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Stimulation of an alpha1-adrenergic receptor downregulates ecto-5' nucleotidase activity on the apical membrane of RPE cells.

Reigada D, Zhang X, Crespo A, Nguyen J, Liu J, Pendrak K, Stone RA, Laties AM, Mitchell C - Purinergic Signal. (2006)

Bottom Line: HPLC measurements indicated norepinephrine reduced levels of adenosine in the bath.In conclusion, RPE cells express ecto-5' nucleotidase, with activity on the apical membrane, and stimulation of alpha-1 adrenergic receptors downregulates activity.As epinephrine is released at light onset, and adenosine can inhibit phagocytosis, the corresponding decrease in subretinal adenosine levels may contribute to the enhanced the phagocytosis of rod outer segments that occurs at this time.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA, 19104-6085, USA.

ABSTRACT
The purines ATP and adenosine play an important role in the communication between the photoreceptors and the retinal pigment epithelium (RPE). While the RPE is known to release ATP into subretinal space, the source of extracellular adenosine is unclear. In other tissues, ecto-nucleotidases mediate the consecutive dephosphorylation of ATP to AMP, and AMP is converted to adenosine by ecto-5' nucleotidase (CD73). This study identifies ecto-5' nucleotidase on RPE cells and investigates modulation of enzyme activity. The RPE was the most active site of 5'AMP dephosphorylation in the posterior rat eye. The ecto-5' nucleotidase inhibitor alphabetamADP prevented the production adenosine by the apical membrane of the bovine RPE. Cultured human ARPE-19 cells expressed mRNA and protein for ecto-5' nucleotidase. The production of phosphate from 5'AMP by ARPE-19 cells was inhibited by alphabetamADP, but the ecto-alkaline phosphatase inhibitor levamisole had no effect. Degradation of 5'AMP was blocked by norepinephrine, epinephrine and phenylephrine, with inhibition by antagonists prazosin and corynanthine implicating the alpha1 adrenergic receptor. The block of enzyme activity by norepinephrine was rapid, occurring within 1 min, and was similar at both 4 and 37 degrees C, consistent with cleavage of the enzyme from its GPI anchor. HPLC measurements indicated norepinephrine reduced levels of adenosine in the bath. In the apical face of the bovine-RPE eyecup, norepinephrine reduced the production of phosphate from 5'AMP, suggesting that both receptor and enzyme face sub-retinal space. In conclusion, RPE cells express ecto-5' nucleotidase, with activity on the apical membrane, and stimulation of alpha-1 adrenergic receptors downregulates activity. As epinephrine is released at light onset, and adenosine can inhibit phagocytosis, the corresponding decrease in subretinal adenosine levels may contribute to the enhanced the phagocytosis of rod outer segments that occurs at this time.

No MeSH data available.


Related in: MedlinePlus

Time and temperature dependence of inhibition. (A) Norepinephrine reduced phosphate production by >60% within the first minute of the reaction, with the reduction remaining throughout 60 min. Circles are 5′AMP alone and stars include 10 µM norepinephrine. *p < 0.05 vs 5′AMP for a given time point, n = 10–12. (B) The decrease in activity associated with 10 µM norepinephrine detected at the usual reaction temperature of 37°C was not affected by performing the reaction at 4°C. *p < 0.05 vs. 5′AMP alone, **not significantly different from Nor at 37°C, n = 14–16.
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Fig5: Time and temperature dependence of inhibition. (A) Norepinephrine reduced phosphate production by >60% within the first minute of the reaction, with the reduction remaining throughout 60 min. Circles are 5′AMP alone and stars include 10 µM norepinephrine. *p < 0.05 vs 5′AMP for a given time point, n = 10–12. (B) The decrease in activity associated with 10 µM norepinephrine detected at the usual reaction temperature of 37°C was not affected by performing the reaction at 4°C. *p < 0.05 vs. 5′AMP alone, **not significantly different from Nor at 37°C, n = 14–16.

Mentions: Phosphate production was determined using the PiPer Phosphate Assay Kit in basic accordance with the manufacturer’s instructions (Molecular Probes/Invitrogen, Eugene, OR). A standard curve constructed with 0 to 400 µM Pi from 20 µM increments indicated that absorbance at 565 nm provided an accurate measure of Pi present in the bath. ARPE-19 cells were grown to confluence in 24 well dishes, washed twice with 0.1 mM TRIS HCl pH 7.5, and incubated with working solution (control), or solution including AMP with or without drugs. All experiments were performed for 30 min at 37°C according to the manufacturer’s protocol except where indicated in Figure 5. α1-adrenergic antagonists were present 30 min before and during the reaction. To test the effect of temperature, all solutions and plates were precooled to 4°C for 30 min before the experiment began, with reaction itself also occurring at the appropriate temperature. After all incubations, 0.3 ml was drawn from each well for analysis. Absorbance of each sample was read at 565 nm using a spectrophotometer (Milton Roy, Ivyland, PA). In experiments involving levamisole, the phosphate signal was assessed on cells grown in 96 well plates with a Fluroskan Ascent fluorimeter (ThermoElectron, Milford, MA). All drugs were tested on the assay in the absence of cells and found to have no effect.Figure 5


Stimulation of an alpha1-adrenergic receptor downregulates ecto-5' nucleotidase activity on the apical membrane of RPE cells.

Reigada D, Zhang X, Crespo A, Nguyen J, Liu J, Pendrak K, Stone RA, Laties AM, Mitchell C - Purinergic Signal. (2006)

Time and temperature dependence of inhibition. (A) Norepinephrine reduced phosphate production by >60% within the first minute of the reaction, with the reduction remaining throughout 60 min. Circles are 5′AMP alone and stars include 10 µM norepinephrine. *p < 0.05 vs 5′AMP for a given time point, n = 10–12. (B) The decrease in activity associated with 10 µM norepinephrine detected at the usual reaction temperature of 37°C was not affected by performing the reaction at 4°C. *p < 0.05 vs. 5′AMP alone, **not significantly different from Nor at 37°C, n = 14–16.
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Related In: Results  -  Collection

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Fig5: Time and temperature dependence of inhibition. (A) Norepinephrine reduced phosphate production by >60% within the first minute of the reaction, with the reduction remaining throughout 60 min. Circles are 5′AMP alone and stars include 10 µM norepinephrine. *p < 0.05 vs 5′AMP for a given time point, n = 10–12. (B) The decrease in activity associated with 10 µM norepinephrine detected at the usual reaction temperature of 37°C was not affected by performing the reaction at 4°C. *p < 0.05 vs. 5′AMP alone, **not significantly different from Nor at 37°C, n = 14–16.
Mentions: Phosphate production was determined using the PiPer Phosphate Assay Kit in basic accordance with the manufacturer’s instructions (Molecular Probes/Invitrogen, Eugene, OR). A standard curve constructed with 0 to 400 µM Pi from 20 µM increments indicated that absorbance at 565 nm provided an accurate measure of Pi present in the bath. ARPE-19 cells were grown to confluence in 24 well dishes, washed twice with 0.1 mM TRIS HCl pH 7.5, and incubated with working solution (control), or solution including AMP with or without drugs. All experiments were performed for 30 min at 37°C according to the manufacturer’s protocol except where indicated in Figure 5. α1-adrenergic antagonists were present 30 min before and during the reaction. To test the effect of temperature, all solutions and plates were precooled to 4°C for 30 min before the experiment began, with reaction itself also occurring at the appropriate temperature. After all incubations, 0.3 ml was drawn from each well for analysis. Absorbance of each sample was read at 565 nm using a spectrophotometer (Milton Roy, Ivyland, PA). In experiments involving levamisole, the phosphate signal was assessed on cells grown in 96 well plates with a Fluroskan Ascent fluorimeter (ThermoElectron, Milford, MA). All drugs were tested on the assay in the absence of cells and found to have no effect.Figure 5

Bottom Line: HPLC measurements indicated norepinephrine reduced levels of adenosine in the bath.In conclusion, RPE cells express ecto-5' nucleotidase, with activity on the apical membrane, and stimulation of alpha-1 adrenergic receptors downregulates activity.As epinephrine is released at light onset, and adenosine can inhibit phagocytosis, the corresponding decrease in subretinal adenosine levels may contribute to the enhanced the phagocytosis of rod outer segments that occurs at this time.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA, 19104-6085, USA.

ABSTRACT
The purines ATP and adenosine play an important role in the communication between the photoreceptors and the retinal pigment epithelium (RPE). While the RPE is known to release ATP into subretinal space, the source of extracellular adenosine is unclear. In other tissues, ecto-nucleotidases mediate the consecutive dephosphorylation of ATP to AMP, and AMP is converted to adenosine by ecto-5' nucleotidase (CD73). This study identifies ecto-5' nucleotidase on RPE cells and investigates modulation of enzyme activity. The RPE was the most active site of 5'AMP dephosphorylation in the posterior rat eye. The ecto-5' nucleotidase inhibitor alphabetamADP prevented the production adenosine by the apical membrane of the bovine RPE. Cultured human ARPE-19 cells expressed mRNA and protein for ecto-5' nucleotidase. The production of phosphate from 5'AMP by ARPE-19 cells was inhibited by alphabetamADP, but the ecto-alkaline phosphatase inhibitor levamisole had no effect. Degradation of 5'AMP was blocked by norepinephrine, epinephrine and phenylephrine, with inhibition by antagonists prazosin and corynanthine implicating the alpha1 adrenergic receptor. The block of enzyme activity by norepinephrine was rapid, occurring within 1 min, and was similar at both 4 and 37 degrees C, consistent with cleavage of the enzyme from its GPI anchor. HPLC measurements indicated norepinephrine reduced levels of adenosine in the bath. In the apical face of the bovine-RPE eyecup, norepinephrine reduced the production of phosphate from 5'AMP, suggesting that both receptor and enzyme face sub-retinal space. In conclusion, RPE cells express ecto-5' nucleotidase, with activity on the apical membrane, and stimulation of alpha-1 adrenergic receptors downregulates activity. As epinephrine is released at light onset, and adenosine can inhibit phagocytosis, the corresponding decrease in subretinal adenosine levels may contribute to the enhanced the phagocytosis of rod outer segments that occurs at this time.

No MeSH data available.


Related in: MedlinePlus