Activation of central angiotensin type 2 receptors suppresses norepinephrine excretion and blood pressure in conscious rats

Abstract

Background: We have previously documented the finding that central angiotensin type 2 receptors (AT2R) negatively modulate sympathetic outflow and arterial blood pressure (BP). In this study, we investigated the effects of intracerebroventricular (icv) infusion of Compound 21 (C21), the first selective nonpeptide AT2R agonist, on norepinephrine (NE) excretion and BP in rats.

Methods: C21 was infused icv for 7 days, using a micro-osmotic pump. Urinary NE concentration was measured using the NE enzyme immunoassay kit. BP was recorded by radiotelemetry. After 7 days, the rats were killed and three relevant samples from sympathetic brain regions and the cerebral cortex were obtained by micro-punching to measure neuronal nitric oxide synthase (nNOS) protein expression by western blot. In addition, the influence of C21 on neuronal potassium current (I(Kv)) was determined by whole-cell patch-clamp in a neuron cell line, CATH.a.

Results: (i) Icv treatment with C21 significantly decreased both the concentration and the amount of NE in night time urine, but had no effect on daytime urine. (ii) C21-treated rats exhibited a slight but significant decrease in BP. (iii) The effects of C21 on NE excretion and BP were abolished by use of the AT2R antagonist, PD123319, and nitric oxide synthase (NOS) inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME). (iv) C21 treatment significantly upregulated nNOS expression in the paraventricular nucleus of the hypothalamus (PVN) and rostral ventrolateral medulla (RVLM), but not in the nucleus of the solitary tract (NTS) and cerebral cortex. (v) In CATH.a neurons, C21 treatment significantly increased I(Kv), and this increase was completely abolished by PD123319 and L-NAME.

Conclusions: These results demonstrate a central inhibitory influence of C21 on sympathetic outflow by means of a nNOS-dependent mechanism that might be mediated by facilitating the neuronal potassium channel.

Figure 1

Panels A and B show NE concentration and NE volume in daytime and nighttime urine. ^#P < 0.05 compared with days 1 to 7; *P < 0.05 compared with the daytime urine of control rats and the nighttime urine of C21 treated rats, n = 8 for the control group and 10 for the C21 treated group. Panels C and D show the blockade of AT2R antagonist and NOS blocker on C21-induced effects at the day 7. *P < 0.05. n = 8 for control, 10 for C21, 9 for C21 plus PD123319, and 8 for C21 plus L-NAME.

Figure 2

Daytime and nighttime mean arterial pressure (MAP). Panel A: MAP in control (n = 8) and C21 treated rats (n = 10). *P < 0.05 compared with control daytime; #P < 0.05 compared with control nighttime. Panel B: MAP at the day 7 after treatment of control (n = 8), C21 (n = 10), C21 plus PD123319 (n = 9), and C21 plus L-NAME (n =8). *P < 0.05 compared with control, #P < 0.05 compared with C21.

Figure 3

nNOS protein expression in the cortex, RVLM, PVN, and NTS from control (n = 8) and C21 treated rats (n = 10) at the day 7 post treatment. *P < 0.05.

Figure 4

I_Kv in CATH.a cells measured by whole cell patch clamp. Top: representative current tracings; Bottom: mean data of I_Kv current densities. *P < 0.001 and ⁺P < 0.05. n = 14 for C21, 10 for C21 plus PD123319, and 9 for C21 plus L-NAME treated cells.