Fetal and postnatal ovine mesenteric vascular reactivity

Abstract

Background: Intestinal circulation and mesenteric arterial (MA) reactivity may play a role in preparing the fetus for enteral nutrition. We hypothesized that MA vasoreactivity changes with gestation and vasodilator pathways predominate in the postnatal period.

Methods: Small distal MA rings (0.5-mm diameter) were isolated from fetal (116-d, 128-d, 134-d, and 141-d gestation, term ~ 147 d) and postnatal lambs. Vasoreactivity was evaluated using vasoconstrictors (norepinephrine (NE) after pretreatment with propranolol and endothelin-1(ET-1)) and vasodilators (NO donors A23187 and s-nitrosopenicillamine (SNAP)). Protein and mRNA assays for receptors and enzymes (endothelin receptor A, alpha-adrenergic receptor 1A (ADRA1A), endothelial NO synthase (eNOS), soluble guanylyl cyclase (sGC), and phosphodiesterase5 (PDE5)) were performed in mesenteric arteries.

Results: MA constriction to NE and ET-1 peaked at 134 d. Relaxation to A23187 and SNAP was maximal after birth. Basal eNOS activity was low at 134 d. ADRA1A mRNA and protein increased significantly at 134 d and decreased postnatally. sGC and PDE5 protein increased from 134 to 141 d.

Conclusion: Mesenteric vasoconstriction predominates in late-preterm gestation (134 d; the postconceptional age with the highest incidence of necrotizing enterocolitis (NEC)) followed by a conversion to vasodilatory influences near the time of full-term birth. Perturbations in this ontogenic mechanism, including preterm birth, may be a risk factor for NEC.

Figure 1. constriction response of ovine mesenteric arteries

(a) Constriction to norepinephrine (NE)—ovine mesenteric arterial (MA) constriction responses to NE 10⁻⁵ and (b) constriction to endothelin-1 10⁻⁷. (c) Constriction to KCl—ovine MA constriction response to 118 mM KCl. (d) Response to l-nitro arginine (LNA)—enhancement of NE induced constriction response by pretreatment with10⁻³ LNA. *P < 0.05 vs. 116 d by Fisher’s protected least significant difference (PLSD). *P < 0.05 vs. 116-d fetus by Fisher’s PLSD. White bars, 116-d fetus; light gray bars, 128-d fetus; dark gray bars, 134-d fetus; black bars, 141-d fetus; and hatched bars, postnatal. **P < 0.05 vs. 128-d fetus by Fisher’s PLSD. ^†P < 0.05 vs. postnatal by Fisher’s PLSD.

Figure 2. relaxation response of ovine mesenteric arteries to nitric oxide donors

(a) A23187—ontogeny of preterm ovine mesenteric arterial relaxation response to nitric oxide donor A23187 and (b) s-nitrosopenicil-lamine (SNAP). ◆, 116-d fetus;□ , 128-d fetus; ▲, 134-d fetus; •, 141-d fetus∙; and ×, postnatal. *P < 0.05 vs. 116-d fetus by ANOVA repeated measures. ^†P < 0.05 vs. postnatal by ANOVA repeated measures. ^‡P < 0.05 vs. 141-d fetus by ANOVA repeated measures. NE, norepinephrine.

Figure 3. changes in mrna and protein expression for constrictor pathway

(a) QRTPCR mRNA analysis of constrictors: real-time PCR assays for alpha-adrenergic receptor 1A (ADRA1A) and endothelin receptor A (ETRA) plotted as fold change compared with postnatal ovine mesenteric arteries. (b). Quantitative protein assay by enzyme-linked immunosorbent assay of constrictors—ADRA1A and ETRA. *P < 0.05 vs. 116-d fetus (white bars).**P < 0.05 vs. 128-d fetus (light gray bars), ^‡P < 0.05 vs. 141-d fetus (black bars), ^†P < 0.05 vs. postnatal (hatched bars) by Fisher’s protected least significant difference. 134-d group is represented by dark gray bars.

Figure 4. changes in mrna and protein expression of enzymes belonging to the no pathway

(a) QRTPCR and (b) western blot analysis of endothelial NO synthase. (c) QRTPCR and (d) western blot analysis of soluble guanylyl cyclase (sGC) and phosphodiesterase5 (PDE5). QRTPCR results plotted as fold change compared with postnatal (hatched bars) ovine mesenteric arteries. Western blot results depicted as values normalized to corresponding actin. *P < 0.05 vs. 116-d fetus (white bars), **P < 0.05 vs. 128-d fetus (light gray bars), ^†P < 0.05 vs. postnatal, ^‡P < 0.05 vs. 141-d fetus (black) by Fisher’s protected least significant difference. 134-d group is represented by dark gray bars.

Figure 5

Summary of the vasoconstrictor and vasorelaxant properties assessed in the study tabulated based on GA. Figure shows the site of action of the various chemical mediators used to assess each specific response. ADRA1A, alpha-adrenergic receptor 1A; eNOS, endothelial NO synthase; ET-1, endothelin-1; ETRA, endothelin receptor A; LNA, l-nitro arginine; NE, norepinephrine; PDE5, phosphodiesterase5; sGC, soluble guanylyl cyclase; SNAP, s-nitrosopenicillamine.