Interaction of substance P antagonists with substance P receptors on dispersed pancreatic acini

In the present study we examined the abilities of three analogs of substance P, [D-Pro2-, D-Phe7-, D-Trp9]-substance P, [D-Pro2-, D- Trp7 ,9]-substance P and [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P to alter substance P-induced changes in pancreatic acinar cell function and to occupy substance P receptors. At 30 microM, each analog of substance P lacked agonist activity and inhibited amylase secretion stimulated by substance P receptor agonists. The inhibition was reversible and specific for peptides that interact with substance P receptors (physalaemin, substance P, eledoisin, kassinin ). The analogs of substance P did not inhibit the actions of cholecystokinin, caerulein, gastrin, carbamylcholine, secretin, vasoactive intestinal peptide, PHI, ionophore A23187 or 8Br -cAMP. At high concentrations, [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P, but not [D-Pro2-, D- Trp7 ,9]-substance P or [D-Pro2-, D-Phe7-, D-Trp9]-substance P, caused a small but significant inhibition of bombesin-stimulated amylase release. For each analog of substance P, the inhibition was competitive in nature in that there was a rightward shift of the dose-response curve for physalaemin-stimulated amylase secretion with no change in efficacy. From Schild plots of the ability of [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P to inhibit either substance p- or physalaemin-stimulated amylase release, the slopes were not different from unity. For each analog of substance P, there was a close correlation between its ability to inhibit substance P- or physalaemin-stimulated amylase release and its ability to inhibit binding of 125I-labeled substance P or 125I-labeled physalaemin. [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P was 2-fold more potent than [D-Pro2-, D- Trp7 ,9]-substance P which was 4-fold more potent than [D-Pro2-, D-Phe7-, D-Trp9]-substance P, (i.e., pA2 6.1, 5.9, and 5.2, respectively). For each analog, the dose-response curve for its ability to inhibit physalaemin-stimulated amylase release was superimpossible on the dose-response curve for its ability to inhibit binding of 125I-labeled physalaemin. These results indicate that each of these analogs of substance P is a specific competitive inhibitor of the action of the substance P on dispersed acini from guinea-pig pancreas, and that their abilities to inhibit substance P-induced changes in acinar cell function can be accounted for by their abilities to occupy the substance P receptor.     

Characterization of bombesin receptors on canine antral gastrin cells

Dispersed canine antral mucosal cells were prepared by sequential steps of collagenase digestion and EDTA treatment. Cell preparations enriched in gastrin cells were made by centrifugal elutriation followed by step density gradient centrifugation. Specific, saturable, and reversible binding of 125I-[Tyr4]-bombesin was found in all preparations. This saturable binding was time, temperature, and cell number dependent. In both velocity (elutriator) and density cell separation experiments, saturable binding of bombesin correlated with the distribution of cells containing gastrin- but not somatostatin-like immunoreactivity. Maximal specific binding to gastrin (G) cell-enriched fractions was reached in 45 min at 37 degrees C and constituted 90% of total binding. Addition of 100 nM nonradioactive bombesin to cells incubated with 50 pM 125I-[Tyr4]-bombesin for 45 min resulted in time-dependent dissociation of specifically bound tracer to about 40% of the maximal equilibrium binding. Analysis of saturable equilibrium binding yielded a best fit to a one-site model of high affinity binding sites with an apparent Kd of 85 +/- 14 pM and a Bmax of 231,000 +/- 71,000 receptors/gastrin cell. Nonradioactive [Tyr4]-bombesin and related analogs inhibited the specific binding of the tracer in a dose-related manner. The rank order of potency, determined at the IC50, of [Tyr4]-bombesin and related analogs for inhibition of specific binding was bombesin greater than [Tyr4]-bombesin = hGRP-27 greater than GRP-10 greater than ranatensin much greater than neuromedin B. Cholecystokinin, somatostatin, substance K, and kassinin each tested at a concentration of 1 microM did not inhibit bombesin binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of protein kinase C isozymes in bombesin-stimulated gastrin release from human antral gastrin cells.

Two of the most effective stimuli of gastrin release from human antral G cells are bombesin and phorbol esters. Both agonists result in activation of the protein kinase C family of isozymes, however, the exact contribution of protein kinase C to the resultant release of gastrin has been difficult to assess, possibly due to the presence of multiple protein kinase C isozymes in the G cells. The results of the present study demonstrated that the human antral G cells expressed 6 protein kinase C isozymes alpha, gamma, theta, epsilon, zeta, and mu. Of these protein kinase C, gamma and theta were translocated by stimulation of the cells by either 10 nM bombesin or 1 nM phorbol ester. Inhibition of protein kinase Cmu (localized to the Golgi complex) did not decrease bombesin-stimulated gastrin release indicating that this isozyme was not involved in the secretory process. The use of selective antagonists of the calcium-sensitive conventional protein kinase C subgroup resulted in an increase in bombesin-stimulated gastrin release and indicated that protein kinase Cgamma was involved in the desensitization of the bombesin response.     

Probing peptide backbone function in bombesin. A reduced peptide bond analogue with potent and specific receptor antagonist activity

Each peptide bond CONH group in the most important COOH-terminal octapeptide region of [Leu14]bombesin was replaced by a CH2NH group using recently developed rapid solid-phase methods. The resulting analogues were then examined for amylase releasing activity in guinea pig pancreatic acini and for their ability to inhibit binding of [125I-Tyr4]bombesin to acinar cells. Replacement of the Trp8-Ala9, Gly11-His12, and His12-Leu13 peptide bonds resulted in about 1000-, 200-, and 300-fold losses in both amylase releasing activity and binding affinity. The Val10-Gly11 replacement, however, retained 30% potency relative to the parent peptide. Ala9-Val10 and Leu13-Leu14 bond replacement analogues exhibited no detectable amylase releasing activity but were still able to bind to acini with Kd values of 1060 and 60 nM, respectively (compared to 15 nM for [Leu14]bombesin itself). Subsequently, both analogues were demonstrated to be competitive inhibitors of bombesin-stimulated amylase release with IC50 values of 937 and 35 nM, respectively. [Leu14-psi-CH2NH-Leu13]Bombesin exhibits a 100-fold improvement in binding affinity compared to previously reported bombesin receptor antagonists and showed no affinity for substance P receptors. It was also a potent inhibitor of bombesin-stimulated growth of murine Swiss 3T3 cells with an IC50 of 18 nM. In terms of a bombesin receptor-binding conformation, these results may aid in the delineation of intramolecular hydrogen-bonding points and the eventual design of improved, conformationally restricted analogues.     

Somatostatin,prostaglandin E2 and atropine inhibition of the gastric actions of bombesin in the dog

Bombesin, acetylcholine, prostaglandins and somatostatin are all thought to be involved in the regulation of gastrin release and gastric secretion. We have studied the effects of low doses of atropine, 16-16(Me)₂-prostaglandin E₂ (PGE₂) and somatostatin-14 on bombesin-stimulated gastrin release and gastric acid and pepsin secretion in conscious fistula dogs. For reference, synthetic gastrin G-17 was studied with and without somatostatin. Bombesin, in a dose-related manner, increased serum gastrin, which in turn stimulated gastric acid and pepsin secretion in a serum gastrin, concentration-dependent manner. Somatostatin inhibited gastrin release by bombesin as well as the secretory stimulation by G-17; the combination of sequential effects resulted in a marked inhibition of bombesin-stimulated gastric acid and pepsin secretion. PGE₂ also strongly inhibited gastrin release and acid and pepsin secretion. Atropine had no significant effect on gastrin release, but greatly inhibited gastric secretion. Thus somatostatin and PGE₂ inhibited at two sites, gastrin release and gastrin effects, while atropine affected only the latter.     

Spantide: failure to antagonize bombesin-induced stimulation of gastrin secretion in dogs

Spantide ([d-Arg1, d-Trp7,9, Leu11] substance P) was shown to function not only as a substance P receptor antagonist but also as a bombesin receptor antagonist. This study examined the effects of spantide on intravenous bombesin-induced stimulation of gastrin and acid secretion. Dogs were infused with spantide (1 or 10 nmol kg 1 hr 1) or saline and bombesin (60 pmol kg-1 hr-1), and the gastric acid and plasma gastrin responses were monitored. Spantide did not significantly modify gastrin or gastric acid secretion induced by bombesin. It is concluded that spantide may not be a useful bombesin antagonist for in vivo studies.     

Bombesin-like peptides elevate cytosolic calcium in small cell lung cancer cells

The ability of bombesin-like peptides to elevate intracellular Ca2+ levels in small cell lung cancer cells was investigated using the fluorescent Ca2+ indicator Fura 2. Nanomolar concentrations of bombesin elevated cytosolic Ca2+ levels in the absence or presence of extracellular Ca2+. Potent bombesin receptor agonists, such as gastrin releasing peptide (GRP) or (GRP)14-27 elevated cytosolic Ca2+ levels whereas inactive compounds such as (D-Trp8)bombesin or (GRP)1-16 did not. Furthermore, the bombesin receptor antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11) substance P (30 microM) had no effect on the Ca2+ levels by itself but antagonized the increase in Ca2+ caused by 10 nM or 100 nM bombesin. These data suggest that bombesin receptors may regulate the release of Ca2+ from intracellular organelles in small cell lung cancer cells.     

Biological effects and receptor binding affinities of new pseudononapeptide bombesin/GRP receptor antagonists with N-terminal D-Trp or D-Tpi.

In an attempt to produce more powerful (effective) bombesin/GRP receptor antagonists, the D forms of Trp or Trp analog (Tpi) were introduced at position 6 in two pseudononapeptides, Leu13 psi (CH2NH)Leu14-bombesin(6-14) and Leu13 psi(CH2NH)Phe14-bombesin (6-14). These antagonists were tested for their ability to inhibit basal and gastrin releasing peptide (GRP) (14-27)-induced amylase release from rat pancreatic acini in a superfusion assay. They were also assessed for the inhibition of 125I-Tyr4-bombesin binding to Swiss 3T3 and small cell lung carcinoma cell line H-345 and the mitogenic response of Swiss 3T3 cells induced by GRP(14-27). The peptides, when given alone, did not stimulate amylase secretion, but were able to inhibit gastrin releasing peptide (14-27)-induced amylase release. All of the antagonists showed strong binding affinities for Swiss 3T3 and H-345 cells and suppressed the GRP(14-27)-induced increase of [3H]thymidine incorporation into DNA of Swiss 3T3 cells at nanomolar concentrations. Antagonist D-Tpi6,Leu13 psi (CH2NH)Leu14-bombesin (6-14)(RC-3095) was slightly more potent in these assays than D-Trp6,Leu13 psi (CH2NH)Leu14-bombesin (6-14)(RC-3125). Nevertheless, D-Trp6,Leu13 psi (CH2NH)Phe14-bombesin (6-14) showed the highest binding affinity for Swiss 3T3 and H345 cells and it was the most potent inhibitor of GRP(14-27)-induced amylase secretion. This antagonist RC-3420 was particularly effective in inhibiting the growth of Swiss 3T3 cells, exhibiting an IC50 value less than 1 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of surfactant secretion from isolated Type II pneumocytes by substance P

Substance P, an eleven amino acid neuropeptide, significantly inhibited release of [3H]phosphatidylcholine from pulmonary Type II epithelial cells in vitro. Basal release and release in response to the beta-adrenergic agonist, terbutaline and 12-O-tetradecanoylphorbol 13-acetate (TPA) were significantly decreased in the presence of substance P. Inhibitory effects of substance P were noted following a 1 h exposure of primary cultures of Type II cells in vitro and persisted up to 3 h in the presence of the secretagogues, TPA and terbutaline. The IC50 values for substance P inhibition of [3H]PC release were 10 microM for basal release, 40 microM for TPA-induced release and 50 microM for terbutaline-induced release. The related neuropeptide, physalaemin and the stable active analog of substance P, [pGlu5, MePhe8, MeGly9]substance P [5-11], had no significant inhibitory effects on surfactant release whether in the presence or absence of TPA or terbutaline. These data support the hypothesis that NH2-terminal basic groups of substance P are necessary for inhibition of surfactant secretion from isolated Type II cells and support the concept that an inhibitory system contributes to mediation of surfactant secretion from Type II epithelial cells.     

Short-chain pseudopeptide bombesin receptor antagonists with enhanced binding affinities for pancreatic acinar and Swiss 3T3 cells display strong antimitotic activity

The high inhibitory potency of the previously developed bombesin antagonist [Leu13, psi CH2NHLeu14]bombesin (analogue I) (IC50 values of 30 and 18 nM for inhibition of bombesin-stimulated amylase secretion from guinea pig acinar cells and Swiss 3T3 cell growth, respectively) diminished considerably when shorter chain lengths were examined. For instance, [Leu13, psi CH2NHLeu14]bombesin-(5-14),[Leu13, psi CH2NHLeu14] bombesin-(6-14), and [Leu9, psi CH2NHLeu10]neuromedin C had IC50 values of 150, 150, and 280 nM, respectively. Incorporation of a D-Phe residue at position 6 of [Leu13, psi CH2NHLeu14] bombesin did not significantly change the various biological parameters. However, its presence in [Leu13, psi CH2NHLeu14]bombesin-(6-14) and at position 2 of psi-neuromedin C-(2-10) resulted in about 10-fold increases in potency up to and above that of the original antagonist. For instance, [D-Phe6,Leu13,psi CH2NHLeu14]bombesin-(6-14) and des-Gly1-[D-Phe2,Leu9,psi CH2NHLeu10]neuromedin C exhibited IC50 values of 5 and 28 nM, respectively. Analogues based on the litorin sequence which contains an NH2-terminal pyroglutamic acid residue at the bombesin position 6 equivalent were also quite potent. The ability of various analogues to interact with bombesin receptors on pancreatic acini correlated reasonably well with potencies derived from inhibition of bombesin-stimulated growth of Swiss 3T3 cells. Additional studies of NH2- and COOH-terminal structure-activity relationships resulted in the synthesis of [D-Phe6,Leu13,psi CH2NHPhe14]bombesin-(6-14), which was particularly effective in inhibiting 3T3 cell growth at high picomolar concentrations (IC50 = 0.72 nM and Ki = 3.1 nM for 3T3 cells; IC50 = 7.5 nM and Ki = 9.9 nM for acini). Detailed investigations with one of the most potent antagonists, [D-Phe6,Leu13,psi CH2NHLeu14]bombesin-(6-14) (Ki = 14 nM for acini cells and 7.1 for 3T3 cells), demonstrated that this analogue was a competitive inhibitor of bombesin and that this activity was specific for the bombesin receptor. Thus, inhibitory potencies have been improved generally up to 25 times over previously reported structures; and, given that bombesin itself has a Ki of 1.2 nM for 3T3 cell binding, some of these analogues are extraordinarily high affinity receptor antagonists. They can also be synthesized more readily and offer fewer proteolytic degradation sites than the original pseudopeptide and should be excellent candidates for in vivo studies aimed at inhibition of bombesin-dependent human small cell lung carcinoma growth.     

Effect of cyclic nucleotides on bombesin-evoked gastrin release from isolated perfused rat stomach

We and others have recently reported an involvement of calcium (Ca2+)-mediated intracellular pathways in the release of antral gastrin in response to bombesin (BBS), while cyclic adenosine 3'5'-monophosphate (cAMP) potentiated the gastrin response to BBS. In this study we examined the effect of cyclic nucleotides on BBS-induced gastrin release from isolated perfused rat stomachs. Dibutyryl cyclic AMP (dbcAMP, 1 mM), and Rolipram (a phosphodiesterase inhibitor, 0.5 microM), stimulated basal gastrin secretion and potentiated BBS-induced gastrin release. The stimulation of gastrin release by BBS was not altered by Wiptide (a cAMP dependent protein kinase inhibitor, 1.0 microM), but was surprisingly inhibited by dbcGMP (1 mM). The cAMP content in antral mucosa or in the perfusates was not changed after infusion of BBS. These findings coupled with previous results suggest that BBS-provoked gastrin release is principally coupled to a Ca2+-mediated intracellular pathway, and that an activation of the adenylate cyclase mediated pathway is not involved. Intracellular cGMP, however, may participate in the negative regulation of gastrin release induced by BBS.     

Intravenous histamine reduces bombesin-stimulated gastrin release in dogs

The effect of histamine on gastrin release was studied in 7 conscious mongrel dogs with chronic gastric and duodenal fistulas. Histamine-2 HCl was infused in doses of 0 (control), 20, 40, 80, and 160 micrograms/kg per h for 2 h on separate days. During the second hour, bombesin 500 ng/kg per h was infused intravenously. Intragastric pH was constantly kept at 2.5 by intragastric titration during each test. Leakage of gastric contents into the duodenum was prevented by a prepyloric balloon passed retrograde through a duodenal fistula. Gastrin release, as expressed by the integrated response during the last 50 min of the bombesin infusion was significantly (P less than 0.05) decreased by all doses of histamine, compared to control. The infusion doses of histamine studied, 20, 40, 80, and 160 micrograms/kg per h reduced bombesin-stimulated gastrin release 16%, 19%, 19%, and 30%, respectively. This effect was blocked by a histamine H-2 but not an H-1 receptor antagonist. We conclude that by an H-2 mechanism, exogenous histamine reduces bombesin-stimulated gastrin release in dog.     

The effect of secretin on acid and pepsin secretion and gastrin release in the totally isolated vascularly perfused rat stomach

The effect of secretin on acid and pepsin secretion and gastrin release in the totally isolated vascularly perfused rat stomach was studied. With the phosphodiesterase inhibitor isobutyl methylxanthine (IMX) added to the vascular perfusate, baseline acid secretion was 4.7 +/- 1.1 (mean +/- S.E.M.) mumol/h and baseline pepsin output 1147 +/- 223 micrograms/h. Secretin significantly inhibited acid output to a minimum of 1.4 +/- 0.2 mumol/h at a concentration of 25 pM in the vascular perfusate (P less than 0.01). Pepsin output was not significantly different from baseline at any of the secretin doses tested. Threshold secretin concentration for acid inhibition was 5 pM. IMX stimulated gastrin output from 48 +/- 9 pM in the basal state to 95 +/- 13 pM after IMX (P less than 0.01). Secretin inhibited gastrin release only at the maximal dose of 625 pM, when gastrin concentration in the venous effluent decreased from 93 +/- 19 to 68 +/- 19 pM after secretin. Thus, in the totally isolated vascularly perfused rat stomach secretin in physiological concentrations inhibits acid secretion by a direct action on the acid secretory process and not via gastrin inhibition. The study also suggests that gastrin release at least in part is mediated via increased intracellular cAMP.     

Effect of substance P/bombesin antagonists on the release of growth hormone by GHRP and GHRH.

The substance P(SP)/bombesin (Bn) antagonists [DArg1DTrp7,9Leu11] SP(P-7482), [DArg1-DPro2DTrp7,9Leu11]SP (P-7483), [DArg1DPhe5DTrp7,9Leu11]SP(P-7492), and the growth hormone releasing hormone (GHRH) antagonist [DArg2Ala8,9,15]GHRH(1-29)(DC21-366) were tested for their in vitro effects on the release of growth hormone (GH) in the presence of GHRH and growth hormone releasing peptide, HisDTrpAlaTrpDPheLysNH2(GHRP). P-7492, P-7483, and P-7482 decreased, dose-dependently, the release of GH by GHRP (IC50 = 0.2 microM, 0.85 microM, and 6 microM, respectively). These antagonists had only a 10-15% inhibitory effect on the stimulated GH release of GHRH even at high dosage. DC21-366 decreased the stimulated release of GH by GHRH (IC50 = 0.16 microM) but not by GHRP. Neither SP nor Bn had GH releasing or inhibitory effects in this system.     

The CCKB/gastrin receptor is coupled to the regulation of enzyme secretion, protein synthesis and p70 S6 kinase activity in acinar cells from ElasCCKB transgenic mice.

In order to determine which physiological functions can be regulated by the pancreatic CCKB/gastrin receptor, studies were carried out on pancreatic acini from mice expressing transgenic CCKB/gastrin receptors in the exocrine pancreas (ElasCCKB mice). Acini were stimulated by sulfated gastrin in the presence of SR 27897 (1.8 microM), blocking endogenous CCKA receptors. After 30 min incubation with gastrin, the secretion of chymotrypsinogen and amylase showed superimposable monophasic dose-response curves. Enzyme secretion was detectable and maximal at 100 pM and 1 nM of gastrin, respectively. No increase in chymotrypsinogen and amylase mRNAs was detected for doses of gastrin which specifically occupy the CCKB/gastrin receptor. In contrast, gastrin stimulated total protein synthesis in isolated acini from ElasCCKB mice. [35S]Methionine incorporation into total proteins was increased dose-dependently to a maximum for 30 pM gastrin and inhibited with higher doses (> 300 pM). Gastrin stimulated p70 S6 kinase activity for concentrations ranging from 10 pM to 1 nM. Gastrin-stimulated p70 S6 kinase activity and protein synthesis were blocked by rapamycin and wortmannin. Therefore, in ElasCCKB mice acinar cells, the CCKB/gastrin receptor mediates enzyme release and protein synthesis. However, a more efficient coupling of the CCKB/gastrin receptor to protein synthesis than to enzyme secretion was demonstrated. CCKB/gastrin receptor-stimulated protein synthesis likely results from an enhancement of mRNA translation and involves phosphatidyl inositol 3-kinase and p70 S6 kinase.     

The bombesin receptor is coupled to a guanine nucleotide-binding protein which is insensitive to pertussis and cholera toxins

The neuropeptide bombesin acts on a variety of target cells to stimulate the processes of secretion and cell proliferation. In this study we determined whether bombesin receptors interact with known guanine nucleotide-binding proteins in four different cell types: GH4C1 pituitary cells, HIT pancreatic islet cells, Swiss 3T3 fibroblasts, and rat brain tissue. Maximal concentrations of nonhydrolyzable GTP analogs decreased agonist binding to bombesin receptors in membranes from all four sources. In GH4C1 and HIT cell membranes GTP analogs inhibited bombesin receptor binding with IC50 values of about 0.1 microM, whereas GDP analogs were approximately 10-fold less potent. In contrast, GMP and the nonhydrolyzable ATP analog adenylyl-imidodiphosphate had no effect at 100 microM. Equilibrium binding experiments in GH4C1 and HIT cell membranes indicated a single class of binding sites with a dissociation constant (Kd) for [125I-Tyr4]bombesin of 24.4 +/- 7.0 pM and a binding capacity of 176 +/- 15 fmol/mg protein. Guanine nucleotides decreased the apparent affinity of the receptors without significantly changing receptor number. Consistent with this observation, guanine nucleotides also increased the rate of ligand dissociation. Pretreatment of GH4C1 or HIT cells with either pertussis toxin (100 ng/ml) or cholera toxin (500 ng/ml) for 18 h did not affect agonist binding to membrane bombesin receptors, its regulation by guanine nucleotides, or bombesin stimulation of hormone release. Although pertussis toxin pretreatment has been reported to block bombesin stimulation of DNA synthesis in Swiss 3T3 cells, it did not alter the binding properties of bombesin receptors in Swiss 3T3 membranes or inhibit the rapid increase in intracellular [Ca2+] produced by bombesin in these cells. In summary, our results indicate that the bombesin receptor interacts with a guanine nucleotide-binding protein which exhibits a different toxin sensitivity from those which regulate adenylate cyclase as well as those which couple some receptors to phospholipases.     

Bombesin and substance P analogues differentially regulate G-protein coupling to the bombesin receptor. Direct evidence for biased agonism

Substance P analogues including [d-Arg1,d-Phe5,d-Trp7,9,Leu11]substance P (SpD) act as "broad spectrum neuropeptide antagonists" and are potential anticancer agents that inhibit the growth of small cell lung cancer cells in vitro and in vivo. However, their mechanism of action is controversial and not fully understood. Although these compounds block bombesin-induced mitogenesis and signal transduction, they also have agonist activity. The mechanism underlying this agonist activity was examined. SpD binds to the ligand-binding site of the bombesin/gastrin-releasing peptide receptor and blocks the bombesin-stimulated increase in [Ca2+]i within the same concentration range that causes sustained activation of c-Jun N-terminal kinase and extracellular signal-regulated protein kinase (ERK). The activation of c-Jun N-terminal kinase by SpD and bombesin is blocked by dominant negative inhibition of G(alpha12). The ERK activation by SpD is pertussis toxin-sensitive in contrast to ERK activation by bombesin, which is pertussis toxin-insensitive but dependent on epidermal growth factor receptor phosphorylation. SpD does not simply act as a partial agonist but differentially modulates the activation of the G-proteins G(alpha12), G(i), and G(q) compared with bombesin. This unique ability allows the bombesin receptor to couple to G(i) and at the same time block receptor activation of G(q). Our results provide direct evidence that SpD is acting as a "biased agonist" and that this has physiological relevance in small cell lung cancer cells. This validation of the concept of biased agonism has important implications in the development of novel pharmacological agents to dissect receptor-mediated signal transduction and of highly selective drugs to treat human disease.     

Analysis of food stimulation of gastrin release in dogs by a panel of inhibitors

The release of gastrin into the serum of five conscious gastric fistula dogs after a meat meal was monitored for 2 hours. Neither the rate of increase in serum gastrin nor the 2 hour cumulative integrated gastrin response was changed by administration of small doses of somatostatin tetradecapeptide (0.5 microgram/kg.hr IV for 2 hr), 16-16 dimethyl prostaglandin E2 (0.25 microgram/kg.hr IV for 2 hr or 1 microgram/kg intragastrically), or bethanechol (20 micrograms/kg.hr IV for 2 hr). Acidification of the food in the antrum to pH 1.2 to 1.4 eliminated serum gastrin release in response to food. In control studies, serum gastrin levels were not altered by IV administration of saline for 2 hr with no food or when a plate of food was held just out of the dogs' reach (teasing). Food-stimulated gastrin release was contrasted with that stimulated by bombesin under identical laboratory conditions [17]. In each case, antral acidification, somatostatin, prostaglandin E2 and bethanechol affected bombesin-stimulated gastrin release differently from that stimulated by food. We conclude that food and bombesin release gastrin by different pathways.     

Role of protein kinase C and phosphorylation in bombesin-evoked gastrin release from isolated perfused rat stomach

We have recently reported that bombesin (BBS)-stimulated gastrin release is principally dependent on a Ca2+/calmodulin intracellular pathway, and that it is independent of the cyclic AMP-mediated pathway. Recently it was demonstrated that stimulation of protein kinase C (PK-C) resulted in increased gastrin release from the isolated canine G-cells in cultures. The role of PK-C in the BBS-evoked gastrin release, however, remains unexamined. In this study we examined a possible role of PK-C in the secretion of BBS-stimulated gastrin from isolated perfused rat stomach. The effect of phosphorylation on gastrin release, in response to BBS, was also determined. Administration of phorbol ester (PMA 10-100 nM, a PK-C activator) alone significantly provoked gastrin release, but markedly inhibited the BBS (1 nM) stimulated gastrin secretion in a dose-dependent manner. Molybdic acid (phosphatase inhibitor), caused an enhancement of BBS-evoked gastrin response at doses of 5 or greater than 5 mM. These results suggest that: (1) diacylglycerol/PK-C pathway may exert a negative feedback control over BBS-induced gastrin release; (2) phosphorylation step is required for gastrin secretion in response to BBS.     

Evidence that bombesin releases extragastric gastrin in man

Bombesin-induced gastrin release from extragastric sources has been investigated in two groups of patients without gastric antrum: 11 patients with total gastrectomy and 11 patients with subtotal (Billroth II) gastrectomy. A 30-min bombesin infusion (5 ng . kg-1 . min-1) caused a prompt significant gastrin increase (P less than 0.05) in both groups of patients. The gastrin response to bombesin was significantly (P less than 0.005) lower in patients without antral tissue than in the control group (n = 7). The individual peak gastrin responses, in totally (TG) and subtotally (SG) gastrectomized patients, were significantly over basal levels (TG: peak 100.3 +/- 12 vs. basal 62.8 +/- 9.1, P less than 0.005; SG: peak 96.9 +/- 9.4 vs. basal 72.4 +/- 6.8, P less than 0.001; pg/ml, mean +/- S.E.M.). These data indicate that bombesin acts not only on antral G cells, but on all gastrin cells in the gastrointestinal tract.