Increased vasoconstrictor receptor desensitization observed during the development of hypertension is mediated by enhanced vascular GRK2 and arrestin expression

CA Nash1,2, RD Rainbow3, CP Nelson2, RAJ Challiss2, JM Willets1. 1Dept of Cancer Studies and Molecular, University of Leicester, Leicester, UK, 2Dept of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK, 3Dept of Cardiovascular Sciences, University of Leicester, Leicester, UK

Blood pressure is regulated by complex interactions between vasoconstrictor and vasodilator signalling pathways, which are mediated by a variety of G protein-coupled receptors (GPCRs), including P2Y and ETA, and whose dysregulation is implicated in the development of hypertension. GPCR signalling is primarily regulated by G protein-coupled receptor kinases (GRKs) and arrestins, and interestingly GRK2 expression increases in hypertensive arterial smooth muscle (Gros et al., 2000). Furthermore, we have shown that both P2Y2 and ETA receptor signalling is regulated by GRK2 and arrestin proteins (Morris et al., 2010; 2011). Here we have utilized mesenteric arteries from 6 week-old (pre-hypertensive) or 12 week-old (hypertensive) spontaneously-hypertensive rats (SHR) or age-matched normotensive (Wistar-Kyoto (WKY)) rats to examine vasoconstrictor-mediated responses using wire myography of intact vessels, as well as primary mesenteric artery smooth muscle cell cultures, to assess a variety of cellular responses.Expression of GRK2 (1.7-fold relative to WKY expression; n=3, p<0.05,), arrestin2 (1.9-fold; n=3, p<0.01) and arrestin3 (2.9-fold; n=3, p<0.01; one-way ANOVA, Bonferroni’s post-hoc test) was significantly increased in 12 week-old SHR animals. No differences in GRK2 or arrestin2/3 expression were observed in 6 week-old SHR versus WKY animals, or in non-vascular smooth muscle at the 12-week time-point. In mesenteric artery myography experiments, responses to high extracellular K+ were increased in vessels from SHR animals at both 6 weeks (1.8 fold; n=16 vessels, p<0.01) and 12 weeks (2.5-fold; n=8 vessels, p<0.01) of age. In contrast, UTP concentration-response experiments displayed a significant leftward shift in 12 week-old (pEC50 3.75±0.15 versus 4.49±0.23 for WKY and SHR; p<0.05; n=8), but not 6 week-old (pEC50 3.69±0.07 versus 3.84±0.22 for WKY and SHR, n=8) animals. Desensitization of UTP-stimulated vessel contractions was similar in vessels from 6 week-old WKY and SHR, but was enhanced in vessels from 12 week-old SHR compared to WKY rats (1.9-fold, n=7, p<0.05). Dual imaging of IP3 and Ca2+ (using eGFP-PHPLC δ and Fura-Red; Morris et al., 2010) in mesenteric arterial cells showed that desensitization responses to UTP and ET1 were similar in 6 week-old animals. In contrast, at 12 weeks desensitization of UTP responses (IP3, 1.9-fold; n=18, p<0.001 and Ca2+, 2.0-fold; n=13, p<0.05; ANOVA; Dunnett’s post-hoc test) and ET-1 (IP3, 2.2-fold; n=10, p<0.05 and Ca2+, 3.3-fold; n=7, p<0.05) was enhanced in arterial cells isolated from SHR compared to WKY rats. siRNA-mediated depletion of GRK2 or arrestin2 for UTP, and GRK2 or arrestin3 for ET1, respectively, abrogated the observed changes in GPCR signalling in 12 week-old SHR-derived mesenteric smooth muscle cells. In conclusion, we show that vasoconstrictor-stimulated vessel contraction is markedly enhanced in SHR animals. Increased GRK2 and arrestin2/3 expression in arterial smooth muscle from hypertensive rats mediates an increased ability to cause P2Y/ETA receptor desensitization in resistance arteries. These changes in the GPCR regulatory machinery may act partially to compensate for increased vasoconstrictor-induced vessel contraction.

We thank the British Heart Foundation for financial support (PG/11/60/29007; PG06/00822062).

Gros R, et al. (2000) Hypertension 35, 38-42

Morris GE, et al. (2010) Cardiovasc Res 85, 424-433

Morris GE et al. (2011) Cardiovasc Res 89, 193-203