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NPR-B natriuretic peptide receptors in human corneal epithelium: mRNA, immunohistochemistochemical, protein, and biochemical pharmacology studies.

Katoli P, Sharif NA, Sule A, Dimitrijevich SD - Mol. Vis. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Research, Alcon Research, Ltd., Fort Worth, TX 76134, USA.

ABSTRACT

Purpose: 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.

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Expression of NPR-A and NPR-B in human p-CEPI cells and CEPI-17-CL4 cells as determined by western blot analysis. A: The presence of NPR-A protein (band observed at 40–55 kDa) in CEPI-17-CL4 and in human p-CEPI cells (from 56, 53, and 56 year old donors) is shown. B: The presence of NPR-B protein (band observed at 24 kDa) is shown for three different donors of human p-CEPI cells (ages 40, 53, and 56) cells and in CEPI-17-CL4 cells. C: The expression of NPR-A and NPR-B in human p-CEPI and CEPI-17-CL4 cells was normalized to GAPDH. The figure shows an apparent lower expression of NPR-A in CEPI-17-CL4 cells than in human p-CEPI cells (p<0.1); the expression of NPR-B was higher in CEPI-17-CL4 than that in human p-CEPI cells (p<0.05). The NPR-B expression was greater than NPR-A expression in CEPI-17-CL4 cells (p<0.05). However, the both receptor subtypes were expressed to the same extent in the p-CEPI cells (p<0.1).
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f4: Expression of NPR-A and NPR-B in human p-CEPI cells and CEPI-17-CL4 cells as determined by western blot analysis. A: The presence of NPR-A protein (band observed at 40–55 kDa) in CEPI-17-CL4 and in human p-CEPI cells (from 56, 53, and 56 year old donors) is shown. B: The presence of NPR-B protein (band observed at 24 kDa) is shown for three different donors of human p-CEPI cells (ages 40, 53, and 56) cells and in CEPI-17-CL4 cells. C: The expression of NPR-A and NPR-B in human p-CEPI and CEPI-17-CL4 cells was normalized to GAPDH. The figure shows an apparent lower expression of NPR-A in CEPI-17-CL4 cells than in human p-CEPI cells (p<0.1); the expression of NPR-B was higher in CEPI-17-CL4 than that in human p-CEPI cells (p<0.05). The NPR-B expression was greater than NPR-A expression in CEPI-17-CL4 cells (p<0.05). However, the both receptor subtypes were expressed to the same extent in the p-CEPI cells (p<0.1).

Mentions: Cellular NPR-A and NPR-B protein expression was confirmed by western blot analysis of p-CEPI and CEPI-17-CL4 cell lysates (Figure 4A,B). Some donor variability was observed in the levels expressed by p-CEPI cells. After normalization of expression to the house-keeping enzyme gene (glycerol-3-phosphate dehydrogenase; GAPDH), CEPI-17-CL4 cells were shown to have a higher expression of NPR-B than p-CEPI cells (p<0.05), while the latter cells had a higher expression of NPR-A than CEPI-17-CL4 cells though this did not reach statistical significance (p<0.1; Figure 4C). In addition, CEPI-17-CL4 cells expressed a greater amount of NPR-B than the p-CEPI cells (p<0.05) but this could have been a reflection of greater variability in the isolated cells from human donor eyes.


NPR-B natriuretic peptide receptors in human corneal epithelium: mRNA, immunohistochemistochemical, protein, and biochemical pharmacology studies.

Katoli P, Sharif NA, Sule A, Dimitrijevich SD - Mol. Vis. (2010)

Expression of NPR-A and NPR-B in human p-CEPI cells and CEPI-17-CL4 cells as determined by western blot analysis. A: The presence of NPR-A protein (band observed at 40–55 kDa) in CEPI-17-CL4 and in human p-CEPI cells (from 56, 53, and 56 year old donors) is shown. B: The presence of NPR-B protein (band observed at 24 kDa) is shown for three different donors of human p-CEPI cells (ages 40, 53, and 56) cells and in CEPI-17-CL4 cells. C: The expression of NPR-A and NPR-B in human p-CEPI and CEPI-17-CL4 cells was normalized to GAPDH. The figure shows an apparent lower expression of NPR-A in CEPI-17-CL4 cells than in human p-CEPI cells (p<0.1); the expression of NPR-B was higher in CEPI-17-CL4 than that in human p-CEPI cells (p<0.05). The NPR-B expression was greater than NPR-A expression in CEPI-17-CL4 cells (p<0.05). However, the both receptor subtypes were expressed to the same extent in the p-CEPI cells (p<0.1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2903464&req=5

f4: Expression of NPR-A and NPR-B in human p-CEPI cells and CEPI-17-CL4 cells as determined by western blot analysis. A: The presence of NPR-A protein (band observed at 40–55 kDa) in CEPI-17-CL4 and in human p-CEPI cells (from 56, 53, and 56 year old donors) is shown. B: The presence of NPR-B protein (band observed at 24 kDa) is shown for three different donors of human p-CEPI cells (ages 40, 53, and 56) cells and in CEPI-17-CL4 cells. C: The expression of NPR-A and NPR-B in human p-CEPI and CEPI-17-CL4 cells was normalized to GAPDH. The figure shows an apparent lower expression of NPR-A in CEPI-17-CL4 cells than in human p-CEPI cells (p<0.1); the expression of NPR-B was higher in CEPI-17-CL4 than that in human p-CEPI cells (p<0.05). The NPR-B expression was greater than NPR-A expression in CEPI-17-CL4 cells (p<0.05). However, the both receptor subtypes were expressed to the same extent in the p-CEPI cells (p<0.1).
Mentions: Cellular NPR-A and NPR-B protein expression was confirmed by western blot analysis of p-CEPI and CEPI-17-CL4 cell lysates (Figure 4A,B). Some donor variability was observed in the levels expressed by p-CEPI cells. After normalization of expression to the house-keeping enzyme gene (glycerol-3-phosphate dehydrogenase; GAPDH), CEPI-17-CL4 cells were shown to have a higher expression of NPR-B than p-CEPI cells (p<0.05), while the latter cells had a higher expression of NPR-A than CEPI-17-CL4 cells though this did not reach statistical significance (p<0.1; Figure 4C). In addition, CEPI-17-CL4 cells expressed a greater amount of NPR-B than the p-CEPI cells (p<0.05) but this could have been a reflection of greater variability in the isolated cells from human donor eyes.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Research, Alcon Research, Ltd., Fort Worth, TX 76134, USA.

ABSTRACT

Purpose: 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.

Show MeSH
Related in: MedlinePlus