Abstract

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 Ca²⁺-dependent isoform of the enzyme, we aimed to examine the effects of gastrin-17 on Ca²⁺ movements. Thus, experiments were performed in Fura-2-acetoxymethyl-ester-loaded PCAEC, where changes of cytosolic Ca²⁺ ([Ca²⁺]_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 Ca²⁺–calmodulin dependent protein kinase II (CaMKII) blockers. The results have shown that gastrin-17 can promote a transient increase in [Ca²⁺]_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 Ca²⁺ pump inhibitor, the gastrin-17-evoked Ca²⁺ transient was long lasting. In conclusion, this study shows that gastrin-17 affected intracellular Ca²⁺ 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.