Evidence that Nitric Oxide is Involved in the Blood Pressure Lowering Effect of the Peptide AVFQHNCQE in Spontaneously Hypertensive Rats

Figure 1. Graphical representation of the experimental design for Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) and indomethacin study in spontaneously hypertensive rats (SHR) (1A) and graphical representation of the experimental design used to study the effects of peptide administration on the renin-angiotensin-aldosterone system (RAAS), endothelial function, and oxidative stress in SHR (1B).

Figure 2. Changes in systolic blood pressure (SBP) caused in spontaneously hypertensive rats (SHR) 6 h post-administration of water or AVFQHNCQE and saline or Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Data are expressed as the mean ± standard error (SEM) of six animals. Different letters represent statistically significant differences (p ≤ 0.05). The value of p was estimated by one-way ANOVA.

Figure 3. Changes in systolic blood pressure (SBP) caused in spontaneously hypertensive rats (SHR) 6 h post-administration of water or AVFQHNCQE and saline or Indomethacin. Data are expressed as the mean ± standard error (SEM) of six animals. Different letters represent statistically significant differences (p ≤ 0.05). The value of p was estimated by one-way ANOVA.

Figure 4. The effect of the peptide AVFQHNCQE on plasma ACE activity 6 hours after administration in spontaneously hypertensive rats. Values are means ± SEM (n = 6 per group). The results were analyzed with Student’s t test. The differences between the means were considered significant when p ≤ 0.05. No significant differences were found.

Figure 5. Hepatic malondialdehyde (MDA) production (A) and hepatic reduced gluthatione (GSH) concentration (B) 6 hours after administration of the peptide AVFQHNCQE in spontaneously hypertensive rats. Results are expressed as the mean ± SEM (n = 6 per group). The results were analyzed with the Student’s t test, and the differences between the means were considered significant when p ≤ 0.05. The asterisk indicates statistically significant differences.

Figure 6. The effect of the peptide AVFQHNCQE on the expression of genes involved in the nitric oxide pathway (A) and on genes related to vasoconstriction and radical oxygen species production (B) in the endothelium 6 hours after administration in spontaneously hypertensive rats. Data are mean ± SEM (n = 6 per group). The results were analyzed with Student’s t test, and the differences between the means were considered significant when p ≤ 0.05. The asterisks indicate statistically significant differences. eNOS: endothelial nitric oxide synthase; Arg-1: L-arginase; Sirt-1: sirtuin-1; KLF-2: Krüpple-like factor 2; ET-1: endothelin 1; NOX4: NADPH oxidase 4.

Figure 7. Cumulative concentration–response curves of AVFQHNCQE (10−8–10−4 M) in methoxamine pre-constricted aortic rings from Sprague-Dawley rats. Tap water was employed as a control. The same volume of water used to carry out the concentration-response curves of the peptide was added. Data are mean values ± SEM (n = 6 per group). No significant differences were observed between the control group and the AVFQHNCQE group.

Figure 8. Hypertension (HTN) is characterized by the presence of endothelial dysfunction. This pathological state is defined as an imbalance between the vasodilator (mainly nitric oxide, NO) and the vasoconstriction (mainly endothelin-1, ET-1) endothelial factors. The administration of AVFQHNCQE produced an NO-mediated anti-hypertensive effect and it was expected to create an increase in NO bioavailability. NADPH oxidase 4 (NOX4) mRNA expression was found to be decreased in rats administered the peptide, which would lead to a decrease in reactive oxygen species (ROS) production and, thus, to an increase in NO availability. The mRNA expression of ET-1, vasoconstrictor, and inhibitor of NO synthesis, was also found to decrease. Lastly, the peptide increased the production of reduced glutathione (GSH), which is the main endogen antioxidant system that decreases ROS and, thus, reduces NO scavenging. All these findings lead to enhance nitric oxide, and, therefore, reduce the vasoconstriction observed under HTN conditions.