Investigation of bombesin peptide as a targeting ligand for the gastrin releasing peptide (GRP) receptor

Gastrin releasing peptide (GRP) receptor (GRPR), a bombesin family receptor, is overexpressed in many cancers including breast, prostate, pancreatic and lung. The targeting of therapeutics to GRPR can be achieved using the full-length (14 amino acid) GRP analogue Bombesin (BBN) or the truncated BBN(6–14) sequence, both of which bind GRPR with high affinity and specificity. In this study, we have investigated the level of GRPR expression in various cancerous (Caco-2, HeLa, LNCap, MDA-MB-231, and PC-3) and non-cancerous (WPMY-1) cell lines using a western blotting approach. Such information is currently lacking in the literature, and is therefore of importance for the in vitro assessment of GRPR targeted therapeutics. Of the cell lines assessed, the PC-3 (prostate cancer) and Caco-2 (colon cancer) cell lines demonstrated the highest and lowest levels of GRPR expression respectively. Using this information, we further investigated the cellular uptake of carboxyfluorescein-labelled BBN and BBN(6–14) peptides by flow cytometry and confocal microscopy using cell lines that express GRPR (Caco-2, HeLa, PC-3). The uptake of each of these peptides was similar, suggesting that the shorter BBN(6–14) peptide is sufficient for GRPR targeting. Further, the uptake of these peptides could be inhibited by competition with unlabelled BBN peptides, suggesting their cellular uptake is GRPR-mediated, while the level of BBN uptake (as measured by flow cytometry) was found to be directly proportional to the level of GRPR expression. Overall, the information obtained from these studies provides useful information for the in vitro assessment of GRPR targeted therapeutics.     

Effects of porcine gastrin releasing peptide (GRP) on canine antral motility and gastrin release in vivo

The effect of GRP on the vivo canine antrum was investigated. GRP caused a dose-dependent increase in antral gastrin output which was not significantly altered by administration of tetrodotoxin. The higher doses of GRP administered also caused excitation of antral motility which was abolished by tetrodotoxin, a finding in contrast to previous in vitro results demonstrating bombesin-induced antral smooth muscle contraction to be tetrodotoxin-resistant. These data suggest that in the vivo canine model GRP causes antral gastrin release via non-reurally mediated mechanisms (probably by acting directly on the G-cell) and excites antral motility via neurally-mediated mechanisms.     

Enzyme immunoassay of gastrin releasing peptide (GRP)-like immunoreactivity in milk

A sensitive and specific enzyme immunoassay (EIA) for gastrin releasing peptide (GRP)-like immunoreactivity was developed using enzyme-labeled antigen. The synthetic carboxy-terminal fragment of human GRP(12-27) was conjugated with beta-D-galactosidase for EIA. The minimum amount of GRP-like immunoreactivity detectable by this method was 0.24 femtomol/well (6 picomol/liter). The level of GRP-like immunoreactive substance in bovine foremilk was about 150 nanomol/liter, the level of which was more than hundredfold higher than that in normal milk or calf serum.     

Inhibition of prolactin secretion by gastrin releasing peptide (GRP) in the rat

Synthetic gastrin releasing peptide (GRP) injected intraventricularly (1 microgram/rat), but not intravenously, suppressed rat prolactin (PRL) release induced by a Met-enkephalin analog, FK33-824 (10 micrograms/100 g body wt., iv). GRP also blunted PRL release induced by a dopamine antagonist, domperidone (1 microgram/100 g body wt., iv). In contrast, GRP did not suppress elevated plasma PRL levels sustained by a large dose of domperidone (10 micrograms/100 g body wt., iv). GRP (10(-5) M) had no effect on PRL release from superfused pituitary cells in vitro. These results suggest that GRP inhibits PRL secretion in the rat by acting through the brain to stimulate the dopaminergic mechanism.     

Aging and gender affect the response of thyrotropin (TSH) to gastrin releasing peptide (GRP) in rats

We had previously shown that GRP acts directly at the pituitary gland inhibiting basal and TRH-stimulated TSH secretion in adult male rats. In this study we showed a gender dimorphism in this response of old animals pituitaries to GRP. In both female and male young adult animals, GRP-incubated pituitaries showed approximately 50% less basal and TRH-stimulated TSH secretion to the medium, without affecting the pituitary content of TSH. However, GRP did not have any significant effect upon TSH secretion in old male rats, but the old female showed the same degree of response to GRP as the young adult female rat, regarding basal and TRH-stimulated TSH secretion, while the TSH pituitary content after GRP incubation was higher than that of the young female group. Our data suggest a loss of thyrotrope responsiveness to GRP in aged male rats that could contribute to the decrease in TSH pituitary stores leading to lower basal and TRH-stimulated TSH secretion. Meanwhile, the preservation of GRP responsiveness could help in the relative maintenance of these parameters in the old female rat.     

Characterization of the murine pancreatic receptor for gastrin releasing peptide and bombesin.

The murine pancreatic receptor for bombesin and gastrin releasing peptide (GRP) has been characterized. Analysis of the binding of 125I-GRP to membranes indicates a single class of sites (10(-13) mol/mg protein) with Kd of 43 pM. A 70 kDa membrane protein was cross-linked to 125I-GRP by bis(sulfosuccinimidyl) suberate; labeling was blocked by GRP, GRP (14-27), AcGRP(20-27), GRP(18-27), bombesin and ranatensin, was partially blocked by [Leu13 psi (CH2NH)Leu14]bombesin and was unaffected by GRP(21-27) and GRP(1-16). The IC50 values for the competitive displacement of 125I-GRP from intact membranes by these peptides were similar to those obtained by the cross-linking experiments showing that the 70 kDa protein is the GRP receptor. The GRP receptor is G-protein coupled; divalent cations are required for high-affinity binding and nonhydrolyzable GTP analogs decrease receptor affinity. In minced pancreas, GRP caused a dose-dependent increase in inositol phosphates implicating phospholipase C in signal transduction. We suggest that the murine pancreatic receptor for bombesin/GRP is a 70 kDa membrane protein, is associated with a G-protein and stimulates phosphatidylinositol turnover.     

Dose-response comparisons of canine plasma gastroenteropancreatic hormone responses to bombesin and the porcine gastrin-releasing peptide (GRP)

This study compares the potencies of the porcine gastrin-releasing peptide (pGRP) and bombesin, in causing elevations of canine plasma gastroenteropancreatic (GEP) levels. In the dose range 0-600 pmol . kg-1 . h-1, infusion of both peptides resulted in obvious dose-related elevations of plasma levels of gastrin, pancreatic polypeptide, enteroglucagon, immunoreactive pancreatic glucagon, and insulin. In this dose range, no significant difference in potency between the two peptides in elevating plasma levels of the above hormones was observed. The results of this study, demonstrating equimolar potency of pGRP and bombesin, are in contrast to previous studies reporting that pGRP was less potent than bombesin in causing certain bioactivities in the rat following intracranial administration of the two peptides.     

Dual effect of bombesin and gastrin releasing peptide on gastric emptying in conscious cats

The effect of bombesin (BBS) and gastrin releasing peptide (GRP) on gastric emptying was studied in conscious cats. This effect was measured simultaneously with antral motility. Acid and pepsin secretions as well as blood hormonal peptide release were additionally measured. A dual effect was observed. First, BBS and GRP slowed gastric emptying of liquids, while antral motility was decreased, then after 60 minutes of continuous intravenous infusion, antral motility returned to basal values and gastric emptying effect reversed. The mechanism of this peculiar action is independent of gastrin, pancreatic polypeptide, somatostatin and motilin release and most probably connected with a cholinergic stimulation induced by the peptides, the late predominance of which counterbalances the inhibitory effect of bombesin-like peptides on antral motility.     

A qualitative comparison of canine plasma gastroenteropancreatic hormone responses to bombesin and the porcine gastrin-releasing peptide (GRP)

The effect on plasma gastroenteropancreatic hormone levels on infusing the porcine gastrin-releasing peptide and bombesin into dogs demonstrated no qualitative difference in the spectrum of activity of the two peptides. Sustained elevations in plasma immunoreactive gastrin, pancreatic polypeptide, enteroglucagon, gastric inhibitory polypeptide, pancreatic glucagon and transient elevations in plasma insulin were seen during infusions of both peptides. The similar spectrum of activities and the structural homology between the two peptides suggests that the porcine gastrin releasing peptide is the porcine counterpart of the amphibian peptide bombesin.     

N-isobutyryl-His-Trp-Ala-Val-D-Ala-His-Leu-NHMe (ICI 216140) a potent in vivo antaconist analogue of bombesin/gastrin releasing peptide (BN/GRP) derived from the C-terminal sequence lacking the final methionine residue

The GRP receptor mediated growth response in Swiss 3T3 cells has been used to identify BN/GRP antagonists. Analysis of bombesin antagonism by substance P analogues and by truncated GRP analogues revealed that deletion of the C-terminal methionine residue was important for antagonism. Des-Met analogues showing potent antagonist activity in the in vitro 3T3 system (IC50 approximately 2nM) were synthesized. Further structural modification of these peptides led to the identification of (CH3)2CHCO-His-Trp-Ala-Val-D-Ala-His-Leu-NHCH3 (ICI 216140) which reduced bombesin-stimulated rat pancreatic amylase secretion to basal levels when administered subcutaneously at 2.0 mg per kg.     

Gastrin-releasing peptide (GRP,mammalian bombesin) in the pathogenesis of lung cancer

Established human lung cancer exhibits a complex pattern of genetic changes as well as several distinct autocrine growth factor loops for regulatory peptides. The best studied example is that of gastrin-releasing peptide (GRP), the mammalian homolog of the amphibian bombesin. It is produced by up to 70% of small cell lung cancers and 10–20% of non-small cell lung cancers. GRP stimulates the growth of normal bronchial epithelium as well as that of small cell lung cancer, and its blockade with the use of antibodies or synthetic antagonists inhibits the growth of these tumors. Study of its molecular biology has revealed a complex pattern of mRNA processing which has lead to the recent isolation of a novel family of peptides termed gastrin-releasing peptide gene-associated peptides (GGAPs), present in normal and malignant human tissues. Additional efforts have been directed at characterizing the GRP receptor as well as its intracellular signaling pathways which have been reported both as G protein phospholipase C coupled events as well as activation of a membrane associated tyrosine kinase. In view of its expression in normal bronchial epithelium and its mitogenic effects on this tissue, GRP appears to play a central role in the early events of pulmonary carcinogenesis.     

The effect of atropine on bombesin and gastrin releasing peptide stimulated gastrin, pancreatic polypeptide and neurotensin release in man

The effects of 1-h infusions of bombesin and gastrin releasing peptide (GRP) at 50 pmol/kg per h and neurotensin at 100 pmol/kg per h on gastrin, pancreatic polypeptide (PP) and neurotensin release in man were determined following either saline or atropine infusion (20 micrograms/kg). Bombesin produced a rise in plasma neurotensin from 32 +/- 6 to 61 +/- 19 pmol/l and of PP from 26 +/- 8 to 36 +/- 7 pmol/l. There was a further rise of plasma PP to 50 +/- 13 pmol/l after cessation of the infusion. GRP had no significant effect on plasma neurotensin, but compared to bombesin, produced a significantly greater rise in plasma PP from 34 +/- 6 to 66 +/- 19 pmol/l during infusion. There was no post-infusional increase. At this dose, GRP was as effective as bombesin in releasing gastrin, although unlike bombesin its effect was enhanced by atropine. Neurotensin produced a rise in plasma PP from 17 +/- 4 to 38 +/- 8 pmol/l. Atropine blocked the release of PP during GRP and neurotensin infusion. Atropine had no effect on neurotensin or PP release during bombesin infusion, but did block the rise in plasma PP following bombesin infusion. We conclude that, in contrast to meal-stimulated neurotensin release, bombesin-stimulated neurotensin release is cholinergic independent. Despite structural homology, bombesin and GRP at the dose used are dissimilar in man in their actions and sensitivity to cholinergic blockade.     

Sequence analysis and feeding responses evoked by the large molecular form of gastrin releasing peptide (GRP) in the rat GRP-29

Microisolation techniques utilizing several reverse phase high performance liquid chromatography (HPLC) steps have resulted in the purification of two rat gastrin releasing peptide (GRP) forms suitable for microsequence and mass spectral analysis. The sequence of the larger form is APVSTGAGGGTVLAKMYPRGSHWAVGHLM-amide and the smaller form is GSHWAVGHLM-amide which is the carboxyl terminal decapeptide of the larger peptide. The peptides were synthesized and their feeding patterns e.g. first meal size (MS), intermeal interval (IMI) and satiety ratio (SR, IMI/MS) were determined in overnight food-, but not water deprived, male Sprague Dawley rats. The peptides were administered in the femoral vein (0, 0.21, 0.41 and 1.03 nmol/kg) immediately before presenting the rats with a 10% sucrose solution. We found that (1) GRP-10 (all doses) and GRP-29 (0.41 nmol/kg) reduced first MS, (2) both peptides prolonged IMI length and (3) both peptides increased the SR to similar extents. In conclusion, GRP-10 and GRP-29 are the two endogenous forms of GRP in the rat intestine and they reduce short term feeding to similar extents when administered intravenously.     

Quantification of the bombesin/gastrin releasing peptide antagonist RC-3095 by liquid chromatography-tandem mass spectrometry

Bombesin (BN) and its mammalian equivalent, gastrin-releasing peptide (GRP), stimulate cell proliferation and are involved in the pathogenesis of several types of human cancer. BN/GRP and their receptors were shown to be critical for the growth of various human malignancies, such as small-cell lung, prostate, ovary, stomach and breast cancers in the human tumor xenograft model. In the present study, a fast, sensitive, robust method was developed for the determination and quantification of a BN/GRP receptor antagonist RC-3095 (D-Tpi-Gln-Trp-Ala-Val-Gly-His-Leupsi(CH2NH)Leu-NH2), in human plasma by liquid chromatography coupled with tandem mass spectrometry. RC-3095 was extracted from 0.2 ml human plasma by protein precipitation using cold acetonitrile (0.4 ml). The method has a chromatographic run of 10 min using a C(8) analytical column (150 mm x 4.6 mm i.d.) and the linear calibration curve over the range was linear from 20 to 10000 ng ml(-1) (r(2)>0.994). The between-run precision, based on the relative standard deviation replicate quality controls, was 5.7% (60 ng ml(-1)), 7.1% (600 ng ml(-1)) and 6.8% (8000 ng ml(-1)). The between-run accuracy was +/-0.0, 2.1 and 3.1% for the above-mentioned concentrations, respectively. The developed procedure allows the quantitative determination of peptide RC-3095 for pharmacokinetics studies in human plasma.     

Effects of bombesin and gastrin releasing peptide on catecholamine secretion from rat adrenal gland, in vitro

The effects of bombesin and gastrin releasing peptide (GRP) on the release of catecholamine were investigated by using isolated rat adrenal gland. Bombesin and GRP stimulated an epinephrine (E) release with dose-dependency. A half maximal effect of bombesin was observed at 1.2 X 10(-9) M, and a maximal release of E occurred at 1 X 10(-6) M of bombesin. The stimulatory effect of GRP on the E release was very similar to that of bombesin. Although both these peptides also stimulated a norepinephrine (NE) release, a significant effect was detected at concentrations of bombesin and GRP above 1 X 10(-7) M. Nicotine and pilocarpine stimulated both E and NE releases dose dependently, but the effect of pilocarpine on E and NE release was 1/100 or less potent than that of nicotine. Bombesin-induced catecholamine releases were not inhibited by hexamethonium or atropine that fully impeded the stimulatory effects of nicotine or pilocarpine. In addition, bombesin had additive effects on the nicotine- or pilocarpine-induced E and NE releases. These data strongly suggest that bombesin or GRP plays a physiological role as one of the important regulators in catecholamine secretion in the adrenal gland.     

Gastrin releasing peptide (GRP): effects on basal and stimulated insulin and glucagon secretion in the mouse

GRP is a pancreatic neuropeptide and may be of importance for the neural control of insulin and glucagon secretion. In this study, we investigated the effects of GRP on basal and stimulated insulin and glucagon secretion in the mouse. Intravenous injections of GRP at dose levels exceeding 2.12 nmol/kg were found to rapidly increase basal plasma levels of both insulin and glucagon. Furthermore, at a low dose level without effect on basal plasma insulin levels, GRP was found to potentiate the insulin response to both glucose (by 40%; p less than 0.05) and to the cholinergic agonist carbachol (by 57%; p less than 0.01). Also, GRP was at this dose level found to potentiate the glucagon response to carbachol (p less than 0.01). Glucose abolished GRP-induced glucagon secretion. Moreover, methylatropine given at a dose level that totally abolishes carbachol-induced insulin secretion inhibited GRP-induced insulin secretion by 39% (p less than 0.05) and GRP-induced glucagon secretion by 25% (p less than 0.01). L-Propranolol at a dose level that totally abolishes beta-adrenergically-induced insulin secretion inhibited GRP-induced insulin secretion by 52% (p less than 0.01) and GRP-induced glucagon secretion by 15% (p less than 0.05). In summary, we have shown that GRP stimulates basal and potentiates stimulated insulin and glucagon secretion in mice, and that the stimulatory effects of GRP on insulin and glucagon secretion are partially inhibited by muscarinic blockade by methylatropine or by beta-adrenoceptor blockade by propranolol. We conclude that GRP activates potently both insulin and glucagon secretion in the mouse by mechanisms that are partially related to the muscarinic and the beta-adrenergic receptors.     

Gastrin-releasing peptide (GRP) is not mammalian bombesin. Identification and molecular cloning of a true amphibian GRP distinct from amphibian bombesin in Bombina orientalis.

On the basis of structural homology and similar biological activity, gastrin-releasing peptide (GRP) has been considered the mammalian equivalent of amphibian bombesin. In this paper we now show this to be incorrect. Chromatography of frog (Bombina orientalis) gut extracts demonstrated two peaks of bombesin-like immunoreactivity (BLI), one similar in size to GRP and one similar in size to amphibian bombesin. These peaks were purified by high pressure liquid chromatography then subjected to mass spectrometric analyses to determine molecular weights and amino acid sequence. Based on the amino acid sequence of the lower molecular weight BLI species, a mixed oligonucleotide probe was prepared and used to screen a B. orientalis stomach cDNA library. Sequence analysis showed that all hybridizing clones encoded a 155-amino acid protein homologous to the mammalian GRP precursor. The mass spectra of the high and low molecular weight peaks of frog gut BLI were consistent with their origin from the processing of the frog GRP (fGRP) precursor into GRP-29 and GRP-10, just like the processing of the rat GRP precursor. Sequence homology showed that the fGRP precursor is more homology showed that the fGRP precursor is more closely related to the mammalian GRP precursors than to either the frog bombesin or frog ranatensin precursors. Northern blot analysis showed that fGRP is encoded by a mRNA of 980 bases, clearly different from the 750-base mRNA which encodes frog bombesin. Northern blot analysis and in situ hybridization showed fGRP mRNA in frog brain and stomach and bombesin mRNA in frog skin, brain, and stomach. That frogs have independent genes for both GRP and bombesin raises the possibility that mammals have an as yet uncharacterized gene encoding a true mammalian bombesin.     

Galanin: an inhibitory neural peptide of the canine small intestine

Galanin injected intraarterially during phasic activity of the canine small intestine in vivo produced inhibition. Fifty percent inhibition occurred at 1.5 +/- 0.5 X 10(-10) mols lasting for 0.7 min. The inhibitory response was not decreased by treatment with atropine, hexamethonium, yohimbine or naloxone, suggesting that muscarinic, nicotinic, alpha 2 adrenergic or opiate receptors were not being stimulated. Since tetrodotoxin blockade of nerves did not reduce the response and galanin at 10(-10) mols was able to eliminate the smooth muscle response to intraarterial acetylcholine, we suggest that galanin acts to inhibit smooth muscle directly. Galanin 10(-9) M added to the muscle bath also inhibited phasic activity of the canine ileum circular muscle in vitro in the presence of tetrodotoxin. These results suggest that the neural peptide galanin may be a non-adrenergic, non-cholinergic, non-opioid neurotransmitter in the canine small intestine.     

Conformational analysis in solution of gastrin releasing peptide

Gastrin releasing peptide (GRP) is the first peptide isolated from porcine gastric and intestinal tissues and is homologous to the carboxyl terminus of bombesin (Bn) isolated from the skin of the frog Bombina bombina. It is a member of the Bn-like peptides, which are important in numerous biological and pathological processes. The Bn-like peptides show high sequence homology in their C-terminal regions, but they have different selectivity for their receptors. In particular, GRP selectively binds to the GRP receptor (GRPR). However, the molecular basis for this selectivity remains largely unknown. Here, we report the three-dimensional structure of GRP. Hopefully, it could be helpful in a better understanding of the binding selectivity between GRP and GRPR.     

Gastrin releasing peptide (GRP) is present in a GRP(1-27) form in anterior pituitary cells of the guinea pig

Immunohistochemical and chromatographic studies were performed on the guinea pig anterior pituitary gland with an antiserum recognizing an epitope within the gastrin releasing peptide (GRP) carboxyterminal amino acid sequence Val-Gly-His-Leu-Met-NH2. Within the anterior pituitary gland GRP-like immunoreactive cells were identified. The GRP-like immunoreactive cells were distributed heterogenously in the gland, predominantly located in ventral aspects of the anterior pituitary. Intracellularly, the immunoreactivity elements were identified as granula-like structures in the cytoplasma. To further characterize the peptide displaying GRP-like immunoreactivity within the pituitary cells, the GRP-like substances were analyzed by radioimmunoassay and gel filtration chromatography. Using this analytical approach it was determined that the guinea pig pituitary extract contained a peptide with characteristics similar to that of authentic porcine GRP(1-27). Only trace amounts of smaller C-terminal fragments were identified. These results indicate, in contrast to findings in other tissues, the GRP(1-27) is not further degraded into smaller peptide fragments.