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Stimulation of Natriuretic Peptide Receptor-C (NPR-C) Induces a Hypoxia-Inducible Factor 1 (HIF-1)-Mediated Response in Alveolar Type II Cells
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A6115 - Stimulation of Natriuretic Peptide Receptor-C (NPR-C) Induces a Hypoxia-Inducible Factor 1 (HIF-1)-Mediated Response in Alveolar Type II Cells
Author Block: R. M. Ryan1, M. K. Paintlia1, D. A. Newton2, J. E. Baatz3; 1Pediatrics-Neonatology, Medical Univ of South Carolina, Charleston, SC, United States, 2Medical University of South Carolina, Charleston, SC, United States, 3Children's Research Inst, Room 307, Med Univ of South Carolina, Charleston, SC, United States.
Background: We have previously shown that the natriuretic peptide pathway plays a role in surfactant secretion; for example, atrial natriuretic peptide (ANP) stimulates surfactant secretion through a cGMP-mediated mechanism, yet higher concentrations of ANP or stimulation of the NPR-C receptor using a specific analog, C-ANP, blocks terbutaline-induced surfactant secretion. We have also shown that hyperoxia suppresses NPR-C expression in type II cells and that surfactant secretion is optimal at physiologic oxygen (13% O2) and lower otherwise, suggesting that the oxygen environment and the NP system are connected. We hypothesized that NPR-C stimulation would alter the expression of genes known to be regulated by oxygen levels.
Methods: Cultures of a model type II cell line, mouse lung epithelial 15 (MLE-15) cells, were maintained in RPMI 1640 medium under ambient air (21% O2) and 5% CO2 at 37°C. We used C-ANP, amino acids 4-23 of ANP, known to be a specific agonist for the NPR-C receptor; C-ANP will not stimulate NRP-A, the classic ANP receptor. Exposures to varying oxygen concentrations were performed by incubation for four days in a Biopherix hypoxia chamber with either 5% O2, 21% O2 or 21% O2 with C-ANP(4-23) at 10-10 M. After four days of exposure to the experimental conditions, cells were lysed for qPCR. Expression of HIF-1α, hemoglobin α (HBA) and the globin transcription factor GATA1 genes were measured by quantitative RT-PCR analysis and normalized to 18S rRNA. Fold-change values for target genes between hypoxia and normoxia groups were then calculated using ΔΔCt analysis to determine expression fold differences.
Results: MLE-15 cells have robust expression of NPR-C by immunohistochemistry. As expected, HIF-1α is induced in 5% oxygen (hypoxia); HBA is also induced. C-ANP stimulation of the NPR-C receptor in MLE-15 cells induced HIF-1α (10-fold), HBA (20-fold), and GATA-1 (20-fold) when cultured in 21% oxygen.
Conclusion: We speculate that activation of various components of the NP system function to alter the oxidative milieu in type II cells, and this may affect surfactant secretion.
Author Block: R. M. Ryan1, M. K. Paintlia1, D. A. Newton2, J. E. Baatz3; 1Pediatrics-Neonatology, Medical Univ of South Carolina, Charleston, SC, United States, 2Medical University of South Carolina, Charleston, SC, United States, 3Children's Research Inst, Room 307, Med Univ of South Carolina, Charleston, SC, United States.
Background: We have previously shown that the natriuretic peptide pathway plays a role in surfactant secretion; for example, atrial natriuretic peptide (ANP) stimulates surfactant secretion through a cGMP-mediated mechanism, yet higher concentrations of ANP or stimulation of the NPR-C receptor using a specific analog, C-ANP, blocks terbutaline-induced surfactant secretion. We have also shown that hyperoxia suppresses NPR-C expression in type II cells and that surfactant secretion is optimal at physiologic oxygen (13% O2) and lower otherwise, suggesting that the oxygen environment and the NP system are connected. We hypothesized that NPR-C stimulation would alter the expression of genes known to be regulated by oxygen levels.
Methods: Cultures of a model type II cell line, mouse lung epithelial 15 (MLE-15) cells, were maintained in RPMI 1640 medium under ambient air (21% O2) and 5% CO2 at 37°C. We used C-ANP, amino acids 4-23 of ANP, known to be a specific agonist for the NPR-C receptor; C-ANP will not stimulate NRP-A, the classic ANP receptor. Exposures to varying oxygen concentrations were performed by incubation for four days in a Biopherix hypoxia chamber with either 5% O2, 21% O2 or 21% O2 with C-ANP(4-23) at 10-10 M. After four days of exposure to the experimental conditions, cells were lysed for qPCR. Expression of HIF-1α, hemoglobin α (HBA) and the globin transcription factor GATA1 genes were measured by quantitative RT-PCR analysis and normalized to 18S rRNA. Fold-change values for target genes between hypoxia and normoxia groups were then calculated using ΔΔCt analysis to determine expression fold differences.
Results: MLE-15 cells have robust expression of NPR-C by immunohistochemistry. As expected, HIF-1α is induced in 5% oxygen (hypoxia); HBA is also induced. C-ANP stimulation of the NPR-C receptor in MLE-15 cells induced HIF-1α (10-fold), HBA (20-fold), and GATA-1 (20-fold) when cultured in 21% oxygen.
Conclusion: We speculate that activation of various components of the NP system function to alter the oxidative milieu in type II cells, and this may affect surfactant secretion.
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