15-hydroxy-5-8-11-13-eicosatetraenoic-acid and 2-(4-amylcinnamoyl)amino-4-chlorobenzoic-acid

We investigated the structural requirements for bombesin (BB)-like peptides to stimulate amylase secretion in rat pancreatic acini and examined the responsible intracellular signal transduction pathways. The tetradecapeptide BB-(1-14) was a full agonist, whereas the heptapeptide BB-(8-14) did not evoke amylase secretion. The mammalian BB analog neuromedin C decapeptide [NMC-(5-14)] was as potent as BB-(1-14) in stimulating amylase secretion, suggesting that Gly5-Asn6-His7 (or Gln7) of the COOH-terminal decapeptide are essential amino acids for full biological activity. BB and NMC equipotently stimulated D-myo-inositol 1,4,5-trisphosphate production, which was inhibited by the phospholipase C (PLC) inhibitor U-73122. BB and NMC also stimulated protein tyrosine kinase (PTK) activities. The half-maximal effective concentration (EC50) for NMC-activated PTK was 2 log units less than the EC50 for BB-activated PTK. NMC was 10-34 times more potent than BB in increasing leukotriene C4 (an index of arachidonic acid production). The production of leukotriene C4 was inhibited by the phospholipase A2 (PLA2) inhibitor ONO-RS-082. NMC is structurally homologous to BB-(5-14) except that Gln7 in BB is replaced by His7 in NMC. Therefore, substitution of Gln7 for His7 may alter the signal transduction systems to include the PTK and PLA2 pathways. U-73122 inhibited Ca2+ spiking and amylase secretion induced by NMC and BB. However, the PTK inhibitor genistein and the PLA2 inhibitor ONO-RS-082 inhibited secretion induced by NMC but not that induced by BB. In contrast to nonmammalian BB receptors, which primarily use the PLC pathway, the rat BB receptor is linked to three different signal transduction systems: PLC, PTK, and PLA2 pathways.

Topics: Amino Acid Sequence; Aminobenzoates; Amylases; Animals; Bombesin; Chlorobenzoates; Cinnamates; Enzyme Inhibitors; Estrenes; Humans; Hydroxyeicosatetraenoic Acids; Inositol 1,4,5-Trisphosphate; Molecular Sequence Data; Neurokinin B; ortho-Aminobenzoates; Pancreas; Peptide Fragments; Phosphodiesterase Inhibitors; Phospholipases A; Phospholipases A2; Protein-Tyrosine Kinases; Pyrrolidinones; Radioimmunoassay; Rats; Receptors, Bombesin; Signal Transduction; Structure-Activity Relationship; Swine; Type C Phospholipases

Recently we have demonstrated that in rat pancreatic acini the high affinity cholecystokinin receptors are coupled to the phospholipase A2 (PLA2)/arachidonic acid (AA) cascades to mediate Ca2+ oscillations and amylase secretion. This intracellular signal transduction system is associated with an unidentified G protein(s) which is neither Gi/Go nor Gq-alpha. Using a newly cloned PLA2-activating protein (PLAP), we further examined the mechanisms by which PLA2 activates Ca2+ oscillation and pancreatic enzyme secretion. In intact acini, 0.1-1 microM PLAP evoked Ca2+ oscillations in a dose-dependent manner (delta [Ca2+]i: 18-121 nM and frequency: 2.3-5.5 cycles/10 min). PLAP elicited a 3-fold increase in monophasic amylase secretion with an EC50 of 0.1 microM. PLAP dose-dependently caused an increase in the AA metabolite 15-HETE. The PLA2 inhibitor, but not inhibitors of lipoxygenase, cytochrome P-450 and cyclooxygenase, inhibited the action of PLAP, suggesting that AA, but not AA metabolites, functions as a signal messenger. In permeabilized acini, a monoclonal antibody of G protein beta subunits inhibited the action of PLAP. Because of the structural similarity between PLAP and Gbeta protein we hypothesize that the PLA2 coupled G protein is Gbeta and it elicits Ca2+ oscillations and monophasic amylase secretion via the AA pathway.

Topics: Aminobenzoates; Amylases; Animals; Arachidonic Acids; Calcium; Chlorobenzoates; Cinnamates; Cloning, Molecular; Dinoprostone; Dose-Response Relationship, Drug; Enzyme Activation; GTP-Binding Proteins; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Indomethacin; Ketoconazole; Kinetics; Macromolecular Substances; Male; ortho-Aminobenzoates; Pancreas; Phospholipases A; Phospholipases A2; Protein Biosynthesis; Proteins; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Time Factors