Alterations in perivascular sympathetic and nitrergic innervation function induced by late pregnancy in rat mesenteric arteries

Abstract

Background and purpose: We investigated whether pregnancy was associated with changed function in components of perivascular mesenteric innervation and the mechanism/s involved.

Experimental approach: We used superior mesenteric arteries from female Sprague-Dawley rats divided into two groups: control rats (in oestrous phase) and pregnant rats (20 days of pregnancy). Modifications in the vasoconstrictor response to electrical field stimulation (EFS) were analysed in the presence/absence of phentolamine (alpha-adrenoceptor antagonist) or L-NAME (nitric oxide synthase-NOS- non-specific inhibitor). Vasomotor responses to noradrenaline (NA), and to NO donor DEA-NO were studied, NA and NO release measured and neuronal NOS (nNOS) expression/activation analysed.

Key results: EFS induced a lower frequency-dependent contraction in pregnant than in control rats. Phentolamine decreased EFS-induced vasoconstriction in segments from both experimental groups, but to a greater extent in control rats. EFS-induced vasoconstriction was increased by L-NAME in arteries from both experimental groups. This increase was greater in segments from pregnant rats. Pregnancy decreased NA release while increasing NO release. nNOS expression was not modified but nNOS activation was increased by pregnancy. Pregnancy decreased NA-induced vasoconstriction response and did not modify DEA-NO-induced vasodilation response.

Conclusions and implications: Neural control of mesenteric vasomotor tone was altered by pregnancy. Diminished sympathetic and enhanced nitrergic components both contributed to the decreased vasoconstriction response to EFS during pregnancy. All these changes indicate the selective participation of sympathetic and nitrergic innervations in vascular adaptations produced during pregnancy.

Fig 1. Vasodilator response to ACh.

ACh-induced vasodilation in endothelium-intact mesenteric segments from control and pregnant rats. Results (mean±SEM) were expressed as a percentage of the previous tone elicited by exogenous NA. n = 6 animals each group.

Fig 2. Vasoconstrictor response to EFS.

EFS-induced vasoconstriction in endothelium intact (A) and endothelium-denuded (B) mesenteric segments from control and pregnant rats. Results (mean±SEM) were expressed as a percentage of the initial contraction elicited by KCl. ANOVA P<0.05 Control vs. pregnant in both endothelium intact (A) and endothelium-denuded (B). *P<0.05 vs. control animals at each frequency (Bonferroni test). n = 10 animals each group.

Fig 3. Effect of pregnancy on sympathetic innervation function.

Effect of preincubation with 1 μmol/L phentolamine on vasonstriction response induced by EFS in endothelium-denuded mesenteric segments from control (A) and pregnant rats (B). Results (mean±SEM) were expressed as a percentage of the initial contraction elicited by KCl. ANOVA P<0.05 vs. conditions without phentolamine in both experimental groups. *P<0.05 vs. conditions without phentolamine at each frequency (Bonferroni test). n = 8 animals per group. (C) Vasoconstriction response to NA in segments of control and pregnant rats. Results (mean±SEM) were expressed as a percentage of the initial contraction elicited by KCl. ANOVA P<0.05 Control vs. pregnant. *P<0.05 vs. control animals at each concentration (Bonferroni test). n = 8 animals per group. (D) EFS-induced NA release in mesenteric segments of control and pregnant rats. Results expressed as ng NA/mL mg tissue. *P<0.05 vs. Control. n = 6 animals per group.

Fig 4. Effect of pregnancy on sensory innervation.

Effect of preincubation with 0.5 μmol/L CGRP (8–37) on the vasoconstrictor response induced by EFS in mesenteric segments from (A) control and (B) pregnant rats. Results (mean±SEM) are expressed as a percentage of the previous contraction elicited by KCl. n = 8 animals per group.

Fig 5. Effect of pregnancy on nitrergic innervation.

Effect of preincubation with 0.1 mmol/L L-NAME on the vasoconstrictor response induced by EFS in mesenteric segments from (A) control and (B) pregnant rats. Results (mean±SEM) are expressed as a percentage of the previous contraction elicited by KCl. ANOVA P<0.05 vs. conditions without L-NAME in both experimental groups. *P<0.05 vs. conditions without L-NAME for each frequency (Bonferroni test). n = 8 animals per group. (C) Differences of area under curve (dAUC) in the absence or presence of 0.1 mmol/L L-NAME, dAUC values are expressed as arbitrary units.*P<0.05 Control vs. pregnant rats.

Fig 6. Effect of pregnancy on neuronal NO synthesis.

(A) Effect of 0.1 mmo/L L-NAME or 0.1 mmol/L 7-NI or 0.1 μmol/L TTX on EFS-induced NO release in segments from control and pregnant rats. Results (mean±SEM) were expressed as arbitrary (A.U.)/mg tissue. *P<0.05 vs. Control; #P<0.05 compared with conditions without specific inhibitor; n = 8 animals per group. (B) Effect of pregnancy on nNOS and P-nNOS expression. The blot is representative of four separate segments from each group. Rat brain homogenates were used as a positive control. Lower panel shows relation between P-nNOS or nNOS expression and β-actin. Results (mean±SEM) are expressed as ratio of the signal obtained for each protein and the signal obtained for β-actin. *P<0.05 Control vs. pregnant rats.

Fig 7. Effect of pregnancy on DEA-NO vasodilation and superoxide anion production.

(A) Vasodilator response to NO donor DEA-NO in segments from control and pregnant rats. Effect of preincubation with tempol (0.1 mmol/L) on vasodilator response to NO donor DEA-NO in segments from (B) control and (C) pregnant rats. Results (mean±SEM) are expressed as a percentage of the previous tone elicited by exogenous NA. ANOVA P<0.05 vs. conditions without tempol in both experimental groups. *P<0.05 vs. conditions without tempol for each concentration (Bonferroni test). n = 7 animals per group. Insert graph shows differences of area under curve (dAUC) in the absence or presence of 0.1 mmol/L tempol; dAUC values are expressed as arbitrary units. (D) Effect of 0.3 mmol/L apocinin or 0.1 mmol/L allopurinol on superoxide anion release in mesenteric segments from control and pregnant rats. Results (mean±SEM) are expressed as chemoluminiscence units (U)/min mg tissue. *P<0.05 vs. Control; #P<0.05 compared with conditions without specific inhibitor; n = 7 animals per group.