Characterization of EP prostanoid receptor subtypes in primary cultures of bovine ciliary epithelial cells by immunofluorescent microscopy and functional studies

T. L. Anthony, K. L. Pierce, John W Regan

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9 Citations (Scopus)

Abstract

Purpose. To determine the expression and functional coupling of EP prostanoid receptor subtypes to second messenger pathways in bovine ciliary epithelium. Methods. Primary cultures of bovine ciliary epithelial (BCE) cells were established and maintained in culture up to four passages. EP receptor protein expression was examined by indirect immunofluorescence microscopy with subtype selective antibodies in both tissue sections and primary cultures of BCE cells. Messenger RNA expression was determined using reverse transcription and polymerase chain reaction. The effects of prostanoid agonists on total inositol phosphate accumulation and cAMP formation were used to assess functional activity. Results. Positive immunoreactivity was obtained in both frozen thin-sections and primary cultures of bovine ciliary process to the prostanoid EP1, EP2, EP3 and EP4 receptor subtypes. Reverse transcription followed by the polymerase chain reaction yielded products corresponding to each of the prostanoid EP subtypes which was confirmed by restriction enzyme analysis. PGE2 dose-dependently stimulated the accumulation of total inositol phosphates in cultured cells with an EC50 value of 100 nM. PGE2, forskolin and isoproterenol produced dose-dependent increases in cAMP formation with EC50 values of 100, 300 and 200 nM, respectively. Isoproterenol-stimulated cAMP formation was attenuated by the EP3 receptor agonist sulprostone in cultured BCE cells. The inhibition elicited by sulprostone was reversed in cells pretreated with pertussis toxin. Conclusions. This study demonstrates the presence of functional prostanoid EP receptor subtypes in the bovine ciliary epithelium. EP1 and EP4 receptor subtypes were found primarily in the NPE cells, whereas, EP2 receptor subtype immunofluorescence was detected in the PE cells. EP3 receptor subtype labeling was observed in both the NPE and the PE cells. PGE2 produces opposing effects on adenylyl cyclase through EP2/EP4 and EP3 receptor activation. The predominant effect of PGE2 is on the adenylyl cyclase stimulatory receptors (EP2/EP4).

Original languageEnglish (US)
Pages (from-to)394-404
Number of pages11
JournalCurrent Eye Research
Volume20
Issue number5
StatePublished - 2000

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Prostaglandins
Microscopy
Epithelial Cells
Dinoprostone
Inositol Phosphates
Isoproterenol
Adenylyl Cyclases
Reverse Transcription
Receptors, Prostaglandin E, EP1 Subtype
Receptors, Prostaglandin E, EP2 Subtype
Receptors, Prostaglandin E, EP4 Subtype
Epithelium
Polymerase Chain Reaction
Restriction Mapping
Pertussis Toxin
Frozen Sections
Second Messenger Systems
Colforsin
Indirect Fluorescent Antibody Technique
Fluorescence Microscopy

Keywords

  • Bovine PGE
  • CAMP
  • Ciliary epithelium
  • EP receptors
  • Prostaglandin

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

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title = "Characterization of EP prostanoid receptor subtypes in primary cultures of bovine ciliary epithelial cells by immunofluorescent microscopy and functional studies",
abstract = "Purpose. To determine the expression and functional coupling of EP prostanoid receptor subtypes to second messenger pathways in bovine ciliary epithelium. Methods. Primary cultures of bovine ciliary epithelial (BCE) cells were established and maintained in culture up to four passages. EP receptor protein expression was examined by indirect immunofluorescence microscopy with subtype selective antibodies in both tissue sections and primary cultures of BCE cells. Messenger RNA expression was determined using reverse transcription and polymerase chain reaction. The effects of prostanoid agonists on total inositol phosphate accumulation and cAMP formation were used to assess functional activity. Results. Positive immunoreactivity was obtained in both frozen thin-sections and primary cultures of bovine ciliary process to the prostanoid EP1, EP2, EP3 and EP4 receptor subtypes. Reverse transcription followed by the polymerase chain reaction yielded products corresponding to each of the prostanoid EP subtypes which was confirmed by restriction enzyme analysis. PGE2 dose-dependently stimulated the accumulation of total inositol phosphates in cultured cells with an EC50 value of 100 nM. PGE2, forskolin and isoproterenol produced dose-dependent increases in cAMP formation with EC50 values of 100, 300 and 200 nM, respectively. Isoproterenol-stimulated cAMP formation was attenuated by the EP3 receptor agonist sulprostone in cultured BCE cells. The inhibition elicited by sulprostone was reversed in cells pretreated with pertussis toxin. Conclusions. This study demonstrates the presence of functional prostanoid EP receptor subtypes in the bovine ciliary epithelium. EP1 and EP4 receptor subtypes were found primarily in the NPE cells, whereas, EP2 receptor subtype immunofluorescence was detected in the PE cells. EP3 receptor subtype labeling was observed in both the NPE and the PE cells. PGE2 produces opposing effects on adenylyl cyclase through EP2/EP4 and EP3 receptor activation. The predominant effect of PGE2 is on the adenylyl cyclase stimulatory receptors (EP2/EP4).",
keywords = "Bovine PGE, CAMP, Ciliary epithelium, EP receptors, Prostaglandin",
author = "Anthony, {T. L.} and Pierce, {K. L.} and Regan, {John W}",
year = "2000",
language = "English (US)",
volume = "20",
pages = "394--404",
journal = "Current Eye Research",
issn = "0271-3683",
publisher = "Informa Healthcare",
number = "5",

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TY - JOUR

T1 - Characterization of EP prostanoid receptor subtypes in primary cultures of bovine ciliary epithelial cells by immunofluorescent microscopy and functional studies

AU - Anthony, T. L.

AU - Pierce, K. L.

AU - Regan, John W

PY - 2000

Y1 - 2000

N2 - Purpose. To determine the expression and functional coupling of EP prostanoid receptor subtypes to second messenger pathways in bovine ciliary epithelium. Methods. Primary cultures of bovine ciliary epithelial (BCE) cells were established and maintained in culture up to four passages. EP receptor protein expression was examined by indirect immunofluorescence microscopy with subtype selective antibodies in both tissue sections and primary cultures of BCE cells. Messenger RNA expression was determined using reverse transcription and polymerase chain reaction. The effects of prostanoid agonists on total inositol phosphate accumulation and cAMP formation were used to assess functional activity. Results. Positive immunoreactivity was obtained in both frozen thin-sections and primary cultures of bovine ciliary process to the prostanoid EP1, EP2, EP3 and EP4 receptor subtypes. Reverse transcription followed by the polymerase chain reaction yielded products corresponding to each of the prostanoid EP subtypes which was confirmed by restriction enzyme analysis. PGE2 dose-dependently stimulated the accumulation of total inositol phosphates in cultured cells with an EC50 value of 100 nM. PGE2, forskolin and isoproterenol produced dose-dependent increases in cAMP formation with EC50 values of 100, 300 and 200 nM, respectively. Isoproterenol-stimulated cAMP formation was attenuated by the EP3 receptor agonist sulprostone in cultured BCE cells. The inhibition elicited by sulprostone was reversed in cells pretreated with pertussis toxin. Conclusions. This study demonstrates the presence of functional prostanoid EP receptor subtypes in the bovine ciliary epithelium. EP1 and EP4 receptor subtypes were found primarily in the NPE cells, whereas, EP2 receptor subtype immunofluorescence was detected in the PE cells. EP3 receptor subtype labeling was observed in both the NPE and the PE cells. PGE2 produces opposing effects on adenylyl cyclase through EP2/EP4 and EP3 receptor activation. The predominant effect of PGE2 is on the adenylyl cyclase stimulatory receptors (EP2/EP4).

AB - Purpose. To determine the expression and functional coupling of EP prostanoid receptor subtypes to second messenger pathways in bovine ciliary epithelium. Methods. Primary cultures of bovine ciliary epithelial (BCE) cells were established and maintained in culture up to four passages. EP receptor protein expression was examined by indirect immunofluorescence microscopy with subtype selective antibodies in both tissue sections and primary cultures of BCE cells. Messenger RNA expression was determined using reverse transcription and polymerase chain reaction. The effects of prostanoid agonists on total inositol phosphate accumulation and cAMP formation were used to assess functional activity. Results. Positive immunoreactivity was obtained in both frozen thin-sections and primary cultures of bovine ciliary process to the prostanoid EP1, EP2, EP3 and EP4 receptor subtypes. Reverse transcription followed by the polymerase chain reaction yielded products corresponding to each of the prostanoid EP subtypes which was confirmed by restriction enzyme analysis. PGE2 dose-dependently stimulated the accumulation of total inositol phosphates in cultured cells with an EC50 value of 100 nM. PGE2, forskolin and isoproterenol produced dose-dependent increases in cAMP formation with EC50 values of 100, 300 and 200 nM, respectively. Isoproterenol-stimulated cAMP formation was attenuated by the EP3 receptor agonist sulprostone in cultured BCE cells. The inhibition elicited by sulprostone was reversed in cells pretreated with pertussis toxin. Conclusions. This study demonstrates the presence of functional prostanoid EP receptor subtypes in the bovine ciliary epithelium. EP1 and EP4 receptor subtypes were found primarily in the NPE cells, whereas, EP2 receptor subtype immunofluorescence was detected in the PE cells. EP3 receptor subtype labeling was observed in both the NPE and the PE cells. PGE2 produces opposing effects on adenylyl cyclase through EP2/EP4 and EP3 receptor activation. The predominant effect of PGE2 is on the adenylyl cyclase stimulatory receptors (EP2/EP4).

KW - Bovine PGE

KW - CAMP

KW - Ciliary epithelium

KW - EP receptors

KW - Prostaglandin

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M3 - Article

VL - 20

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JO - Current Eye Research

JF - Current Eye Research

SN - 0271-3683

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