Novel model of integration of signaling pathways in rat pancreatic acinar cells

Abstract

Cholecystokinin (CCK) is the major pancreatic secretagogue and acinar cell mitogen. This study was performed to determine by which effector systems CCK regulates tyrosine kinases, phosphatidylinositol (PtdIns) 3-kinase, and phospholipase D (PLD) activities. Pancreatic acini loaded with [3H]myristic acid or [3H]inositol were used to assay PLD and PtdIns 3-kinase. G protein activation with NaF increased particulate and crude cytosolic tyrosine kinase and PLD activities. PLD activation was pertussis toxin sensitive. Inhibition of phospholipase C (PLC) slightly reduced caerulein-stimulated particulate tyrosine kinase and blocked crude cytosolic tyrosine kinase activity without affecting caerulein-induced PLD activity. Ca2+ is an important factor in caerulein stimulation of tyrosine kinase and PLD activities. Protein kinase C and tyrosine kinase inhibition abolished caerulein-activated particulate and crude cytosolic tyrosine kinase and PtdIns 3-kinase activities without any effect on PLD. Wortmannin inhibited PLD and PtdIns 3-kinase activation. Caerulein-induced amylase secretion was partially reduced by tyrosine kinase inhibition, with no effect from wortmannin. Caerulein can stimulate a pertussis toxin-insensitive G protein, leading to particulate tyrosine kinase activation and a Ca(2+)-sensitive cytosolic tyrosine kinase through PLC activation. However, PLD activation by caerulein is pertussis toxin sensitive, cytosolic Ca2+ sensitive, and independent of previous PLC and tyrosine kinase activation.

Phosphatidylinositol 3-kinase regulates Ca2+signaling in pancreatic acinar cells through inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase

“…Of note, we found that the regulatory effect of PI3-kinase on CCK-induced [Ca 2ϩ ] i responses is observed in a wide range of CCK concentrations. At present, there is conflicting evidence on whether CCK (or carbachol) activates PI3-kinase in pancreatic acinar cells (10,20,46,49,55); alternatively, the role of PI3-kinase in Ca 2ϩ signaling may be permissive in these cells.…”

“…Ionomycin, LY-294002, and creatine phosphate were from Calbiochem (La Jolla, CA To gain an insight into the mechanism(s) of this regulation, we first measured the effect of the PI3-kinase inhibitors on [Ca 2ϩ ] i responses to various doses of CCK in isolated rat pancreatic acini. We used concentrations of LY-294002 and wortmannin that have been previously shown to inhibit PI3-kinase-mediated pathways in acinar cells (20,46,49,55). The results in Fig.…”

Stimulatory effects of bilirubin on amylase release from isolated rat pancreatic acini

“…Furthermore, PLC activates both PKC and TK (34), and PLA 2 activates PKC (1). When treating bilirubin-stimulated acini with PLC inhibitor U-73122 or PLA 2 inhibitor indoxam, both 1 M U-73122 and 1 M indoxam partially but significantly inhibited bilirubinevoked amylase release (Fig.…”

“…The supernatant was saved for assay of PKC or TK enzyme activity (cytosolic fraction), while the pellet was further homogenized with 1 ml of ice-cold solution A containing 1% Nonidet P-40 and left on ice for 1 h. The homogenate was centrifuged at 100,000 g for 60 min at 4°C, and the supernatant was taken for assay of PKC or TK enzyme activity (membrane fraction). PKC enzyme activity was measured using an enzyme assay kit, whereas TK enzyme activity was measured as reported previously using poly(L-Glu,L-Tyr) (4:1) as a substrate (34). The reaction mixture in a final volume of 50 l contained 2.5 M Tris ⅐ HCl, 2.5 M MgCl 2, 0.5 nM orthovanadate, 0.02% Triton X-100, 3 nM ATP, 0.4 Ci [␥-32 P]ATP, and 100 g Glu-Tyr polymer.…”

Inhibitory and Stimulatory Effects of Somatostatin on Two Human Pancreatic Cancer Cell Lines: A Primary Role for Tyrosine Phosphatase SHP-11

“…Previous observations indicated that tyrosine kinases and MAPKs were associated with growth-related processes (31,34). We therefore investigated their potential involvement in MIA PaCa-2 cell growth by measuring their specific activation in response to SS-14 and SMS.…”