Calcium handling in porcine coronary endothelial cells by gastrin-17.
In porcine coronary artery endothelial cells (PCAEC), gastrin-17 has recently been found to increase nitric oxide (NO) production by the endothelial NO synthase (eNOS) isoform through cholecystokinin 1/2 (CCK1/2) receptors and the involvement of protein kinase A (PKA), PKC and the β2-adrenoreceptor-related pathway. As eNOS is the Ca(2)(+)-dependent isoform of the enzyme, we aimed to examine the effects of gastrin-17 on Ca(2)(+) movements. Thus, experiments were performed in Fura-2-acetoxymethyl-ester-loaded PCAEC, where changes of cytosolic Ca(2)(+) ([Ca(2)(+)]c) caused by gastrin-17 were analysed and compared with those of CCK receptors and β2-adrenoreceptors agonists/antagonists. In addition, some experiments were performed by stimulating cells with gastrin-17 in the presence or absence of cAMP/PKA activator/inhibitor and of phospholipase C (PLC) and Ca(2)(+)-calmodulin dependent protein kinase II (CaMKII) blockers. The results have shown that gastrin-17 can promote a transient increase in [Ca(2)(+)]c mainly originating from an intracellular pool sensitive to thapsigargin and from the extracellular space. In addition, the response of cells to gastrin-17 was increased by the adenylyl cyclase activator and the β2-adrenoreceptor agonists and affected mainly by the CCK2 receptor agonists/antagonists. Moreover, the effects of gastrin-17 were prevented by β2-adrenoreceptors and CaMKII blockers and the adenylyl cyclase/PKA and PLC inhibitors. Finally, in PCAEC cultured in Na(+)-free medium or loaded with the plasma membrane Ca(2)(+) pump inhibitor, the gastrin-17-evoked Ca(2)(+) transient was long lasting. In conclusion, this study shows that gastrin-17 affected intracellular Ca(2)(+) homeostasis in PCAEC by both promoting a discharge of an intracellular pool and by interfering with the operation of store-dependent channels through mainly CCK2 receptors and PKA/PLC- and CaMKII-related signalling downstream of β2-adrenoreceptor stimulation.
Department of Translational Medicine, Biotechnology Centre for Applicated Medical Research (BRMA), University of East Piedmont A. Avogadro, Novara, Italy. grossini@med.unipmn.it
E
E
Grossini
C
C
Molinari
L
L
Sigaudo
M
M
Biella
D A S G
DA
Mary
G
G
Vacca
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000187:drug effects* / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D005755:Gastrins / Q000494:pharmacology*; D011949:Receptors, Cholecystokinin / Q000819:agonists / Q000378:metabolism; D013552:Swine
D005755:Gastrins; D011949:Receptors, Cholecystokinin; C013550:gastrin 17; D002118:Calcium