Early life stress enhances angiotensin II-mediated vasoconstriction by reduced endothelial nitric oxide buffering capacity

Abstract

We reported previously that maternal separation (MS) sensitizes adult rats to angiotensin II (Ang II)-induced hypertension. The aim of this study was to investigate the vascular reactivity to Ang II and the role of renin-angiotensin system components, reactive oxygen species production, and NO synthase (NOS) buffering capacity mediating the exacerbated Ang II-induced responses. MS rats were separated from their mothers for 3 h/d from days 2 to 14 of life. Controls were nonhandled littermates. At 12 weeks of age, aortic Ang II-induced constriction was greater from MS rats compared with controls (P<0.05); moreover, endothelial denudation abolished this difference. The response to other constrictors was unchanged. Angiotensin type 2 receptor function was reduced in aortic Ang II-induced constriction from MS rats compared with controls. Angiotensin type 1 receptor function was similarly abolished in both groups. However, protein expressions of angiotensin type 1 and angiotensin type 2 receptors were similar in aortic rings from MS and control rats. Preincubation with superoxide inhibitor or scavenger attenuated the Ang II-induced vasoconstriction in control but not in MS rats. However, acute preincubation with an NOS inhibitor enhanced aortic Ang II-induced constriction in aorta from control rats, but this effect was significantly reduced in MS rats compared with control rats. Accordingly, a further increase in Ang II-induced hypertension attributed to chronic NOS inhibition (days 10 to 13) was blunted in MS rats compared with control rats. Similar NOS expression and activity were observed in control and MS rats. In conclusion, MS induces a phenotype with reduced endothelial NOS buffering capacity leading to dysfunctional endothelial Ang II-mediated signaling and sensitization to Ang II-induced vasoconstriction.

Figure 1

Vascular reactivity in intact thoracic aortic rings. A) Cumulative concentration-response curves of AngII from MS (n=17) and control (n=17) rats. B) AngII-induced constriction in endothelium denuded rings of MS (n=6) and control (n=6) rats. * p< 0.05.

Figure 2

AngII receptor pharmacological blockade. A) Pre-incubation with candesartan (specific AT₁ receptor blocker, 10^-5 M) blunted AngII-induced constriction in MS (n=4) and control (n=4) rats. B) Pre-incubation with PD123,319 (specific AT₂ receptor blocker, 10^-6 M) increased the response to AngII in control rats (n=7), but had no effect in MS rats (n= 7). Data are expressed in percentage of increase from the maximal response to AngII 10^-7 M. Values from the control group represent the baseline (100%). * p<0.05 vs. control.

Figure 3

Expression and localization of AT₁ and AT₂ receptor. A) Representative Western blot of AT₁ and AT₂ protein expression in aortic tissue of control and MS rats with densitometric analysis (n=5-6). Representative immunolocalization of AT₁ receptor (B and C) and AT₂ receptor (E and F) in control and MS aortae. Negative control was assessed by antigen blockade (D and F). Magnification=100X.

Figure 4

Participation of ROS in AngII-induced aortic constriction. A) AngII-induced aortic constriction in control rats (n=12) was reduced after pre-incubation with apocynin (300uM, n=7), B) MS rats showed similar AngII-induced constriction in the presence (n=5) or absence (n=12) of apocynin. C) AngII-induced aortic constriction in control rats (n=12) was reduced after pre-incubation with PEG-SOD (1000 Units/mL, n=6), D) MS rats (n=12) showed similar AngII-induced constriction using PEG-SOD. * p<0.05 vs. untreated.

Figure 5

AngII-induced aortic constriction during NOS blockade. Pre-incubation of intact aortic rings with a NOS inhibitor (L-NAME, 100uM) enhanced AngII-induced constriction in both A) control (n=7) and B) MS (n=6) rats. C) The increase in the percentage of contraction in the L-NAME-treated rings was significantly less in MS compared to control rats. * p<0.05 vs. control.

Figure 6

MAP responses to chronic L-NAME treatment during AngII infusion. MS rats (n=6) have exaggerated MAP to chronic AngII infusion and a blunted pressor response to chronic L-NAME compared to control rats (n=6). A) MAP expressed as 24 hr average. B) Change in the MAP in response to L-NAME treatment after 24 hours. * p<0.05 vs. AngII-infused control rats, # p<0.05 vs. AngII-infused+L-NAME control rats.

Figure 7

Endothelial-dependent and –independent vasorelaxation of aortic rings. A) ACh and B) SNP-induced relaxation were similar in control (n=5) and MS (n=9) rats. C) Total NOS and NOS isoform enzymatic activity were similar in MS (n=7) and control (n=7) rats. D) Representative Western blots for NOS1 and NOS3 from aortic tissue of MS and control rats (top) and densitometric analysis of NOS1 and NOS3 immunoreactivity revealed similar expression in aortic tissue between MS and control rats (bottom, n=4).