Modulation in G protein coupled receptor kinase 2 expression/catalytic activity differentially change vasoconstrictor-induced arterial smooth muscle cell proliferation

A. ALONAZI^1,2, J. Willets¹. ¹Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom, ²Pharmacology and Toxicology Department, King Saud University, Pharmacy College, Riyadh, Saudi Arabia.

Introduction: Vascular smooth muscle cell proliferation plays a key role in the development of hypertensive vascular remodelling¹, a process strongly associated with increased circulating vasoconstrictor concentrations², leading to continuous activation of their cognate Gα_q-coupled/G protein-coupled receptors (GPCR). Hypertension is associated with elevated G protein coupled receptor kinase 2 (GRK2) expression in arterial smooth muscle cells (ASMC)³, which negatively regulates Gα_q/GPCR signalling³. Here we investigated the potential roles that GRK2 plays in vasoconstrictor-stimulated ASMC proliferation, and its effects on phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt signalling pathway as a possible molecular mechanism underlying GRK2-mediated regulation of ASMC proliferation.

Methods: ASMC were prepared from isolated rat aortae exactly as described previously. [³H]-thymidine incorporation assays and specific siRNA techniques were utilised to determine whether GRK2 played a role in vasoconstrictor-stimulated ASMC growth. Furthermore, the effects of GRK2 depletion/inhibition on Akt signalling using immunoblotting techniques were examined.

Results: Vasoconstrictors increased ASMCs proliferation (angiotensin-II 48425±2826, ET1 52251± 3634 vs. 36245± 1316 FCS stimulated cells; one-way ANOVA; mean ± SEM, n=16). Western blotting experiments indicate GRK2 siRNA mediated (>80%, n=9) depletion in comparison to NC siRNA transfected cells. In GRK2 depleted cells, ASMCs proliferation was significantly decreased in angiotensin-II (p<0.05) and ET1 (p<0.05) stimulated cells [164.6±22.3 GRK2 depleted cells vs. 249.7±38.7 NC transfected cells; two-way ANOVA, n=16]. Following COMP101 (30 μM; 30 min) pre-treatment ASMCs proliferation was significantly (p<0.05) decreased [ET1 68424±38741 vs. DMSO 127747±17117; two-way ANOVA, n=12]. Moreover, we examined the effects of GRK2 depletion on Akt signalling. In NC siRNA transfected cells angiotensin-II or ET-1 induced a time-dependent increase in Akt phosphorylation, that peaked at 5 min and remained elevated for >60 min. Knockdown of GRK2 attenuated the sustained phase (>5 min) of both angiotensin-II (p<0.001) and ET-1 (p<0.05) stimulated Akt phosphorylation.

Conclusion: These data suggest that the presence of GRK2 protein and its catalytic activity are required to mediate vasoconstrictor-stimulated ASMC proliferation. Moreover, since Akt signalling is known to mediate vasoconstrictor-stimulated ASMC growth and as GRK2 is required for angiotensin-II and ET-1 mediated Akt signalling, this may explain why knockdown of GRK2 prevents vasoconstrictor-stimulated ASMC growth.

References:

(1) Agabiti-Rosei E and Rizzoni D. (2010) Curr Hypertens Rep. 12:80-5.

(2) Harris DM et al. (2008) Clin Sci (Lond). 115:79-89.

(3) Willets JM et al. (2015) Am J Physiol Cell Physiol. 309:C179-C189.