Renin activates PI3K-Akt-eNOS signalling through the angiotensin AT₁ and Mas receptors to modulate central blood pressure control in the nucleus tractus solitarii

Abstract

Background and purpose: The renin-angiotensin system (RAS) is critical for the control of blood pressure by the CNS. Recently, direct renin inhibitors were approved as antihypertensive agents. However, the signalling mechanism of renin, which regulates blood pressure in the nucleus tractus solitarii (NTS) remains unclear. Here we have investigated the signalling pathways involved in renin-mediated blood pressure regulation, at the NTS.

Experimental approach: Depressor responses to renin microinjected into the NTS of Wistar-Kyoto rats were elicited in the absence and presence of the endothelial nitric oxide synthase (eNOS)-specific inhibitor, N(5)-(-iminoethyl)-L-ornithine, Akt inhibitor IV and LY294002, a PI3K inhibitor and GP antagonist-2A [G(q) inhibitor]. Lisinopril (angiotensin converting enzyme inhibitor), losartan, valsartan (angiotensin AT(1) receptor antagonists), D-Ala7-Ang-(1-7) (angiotensin-(1-7) receptor antagonist) were used to study the involvement of RAS on renin-induced depressor effects.

Key results: Microinjection of renin into the NTS produced a prominent depressor effect and increased NO production. Pretreatment with G(q) -PI3K-Akt-eNOS pathway-specific inhibitors significantly attenuated the depressor response evoked by renin. Immunoblotting and immunohistochemical studies further showed that inhibition of PI3K significantly blocked renin-induced eNOS-Ser ¹¹⁷ and Akt-Ser⁴⁷³ phosphorylation in situ. In addition, pre-treatment of the NTS with RAS inhibitors attenuated the vasodepressor effects evoked by renin. Microinjection of renin also increased Ras activation in the NTS.

Conclusions and implications: Taken together, these results suggest renin modulated blood pressure at the NTS by AT₁ and Mas receptor-mediated activation of G(q) and Ras to evoke PI3K-Akt-eNOS signalling.

Figure 1

Systemic blood pressure (BP) and NO production in the NTS after administration of renin to the NTS. (A) Representative tracing show the depressor effect after unilateral microinjection of renin (240 fg) into the NTS. Artificial cerebrospinal fluid (aCSF) as the drug vehicle did not change blood pressure. Summary data (means ± SEM, n= 6) are shown in the graph. *P < 0.05, significantly different from the aCSF group. (B) Levels of NO in samples of the NTS after microinjection of renin. The bar graph shows the NO concentration (as µM nitrate per µg of total protein). Pretreatment with renin significantly elevated NO levels in the NTS compared with aCSF. *P < 0.05, significantly different from the aCSF group.

Figure 2

Microinjection of renin induces eNOS-Ser¹¹⁷⁷ phosphorylation in the NTS. (A) Representative tracing demonstrates that the depressor effect of renin was significantly attenuated by a non-selective NOS inhibitor, L-NAME (33 nmol). Summary data (means ± SEM, n= 6) are shown in the graph. *P < 0.05 significantly different from the renin group. (B) Representative tracing demonstrates that the depressor effect of renin was significantly attenuated by the specific eNOS inhibitor, L-NIO (6 nmol). Summary data (means ± SEM, n= 6) are shown in the graph. *P < 0.05, significantly different from the renin group. (C) The quantitative immunoblotting analysis demonstrates that renin treatment increased the level of P-eNOS-Ser¹¹⁷⁷ protein in the NTS. Densitometric analysis of P-eNOS-Ser¹¹⁷⁷ protein levels (means ± SEM, n= 6) after administration of aCSF or renin. *P < 0.05, significantly different from the aCSF group. (D) Immunohistochemical staining of the brainstem for P-eNOS-Ser¹¹⁷⁷ showed that injection of renin into the NTS induced P-eNOS-Ser¹¹⁷⁷ (c vs. d). Arrows indicate P-eNOS-Ser¹¹⁷⁷-positive cells. The scale bar represents 200 µm. Summary data (means ± SEM, n= 6) are shown in the graph. The percentage of P-eNOS-Ser¹¹⁷⁷-positive cells was determined by counting P-eNOS-Ser¹¹⁷⁷-expressing cells in each hemisphere of the NTS at 200 × magnification. These counts were divided by all of the cells in the same paraffin section. *P < 0.05, significantly different from the a CSF group.

Figure 3

PI3K and Akt participate in renin-mediated eNOS-Ser¹¹⁷⁷ phosphorylation in the NTS. (A) The quantitative immunoblotting analysis demonstrates that renin treatment increased the level of P-Akt-Ser⁴⁷³ protein in the NTS. Densitometric analysis of P-Akt-Ser⁴⁷³ protein levels (means ± SEM, n= 6) after administration of aCSF or renin. *P < 0.05, significantly different from the aCSF group. (B) Representative tracings reveal the effects of BP by microinjection renin (240 fg) into the NTS pretreated with Akt inhibitor, Akt inhibitor IV (375 fmol). *P < 0.05, significantly different from the renin group. (C) The blood pressure of renin (240 fg) injection into the NTS after administration of the PI3K inhibitor, LY294002 (6 pmol). Representative tracings demonstrate that the depressor effect of renin was significantly attenuated by LY294002. Summary data (means ± SEM, n= 6) are shown in the graph. *P < 0.05, significantly different from the renin group. (D) Immunoblotting analysis reveals that the P-eNOS-Ser¹¹⁷⁷ protein level was increased after renin administration in the NTS. Phosphorylation of eNOS-Ser¹¹⁷⁷ was reduced by pretreatment with LY294002. Densitometric analysis of P-eNOS-Ser¹¹⁷⁷ protein levels (means ± SEM, n= 6) after treatment with aCSF, renin or LY294002. *P < 0.05, significantly different from the aCSF group; ^#P < 0.05, significantly different from the renin group.

Figure 4

Angiotensin AT₁ and Mas receptors participate in renin-mediated depressor effects at the NTS. (A) Blood pressure response to renin (240 fg) injection into the NTS after administration of the ACE inhibitor, lisinopril (2.4 fmol). Representative tracings demonstrate that the depressor effect of renin was significantly attenuated by lisinopril. Summary data (means ± SEM, n= 6) are shown in the graph. *P < 0.05, significantly different from the renin group. (B) Blood pressure response to renin (240 fg) injection of into the NTS after administration of losartan (4 nmol). Representative tracings demonstrate that the depressor effect of renin was significantly attenuated by losartan. Summary data (means ± SEM, n= 6) are shown in the graph. *P < 0.05, significantly different from the renin group. (C) Blood pressure response to renin (240 fg) injection of into the NTS after administration of valsartan (7.5 pmol). Representative tracings demonstrate that the depressor effect of renin was significantly attenuated by valsartan. Summary data (means ± SEM, n= 6) are shown in the graph. *P < 0.05, significantly different from the renin group. (D) Blood pressure response to renin (240 fg) injection of into the NTS after administration of the angiotensin-(1-7) antagonist, D-Ala7-Ang-(1-7) (144 fmol). Representative tracings demonstrate that the depressor effect of renin was significantly attenuated by D-Ala7-Ang-(1-7). Summary data (means ± SEM, n= 6) are shown in the graph. *P < 0.05, significantly different from the renin group.

Figure 5

Microinjection of renin induces G_q-Ras signalling in the NTS. (A) Blood pressure response to renin (240 fg) injection into the NTS after administration of the G_q inhibitor, GPA-2A (1.98 pmol). Representative tracings demonstrate that the depressor effect of renin was significantly attenuated by GPA-2A. Summary data (means ± SEM, n= 6) are shown in the graph. *P < 0.05, significantly different from the renin group. (B) Bar graph showing the activation ratio of Ras after microinjection of renin into the NTS.. Note the significant increase in Ras activation after treatment with renin. *P < 0.05, significantly different from the aCSF group.

Figure 6

The proposed renin-induced signalling pathway in the NTS to regulate systemic blood pressure in WKY rats. Microinjection of renin stimulates the PI3K-Akt-eNOS cascade via activating AT₁ and Mas receptors, ultimately leading to elevated NO concentrations in the NTS and decreased blood pressure. ACE2, angiotensin converting enzyme 2; Agt, angiotensinogen; Ang I, angiotensin I.