Abstract
The plasma membrane Ca2+-ATPase pump (PMCA) is an integral component of the Ca2+ signaling system which participates in signal transduction during agonist stimulated cell activation. To better understand the physiological function of the pump, isoform 1a (PMCA1a) was over-expressed in rat aortic endothelial cells using a stable transfection system under the control of a cytomegalovirus promoter. The cell lines selected after transfection with PMCA1a construct, expressed 3-4-fold increased pump protein which was mostly targeted to the plasma membrane as indicated by immunoperoxidase staining. Ca2+ uptake assays in a membrane preparation indicated a 3-4-fold increase in Ca2+ pumping activity in the transfected cells, and the expressed PMCA1a showed typical dependence on Ca2+ and calmodulin for stimulation of activity. Measurement of [Ca2+]i and [Ca2+]out showed that expression of PMCA1a had a profound effect on different aspects of the Ca2+ signal. The peak increase in [Ca2+]i evoked by ATP and/or thapsigargin was lower but the plateau phase was similar in the PMCA1a expressing cells. Accordingly, titration with ionomycin of Ca2+ content of internal stores, measurement of Ca2+ uptake into the thapsigargin- and oxalate-sensitive pool (endoplasmic reticulum) of isolated microsomes, Ca2+ uptake into streptolysin O-permeabilized cells, and analysis of SERCA mRNA and protein, showed that expression and activity of the SERCA pump was down-regulated in cells expressing PMCA1a pump. Expression of PMCA1a also down-regulated expression of the inositol 1,4,5-trisphosphate (IP3)-activated Ca2+ channel and the rate of IP3-mediated Ca2+ release in permeable cells, without affecting the affinity of the channel for IP3. On the other hand the rate of store depletion-dependent Ca2+ and Mn2+ influx (Ca2+ entry) into PMCA1a expressing cells was increased by about 2.6-fold. These changes prevented estimating the rate of pump-mediated Ca2+ efflux from changes in [Ca2+]i. Measurement of [Ca2+]out showed that the rate of Ca2+ efflux in cells expressing PMCA1a was about 1.45-fold higher than Neo controls, despite the 4-fold increase in the amount of functional pump protein. The overall study points to the flexibility, interdependence, and adaptability of the different components of the Ca2+ signaling systems to regulate the expression and activity of each component and maintain a nearly constant Ca2+ signal.
Highlights
(PMCA1a) was over-expressed in rat aortic endothelial cells using a stable transfection system under the control of a cytomegalovirus promoter
In this report we focus on the stable transfection of the plasma membrane Ca2ϩ-ATPase isoform 1a (PMCA1a) isoform of the pump in rat aortic endothelial cells (RAECs), which endogenously contain a low level of PMCA1b as the predominant species, while isoform 1a is either absent or undetectable by reverse transcriptase-polymerase chain reaction [13]
Over-expression of Rat PMCA Isoform 1a in RAEC—Following G418 selection, 36 neomycin-resistant clones were isolated from the culture transfected with the PMCA1a construct
Summary
(PMCA1a) was over-expressed in rat aortic endothelial cells using a stable transfection system under the control of a cytomegalovirus promoter. In this report we focus on the stable transfection of the PMCA1a isoform of the pump in rat aortic endothelial cells (RAECs), which endogenously contain a low level of PMCA1b as the predominant species, while isoform 1a is either absent or undetectable by reverse transcriptase-polymerase chain reaction [13]. This over-expression system allowed us to study the physiological function of PMCA during agonist stimulation. The overall findings demonstrate the flexibility and coordinated regulation of all pathways, possibly on the gene level, to maintain constant Ca2ϩ signaling
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