Whereas during the agonist-dependent opening phase of the IP3 receptor the activity of the SERCA pump was likely to become short-circuited by that of the Ca2+ release channel, the PMCA pump, whose essential task under resting conditions is that of extruding Ca2+ penetrating from the extracellular medium, would rapidly increase the rate of Ca2+ ejection as soon as the ER Ca2+ channels open and some of the released Ca2+ trickles over to the cytosol at large (see Camello em et al /em ., 1996). The slow decline of the [Ca2+]c transient after the agonist-induced peak evidently was the algebraic sum of two opposite components: the Ca2+ removal from the cytosol (into the ER or the extracellular medium) and its entry from the external space by the CCE. be limiting. = 8) for PMCA4 and 45% (= 6) for SERCA2b, over the respective controls (lanes 3 and 4). Since the average transfection efficiency approached 25%, the increase of the pump protein in overexpressing cells, corrected for the whole cell population, would correspond to 200%, i.e. the total amount of LG-100064 pump would be 3-fold the endogenous level. The overexpression of one pump apparently failed to influence the expression of the other, at variance with what has been reported for stable clones (Guerini et al., 1995; Liu et al., 1996). Open in a separate window Fig. 2. Western blot analysis of PMCA- and SERCA-overexpressing cells. Lysates from the transiently transfected CHO cells were prepared and separated by SDSCPAGE as described in Materials and methods. Lane?1 corresponds to CHO cells co-transfected with erAEQ/PMCA4 expression plasmids, lane?2 to cells co-transfected with erAEQ/SERCA2b, lane?3 to cells transfected with the empty bHLHb38 plasmid and lane?4 to cells transfected with the erAEQ expression vector. The blot was probed with four different monoclonal antibodies: 5F10?(A), JA9?(B), IID8?(C) and 12CA5?(D). The resting [Ca2+] in the ER erAEQ was used to measure the intralumenal Ca2+ concentration, [Ca2+]er (Figure?3). Functional aequorin was reconstituted with a modified prosthetic group, coelenterazine?n, in order to decrease the affinity of the photoprotein and thus monitor the high values of [Ca2+]er (Barrero = 16) than control cells (trace?b; 556.46 74?M, = 13). Interestingly, PMCA-overexpressing cells (trace?c) showed a lower plateau value of Ca2+ in the ER lumen (451.00 77?M, = 14) (Figure?3A). The initial kinetics LG-100064 of ER refilling in the experiments of Figure?3A was comparable in the three cell batches; however, differences may have gone undetected since the concentration of Ca2+ in the perfused chamber equilibrated in seconds, and cells could have responded asynchronously, affecting the accuracy of the measurements and obscuring possible differences in Ca2+ pumping rate. The rate of Ca2+ accumulation clearly fell as [Ca2+]er rose, and came to a stop when the store was full (Mogami et al., 1998). The specific SERCA pump inhibitor thapsigargin (TG) (Thastrup = 9) in the former case (Figure?4B), and 2.84 0.46?M (= 9) in the latter case (Figure?4C), as compared with 3.95 0.43?M (= 9) for control cells (Figure?4A). While the faster clearance of the cytosolic Ca2+ signal could be easily explained by the increased Ca2+ pumping activity (see below), the LG-100064 decrease in its peak height suggests decreased release from the ER. This was largely expected in PMCA overexpressers, which have a lower [Ca2+]er, but could appear surprising in SERCA overexpressers, which contain higher [Ca2+]er. The scenario was clarified by the direct monitoring of [Ca2+]er, which revealed major differences in the dynamics of Ca2+ release between control cells and PMCA and SERCA overexpressers. Figure?4 shows that a rapid and nearly complete Ca2+ release was induced by agonist stimulation in control (Figure?4A) and PMCA-overexpressing (Figure?4B) cells. Conversely, in SERCA overexpressers (Figure?4C) the release rate rapidly slowed down (half decay LG-100064 time of the steady-state plateau phase was 21.5 5.95?s, = 6 as opposed to 7.02 1.71?s, = 8 and 6.29 1.68?s, = 9 for controls and PMCA overexpressers, respectively), most likely due to the Ca2+-dependent inhibition of IP3-gated channels (see Discussion). As a consequence, [Ca2+]er stabilized at an intermediate plateau level (215 42.5?M, = 12), also due to the larger Ca2+ re-uptake into the ER of SERCA overexpressers. The overexpression of the PMCA and SERCA pumps had an obvious effect on the Ca2+ influx across the plasma membrane elicited by the emptying of intracellular Ca2+ stores [the capacitative Ca2+ entry (CCE); Clapham, 1995; Putney and McKay, 1999]. In LG-100064 the experiments described here, the contribution of this influx pathway to the [Ca2+]c rise elicited by the agonist corresponded to the phase of slow decline of the trace after its peak. This long-lasting decay phase was accelerated in both PMCA- and SERCA-overexpressing cells. While the increased activity of the PMCA pump could be expected to offset the capacitative Ca2+ influx in the former, in SERCA-overexpressing cells [Ca2+]er was decreased to a level comparable to that of control cells at rest (i.e. 550?M) at the peak of [Ca2+]c. Apparently, the capacitative influx mechanism became activated only when a critical threshold of [Ca2+]er was reached, rather.