ARRB2 (β-Arrestin-2) Deficiency Alters Fluid Homeostasis and Blood Pressure Regulation

Abstract

Background: GPCRs (G protein-coupled receptors) are implicated in blood pressure (BP) and fluid intake regulation. There is a developing concept that these effects are mediated by both canonical G protein signaling and noncanonical β-arrestin mediated signaling, but the contributions of each remain largely unexplored. Here, we hypothesized that β-arrestin contributes to fluid homeostasis and blood pressure (BP) regulation in deoxycorticosterone acetate (DOCA) salt hypertension, a prototypical model of salt-sensitive hypertension.

Methods: Global β-arrestin1 (Arrb1) and β-arrestin2 (Arrb2) knockout mice were employed to evaluate drinking behavior, and BP was evaluated in Arrb2-knockout mice. Age- and sex-matched C57BL/6 mice served as controls. We measured intake of water and different sodium chloride solutions and BP employing a 2-bottle choice paradigm with and without DOCA.

Results: Without DOCA (baseline), Arrb2-knockout mice exhibited a significant elevation in saline intake with no change in water intake. With DOCA treatment, Arrb2-knockout mice exhibited a significant increase in both saline and water intake. Although Arrb2-knockout mice exhibited hypernatremia at baseline conditions, we did not find significant changes in total body sodium stores or sodium palatability. In a separate cohort, BP was measured via telemetry in Arrb2-knockout and C57BL/6 mice with and without DOCA. Arrb2-knockout did not exhibit significant differences in BP before DOCA treatment when provided water alone, or when provided a choice of water and saline. However, Arrb2-knockout exhibited an increased pressor response to DOCA-salt.

Conclusions: These findings suggest that in salt-sensitive hypertension, ARRB2, but not ARRB1 (β-arrestin 1), might counterbalance the canonical signaling of GPCRs.

Keywords: angiotensin; arrestin; blood pressure; homeostasis; hypertension.

Figure 1.. Ablation of β-Arrestin2 Exacerbates Water Intake with DOCA-salt.

A) Schematic representation of the two-bottle choice experimental protocol. Mice were subjected to the two-bottle paradigm under baseline and DOCA conditions. At each condition, mice were subjected to 4 different trials in which mice were presented to two burettes containing: 1) water vs. water, 2) water vs. 0.15 M saline, 3) water vs. 0.30 M saline, and 4) water vs. 0.45 M saline. Burettes were switched every 24h to avoid a side bias and data were calculated as the average of 2 consecutive days. B and C) Side bias (B) and total daily water intake (C) when animals were presented with 2 burettes filled with water (water vs. water). Data are expressed as mean±SEM. Data were analyzed by 2-way ANOVA with Tukey multiple comparisons procedure. *P<0.05 compared with C57BL/6; ^#P<0.05 compared to same genetic groups at baseline. C57BL/6 (n=21), Arrb1-KO (n=19), and Arrb2-KO (n=24).

Figure 2.. Drinking Responses to Two-bottle Choice: Water vs. 0.15M Saline

Female (A) and male (B) C57BL/6J, Arrb1-KO and Arrb2-KO mice were presented with a choice of water vs. 0.15 M saline at baseline and after 2 weeks of DOCA-salt. Total water intake, saline intake, fluid intake (calculated as total water intake plus total saline intake), and saline preference (calculated as the percentage of total saline intake over total fluid intake) are shown. Data are expressed as mean±SEM. Data were analyzed by 3-way ANOVA with Sidak multiple comparisons procedure. *P<0.05 compared to same genetic group on baseline condition; ^$P<0.05 Arrb2-KO+DOCA vs. C57BL/6+DOCA; ^P<0.05 one sample t-test compared to 50%. C57 females (n=12), C57 males (n=9), Arrb1-KO females (n=6), Arrb1-KO males (n=13), Arrb2-KO females (n=12), and Arrb2-KO males (n=12).

Figure 3.. Summary of Drinking Responses to 0.15 M, 0.30 M, and 0.45 M Saline.

Summary of combined data of male and female C57BL/6, Arrb1-KO and Arrb2-KO mice subjected to the 2-bottle choice paradigm at Baseline (A and B) and after 2 weeks of DOCA (C and D). Total water intake, saline intake, fluid intake (calculated as total water intake plus total saline intake), saline preference (calculated as the percentage of total saline intake over total fluid intake) and sodium concentration are shown. Data are expressed as mean±SEM. Data were analyzed by 2-way ANOVA with Tukey multiple comparisons procedure. *P<0.05 Arrb2-KO vs. C57BL/6; ^φP<0.05 Arrb1-KO vs. C57BL/6J; ^P<0.05 one sample t-test compared to 50%. C57BL/6 (n=21), Arrb1-KO (n=19), and Arrb2-KO (n=24).

Figure 4.. Plasma and Urine Electrolytes.

Mice were placed in metabolic cages for 24-hour urine collection. A and B) Urine Na, K and osmolality (A); and plasma Na, K and osmolality (B) were measured in Arrb1-KO, Arrb2-KO and wildtype controls. C-E) Capacity to excrete Na was assessed in Arrb2-KO mice and wildtype controls. C) Urine Na excretion 4-hours post intraperitoneal injection of isotonic saline solution. D) Urine K excretion 4-hours post intraperitoneal injection of isotonic saline solution. E)Transcutaneous measurement of glomerular filtration (tGFR) rate was assessed in Arrb2-KO mice and wildtype controls. Data are expressed as mean±SEM. Data were analyzed by t-test. *P<0.05 compared with C57BL/6 or Arrb1-KO. C57BL/6 (n=7–14), Arrb1-KO (n=7–11), and Arrb2-KO (n=7–15).

Figure 5.. Saline Intake in a Brief-Access Paradigm.

Voluntary saline intake was assessed in Arrb2-KO mice and C57BL/6. Animals were subjected to the two-bottle paradigm in lickometer-equipped cages. Mice were presented with 2 burettes, one containing water and one containing 0.15 M saline. Licking events from each burette were recorded for 2 hours. Number of licking events during the first bout (first minute) were totaled during the 2-hour experiment. Data are expressed as mean±SEM. One point was identified as an outlier in each of the C57 and Arrb2-KO (Saline) by Grubb’s test and were excluded from the analysis. Data were analyzed by one sample t-test. C57BL/6 (n=5) and Arrb2-KO males (n=7).

Figure 6.. Ablation of β-Arrestin2 Exacerbates DOCA-salt Hypertension.

A) Arrb2-KO mice and wildtype controls were instrumented with radiotelemeters. Subsequently, mice were subjected to 3 separate conditions and blood pressure (BP) and heart rate (HR) were continuously recorded. First, animals were presented with 2 burettes of water. Second, animals were presented with water vs. 0.15M saline. Third, animals were implanted with DOCA pellets and given water vs. 0.15 M saline. B and C) The daily average systolic blood pressure (SBP, B) and averaged per condition (C) are plotted. D) Hourly SBP average during water, saline and DOCA conditions. E) Average SBP during water vs. water, water vs. 0.15M saline and DOCA conditions are shown separated into the light and dark (shadowed region) cycles. Data are expressed as mean±SEM. Data were analyzed by 2-way ANOVA with Tukey multiple comparisons procedure (B-D) or Sidak multiple comparisons procedure (E). *P<0.05 compared to C57BL/6. C57BL/6 (n=8), and Arrb2-KO (n=9).