Intermedin in Paraventricular Nucleus Attenuates Sympathoexcitation and Decreases TLR4-Mediated Sympathetic Activation via Adrenomedullin Receptors in Rats with Obesity-Related Hypertension

Abstract

Intermedin/adrenomedullin-2 (IMD/AM2), a member of the calcitonin gene-related peptide/AM family, plays an important role in protecting the cardiovascular system. However, its role in the enhanced sympathoexcitation in obesity-related hypertension is unknown. In this study, we investigated the effects of IMD in the paraventricular nucleus (PVN) of the hypothalamus on sympathetic nerve activity (SNA), and lipopolysaccharide (LPS)-induced sympathetic activation in obesity-related hypertensive (OH) rats induced by a high-fat diet for 12 weeks. Acute experiments were performed under anesthesia. The dynamic alterations of sympathetic outflow were evaluated as changes in renal SNA and mean arterial pressure (MAP) in response to specific drugs. Male rats were fed a control diet (12% kcal as fat) or a high-fat diet (42% kcal as fat) for 12 weeks to induce OH. The results showed that IMD protein in the PVN was downregulated, but Toll-like receptor 4 (TLR4) and plasma norepinephrine (NE, indicating sympathetic hyperactivity) levels, and systolic blood pressure were increased in OH rats. LPS (0.5 µg/50 nL)-induced enhancement of renal SNA and MAP was greater in OH rats than in obese or control rats. Bilateral PVN microinjection of IMD (50 pmol) caused greater decreases in renal SNA and MAP in OH rats than in control rats, and inhibited LPS-induced sympathetic activation, and these were effectively prevented in OH rats by pretreatment with the AM receptor antagonist AM22-52. The mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) inhibitor U0126 in the PVN partially reversed the LPS-induced enhancement of SNA. However, IMD in the PVN decreased the LPS-induced ERK activation, which was also effectively prevented by AM22-52. Chronic IMD administration resulted in significant reductions in the plasma NE level and blood pressure in OH rats. Moreover, IMD lowered the TLR4 protein expression and ERK activation in the PVN, and decreased the LPS-induced sympathetic overactivity. These results indicate that IMD in the PVN attenuates SNA and hypertension, and decreases the ERK activation implicated in the LPS-induced enhancement of SNA in OH rats, and this is mediated by AM receptors.

Keywords: Intermedin; Obesity-related hypertension; Paraventricular nucleus; Sympathoexcitation; Toll-like receptor 4.

Fig. 1

A representative image of microinjection sites in the PVN evaluated by Evans blue diffusion. PVN, paraventricular nucleus; 3V, third ventricle.

Fig. 2

Plasma norepinephrine (NE) (A, n = 6–8), systolic blood pressure (B, n = 6–8), and relative values of intermedin (IMD) in the paraventricular nucleus (C, n = 3–4) in control, obese (OB), and obesity-related hypertensive (OH) rats. Values are mean ± SEM. *P < 0.05 vs control; ^#P < 0.05 vs OB.

Fig. 3

Effects of paraventricular nucleus (PVN) microinjection of saline or 3 doses of intermedin (IMD, 5, 25, or 50 pmol) on the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) in control (A), obese (B), and obesity-related hypertensive (OH) rats (C), and a comparison of the effects of IMD (50 pmol) on RSNA and MAP in control, obese, and OH rats (D). Values are mean ± SEM. *P < 0.05 vs saline; ^#P < 0.05 vs IMD 5 pmol (A–C); *P < 0.05 vs control; ^#P < 0.05 vs OB (D); n = 6 for each group.

Fig. 4

Relative levels of Toll-like receptor 4 (TLR4) protein in the paraventricular nucleus (PVN) (A) and effects of PVN microinjection of saline or LPS (0.5 µg/50 nL) on renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) in control, obese (OB), and obesity-related hypertensive (OH) rats (B). Values are mean ± SEM. *P < 0.05 vs control; ^#P < 0.05 vs OB (A, n = 3–4); *P < 0.05 vs saline; ^#P < 0.05 vs control; ^$P < 0.05 vs OB (B); n = 6 for each group.

Fig. 5

A Representative recordings showing the effects of saline or IMD (50 pmol) pretreatment of the paraventricular nucleus (PVN) on renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to LPS (0.5 µg/50 nL) in the PVN in obesity-related hypertensive (OH) rats. B Statistical analysis of data as in A. LPS was administered 10 min after the pretreatment. Values are mean ± SEM. *P < 0.05 vs control; ^#P < 0.05 vs saline + saline, ^$P < 0.05 vs saline + LPS; n = 6 for each group.

Fig. 6

Effect of paraventricular nucleus (PVN) pretreatment with saline or IMD (50 pmol) on the LPS (0.5 µg/50 nL)-induced phosphorylated and total MAP kinases (MAPKs). A–C Activation of p38 (A), JNK (B), and ERK (C) MAPKs in the PVN of obesity-related hypertensive (OH) rats. S, Saline. Values are mean ± SEM. *P < 0.05 vs S + S; ^#P < 0.05 vs S + LPS; n = 3–4 for each group.

Fig. 7

Effects of paraventricular nucleus (PVN) pretreatment with saline, the ERK inhibitor U0126 (50 µmol/L) (A), or the AM receptor antagonist AM22-52 and CGRP receptor antagonist CGRP8-37 (B) on the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to LPS (0.5 µg/50 nL) or IMD (50 pmol) in the PVN in obesity-related hypertensive (OH) rats. Values are mean ± SEM. *P < 0.05 vs saline + saline; ^#P < 0.05 vs saline + LPS (A); *P < 0.05 vs saline + saline; ^#P < 0.05 vs saline + IMD (B); n = 6 for each group.

Fig. 8

Effects of paraventricular nucleus (PVN) pretreatment with saline or the AM receptor antagonist AM22-52 on IMD responses to LPS (0.5 µg/50 nL)-induced changes of renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and ERK activation in the PVN in obesity-related hypertensive (OH) rats. Values are mean ± SEM. *P < 0.05 vs S + S + LPS; ^#P < 0.05 vs S + IMD + LPS (A). n = 6 for each group. *P < 0.05 vs S + S + S; ^#P < 0.05 vs S + S + LPS; ^$P < 0.05 vs S + IMD + LPS (B). n = 3–4 for each group. S: Saline.

Fig. 9

Effects of chronic systemic application of intermedin (IMD, 300 ng/kg per hour) for 28 days on the plasma NE level (A), systolic blood pressure (B), and PVN TLR4 protein expression and ERK activation (D, E. n = 3–4), and the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to LPS (0.5 µg/50 nL) in the PVN (C) in obesity-related hypertensive (OH) rats. Values are mean ± SEM. *P < 0.05 vs OH + saline. n = 6–8 for (A–C).