Atrial natriuretic factor increases splenic microvascular pressure and fluid extravasation in the rat

Abstract

The spleen is an important site of atrial natriuretic factor (ANF)-induced fluid extravasation into the systemic lymphatic system. The mechanism underlying this process was studied in a blood-perfused (1 ml min(-1)) rat spleen using the double occlusion technique. To ensure that our observations were spleen specific, a similar protocol was repeated in the hindquarters. Rat ANF(1-28), infused into the splenic artery of anaesthetized male rats, caused a dose-dependent (0.3-59 pmol min(-1)) increase in microvascular pressure from 11.3 +/- 0.7 to 14.9 +/- 0.5 mmHg and in post-capillary resistance from 7.2 +/- 0.6 to 10.1 +/- 1.1 mmHg ml(-1). ANF elicited no change in splenic pre-capillary resistance or in hindquarter haemodynamics. Intrasplenic ANF (6.5 pmol min(-1)) caused a sustained increase in intrasplenic fluid efflux from 0.1 +/- 0.1 to 0.3 +/- 0.1 ml min(-1), and in capillary filtration coefficient (Kf) from 1.2 +/- 0.5 to 2.4 +/- 0.6 ml mmHg-1 min-1 (100 g tissue)-1. Mechanical elevation of splenic intravascular pressure (from 11.3 +/- 0.7 to 22.4 +/- 0.2 mmHg) significantly increased intrasplenic fluid extravasation (from 0.4 +/- 0.3 to 1.4 +/- 0.3 ml min(-1)). The natriuretic peptide receptor-C (NPRC)-specific agonist C-ANF(4-23) (12.5 and 125 pmol min(-1)) did not alter splenic intravascular pressure or pre-/post-capillary resistance. The ANF antagonist A71915 (8.3 and 83 pmol min-1), which blocks ANF-stimulated cGMP production via natriuretic peptide receptor-A (NPRA), inhibited the ANF-induced changes in splenic microvascular pressure and post-capillary resistance. It is concluded that ANF enhances the extravasation of isoncotic fluid from the splenic vasculature both by raising intrasplenic microvascular pressure (increased post-capillary resistance) and by increasing filtration area. The constrictive activity of ANF on the splenic vasculature is mediated through NPRA.

Figure 1. Anatomy of abdominal blood vessels associated with the spleen

This diagram shows the surgical interventions and cannulation sites related to the preparation of the blood-perfused spleen. The stomach has been reflected out of the peritoneal cavity and placed on the thorax to allow access to the gastric vasculature.

Figure 2. Double vascular occlusion technique for determining microvascular pressure

Pressure-time tracings are shown during the double occlusion, where arterial pressure (P_a) and venous pressure (P_v) are shown from the isolated, blood-perfused spleen in control (A) and ANF-infused (59 pmol min⁻¹) animals (B).

Figure 3. Effect of ANF on microvascular pressure (A), pre-capillary resistance (B) and post-capillary resistance (C), in the spleen (•) and hindquarters (○)

ANF was infused continuously intra-arterially into the respective vascular beds, and the responses were measured after 5, 10 and 20 min using the double occlusion technique. Isotonic saline was infused into the control animals (0 pmol min⁻¹). Separate animals were used for each dose of ANF. For the splenic measurement, the sample sizes for each dose were n = 8, 3, 5, 5, 6 and 3, in order of increasing ANF dose. For the hindquarters measurements, the sample sizes for each dose were n = 4, 3, 3 and 3). The vertical error bars indicate s.e.m. *P < 0.05 relative to saline values (ANOVA plus Student-Neuman-Keuls post hoc test for multiple comparisons).

Figure 4. Effect of ANF and portal hypertension on splenic microvascular pressure

ANF was infused continuously via the splenic artery at doses of 6.5 pmol min⁻¹(n = 5) and 59 pmol min⁻¹(n = 3). The control animals were infused with isotonic saline (n = 8). Portal hypertension (PHT) was achieved by tightening a ligature around the portal vein until upstream intraluminal pressure reached 15 mmHg (n = 4). The responses were measured at 5, 10 and 20 min using the double occlusion technique. The vertical error bars indicate s.e.m. *P < 0.05 relative to saline values (ANOVA plus Student-Neuman-Keuls post hoc test for multiple comparisons).

Figure 5. Effects of C-ANF on splenic microvascular pressure (A) and post-capillary resistance (B)

C-ANF, a NPR_C-specific agonist, was infused continuously via the splenic artery at doses of 12.5 pmol min⁻¹(n = 3) and 125 pmol min⁻¹(n = 3). The control animals were infused with isotonic saline. Intrasplenic haemodynamics were measured after 5, 10 and 20 min using the double occlusion technique. The vertical error bars indicate s.e.m. There were no significant differences between the groups.

Figure 6. Effects of the ANF antagonist A71915 on ANF-induced changes in splenic microvascular pressure (A) and post-capillary resistance (B)

A71915 (8.3 pmol min⁻¹,; and 83 pmol min⁻¹, ▪) was co-infused with ANF (6.5 and 65 pmol min⁻¹) continuously via the splenic artery. The control animals were infused with isotonic saline (□). The haemodynamic responses were measured at 5, 10 and 20 min using the double occlusion technique. Separate animals were used for each dose (n = 4 for each group). The vertical error bars indicate s.e.m. *P < 0.05 (ANOVA plus Student-Neuman-Keuls post hoc test for multiple comparisons).