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 AT₁ 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-Ser¹¹⁷ 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.

Introduction

Renin is a critical component of the renin-angiotensin system (RAS) and catalyzes the rate-limiting step of converting angiotensinogen to angiotensin I. Angiotensin I is converted to angiotensin II (Ang II) by angiotensin converting enzyme (ACE). Dzau and his colleagues have demonstrated the expression of renin gene in mouse and rat brains. (Dzau et al., 1986) Local synthesis of angiotensin, independent of angiotensin generation in the circulation, is now widely accepted, indicating that renin can function in tissue sites, including the brain. (Dampney, 1994; Davisson et al., 2000) Taken together, these studies indicate that renin is expressed locally in the brain and may functionally regulate brain RAS through the generation of angiotensin peptides.

The RAS plays an important role in blood pressure regulation and cardiovascular disease risk profile. ACE inhibitors and antagonists of the angiotensin AT₁ receptor (receptor nomenclature follows Alexander et al., 2011) are current antihypertensive therapies. In 2007, direct renin inhibitors were released as a new class of antihypertensive agents (Brown, 2008). Direct renin inhibitors can reduce renin activity, even in the presence of elevated renin activity induced by ACE inhibitors or AT₁ receptor antagonists. Direct renin inhibitors not only lower blood pressure but also have renoprotective effects and reduce the risk of heart failure (McMurray et al., 2008; Parving et al., 2008). These findings suggest that renin plays a critical role in blood pressure regulation. However, the role of renin itself and its mechanism of action in the brainstem remain unclear.

The nucleus tractus solitarii (NTS), which is located in the dorsal medulla of the brain stem, participates in the central control of blood pressure and sympathetic nerve activity. Our previous studies demonstrated that several neuromodulators are involved in cardiovascular control by the NTS, including Ang II (Mosqueda-Garcia et al., 1990; Cheng et al., 2010) and NO (Tseng et al., 1996).

NO is synthesized by nitric oxide synthase (NOS) and there are three different types of NOS: neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS; Calabrese et al., 2007) In vivo, eNOS gene transfer experiments in the NTS caused hypotension and bradycardia (Sakai et al., 2000; Hirooka et al., 2003; Tai et al., 2004) and eNOS and nNOS both participated in central cardiovascular regulation by the NTS. (Ho et al., 2008; Chiang et al., 2009; Cheng et al., 2010; 2011) Nevertheless, the relationship between renin, NO synthase activity and the corresponding modulation of blood pressure by the NTS has not been established.

In this study, we investigated whether microinjection of renin into the NTS might regulate blood pressure, and we determined which form of NOS could be activated by renin administration. In addition, we investigated which receptors and downstream signalling pathways were involved in renin-induced effects in the NTS. Our results suggest that the G_q- PI3K-Akt-eNOS signalling pathway was involved in the modulation of blood pressure by activation of AT₁ and Mas receptors in the NTS.

Methods

Animals

All animal care and experimental research protocols had been approved by the Research Animal Facility Committee of Kaohsiung Veterans General Hospital. Male Wistar-Kyoto (WKY) rats of 250 to 300 g were obtained from National Science Council Animal Facility and housed in the animal room of Kaohsiung Veterans General Hospital (Kaohsiung, Taiwan). The rats were kept in individual cages in a room in which lighting was controlled (12 h on/12 h off), and the temperature was maintained at 23 to 24°C. The rats were allowed to acclimatise to the housing conditions for 1 week.

Intra-NTS microinjection

The preparation of animals for intra-NTS microinjection and the methods used to locate the NTS have been described previously (Tseng et al., 1996). In the present study, each injection volume in the NTS was restricted to 60 nL. Rats were anaesthetized with urethane (1.0 g·kg⁻¹ i.p., supplemented with 300 mg·kg⁻¹ i.v. as necessary). A polyethylene catheter was placed in the femoral vein for drug administration. blood pressure was measured directly through a catheter placed in the femoral artery and connected to a pressure transducer (P23 ID, Gould Electronics, Eichstetten, Germany) and polygraph (RS3800, Gould Electronics). Heart rate (HR) was monitored continuously by a tachograph preamplifier (13-4615-65, Gould Electronics). Tracheostomy was performed to maintain airway patency during the experiment. For microinjection into brain stem nuclei, the rats were placed in a stereotaxic instrument (Kopf), with the head flexed downward at a 45° angle. The dorsal surface of the medulla was exposed by limited craniotomy, and the rats were rested for at least 1 h before experiments. Single-barrel glass catheter were prepared (0.031-inch OD, 0.006 inch ID; Richland Glass Co, Vineland, NJ, USA) that had external tip diameters of 40 µm. The volume of microinjection (60 nL) was directly measured by the displacement of fluid meniscus in the micropipette barrel and confirmed visually under a microscope with a fine reticule (Tseng et al., 1989; Dhar et al., 2000). To verify that the needle tip of the glass electrode was exactly in the NTS, L-glutamate (0.154 nmol/60 nL) was microinjected. This would induce a characteristically abrupt fall in blood pressure (ΔBP ≥−35 mmHg) and HR (ΔHR ≥−50 bpm) if the needle tip was located precisely in the medial site of the intermediate one third of the NTS with the coordinates of anteroposterior, 0.0 mm; mediolateral, 0.5 mm; and vertical, 0.4 mm with the obex as reference (Tseng et al., 1996).

The injected renin (2.4 fg, 24 fg, 240 fg and 2400 fg) was dissolved in artificial cerebrospinal fluid (aCSF; 142 mmol·L⁻¹ NaCl, 5 mmol·L⁻¹ KCl, 10 mmol·L⁻¹ glucose and 10 mmol·L⁻¹ HEPES, pH 7.4). Other compounds – L-NAME (33 nmol; non-selective NOS inhibitor, Huang et al., 2004); L-NIO (6 nmol; inhibitor of eNOS, Ho et al., 2008); vinyl-L-NIO (600 pmol; selective nNOS inhibitor Chiang et al., 2009); lisinopril (2.4 fmol; ACE inhibitor); losartan (4 nmol; AT₁ receptor antagonist); D-Ala7-Ang-(1-7) (144 fmol; angiotensin-(1-7) (Ang-(1-7)) antagonist) and GPA-2A (1.98 pmol, G protein q subunit (Gq) inhibitor) were dissolved in aCSF. PD98059 (6 pmol; MEK inhibitor, Ho et al., 2008); LY294002 (6 pmol; PI3K inhibitor, Huang et al., 2004); valsartan (7.5 pmol; AT₁ receptor antagonist); gallein (240 fmol; G protein βγ subunit (Gβγ) inhibitor); 7-NI (66 pmol; nNOS inhibitor) and Akt inhibitor IV (375 fmol) were dissolved in DMSO, then diluted with aCSF.

Determination of NO in NTS

Renin and aCSF groups of rats (6 rats per group) were enrolled in the experiment. Rats were anesthetized with urethane (1.0 g kg⁻¹ i.p., supplemented with 300 mg kg⁻¹ i.v., if necessary).The NTS was dissected by micropunch (1-mm inner diameter) from a 1-mm thick brainstem slice at the level of the obex under a microscope. Total protein was prepared by homogenizing (BBX24 Bullet Blender homogenizer, Next Advance, Inc., Averill Park, NY, USA) the NTS tissue in lysis buffer (CelLytic MT Cell Lysis Reagent, Sigma-Aldrich, St. Louis, MO, USA), and incubating for 1 hour at 4 ^oC. Samples were then deproteinized using Microcon YM-30 centrifugal filter units (Millipore, Bedford, MA, USA). The amount of total NO (as nitrite and nitrate) in the samples was determined by a modification of the procedure based on the purge system of Sievers Nitric Oxide Analyzer (NOA 280i; Sievers Instruments, Boulder, CO, USA), which involves the use of chemiluminescence. Samples (10 µL) were injected into a reflux column containing 0.1 mol·L⁻¹ VCl₃ in 1 mol·L⁻¹ HCl at 90°C to reduce any nitrates and nitrites into NO. The NO was then combined with the O₃ produced by the analyzer to form NO₂. The resulting emission from the excited NO₂ was detected by a photomultiplier tube and recorded digitally (mV). The values were then interpolated to a standard curve of NaNO₃ concentrations determined concurrently. The measurements were made in triplicate for each sample. The levels of NO were expressed as concentrations of nitrate in each sample, corrected for the total protein in the NTS samples from each rat (as µM nitrate per µg protein).

Immunoblotting analysis

The NTS of rats were removed after injection of renin or vehicle. Total protein was prepared by homogenizing the NTS for 1 h at 4°C in lysis buffer and proteinase inhibitor cocktail. Protein extracts (20 µg per sample assessed by bicinchoninic acid protein assay,. Pierce Chemical Co., IL, USA) were subjected to 6% to 12.5% SDS-Tris glycine gel electrophoresis and transferred to a polyvinylidene fluoride membrane (GE Healthcare, Buckingamshire, UK). The membranes were blocked and incubated at 4°C overnight with the appropriate antibody: rabbit anti-P-ERK1/2-Thr²⁰²_/Tyr²⁰⁴ (1:1000; Cell Signaling Technology, Denvers, MS, USA), rabbit anti-ERK1/2 (1:1000; Cell Signaling Technology), rabbit anti-P-Akt-Ser⁴⁷³ (1:1000; Cell Signaling Technology), rabbit anti-Akt (1:1000; Cell Signaling Technology), mouse anti-P-eNOS-Ser¹¹⁷⁷ (1:1000; BD Biosciences, San Jose, CA, USA) and mouse anti-eNOS (1:1000; BD Biosciences). Also, mouse anti-actin (1:10 000; Millipore), anti-P-nNOS-Ser¹⁴¹⁶ (1:1000; Abcam, Cambridge, UK), anti-nNOS (1:2000; Millipore), and anti-iNOS (1:1000; Millipore) were diluted in PBST with 5% bovine serum albumin. Horseradish peroxidase (HRP)-conjugated anti-mouse (1:10 000) or anti-rabbit antibody (1:10 000) was used as the secondary antibody at room temperature for 1 h.

Immunohistochemistry analysis

After perfusion with 0.9% normal saline, the rat brainstems were fixed immediately in 4% formaldehyde overnight and embedded in paraffin. The brainstems were sectioned coronally at a 5 µm thickness. The sections were deparaffinized, quenched in 3% H₂O₂/methanol, heated (microwave) in citrate buffer (10 mmol·L⁻¹, pH 6.0), blocked in 5% goat serum and incubated with an anti-P-eNOS-Ser¹¹⁷⁷ antibody overnight at 4°C. Next, the sections were incubated with biotinylated secondary antibodies (1:200; Vector Laboratories, Burlingame, CA, USA) for 1 h and in AB complex (1:100) for 30 min at room temperature. The sections were visualized with a DAB substrate kit (Vector Laboratories) and counterstained with haematoxylin. The sections were then photographed with a microscope mounted with a charge-coupled device camera.

Ras activation assay

Ras activation was measured using a Ras Activation elisa Assay Kit (Millipore) following the manufacturer's instructions. The assay was initiated by the addition of recombinant Ras-binding domain of Raf-1 to the glutathione-coated elisa plate via a glutathione-S-transferase/glutathione interaction, thus capturing the active Ras and allowing the inactive/GDP-bound Ras to be washed away. The captured, active Ras was detected and measured quantitatively through the addition of a monoclonal anti-Ras antibody that detects K-, H-, N-Ras isoforms from rat. A HRP-conjugated secondary antibody was then added for the detection. Following the addition of the chemiluminescent substrate, signals can be measured using a luminometer (Promega, Madison, WI, USA).

Data analysis

A paired Student's t-test was used for comparing blood pressure measurements and counting cells expressing P-eNOS-Ser¹¹⁷⁷. One-way anova with Scheffe's post hoc test were applied to compare group differences. Differences with P < 0.05 were considered significant. All data are expressed as the means ± SEM.

Materials

Experimental drugs, such as urethane, Triton-X100, L-glutamate, heparin, human renin, lisinopril, losartan, N-nitro-L-arginine methyl ester (L-NAME), 7-nitroindazole (7-NI), LY294002, PD98059 and D-Ala7-Ang-(1-7), were obtained from Sigma-Aldrich (St. Louis, MO, USA). N(5)-(-iminoethyl)-L-ornithine (L-NIO), GP antagonist-2A (GPA-2A) and Akt inhibitor IV were obtained from Calbiochem (Darmstadt, Germany). N(5)-(1-imino-3-butenyl)-ornithine (vinyl-L-NIO) was obtained from ALEXIS (Lausen, Switzerland). Gallein and valsartan were obtained from Tocris (Bristol, UK).

Results

Renin induces systemic vasodepressor effect and NO release in the NTS

In this study, we utilized urethane-anaesthetized WKY rats to investigate the cardiovascular effects of microinjection of renin into the NTS. Tachyphylaxis was observed after repeated administration of similar doses of renin in the same site of the NTS under these experimental conditions (data not shown). Different concentrations of renin were then injected into the NTS of several rats. All the doses subsequently used (2.4 fg, 24 fg, 240 fg and 2400 fg) decreased mean blood pressure and heart rate (Supporting Information Figure S1). The response to renin (240 fg) unilaterally injected into the NTS was a fall in blood pressure (Figure 1A; mean values in Figure 1B, n= 6). aCSF was used as the drug vehicle and for control experiments. Interestingly, NO production in the NTS was significantly increased after the administration of renin.

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.

Renin induces eNOS phosphorylation in the NTS

We further investigated which NOS isoform contributes to depressor effects and NO release induced by renin in the NTS of WKY rats. Pretreatment with the non-selective NOS inhibitor, L-NAME, attenuated the depressor effect of renin (Figure 2A, n= 6). Prior treatment with the selective eNOS inhibitor, L-NIO, also significantly attenuated the depressor effect of renin (Figure 2B, n= 6). In contrast, administration of the specific nNOS inhibitor, vinyl-L-NIO or 7-NI, did not diminish the depressor effect of renin in the NTS (Figure S2A and 2B, n= 6).

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.

In addition, immunoblotting analyses of proteins extracted from the NTS demonstrated that treatment with renin did not increase nNOS-Ser¹⁴¹⁶ phosphorylation (Figure S2C, n= 6) or iNOS protein expression (Figure S2D, P > 0.05, n= 6) in the NTS. However, treatment with renin significantly increased the level of eNOS-Ser¹¹⁷⁷ phosphorylation (Figure 2C, n= 6) and (Figure 2D, n= 6) the proportion of P- eNOS-Ser¹¹⁷⁷-positive cells in the NTS, compared with the corresponding values in the group treated with aCSF.

PI3K inhibitor attenuated renin-induced eNOS phosphorylation in the NTS

Our previous studies demonstrated that Akt and ERK could regulate NOS activity in the NTS (Huang et al., 2004; Ho et al., 2008). To test whether Akt or ERK may induce eNOS phosphorylation, the phosphorylation of Akt was determined by immunoblotting analyses with an antibody against P-Akt-Ser⁴⁷³. After administration of renin into the NTS, the phosphorylation level of Akt in the NTS was significantly elevated, compared with that after aCSF (Figure 3A, n= 6). However, there were no differences in ERK phosphorylation levels between the renin-treated and aCSF groups (Figure S3A, n= 6). Furthermore, pretreatment with the PI3K inhibitor, LY294002, attenuated renin-induced eNOS-Ser¹¹⁷⁷ phosphorylation in the NTS (Figure 3D, lane 2 and 3, n= 6).

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.

We then used specific pharmacological inhibitors to confirm these results. Prior microinjection of the Akt-specific inhibitor, Akt inhibitor IV, significantly diminished the vasodepressor response evoked by renin (Figure 3B, n= 6). Pretreatment with LY294002 similarly attenuated the response to renin (Figure 3C, n= 6). In contrast, pretreatment of the NTS with the MEK inhibitor, PD98059, did not attenuate the depressor effect of renin (Figure S3B, n= 6).

Central administration of renin may regulate blood pressure through AT₁ and Mas receptors in the NTS

To assess the connection between renin and the downstream PI3K-Akt-eNOS signalling cascade in the NTS, we tested the effects of lisinopril (ACE inhibitor), losartan, valsartan (AT₁ receptor antagonists) and D-Ala7-Ang(1-7) [Ang-(1-7) antagonist] on blood pressure responses to renin, in the NTS. Pre-treatment with lisinopril, losartan or valsartan all attenuated the depressor effect of renin (Figures 4A, 4B and 4C, n= 6). In addition, Figure 4D shows that injection of the Mas receptor antagonist, D-Ala7-Ang-(1-7) also attenuated the depressor effect of renin (n= 6).

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.

Renin may activate the PI3K-Akt-eNOS cascade via Gα_q and Ras signalling in the NTS

These results showed that renin could activate PI3K-Akt-eNOS signalling in the NTS. Previous studies have demonstrated that Ang II and Ang-(1-7) may activate PI3K and Akt through AT₁ receptors coupled to Gβ (Quignard et al., 2001), AT₁ receptors coupled to G_q (Dugourd et al., 2003) or Mas receptors (Sampaio et al., 2007) Therefore, we investigated the G-protein coupling involved in the effects of the PI3K-Akt-eNOS pathway within the NTS and its relevance to the response to renin. Pretreatment with the Gβγ inhibitor, gallein, did not diminish the depressor effect of renin in the NTS (Figure S4, n= 6). However, pre-treatment with the G_q inhibitor, GPA-2A, attenuated the depressor effect of renin at the NTS (Figure 5A, n= 6). We further assessed the involvement of Ras in the renin-induced depressor effect and found that administration of renin to the NTS enhanced Ras activation (Figure 5B, n= 6).

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.

Discussion and conclusions

In the medulla of the brainstem, the nuclei have extensive connections to each other, and their major function is regulation of sympathetic nerve activity to modulate blood pressure. Ang II activates AT₁ and AT₂ receptors to modulate sympathetic nerve activity, in the brainstem but there is high expression of only AT₁ receptors in the medial NTS (Lenkei et al., 1997). Our present data show that microinjection of renin into the NTS of WKY rats induced a depressor effect and increased NO production (Figure 1). This renin-mediated depressor response was similar to the effect induced by Ang II in the NTS (Mosqueda-Garcia et al., 1990; Fow et al., 1994; Tan et al., 2005). In addition, pre-treatment with an ACE inhibitor or AT₁ receptor antagonists, separately attenuated the depressor effect of renin, suggesting that renin induced depressor responses through Ang II and AT₁ receptors in the NTS (Figure 4). The Mas receptor is a G-protein-coupled receptor and is the endogenous receptor for Ang-(1-7) (Jackson et al., 1988). Mas receptors can antagonize AT₁ receptor function by hetero-oligomeric interaction (Kostenis et al., 2005) and unilateral injections of Ang-(1-7) into the NTS caused depressor and bradycardic effects (Campagnole-Santos et al., 1989). In addition, the depressor effect evoked by Ang-(1-7) also involves a NO-mediated signalling (Alzamora et al., 2002). Moreover, Ang-(1-7) up-regulated the PI3K-Akt-eNOS pathway to increase NO production, through Mas receptors (Sampaio et al., 2007). However, we observed that pre-treatment with AT₁ receptor antagonists and a Ang-(1-7) antagonist both attenuated the depressor effect produced by renin (Figure 4). Thus, we would propose that both AT₁ and Mas receptors may contribute to the regulation of blood pressure by renin, at the NTS. The mechanisms connecting Mas receptors and Ras signalling in the NTS remain to be elucidated.

In the NTS, nNOS plays an important role in the modulation of blood pressure (Chiang et al., 2009; Cheng et al., 2010) and block of its expression in the NTS increased blood pressure (Maeda et al., 1999). Also, overexpression of eNOS induced a depressor response (Sakai et al., 2000; Hirooka et al., 2003; Tai et al., 2004). In Figure 2 and Figure S2, we found increased eNOS-Ser¹¹⁷⁷ phosphorylation levels in the NTS after administration of renin, whereas no activity or expression reductions were detected for nNOS or iNOS. Therefore, our results indicated that eNOS acts as a key factor in the production of NO in the NTS of WKY rats (Figure 1B). Because our previous study revealed that eNOS might play a key role in NTS cardiovascular function via the MAP kinase-ERK kinase (MEK)-ERK1/2 and eNOS signalling (Ho et al., 2008), we decided to further investigate this possibility. Our present data indicated that renin might regulate blood pressure through AT₁ receptors. We propose that eNOS might act as a downstream target of the AT₁ and Mas receptors and may be involved in renin-induced NO production in the NTS, thereby regulating blood pressure.

It has been reported that Akt, AMP-activated protein kinase, PKA, protein phosphatase 2A, protein kinase G, Ca²⁺/calmodulin-dependent protein kinases and ERK can all phosphorylate eNOS at Ser¹¹⁷⁷ (Mount et al., 2007; Ho et al., 2008). Previously, we revealed the crucial role of the PI3K-Akt-NOS pathway in regulating cardiovascular effects in the NTS (Huang et al., 2004). In the present study, LY294002 inhibited eNOS-Ser¹¹⁷⁷ phosphorylation induced by renin (Figure 3D) suggesting that renin may phosphorylate eNOS via a PI3K-Akt cascade in the NTS. We tested the effect of the G_q inhibitor, GPA-2A and the Gβγ inhibitor, gallein, on the renin-induced depressor effect in the NTS to provide further evidence that renin may produce Ang II, which in turn activates AT₁ receptor-G_q signalling, as described previously (Wong et al., 2002). Ang II stimulated PI3K-Akt signalling via AT₁ receptors in the brain (Yang and Raizada, 1999) and chronic blockade of PI3K in the NTS of spontaneously hypertensive rat (SHR) resulted in hypertension (Zubcevic et al., 2009). Furthermore, renin induced Ras activation in the NTS (Figure 5B) and similar results have been demonstrated in cardiac myocytes and vascular smooth muscle cells (Eguchi et al., 1996; Sadoshima and Izumo, 1996). Activation of the epidermal growth factor receptors (EGFR) leads to signalling mediated by the PI3K-Akt and Ras-MAPK pathways. The EGFRs also can be transactivated by AT₁ receptors and lead to the activation of PI3K-Akt cascade or Ras-MAPK pathway (Eguchi et al., 2001; Kippenberger et al., 2005; Mifune et al., 2005). Thus, further studies are needed to elucidate this possible mechanism in the NTS. However, we did not observe MEK-ERK signalling in the renin-mediated depressor response (Figure S2). Our data support the hypothesis that, in the NTS, renin activated the PI3K-Akt-eNOS signalling pathway, and consequently, the Ras pathway.

The (pro)renin receptor (PRR) consists of a 350-amino-acid protein with a single transmembrane domain that has comparable affinity for (pro)renin and renin (Nguyen et al., 2002) The PRR is highly expressed in brain regions known to modulate the cardiovascular system, such as the cortex, hippocampus, subfornical organ and NTS (Shan et al., 2008; Contrepas et al., 2009). Levels of mRNA for PRR were 45% and 70% higher in the NTS and supraoptic nucleus of SHR, compared to WKY rats (Shan et al., 2010), indicating that PRR may contribute to central blood pressure regulation. In our study, inhibition of RAS activity attenuated renin-mediated depressor effects in the NTS (Figure 4 and Figure S3). Renin binds PRR with affinity in the nanomolar range (Nabi et al., 2006) and in the present study, we have microinjected renin into NTS in the picomolar range. However, further studies are needed to establish the mechanisms connecting PRRs and their downstream signalling cascade in the NTS.

The study herein represents a key step in understanding how renin may participate in regulating the cardiovascular system through effects exerted in the NTS. We have utilized SensoLyte® 520 Rat Renin Assay Kit (ANASPEC, San Jose, CA, USA) to determine the concentration of renin in the NTS of WKY rats as 56 pM (data not shown). In Figure 1A, we microinjected 240 fg of renin into the NTS of WKY rats, equivalent to a concentration of 100 pM. However, the levels of angiotensin I, Ang II and Ang-(1-7), after administration of renin to the NTS are not known and these values should be determined. Another limitation of this study derives from the use of pharmacological inhibitors and their specificity. For instance, administration of losartan at higher doses induced non-specific effects in the RVLM (Averill et al., 1994). Moreover, losartan may bind to non-Ang II binding sites and valsartan has approximately five-fold greater affinity than losartan, for the AT₁ receptor (de Gasparo and Whitebread, 1995). We used both these AT₁ receptor antagonists to assess the contribution of AT₁ receptors to the modulation of renin-induced depressor effect in this study (Figure 4B and C). We intend to utilize shRNA-based gene delivery to validate the role of central cardiovascular effects more precisely, which may help us overcome the limitations imposed in animals.

In conclusion, we propose that renin could mediate central control of blood pressure at the NTS, through Ras-PI3K-Akt signalling-regulated phosphorylation of eNOS (Figure 6). The present data further show that renin may modulate blood pressure via centrally located AT₁ and Mas receptors, and via G_q, to activate Ras, Akt and eNOS. The RAS modulates sympathetic function in the NTS, where integration of autonomic control of the cardiovascular system occurs. Direct renin inhibitors have been approved for clinical use, however, the mechanisms by which direct renin inhibitors regulate blood pressure in the CNS have not been clarified. Our findings provide new insights into the mechanism(s) of central regulation of blood pressure by renin and may be of help in further development of direct renin inhibitors in therapy.

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.

Glossary

7-NI

7-nitroindazole

ACE

angiotensin converting enzyme

aCSF

artificial cerebrospinal fluid

Ang II

angiotensin II

Ang-(1-7)

angiotensin-(1-7)

DAB

diaminobenzidine

EGFR

epidermal growth factor receptor

eNOS

endothelium nitric oxide synthase

GPA-2A

GP Antagonist-2A

HR

heart rate

iNOS

inducible nitric oxide synthase

L-NAME

N-nitro-L-arginine methyl ester

L-NIO

N(5)-(-iminoethyl)-L-ornithine

MEK

MAP kinase-ERK kinase

nNOS

neuronal nitric oxide synthase

NTS

nucleus tractus solitarii

PBST

phosphate-buffered saline with Tween 20

PRR

(pro)renin receptor

RAS

renin-angiotensin system

RVLM

rostral ventrolateral medulla

SDS

sodium dodecyl sulfate

SFO

subfornical organ

SHR

spontaneously hypertensive rats

shRNA

short hairpin RNA

vinyl-L-NIO

N(5)-(1-imino-3-butenyl)-ornithine

WKY

Wistar-Kyoto rat

Sources of funding

This work was supported by funding from the National Science Council NSC98-2321-B-075B-002 (to Dr. C.-J. Tseng), NSC100-2320-B-283-001 (to Dr. W.-Y. Ho) and Kaohsiung Veterans General Hospital VGHKS97-080, VGHKS98-099. (to Dr. C.-J. Tseng)

Conflicts of interest

None.

Supporting information

Additional Supporting information may be found in the online version of this article:

Figure S1 The bar graphs reveal hypotensive and bradycardic effects of unilateral administration of different doses of renin in the nucleus tractus solitarii (NTS). Both the mean blood pressure (MBP) and heart rate (HR) effects were significantly changed by renin. *P < 0.05 versus the 0 group.

Figure S2 (A) The cardiovascular effect of injection of renin (240 fg) into the NTS after administration of the selective nNOS inhibitor, vinyl-L-NIO (600 pmol). Representative tracings demonstrate that vinyl-L-NIO did not diminish the depressor effect of renin in the NTS. Summary data (means ± SEM, n = 6) are shown in the graph (B) The cardiovascular effect of injection of renin (240 fg) into the NTS after administration of the potent nNOS inhibitor, 7-NI (66 pmol). Representative tracings demonstrate that 7-NI did not diminish the depressor effect of renin in the NTS. Summary data (means ±SEM, n = 6) are shown in the graph. (C) Immunoblot depicts the levels of P-nNOS-Ser¹⁴¹⁶ protein in the NTS. Densitometric analysis of P-nNOS-Ser¹⁴¹⁶ protein levels (means ± SEM, n = 6) before and after treatment with renin. Note that there are no significant differences in nNOS-Ser¹⁴¹⁶ phosphorylation levels in the NTS betweenthe aCSF and renin-treated groups. (D) Immunoblot shows iNOS protein levels (means ± SEM, n = 6) in the NTS. Note that there are no significant differences in iNOS protein levels in the NTS between aCSF and the renin-treated groups.

Figure S3 (A) Immunoblots depicting the levels of P-ERK1/2-Thr²⁰²/Tyr²⁰⁴ protein in the NTS. There were no significant differences in ERK1/2 phosphorylation levels in the NTS between the aCSF and renin-treated groups. Densitometric analysis shows P-ERK1/2-Thr²⁰²/Tyr²⁰⁴ protein levels (means ± SEM, n = 6) after treatment with renin. (B) The cardiovascular effect of injection of renin (240 fg) into the NTS after administration of the MEK inhibitor, PD98059 (10 pmol). Representative tracings demonstrate that PD98059 did not diminish the depressor effect of renin in the NTS. Summary data (means ± SEM, n = 6) are shown in the graph.

Figure S4 Blood pressure (BP) after injection of renin (240 fg) into the NTS following administration of the Gβγ inhibitor, gallein (240 fmol). Representative tracings demonstrate that the depressor effect of renin was not attenuated by gallein. Summary data (means ± SEM, n = 6) are shown in the graph.

Table S1 Cardiovascular response to microinjection of renin (240 fg) into the NTS in rats before and after microinjection of the pharmacological inhibitors

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