Downregulation of Brain Gα12 Attenuates Angiotensin II-Dependent Hypertension

Abstract

BACKGROUND

Angiotensin II (Ang II) activates central Angiotensin II type 1 receptors to increase blood pressure via multiple pathways. However, whether central Gα proteins contribute to Ang II-induced hypertension remains unknown. We hypothesized that Angiotensin II type 1 receptors couple with Gα12 and/or Gαq to produce sympatho-excitation and increase blood pressure and downregulation of these Gα-subunit proteins will attenuate Ang II-dependent hypertension.

METHODS AND RESULTS

After chronic infusion of Ang II (s.c. 350 ng/kg/min) or vehicle for 2 weeks, Ang II evoked an increase in Gα12 expression, but not Gαq in the rostral ventrolateral medulla of Sprague-Dawley rats. In other studies, rats that received Ang II or vehicle infusion s.c. were simultaneously infused i.c.v. with a scrambled (SCR) or Gα12 oligodeoxynucleotide (ODN; 50 µg/day). Central Gα12 ODN infusion lowered mean blood pressure in Ang II infused rats compared with SCR ODN infusion (14-day peak; 133 ± 12 vs. 176 ± 11 mm Hg). Compared to the SCR ODN group, Ang II infused rats that received i.c.v. Gα12 ODN showed a greater increase in heart rate to atropine, an attenuated reduction in blood pressure to chlorisondamine, and an improved baroreflex sensitivity. In addition, central Gα12 and Gαq ODN pretreatment blunted the pressor response to an acute i.c.v. injection of Ang II (i.c.v., 200 ng).

CONCLUSIONS

These findings suggest that central Gα12 protein signaling pathways play an important role in the development of chronic Ang II-dependent hypertension in rats.

Angiotensin II (Ang II) has an established role in contributing to the development of hypertension and the pathology of several different cardiovascular and kidney disease states.¹ Ang II produces a majority of its normal regulatory and pathological effects by binding to the Ang II type 1 (AT1) receptor, which is a G-protein-coupled receptor.² The superfamily of seven transmembrane-domain G-protein-coupled receptors plays a pivotal role in initiating the downstream cellular signaling processes evoked by neurotransmitters, hormones, and drugs, including those evoked by Ang II.^3,4 The downstream signaling pathways by which Ang II contributes to vascular remodeling and endothelial dysfunction, cardiac hypertrophy, hypertension, and acute/chronic kidney disease are highly complex and are often cell/tissue specific.⁵ Importantly, heterotrimeric G-protein signaling pathways can also contribute to Ang II evoked responses, although the role(s) that specific Gα proteins play in different disease states remains to be fully understood.⁶

G-proteins are heterotrimeric signal transduction coupling proteins which are composed of a guanine nucleotide binding α-subunit, and a βγ dimer.⁷ Based on their sequence homology, Gα-subunit proteins have been divided into four major classes, Gαs, Gαi, Gαq, and Gα12, and their respective subtypes.^8,9 Subsequent to agonist binding, the AT1 receptor (and other G-protein-coupled receptors) undergoes a conformational change which triggers substitution of guanosine triphosphate for guanosine diphosphate on the Gα-subunit. The dissociated Gα- and βγ-subunits then activate or inhibit different downstream effectors and subsequent second messengers, which together contribute to Ang II-mediated physiological responses. AT1 receptors are recognized to couple with Gαq, Gα12, and Gαi/o protein subtypes to mediate their G-protein signaling responses.² In vascular smooth muscle cells, Gαq and Gα12 proteins couple with AT1 receptors to mediate vasoconstriction by stimulating phosphorylation of myosin light chain via Ca²⁺/myosin light chain kinase and Rho/Rho kinase pathways, respectively.¹⁰ Studies performed in cell culture have shown that both Gαq and Gα12 signaling pathways are involved in Ang II-induced activation of superoxide generation.^11,12 Ang II augments expression of Gα12 in vascular smooth muscle cells, which is of merit since other studies have shown that Gα12 protein signaling pathways are involved in mediating endothelial dysfunction produced by Ang II.^13,14 Finally, inhibition of vascular smooth muscle Gαq signaling attenuates Ang II-dependent hypertension induced by renal artery stenosis.¹⁵

Together, these findings provide strong evidence that both Gα12 and Gαq protein signaling pathways couple with AT1 receptors in the periphery and contribute to the deleterious pathological effects of Ang II in vascular smooth muscle cell as well as the heart and kidneys. The neurogenic component to hypertension is well established^16,17 and the enhanced renin–angiotensin–aldosterone system in the brain has been regarded as a major contributor to neurogenic hypertension. However, whether Ang II increases blood pressure via activation of central AT1 receptors, which are coupled to a downstream Gα protein signaling pathway(s) remains unknown. Therefore, the present studies were performed to investigate the role(s) of central Gα12 and Gαq proteins in the development of Ang II-dependent hypertension. We hypothesize that Gα12 and Gαq each play a significant, but differential role in mediating the cardiovascular responses produced by stimulation of AT1 receptors in the central nervous system, potentially by modifying autonomic function.

METHODS

Please see Supplementary Material.

RESULTS

Subcutaneous Ang II infusion significantly increases Gα12, but not Gαq expression in the RVLM

To understand the potential physiological role(s) of brain Gα12 and/or Gαq protein signaling pathways in mediating Ang II-dependent hypertension, we first examined whether the expression levels for these Gα proteins in the rostral ventrolateral medulla (RVLM), paraventricular nucleus, subfornical organ, and cortex are altered in rats with Ang II hypertension. As shown in Figure 1, the Gα12 protein expression level in the RVLM was significantly increased in rats infused with Ang II for 14 days as compared with rats that received vehicle (Figure 1). In contrast, there was no difference in Gαq protein expression levels in RVLM between the two groups. Further, expression levels for Gα12 and Gαq protein were not different in paraventricular nucleus, subfornical organ, or cortex between the two groups of rats.

Figure 1.

Protein expression of Gα12 and Gαq in rostral ventrolateral medulla (RVLM), paraventricular nucleus (PVN), subfornical organ (SFO), and cortex of rats infused with Ang II (350 ng/kg/min, s.c.) or isotonic saline vehicle for 2 weeks. (a) Representative blots for Gα12 and Gαq in each nucleus; (b, c), quantified protein expression levels of Gα12 and Gαq. n = 4–7 in each group. *P < 0.05, compared with saline infused rats.

Continuous i.c.v. Gα12 oligodeoxynucleotide (ODN) infusion significantly attenuates Ang II-dependent hypertension

To determine whether the increased brain levels of Gα12 proteins may participate in mediating Ang II-dependent hypertension, we examined the effects of continuous i.c.v. miniosmotic pump infusion of a scrambled (SCR), or Gα12 ODN, on the development of hypertension in rats infused for 14 days with s.c. Ang II (350 ng/kg/min). As shown in Figure 2a, rats that received Ang II (s.c.) + SCR ODN (i.c.v.) developed a gradual increase in mean arterial pressure (MAP) that reached a plateau on the eighth day of infusion and which lasted until the end of the study. In contrast, in rats that received i.c.v. Gα12 ODN infusion concurrent with s.c. Ang II, there was a delay in the increase in MAP (days 3–5) and thereafter, MAP increased; however, the magnitude of the Ang II-induced hypertension was significantly attenuated over the remainder of the study (days 6–14), with MAP actually being the lowest over the last 3 days. Infusion of isotonic saline (s.c.) + SCR ODN (i.c.v.) or isotonic saline (s.c.) + Gα12 ODN (i.c.v.) did not alter blood pressure in rats. Compared to the SCR ODN group, Ang II infused rats that received i.c.v. Gα12 ODN showed a significantly lower basal heart rate (344 ± 8 vs. 397 ± 16 bpm).

Figure 2.

(a) Changes in mean arterial pressure (MAP) produced by i.c.v. infusion of a scrambled (SCR) or Gα12 ODN (50 µg/day) in rats that received s.c. Ang II (350 ng/kg/min) or isotonic saline vehicle for 14 days. *P < 0.05, compared with Saline + SCR ODN and Saline + Gα12 ODN infused rats. #P < 0.05, compared with both Ang II + SCR ODN infused rats. There is interaction between treatments and time. (b–e) Spontaneous baroreflex gain (b), changes in heart rate (HR) produced by i.p. injection of atropine (c) and propranolol (d) and change in mean blood pressure (BP) produced by chlorisondamine (e) measured in four chronic treatment groups. *P < 0.05. n = 5–7 in each group. Abbreviation: ODN, oligodeoxynucleotide.

I.c.v. Gα12 ODN infusion partially restores spontaneous baroreflex function in rats with Ang II-induced hypertension

After 14 days of treatment, spontaneous baroreflex function was significantly impaired in Ang II-induced hypertensive rats (Figure 2b). The decrease in spontaneous baroreflex gain observed in rats that received 2-week Ang II infusion was partially reversed by central Gα12 ODN administration. Intracerebroventricle (i.c.v.) infusion of the Gα12 ODN did not alter basal level of spontaneous baroreflex function in rats that received vehicle administration. Further, after 2 weeks of treatment rats that received Ang II (subcutaneous (s.c.)) + SCR ODN (i.c.v.) exhibited a blunted increase in heart rate to intraperitoneal (i.p.) atropine as compared with normal saline (s.c.) + SCR ODN (i.c.v.) treated rats (Figure 2c). However, the reduced tachycardic response to atropine was partially restored in rats that received i.c.v. Gα12 ODN during Ang II administration. In these animals, there were no significant differences between groups in the magnitude change in heart rate produced by i.p. propranolol (Figure 2d). However, Ang II (s.c.) + SCR ODN (i.c.v.) infused rats showed a significantly greater decrease in MAP to i.p. administration of the ganglionic blocker, chlorisondamine (Figure 2e). This enhanced chlorisondamine-induced hypotensive response was attenuated in rats that received Gα12 ODN infusion.

I.c.v. Gα12 ODN infusion prevents upregulation of RVLM Gα12 protein levels in rats receiving s.c. Ang II

Figure 3 illustrates Gα12 and Gαq protein expression from rats for which data was presented in Figure 2. As shown in Figure 3a, as compared to rats that received normal saline (s.c.) + SCR ODN (i.c.v.), the Gα12 protein expression level was significantly increased in the RVLM of Ang II (s.c.) + SCR ODN (i.c.v.) treated rats. In contrast, the Ang II-induced increase in Gα12 expression was prevented in rats that concurrently received central Gα12 ODN infusion. No difference in Gα12 protein levels was found in paraventricular nucleus or cortex between s.c. Ang II + i.c.v. SCR ODN and s.c. Ang II + i.c.v. Gα12 ODN infused rats (Figure 3b,c). Gαq expression in RVLM was not altered by Gα12 ODN i.c.v. infusion (Figure 3d).

Figure 3.

Representative blots and Gα12 protein expression levels in rostral ventrolateral medulla (RVLM) (a), paraventricular nucleus (PVN) (b), cortex (c) and Gαq protein level in RVLM (d) of rats (n = 5–7/group) that received continuous i.c.v. infusion of a scrambled (SCR) or Gα12 ODN (50 µg/day) during 14 days s.c. isotonic saline or Ang II (350 ng/kg/min) administration. n = 5–7 in each group. *P < 0.05. Abbreviation: ODN, oligodeoxynucleotide.

I.c.v. Gα12 and Gαq ODN pretreatment attenuates the pressor response to acute i.c.v. Ang II

Considering that chronic downregulation of Gα12 proteins in the brain significantly attenuates the magnitude of chronic (14 days) Ang II-dependent hypertension, studies were performed to determine whether brain Gα12, and potentially Gαq, protein pathways are also involved in mediating the pressor response to acute i.c.v. Ang II administration. As shown in Supplementary Figure S1A, i.c.v. injection of Ang II (200 ng) into conscious rats produced a rapid increase in pulsatile blood pressure and MAP, and a concurrent decrease in heart rate. The magnitude of the pressor response to i.c.v. Ang II injection was significantly attenuated by i.c.v. pretreatment (48 h) of either a Gα12 or Gαq ODN (Supplementary Figure S1B,C).

Concurrent i.c.v. Gα12 and Gαq ODN infusion attenuates Ang II-dependent hypertension

Based on the finding that brain Gαq protein signaling pathways are also involved in producing the acute pressor response to i.c.v. Ang II, studies were performed to investigate whether Gαq proteins also contribute in mediating Ang II-dependent hypertension. Therefore, additional studies were performed to determine whether brain Gαq proteins are involved in mediating hypertension to chronic (14 days) Ang II administration. Continuous i.c.v. Gαq ODN infusion blunted the time course and magnitude increase in MAP to Ang II (s.c.) as compared to the level of MAP attained in Ang II infused rats that received SCR ODN (i.c.v., Figure 4). At the end of the 14-day study, the final level of MAP in Ang II infused rats that received the central Gαq ODN infusion was, however, higher than that attained in rats that received i.c.v. Gα12 infusion (164 ± 7 vs. 133 ± 11 mm Hg). Note, however, that the combination of continuous i.c.v. Gα12 + Gαq ODN infusion markedly inhibited the magnitude of Ang II-induced hypertension throughout the duration of the 14-day study.

Figure 4.

Changes in mean arterial pressure (MAP) produced by s.c. Ang II (350 ng/kg/min) in rats that concurrently received i.c.v. infusion of a scrambled (SCR) ODN, a Gαq ODN, or combined Gα12 and Gαq ODN (50 µg/day/ODN). n = 6–7 in each group. #P < 0.05, compared with Ang II + SCR ODN. There is no interaction between treatment and time. Abbreviation: ODN, oligodeoxynucleotide.

DISCUSSION

A key finding of this study is demonstration that brain Gα12 and Gαq protein signaling pathways both play a role in the development of Ang II-dependent hypertension. Support for this premise comes from the observation that chronic i.c.v. infusion for 2 weeks with either a Gα12 or Gαq ODN, which were used to downregulate expression of each respective protein, markedly slowed the onset and attenuated the magnitude increase in blood pressure produced by the concurrent s.c. infusion of Ang II. Further, when infused into the brain together, the combined Gα12 and Gαq ODN produced an even further reduction in blood pressure over the course of the 2-week Ang II study. To our knowledge, these findings are the first to demonstrate the specific and interactive role that brain Gα12 and Gαq protein pathways have in mediating Ang II-induced hypertension.

In the present studies, the level of Gα12, but not Gαq, protein expression in the RVLM was significantly increased in rats that became hypertensive after continuous s.c. Ang II administration for 2 weeks as compared to respective protein levels measured in normotensive rats that received s.c. isotonic saline vehicle. Interestingly, the Ang II-induced upregulation of Gα12 protein expression was selective for the RVLM and not observed in other brain regions explored in this study (paraventricular nucleus, subfornical organ, and cortex). Also, the Ang II-induced increase in RVLM Gα12 protein expression was completely prevented in rats that concurrently received chronic i.c.v. infusion of Gα12 ODN. While it is possible that chronic Ang II administration also increased AT1 receptor expression in the RVLM, the selective increase in Gα12, but not Gαq protein expression in this same brain site provides strong support for augmentation of an AT1/Gα12 signaling pathway involved in mediating the altered cardiovascular and autonomic responses to sustained Ang II administration.

These findings are of considerable interest and suggest that upregulation of Gα12 protein signaling pathways in the RVLM may contribute to the sympatho-excitatory effects that occur during the development of Ang II hypertension. In line with our findings, Parker et al. reported that Gα12 mRNA expression in the RVLM of spontaneously hypertensive rats (SHRs) was significantly elevated as compared to that in normotensive Wistar-Kyoto rats.¹⁸ The spontaneously hypertensive rat is an established animal model of essential and neurogenic hypertension, which has a blunted baroreflex control of heart rate and sympathetic nerve activity.¹⁹ Thus, it is possible that an increase in Gα12 mRNA, and presumably Gα12 protein levels, observed in the RVLM of spontaneously hypertensive rat could also contribute to the markedly enhanced activation of the sympathetic nervous system, which is characteristic of this rat strain. Prior studies have shown that brain Ang II contributes in mediating the increased sympathetic and hypertensive responses in spontaneously hypertensive rat.²⁰ The findings of this study lend support to this speculation since we observed that rats infused with Ang II had an impaired baroreflex as demonstrated by a dampened tachycardic response to atropine. Moreover, rats infused s.c. with Ang II showed an enhanced hypotensive response to chlorisondamine. Together, these findings suggest that the sympathetic control of the vasculature is augmented, and the parasympathetic control of the heart is attenuated in Ang II hypertensive rats. These findings are in agreement with other studies, which have also demonstrated that there is an imbalance in the autonomic control of cardiovascular function in Ang II-dependent hypertension in rats and other species.^21,22 Extending these observations, our current findings suggest that upregulation of brain (RVLM) Gα12 protein pathways may contribute to these derangements in sympathetic and parasympathetic activity.

In addition to Gα12, Gαq is also coupled to AT1 receptors in vascular smooth muscle cells.²³ Inhibition of Gαq and its intracellular signaling cascade prevents vascular dysfunction, remolding, and hypertension.^24,25 However, whether brain Gαq is involved in mediating Ang II-induced hypertension in rats has remained unclear. As shown by Veerasingham and Raizada,²⁶ activation of AT1 receptor signal transduction pathways results in transcriptional norepinephrine neuromodulation and increased neuronal firing rate in neuronal cultures. Importantly, other studies have shown that in an unanesthetized decerebrate rat model, Ang II acts in the nucleus tractus solitaries to depress the baroreflex via a Gαq protein signaling pathway.²⁷ Considering these and related findings, we speculated that brain Gαq signaling pathways may participate in mediating the pressor response to either an acute i.c.v. injection or chronic s.c. infusion (2 weeks) of Ang II. Our studies showed that central pretreatment of rats (48 h) with either a Gαq or Gα12 ODN significantly blunted the peak pressor response to an acute i.c.v. injection of Ang II. These findings demonstrate that both brain Gαq and Gα12 protein pathways are involved in producing the increase in blood pressure to an acute i.c.v. injection of Ang II. Likewise, our findings also showed that the time course and magnitude increase in blood pressure produced by 2-week s.c. Ang II infusion was significantly attenuated in rats that received a continuous i.c.v. infusion of the Gαq ODN. Regarding the time course, administration of Gαq ODN in the brain tended to have the most impact to initially delay, by approximately 5 days, the onset of the Ang II-mediated hypertension. Thereafter, blood pressure increased gradually and by the 14th day of treatment the level of blood pressure was nearly the same as that observed in animals that received i.c.v. SCR ODN during the development of Ang II hypertension. Alternatively, chronic i.c.v. infusion of rats with the Gα12 ODN had a biphasic effect on blood pressure in response to s.c. Ang II; Initially the onset of the hypertension was delayed (days 2–5) and then after approximately 10 days blood pressure significantly decreased well below levels observed in SCR or Gα12 ODN treated rats. One explanation for the differences in time course is that central Gαq vs. Gα12 protein pathways may affect different hormonal (vasopressin) vs. neural (sympathetic) pathways, which collectively contribute in mediating Ang II hypertension.²⁸ We have previously shown that central Gαq protein pathways have a critical role in stimulating the release/secretion of vasopressin (AVP) into the circulation.²⁹ AVP is known to have a major role in the hypertensive response to hyperactivity of the brain renin–angiotensin system.³⁰ In contrast, and as supported by the present data, central Gα12 protein pathways appear to have a preferential role in activating sympathetic outflow during development of Ang II hypertension. Notably, chronic i.c.v. infusion of the combination of the Gα12 and Gαq ODN had the greatest effect to attenuate the pressor response to Ang II throughout the entire 2 weeks of study. Thus, both of these central Gα proteins signaling pathways play important and differential roles in mediating the development of Ang II-induced hypertension.

A limitation of these studies is that the intracellular signaling pathways activated downstream of brain Gα12 and Gαq proteins and mediate the cardiovascular and autonomic responses to Ang II remain to be elucidated. The Gα12/Src signaling pathway has been shown to be activated by excess mitochondrial reactive oxygen species, lead to disruption of tight junction integrity, and cause epithelial dysfunction.³¹ In the brian, Src family kinases participate in the regulation of a variety of neuronal functions by catalyzing the tyrosine phosphorylation of specific substrates, including neurotransmitter receptors and proteins that regulate receptor function.³² It is also possible that a switching of activity of Gα (i.e., Gα12 and Gαq to only Gα12) may occur during the development of Ang II hypertension, which could explain why selective downregulation of Gαq or Gα12 blunted the pressor response to Ang II at different times points. Further, we did not explore a potential role of Gβγ or assessor proteins, which may have significant effects to alter blood pressure through AT1 receptors. Whether central Gα12/Src, Gβγ, or assessor protein signaling pathways contribute to Ang II-dependent hypertension are important questions that have yet to be answered.

Together, the results of these investigations demonstrate that both brain Gα12 and Gαq protein signaling pathways have important roles in mediating the pressor response to acute and chronic Ang II administration in Sprague-Dawley rats. Of merit, during chronic s.c. administration, Ang II evoked a marked increase in Gα12, but not Gαq protein levels in the RVLM. This is an important finding when taken together with our data showing that central Gα12 protein signaling pathways contribute to the impaired autonomic responses (blunted cardiac parasympathetic and augmented vascular sympathetic) observed in Ang II-dependent hypertensive rats. These findings indicate that central Gα12 might be a potential target for treatment of neurogenic hypertension. However, our data show that it is necessary to simultaneously target both brain Gα12 and Gαq protein pathways to maximally inhibit the brain Gα signaling component, which mediates Ang II hypertension.

DISCLOSURE

The authors declared no conflict of interest.