Differential expression of angiotensin II type 1 receptor subtypes within the cerebral microvasculature

Ethical approvals.

Husbandry and all experimental protocols involving animals were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of Nevada, Reno School of Medicine.

Animals.

Young adult male (8–20 wk old) wild-type (WT) C57BL/6J mice (Jackson Laboratories Bar Harbor, ME) and homozygous global AT₁R_a-knockout (Agtr1a^−/−) mice (B6.129P2-Agtra1^tm1Unc/J; Jackson Laboratories) were used for these studies. A prior study reported that the systolic blood pressure of Agtr1a^−/− mice is lower compared with WT controls when measured using the tail-cuff method (WT: 113 ± 2 mmHg; Agtr1a^−/−: 89 ± 4 mmHg) and intra-arterial catheters (WT: 133 ± 4 mmHg; Agtr1a^−/−: 90 ± 10 mmHg) (17). Animals were maintained in plastic cages (≤5 mice/cage) with free access to food and water in a room with a controlled 12-h:12-h light-dark cycle. Mice were euthanized with 5% isoflurane (Baxter Healthcare, Deerfield, IL) followed by decapitation before experimentation.

Isolation of pial arteries and parenchymal arterioles.

Cerebral pial arteries were gently removed from the brain in ice-cold (~4°C), Mg²⁺-physiological saline solution (Mg²⁺-PSS) consisting of 134 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 10 mM HEPES, 10 mM glucose, and 0.5% bovine serum albumin (BSA), pH 7.4. Parenchymal arterioles were isolated using our previously described procedure (29). Briefly, a 5 × 3-mm section of brain surrounding the middle cerebral artery was removed. The pia containing the middle cerebral artery was then gently separated by blunt dissection, and parenchymal arterioles were carefully dissected free of the pia.

Pressure myography.

Isolated, endothelium-intact cerebral parenchymal arterioles and pial arteries from WT and Agtr1a^−/− mice were mounted between two glass cannulas (outer diameter: 30–50 μm) in a pressure myography chamber (Living Systems Instrumentation, Burlington, VT) and secured using nylon monofilament thread. Cannulated parenchymal arterioles were bathed in warmed (37°C), oxygenated (21% O₂-6% CO₂-73% N₂) artificial cerebrospinal fluid (aCSF; in mM: 124 NaCl, 3 KCl, 2 CaCl₂, 2 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, and 4 glucose) at an initial intraluminal pressure of 5 mmHg using aCSF as the intraluminal solution and were allowed to equilibrate for 15 min. Cannulated pial arteries were bathed in warmed, oxygenated PSS (in mM: 119 NaCl, 4.7 KCl, 1.8 CaCl₂, 1.17 MgSO₄, 1.18 KH₂PO₄, 21 NaHCO₃, 4 glucose, and 0.026 EDTA) at an initial intraluminal pressure of 5 mmHg using PSS as the intraluminal solution and were allowed to equilibrate for 15 min. Pressurized parenchymal arterioles and pial arteries were visualized, and luminal diameter was recorded using an inverted microscope (Accu-Scope, Commack, NY) coupled to a CCD camera (The Imaging Source, Charlotte, NC) controlled by IonWizard software (version 6.4.1.73; IonOptix, Westwood, MA). The viability of preparations was verified by measuring vasoconstriction in response to superfusion with isotonic aCSF or PSS containing elevated K⁺ concentration ([K⁺]; 60 mM). Arterioles that showed <10% constriction in response to elevated [K⁺] were considered nonviable and were excluded from further study.

Myogenic response.

The myogenic response of parenchymal arterioles to acute increases in intraluminal pressure was assessed by slowly raising intraluminal pressure in a stepwise fashion from 5 to 20 mmHg and then to 140 mmHg in 20-mmHg increments. Arterioles were maintained at each pressure step until a steady-state diameter was reached. Following completion of initial control pressure steps, intraluminal pressure was lowered to 5 mmHg and the vessels were allowed to equilibrate for 15 min before losartan, captopril, or vehicle (water) was added to superfusing bath. Vessels were pretreated with drugs (or vehicle) for 15 min before the initiation of pressure challenges and for the remainder of the experiment. Following completion of these procedures, the intraluminal pressure was lowered to 5 mmHg and arterioles were superfused with Ca²⁺-free aCSF supplemented with EGTA (2 mM) and the voltage-dependent Ca²⁺ channel blocker diltiazem (10 μM) to inhibit SMC contraction and reveal the passive diameter of the arteriole segment. Passive luminal diameter was recorded under these conditions as the intraluminal pressure was raised from 5 to 20 mmHg and then to 140 mmHg in 20-mmHg increments. Myogenic tone at each pressure step was calculated as myogenic tone (%) = [1 – (active lumen diameter/passive lumen diameter)] × 100.

Acute administration of losartan.

The impact of AT₁R inhibition on preestablished myogenic tone in parenchymal arterioles was assessed by first slowly raising intraluminal pressure from 5 to 20 mmHg and then to 60 mmHg in 20-mmHg increments to induce spontaneous myogenic tone. After arterioles reached a steady-state diameter at 60 mmHg, losartan (1 µM) was added to the superfusing bath and was maintained for at least 5 min or until the vessels reached a steady-state diameter. The arterioles were subsequently superfused with Ca²⁺-free aCSF supplemented with EGTA (2 mM) and diltiazem (10 μM) to reveal the passive diameter of the arteriole segment. Myogenic tone in the absence and presence of losartan was calculated as myogenic tone (%) = [1 – (active lumen diameter/passive lumen diameter)] × 100.

ANG II-induced contractility.

The contractility of parenchymal arterioles and pial arteries in response to ANG II was assessed at an intraluminal pressure of 20 mmHg to minimize the development of spontaneous myogenic tone. Following a 15-min equilibration period, vessels were superfused with aCSF (parenchymal arterioles) or PSS (pial arteries) containing different concentrations of ANG II (0.1–100 nM) until a steady-state diameter was reached. Luminal diameter was recorded before addition of ANG II (baseline) and at the peak response. ANG II response was calculated as vasoconstriction (%) = [(baseline – peak response)/baseline] × 100. The possibility that AT₁Rs in pial arteries desensitize in response to repeated application of ANG II was evaluated by repeating administration of ANG II at the same concentration after a 20-min washout with PSS. These studies suggested that desensitization of AT₁Rs persisted for at least 20 min. Therefore, ANG II treatment of arteries and arterioles was limited to a single exposure.

RNA isolation, reverse transcription, and end point/quantitative PCR.

Isolated whole pial arteries or parenchymal arterioles were suspended in TRIzol Reagent (Invitrogen, Carlsbad, CA) and homogenized using a syringe and 20-gauge needle. The homogenate was centrifuged at 20,800 g for 5 min, and the supernatant was transferred to a new tube. RNA was isolated using Direct-zol RNA MicroPrep (Zymo Research, Irvine, CA), quantified using a Nanodrop 2000 spectrophotometer (ThermoFisher, Waltham, MA), and treated with OPTIZYME DNase I (Fisher BioReagents, Pittsburgh, PA) to remove contaminating genomic DNA. First-strand cDNA was synthesized using qScript cDNA SuperMix (Quanta, Gaithersburg, MD).

Quantitative PCR was performed on a QuantStudio 3 Real-Time PCR System (ThermoFisher) using Fast SYBR Green Master Mix (ThermoFisher) and the following specific primer pairs: β-actin (Actb; NM_007393): 5′-CCAGCCTTCCTTCTTGGGTA-3′ (sense) and 5′-AGAGGTCTTTACGGATGTCAACG-3′ (antisense); AT₁R_a (Agtr1a; NM_177322): 5′-GACCAACTCAACCCAGAAAAGC-3′ (sense) and 5′-GGGGCAGTCATCTTGAATTCTT-3′ (antisense); AT₁R_b (Agtr1b; NM_175086): 5′- GCAGCATTTAGCTAGACAGTTC-3′ (sense) and 5′-AACAGTAGCTTTGCTCTGGGG-3′ (antisense); and total AT₁R (Agtr1a and Agtr1b): 5′-GATCGCTTCGGCCAGCGTCA-3′ (sense) and 5′-GCCAAGCCAGCCATCAGCCA-3′ (antisense). Reactions without template cDNA served as contamination controls. Quantitative PCR data were normalized to Actb expression levels and analyzed using the ΔΔC_T method (21). All RT-PCR assays were repeated three times for each sample and were independently replicated using RNA extracted from three different animals in each experimental group.

Statistical analysis.

All summary data are expressed as means ± SE. Prism 8.1.2 (GraphPad Software, La Jolla, CA) was used to perform statistical analyses and construct graphs. The significance of differences in ANG II contractile responses and myogenic reactivity were analyzed by two-way ANOVA with a Sidak post hoc test. Differences in the constriction of pial arteries in response to repeated administration of ANG II was analyzed by paired two-tailed t-test. The constriction of parenchymal arterioles and pial arteries in response to elevated extracellular [K⁺] was analyzed by unpaired two-tailed t-test. mRNA expression levels were analyzed by unpaired two-tailed t-test. Values of P < 0.05 were considered statistically significant for all experiments.

Agonist-independent AT₁R activity is essential for the initiation of myogenic tone in parenchymal arterioles.

Pressure myography was used to investigate the importance of AT₁R-activated signaling pathways in the development of myogenic tone in parenchymal arterioles. We found that pretreatment with losartan (1 µM), a selective AT₁R antagonist, significantly impaired the development of myogenic tone across a range of intraluminal pressures in parenchymal arterioles isolated from WT mice (Fig. 1A). To determine if this effect was related to local generation of ANG II by angiotensin-converting enzyme (ACE), we investigated the impact of the selective ACE inhibitor captopril (10 µM). We found that captopril had no effect on the development of myogenic tone in parenchymal arterioles (Fig. 1B). As expected, control studies showed that water (vehicle for both losartan and captopril) had no effect on the development of myogenic tone (Fig. 1C). However, in agreement with a previous study (4), we found that acute application of losartan to untreated parenchymal arterioles with spontaneously established myogenic tone had no effect on luminal diameter (Fig. 1D). Together, these data indicate that the activity of AT₁Rs, which is independent of ACE activity, is required for the initiation of myogenic constriction in parenchymal arterioles, but once established, myogenic tone is maintained when these receptors are blocked.

Myogenic constriction is impaired in parenchymal arterioles from Agtr1a^−/− mice.

Our previous study showed that genetic knockout of the AT₁R_a isotype has no effect on the myogenic tone of cerebral pial arteries, likely due to high expression levels of AT₁R_b (31). To determine the specific role of AT₁R_a in pressure-induced constriction of parenchymal arterioles, we compared myogenic tone between parenchymal arterioles isolated from WT and Agtr1a^−/− mice. These experiments showed that parenchymal arterioles isolated from Agtr1a^−/− mice developed significantly less myogenic tone across a range of intraluminal pressures compared with arterioles from WT mice (Fig. 2A). Control experiments demonstrated that constriction of parenchymal arterioles from WT and Agtr1a^−/− mice in response to direct depolarization of the SMC plasma membrane by elevation of extracellular [K⁺] did not differ, suggesting that knockout of AT₁R_a does not grossly alter voltage-dependent Ca²⁺-influx pathways or the contractile apparatus (Fig. 2B). Expression level analysis of Agtr1b, encoding AT₁R_b, in parenchymal arterioles isolated from WT and Agtr1a^−/− mice exhibited that Agtr1a^−/− did not impact AT₁R_b expression levels in parenchymal arterioles (Fig. 2C). These data indicate that AT₁R_a expression is critically important for the development of myogenic tone in parenchymal arterioles.

AT₁R is expressed at much lower levels in parenchymal arterioles compared with cerebral pial arteries.

Expression levels of Agtr1a and Agtr1b, encoding AT₁R_a and AT₁R_b, respectively, were independently assessed in parenchymal arterioles and pial arteries using quantitative RT-PCR. We found that AT₁R_a and AT₁R_b mRNA were expressed at similar levels in parenchymal arterioles (Fig. 3A). In contrast, and in agreement with our prior study (31), we found that AT₁R_b mRNA was expressed at much higher levels compared with AT₁R_a mRNA in pial arteries (Fig. 3A). A comparison of isotypes between vascular segments showed that AT₁R_a expression levels were similar in parenchymal arterioles and pial arteries (Fig. 3A), but AT₁R_b expression levels were ~15-fold higher in pial arteries compared with parenchymal arterioles (Fig. 3A). Overall, total AT₁R expression was 11-fold higher in pial arteries compared with parenchymal arterioles (Fig. 3B).

Parenchymal arterioles are less sensitive to the vasoconstrictor effects of ANG II compared with pial arteries.

The fact that total AT₁R mRNA expression levels were much lower in parenchymal arterioles compared with cerebral pial arteries suggested that parenchymal arterioles might be less sensitive to the vasoconstrictor effects of ANG II. In initial experiments designed to test this possibility, we found that the contractile response of cerebral pial arteries to a second administration of ANG II (100 nM) was greatly diminished compared with the first administration, even after a 20-min interval between challenges (Fig. 4). These results are consistent with previous studies showing that AT₁Rs are rapidly internalized and desensitized following stimulation with ANG II (9, 14, 28, 33). Therefore, in subsequent concentration-response studies, vessels were exposed once to a single concentration of ANG II.

At low concentrations, ANG II had little effect on the luminal diameter of either parenchymal arterioles or pial arteries (Fig. 5A). At a higher concentration (100 nM), ANG II induced significant constriction of cerebral pial arteries but had little effect on the luminal diameter of parenchymal arterioles (Fig. 5A), demonstrating that parenchymal arterioles are less sensitive to the constrictor effects of ANG II compared with pial arteries. In control experiments, constriction of parenchymal arterioles in response to elevated extracellular [K⁺] was greater than that of pial arteries (Fig. 5B), indicating that the parenchymal arterioles used for this study show robust contractile responses to direct SMC depolarization.