Ca2+ uptake by the endoplasmic reticulum Ca2+-ATPase in rat microvascular endothelial cells.
In non-excitable cells, many agonists increase the intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing an inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from the intracellular stores. Ca(2+) influx from the extracellular medium may then sustain the Ca(2+) signal. [Ca(2+)](i) recovers its resting level as a consequence of Ca(2+)-removing mechanisms, i.e. plasma-membrane Ca(2+)-ATPase (PMCA) pump, Na(+)/Ca(2+) exchanger (NCX) and sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump. In a study performed in pancreatic acinar cells, evidence has been provided suggesting that, during the decay phase of the agonist-evoked Ca(2+) transients, the Ca(2+) concentration within the intracellular stores remains essentially constant [Mogami, Tepikin and Petersen (1998) EMBO J. 17, 435-442]. It was therefore hypothesized that, in such a situation, intracellular Ca(2+) is not only picked up by the SERCA pump, but is also newly released through IP(3)-sensitive Ca(2+) channels, with the balance between these two processes being approximately null. The main aim of the present work was to test this hypothesis by a different experimental approach. Using cardiac microvascular endothelial cells, we found that inhibition of the SERCA pump has no effect on the time course of agonist-evoked Ca(2+) transients. This result was not due to a low capacity of the SERCA pump since, after agonist removal, this pump proved to be very powerful in clearing the excess of intracellular Ca(2+). We showed further that: (i) in order to avoid a rapid removal of Ca(2+) by the SERCA pump, continuous IP(3) production appears to be required throughout all of the decay phase of the Ca(2+) transient; and (ii) Ca(2+) picked up by the SERCA pump can be fully and immediately released by agonist application. All these results support the model of Mogami, Tepikin and Petersen [(1998) EMBO J. 17, 435-442]. Since the SERCA pump did not appear to be involved in shaping the decay phase of the agonist-evoked Ca(2+) transient, we inhibited the PMCA pump with carboxyeosin, and NCX with benzamil and by removing extracellular Na(+). The results indicate that, during the decay phase of the agonist-evoked Ca(2+) transient, the intracellular Ca(2+) is removed by both the PMCA pump and NCX. Finally, we provide evidence indicating that mitochondria have no role in clearing intracellular Ca(2+) during agonist-evoked Ca(2+) transients.
Moccia
Francesco
F
Department of Physiological and Pharmacological Sciences, University of Pavia, Via Forlanini 6, 27100 Pavia, Italy.
Berra-Romani
Roberto
R
Baruffi
Silvana
S
Spaggiari
Santina
S
Signorelli
Silvia
S
Castelli
Loretta
L
Magistretti
Jacopo
J
Taglietti
Vanni
V
Tanzi
Franco
F
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004730:Endothelium, Vascular / Q000201:enzymology*; D007700:Kinetics; D008833:Microcirculation / Q000201:enzymology*; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D012964:Sodium / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism
D012964:Sodium; D011493:Protein Kinase C; D010738:Type C Phospholipases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium; D014544:Uridine Triphosphate