Prostanoid receptor gene expression profile in human trabecular meshwork: a quantitative real-time PCR approach.
OBJECTIVE
To assess the expression pattern of prostanoid receptor-encoding genes in trabecular meshwork (TM) of human donor eyes.


METHODS
Disposed human donor eyes (n = 10) were obtained from the Cornea Bank, Amsterdam. The TM was dissected from the scleral tissue and homogenized in lysis buffer, and total RNA was isolated. The RNA was converted into cDNA and used as a template for noncompetitive quantitative real-time polymerase chain reaction (PCR) using green fluorescent dye to quantify the accumulation of double-stranded PCR product. Specific primers for four housekeeping genes and DP, EP(1), EP(2), EP(3,) EP(4), FP, IP, and TP receptor-encoding transcripts were developed and tested for their efficiency.


RESULTS
The characterized expression profile was highly reproducible in all samples, with the EP(2) receptor-encoding transcript in the highest abundance, followed by FP, TP, IP, and EP(4) at levels that were approximately 10 to 15 times lower than that of the EP(2) subtype. DP and EP(3) were at the lowest levels, which were, on average, 45 times and 228 times lower than EP(2), respectively.


CONCLUSIONS
These data show that all prostanoid receptors are expressed at different levels in human TM tissue. Because the gene expression of the EP(2) receptor is, on average, 15 times more abundant than that of the EP(4) receptor, it may be expected that the increase in flow and cAMP levels in response to the activation of the EP receptors by application of prostaglandin E(1) (PGE(1)), is primarily mediated by the EP(2) receptor. These data should be considered when designing prostanoid receptor mimetics intended to enhance the aqueous humor outflow through the TM and Schlemm's canal.
Kamphuis
W
W
Glaucoma Research Group, Netherlands Ophthalmic Research Institute (NORI)-KNAW, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands. w.kamphuis@ioi.knaw.nl
Schneemann
A
A
van Beek
L M
LM
Smit
A B
AB
Hoyng
P F
PF
Koya
E
E
D000199:Actins / Q000235:genetics; D000368:Aged; D003199:Computer Systems; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods; D011982:Receptors, Prostaglandin / Q000235:genetics*; D015203:Reproducibility of Results; D014129:Trabecular Meshwork / Q000502:physiology*
D000199:Actins; D011982:Receptors, Prostaglandin