NPR-B natriuretic peptide receptors in human corneal epithelium: mRNA, immunohistochemistochemical, protein, and biochemical pharmacology studies.
OBJECTIVE
To demonstrate the presence of natriuretic peptide receptors (NPRs) in primary human corneal epithelial cells (p-CEPI), SV40-immortalized CEPI cells (CEPI-17-CL4) and in human corneal epithelium, and to define the pharmacology of natriuretic peptide (NP)-induced cGMP accumulation.


METHODS
NPR presence was shown by RT-PCR, western blot analysis, and indirect immunofluoresence. cGMP accumulation was determined using an enzyme immunoassay.


RESULTS
p-CEPI and CEPI-17-CL4 cells expressed mRNAs for NPR-A and NPR-B. Proteins for both NPRs were present in these cells and in human corneal epithelium. C-type NP (CNP), atrial NP (ANP) and brain NP (BNP) stimulated the accumulation of cGMP in a concentration-dependent manner in p-CEPI cells (potency; EC(50s)): CNP (1-53 amino acids) EC(50)=24+/-5 nM; CNP fragment (32-53 amino acids) EC(50)=51+/-8 nM; ANP (1-28 amino acids) EC(50)=>10 microM; BNP (32 amino acids) EC(50)>10 microM (all n=3-4). While the NPs were generally more potent in the CEPI-17-CL4 cells than in p-CEPI cells (n=4-9; p<0.01), the rank order of potency of the peptides was essentially the same in both cell types. Effects of CNP fragment in p-CEPI and CEPI-17-CL4 cells were potently blocked by HS-142-1, an NPR-B receptor subtype-selective antagonist (K(i)=0.25+/-0.05 microM in CEPI-CL4-17; K(i)=0.44+/-0.09 microM in p-CEPIs; n=6-7) but less so by an NPR-A receptor antagonist, isatin (K(i)=5.3-7.8 microM, n=3-7).


CONCLUSIONS
Our studies showed the presence of NPR-A and NPR-B (mRNAs and protein) in p-CEPI and CEPI-17-CL4 cells and in human corneal epithelial tissue. However, detailed pharmacological studies revealed NPR-B to be the predominant functionally active receptor in both cell-types whose activation leads to the generation of cGMP. While the physiologic role(s) of the NP system in corneal function remains to be delineated, our multidisciplinary findings pave the way for such future investigations.
Katoli
Parvaneh
P
Pharmaceutical Research, Alcon Research, Ltd., Fort Worth, TX 76134, USA.
Sharif
Najam A
NA
Sule
Anupam
A
Dimitrijevich
Slobodan D
SD
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015153:Blotting, Western; D006152:Cyclic GMP / Q000096:biosynthesis; D019573:Epithelium, Corneal / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008875:Middle Aged; D012333:RNA, Messenger / Q000378:metabolism; D017461:Receptors, Atrial Natriuretic Factor / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D000099242:Atrial Natriuretic Factor Receptor B
D006152:Cyclic GMP; D012333:RNA, Messenger; D017461:Receptors, Atrial Natriuretic Factor; D000099242:Atrial Natriuretic Factor Receptor B