Expression of plasma membrane calcium ATPases in phenotypically distinct canine vascular smooth muscle cells

Our laboratory has identified at least two types of vascular smooth muscle cells (VSMCs) that exist in canine arteries and veins: type 1 cells, locate d in the media express muscle specific proteins but do not proliferate in c ulture; and type 2 cells, located in both media and adventitia, do not expr ess muscle specific protein but proliferate in culture. Plasma membrane Ca2 +-ATPases (PMCAs) have been implicated in proliferation control. The presen t study examines the expression of PMCA isoforms and calmodulin-binding dom ain splice variants in these two types of canine VSMCs. PMCA protein was fo und in both type 1 and type 2 cells, Reverse transcriptase-polymerase chain reaction assays were developed for canine PMCA calmodulin-binding domain s plice variants. We cloned and sequenced isolates corresponding to PMCA1b, 4 a and 4b from canine VSMCs. PMCA 2 and 3 were not detected. Freshly isolate d type 1 cells expressed PMCA Ib, 4a and 4b, while freshly isolated type 2 cells expressed PMCA1b and 4b, Upon placement in culture, type 2 cells orig inating from either carotid artery or saphenous vein demonstrated a time-de pendent upregulation of PMCA4a mRNA, Treatment with the phosphoinositide 3- kinase inhibitor wortmannin produced concentration-dependent inhibition of both PMCA4a upregulation and [H-3]thymidine incorporation. These findings s uggest a role for phosphoinositide 3-kinase in regulating PMCA expression, which may be important in the control of Ca2+-sensitive VSMC functions. (C) 2000 Academic Press.