Receptor-operated Ca2+ influx and its association with the Src family in secretagogue-stimulated pancreatic acini

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Abstract

We investigated signal transduction between receptor-operated Ca2+ influx (ROCI) and Src-related nonreceptor protein tyrosine kinase (PTK) in rat pancreatic acini. CCK and the Ca2+ ionophore enhanced the Src-related PTK activity, whereas the high-affinity CCK-A receptor agonists, fibroblast growth factor (FGF), and the protein kinase C (PKC) activator had no or little effect. This increase was abolished by eliminating [Ca2+]o, loading of the intracellular Ca2+ chelator, and administering the PTK inhibitor genistein. While genistein inhibited extracellular Ca2+ or Mn2+ entry induced by CCK and carbachol, it did not affect intracellular Ca2+ release and oscillations. CCK dose-dependently increased the Src phosphotransferase activity, which was abolished by inhibitors of Gq protein, phospholipase C (PLC), and Src, but not by the calmodulin kinase (CaMK) inhibitor. Intensities of the Src band and amounts of tyrosine phosphorylated Src were enhanced by CCK stimulation. Thus, Src cascades appear to be coupled to the low-affinity CCK-A receptor and utilize Gq-PLC pathways for their activation, independent of PKC and CaMK cascades. The low-affinity CCK-A receptor regulates ROCI via mediation of Src-related PTK and activates Src pathways to cause [Ca2+]o-dependent pancreatic exocytosis.

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.

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