Src-family tyrosine kinases in activation of ERK-1 and p85/p110-phosphatidylinositol 3-kinase by G/CCKB receptors.

We have analyzed in Chinese hamster ovary cells the upstream mediators by which the G protein-coupled receptor, gastrin/CCKB, activates the extracellular-regulated kinases (ERKs) and p85/p110-phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Overexpression of an inhibitory mutant of Shc completely blocked gastrin-stimulated Shc.Grb2 complex formation but partially inhibited ERK-1 activation by this peptide. Expression of Csk, which inactivates Src-family kinases, totally inhibited gastrin-induced Src-like activity detected in anti-Src and anti-Shc precipitates but diminished by 50% Shc phosphorylation and ERK-1 activation. We observed a rapid tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and an increase in Src-like kinase activity in anti-IRS-1 immunoprecipitates from gastrin-stimulated cells, suggesting that IRS-1 may be a direct substrate of Src. This hypothesis was supported by the inhibition of gastrin-induced Src. IRS-1 complex formation and IRS-1 phosphorylation in Csk-transfected cells. In addition, the increase in PI 3-kinase activity measured in anti-p85 or anti-IRS-1 precipitates following gastrin stimulation was abolished by Csk. Our results demonstrate the existence of two mechanisms in gastrin-mediated ERKs activation. One requires Shc phosphorylation by Src-family kinases, and the other one is independent of these two proteins. They also indicate that tyrosine phosphorylation of IRS-1 by Src-family kinases could lead to the recruitment and the activation of the p85/p110-PI 3-kinase in response to gastrin.     

Autoregulation of enterochromaffin-like cell histamine secretion via the histamine 3 receptor subtype.

INTRODUCTION: The neuroendocrine histamine-secreting cell of the gastric fundus, the enferochromaffin-like cell, is the principal regulator of parietal cell acid secretion. We have proposed that histamine may regulate its own synthesis and release via an autocrine mechanism. The purpose of this study was to evaluate the role of the histamine receptor subtypes H1, H2 and H3 in the regulation of this phenomenon. METHODS: Purified ECL cells were isolated by pronase digestion and EDTA exposure of the rat stomach, followed by particle size and density separation using counterflow elutriation and Nycodenz gradient centrifugation, 24-hr cultured cells were pretreated for 30 min with the agents; H1 receptor agonist (2-[(3-trimethyl)-diphenyl] histamine) (TMPH), H1 receptor antagonist (terfenadine); H2 receptor agonist (dimaprit) or antagonist (cimetidine or loxitidine); or H3 receptor agonist (imetit) or antagonist (thioperamide) (all tested, 10(-10)-10(-6) M). Gastrin was then used to stimulate histamine secretion. Histamine secretion was quantified by specific enzyme-immunoassay. RESULTS: Basal histamine secretion was 2.7 +/- 0.14 nmol/10(3) cells. Gastrin-stimulated (10 nM) levels were 4.6 +/- 0.4 nmol/10(3) cells (p < .01). TMPH inhibited both basal and gastrin driven histamine secretion with a maximal effect (34 percent) (1.78 +/- 0.08 nmol/10(3) cells) and an IC50 of > 5 x 10(-7) M. H1 receptor antagonism did not alter histamine secretion alone or in combination with gastrin. Neither H2 receptor stimulation nor antagonism had any effect on histamine secretion alone or in combination with gastrin. Gastrin-induced histamine secretion was dose-dependently inhibited by imetit (H3 agonist) with a maximal effect (2.4 +/- 0.6 nmol/10(3) cells) (p < .05) and an IC50 of 10(-9) M. Conversely, Thioperamide (H3 antagonist) dose-dependently augmented gastrin-stimulated histamine secretion with a maximum effect (5.7 +/- 0.5 nmol/10(3) cells) (p < .05) at 10(-8) M and an EC50 of 7 x 10(-10) M. CONCLUSION: These data are consistent with the presence of an H3 receptor on the ECL cell which modulates gastrin-stimulated histamine secretion. Our observations support the proposal that a histamine-mediated short-loop autocrine regulatory mechanism of ECL cell secretion exists.     

A novel effect of bismuth ions: selective inhibition of the biological activity of glycine-extended gastrin

Although bismuth salts have been used for over two centuries for the treatment of various gastrointestinal disorders, the mechanism of their therapeutic action remains controversial. Because gastrins bind two trivalent ferric ions with high affinity, and because ferric ions are essential for the biological activity of glycine-extended gastrin 17, we have investigated the hypothesis that trivalent bismuth ions influence the biological activity of gastrins. Binding of bismuth ions to gastrins was measured by fluorescence quenching and NMR spectroscopy. The effects of bismuth ions on gastrin-stimulated biological activities were measured in inositol phosphate, cell proliferation, and cell migration assays. Fluorescence quenching experiments indicated that both glycine-extended and amidated gastrin 17 bound two bismuth ions. The NMR spectral changes observed on addition of bismuth ions revealed that Glu-7 acted as a ligand at the first bismuth ion binding site. In the presence of bismuth ions the ability of glycine-extended gastrin 17 to stimulate inositol phosphate production, cell proliferation, and cell migration was markedly reduced. In contrast, bismuth ions had little effect on the affinity of the CCK-2 receptor for amidated gastrin 17, or on the stimulation of inositol phosphate production by amidated gastrin 17. We conclude that bismuth ions may act, at least in part, by blocking the effects of glycine-extended gastrin 17 on cell proliferation and cell migration in the gastrointestinal tract. This is the first report of a specific inhibitory effect of bismuth ions on the action of a gastrointestinal hormone.     

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.     

Divergent proliferative responses to a gastrin receptor ligand in synchronized and unsynchronized rat pancreatic AR42J tumour cells

Depending upon experimental model, the CCK-B/gastrin receptor ligand CI-988 exhibits either agonist or antagonist activity. To confirm that CI-988 behaves as an antagonist toward gastrin-stimulated growth, its effects on cell proliferation were investigated in unsynchronized and synchronized AR42J rat pancreatic tumour cells. In unsynchronized cultures CI-988 alone had no effect, but inhibited gastrin-stimulated cell proliferation. In contrast, in synchronized cultures, CI-988 stimulated cell proliferation. Similarly, CI-988 inhibited gastrin-stimulated cAMP production in unsynchronized cells, but stimulated cAMP formation in synchronized cultures. Therefore, CI-988 stimulation of cAMP production and proliferation in AR42J cell cultures appears to be cell cycle-dependent. CI-988 inhibited gastrin-stimulated intracellular calcium ([Ca2+]i) mobilization in both populations and thus acted as an antagonist toward this pathway. Because CCK receptor densities and affinities were similar in both cell populations, the data suggest that CI-988's divergent effects on cell proliferation are governed by postreceptor signalling events which vary with cell cycle.     

Signaling pathways mediating gastrin's growth-promoting effects.

In addition to its fundamental role in stimulating gastric acid secretion, the peptide hormone gastrin induces growth-promoting effects on diversity of target cells. Various mechanisms, including endocrine, paracrine, and autocrine, have been proposed for gastrin's growth-promoting actions. The mitogenic effects of gastrin are mediated by specific cell surface receptors activated after gastrin binding. The functionally defined receptors for gastrin include cholecystokinin A (CCKA) receptor, which is discriminating for sulfated CCK8; cholecystokinin B (CCKB)/gastrin receptor, which binds gastrin17 sulfated, and nonsulfated CCK8 with nearly equal affinities; cholecystokinin C (CCKC), which is a low-affinity gastrin binding protein; and novel, high-affinity receptors selective for amidated gastrin, processing intermediates of gastrin, or both. The signaling pathways mediating gastrin's stimulation of the CCKB/gastrin receptor have been progressively outlined, and the pathways mediating other receptors have been slowly emerging. Engagement of the gastrin receptor initiates various biochemical and molecular events, including recruitment and activation of tyrosine kinases, activation of the phospholipase C signaling pathway leading to phosphoinositide breakdown, intracellular calcium mobilization and protein kinase C stimulation, activation of the mitogen-activated protein kinase pathway, and induction of early response genes. Current emphasis is on understanding the functional significance of processing intermediate forms of gastrin, and the receptor subtypes and pathways that promote the trophic/mitogenic effects of the different molecular forms of gastrin.     

Glycine-extended gastrin exerts growth-promoting effects on human colon cancer cells.

BACKGROUND: Since human colon cancers often contain significant quantities of progastrin-processing intermediates, we sought to explore the possibility that the biosynthetic precursor of fully processed amidated gastrin, glycine-extended gastrin, may exert trophic effects on human colonic cancer cells. MATERIALS AND METHODS: Binding of radiolabeled glycine-extended and amidated gastrins was assessed on five human cancer cell lines: LoVo, HT 29, HCT 116, Colo 320DM, and T 84. Trophic actions of the peptides were assessed by increases in [3H]thymidine incorporation and cell number. Gastrin expression was determined by northern blot and radioimmunoassay. RESULTS: Amidated gastrin did not bind to or stimulate the growth of any of the five cell lines. In contrast, saturable binding of radiolabeled glycine-extended gastrin was seen on LoVo and HT 29 cells that was not inhibited by amidated gastrin (10(-6) M) nor by a gastrin/CCKB receptor antagonist (PD 134308). Glycine-extended gastrin induced a dose-dependent increase in [3H]thymidine uptake in LoVo (143 +/- 8% versus control at 10(-10) M) and HT 29 (151 +/- 11% versus control at 10(-10) M) cells that was not inhibited by PD 134308 or by a mitogen-activated protein (MAP) or ERK kinase (MEK) inhibitor (PD 98509). Glycine-extended gastrin did stimulate jun-kinase activity in LoVo and HT 29 cells. The two cell lines expressed the gastrin gene at low levels and secreted small amounts of amidated gastrin and glycine-extended gastrin into the media. CONCLUSIONS: Glycine-extended gastrin receptors are present on human colon cancer cells that mediate glycine-extended gastrin's trophic effects via a MEK-independent mechanism. This suggests that glycine-extended gastrin and its novel receptors may play a role in colon cancer cell growth.     

Calcium influx: an intracellular message of the mitogenic action of insulin-like growth factor-I

When G0-arrested BALB/c 3T3 cells were treated sequentially with platelet-derived growth factor and epidermal growth factor, cells became responsive to insulin-like growth factor-I (IGF-I). In these primed competent cells, 1 nM IGF-I elicited an approximately 3-fold increase in the calcium influx rate. IGF-I-induced calcium influx was relatively slow in onset and continued for at least 2 h in the presence of IGF-I. When a single Ca2+ channel current was studied by the patch-clamp technique using the cell-attached mode, inward currents with unitary conductance of 19 pS were observed in the presence of 1 nM IGF-I in the patch pipette. IGF-I-sensitive inward current was independent of membrane potential and was activated by a high concentration of insulin. Accordingly, 1 nM IGF-I caused a gradual increase in cytoplasmic free calcium concentration measured by fura2. The action of IGF-I on calcium influx was dependent on extracellular calcium, and IGF-I did not stimulate calcium influx when extracellular calcium concentration was reduced to 10 microM. Both cobalt and tetramethrin blocked the action of IGF-I on calcium influx without affecting the binding of 125I-IGF-I. In primed competent cells, IGF-I-stimulated [3H]thymidine incorporation was dependent on extracellular calcium and was attenuated by cobalt and tetramethrin. When cell-bound 125I-IGF-I was cross-linked by use of disuccinimidyl suberate, a 130-kDa protein was radiolabeled. Affinity labeling of the 130-kDa protein, presumably the alpha-subunit of the IGF-I receptor, was blocked by excess amount of unlabeled IGF-I. These results suggest that relatively low concentrations of IGF-I stimulate calcium influx in primed competent BALB/c 3T3 cells by activating a calcium-permeable cation channel via the IGF-I receptor and that calcium influx may be a critical intracellular message of the progression activity of IGF-I.     

Phospholipase C-gamma, a substrate for PDGF receptor kinase, is not phosphorylated on tyrosine during the mitogenic response to CSF-1.

Quiescent mouse NIH3T3 cells expressing a transduced human c-fms gene encoding the receptor for colony stimulating factor-1 (CSF-1) were stimulated with mitogenic concentrations of platelet-derived growth factor (PDGF) or CSF-1. Immunoprecipitated phospholipase C-gamma (PLC-gamma) was phosphorylated on tyrosine and calcium was mobilized following treatment of intact cells with PDGF. In contrast, only trace amounts of phosphotyrosine were incorporated into PLC-gamma and no intracellular calcium signal was detected after CSF-1 stimulation. Similarly, CSF-1 treatment did not stimulate phosphorylation of PLC-gamma on tyrosine in a CSF-1-dependent. SV40-immortalized mouse macrophage cell line that expresses high levels of the CSF-1 receptor. In fibroblasts, antiserum to PLC-gamma co-precipitated a fraction of the tyrosine phosphorylated form of the PDGF receptor (PDGF-R) after ligand stimulation, implying that phosphorylated PDGF-R and PLC-gamma were associated in a stable complex. Pre-treatment of cells with orthovanadate also led to tyrosine phosphorylation of PLC-gamma which was significantly enhanced by PDGF, but not by CSF-1. Thus, although the PDGF and CSF-1 receptors are structurally related and appear to be derived from a single ancestor gene, only PDGF-induced mitogenesis in fibroblasts correlated with tyrosine phosphorylation of PLC-gamma.     

CCKB/gastrin receptor antagonists: recent advances and potential uses in gastric secretory disorders.

Cholecystokinin (CCK) and the structurally related peptide, gastrin, have numerous effects on tissues in the central nervous system and gastrointestinal tract. Recent studies show these effect are mediated by a CCKA and CCKB receptor. Knowledge of the physiological role and role of CCKB receptors in pathologic processes has been particularly limited by the availability of selective, potent receptor antagonists. Recently, new members of five different classes of non-peptide CCKB receptor antagonists are reported and are reviewed briefly. these include compounds isolated from Streptomyces (tetronothiodin, virginiamycin analogues), ureido-acetamide analogues (RP 69758, RP 72540, RP 73870), newer benzodiazepine analogues (L-368,935, L-740,093, YM022), pyrazolidimine analogues (LY 262,691) and glutamic acid analogues (CR2194). Many of these compounds have greater than 1000-fold selectivity for the CCKB over the CCKA receptor and some have greater than 10,000-fold selectivity. The pharmacology and effects of CCKB receptor antagonists on gastric acid secretion is briefly reviewed. Furthermore, the possible clinical usefulness of CCKB receptor antagonists in treating disorders of gastric acid secretion, in inhibiting the trophic effects of gastrin and in other clinical conditions is briefly discussed.     

Characterization of the growth of murine fibroblasts that express human insulin receptors. II. Interaction of insulin with other growth factors

The effects of insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin on DNA synthesis were studied in murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental NIH 3T3 cells. In NIH 3T3/HIR cells, individual growth factors in serum-free medium stimulated DNA synthesis with the following relative efficacies: insulin greater than or equal to 10% fetal calf serum greater than PDGF greater than IGF-1 much greater than EGF. In comparison, the relative efficacies of these factors in stimulating DNA synthesis by NIH 3T3 cells were 10% fetal calf serum greater than PDGF greater than EGF much greater than IGF-1 = insulin. In NIH 3T3/HIR cells, EGF was synergistic with 1-10 ng/ml insulin but not with 100 ng/ml insulin or more. Synergy of PDGF or IGF-1 with insulin was not detected. In the parental NIH 3T3 cells, insulin and IGF-1 were found to be synergistic with EGF (1 ng/ml), PDGF (100 ng/ml), and PDGF plus EGF. In NIH 3T3/HIR cells, the lack of interaction of insulin with other growth factors was also observed when the percentage of cells synthesizing DNA was examined. Despite insulin's inducing only 60% of NIH 3T3/HIR cells to incorporate thymidine, addition of PDGF, EGF, or PDGF plus EGF had no further effect. In contrast, combinations of growth factors resulted in 95% of the parental NIH 3T3 cells synthesizing DNA. The independence of insulin-stimulated DNA synthesis from other mitogens in the NIH 3T3/HIR cells is atypical for progression factor-stimulated DNA synthesis and is thought to be partly the result of insulin receptor expression in an inappropriate context or quantity.     

Effects of the amphiphilic peptides mastoparan and adenoregulin on receptor binding,G proteins,phosphoinositide breakdown,cyclic AMP generation,and calcium influx

Summary 1. The amphiphilic peptide mastoparan is known to affect phosphoinositide breakdown, calcium influx, and exocytosis of hormones and neurotransmitters and to stimulate the GTPase activity of guanine nucleotide-binding regulatory proteins. Another amphiphilic peptide, adenoregulin was recently identified based on stimulation of agonist binding to A₁-adenosine receptors.2. A comparison of the effects of mastoparan and adenoregulin reveals that these peptides share many properties. Both stimulate binding of agonists to receptors and binding of GTPS to G proteins in brain membranes. The enhanced guanyl nucleotide exchange may be responsible for the complete conversion of receptors to a high-affinity state, complexed with guanyl nucleotide-free G proteins.3. Both peptides increase phosphoinositide breakdown in NIH 3T3 fibroblasts. Pertussis toxin partially inhibits the phosphoinositide breakdown elicited by mastoparan but has no effect on the response to adenoregulin.N-Ethylmaleimide inhibits the response to both peptides.4. In permeabilized 3T3 cells, both adenoregulin and mastoparan inhibit GTPS-stimulated phosphoinositide breakdown. Mastoparan slightly increases basal cyclic AMP levels in cultured cells, followed at higher concentrations by an inhibition, while adenoregulin has minimal effects.5. Both peptides increase calcium influx in cultured cells and release of norepinephrine in pheochromocytoma PC12 cells. The calcium influx elicited by the peptides in 3T3 cells is not markedly altered byN-ethylmaleimide.6. Multiple sites of action appear likely to underlie the effects of mastoparan/adenoregulin on receptors, G proteins, phospholipase C, and calcium.     

Calcium signaling mechanisms in the gastric parietal cell.

Gastric hydrochloric acid (HCl) secretion is stimulated in vivo by histamine, acetylcholine, and gastrin. In vitro studies have shown that histamine acts mainly via a cAMP-dependent pathway, and acetylcholine acts via a calcium-dependent pathway. Histamine also elevates intracellular calcium ([Ca2+]i) in parietal cells. Both gastrin and acetylcholine release histamine from histamine-containing cells. In humans, rats, and rabbits, there is considerable controversy as to whether or not gastrin receptors are also present on the parietal cell. We utilized digitized video image analysis techniques in this study to demonstrate gastrin-induced changes in intracellular calcium in single parietal cells from rabbit in primary culture. Gastrin also stimulated a small increase in [14C]-aminopyrine (AP) accumulation, an index of acid secretory responsiveness in cultured parietal cells. In contrast to histamine and the cholinergic agonist, carbachol, stimulation of parietal cells with gastrin led to rapid loss of the calcium signaling response, an event that is presumed to be closely related to gastrin receptor activation. Moreover, different calcium signaling patterns were observed for histamine, carbachol, and gastrin, Previous observations coupled with present studies using manganese, caffeine, and ryanodine suggest that agonist-stimulated increases in calcium influx into parietal cells do not occur via voltage-sensitive calcium channels or nonspecific divalent cation channels. It also appears to be unlikely that release of intracellular calcium is mediated by a muscle or neuronal-type ryanodine receptor. We hypothesize that calcium influx may be mediated by either a calcium exchange mechanism or by an unidentified calcium channel subtype that possesses different molecular characteristics as compared to muscle, nerve, and certain secretory cell types such as, for example, the adrenal chromaffin cell. Release of intracellular calcium may be mediated via both InsP3-sensitive and -insensitive mechanisms. The InsP3-insensitive calcium pools, if present, do not appear, however, to possess ryanodine receptors capable of modulating calcium efflux from these storage sites.     

Expression of PTEN and Akt phosphorylation in lipopolysaccharide-treated NIH3T3 cells

PTEN is a tumor suppressor gene encoding a phosphatase, and it negatively regulates cell survival mediated by the phosphoinositol 3-kinase (PI3-Kinase)-Akt pathway. To elucidate PTEN expression and its effect on the PI3-kinase-Akt pathway in fibroblasts and macrophages, we investigated the expression of PTEN and the phosphorylation status of Akt in NIH3T3 and RAW264.7 cells treated with LPS. Phosphorylation of Akt was induced by LPS treatment in a dose-dependent manner in RAW264.7 cells, but not in NIH3T3 cells. LPS induced the expression of PTEN in a dose and time-dependent manner in NIH3T3 cells (0-1 microg/ml, 0-6h). However, LPS did not stimulate PTEN expression in RAW264.7 cells. These data indicate the existence of diverse mechanisms for PTEN expression and Akt activation in fibroblasts and macrophages. RNA interference using double-stranded RNA specific for the PTEN gene reduced both mRNA and protein levels of PTEN in NIH3T3 cells treated or not with LPS. The phosphorylation status of Akt in NIH3T3 cells stimulated with LPS did not change when the PTEN expression had been inhibited by RNA interference. The present results suggest that the up-regulation of PTEN expression by LPS is not involved in the activation of Akt in NIH3T3 cells. PTEN expression might be involved in the diverse inflammatory responses to LPS in fibroblasts and macrophages.     

Stimulation of canine pancreatic polypeptide, gastrin, and gastric acid secretion by ranatensin, litorin, bombesin nonapeptide and substance P

Four dogs with chronic gastric fistulas were give intravenous bombesin nonapeptide (B9), ranatensin, and litorin by constant infusion for 90 min at 1.2 micrograms x kg-1 on separate days. A dose response study with substance P (1.5, 3.0, 60, 18 and 54 micrograms x kg-1 x h-1) was also carried out and all tests compared to a standard protein meal (10g x kg-1). Plasma gastrin and PP were measured by radioimmunoassay and gastric acid by autobiuret titration. Substance P failed to stimulate gastric acid secretion or release either pancreatic polypeptide (PP) or gastrin. Basal gastrin levels were 8 +/-2 fmol/ml. The peak increment of gastrin released by bombesin was 95 +/- 16, ranatensin 22 +/- 6, litorin 18 +/- 4, and meal 39 +/- 5 fmol/ml. Bombesin caused significantly greater release of gastrin than a meal, litorin or ranatensin (P less than 0.01). Basal gastric secretion was 23 +/- 4 microequiv./min. B9 produced a peak acid secretion of 356 +/- 124 muequiv./min. There was no significant difference between the bombesin-like peptides (P less than 0.01). Basal plasma PP was 38 +/- 12 fmol/ml. B9 produced a peak PP increment of 600 +/- 50, litorin 137 +/- 36, ranatensin 98 +/- 11, and a meal 305 +/- 58 fmol/ml. B9 released significantly more PP than either litorin of ranatensin (P less than 0.01). The different amino acid sequences of the peptides are probably responsible for their potency. The substitution of a penultimate phenylalanine residue in litorin and ranatensin for leucine in bombesin does not prevent PP or gastrin release by bombesin-like peptides. Since bombesin-like peptides are widely distributed in the gastrointestinal tract of man and stimulate both acid and gut hormone secretion, it is possible that they might play a physiological role in the modulation of gastrointestinal function.     

Cell cycle-related differences in susceptibility of NIH/3T3 cells to ribonucleases

Microinjection of Onconase or RNase A into NIH/3T3 cells was used to study the intracellular actions of these two proteins. Onconase preferentially killed actively growing cells in both microinjection and cell culture experiments. Moreover, agents that increased the number of cells in S phase such as serum or microinjected signal transduction mediators (Ras, protein kinase C, and mitogen-activated protein kinase) enhanced Onconase cytotoxicity. Conversely, agents that decreased these proliferative pathways (dibutyryl cAMP and protein kinase A) correspondingly diminished Onconase cytotoxicity in microinjection experiments. These results were also mimicked in cell culture experiments since log-phase v-ras-transformed NIH/3T3 cells were more sensitive to Onconase (IC50 of 7 microg/ml) than parental NIH/3T3 fibroblasts (IC50 of 40 microg/ml). Based on those data we postulated that Onconase-mediated cell death in NIH/3T3 cells was related to events occurring at two or more points in the cell cycle preferentially associated with late G1/S and S phases. In contrast, quiescent NIH/3T3 cells were more sensitive to microinjected RNase A than log phase cells and positive mediators of proliferative signal transduction did not enhance RNase A-mediated cytotoxicity. Taken together, these results demonstrate that these two RNases use different pathways and/or mechanisms to elicit cytotoxic responses in NIH/3T3 cells. Predictions formulated from these studies can be tested for relevance to RNase actions in different target tumor cells.