Effects of atrial natriuretic peptide on the extrasplenic microvasculature and lymphatics in the rat in vivo

Abstract

We developed a novel model using fluorescent intravital microscopy to study the effect of atrial natriuretic peptide (ANP) on the extrasplenic microcirculation. Continuous infusion of ANP into the splenic artery (10 ng min(-1) for 60 min) of male Long-Evans rats (220-250 g, n = 24) induced constriction of the splenic arterioles after 15 min (-7.2 +/- 6.6% from baseline diameter of 96 +/- 18.3 microm, mean +/- S.E.M.) and venules (-14.4 +/- 4.0% from 249 +/- 25.8 microm; P < 0.05). At the same time flow did not change in the arterioles (from 1.58 +/- 0.34 to 1.27 +/- 0.27 ml min(-1)), although it decreased in venules (from 1.67 +/- 0.23 to 1.15 +/- 0.20 ml min(-1)) and increased in the lymphatics (from 0.007 +/- 0.001 to 0.034 +/- 0.008 ml min(-1); P < 0.05). There was no significant change in mean arterial pressure (from 118 +/- 5 to 112 +/- 5 mmHg). After continuous ANP infusion for 60 min, the arterioles were dilated (108 +/- 16 microm, P < 0.05) but the venules remained constricted (223 +/- 24 microm). Blood flow decreased in both arterioles (0.76 +/- 0.12 ml min(-1)) and venules (1.03 +/- 0.18 ml min(-1); P < 0.05), but was now unchanged from baseline in the lymphatics (0.01 +/- 0.001 ml min(-1)). This was accompanied by a significant decrease in MAP (104 +/- 5 mmHg; P < 0.05). At 60 min, there was macromolecular leak from the lymphatics, as indicated by increased interstitial fluorescein isothiocyanate-bovine serum albumin fluorescence (grey level: 0 = black; 255 = white; from 55.8 +/- 7.6 to 71.8 +/- 5.9, P < 0.05). This study confirms our previous proposition that, in the extrasplenic microcirculation, ANP causes greater increases in post- than precapillary resistance, thus increasing intrasplenic capillary hydrostatic pressure (P(c)) and fluid efflux into the lymphatic system. Longer-term infusion of ANP also increases Pc, but this is accompanied by increased 'permeability' of the extrasplenic lymphatics, such that fluid is lost to perivascular third spaces.

Figure 1

A, extrasplenic lymphatics can be observed following intravascular administration of FITC–BSA and epi-illumination with blue light (Hitachi KP-M2 S1 camera). B, arteriole and venule (∼30 μm) accompanying lymphatic vessel (in centre; Hitachi KP-161 camera). C, arteriole, venule and lymphatic vessel illuminated with transmitted light. D, same specimen as C, but taken approximately 3 s later. Arrow shows direction of flow. In C and D, lymphocytes are within the box and are clearly seen moving at a continuous steady rate within the lymphatic vessels.

Figure 2

The effect of ANP infusion (10 ng min⁻¹; shaded bars) or 1.5 ml h⁻¹ saline (open bars) on the percentage change (mean ± s.e.m.) from baseline in the diameters of large extrasplenic arterioles (A, baseline diameter for ANP group 96.3 ± 18.0 μm, and for saline group 101.1 ± 9.6 μm) and venules (C, baseline for ANP group 249.0 ± 25.8 μm, and for saline group 269.0 ± 49.7 μm) and centreline erythrocyte velocity within extrasplenic large arterioles (B) and venules (D). *P < 0.05 ANP versus baseline, †P < 0.05 versus control.

Figure 3

The effect of ANP infusion (10 ng min⁻¹; shaded bars) or 1.5 ml h⁻¹ saline (open bars) on the percentage change (mean ± s.e.m.) from baseline in the diameters of small extrasplenic arterioles (A, baseline diameter for ANP group 22.5 ± 3.6 μm, and for saline group 24.7 ± 0.9 μm) and venules (B, baseline for ANP group 32.1 ± 6.9 μm, and for saline group 33.7 ± 6.3 μm) *P < 0.05 ANP versus baseline, †P < 0.05 versus control.

Figure 4

The effect of ANP infusion (10 ng min⁻¹; shaded bars) or 1.5 ml h⁻¹ saline (open bars) on the percentage change (mean ± s.e.m.) from baseline in diameter of extrasplenic lymphatics (A, baseline diameter for ANP group 89.4 ± 10.2 μm, and for saline group 89.7 ± 11.1 μm) and fluid velocity within the extrasplenic lymphatics. *P < 0.05 ANP versus baseline, †P < 0.05 ANP versus control.

Figure 5

The effect of ANP infusion (10 ng min⁻¹; shaded bars) or 1.5 ml h⁻¹ saline (open bars) on interstitial fluorescence (0 = black, 255 = white) within the interstitium adjacent to extrasplenic postcapillary venules and lymphatics. Increased grey level indicates macromolecular leak and a compromise of the lymphatic endothelium. *P < 0.05 ANP versus baseline, †P < 0.05 ANP versus control.