Role of SERCA3 in calcium signaling mediated by α1-adrenoceptors and potential interaction with the NO pathway in mouse aorta

Introduction

Regulation of vascular tone is a balance between contraction and relaxation of smooth muscle cells (SMC). Calcium plays a key role in these processes, and the Sarco/Endoplasmic Reticulum Ca2 ± ATPases (SERCAs) pumps are crucial to regulate calcium homeostasis. While SERCA2 is the predominant isoform in the vascular system, SERCA3 is also co-localized in both SMC and endothelial cells, raising the question of the specific role of SERCA3 in vascular calcium regulation.

Objective

Our objective is to investigate the role of SERCA3-dependent calcium signaling in the regulation of mouse aorta vasomotion.

Method

Aortas were collected from wild-type (WT) and SERCA3-deficient (KO) male mice and vascular tone was studied by ex vivo myography. Western blot was used to evaluate protein expression in aorta lysates. Cytosolic calcium levels were studied by epifluorescence microscopy in freshly-isolated aortic SMC loaded with the Fura-2 probe (1 μM).

Results

Deletion of SERCA3 leads to a decrease in contractile response specifically to α1-adrenoceptor agonist (phenylephrine, PE) without alteration of the α1-adrenoceptor expression. Interestingly, inhibition of eNOS with L-NAME abolishes this difference in PE-induced contraction between WT and KO aorta. In addition, eNOS phosphorylation (S1177) during PE stimulation is higher in KO compared to WT aortas. Overall, this suggests an increase in the NO-dependent inhibitory control of PE response in KO arteries. Furthermore, contribution of intracellular calcium stores and extracellular calcium influx to PE contractile response were investigated. Using a calcium free extracellular solution and calcium channel blockers (verapamil for L-type calcium channels (LTCC) and 2APB for Store-Operated Calcium Entry (SOCE) inhibition, respectively), we propose that the decreased PE-contraction in KO is due to a reduced calcium influx through LTCC and SOCE. This alteration is not associated to changes in LTCC or TRPC5 expression. Finally, we showed that calcium mobilization in response to PE is reduced in freshly-isolated SMC from SERCA3-KO compared to WT aorta.

Conclusion

Our findings suggest that in mouse aorta, SERCA3 regulates the α1-adrenergic-dependent contractile function through modulation of calcium influx (by LTTCs and SOCEs) and the endothelial NO-related relaxing pathway.