Endothelin-1 Synthesis and Secretion in Human Retinal Pigment Epithelial Cells (ARPE-19)

Differential Regulation by Cholinergics and TNF-α

Santosh Narayan, Ganesh Prasanna, Raghu Krishnamoorthy, Xinyu Zhang, Thomas Yorio

Research output: Contribution to journalArticleResearchpeer-review

48 Citations (Scopus)

Abstract

PURPOSE. Endothelin (ET)-1 can produce nerve damage analogous to that in optic neuropathies such as glaucoma. The precise source of endothelin in the posterior segment of the eye remains unclear. The present study was conducted to determine whether the retinal pigment epithelium (RPE), which helps maintain the outer blood-retinal barrier, is a local source for ET-1 and whether the amount of ET-1 secreted by the RPE is be differentially regulated by cholinergics and the cytokine TNF-α. METHODS. Human retinal pigment epithelial cells (ARPE-19) were cultured either to a mature state (mRPE) for 4 weeks, with well-defined tight junctions, or to a young state (yRPE) for 4 days, with incompletely formed tight junctions. ET-1-like immunoreactivity was determined by immunocytochemistry, and secreted ET-1 was quantified by radioimmunoassay in both cell types. Cells were stimulated with the cholinergic agonist carbachol or with the cytokine TNF-α for specific periods. The expression of muscarinic receptor subtypes M1 and M3 and the peripheral membrane protein ZO-1 were analyzed by immunoblot and immunocytochemistry, respectively. Expression of preproendothelin-1 (ppET-1) mRNA after application of different stimuli at specific time points was determined by real-time RT-PCR. Carbachol-mediated elevation in intracellular calcium ([Ca2+]i) was also measured in the presence or absence of a selective muscarinic receptor antagonist. RESULTS. Constitutive synthesis and secretion of ET-1 was greater in mRPE than in yRPE cells. TNF-α caused a significant increase in ppET-1 mRNA levels and ET-1 secretion in both phenotypes. The disruption and subsequent breakdown of the tight junction barrier was evident in either phenotype after treatments with TNF-α. There was a concentration-dependent increase in [Ca 2+]i in both y- and mRPE cells in response to CCh. CCh at 1 μM significantly increased ET-1 secretion, a response observed in yRPE but not in mRPE cells. This effect may be mediated primarily by the M3 receptor subtype and is phospholipase C (PLC) dependent. CONCLUSIONS. Regulation of ET-1 release in ARPE-19 cells was differentially regulated by TNF-α and CCh and was dependent on the age of the culture. RPE may be a source for ET-1 in the retina, and its increased release may become more important during breakdown of the blood-retinal barrier, as seen after TNF-α treatment.

Original languageEnglish
Pages (from-to)4885-4894
Number of pages10
JournalInvestigative Ophthalmology and Visual Science
Volume44
Issue number11
DOIs
StatePublished - 1 Nov 2003

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Retinal Pigments
Endothelin-1
Cholinergic Agents
Epithelial Cells
Retinal Pigment Epithelium
Tight Junctions
Blood-Retinal Barrier
Carbachol
Posterior Eye Segment
Immunohistochemistry
Muscarinic M3 Receptors
Muscarinic M1 Receptors
Cytokines
Phenotype
Cholinergic Agonists
Optic Nerve Diseases
Messenger RNA
Muscarinic Antagonists
Endothelins
Type C Phospholipases

Cite this

@article{5dc22024282f40b1b528a3a3f7edc538,
title = "Endothelin-1 Synthesis and Secretion in Human Retinal Pigment Epithelial Cells (ARPE-19): Differential Regulation by Cholinergics and TNF-α",
abstract = "PURPOSE. Endothelin (ET)-1 can produce nerve damage analogous to that in optic neuropathies such as glaucoma. The precise source of endothelin in the posterior segment of the eye remains unclear. The present study was conducted to determine whether the retinal pigment epithelium (RPE), which helps maintain the outer blood-retinal barrier, is a local source for ET-1 and whether the amount of ET-1 secreted by the RPE is be differentially regulated by cholinergics and the cytokine TNF-α. METHODS. Human retinal pigment epithelial cells (ARPE-19) were cultured either to a mature state (mRPE) for 4 weeks, with well-defined tight junctions, or to a young state (yRPE) for 4 days, with incompletely formed tight junctions. ET-1-like immunoreactivity was determined by immunocytochemistry, and secreted ET-1 was quantified by radioimmunoassay in both cell types. Cells were stimulated with the cholinergic agonist carbachol or with the cytokine TNF-α for specific periods. The expression of muscarinic receptor subtypes M1 and M3 and the peripheral membrane protein ZO-1 were analyzed by immunoblot and immunocytochemistry, respectively. Expression of preproendothelin-1 (ppET-1) mRNA after application of different stimuli at specific time points was determined by real-time RT-PCR. Carbachol-mediated elevation in intracellular calcium ([Ca2+]i) was also measured in the presence or absence of a selective muscarinic receptor antagonist. RESULTS. Constitutive synthesis and secretion of ET-1 was greater in mRPE than in yRPE cells. TNF-α caused a significant increase in ppET-1 mRNA levels and ET-1 secretion in both phenotypes. The disruption and subsequent breakdown of the tight junction barrier was evident in either phenotype after treatments with TNF-α. There was a concentration-dependent increase in [Ca 2+]i in both y- and mRPE cells in response to CCh. CCh at 1 μM significantly increased ET-1 secretion, a response observed in yRPE but not in mRPE cells. This effect may be mediated primarily by the M3 receptor subtype and is phospholipase C (PLC) dependent. CONCLUSIONS. Regulation of ET-1 release in ARPE-19 cells was differentially regulated by TNF-α and CCh and was dependent on the age of the culture. RPE may be a source for ET-1 in the retina, and its increased release may become more important during breakdown of the blood-retinal barrier, as seen after TNF-α treatment.",
author = "Santosh Narayan and Ganesh Prasanna and Raghu Krishnamoorthy and Xinyu Zhang and Thomas Yorio",
year = "2003",
month = "11",
day = "1",
doi = "10.1167/iovs.03-0387",
language = "English",
volume = "44",
pages = "4885--4894",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
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number = "11",

}

Endothelin-1 Synthesis and Secretion in Human Retinal Pigment Epithelial Cells (ARPE-19) : Differential Regulation by Cholinergics and TNF-α. / Narayan, Santosh; Prasanna, Ganesh; Krishnamoorthy, Raghu; Zhang, Xinyu; Yorio, Thomas.

In: Investigative Ophthalmology and Visual Science, Vol. 44, No. 11, 01.11.2003, p. 4885-4894.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Endothelin-1 Synthesis and Secretion in Human Retinal Pigment Epithelial Cells (ARPE-19)

T2 - Differential Regulation by Cholinergics and TNF-α

AU - Narayan, Santosh

AU - Prasanna, Ganesh

AU - Krishnamoorthy, Raghu

AU - Zhang, Xinyu

AU - Yorio, Thomas

PY - 2003/11/1

Y1 - 2003/11/1

N2 - PURPOSE. Endothelin (ET)-1 can produce nerve damage analogous to that in optic neuropathies such as glaucoma. The precise source of endothelin in the posterior segment of the eye remains unclear. The present study was conducted to determine whether the retinal pigment epithelium (RPE), which helps maintain the outer blood-retinal barrier, is a local source for ET-1 and whether the amount of ET-1 secreted by the RPE is be differentially regulated by cholinergics and the cytokine TNF-α. METHODS. Human retinal pigment epithelial cells (ARPE-19) were cultured either to a mature state (mRPE) for 4 weeks, with well-defined tight junctions, or to a young state (yRPE) for 4 days, with incompletely formed tight junctions. ET-1-like immunoreactivity was determined by immunocytochemistry, and secreted ET-1 was quantified by radioimmunoassay in both cell types. Cells were stimulated with the cholinergic agonist carbachol or with the cytokine TNF-α for specific periods. The expression of muscarinic receptor subtypes M1 and M3 and the peripheral membrane protein ZO-1 were analyzed by immunoblot and immunocytochemistry, respectively. Expression of preproendothelin-1 (ppET-1) mRNA after application of different stimuli at specific time points was determined by real-time RT-PCR. Carbachol-mediated elevation in intracellular calcium ([Ca2+]i) was also measured in the presence or absence of a selective muscarinic receptor antagonist. RESULTS. Constitutive synthesis and secretion of ET-1 was greater in mRPE than in yRPE cells. TNF-α caused a significant increase in ppET-1 mRNA levels and ET-1 secretion in both phenotypes. The disruption and subsequent breakdown of the tight junction barrier was evident in either phenotype after treatments with TNF-α. There was a concentration-dependent increase in [Ca 2+]i in both y- and mRPE cells in response to CCh. CCh at 1 μM significantly increased ET-1 secretion, a response observed in yRPE but not in mRPE cells. This effect may be mediated primarily by the M3 receptor subtype and is phospholipase C (PLC) dependent. CONCLUSIONS. Regulation of ET-1 release in ARPE-19 cells was differentially regulated by TNF-α and CCh and was dependent on the age of the culture. RPE may be a source for ET-1 in the retina, and its increased release may become more important during breakdown of the blood-retinal barrier, as seen after TNF-α treatment.

AB - PURPOSE. Endothelin (ET)-1 can produce nerve damage analogous to that in optic neuropathies such as glaucoma. The precise source of endothelin in the posterior segment of the eye remains unclear. The present study was conducted to determine whether the retinal pigment epithelium (RPE), which helps maintain the outer blood-retinal barrier, is a local source for ET-1 and whether the amount of ET-1 secreted by the RPE is be differentially regulated by cholinergics and the cytokine TNF-α. METHODS. Human retinal pigment epithelial cells (ARPE-19) were cultured either to a mature state (mRPE) for 4 weeks, with well-defined tight junctions, or to a young state (yRPE) for 4 days, with incompletely formed tight junctions. ET-1-like immunoreactivity was determined by immunocytochemistry, and secreted ET-1 was quantified by radioimmunoassay in both cell types. Cells were stimulated with the cholinergic agonist carbachol or with the cytokine TNF-α for specific periods. The expression of muscarinic receptor subtypes M1 and M3 and the peripheral membrane protein ZO-1 were analyzed by immunoblot and immunocytochemistry, respectively. Expression of preproendothelin-1 (ppET-1) mRNA after application of different stimuli at specific time points was determined by real-time RT-PCR. Carbachol-mediated elevation in intracellular calcium ([Ca2+]i) was also measured in the presence or absence of a selective muscarinic receptor antagonist. RESULTS. Constitutive synthesis and secretion of ET-1 was greater in mRPE than in yRPE cells. TNF-α caused a significant increase in ppET-1 mRNA levels and ET-1 secretion in both phenotypes. The disruption and subsequent breakdown of the tight junction barrier was evident in either phenotype after treatments with TNF-α. There was a concentration-dependent increase in [Ca 2+]i in both y- and mRPE cells in response to CCh. CCh at 1 μM significantly increased ET-1 secretion, a response observed in yRPE but not in mRPE cells. This effect may be mediated primarily by the M3 receptor subtype and is phospholipase C (PLC) dependent. CONCLUSIONS. Regulation of ET-1 release in ARPE-19 cells was differentially regulated by TNF-α and CCh and was dependent on the age of the culture. RPE may be a source for ET-1 in the retina, and its increased release may become more important during breakdown of the blood-retinal barrier, as seen after TNF-α treatment.

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U2 - 10.1167/iovs.03-0387

DO - 10.1167/iovs.03-0387

M3 - Article

VL - 44

SP - 4885

EP - 4894

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 11

ER -