GI side-effects of a possible therapeutic GRF analogue in monkeys are likely due to VIP receptor agonist activity.

Growth hormone (GH) is used or is being evaluated for efficacy in treatment of short stature, aspects of aging, cardiac disorders, Crohn's disease, and short bowel syndrome. Therefore, we synthesized several stable growth hormone-releasing factor (GRF) analogues that could be therapeutically useful. One potent analog, [D-Ala(2),Aib(8, 18,)Ala(9, 15, 16, 22, 24-26,)Gab(27)]hGRF(1-27)NH(2) (GRF-6), with prolonged infusion caused severe diarrhea in monkeys; however, it had no side-effects in rats. Because GRF has similarity to VIP/PACAP and VIPomas cause diarrhea, this study investigated the ability of this and other GRF analogues to interact with the VIP/PACAP receptors. Rat VPAC(1)-R (rVPAC(1)-R), human VPAC(1)-R (hVPAC(1)-R), rVPAC(2)-R and hVPAC(2)-R stably transfected CHO and PANC 1 cells were made and T47D breast cancer cells containing native human VPAC(1)-R and AR4-2J cells containing PAC(1)-R were used. hGRF(1-29)NH(2) had low affinity for both rVPAC(1)-R and rVPAC(2)-R while VIP had a high affinity for both receptors. GRF-6 had a low affinity for both rVPAC(1)-R and rVPAC(2)-R and very low affinity for the rPAC(1)-R. VIP had a high affinity, whereas hGRF(1-29)NH(2) had a low affinity for both hVPAC(1)-R and hVPAC(2)-R. In contrast GRF-6, while having a low affinity for hVPAC(2)-R, had relatively higher affinity for the hVPAC(1)-R. In guinea pig pancreatic acini, all GRF analogues were full agonists at the VPAC(1)-R causing enzyme secretion. These results demonstrate that in contrast to native hGRF(1-29)NH(2,) GRF-6 has a relatively high affinity for the human VPAC(1)-R but not for the human VPAC(2)-R, rat VPAC(1)-R, rat VPAC(2)-R or rat PAC(1)-R. These results suggest that the substituted GRF analog, GRF-6, likely causes the diarrheal side-effects in monkeys by interacting with the VPAC(1)-R. Furthermore, they demonstrate significant species differences can exist for possible therapeutic peptide agonists of the VIP/PACAP/GRF receptor family and that it is essential that receptor affinity assessments be performed in human cells or from a closely related species.

Rational design of a peptide agonist that interacts selectively with the orphan receptor, bombesin receptor subtype 3.

The orphan receptor, bombesin (Bn) receptor subtype 3 (BRS-3), shares high homology with bombesin receptors (neuromedin B receptor (NMB-R) and gastrin-releasing peptide receptor (GRP-R)). This receptor is widely distributed in the central nervous system and gastrointestinal tract; target disruption leads to obesity, diabetes, and hypertension, however, its role in physiological and pathological processes remain unknown due to lack of selective ligands or identification of its natural ligand. We have recently discovered (Mantey, S. A., Weber, H. C., Sainz, E., Akeson, M., Ryan, R. R. Pradhan, T. K., Searles, R. P., Spindel, E. R., Battey, J. F., Coy, D. H., and Jensen, R. T. (1997) J. Biol. Chem. 272, 26062-26071) that [d-Tyr(6),beta-Ala(11),Phe(13),Nle(14)]Bn-(6-14) has high affinity for BRS-3 and using this ligand showed BRS-3 has a unique pharmacology with high affinity for no known natural Bn peptides. However, use of this ligand is limited because it has high affinity for all known Bn receptors. In the present study we have attempted to identify BRS-3 selective ligands using a strategy of rational peptide design with the substitution of conformationally restricted amino acids into the prototype ligand [d-Tyr(6),beta-Ala(11),Phe(13),Nle(14)]Bn-(6-14) or its d-Phe(6) analogue. Each of the 22 peptides synthesized had binding affinities determined for hBRS-3, hGRPR, and hNMBR, and hBRS-3 selective ligands were tested for their ability to activate phospholipase C and increase inositol phosphates ([(3)H]inositol phosphate). Using this approach we have identified a number of BRS-3 selective ligands. These ligands functioned as receptor agonists and their binding affinities were reflected in their potencies for altering [(3)H]inositol phosphate. Two peptides with an (R)- or (S)-amino-3-phenylpropionic acid substitution for beta-Ala(11) in the prototype ligand had the highest selectivity for the hBRS-3 over the mammalian Bn receptors and did not interact with receptors for other gastrointestinal hormones/neurotransmitters. Molecular modeling demonstrated these two selective BRS-3 ligands had a unique conformation of the position 11 beta-amino acid. This selectivity was of sufficient magnitude that these should be useful in explaining the role of hBRS-3 activation in obesity, glucose homeostasis, hypertension, and other physiological or pathological processes.

Rat and guinea pig pancreatic acini possess both VIP(1) and VIP(2) receptors, which mediate enzyme secretion.

Pancreatic acini from most species possess vasoactive intestinal peptide (VIP) receptors. Recently, two subtypes of VIP receptors, VIP(1)-R and VIP(2)-R, were cloned. Which subtype exists on pancreatic acini or mediates secretion is unclear. To address this, we examined pancreatic acini from both rat and guinea pig. VIP(1)-R and VIP(2)-R mRNA were identified in dispersed acini from both species by Northern blot analysis and in rat by Southern blot analysis. With the use of the VIP(2)-R-selective ligand Ro-25-1553 in both species, inhibition of binding of (125)I-labeled VIP to acini showed a biphasic pattern with a high-affinity component (10%) and a second representing 90%. The VIP(1)-R-selective ligand, [Lys(15),Arg(16),Leu(27)]VIP-(1-7)-GRF-(8-27), gave a monophasic pattern. Binding of Ro-25-1553 was better fit by a two-site model. In both rat and guinea pig acini, the dose-response curve of Ro-25-1553 for stimulation of enzyme secretion was biphasic, with a high-affinity component of 10-15% of the maximal secretion and a low-affinity component accounting for 85-90%. At low concentrations (10 nM) of Ro-25-1553 and [Lys(15),Arg(16), Leu(27)]VIP-(1-7)-GRF(8-27), which only occupy VIP receptors, a 4-fold and a 56-fold increase in cAMP occurred, respectively. These results show that both VIP(1)-R and VIP(2)-R subtypes exist on pancreatic acini of rat and guinea pig, their activation stimulates enzyme secretion by a cAMP-mediated mechanism, and the effects of VIP are mediated 90% by activation of VIP(1)-R and 10% by VIP(2)-R. Because VIP has a high affinity for both VIP-R subtypes, its effect on pancreatic acini is mediated by two receptor subtypes, which will need to be considered in future studies of the action of VIP in the pancreas.

Comparative pharmacology of the nonpeptide neuromedin B receptor antagonist PD 168368.

The mammalian peptide neuromedin B (NMB) and its receptor are expressed in a variety of tissues; however, little is definitively established about its physiological actions because of the lack of potent, specific antagonists. Recently, the peptoid PD 168368 was found to be a potent human NMB receptor antagonist. Because it had been shown previously that either synthetic analogs of bombesin (Bn) or other receptor peptoid or receptor antagonists function as an antagonist or agonist depends on animal species and receptor subtype studied, we investigated the pharmacological properties of PD 168368 compared with all currently known Bn receptor subtypes (NMB receptor, gastrin-releasing peptide receptor, Bn receptor subtype 3, and Bn receptor subtype 4) from human, mouse, rat, and frog. In binding studies, PD 168368 had similar high affinities (K(i) = 15-45 nM) for NMB receptors from each species examined, 30- to 60-fold lower affinity for gastrin-releasing peptide receptors, and >300-fold lower affinity for Bn receptor subtype 3 or 4. It inhibited NMB binding in a competitive manner. PD 168368 alone did not stimulate increases in either intracellular calcium concentration or [(3)H]inositol phosphates in any of the cells studied but inhibited NMB-induced responses with equivalent potencies in cells containing NMB receptors. PD 168368 was only minimally soluble in water. When hydroxypropyl-beta-cyclodextrin rather than dimethyl sulfoxide was used as the vehicle, both the affinity and the antagonist potency of PD 168368 were significantly greater. The results demonstrate that PD 168368 is a potent, competitive, and selective antagonist at NMB receptors, with a similar pharmacology across animal species. PD 168368 should prove useful for delineating the biological role of NMB and selectively blocking NMB signaling in bioassays and as a lead for the development of more selective nonpeptide antagonists for the NMB receptor.

Pharmacology and cell biology of the bombesin receptor subtype 4 (BB4-R).

Recently, a fourth member of the bombesin (Bn) receptor family (fBB4-R) was isolated from a cDNA library from the brain of the frog, Bombina orientalis. Its pharmacology and cell biology are largely unknown, and no known natural cell lines or tissues possess sufficient numbers of fBB4-R's to allow either of these to be determined. To address these issues, we have used three different strategies. fBB4-R expression in cells widely used for other Bn receptor subtypes was unsuccessful as was expression in two frog cell lines. However, stable fBB4-R cell lines were obtained in CHO-K1 cells which were shown to faithfully demonstrate the correct pharmacology of the related Bn receptor, the GRP receptor, when expressed in these cells. [DPhe6,betaAla11,Phe13,Nle14]Bn(6-14) was found to have high affinity (Ki = 0.4 nM) for the fBB4 receptor and 125I-[DTyr6,betaala11,Phe13,Nle14]Bn(6-14) to be an excellent ligand for this receptor. The fBB4-R had a unique pharmacology for naturally occurring Bn-related agonists, with the presence of a penultimate phenylalanine being critical for high-affinity interaction. It also had a unique profile for six classes of Bn antagonists. The fBB4-R was coupled to phospholipase C with activation increasing [3H]inositol phosphates and mobilizing Ca2+ almost entirely from cellular sources. There was a close correlation between agonist the receptor occupation and the receptor activation. Three of the five classes of Bn receptor antagonists that interacted with higher affinity with the fBB4-R functioned as fBB4-R antagonists and two as partial agonists. fBB4-R activation stimulated increases in phospholipase D (PLD) over the same range of concentrations at which it activated phospholipase C. These results demonstrate that the fBB4 receptor has a unique pharmacology for agonists and antagonists and is coupled to phospholipase C and D. The availability of these cell lines, this novel ligand, and the identification of three classes of antagonists that can be used as lead compounds should facilitate the further investigation of the pharmacology and cell biology of the BB4 receptor.

Pharmacology and intracellular signaling mechanisms of the native human orphan receptor BRS-3 in lung cancer cells.

Neither the native ligand nor the cell biology of the bombesin (Bn)-related orphan receptor subtype 3 (BRS-3) is known. In this study, we used RT-PCR to identify two human lung cancer lines that contain sufficient numbers of native hBRS-3 to allow study: NCI-N417 and NCI-H720. In both cell lines, [DPhe6,betaAla11,Phe13, Nle14]Bn(6-14) stimulates [3H]inositol phosphate. In NCI-N417 cells, binding of 125I-[DTyr6,betaAla11,Phe13,Nle14]Bn(6-14) was saturable and high-affinity. [DPhe6,betaAla11,Phe13,Nle14]Bn(6-14) stimulated phospholipase D activity and a concentration-dependent release of [3H]inositol phosphate (EC50 = 25 nM) and intracellular calcium (EC50 = 14 nM); the increases in intracellular calcium were primarily from intracellular stores. hBRS-3 activation was not coupled to changes in adenylate cyclase activity, [3H]-thymidine incorporation or cell proliferation. No naturally occurring Bn-related peptides bound or activated the hBRS-3 with high affinity. Four different bombesin receptor antagonists inhibited increases in [3H]inositol phosphate. Using cytosensor microphysiometry, we found that [DPhe6,betaAla11,Phe13, Nle14]Bn(6-14) caused concentration-dependent acidification. The results show that native hBRS-3 receptors couple to phospholipases C and D but not to adenylate cyclase and that they stimulate mobilization of intracellular calcium and increase metabolism but not growth. The discovery of human cell lines with native, functional BRS-3 receptors, of new leads for a more hBRS-3-specific antagonist and of the validity of microphysiometry as an assay has yielded important tools that can be used for the identification of a native ligand for hBRS-3 and for the characterization of BRS-3-mediated biological responses.

Identification of a unique ligand which has high affinity for all four bombesin receptor subtypes.

Four subtypes of bombesin receptors are identified (gastrin-releasing peptide receptor, neuromedin B receptor, the orphan receptor bombesin receptor subtype 3 (BB3 or BRS-3) and bombesin receptor subtype 4 (BB4)), however, only the pharmacology of the gastrin-releasing peptide receptor has been well studied. This lack of data is due in part to the absence of a general ligand. Recently we have discovered a ligand, 125I-[D-Tyr6,betaAla11,Phe13,Nle14]bombesin-(6-1 4) that binds to BRS-3 receptors. In this study we investigate its ability to interact with all four bombesin receptor subtypes. In rat pancreatic acini containing only gastrin-releasing peptide receptor and in BB4 transfected BALB cells, this ligand and 125I-[Tyr4]bombesin, the conventional gastrin-releasing peptide receptor ligand, gave similar results for receptor number, affinity for bombesin and affinity for the unlabeled ligand. In neuromedin B receptor transfected BALB cells, this ligand and 125I-[D-Tyr0]neuromedin B, the generally used neuromedin B receptor ligand, gave similar results for receptor number, neuromedin B affinity or the unlabeled ligand affinity. Lastly, in BRS-3 transfected BALB cells, only this ligand had high affinity. For all four bombesin receptors this ligand had an affinity of 1-8 nM and was equal or greater in affinity than any other specific ligands for any receptor. The unlabeled ligand is specific for gastrin-releasing peptide receptors on rat pancreatic acini and did not inhibit binding of 125I-cholecystokinin octapeptide (125I-CCK-8), 125I-vasoactive intestinal peptide (125I-VIP) or 125I-endothelin to their receptors. The unlabeled ligand was an agonist only at the gastrin-releasing peptide receptor in rat acini and did not interact with CCK(A) receptors or muscarinic M3 acetylcholine receptors to increase [3H]inositol phosphates. These results demonstrate 125I-[D-Tyr6,betaAla11,Phe13,Nle14]bombesin-(6-1 4) is a unique ligand with high affinity for all subtypes of bombesin receptors. Because of the specificity for bombesin receptors, this ligand will be a valuable addition for such pharmacological studies as screening for bombesin receptor agonists or antagonists and, in particular, for investigating BRS-3 cell biology, a receptor for which no ligand currently exists.

Discovery of a high affinity radioligand for the human orphan receptor, bombesin receptor subtype 3, which demonstrates that it has a unique pharmacology compared with other mammalian bombesin receptors.

An orphan receptor discovered in 1993 was called bombesin receptor subtype 3 (BRS-3) because of 47-51% amino acid identity with bombesin (Bn) receptors. Its pharmacology is unknown, because no naturally occurring tissues have sufficient receptors to allow studies. We made two cell lines stably expressing the human BRS-3 (hBRS-3). hBRS-3 was overexpressed in the human non-small cell lung cancer cells, NCI-H1299, and the other was made in Balb 3T3 cells, which lack endogenous BRS-3. [D-Phe6,beta-Ala11,Phe13, Nle14]Bn-(6-14) (where Nle represents norleucine) was discovered to have high potency for stimulating inositol phosphate formation in both cell lines. [125I-D-Tyr6,beta-Ala11,Phe13, Nle14]Bn-(6-14) bound to both cell lines with high affinity. Neither Bn nor 14 other naturally occurring Bn peptides bound to hBRS-3 with a Kd <1000 nM. Twenty-six synthetic peptides that are high affinity agonists or antagonists at other bombesin receptors had an affinity >1000 nM. Guanosine 5'-(beta,gamma-imido)triphosphate inhibited binding to both cells due to a change in receptor affinity. These results demonstrate hBRS-3 has a unique pharmacology. It does not interact with high affinity with any known natural agonist or high affinity antagonist of the Bn receptor family, suggesting the natural ligand is either an undiscovered member of the Bn peptide family or an unrelated peptide. The availability of these cell lines and the hBRS-3 ligand should facilitate identification of the natural ligand for BRS-3, its pharmacology, and cell biology.

The gastrin-releasing peptide receptor is differentially coupled to adenylate cyclase and phospholipase C in different tissues.

Recent studies suggest that in some tissues GRP receptor activation can both stimulate phospholipase C and the adenylate cyclase pathway and that activation of the latter pathway may be important in mediating some of its well-described growth effects. However, other studies suggest GRP-R may not be coupled to adenylate cyclase. To investigate this possibility, in the present study we determined the coupling of the GRP receptors to each pathway in mouse, rat, and guinea pig pancreatic acini and compared it to that in mouse Swiss 3T3 cells and human SCLC cells, all of which possess well-characterized GRP receptors. Moreover, we tested the effect of PKC activation on the ability of GRP-related peptides to increase cAMP accumulation in these tissues. Changes in cAMP levels were determined with or without IBMX present, with or without forskolin, or both to amplify small increases in cAMP. In mouse, rat and guinea pig pancreatic acini, murine Swiss 3T3 cells and human SCLC cells, GRP-related peptides caused a 600%, 500%, 250%, 300% and 60% increase, respectively, in [3H]IP with 1-3 nM causing a half-maximal effect. In murine Swiss 3T3 cells, IBMX, forskolin, and IBMX plus forskolin caused a 300%, 3500% and 10500% increase in cAMP, respectively. GRP-related peptides and VIP caused an additional 70% increase in cAMP with GRP causing a half-maximal (EC50) increase in cAMP at 2.1 +/- 0.5 nM, which was not significantly different from the EC50 of 3.1 +/- 0.9 nM for increasing [3H]IP in these cells. GRP-related peptides did not stimulate increases in cAMP in mouse, rat or guinea pig pancreatic acini or in SCLC cells either alone, with IBMX or forskolin or both. However, in pancreatic acini IBMX, forskolin or both increased cAMP 3 to 8-, 10 to 500-, and 100 to 1000-fold increase and the addition of VIP caused an additional 20-, 2-, and 3-fold increase in cAMP in the different species. In mouse pancreatic acini with TPA alone or IBMX plus TPA, neither bombesin nor GRP increased cAMP. Furthermore, in mouse pancreatic acini, neither TPA nor TPA plus IBMX altered basal or VIP-stimulated increases in cAMP. In mouse Swiss 3T3 cells TPA significantly increased cAMP stimulated by Bn, GRP or VIP. These results demonstrated that GRP receptor activation in normal tissues from three different species and a human tumoral cell line do not result in adenylate cyclase activation, whereas in Swiss 3T3 cells it causes such activation. The results suggest that the difference in coupling to adenylate cyclase is likely at least partially due to a difference in coupling to an adenylate cyclase subtype whose activation is regulated by PKC. Therefore, the possible growth effects mediated by this receptor in different embryonic or tumoral cells through activation of adenylate cyclase are not likely to be an important intracellular pathway for these effects in normal tissues.

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.

Identification of CCK-A receptors on chief cells with use of a novel, highly selective ligand.

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.

Expression and characterization of cloned human bombesin receptors.

Little is known about the pharmacology or cell biology of human bombesin (Bn) receptors, because they are usually present at low levels and both subtypes are frequently present in the same tissues. Human gastrin-releasing peptide (GRP) receptors (huGRP-R) and human neuromedin B (NMB) receptors (huNMB-R) were stably transfected into BALB/3T3 fibroblasts. Both receptor types were glycosylated, with 35% of the huGRP-R and 38% of the huNMB-R representing carbohydrate residues. The extent of glycosylation of the transfected huGRP-R was the same as that seen in the human glioblastoma cell line U-118. Radiolabeled agonist ligands were rapidly internalized, whereas noninternalized ligand readily dissociated in a temperature-dependent fashion. The affinities of various agonists for binding to the huGRP-R were Bn (Ki = 1.4 +/- 0.2 nM) = 4 x GRP = 300 x NMB. In contrast, affinities for the huNMB-R were NMB (Ki = 8.1 +/- 5.2 nM) = 4 x Bn = 600 x GRP. [F5-D-Phe6,D-Ala11]Bn(6-13)methyl ester was the most potent huGRP-R antagonist, whereas D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Nal-NH2 was the most potent huNMB-R antagonist. Agonist binding to either receptor type caused activation of phospholipase C and increased cellular [3H]inositol phosphate levels. GRP was potent at increasing [3H]inositol phosphate generation in cells expressing the huGRP-R (EC50 = 13.6 +/- 1.3 nM), whereas NMB was similarly potent when acting upon cells expressing the huNMB-R (EC50 = 9.3 +/- 1.4 nM). However, neither receptor type, when stimulated with agonist, caused an increase in cAMP levels. These data show that stably transfected huGRP-R exhibit similar pharmacology for agonists and antagonists, are appropriately glycosylated, and function similarly with respect to their ability to alter biological activity, compared with natively expressed receptors. Minimal native huNMB-R data are available for comparison, but in general the huNMB-R is similar to the rat NMB receptor in its pharmacology and cell biology.

Bombesin does not stimulate pepsinogen release in isolated gastric chief cells.

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.

Interaction of galanin fragments with galanin receptors on isolated smooth muscle cells from guinea pig stomach: identification of a novel galanin receptor subtype.

From structure-function studies it has been proposed that two subtypes of receptors may mediate galanin's actions in the gastrointestinal tract and other tissues and their effects can be either direct or neurally mediated. We have recently demonstrated that isolated gastric smooth muscle cells possess high-affinity galanin receptors, activation of which increases AMP and causes relaxation. Because this cell system contains no neural elements, contains a single class of galanin receptors and allows binding to be correlated with function, it is a good system to investigate peptide requirements for cell activation. Porcine galanin (p-Gal) and rat galanin (r-Gal) were equipotent to the NH2-terminal fragments r,p-Gal(1-10), r,p-Gal(1-15), r,p-Gal(1-20), p-Gal(2-29) and p-Gal(3-29) at inhibiting binding of 125I-galanin to gastric smooth muscle cells from guinea pig (Kd 5-8 nM). The midmolecule fragment p-Gal(9-25) and the long COOH-terminal fragment r-Gal(9-29) were equipotent and 25-fold less potent than p-Gal. Acetylation of r-Gal(9-29) increased potency to that of p-Gal. The short COOH-terminal fragment, p-Gal(21-29) and r-Gal(21-29), had very low affinity (Kd 3 microM). Each peptide alone (1 microM) caused no effect on cell length, but inhibited carbachol-induced contraction. The inhibition was 81% to 92% for r-Gal or p-Gal, r,p-Gal(1-10), r,p-Gal(1-15), r,p-Gal(1-20), p-Gal(2-29), p-Gal(3-29) and Ac-r-Gal(9-29); 47% for p-Gal(9-25) and r-Gal(9-29); and 16% to 17% for p-Gal(21-29).(ABSTRACT TRUNCATED AT 250 WORDS)

Chief cells possess somatostatin receptors regulated by secretagogues acting through the calcium or cAMP pathway.

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.

Smooth muscle cells from guinea pig stomach possess high-affinity galanin receptors that mediate relaxation.

Galanin-like immunoactivity occurs in nerves and plexi in muscle layers throughout gastrointestinal tract including the stomach. Galanin can affect gastric emptying and contraction or relaxation of gastric muscle in different species. The aim of this study was to investigate the direct effect of galanin on dispersed gastric smooth muscle cells and to characterize any galanin receptors that mediated any effect. Dispersed gastric smooth muscle cells were prepared from guinea pig stomach by collagenase digestion. Porcine galanin (p-galanin; 1 microM) did not stimulate contraction when present alone; however, p-galanin (1 microM) inhibited carbachol-induced contraction with a half-maximal effect at 7 nM. p-Galanin (1 microM) increased cellular adenosine 3',5'-cyclic monophosphate (cAMP) content by 10 s and caused a maximal increase of 80% over basal. 125I-galanin (porcine) bound to dispersed cells in a time- and temperature-dependent manner. Binding was saturable, reversible, and specific. Binding of 125I-galanin was inhibited almost equally by porcine and rat galanin (Ki = 6-8 nM) but was not inhibited by the galanin-associated peptide [preprogalanin-(108-123)]. The fragment galanin-(1-16) was equally potent to rat galanin; however, the fragment galanin-(9-29) was 56-fold less potent (Ki = 370 nM). Computer analysis demonstrated there were two binding sites for p-galanin on gastric smooth muscle cells, a high-affinity site (Kd = 2.6 nM) with low capacity (Bmax = 175 fmol/mg protein) and a low-affinity site (Kd = 150 nM) with large capacity (Bmax = 3,611 fmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Galanin-induced relaxation in gastric smooth muscle cells is mediated by cyclic AMP.

Galanin has numerous effects on gastrointestinal motility in different species; however, its cellular basis of action in mediating these effects is unclear. Dispersed gastric smooth muscle cells have been shown to possess high-affinity galanin receptors that increase cAMP and cause relaxation. Recent studies show some smooth muscle relaxants such as VIP cause relaxation by both cAMP-dependent and -independent mechanisms. It is unknown if galanin's cellular basis of relaxation is similar or different from that of VIP. To investigate galanin's relaxant effect and compare it to VIP's effect, dispersed smooth muscle cells from guinea pig stomach were prepared by collagenase digestion. The mean length in resting cells was 110 +/- 2 microns and, with carbachol treatment, contracted to 89 +/- 2 microns. VIP and galanin alone had no effect on cell length, but each caused a dose-dependent inhibition of carbachol-induced contraction and both had an EC50 of 3-7 nM. Galanin (1 microM) and VIP (1 microM) increased cellular cAMP from 118 +/- 10 pmol/10(6) cells in control to 212 +/- 14 and 214 +/- 12 pmol/10(6) cells, respectively. The protein kinase A inhibitor, Rp-cAMPS, at 100 microM, completely inhibited the relaxant effect of an EC50 concentration of galanin (3 nM), but only inhibited that by VIP by 80% (p < 0.05). Adding the nitric oxide inhibitor, L-NNA (NG-nitro-L-arginine), at 100 microM did not alter the length of resting cells or inhibit carbachol-induced contraction. However, L-NNA (100 microM) decreased VIP-induced relaxation by 45%, whereas it had no effect on galanin-induced relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of inhibition of microsomal Ca(2+)-ATPase on cytoplasmic calcium and enzyme secretion in pancreatic acini.

We used thapsigargin (TG), 2,5-di-tert-butyl-1,4-benzohydroquinone (BHQ) and cyclopiazonic acid (CPA), each of which inhibits microsomal Ca(2+)-ATPase, to evaluate the effects of this inhibition on cytoplasmic free calcium ([Ca2+]i) and secretagogue-stimulated enzyme secretion in rat pancreatic acini. Using single-cell microspectrofluorimetry of fura-2-loaded acini we found that all three agents caused a sustained increase in [Ca2+]i by mobilizing calcium from inositol-(1,4,5)-trisphosphate-sensitive intracellular calcium stores and by promoting influx of extracellular calcium. Concentrations of all three agents that increased [Ca2+]i potentiated the stimulation of enzyme secretion caused by secretagogues that activate adenylate cyclase but inhibited the stimulation of enzyme secretion caused by secretagogues that activate phospholipase C. With BHQ, potentiation of adenylate cyclase-mediated enzyme secretion occurred immediately whereas inhibition of phospholipase C-mediated enzyme secretion occurred only after several min of incubation. In addition, the effects of BHQ and CPA on both [Ca2+]i and secretagogue-stimulated enzyme secretion were reversed completely by washing whereas the actions of TG could not be reversed by washing. Concentrations of BHQ in excess of those that caused maximal changes in [Ca2+]i inhibited all modes of stimulated enzyme secretion by a mechanism that was apparently unrelated to changes in [Ca2+]i. Finally, in contrast to the findings with TG and BHQ, CPA inhibited bombesin-stimulated enzyme secretion over a range of concentrations that was at least 10-fold lower than the range of concentrations over which CPA potentiated VIP-stimulated enzyme secretion.

Chimeric galanin analogs that function as antagonists in the CNS are full agonists in gastrointestinal smooth muscle.

Galanin has numerous effects on gastrointestinal smooth muscle. However, because of the lack of specific inhibitors, it is not known which are physiological and which are pharmacological. This study investigates the ability of two chimeric galanin analogs, [# 1-galantide = (M-15) = [galanin (1-13)-substance P(5-11)] and #2-M-35[galanin(1-13)bradykinin (2-9)], which were recently reported to function as galanin-receptor antagonists in the CNS, to interact with galanin receptors on rat jejunal muscle strips or dispersed smooth muscle cells from guinea pig stomach. In both systems each chimeric analog had agonist activity and was as efficacious as galanin. Cross-desensitization experiments demonstrated that in the jejunal muscle strips, both chimeric analogs were causing muscle contraction by interacting with the galanin receptor. In dispersed smooth muscle cells, galanin, as well as each chimeric analog, caused muscle relaxation, whereas substance P and bradykinin both caused muscle contraction. Each chimeric analog was equipotent to galanin in inhibiting binding of 125I-galanin, and there was close agreement between their abilities to occupy the galanin receptor and cause relaxation. Each chimeric analog also activated adenylate cyclase and increased cAMP characteristic of relaxants. These studies demonstrate these chimeric analogs will not be useful for defining the physiological role of galanin in altering gastrointestinal motility, because they function as full galanin-receptor agonists instead of as galanin-receptor antagonists.

8-Methoxypsoralen levels in blood of vitiligo patients and in skin, ophthalmic fluids, and ocular tissues of the guinea pig.

8-Methoxypsoralen (8-MOP) levels in the blood of vitiligo patients were determined through the use of a reverse-phase high-performance liquid chromatographic method. The overall recovery of the internal standards was 85-94%, with the lower detection limit of 8-MOP at 2 ng. Peak blood levels as low as 130 ng/ml and as high as 3892 ng/ml were obtained in patients at 1-3 h following the oral administration of 0.6 mg/kg body weight of Oxsoralen capsules (Elder Pharmaceuticals Co.). These results are consistent with the clinical observation that maximum response in phototherapy is obtained at about 2 h after oral administration of the drug. Two hours after oral administration of 0.6 mg/kg of Oxsoralen, 8-MOP levels in the epidermis, dermis, and whole skin of the guinea pig (in ng/g) were: epidermis, 330 +/- 20; dermis, 89 +/- 16; whole skin, 379 +/- 19. Also detected were 8-MOP levels of 441 +/- 22 ng/ml in aqueous humor, 166 +/- 18 ng/ml in vitreous gel, 355 +/- 15 ng/g in lens, and 410 +/- 26 ng/g in retina. These results point to the fact that the eyes of the patient must be protected from exposure to sunlight after psoralen UV treatment, and that 8-MOP is absorbed in blood unevenly and varies from patient to patient. The fact that only 50-60% of the patients responded to psoralen photochemotherapy for vitiligo may be related to the variation of absorption of the drug in individual patients.