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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.

Functional studies suggest that guinea pig chief cells have both cholecystokinin-A (CCK-A) and CCK-B receptors (CCK-A-R and CCK-B-R, respectively). However, all efforts to directly characterize the specific CCK-A-R using binding have been unsuccessful. Recent studies describe specific CCK-A-R agonists such as A-71378 ([desamino-Nle28,31-(N-methyl)Asp32]CCK heptapeptide]. In the present study, [D-Tyr-Gly]A-71378 was synthesized, which has > 300-fold selectivity for CCK-A-R and can be iodinated. [D-Tyr-Gly]A-71378 was equipotent to A-71378 in stimulating pepsinogen release from purified guinea pig chief cells. Binding of 125I-labeled [D-Tyr-Gly]A-71378 was saturable and specific. Potencies for inhibiting binding were as follows: [D-Tyr-Gly]A-71378 = A-71378 = 4x CCK octapeptide (CCK-8) > 1,000x des(SO4)-CCK-8, gastrin. In contrast, for 125I-gastrin binding they were CCK-8 > gastrin-17-I > des(SO4)-CCK-8 >> A-71378 or [D-Tyr-Gly]A-71378. Binding of [D-Tyr-Gly]A-71378 was best fitted by a two-site model. In contrast, 125I-gastrin binding was fitted with a single-site model. For inhibiting binding of 125I-[D-Tyr-Gly]A-71378, the CCK antagonists had relative affinities of L-364,718 >> L-365,260, and the reverse was true with 125I-gastrin. Correlation of binding with changes in biological activity suggested low-affinity CCK-A-R were mediating these changes. These results demonstrate directly for the first time that guinea pig chief cells possess CCK-A-R and CCK-B-R. The pharmacology of these CCK-A-R resembles those on other tissues. This novel, highly selective CCK-A ligand should be useful because it will identify CCK-A-R when they make up as little as 0.2% of the total CCK receptor number.

Bombesin (BN)-related peptides, such as gastrin-releasing peptide (GRP), have been shown in vivo to stimulate release of pepsinogen. However, whether this is due to a direct interaction with chief cells is not clear. To clarify this we prepared isolated chief cells (> 90% pure) from guinea pig stomach. BN, GRP, or neuromedin B (NMB), at concentrations up to 1 microM, did not stimulate pepsinogen release or affect the stimulation caused by vasoactive intestinal peptide (VIP) (100 nM) or CCK-8 (10 nM), respectively. In addition, BN, GRP, or NMB at a concentration of 1 microM did not increase cAMP nor did they alter the increase in cAMP caused by VIP or secretin. BN (1 microM) did not alter basal cytosolic calcium [Ca2+]i or affect the increase in [Ca2+]i caused by CCK-8 (1 microM). Furthermore, BN, GRP, or NMB at a concentration of 1 microM did not increase the generation of inositol phosphates (IP) or alter the increase in [3H]IP1, [3H]IP2, or [3H]IP3, caused by CCK-8 (1 microM) or carbachol (1 mM). Binding studies demonstrated no saturable binding of either [125I][Tyr4]BN or [125I][D-Tyr0]NMB using experimental conditions where binding with other peptide ligands to other receptors on chief cells is seen. We conclude that BN-related peptides do not interact directly with specific receptors on chief cells to stimulate or alter stimulated pepsinogen secretion, increase the breakdown of inositol phosphates, or alter [Ca2+]i or cAMP.

Inhibition both in vivo and in vitro of pepsinogen secretion by somatostatin (SS) and the histological demonstration that fundic D-cells contain long cytoplasmic processes extending to chief cells suggest a possible direct effect of SS on chief cell function. The aim of the present study was to determine whether SS interacts directly with receptors on isolated gastric chief cells and, if so, how SS alters cell function. Binding of 125I-[Tyr11]SS14 to chief cells was saturable, time and temperature dependent, and was inhibited by both SS14 (Ki 1.6 nM) and SS28 (Ki 5.2 nM). SMS-201-995 was 1,300-fold less potent than SS14. Calcium-mobilizing secretagogues reduced binding of 125I-[Tyr11]SS14 with efficacies of cholecystokinin octapeptide (CCK-8) > carbachol > gastrin. Adenosine 3',5'-cyclic monophosphate (cAMP)-activating secretagogues also inhibited binding with efficacies of secretin > vasoactive intestinal polypeptide (VIP). 12-O-tetradecanoylphorbol 13-acetate (TPA) or A-23187 also decreased binding. Analyses demonstrated that CCK-8 and TPA were decreasing the affinity of SS receptors for 125I-[Tyr11]SS14 without affecting their binding capacity. Both SS14 and SS28 at a maximally effective concentration inhibited cAMP production caused by VIP or secretin (20-30%) but did not alter cytosolic calcium ([Ca2+]i), inositol phosphates, or pepsinogen release. We conclude that chief cells possess SS receptors with a high affinity for both SS14 and SS28 but low affinity for SMS-201-995 and thus resemble the SSB receptors described in the rat cerebral cortex. Although occupation of these receptors by SS has no effect on pepsinogen release induced by secretagogues acting through either the calcium or the cAMP pathway, SS receptor occupation is regulated by agents activating phospholipase C, adenylate cyclase, protein kinase C, and [Ca2]i.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide of the secretin-vasoactive intestinal peptide (VIP) family. To investigate whether PACAP alters chief cell function, we prepared isolated chief cells (> 90% pure) from guinea pig stomach. PACAP-38, PACAP-27, VIP, and secretin all caused a threefold increase in pepsinogen release. The dose-response curves of PACAP-38, PACAP-27, and VIP were biphasic, whereas with secretin it was not. The first phase comprised 40% of maximal release, and each of the three peptides (PACAP-38, PACAP-27, and VIP) were equipotent (EC50 0.1-0.3 nM). For the second phase, comprising 60% of maximal release, the relative potencies were PACAP-38 > PACAP-27 = VIP. 125I-labeled secretin, 125I-VIP, and 125I-PACAP-27 all demonstrated saturable binding to chief cells. Binding of both 125I-PACAP-27 and 125I-VIP was inhibited completely and with similar potencies by PACAP-38, PACAP-27, and VIP. Secretin had a > 500-fold lower affinity than PACAP-38 for displacing both 125I-PACAP-27 and 125I-VIP. With 125I-secretin, secretin was the most potent, and was 197 times more potent than PACAP-38, which was 6-8 times more potent than both PACAP-27 and VIP. We conclude that both PACAP-38 and PACAP-27 stimulate pepsinogen secretion from dispersed chief cells. In contrast to a number of other tissues, no evidence for a high-affinity receptor that interacted only with PACAP was found. PACAP and VIP interact with equal high affinity with a common receptor and with low affinity with the secretin receptor.

On the basis of opioid-stimulated contraction of dispersed gastric smooth muscle cells it has been suggested that these cells possess opioid receptors of three subtypes: kappa (kappa), mu (mu), and delta (delta). We have used selective peptidase-resistant radioligands, agonists and antagonists, to examine receptor subtypes on dispersed gastric smooth muscle cells from guinea pigs prepared by collagenase digestion. The kappa-agonist U-50488H, the mu-agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAGO), and the delta-agonist [D-Pen2,Pen5]enkephalin (DPDPE) each caused muscle contraction. The concentrations required to caused half-maximal contraction were U50488H (6 pM) greater than DAGO (13 pM) greater than DPDPE (6 nM). The abilities of these agonists to inhibit binding of [3H]U-69593 (kappa-preferring) by 50% were U50488H (43 nM) greater than DAGO (43 microM) greater than DPDPE (200 microM). Their abilities to inhibit binding of [3H]naloxone (mu-preferring) by 50% were DAGO (0.2 microM) greater than U50488H (10 microM) greater than DPDPE (greater than 100 microM). No binding could be detected with the delta-selective ligand [3H]DPDPE. The kappa-preferring antagonist Mr2266 (10 nM) preferentially inhibited contraction stimulated by the kappa-agonist U50488H, and naltrexone (10 nM) (mu-selective antagonist) preferentially inhibited contraction stimulated by the mu-agonist DAGO. ICI 174864 (200 microM; delta-selective antagonist) had no effect on contraction stimulated by mu-, kappa-, or delta-agonists. Contraction stimulated by the delta-agonist DPDPE was inhibited by both kappa- and mu-receptor antagonists. Studies on the effect of the antagonists on binding of [3H]naloxone and [3H]U69593 also provided evidence for kappa- and mu-sites but nor for delta-sites.(ABSTRACT TRUNCATED AT 250 WORDS)