Vasopressin-dependent Inhibition of the C-type Natriuretic Peptide Receptor, NPR-B/GC-B, Requires Elevated Intracellular Calcium Concentrations

Abstract

Natriuretic peptides bind their cognate cell surface guanylyl cyclase receptors and elevate intracellular cGMP concentrations. In vascular smooth muscle cells, this results in the activation of the type I cGMP-dependent protein kinase and vasorelaxation. In contrast, pressor hormones like arginine-vasopressin, angiotensin II, and endothelin bind serpentine receptors that interact with G q and activate phospholipase C␤. The products of this enzyme, diacylglycerol and inositol trisphosphate, activate the conventional and novel forms of protein kinase C (PKC) and elevate intracellular calcium concentrations, respectively. The latter response results in vasoconstriction, which opposes the actions of natriuretic peptides. Previous reports have shown that pressor hormones inhibit natriuretic peptide receptors NPR-A or NPR-B in a variety of different cell types. Although the mechanism for this inhibition remains unknown, it has been universally accepted that PKC is an obligatory component of this pathway primarily because pharmacologic activators of PKC mimic the inhibitory effects of these hormones. Here, we show that in A10 vascular smooth muscle cells, neither chronic PKC down-regulation nor specific PKC inhibitors block the AVP-dependent desensitization of NPR-B even though both processes block PKC-dependent desensitization. In contrast, the cell-permeable calcium chelator, BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N,Ntetraacetic acid, tetraacetoxymethyl ester), abrogates the AVP-dependent desensitization of NPR-B, and ionomycin, a calcium ionophore, mimics the AVP effect. These data show that the inositol trisphosphate/calcium arm of the phospholipase C pathway mediates the desensitization of a natriuretic peptide receptor in A10 cells. In addition, we report that CNP attenuates AVPdependent elevations in intracellular calcium concentrations. Together, these data reveal a dominant role for intracellular calcium in the reciprocal regulation of these two important vasoactive signaling systems.

Sphingosine-1-Phosphate Inhibits C-Type Natriuretic Peptide Activation of Guanylyl Cyclase B (GC-B/NPR-B)

“…To block the activation of PKC, the cells were incubated for 1 hour with 1 mol/L GF-109203X, a cell permeable inhibitor of the "classic" (␣1, ␤1, ␤2, ␦, ⑀, and ␥) PKC isoforms, 23 prior to the addition of S1P or PMA. As previously reported, 14 pretreatment with the PKC inhibitor completely blocked the PMA-dependent desensitization. In contrast, the PKC inhibitor failed to prevent the majority of S1P-dependent desensitization of NPR-B, indicating that the GF-109203X sensitive forms of PKC are not involved in this process ( Figure 6).…”

“…Cells were stimulated with various agents, and intracellular cGMP concentrations were determined as previously described. 14,16 …”

“…As a model system, we used rat aortic smooth muscle A10 cells that we have previously shown to express NPR-B but not NPR-A. 14 We incubated confluent, serum-starved A10 cells with or without 1 mol/L S1P for 30 minutes and then stimulated cGMP synthesis with increasing concentrations of CNP ( Figure 2). At each CNP concentration tested, S1P reduced the amount of intracellular cGMP detected.…”

“…12,13 A third natriuretic peptide binding protein, natriuretic peptide receptor-C (NPR-C), is structurally related to NPR-A and NPR-B, but it does not possess any known enzymatic activity. 10,11 NPR-B activity is inhibited by hormones such as argininevasopressin 14 or growth factors like platelet-derived growth factor (PDGF) 15 that activate phospholipase C. These agents counter the actions of natriuretic peptides by stimulating vasoconstriction and cell proliferation. Fetal bovine serum elicits the most dramatic inhibitory effect on NPR-B activity, 15 but the serum factors responsible for this effect are currently unknown.…”

Furin-mediated Processing of Pro-C-type Natriuretic Peptide

“…To determine whether the furin-processed recombinant CNP is biologically active, an aortic smooth muscle cell-based cGMP assay was performed (37). As shown in Fig.…”