Alterations in perivascular innervation function in mesenteric arteries from offspring of diabetic rats

Abstract

Background and purpose: We have reported that exposure to a diabetic intrauterine environment during pregnancy increases blood pressure in adult offspring, but the mechanisms involved are not completely understood. This study was designed to analyse a possible role of perivascular sympathetic and nitrergic innervation in the superior mesenteric artery (SMA) in this effect.

Experimental approach: Diabetes was induced in pregnant Wistar rats by a single injection of streptozotocin. Endothelium-denuded vascular rings from the offspring of control (O-CR) and diabetic rats (O-DR) were used. Vasomotor responses to electrical field stimulation (EFS), NA and the NO donor DEA-NO were studied. The expressions of neuronal NOS (nNOS) and phospho-nNOS (P-nNOS) and release of NA, ATP and NO were determined. Sympathetic and nitrergic nerve densities were analysed by immunofluorescence.

Key results: Blood pressure was higher in O-DR animals. EFS-induced vasoconstriction was greater in O-DR animals. This response was decreased by phentolamine more in O-DR animals than their controls. L-NAME increased EFS-induced vasoconstriction more strongly in O-DR than in O-CR segments. Vasomotor responses to NA or DEA-NO were not modified. NA, ATP and NO release was increased in segments from O-DR. nNOS expression was not modified, whereas P-nNOS expression was increased in O-DR. Sympathetic and nitrergic nerve densities were similar in both experimental groups.

Conclusions and implications: The activity of sympathetic and nitrergic innervation is increased in SMA from O-DR animals. The net effect is an increase in EFS-induced contractions in these animals. These effects may contribute to the increased blood pressure observed in the offspring of diabetic rats.

Figure 1

(A) ACh-induced vasodilatation in endothelium-intact mesenteric segments from O-CR and O-DR rats. Results (mean ± SEM) are expressed as a percentage of the previous tone elicited by exogenous NA. anova *P < 0.05 O-CR versus O-DR. n = 12 animals in each group. (B) EFS-induced vasoconstriction in endothelium-intact mesenteric segments from O-CR and O-DR rats. Results (mean ± SEM) are expressed as a percentage of the initial contraction elicited by KCl. *P < 0.05 versus O-CR animals at each frequency (Bonferroni test). n = 12 animals per group. Effect of endothelium removal on the vasoconstrictor response to EFS in mesenteric segments from O-CR (C) and O-DR (D) animals. Results (mean ± SEM) are expressed as a percentage of the initial contraction elicited by KCl. anova *P < 0.05 endothelium-intact versus endothelium-denuded arteries. n = 12 animals per group. (E) dAUC in the absence or presence of endothelium.

Figure 2

Effect of pre-incubation with 1 μmol·L⁻¹ phentolamine or phentolamine plus 0.1 mmol·L⁻¹ suramin on the vasoconstrictor response induced by EFS in endothelium-denuded mesenteric segments from O-CR (A) and O-DR animals (B). Results (mean ± SEM) are expressed as a percentage of the initial contraction elicited by KCl. anova P < 0.05 versus conditions without phentolamine or suramin in both experimental groups. *P < 0.05 versus conditions without phentolamine at each frequency (Bonferroni test). #P < 0.05 conditions phentolamine plus suramin versus conditions without suramin at each frequency (Bonferroni test). n = 8 animals each group. (C) dAUC in the absence or presence of 1 μmol·L⁻¹ phentolamine. dAUC values are expressed as arbitrary units. (D) Representation of the vasoconstriction remaining after pre-incubation with 1 μmol·L⁻¹ phentolamine, expressed as AUC (in arbitrary units).

Figure 3

(A) Vasoconstrictor response to NA in segments from O-CR and O-DR. Results (mean ± SEM) are expressed as a percentage of the initial contraction elicited by KCl. n = 8 animals each group. EFS-induced NA (B) and ATP (C) release in mesenteric segments from O-CR and O-DR animals. Results (mean ± SEM) are expressed as ng NA·mL⁻¹ mg⁻¹ tissue or nmol ATP·mL⁻¹ mg⁻¹ tissue. n = 8 animals per group.

Figure 4

Dopamine β-hydroxylase immunoreactivity in the adventitia of mesenteric arteries from O-CR and O-DR animals (upper panel). Tissues were stained with primary monoclonal dopamine β-hydroxylase (DβH) antibody and a species-specific secondary Alexa 647 antibody. Bar = 75 μm. All images are reconstructions from 10 serial optical sections obtained by LSCM. Lower panel shows percentage of area occupied by sympathetic nerve fibres. n = 6 animals per group. (For a better visualization, brightness and contrast were modified equally in both experimental groups using ImageJ software.)

Figure 5

Effect of pre-incubation with 0.5 μmol·L⁻¹ CGRP (8–37) on the vasoconstrictor response induced by EFS in mesenteric segments from O-CR (A) and O-DR (B) animals. Results (mean ± SEM) are expressed as a percentage of the previous contraction elicited by KCl. n = 8 animals each group.

Figure 6

Effect of pre-incubation with 0.1 mmol·L⁻¹ L-NAME on the vasoconstrictor response induced by EFS in mesenteric segments from O-CR (A) and O-DR (B) rats. Results are expressed as a percentage of the previous contraction elicited by KCl. anova P < 0.05 versus conditions without phentolamine or suramin in both experimental groups. *P < 0.05 versus conditions without L-NAME for each frequency (Bonferroni test). n = 8 animals each group. (C) dAUC in the absence or presence of 0.1 mmol·L⁻¹ L-NAME. dAUC values are expressed as arbitrary units. *P < 0.05.

Figure 7

(A) EFS-induced NO release in segments from O-CR and O-DR rats. Results (mean ± SEM) are expressed as arbitrary (A.U.)·mg⁻¹ tissue; n = 8 animals per group. (B) Effect of exposure to maternal hyperglycaemia on nNOS and P-nNOS expression. The blot is representative of eight separate segments from each group. Rat brain homogenates were used as a positive control. Lower panel shows relationship 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. (C) Vasodilator response to NO donor DEA-NO in segments from O-CR and O-DR rats. Results (mean ± SEM) are expressed as a percentage of the previous tone elicited by exogenous NA. n = 8 animals each group. (D) Superoxide anion release in mesenteric segments from O-CR and O-DR rats. Results (mean ± SEM) are expressed as chemiluminescence U·min⁻¹ mg tissue. n = 8 animals each group.

Figure 8

nNOS immunoreactivity in the adventitia of mesenteric arteries from O-CR and O-DR animals (upper panel). Tissues were stained with primary polyclonal nNOS antibody and a species-specific secondary Alexa 647 antibody. Bar = 75 μm. All images are reconstructions from 10 serial optical sections obtained by LSCM. Lower panel shows percentage of area occupied by nitrergic nerve fibres. n = 6 animals per group.