Endocrine Abstracts

We have recently demonstrated expression of a functional calcium sensing receptor (CaSR) in human aortic smooth muscle cells (HAoSMC) and human arteries and demonstrated a correlation between CaSR expression and medial calcification (a major cause of cardiovascular mortality in chronic kidney disease). Apoptosis of vascular SMC occurs early in the development of calcification and apoptotic bodies accumulate calcium. Here, we have examined CaSR-mediated signalling pathways in HAoSMC and the role of the CaSR in proliferation and apoptosis. Incubation of HAoSMC with the CaSR agonist neomycin (300 μM), caused a significant increase in ERK1,2 phosphorylation (748±130% of control, P<0.05). Neomycin mediated ERK1,2 stimulation was inhibited by pre-treatment with PD-98059 (10 μM; ERK1 inhibitor) (32% of neomycin induction, P<0.01) and U-73122 (5 μM; PLC inhibitor) (23% of neomycin induction, P<0.01) suggesting that PLC-signalling was important for MEK1/ERK1,2 activation. No changes were seen with PKC and PI3K inhibitors. To confirm ERK1,2 stimulation was mediated via the CaSR, HAoSMC were transfected with CaSR siRNA (CaSR-knockdown). In CaSR-knockdown cells, CaSR expression was reduced by >80% (P<0.05 compared to cells transfected with control siRNA). CaSR-knockdown cells demonstrated an attenuated response to neomycin (44% of neomycin induction in control siRNA cells, P<0.01). Treatment with neomycin increased HAoSMC proliferation (365±33%, P<0.001). This was attenuated in CaSR-knockdown cells (111±6%, P<0.01) and was further inhibited by PD-98059 and U-73122 (P<0.05) confirming the importance of CaSR mediated MEK/ERK1,2 and PLC signalling in HAoSMC proliferation. Apoptosis was not affected by neomycin or CaSR siRNA, but incubation with U-73122 increased apoptotic cell death (3.5-fold vs control P<0.05), which was further increased by CaSR-knockdown (4.8-fold vs control siRNA, P<0.05). Taken together these data demonstrate that agonist stimulation of the CaSR activates the MEK/ERK1,2 pathway and PLC signalling is involved. In addition, CaSR-mediated PLC activation is important for HAoSMC proliferation and protection against apoptosis.