Atrial natriuretic peptide enhances microvascular albumin permeability by the caveolae-mediated transcellular pathway

Abstract

Aims: Cardiac atrial natriuretic peptide (ANP) participates in the maintenance of arterial blood pressure and intravascular volume homeostasis. The hypovolaemic effects of ANP result from coordinated actions in the kidney and systemic microcirculation. Hence, ANP, via its guanylyl cyclase-A (GC-A) receptor and intracellular cyclic GMP as second messenger, stimulates endothelial albumin permeability. Ultimately, this leads to a shift of plasma fluid into interstitial pools. Here we studied the role of caveolae-mediated transendothelial albumin transport in the hyperpermeability effects of ANP.

Methods and results: Intravital microscopy studies of the mouse cremaster microcirculation showed that ANP stimulates the extravasation of fluorescent albumin from post-capillary venules and causes arteriolar vasodilatation. The hyperpermeability effect was prevented in mice with conditional, endothelial deletion of GC-A (EC GC-A KO) or with deleted caveolin-1 (cav-1), the caveolae scaffold protein. In contrast, the vasodilating effect was preserved. Concomitantly, the acute hypovolaemic action of ANP was abolished in EC GC-A KO and Cav-1(-/-) mice. In cultured microvascular rat fat pad and mouse lung endothelial cells, ANP stimulated uptake and transendothelial transport of fluorescent albumin without altering endothelial electrical resistance. The stimulatory effect on albumin uptake was prevented in GC-A- or cav-1-deficient pulmonary endothelia. Finally, preparation of caveolin-enriched lipid rafts from mouse lung and western blotting showed that GC-A and cGMP-dependent protein kinase I partly co-localize with Cav-1 in caveolae microdomains.

Conclusion: ANP enhances transendothelial caveolae-mediated albumin transport via its GC-A receptor. This ANP-mediated cross-talk between the heart and the microcirculation is critically involved in the regulation of intravascular volume.

Figure 1

Endothelium-restricted disruption of GC-A preserves the vasodilating but prevents the permeability actions of ANP in the mouse cremaster microcirculation. (A) Effect of ANP (100 nM) and SNP (30 μM) on arteriolar diameters in control and EC GC-A KO mice. Left, absolute changes in vessel diameter; right, the effect of ANP is expressed as a percentage of the maximal vasodilatation evoked by subsequent topical application of SNP. (B and C) Time course of changes in net integrated optical intensity (IOI; an index of permeability) during 30 min of continuous local ANP (100 nM) or vehicle superfusion in control mice (B) and in mice with conditional, endothelial GC-A deletion (EC GC-A KO; C; n = 6 mice per genotype and treatment; *P < 0.05 vs. vehicle). As shown in the original photographs, the hyperpermeability effects of histamine are much greater than the effects of ANP.

Figure 2

ANP, in contrast to histamine, does not stimulate the extravasation of FITC–dextran within the cremaster microcirculation. Net IOI (an index of permeability) was evaluated during 40 min of continuous local vehicle (saline) or ANP (100 nM) superfusion. Thereafter, histamine (10 μM) was superfused for an additional 10 min (n = 5 mice per treatment; *P < 0.05 vs. vehicle).

Figure 3

ANP increases cGMP content and stimulates endocytosis and transcellular transport of albumin in microvascular rat fat pad endothelial cells (RFPECs). Effect of ANP on cyclic GMP content (A), uptake of Alexa 488-labelled albumin (B), transendothelial FITC–BSA permeability (C), and TER (D). In (B), ANP effects were abolished after pre-treatment of RFPECs with MβCD (2 mM, 15 min). In (C and D), thrombin was used for comparison. (B) Confocal images showing ANP-induced concentration-dependent increases in the uptake of Alexa 488-labelled albumin (green). The nucleus (blue) was stained with DAPI. Results are typical of three to four experiments. *P < 0.05 compared with vehicle.

Figure 4

Murine microvascular lung endothelial cells (MLECs) express GC-A receptors, a subpopulation being present in caveolin-enriched lipid rafts. (A) Effect of ANP on intracellular cyclic GMP content. ANP increases cGMP in WT but not in GC-A-deficient MLECs. In Cav-1-deficient MLECs, the maximal cGMP responses are slightly but not significantly diminished. (B) Peripheral lung tissues were homogenized and subfractionated to isolate light buoyant density membranes (rafts). Western blotting demonstrates the localization of GC-A and cGKI in caveolin-enriched lipid rafts (fraction 5). Immunoblots are representative of three separate experiments.

Figure 5

ANP, via GC-A, stimulates caveolae-mediated albumin endocytosis in MLECs. (A) Left, confocal images showing the uptake of Alexa 488-labelled albumin (green). The nucleus (blue) was stained with DAPI. Right, fluorescence, quantified as pixel intensity per cell using confocal microscopy, showed that ANP induced concentration-dependent increases in Alexa 488-albumin uptake by WT MLECs. These responses were abolished after pre-treatment of WT MLECs with MβCD (2 mM, 15 min; A) as well as in GC-A-deficient (B) or in Cav-1-deficient MLECs (C). For each genotype, n = 3–4. *P < 0.05 compared with vehicle.

Figure 6

The microvascular hyperpermeability effects of ANP are abolished in Cav-1-deficient mice, while the vasodilatating effects are preserved. (A) Effect of ANP (100 nM) and SNP (30 μM) on arteriolar diameters in control and Cav-1^−/− mice. Left, absolute changes in vessel diameter; right, the effect of ANP is expressed as a percentage of the maximal vasodilatation evoked by subsequent topical application of SNP. (B and C) Time course of changes in net IOI during 30 min of continuous local ANP (100 nM) or vehicle superfusion. (B) ANP increases FITC–BSA extravasation in control mice. (C) The permeability responses to ANP are abolished in mice with deleted Cav-1 (n = 8 mice per genotype and treatment; *P < 0.05 vs. vehicle).