Gastric chief cells possess NK1 receptors which mediate pepsinogen secretion and are regulated by agents that increase cAMP and phospholipase C.
In order to determine whether tachykinins alter the function of chief cells and to characterize the receptors mediating the effect, we investigated the abilities of various substance P (SP)-related peptides to inhibit the binding of 125I-Bolton-Hunter labeled substance P (125I-BH-SP) and their abilities to alter cell function in dispersed chief cells from guinea pig stomach. Binding of 125I-BH-SP was saturable, reversible, time- and temperature-dependent and was inhibited by several SP-related peptides with relative potencies of SP = physalaemin (IC50:0.19 nM) > SP methyl ester (SP-ME) (IC50:3.3 nM) > eledoisin (IC50:6.1 nM) > neurokinin A (NKA) (IC50: 65 nM) > neurokinin B (NKB) (IC50:80 nM). Analyses of these binding data demonstrated that chief cells possess a high and low affinity class of binding sites. Neither 125I-NKA nor [phenylalanyl-3,4,5-3H]senktide demonstrated saturable binding to chief cells. Acid stripping experiments demonstrated rapid ligand internalization with 55% of the bound radioligand internalized by 10 min. Phospholipase C activating agents (carbachol, CCK-8), adenylate cyclase activating agents (secretin, VIP), TPA and the calcium ionophore, A23187, all inhibited the binding of 125I-BH-SP and it was due to inhibition of ligand internalization with no change in surface bound parameters. SP (0.1 microM) stimulated pepsinogen secretion but was 4-times less efficacious than CCK-8 (10 nM) or carbachol (1 mM). 10 nM SP stimulated a rapid increase in cytoplasmic free calcium concentration ([Ca2+]i) followed by a sustained elevation lasting 2 min. Single cell spectroscopy demonstrated SP (10 pM to 1 microM) did not cause calcium oscillations. The NK1 receptor antagonist, CP96,345 specifically inhibited the SP-stimulated changes in [Ca2+]i and pepsinogen secretion. The relative potencies of SP-related peptides to stimulate pepsinogen secretion and [Ca2+]i demonstrated a close agreement with their abilities to inhibit the binding of 125I-BH-SP, and comparison of the dose-response curves suggests occupation of the low affinity sites mediate changes in biologic activity. In conclusion, the present study demonstrates that chief cells possess a NK1 subtype of tachykinin receptor, occupation of the low affinity sites of this receptor cause calcium mobilization and pepsinogen secretion, and that binding to this receptor is regulated by agents that activate phospholipase C, adenylate cyclase, protein kinase C and calcium mobilization.
Kitsukawa
Y
Y
Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1804, USA.
Turner
R J
RJ
Pradhan
T K
TK
Jensen
R T
RT
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects; D005753:Gastric Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006168:Guinea Pigs; D007700:Kinetics; D008297:Male; D064729:Neurokinin-1 Receptor Antagonists; D010435:Pepsinogens / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D018040:Receptors, Neurokinin-1 / Q000819:agonists / Q000378:metabolism*; D012633:Secretin / Q000494:pharmacology; D012844:Sincalide / Q000494:pharmacology; D013373:Substance P / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D015320:Tachykinins / Q000494:pharmacology*; D013696:Temperature; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism*
D064729:Neurokinin-1 Receptor Antagonists; D010435:Pepsinogens; D010455:Peptides; D018040:Receptors, Neurokinin-1; D015320:Tachykinins; D012633:Secretin; D013373:Substance P; D000242:Cyclic AMP; D010738:Type C Phospholipases; D012844:Sincalide; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium