The heart communicates with the endothelium through the guanylyl cyclase-A receptor: acute handling of intravascular volume in response to volume expansion

Abstract

Atrial natriuretic peptide (ANP) regulates arterial blood pressure and volume. Its guanylyl cyclase-A (GC-A) receptor is expressed in vascular endothelium and mediates increases in cGMP, but the functional relevance is controversial. Notably, mice with endothelial-restricted GC-A deletion [EC GC-A knockout (KO) mice] exhibit significant chronic hypervolemic hypertension. The present study aimed to characterize the endothelial effects of ANP and their relevance for the acute regulation of intravascular fluid volume. We studied the effect of ANP on microvascular permeability to fluorescein isothiocyanate-labeled albumin (BSA) using intravital microscopy on mouse dorsal skinfold chambers. Local superfusion of ANP (100 nm) increased microvascular fluorescein isothiocyanate-BSA extravasation in control but not EC GC-A KO mice. Intravenous infusion of synthetic ANP (500 ng/kg x min) caused immediate increases in hematocrit in control mice, indicating intravascular volume contraction. In EC GC-A KO mice, the hematocrit responses were not only abolished but even reversed. Furthermore, acute vascular volume expansion, which caused release of endogenous cardiac ANP, did not affect resting central venous pressure of control mice but rapidly and significantly increased central venous pressure of EC GC-A KO mice. In cultured lung endothelial cells, ANP provoked cGMP-dependent protein kinase I-mediated phosphorylation of vasodilator-stimulated phosphoprotein. We conclude that ANP, via GC-A, enhances microvascular endothelial macromolecule permeability in vivo. This effect might be mediated by cGMP-dependent protein kinase I-dependent phosphorylation of vasodilator-stimulated phosphoprotein. Modulation of transcapillary protein and fluid transport may represent one of the most important hypovolemic actions of ANP.

Figure 1

Effects of ANP on microvascular permeability to FITC-albumin in mice with dorsal skinfold chambers. Relative changes in interstitial fluorescence intensity were evaluated during 30 min of continuous local ANP (100 nm) or vehicle superfusion. A, ANP increases albumin extravasation in wild-type (GC-A^+/+) mice. B, The permeability responses to ANP are abolished in mice with global GC-A deletion (GC-A^−/−). C, ANP increases albumin extravasation in control mice (floxed GC-A, with normal GC-A expression levels). D, The permeability responses to ANP are abolished in mice with conditional, endothelial GC-A deletion (EC GC-A KO) (n = 7–9 mice per genotype and treatment). *, P < 0.05 vs. vehicle.

Figure 2

Effects of histamine on microvascular permeability to FITC-albumin in EC GC-A KO and corresponding control mice. Relative changes in interstitial fluorescence intensity were evaluated during 30 min of continuous local histamine (80 μm) superfusion (n = 7 mice per genotype).

Figure 3

A, Effect of iv ANP (500 ng/kg BW· min, as an infusion) on hematocrit values of control mice (with unaltered GC-A expression levels) and mice with either endothelial (EC GC-A KO) or global GC-A deletion (GC-A^−/−). Note that at 30 and 60 min of ANP infusion, control mice show significant increases in hematocrit. Notably, this effect was not only abolished but also even reversed in EC GC-A KO and GC-A^−/− mice. B, The selective NPR-C ligand cANP_(4–23) (250 ng/kg BW·min, as an infusion) significantly decreased hematocrit in mice from all three genotypes (n = 6–8 per genotype). *, P < 0.05, compared with basal values at time 0.

Figure 4

Effect of acute vascular volume expansion on jugular venous blood pressure. A, Volume expansion in wild-type (GC-A^+/+) mice had no effect on jugular venous pressure. In contrast, in mice with global GC-A deletion (GC-A^−/−), volume expansion caused a rapid and significant increase in venous pressure, which did not fully reverse during the experimental observation time. B, Effect of acute vascular volume expansion on jugular venous blood pressure of control and EC GC-A KO mice. Whereas the former tolerated intravascular volume expansion without changes in jugular venous pressure, EC GC-A KO mice responded with significant increases in venous pressure, which partly reversed during the following observation time (n = 7–9 per genotype). *, P < 0.05 vs. baseline; #, P < 0.05 vs. respective GC-A^+/+ or control mice.

Figure 5

A. Effect of ANP on intracellular cGMP content of MLECs. ANP increased cGMP in wild-type (GC-A^+/+) but not GC-A^−/− MLECs. B and C, Western blot analyses. GC-A protein was present in primary cultures of GC-A^+/+ and absent in GC-A^−/− cells. PKG I was present in MLECs from both genotypes, expression levels being significantly increased in GC-A-deficient cells. ANP stimulates the phosphorylation of VASP at the PKG I preferred site, Ser₂₃₅, in GC-A^+/+ but not GC-A^−/− MLECs.