Expression of prostaglandin receptors EP4 and FP in human lens epithelial cells.
OBJECTIVE
To examine the expression of prostaglandin (PG) receptors EP2, EP4, and FP in a human lens epithelial cell line (HLE-B3) at molecular and pharmacologic levels.


METHODS
Reverse transcription-polymerase chain reactions (RT-PCR) were performed with total RNA preparations from HLE-B3 cells using sense and antisense primers for each of the three prostaglandin receptors. The PCR products were hybridized with specific 32P-labeled probes and, for further confirmation, digested with appropriate restriction enzymes. At the pharmacologic level, the expression of EP4 receptors was determined by measuring intracellular cyclic adenosine monophosphate (cAMP) formation in response to PGE2 (EP1, EP2, EP3, and EP4 agonist) and the EP4 receptor-selective antagonist AH23848. The expression of FP receptors in HLE-B3 cells was explored by measuring intracellular [Ca2+]i mobilization.


RESULTS
RT-PCR generated DNA products of predicted sizes corresponding to the EP2, EP4, and FP receptors. Hybridization of the PCR products with specific 32P-labeled probes and restriction digestion of the PCR products further confirmed that they were generated from the respective EP2, EP4, and FP mRNAs. The EP receptor agonist PGE2 significantly increased the cAMP level in HLE-B3 cells. The formation of cAMP by PGE2 was concentration-dependently inhibited by the EP4 receptor-selective antagonist AH23848. Stimulation of HLE-B3 cells by the FP receptor agonist fluprostenol increased [Ca2+]i in a time-dependent manner.


CONCLUSIONS
The results of the molecular biologic and pharmacologic experiments showed conclusively the presence of EP4 and FP receptor messenger RNAs and proteins, respectively, in HLE-B3 cells.
Mukhopadhyay
P
P
Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, Kentucky 40202, USA.
Bhattacherjee
P
P
Andom
T
T
Geoghegan
T E
TE
Andley
U P
UP
Paterson
C A
CA
D000199:Actins / Q000235:genetics / Q000378:metabolism; D001713:Biphenyl Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D015232:Dinoprostone / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011461:Prostaglandins F, Synthetic / Q000494:pharmacology; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D011982:Receptors, Prostaglandin / Q000235:genetics / Q000378:metabolism*; D018078:Receptors, Prostaglandin E / Q000235:genetics / Q000378:metabolism*; D058307:Receptors, Prostaglandin E, EP2 Subtype; D058309:Receptors, Prostaglandin E, EP4 Subtype; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000199:Actins; D001713:Biphenyl Compounds; D017931:DNA Primers; D015342:DNA Probes; C545644:PTGER2 protein, human; C545650:PTGER4 protein, human; D011461:Prostaglandins F, Synthetic; D012333:RNA, Messenger; D011982:Receptors, Prostaglandin; D018078:Receptors, Prostaglandin E; D058307:Receptors, Prostaglandin E, EP2 Subtype; D058309:Receptors, Prostaglandin E, EP4 Subtype; C034671:prostaglandin F2alpha receptor; C006326:fluprostenol; D012313:RNA; C046926:AH 23848; D000242:Cyclic AMP; D015232:Dinoprostone; D002118:Calcium