Biochemical and Biophysical Research Communications
Receptor-operated Ca2+ influx and its association with the Src family in secretagogue-stimulated pancreatic acini☆
Section snippets
Materials and methods
Materials. Chemicals were purchased from the following sources. CCK-8, MnCl2, NiCl2, and wortmannin from Sigma (St. Louis, MO). Genistein from GIBCO-BRL (Grand Island, NY). Herbimycin A, GP antagonist-2A, and U-73122 from Biomol (Plymouth Meeting, PA). Ionomycin, BAPTA/AM, 4β-phorbol 12,13-dibutyrate (4β-PDBu), and K252-a from Calbiochem (La Jolla, CA). Fura-2/AM from Molecular Probes (Eugene, OR). JMV-180 and CCK-OPE from Research Plus (Bayonne, NJ). Human recombinant basic FGF (hFGF) and
CCK enhances Src-related PTK activities
Synthetic RR-Src is a tyrosine kinase substrate derived from v-Src autophosphorylation site. Therefore, this kinase assay seems to measure autophosphorylation of the Src family. CCK-8 at 10 pM–10 nM increased PTK activities by 1.5- to 3-fold over basal after 3 min cell stimulation (Fig. 1A). Basal PTK activity was 0.86 ± 0.43 pmol/min/mg protein (n=13). CCK at 10 nM increased PTK activity to 2.44 ± 1.12 pmol/min/mg protein (n=13). This was abolished by either genistein (100 μM) or eliminating [Ca2+]o.
Discussion
In voltage-dependent Ca2+ channels, Src has a fundamental role in Ca2+ signal transduction. Neuronal differentiation of PC12 cells elicited by v-Src is accompanied by an enhanced expression of the N-type Ca2+ channels [33]. The noncatalytic and unique domain of tyrosine phosphorylated c-Src(40-58) may be a member of the N-methyl-d-aspartate (NMDA) channel complex and Src may directly regulate NMDA/Ca2+ channel activity [34]. NMDA receptor subunits 2A and 2B may be phosphorylated on tyrosine;
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Abbreviations: CCK, cholecystokinin; [Ca2+]o, extracellular Ca2+ concentration; [Ca2+]i, intracellular Ca2+ concentration; trp, transient receptor potential; ICRAC, Ca2+ release-activated Ca2+currents; IP3, inositol 1,4,5-trisphosphate.