Myogenic Vasoconstriction Requires Canonical Gq/11 Signaling of the Angiotensin II Type 1a Receptor in the Murine Vasculature

Background The myogenic response is an inherent vasoconstrictive property of resistance arteries to keep blood flow constant in response to increases in intravascular pressure. Angiotensin II (Ang II) type 1 receptors (AT1R) are broadly distributed, mechanoactivated receptors, which have been proposed to transduce myogenic vasoconstriction. However, the AT1R subtype(s) involved and their downstream G protein- and β-arrestin-mediated signaling pathways are still elusive. Objective To characterize the function of AT1aR and AT1bR in the regulation of the myogenic response of resistance size arteries and possible downstream signaling cascades mediated by Gq/11 and/or β-arrestins. Methods We used Agtr1a-/-, Agtr1b-/- and tamoxifen-inducible smooth muscle-specific AT1aR knockout mice (SM-Agtr1a mice). FR900359, [Sar1, Ile4, Ile8] Ang II (SII) and TRV120055 were used as selective Gq/11 protein inhibitor and biased agonists to activate non-canonical β-arrestin and canonical Gq/11 signaling of the AT1R, respectively. Results Myogenic and Ang II-induced vasoconstrictions were diminished in the perfused renal vasculature of Agtr1a-/- and SM-Agtr1a mice. Similar results were observed in isolated pressurized mesenteric and cerebral arteries. Myogenic tone and Ang II-induced vasoconstrictions were normal in arteries from Agtr1b-/- mice. The Gq/11 blocker FR900359 decreased myogenic tone and Ang II vasoconstrictions while selective biased targeting of AT1R β-arrestin signaling pathways had no effects. Conclusion The present study demonstrates that myogenic arterial constriction requires Gq/11-dependent signaling pathways of mechanoactivated AT1aR but not G protein-independent, noncanonical alternative signaling pathways in the murine mesenteric, cerebral and renal circulation.


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Myogenic vasoconstriction reflects the inherent ability of resistance arteries to adapt their 53 diameter in response to alterations of intraluminal pressure. This response was first described pressure has yet to be fully clarified.

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Whether this noncanonical ß-arrestin effector pathway plays a role in myogenic and 81 ligand-dependent vasoconstriction has yet to be ascertained.

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This study explored the specific function of AT1R subtypes in the regulation of myogenic 83 tone and whether downstream signaling pathways are dependent on canonical G q/11 and/or 84 noncanonical alternative signaling pathways. In this regard, we generated mice with cell 85 specific deletion of smooth muscle AT1a receptors (SM-Agtr1a mice) and studied the effects 86 of biased GPCR agonists and G q/11 protein inhibition on tone development in three distinct 87 vascular beds (renal, cerebral and mesenteric circulation). We found that the AT1aR coupled 88 towards the canonical G q/11 signaling pathway is required for the myogenic response in all 89 three vascular beds. Our data argue against involvement of noncanonical G

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(PCR) analysis of tail DNA as described previously (26

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We evaluated myogenic tone in mouse renal circulation, a highly myogenic bed regulating 166 blood flow to the kidneys and consequently sodium excretion and systemic blood pressure.

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Renal vascular resistance of isolated perfused kidneys was determined by measuring 168 perfusion pressure at fixed levels of flow. The perfusion pressure increased with flow rate in 169 kidneys of wild-type Agtr1a +/+ mice, reaching a value of about 160 mmHg at a flow rate of 1.9 170 ml/min (Figure 2A). Kidneys from Agtr1a -/mice developed significantly less pressure at the 171 same flow rate (Figure 2B, E). 60 mmol/L KCl-induced increases in perfusion pressure were 172 normal in Agtr1a -/kidneys (Figure 2 F). At a flow rate of 1.9 ml/min, pressure in Agtr1a -/-173 kidneys was ~100 mmHg lower than in Agtr1a +/+ kidneys. Angiotensin II (Ang II, 10 nmol/L) 174 increased perfusion pressure by ~80 mmHg in kidneys of Agtr1a +/+ mice, but had no effect in

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Next, we studied the function of AT1aRs in cerebral arteries. Vessels were equilibrated at 15 209 mmHg (30 min) and following an assessment of KCl-induced constriction, arteries were 210 pressurized to 80 mmHg ( Figure 6A). Ang II constrictions and myogenic constriction was 211 significantly decreased in SM-Agtr1a -/arteries compared to wild-type ( Figure 6A, B, C, D).

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Both wild-type and SM-Agtr1a -/arteries produced similar constrictions when exposed to 60 213 mmol/L KCl ( Figure 6E). The results demonstrate a key role of AT1aR in the myogenic 214 response of mouse cerebral arteries.

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The study found that the canonical G q/11 signaling of mechanoactivated AT1aR is responsible 231 for myogenic vasoconstriction in mesenteric, renal arteries and cerebral arteries. We 232 observed a loss of myogenic autoregulation in the renal circulation of Agtr1a -/mice, an effect 233 which was normal in Agtr1b -/mice. Similarly, we found that myogenic tone was strongly 234 reduced in two other myogenic arteries (mesentery and cerebral) from smooth muscle 235 specific AT1aR-deficient (SM-Agtr1a -/-) mice compared to wild-type. Using the 236 pharmacological G q/11 inhibitor FR900359 and several GPCR biased agonists, we showed 237 that AT1Rs cause vasoconstriction via canonical G q/11 signaling but not alternative G protein   (27) (28) (30), the AT1R remains one of the best characterized mechanosensor 244 in the vasculature (49) (55). Humans express a single type of AT1R, whereas two isoforms 245 (AT1aR and AT1bR) are present in rodents (36) (53). Using Agtr1a -/mice and inverse AT1R 246 agonist, our previous data suggested that ligand-independent AT1aR activation is required for 247 myogenic response in resistance mesenteric arteries and renal arterioles (46). However, two 248 recent studies reported that myogenic tone was diminished in Agtr1b -/mesenteric and 249 cerebral arteries, which implies a possible role of AT1bRs in mechanosensation (42) (3). In 250 contrast, we found that myogenic tone was normal in Agtr1b -/perfused kidneys, which argues 251 against a role of AT1bR in myogenic constriction in the renal circulation. This data was, 252 however, obtained in global mutant mice, which often display compensatory mechanisms for 253 the lack of AT1Rs. Moreover, AT1aR and AT1bR are expressed at similar levels in cerebral 254 parenchymal arterioles and genetic knockout of AT1aR (but not AT1bR) blunted the ability of 255 these vessels to generate myogenic tone (52). The latter effect is opposite to cerebral arteries 256 where genetic knockout of AT1bR blunted the ability to develop myogenic tone (42). To 257 overcome these potential limitations, we generated tamoxifen-inducible SM-Agtr1a 258 (SMMHC-Cre+Agtr1a flox/flox ) mice for careful phenotypic investigation. We found that myogenic 259 constriction was impaired in cerebral, mesenteric and renal arteries isolated from smooth 260 muscle AT1aR-deficient mice. The data provide firm evidence that AT1aRs play a key role as 261 9 mechanosensors mediating myogenic constriction in the murine vasculature.

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AT1aRs downstream signaling to cause vasoconstriction 263 We next explored downstream signaling pathways mediated by G q/11 and/or β arrestins of the 264 AT1R in the vascular response. In cell culture, osmotic cell stretch has been found to increase 265 the binding affinity and potency of the β-arrestin-biased agonist TRV120023 with no effect on 266 the balanced agonist Ang II through AT1R to induce a conformation change of β-arrestin 2, 267 similar to that induced by β-arrestin-biased agonists (50). Similarly, hypo-osmotic stretch 268 induced β-arrestin-biased signaling of AT1Rs in the absence of G protein activation (44). We

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We hypothesized that G q/11 signaling contributes to myogenic tone in mesenteric and 283 renal arteries and consistent with this idea, we found that the vasoconstrictor responses were 284 strongly increased by the G q/11 AT1R biased agonists TRV120055 and TRV20056 (Figure 10).

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Moreover, we found that the G q/11 blocker FR900359 inhibited both myogenic tone and Ang II 286 induced constrictions in mesenteric arteries and renal arterioles (Figure 10). The data imply 287 that myogenic vasoconstriction requires canonical G q/11 signaling of the AT1aR. Consistently, 288 myogenic tone is increased in the absence of regulator of G-protein signaling 2 (RGS2), 289 which is an endogenous terminator of Galpha q/11 (Gα q/11 ) signaling (19) (37). The data align 290 with findings indicating that mechanically activated AT1R generate diacylglycerol, which in 291 turn activates protein kinase C (PKC) and induces the actin cytoskeleton reorganization 292 necessary for pressure-induced vasoconstriction (22). Finally, our conclusions are supported 293 by findings indicating that another G q/11 -protein inhibitor YM 254890 profoundly reduced 294 10 myogenic tone in mesenteric arteries (49). Note, this data contrast with recent findings, which 295 proposed that G 12/13 -and Rho/Rho kinase-mediated signaling is required in myogenic 296 vasoconstriction by inhibition of myosin phosphatase (5). The reason for the discrepancy is 297 presently unknown, but may depend on which vessel order was utilized, i.e. 3 rd or 4 th order 298 mesenteric versus 1 st or 2 nd order mesenteric arteries. Moreover, the myogenic response was 299 only reduced by 50% in G 12/13 -deficient cerebral arteries (5), which may indicate that this 300 pathway may play a role in some but not all vessels. Thus, it is possible that the relevance to 301 the two signaling pathway differs between various vascular beds and artery branches. Our 302 study provides firm evidence that AT1aRs coupled to G q/11 signaling is an essential 303 component of dynamic mechanochemical signaling in arterial vascular smooth muscle cells 304 causing myogenic tone (Figure 10).

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Signaling of most GPCRs via G proteins is terminated (desensitization) by the 306 phosphorylation of active receptor by specific kinases (GPCR kinases, or GRKs) and 307 subsequent binding of ß-arrestins that selectively recognize active phosphorylated receptors.

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Although, GRKs and ß-arrestins play also a role in multiple noncanonical signaling pathways 309 in the cell, both GPCR-initiated and receptor-independent (32) (15), our study failed to 310 demonstrate that this pathway plays an important role in the myogenic response (Figure 10).

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Thus, it is unlikely that blood pressure lowering effects of ß-arrestin biased AT1R agonists,

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In summary, we provide new and firm evidence for a mechanosensitive function of 315 AT1aR in myogenic vasoconstriction in mesenteric, renal and cerebral arteries, i.e. in three 316 different highly myogenic vascular beds. Our study clearly shows that mechanical stress 317 activates AT1R in arterial smooth muscle cells, which subsequently triggers canonical G q/11 318 signaling, irrespective of GRK/β-arrestin signaling, to cause myogenic vasoconstriction. Our 319 results argue against the idea of multiple mechanosensors coupled to noncanonical β-arrestin 320 pathways generating myogenic arterial tone. These findings lay ground for additional studies 321 to characterize the molecular mechanisms of mechanoactivated AT1aR coupled to G q/11 322 signaling in intact arteries, which may reveal new molecular targets for drug development to