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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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Related in: MedlinePlus

The expression of NPR-A and NPR-B in formalin-fixed human p-CEPI and CEPI-17-CL4 cells by indirect immunofluorescence is shown. A shows a strong expression of NPR-A in human p-CEPI cells, while B shows expression of NPR-A in CEPI-17-CL4 cells. C shows expression of NPR-B in human p-CEPI cells while D shows expression of NPR-B in CEPI-17-CL4 cells. E and F are DAPI labeled controls (treated with secondary antibody only) for human p-CEPI and CEPI-17-CL4 cells, respectively. Each panel is at 40× magnification.
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f3: The expression of NPR-A and NPR-B in formalin-fixed human p-CEPI and CEPI-17-CL4 cells by indirect immunofluorescence is shown. A shows a strong expression of NPR-A in human p-CEPI cells, while B shows expression of NPR-A in CEPI-17-CL4 cells. C shows expression of NPR-B in human p-CEPI cells while D shows expression of NPR-B in CEPI-17-CL4 cells. E and F are DAPI labeled controls (treated with secondary antibody only) for human p-CEPI and CEPI-17-CL4 cells, respectively. Each panel is at 40× magnification.

Mentions: Expression of NPR-A and NPR-B was observed in the epithelium of human corneas and was particularly strong in the superficial layers of the central region (Figure 2A-D). NPR-B appeared to be expressed more strongly than NPR-A in the epithelium of the limbus and the expression of both was confined to the superficial layers. Similarly, immunocytochemical analysis of the cultured cell monolayers showed what appeared be cytoplasmic expression of both NPR-A and NPR-B (Figure 3).


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)

The expression of NPR-A and NPR-B in formalin-fixed human p-CEPI and CEPI-17-CL4 cells by indirect immunofluorescence is shown. A shows a strong expression of NPR-A in human p-CEPI cells, while B shows expression of NPR-A in CEPI-17-CL4 cells. C shows expression of NPR-B in human p-CEPI cells while D shows expression of NPR-B in CEPI-17-CL4 cells. E and F are DAPI labeled controls (treated with secondary antibody only) for human p-CEPI and CEPI-17-CL4 cells, respectively. Each panel is at 40× magnification.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: The expression of NPR-A and NPR-B in formalin-fixed human p-CEPI and CEPI-17-CL4 cells by indirect immunofluorescence is shown. A shows a strong expression of NPR-A in human p-CEPI cells, while B shows expression of NPR-A in CEPI-17-CL4 cells. C shows expression of NPR-B in human p-CEPI cells while D shows expression of NPR-B in CEPI-17-CL4 cells. E and F are DAPI labeled controls (treated with secondary antibody only) for human p-CEPI and CEPI-17-CL4 cells, respectively. Each panel is at 40× magnification.
Mentions: Expression of NPR-A and NPR-B was observed in the epithelium of human corneas and was particularly strong in the superficial layers of the central region (Figure 2A-D). NPR-B appeared to be expressed more strongly than NPR-A in the epithelium of the limbus and the expression of both was confined to the superficial layers. Similarly, immunocytochemical analysis of the cultured cell monolayers showed what appeared be cytoplasmic expression of both NPR-A and NPR-B (Figure 3).

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