Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are endogenous cardiac hormones that are essential for cardiovascular homeostasis. To decrease blood pressure and cardiac hypertrophy, ANP and BNP bind to the transmembrane guanylyl cyclase natriuretic peptide receptor-A (NPR-A). Activation of NPR-A leads to the synthesis of the intracellular second messenger cGMP, which mediates the physiological effects of the natriuretic peptides. Under cardiovascular stress serum ANP and BNP concentrations are elevated. Initially, these cardiac peptides stimulate compensatory hemodynamic functions, but over time their cardiac unloading effects wane despite continued elevation of serum ANP and BNP levels. Unfortunately, the underlying molecular mechanisms responsible for the diminished effect are poorly understood.
Transaortic banding was performed to induce congestive heart failure in mice; failed hearts had both reduced ANP-dependent guanylyl cyclase activity and NPR-A protein levels compared to control hearts, indicating that NPR-A is downregulated in the failed heart. Consistent with the in vivo studies, cell culture experiments demonstrated that prolonged ANP exposure resulted in degradation of NPR-A in multiple cell lines. To investigate potential mechanisms involved in NPR-A downregulation, a novel antibody-based intracellular accumulation assay was developed. The assay revealed that NPR-A is basally internalized by a relatively slow clathrin- and dynamin-independent process that is stimulated by ANP. Dynamin inactivation increased intracellular accumulation of NPR-A at long, but not short, time periods after initiation of internalization, which is consistent with the notion of NPR-A recycling in a dynamin-dependent process. Surprisingly, the rate of NPR-A internalization was accelerated in clathrin-depleted cells. Understanding the molecular mechanisms underlying NPR-A downregulation will aid in the development of potential therapeutic strategies that disrupt this process and prolong the beneficial compensatory effects of natriuretic peptides in patients with cardiovascular disease.
University of Minnesota. Ph.D. dissertation. December 2009. Major: Pharmacology. Advisor: Lincoln R. Potter. 1 computer file (PDF); ix, 182 pages.
Flora, Darcy Rae.
Downregulation of natriuretic peptide receptor-A: evidence for clathrin- and dynamin-independent internalization..
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