Angiotensin-(1-7) inhibits neuronal activity of dorsolateral periaqueductal gray via a nitric oxide pathway

Abstract

The midbrain periaqueductal gray (PAG) is a neural site for several physiological functions related to cardiovascular regulation, pain modulation and behavioral reactions. Recently, angiotensin-(1-7) [Ang-(1-7)] has been considered as an important biologically active component of the renin-angiotensin system in the CNS. The purpose of this study was to determine (1) existence of Ang-(1-7) receptor, Mas-R, within the dorsolateral PAG (dl-PAG), (2) the role for Ang-(1-7) in modulating activity of dl-PAG neurons, and (3) the mechanisms by which Ang-(1-7) plays a regulatory role. Western blot analysis showed that Mas-R appears within the dl-PAG. Whole cell patch-clamp recording demonstrated that the discharge rates of dl-PAG neurons were decreased from 4.35±0.32 Hz of control to 1.06±0.34 Hz (P<0.05, vs. control) by 100 nM of Ang-(1-7). With pretreatment of A-779, a Mas-R inhibitor, the discharge rate was 4.66±0.62 Hz (P>0.05, vs. control) during infusion of Ang-(1-7). Additionally, neuronal nitric oxide synthase (nNOS) was largely localized within the dl-PAG among the three isoforms. The effects of Ang-(1-7) on neuronal activity of the PAG were attenuated in the presence of S-methyl-L-thiocitrulline (SMTC), a nNOS inhibitor. The discharge rates were 4.21±0.39 Hz in control and 4.09±0⋅47 Hz (P>0.05, vs. control) when Ang-(1-7) was applied with pretreatment of SMTC. Those findings suggest that Ang-(1-7) plays an inhibitory role in the dl-PAG via a NO dependent signaling pathway. This offers the basis for the physiological role of Ang-(1-7) and Mas R in the regulation of various functions in the CNS.

Figure 1

Dual representative bands illustrate Mas-R and three isoforms of NOS expression. Mas-R is apparently present in the dl-PAG. Among the three isoforms, expression of nNOS is greater than eNOS and iNOS. Note that the same amount of protein was sampled to examine their individual expression. Beta-actin was used as control for equal loading of protein.

Figure 2

Ang-(1-7) had an inhibitory effect on the firing activity of dl-PAG neurons. A:Original tracings from a dl-PAG neuron show the spontaneous discharge during control, Ang-(1-7) (10 nM -1μM) perfusion and washout for a recovery. B: Average data (n=33) for three dosages of Ang-(1-7). *P < 0.05, vs. control and recovery.

Figure 3

A prior application of A-779 to block Mas-R antagonized the effects of Ang-(1-7) on the firing activity of dl-PAG neurons. A: Original tracings from a dl-PAG neuron. B: average data (n=19) show the spontaneous discharge during control, Ang-(1-7), recovery, A-779 (10 μM), and Ang-(1-7) perfusion in the presence of A-779. The effect of Ang-(1-7) was abolished after application of Mas-R antagonist. *P < 0.05, vs. control and recovery. There were no significant differences among control, recovery, A-779 and A-779 plus Ang-(1-7).

Figure 4

An inhibitory effect of Ang-(1-7) on the firing activity dl-PAG neurons was abolished with prior application of SMTC to attenuate nNOS. A: Original tracings from a dl-PAG neuron show the spontaneous discharge during control, Ang-(1-7) perfusion (100 nM), washout, SMTC (1 μM) and Ang-(1-7) perfusion in the presence of nNOS blocker. B: Average data (n=8). *P < 0.05, vs. control and recovery. No significant differences were observed among control, recovery, SMTC and SMTC plus Ang-(1-7).