Abstract 15766: Protein Disulfide Isomerase (PDI) Activity Enhances cGMP Generation by Natriuretic Peptides
Background: The natriuretic peptides (NPs), atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), alternatively spliced BNP (ASBNP) and C-type natriuretic peptide (CNP) exert important and diverse beneficial actions on cardiovascular and renal homeostasis. The autocrine/paracrine functions of the NPs are mediated through the cellular membrane bound guanylate cyclase receptors NPR-A and NPR-B. Here we report for the first time that membrane bound protein disulfide isomerase (PDI) regulates natriuretic peptide stimulation of cGMP.
Methods and Results: To detect the interaction between NPs and receptors on human mesangial cells (MC) and porcine kidney epithelial cells (LLC-PK1), one dimensional far western blotting was performed using cell membrane preparation as prey protein and BNP as bait protein. An interactive band (approx 60 kd) was identified. Further two dimensional far western blotting was performed, and binding spots were identified and analyzed by mass spectrometry. PDI was identified as a peptide binding partner at size of 60 kd. Western blotting and immunostaining analysis confirmed PDI expression in these cells with MCs expressing greater levels than LLC-PK1 cells. To study the roles of PDI on NPs stimulation of cGMP, bacitracin, a PDI inhibitor, was used in cGMP analysis. In human mesangial cells, cGMP generation of ANP, CNP, and truncated ASBNP (ANX042) was reduced by 83%, 80%, and 83% respectively. In LLC-PK1 cells, the reductions of cGMP generation for ANP and ANX042 were 28% and 37% respectively. These data suggest that PDI is involved in NP stimulation of cGMP and that the impact of PDI varies among cell types.
Conclusions: We present for the first time that PDI acts as a mediator of the interaction of NPs and their receptors to regulate cGMP generation. PDI expression and activity should be further considered in understanding the nature of the autocrine, paracrine and endocrine functions of the NP family.
- © 2011 by American Heart Association, Inc.