peptide-phi and vasoactive-intestinal-peptide-(10-28)

The proliferation of human lymphoblastoma cell line (H9) was differently stimulated by Peptide Histidine Methionine (PHM) and Vasoactive Intestinal Peptide (VIP). PHM induced a cyclic AMP (cAMP) accumulation, abolished by Adenylate Cyclase (AC) inhibitors leading to a loss of proliferative effect. VIP mitogenic activity was Pertussis toxin (PTX) sensitive and AC inhibitors insensitive. Pharmacological experiments performed on H9 membranes with or without a GTP analogue indicated expression of both GTP-insensitive and -sensitive PHM/VIP high-affinity binding sites (HA). H9 cells expressed only the VPAC1 receptor. VIP(10-28), known as a VPAC1 antagonist, bond to all GTP-insensitive PHM sites and inhibited evenly the PHM and VIP mitogenic actions. These data strongly suggested different mechanisms initiated by VIP and PHM and highlighted the key role of GTP-insensitive binding sites in the control of cell proliferation.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Analysis of Variance; Blotting, Southern; Bromodeoxyuridine; Cell Line, Tumor; Cell Proliferation; Cyclic AMP; Dose-Response Relationship, Drug; Drug Interactions; Gene Expression; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; Imines; Iodine Isotopes; Lymphoma; Peptide Fragments; Peptide PHI; Pertussis Toxin; Protein Binding; Radioligand Assay; Receptors, Cell Surface; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Peptide, Type II; Receptors, Vasoactive Intestinal Polypeptide, Type I; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) receptors were characterized in freshly isolated and cultured smooth muscle cells from guinea pig stomach by radioligand binding and by measurement of relaxation in single isolated and cultured cells. 125I-VIP bound to both freshly isolated and cultured muscle cells: binding was rapid, specific, saturable and temperature-dependent, and was inhibited in a concentration-dependent fashion by VIP, VIP10-28, PHI and secretin, in this order. Competition curves for VIP could be resolved into high- and low-affinity components, yielding similar binding constants in freshly isolated and cultured cells (high-affinity Kd 0.11 and 0.22 nM; low-affinity Kd 59 and 37 nM; high-affinity binding sites: 1183 and 1021 per cell, representing about 1% of total binding sites). VIP10-28 inhibited 125I-VIP binding completely and acted as potent competitive antagonist of VIP-induced relaxation (Ki 0.5 nM). PHI and secretin, however, inhibited partly 125I-VIP binding: the pattern of inhibition implied that VIP interacts with VIP-preferring receptors that are recognized by PHI and secretin as well as with VIP-specific receptors. The pattern of binding is consistent with recent evidence indicating that VIP activates two signalling pathways, a VIP-specific, nitric oxide/cGMP-dependent pathway and a common cAMP-dependent pathway shared by all three peptides. PHI and secretin were relatively more potent as relaxant agents than as inhibitors of 125I-VIP binding raising the possibility that PHI and secretin could interact additionally with PHI- and secretin-preferring receptors in mediating relaxation.

Topics: Animals; Binding, Competitive; Cell Separation; Cells, Cultured; Gastric Mucosa; Guinea Pigs; Muscle Relaxation; Muscle, Smooth; Peptide Fragments; Peptide PHI; Radioligand Assay; Receptors, Vasoactive Intestinal Peptide; Secretin; Stomach; Vasoactive Intestinal Peptide