as a protein loading and integrity control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3198499&req=5

f5: Western blot analyses of smelt orthologs of mammalian Tbdn and ZO-1 proteins. A: A Tbdn western blot analysis showing a major band of approximately 103 kDa in smelt and 100 kDa in primate cells reacting with affinity purified rabbit anti-mouse Tbdn C10–20 antibody, which are completely competed out by the addition of the C10–20 Tbdn peptide, but not by a control peptide, as indicated. The bottom panel shows Tubulin as a protein loading and integrity control. B: A ZO-1 western blot analysis showing a doublet in primate and smelt cells reacting with the anti-ZO-1 antibody, as indicated. The bottom panel shows Tubulin as a protein loading and integrity control.
Mentions: A teleost ortholog of the mouse Tbdn gene, the protein product of which represents a marker of the endothelial transcellular permeability pathway, exists [National Center for Biotechnology Information resources: Expressed sequence tags (EST): GE781036.1, EG915740.1, DY704791.1, DY734706.1, CX355128.1, and CX066490.1]. In addition, the mouse Tbdn peptide epitope (C10–20) against which we raised a Tbdn specific monoclonal antibody reagent (OE5) displays approximately 90% homology with salmonid and osmerus mordax expressed sequence tags (EST: CX355128.1, CX066490.1, EL547336 and EL536341.1). Moreover, our anti-Tbdn monoclonal antibody (OE5) stained retinal choriocapillaris and rete mirabile blood vessels in smelt eyes, and these have similar anatomic structures to those in which we observed Tbdn immunostaining in mammals [9–12] (Figure 4). To further confirm the specificity of mouse Tbdn peptide epitope C10–20 and the OE5 monoclonal antibody for a putative smelt Tbdn protein, western blot analyses on smelt retinal tissues were performed. Since our Tbdn monoclonal antibody OE5 is not useful for western blotting applications, our affinity purified rabbit anti-Tbdn antibody C10–20, which is raised against the same epitope as our OE5 antibody, was used. A western blot analysis of smelt retinal tissue using the anti-Tbdn antibody C10–20 revealed a major band of ~103 kDa (Figure 5). Moreover, the reactivity of the C10–20 Tbdn antibody with the 103 kDa protein present in smelt retinal tissues was competed away by the presence of the competing peptide Tbdn C10–20 as compared to a control peptide (Figure 5). Similar results were obtained with primate retinal endothelial cell line protein extracts (Figure 5). This data strongly suggests that smelt harbor a Tbdn ortholog. The slight difference in molecular weight between the smelt (103 kDa) and primate (100 kDa) Tbdn proteins might reflect their evolutionary divergence. Additional studies are required to clarify these differences. However, this is the first report of a putative smelt ortholog for the mammalian Tbdn protein.

Bottom Line: The effects that such massive changes in osmolarity have on both its visual system and its highly evolved and specialized circulation are not known.We propose a hypothesis that in a state of cold-induced hyperosmolarity, changes in ZO-1 expression are associated with the passage of small solutes from the plasma space to ocular fluid, while changes in Tbdn expression regulate the passage of proteins between the ocular fluid and plasma space.This work also provides fundamental insight into the mechanisms underlying the adaptation of the blood-retinal barrier to metabolically relevant compounds such as glycerol.

View Article: PubMed Central - PubMed

Affiliation: Division of BioMedical Sciences, Faculty of Medicine, Memorial University, St. John’s, NL, A1B 3V6, Canada. rgendron@mun.ca

ABSTRACT

Purpose: The rainbow smelt (Osmerus mordax), is a teleost fish, which avoids freezing by becoming virtually isosmotic with seawater. The effects that such massive changes in osmolarity have on both its visual system and its highly evolved and specialized circulation are not known. New knowledge about the osmotic adaptation of the rainbow smelt eye is highly relevant to the adaptation and survival of this species and to its ability to feed as a visual predator in the face of environmental pressures. Moreover, the molecular physiologic response of the smelt to osmotic stress might provide valuable insights into understanding and managing mammalian pathological hyperosmolarity conditions, such as diabetes. We undertook the present study to provide an initial assessment of gene expression in ocular vasculature during osmotic adaptation in rainbow smelt.

Methods: Immunohistochemistry with species cross reactive antibodies was used to assess blood vessel protein expression in paraffin sections. Western blotting was used to further verify antibody specificity for orthologs of mammalian blood vessel proteins in rainbow smelt. Thermal hysteresis and the analysis of glycerol concentrations in vitreous fluid were used to assess the physiologic adaptive properties of cold stressed eyes.

Results: Glycerol levels and osmotic pressure were significantly increased in the vitreal fluid of smelt maintained at <0.5 °C versus those maintained at 8-10 °C. Compared to the 8-10 °C adapted specimens, the rete mirabile blood vessels and connecting regions of the endothelial linings of the choroidal vessels of the <0.5 °C adapted specimens showed a higher expression level of Tubedown (Tbdn) protein, a marker of the endothelial transcellular permeability pathway. Expression of the zonula occludens protein ZO-1, a marker of the endothelial paracellular permeability pathway showed a reciprocal expression pattern and was downregulated in rete mirabile blood vessels and connecting regions in the endothelial linings of choroidal vessels in <0.5 °C adapted specimens. Smelt orthologs of the mammalian Tbdn and zoluna occludens protein 1 (ZO-1) proteins were also detected by western blotting using anti-mammalian antibodies raised against the same epitopes as those used for immunohistochemistry.

Conclusions: This work provides the first evidence that molecules known to play a role in ocular vascular homeostasis are expressed and may be differentially regulated during anti-freezing cold adaptation in smelt eyes. We propose a hypothesis that in a state of cold-induced hyperosmolarity, changes in ZO-1 expression are associated with the passage of small solutes from the plasma space to ocular fluid, while changes in Tbdn expression regulate the passage of proteins between the ocular fluid and plasma space. This work also provides fundamental insight into the mechanisms underlying the adaptation of the blood-retinal barrier to metabolically relevant compounds such as glycerol.

Show MeSH
Related in: MedlinePlus