Abstract
RationaleThe family of natriuretic peptides (NPs), including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), exert important and diverse actions for cardiovascular and renal homeostasis. The autocrine and paracrine functions of the NPs are primarily mediated through the cellular membrane bound guanylyl cyclase-linked receptors GC-A (NPR-A) and GC-B (NPR-B). As the ligands and receptors each contain disulfide bonds, a regulatory role for the cell surface protein disulfide isomerase (PDI) was investigated.ObjectiveWe utilized complementary in vitro and in vivo models to determine the potential role of PDI in regulating the ability of the NPs to generate its second messenger, cyclic guanosine monophosphate.Methods and ResultsInhibition of PDI attenuated the ability of ANP, BNP and CNP to generate cGMP in human mesangial cells (HMCs), human umbilical vein endothelial cells (HUVECs), and human aortic smooth muscle cells (HASMCs), each of which were shown to express PDI. In LLC-PK1 cells, where PDI expression was undetectable by immunoblotting, PDI inhibition had a minimal effect on cGMP generation. Addition of PDI to cultured LLC-PK1 cells increased intracellular cGMP generation mediated by ANP. Inhibition of PDI in vivo attenuated NP-mediated generation of cGMP by ANP. Surface Plasmon Resonance demonstrated modest and differential binding of the natriuretic peptides with immobilized PDI in a cell free system. However, PDI was shown to co-localize on the surface of cells with GC-A and GC-B by co-immunoprecpitation and immunohistochemistry.ConclusionThese data demonstrate for the first time that cell surface PDI expression and function regulate the capacity of natriuretic peptides to generate cGMP through interaction with their receptors.
Highlights
Members of the natriuretic peptide (NP) family, atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) are central regulators of sodium and water balance, blood volume, and arterial pressure as well as myocardial and vascular structure and function. [1] The actions of the natriuretic peptides (NPs) are mediated through the guanylyl cyclase (GC)-associated receptors GC-A and GC-B. [2] Both receptors contain three different functional domains, an extracellular ligand-binding domain, a transmembrane domain and an intracellular domain which consists of a kinase homology domain and a GC domain
We defined the role of Protein disulfide isomerase (PDI) on NP-mediated cGMP stimulation in cells using bacitracin, PDI small interfering RNA (siRNA) and an anti-PDI monoclonal antibody, RL90. [17, 18, 19]
To exclude the effects of protease contamination from bacitracin, [23] we determined that bacitracin did not affect the expression of CG-Bin human mesangial cells (HMCs) (Figure S2), and that it did not lose its inhibitory effects on cGMP levels in the presence of protease inhibitors (Figure S3)
Summary
Members of the natriuretic peptide (NP) family, atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) are central regulators of sodium and water balance, blood volume, and arterial pressure as well as myocardial and vascular structure and function. [1] The actions of the NPs are mediated through the guanylyl cyclase (GC)-associated receptors GC-A and GC-B. [2] Both receptors contain three different functional domains, an extracellular ligand-binding domain, a transmembrane domain and an intracellular domain which consists of a kinase homology domain and a GC domain. [3, 4, 5] When ligands bind to the ligand-binding pocket of the dimer, the conformation of receptors is changed and signaling through the transmembrane domain results in ATP binding. These steps are essential for generation of the second-messenger, cGMP. [11, 12, 13] Membrane-bound PDI can catalyze reduction of disulfide bonds in cell surface proteins and augment cell adhesion and migration. The current studies investigate for the first time, the role of PDI as a novel regulator of natriuretic peptide activity specially in the regulation of cGMP generation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
