The Release of Nitric Oxide Is Involved in the β-Arrestin1-Induced Antihypertensive Effect in the Rostral Ventrolateral Medulla

Abstract

β-Arrestin1 is a multifunctional scaffold protein with the ability to interact with diverse signaling molecules independent of G protein-coupled receptors. We previously reported that overexpression of β-arrestin1 in the rostral ventrolateral medulla (RVLM) decreased blood pressure (BP) and renal sympathetic nerve activity (RSNA) in spontaneously hypertensive rats (SHRs). Nitric oxide (NO) is widely reported to be involved in central cardiovascular regulation. The goal of this study was to investigate whether NO signaling contributes to the β-arrestin1-mediated antihypertensive effect in the RVLM. It was found that bilateral injection of adeno-associated virus containing Arrb1 gene (AAV-Arrb1) into the RVLM of SHRs significantly increased NO production and NO synthase (NOS) activity. Microinjection of the non-selective NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME; 10 nmol) into the RVLM prevented the β-arrestin1-induced cardiovascular inhibitory effect. Furthermore, β-arrestin1 overexpression in the RVLM significantly upregulated the expression of phosphorylated neuronal NOS (nNOS) by 3.8-fold and extracellular regulated kinase 1/2 (ERK1/2) by 5.6-fold in SHRs. The β-arrestin1-induced decrease in BP and RSNA was significantly abolished by treatment with ERK1/2 small interfering RNA (ERK1/2 siRNA). Moreover, ERK1/2 siRNA attenuated the β-arrestin1-induced NO production, NOS activity, and nNOS phosphorylation in the RVLM. Taken together, these data demonstrate that the antihypertensive effect of β-arrestin1 in the RVLM is mediated by nNOS-derived NO release, which is associated with ERK1/2 activation.

Keywords: ERK1/2; NO; RVLM; hypertension; β-arrestin1.

FIGURE 1

Effects of overexpression of β-arrestin1 on NO production and NOS activity in the RVLM. Representative gel bands (top) and quantification data (bottom) of β-arrestin1 (A). (B) Relative changes of nitrate and nitrite content (top) and total NOS activity (bottom) in the RVLM in response to administration of AAV-GFP and AAV-Arrb1 in WKY rats or SHRs. n = 6 per group in A and n = 5 per group in B. ****P < 0.0001, *P < 0.05 vs. WKY; ^#P < 0.05 vs. AAV-GFP. Original tracings (C) and quantification data (D,E) of BP, HR, and RSNA in response to unilateral microinjection of L-NAME (10 nmol) into the RVLM of SHRs pretreated with AAV transfection (AAV-GFP or AAV-Arrb1). n = 5 per group. *P < 0.05 vs. aCSF; ^#P < 0.05 vs. AAV-GFP. MAP, mean arterial pressure; NO, nitric oxide; NOS, nitric oxide synthase; RVLM, rostral ventrolateral medulla; AAV, adeno-associated virus; GFP, green fluorescent protein; WKY, Wistar-Kyoto; SHRs, spontaneously hypertensive rats; BP, blood pressure; HR, heart rate; RSNA, renal sympathetic nerve activity; L-NAME, N-nitro-L-arginine methyl ester; aCSF, artificial cerebrospinal fluid.

FIGURE 2

Effects of overexpression of β-arrestin1 on nNOS and ERK1/2 phosphorylation in the RVLM of SHRs. Representative gel bands (top) and quantification data (bottom) of phosphorylated nNOS (A) and ERK1/2 (B) in response to β-arrestin1 overexpression in the RVLM in SHRs. (C) Confocal microscopy photographs represent p-ERK1/2 (red fluorescence) and nucleus (blue fluorescence) in the RVLM. Scale bar = 200 μm. n = 5 per group in A and n = 4 per group in B, *P < 0.05 vs. WKY; ^#P < 0.05 vs. AAV-GFP. Relative level, standardization of dividing phosphorylated values by total values. nNOS, neuronal nitric oxide synthase; ERK1/2, extracellular regulated kinase; RVLM, rostral ventrolateral medulla; SHRs, spontaneously hypertensive rats; WKY, Wistar-Kyoto; AAV, adeno-associated virus; GFP, green fluorescent protein.

FIGURE 3

Effects of knockdown of ERK1/2 in the RVLM on the β-arrestin1-induced changes in BP and RSNA. (A) Representative gel bands (top) and quantification data (bottom) show the changes in phosphorylated and total ERK1/2 after treatment of ERK1/2 siRNA in the RVLM. n = 4 per group. *P < 0.05 vs. Ctrl. (B) Confocal microscopy photographs consecutively represent the potency and stability of siRNA-Cy3 (red fluorescence) in the RVLM from day 1 to day 5. (C) Levels of BP in the conscious groups (GFP + CON siRNA, GFP + ERK1/2 siRNA, Arrb1 + CON siRNA, and Arrb1 + ERK1/2 siRNA). The treatment of siRNA injection into the RVLM of SHRs was performed at day 9 post AAV transfection. n = 5 per group. *P < 0.05 vs. day 7 in the corresponding group; ^#P < 0.05 vs. CON siRNA. (D) Changes in BP (left) and basal RSNA (right) of anaesthetized SHRs in response to siRNA injections. n = 5 per group. *P < 0.05 vs. AAV-GFP; ^#P < 0.05 vs. CON siRNA. MAP, mean arterial pressure; Ctrl, rats that received transfection reagent without siRNA; ERK1/2, extracellular regulated kinase; RVLM, rostral ventrolateral medulla; BP, blood pressure; RSNA, renal sympathetic nerve activity; siRNA, small interfering RNA; GFP, green fluorescent protein; SHR, spontaneously hypertensive rat; AAV, adeno-associated virus.

FIGURE 4

Confirmation of the same injection site of both AAV and siRNA delivered in the RVLM. (A) Standard rat atlas of the RVLM region. (B,C) Green fluorescent protein (GFP) and Cy3 (red fluorescence) confirmed in the RVLM. (D) Merged image of (B,C). (E–G) Enlarged images of (B–D). Scale bars = 200 μm in (B–D) and 50 μm in (E–G). AAV, adeno-associated virus; siRNA, small interfering RNA; RVLM, rostral ventrolateral medulla.

FIGURE 5

Effects of knockdown of ERK1/2 in the RVLM on the β-arrestin1-induced NO production and nNOS phosphorylation. (A) Relative changes of nitrate and nitrite content (top) as well as total NOS activity (bottom) in the RVLM among the four groups. (B) Representative gel bands (top) and quantification data (bottom) of changes in phosphorylated nNOS after treatment of ERK1/2 siRNA in the RVLM among the four groups. n = 5 per group, *P < 0.05 vs. AAV-GFP; ^#P < 0.05 vs. CON siRNA. Relative level, standardization of dividing phosphorylated values by total values. ERK1/2, extracellular regulated kinase; RVLM, rostral ventrolateral medulla; NO, nitric oxide; nNOS, neuronal nitric oxide synthase; NOS, nitric oxide synthase; siRNA, small interfering RNA.