Hypoxia stimulates atrial natriuretic peptide gene expression in cultured atrial cardiocytes

The current study tested the hypothesis that hypoxia stimulates atrial natriuretic peptide (ANP) gene expression and secretion in cultured atrial myocytes (AT-1 cells). AT-1 cells were obtained from a transplantable mouse atrial cardiomyocyte tumor lineage. Confluent AT-1 cells were exposed to hypoxia (1% oxygen) or normoxia (21% oxygen) as controls for 6 hours to 7 days. Medium ANP levels were measured by radioimmunoassay, and intracellular ANP gene transcripts were quantified by Northern and slot blot analyses. Exposure to hypoxia resulted in a significant increase in cellular ANP mRNA levels within 36 hours, which peaked (3.6-fold increase) at 2 days after hypoxic exposure, and produced a time-dependent increase in the release of ANP from AT-1 cells for 2 to 7 days. Transfection studies with recombinant DNA constructs that contained fragments of the -3003/+62 sequence of the ANP promoter and the luciferase reporter gene revealed that the regulatory sequences that mediate the hypoxia-induced increase in transcription are located within a region that extends from -638 to -518 bp to the transcriptional start site of the ANP gene. Gel mobility shift assays demonstrated that hypoxia-inducible nuclear proteins that bound to the 120-bp putative hypoxia-responsive elements of the ANP gene were produced during hypoxic exposure. We have thus defined a 120-bp region within the ANP gene promoter that contains hypoxia-responsive elements that might be responsible for the enhancement of ANP gene expression in atrial myocytes during hypoxic exposure.