The angiotensin II receptor type 1b is the primary sensor of intraluminal pressure in cerebral artery smooth muscle cells

Abstract

The angiotensin II receptor type 1b (AT₁ R_b ) is the primary sensor of intraluminal pressure in cerebral arteries. Pressure or membrane-stretch induced stimulation of AT₁ R_b activates the TRPM4 channel and results in inward transient cation currents that depolarize smooth muscle cells, leading to vasoconstriction. Activation of either AT₁ R_a or AT₁ R_b with angiotensin II stimulates TRPM4 currents in cerebral artery myocytes and vasoconstriction of cerebral arteries. The expression of AT₁ R_b mRNA is ∼30-fold higher than AT₁ R_a in whole cerebral arteries and ∼45-fold higher in isolated cerebral artery smooth muscle cells. Higher levels of expression are likely to account for the obligatory role of AT₁ R_b for pressure-induced vasoconstriction_. ABSTRACT: Myogenic vasoconstriction, which reflects the intrinsic ability of smooth muscle cells to contract in response to increases in intraluminal pressure, is critically important for the autoregulation of blood flow. In smooth muscle cells from cerebral arteries, increasing intraluminal pressure engages a signalling cascade that stimulates cation influx through transient receptor potential (TRP) melastatin 4 (TRPM4) channels to cause membrane depolarization and vasoconstriction. Substantial evidence indicates that the angiotensin II receptor type 1 (AT₁ R) is inherently mechanosensitive and initiates this signalling pathway. Rodents express two types of AT₁ R - AT₁ R_a and AT₁ R_b - and conflicting studies provide support for either isoform as the primary sensor of intraluminal pressure in peripheral arteries. We hypothesized that mechanical activation of AT₁ R_a increases TRPM4 currents to induce myogenic constriction of cerebral arteries. However, we found that development of myogenic tone was greater in arteries from AT₁ R_a knockout animals compared with controls. In patch-clamp experiments using native cerebral arterial myocytes, membrane stretch-induced cation currents were blocked by the TRPM4 inhibitor 9-phenanthrol in both groups. Further, the AT₁ R blocker losartan (1 μm) diminished myogenic tone and blocked stretch-induced cation currents in cerebral arteries from both groups. Activation of AT₁ R with angiotensin II (30 nm) also increased TRPM4 currents in smooth muscle cells and constricted cerebral arteries from both groups. Expression of AT₁ R_b mRNA was ∼30-fold greater than AT₁ R_a in cerebral arteries, and knockdown of AT₁ R_b selectively diminished myogenic constriction. We conclude that AT₁ R_b , acting upstream of TRPM4 channels, is the primary sensor of intraluminal pressure in cerebral artery smooth muscle cells.

Keywords: GPCR; cation channel; mechanosensitivity; myogenic response; vasoconstriction.

Figure 1. Cerebral arteries from AT₁R_a ^−/− mice exhibit enhanced myogenic responses

A, representative endpoint RT‐PCR gel from three independent experiments showing that mRNAs encoding AT₁R_a and AT₁R_b are present in cerebral arteries from WT mice, whereas mRNA encoding AT₁R_a is not detected in cerebral arteries from AT₁R_a ^−/− mice. NTC, no template control. B, summary data showing that vasoconstriction in response to elevated extracellular levels of KCl (60 mm) does not differ between cerebral arteries from WT and AT₁R_a ^−/− mice (n = 13 for WT and AT₁R_a ^−/−). C, representative traces (left) and summary data (right) showing that vasoconstriction in response to the GPCR agonist ET‐1 does not differ between cerebral arteries from WT and AT₁R_a ^−/− mice (n = 4 arteries per group). D, representative traces (left) and summary data (right) showing that the development of myogenic tone in response to increases in intraluminal pressure is greater in cerebral arteries from AT₁R_a ^−/− mice (n = 6 arteries) than in WT mice (n = 5 arteries; ^* P < 0.05).

Figure 2. Myogenic tone of mesenteric resistance arteries from WT and AT₁R_a ^−/− mice does not differ

A, representative traces of changes in luminal diameter of third‐order mesenteric arteries from WT and AT₁R_a ^−/− mice in response to increasing intraluminal pressure. B, summary data. No significant differences in myogenic reactivity were observed between WT and AT₁R_a ^−/− mice (n = 5 arteries per group).

Figure 3. The structure of cerebral and mesenteric resistance arteries does not differ between WT and AT₁R_a ^−/− mice

A, lumen diameter, outer diameter and wall thickness of cerebral arteries were not different between WT and AT₁R_a ^−/−mice (n = 11 arteries per group). B, no differences in lumen and outer diameters or wall thickness were observed in third‐order mesenteric arteries between WT and AT₁R_a ^−/− mice (n = 5 arteries per group). Passive diameters were assessed by bathing the arteries in Ca²⁺‐free PSS supplemented with EGTA (2 mm) and the Ca_V1.2 channel inhibitor diltiazem (10 μm) to assure maximal dilatation at each intraluminal pressure.

Figure 4. Stretch‐induced activation of cation currents in smooth muscle cells is not altered by AT₁R_a deletion

A, representative traces from perforated patch‐clamp experiments demonstrating that increasing negative pressure on the patch pipette from −3 to −20 mmHg increases TICC activity in cerebral artery smooth muscle cells from WT and AT₁R_a ^−/− mice are shown on the left. These currents were nearly abolished by the TRPM4 blocker 9‐phenanthrol (30 μm). The right panels show summary data (WT, n = 9–10 cells per group; AT₁R_a ^−/−, n = 5–7 cells per group); ^* P < 0.05. B, summary data demonstrating that increases in TICC NP _o in response to increasing negative pressure applied to the patch pipette are not different between cerebral artery smooth muscle cells from WT (n = 9–11 cells per group) and AT₁R_a ^−/− (n = 7–16 cells per group) mice.

Figure 5. Losartan inhibits myogenic responses in cerebral arteries

A and B, representative traces (left) and summary data (right) demonstrating that the development of myogenic tone in cerebral arteries from WT and AT₁R_a ^−/− mice is significantly reduced by losartan (1 μm). ^* P < 0.05, for WT + vehicle (n = 7 arteries) vs. WT + losartan (n = 5 arteries) and AT₁R_a ^−/− + vehicle (n = 7 arteries) vs. AT₁R_a ^−/− + losartan (n = 6 arteries).

Figure 6. Losartan inhibits stretch‐induced TRPM4 currents

A, representative traces (left) and summary data (right) showing that TICC activity, stimulated by application of suction to the patch pipette (−20 mmHg), is significantly diminished by losartan (1 μm) in cerebral artery smooth muscle cells isolated from WT and AT₁R_a ^−/− mice. ^* P < 0.05 for WT (n = 5–10 cells) vs. AT₁R_a ^−/− (n = 5–7 cells). B, losartan did not block UTP‐induced TICCs (^* P ≤ 0.05 vs. losartan; n = 6 cells per group), but UTP‐induced TICCs were blocked by 9‐phenanthrol (^* P ≤ 0.05 vs. UTP; n = 6 cells per group).

Figure 7. Knockdown of AT₁R_b expression selectively blunts myogenic constriction of cerebral arteries

A, on the left are representative confocal images (from four independent experiments) of freshly isolated cerebral artery myocytes treated with Lissamine‐tagged morpholinos (red). Smooth muscle cells from arteries treated with non‐tagged, standard control morpholinos are shown on the right. These cells showed no red fluorescence. Plasma membranes were stained (green) to show the outline of the cells. Scale bar = 10 μm. B–D, vasoconstriction in response to Ang II (30 nm; n = 4–5 arteries per group; B and C) and increased extracellular [K⁺] (60 mm; n = 8 arteries per group; D) does not differ between arteries treated with standard control or AT₁R_b‐targeting morpholinos (bottom). E, representative traces (left) and summary data (right) demonstrating that development of myogenic tone is suppressed in arteries treated with AT₁R_b‐targeting morpholinos compared with standard control morpholinos (^* P < 0.05; n = 9 arteries per group).

Figure 8. Knockdown of AT₁R_b expression in cerebral arteries from AT₁R_a ^−/− mice blunts constriction to Ang II and reduces myogenic reactivity

A and B, representative traces (A) and summary data (B) demonstrating that constriction in response to Ang II (30 nm) was nearly abolished after AT₁R_b knockdown in cerebral arteries from AT₁R_a ^−/− mice. (^* P < 0.05 vs. AT1R_a ^−/−, n = 6–7 arteries per group). C, summary data showing that vasoconstriction of cerebral arteries from AT₁R_a ^−/− mice in response to elevated extracellular levels of KCl (60 mm) is not altered by AT₁R_b knockdown (n = 6–7 arteries per group). D and E, representative recordings (D) and summary data (E) demonstrating that myogenic constriction of cerebral arteries from AT₁R_a ^−/− mice is nearly abolished by AT₁R_b knockdown. (^* P < 0.05, n = 6–7 arteries per group).

Figure 9. AT₁R_b mRNA is more abundant than AT₁R_a mRNA in cerebral and mesenteric arteries

A, levels of AT₁R_b mRNA are ∼30‐fold greater than those of AT₁R_a in cerebral arteries from WT mice (^* P < 0.05; n = 5 separate experiments). B, mRNA expression of AT₁R_b is ∼45‐fold higher than that of AT₁R_a in isolated cerebral artery smooth muscle cells (^* P < 0.05; n = 3 separate experiments). Smooth muscle cells were isolated using FACS flow cytometry as described. C, levels of AT₁R_b mRNA are ∼2.4‐fold greater than those of AT₁R_a in mesenteric arteries from WT mice (^* P < 0.05; n = 3 separate experiments). C, levels of AT₁R_a mRNA are ∼7‐fold greater than those of AT₁R_b in samples from whole kidney (^* P < 0.05; n = 3 separate experiments).

Figure 10. Ang II stimulates cation current activity in cerebral artery smooth muscle cells

A and B, representative traces (left) and summary data (right), demonstrating that cation current activity induced by Ang II (30 nm) in cerebral artery myocytes from WT (A) and AT₁R_a ^−/− (B) mice is attenuated by the TRPM4 inhibitor 9‐phenanthrol (30 μm). ^* P < 0.05 vs. all other groups; n = 6–13 (WT) and 5–7 (AT₁R_a ^−/−) cells per group. 3 mmHg of negative pressure was maintained at the patch pipette during these experiments. C, representative traces (left) and summary data (right), demonstrating that Ang II (30 nm)‐induced vasoconstriction of pressurized cerebral arteries from WT (top) and AT₁R_a ^−/− (bottom) mice is attenuated by losartan (1 μm, ^* P < 0.05 vs. Ang II; n = 5–6 arteries per group).