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.     

Des-Met14]bombesin analogues function as small cell lung cancer bombesin receptor antagonists.

A series of bombesin (BN) analogues lacking the C-terminal methionine at the 14 position were evaluated as BN receptor antagonists. [D-Phe6]BN(6-13)amide inhibited specific 125I-GRP binding to lung cancer cell line NCI-H720 with an IC50 value of 12 nM. In contrast, [D-Phe6]BN(6-13)propylamide, butylamide and methylester were more potent with IC50 values of 3, 5 and 5 nM whereas [D-Phe6,Sta13]BN(6-13)amide was less potent with an IC50 value of 180 nM. [D-Phe6]BN(6-13)propylamide antagonized the ability of BN to elevate cytosolic Ca2+, whereas [D-Phe6]BN(6-13)butylamide was a partial agonist. In a small cell lung cancer (SCLC) growth assay, [D-Phe6]BN(6-13)propylamide inhibited colony formation. In summary, BN analogues which lack a C-terminal methionine may function as useful SCLC BN receptor antagonists.     

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 the bombesin receptor blockers [Leu13, psi CH2NH-Leu14]bombesin and N-pivaloyl GRP(20-25) alkylamide (L 686,095-001C002) on basal and neuromedin C-stimulated PRL and GH release in pituitary cell aggregates.

Perifusion of rat anterior pituitary cell aggregates, cultured in estrogen-supplemented serum-free medium with 1 nM of the bombesin (BBN)-like peptide, neuromedin C (NMC), significantly stimulates GH and PRL release. This effect is dose-dependently inhibited by the BBN receptor blocker L 686,095-001C002 [an N-pivaloyl-gastrin-releasing-peptide(20-25) alkylamide]. The IC50 was 0.20 nM in the case of the GH response and 0.16 nM in the case of the PRL response. The antagonist has no effect on basal PRL or GH release. [Leu13, psi CH2NH-Leu14]BBN (psi BBN) displays an IC50 of 0.41 microM for inhibiting the GH response and 0.36 microM for inhibiting the PRL response to NMC. At a concentration of 0.5 microM or 5 microM, however, the latter antagonist stimulates PRL and GH release when perifused alone. This stimulatory effect is dose dependent, augments when aggregates are cultured in 1 nM E2 (as is the case for NMC) and is abolished by 2 nM L 686,095-001C002. It is concluded that L 686,095-001C002 is a potent and pure antagonist of pituitary BBN receptors mediating PRL and GH release, whereas psi BBN is a relatively weak antagonist with considerable partial agonist activity.     

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)     

Inhibition of bombesin-stimulated gastrin release from isolated canine G cells by bombesin antagonists

This study investigated the effects of two putative bombesin antagonists, [D-Arg1,D-Pro2,D-Trp7,9,Leu11]substance P and [Leu13-psi-CH2NH-Leu14]bombesin, on bombesin-stimulated gastrin release from isolated canine G cells following short-term culture. Canine antral tissue was dispersed by sequential collagenase and EDTA treatment, and counterflow elutriation was used to enrich for G cells. Plates were seeded with 2 x 10(6) cells/mL in each well and cultured for 2 days prior to testing. Gastrin-containing and somatostatin-containing cells were identified by immunocytochemistry using the biotin-avidin-peroxidase method and accounted for 8.5 and 1%, respectively, of adhered cells. Basal gastrin secretion was 1.91 +/- 0.48% of total cell content. After a 2-h incubation period, bombesin (0.01-100 pM) stimulated gastrin release in a concentration-dependent fashion. The substance P analog, at a concentration of 1 microM, modestly inhibited bombesin-stimulated gastrin release from canine G cells. This analog also produced weak stimulation of basal gastrin release. In contrast, the bombesin analog, at a concentration of 1 microM, did not affect basal gastrin secretion. The bombesin analog completely blocked bombesin-stimulated gastrin release from 0.01 to 1 pM and produced greater than 50% inhibition at higher doses. The ability of the bombesin analog to directly inhibit bombesin-stimulated gastrin release from cultured canine G cells underscores its usefulness in studies involving the role of bombesin and its mammalian counterpart, gastrin-releasing peptide, in the control of gastrin cell function.     

Pseudononapeptide bombesin antagonists containing C-terminal Trp or Tpi.

Seven new antagonists of bombesin (Bn)/gastrin-releasing peptide (GRP) containing C-terminal Trp or Tpi (2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-3-carboxylic acid) in a reduced peptide bond were synthesized by solid phase methods and evaluated biologically. The reduced bond in four [Leu13 psi(CH2NH)Trp14]Bn(6-14) analogs was formed by reductive alkylation at the dipeptide stage. In the case of three [Leu13 psi(CH2N)Tpi14]Bn(6-14) analogs, the Trp dipeptide with reduced bond was reacted with formaldehyde to form the corresponding Tpi derivative. These Tpi-containing analogs have a new reduced bond which is structurally more constrained. Leu13 psi(CH2N)Tpi14 analogs inhibit [125I][Tyr4]bombesin binding to Swiss 3T3 cells with IC50 values of 2-4 nM, compared to 5-10 nM for Leu13 psi(CH2NH)Trp14 analogs. Leu13 psi(CH2N)Tpi14 analogs are also more potent than Leu13 psi(CH2NH)Trp14 analogs in growth inhibition studies using Swiss 3T3 cells. The two best bombesin antagonists of this series, [D-Trp6,Leu13 psi(CH2N)Tpi14]Bn(6-14) (RC-3415) and [Tpi6,Leu13 psi(CH2N)Tpi14]Bn(6-14) (RC-3440), inhibited GRP-stimulated growth of Swiss 3T3 cells with IC50 values less than 1 nM. RC-3440 was also active in vivo, suppressing GRP(14-27)-stimulated serum gastrin secretion in rats. Bombesin/GRP antagonists, such as RC-3440, containing the new reduced bond (CH2N) reported herein are very potent.     

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.     

Small cell lung cancer bombesin receptors are antagonized by reduced peptide bond analogues

The potency of 3 reduced peptide bond analogues of bombesin (BN) was investigated using small cell lung cancer (SCLC) cell lines. (Psi13,14, Leu14)BN, (Psi9,10, Leu14)BN and (Psi12,13, Leu14)BN inhibited specific binding of 125I-GRP with IC50 values of 15, 90, and 600 nM. (Psi13,14, Leu14)BN and (Psi9,10, Leu14)BN did not elevate cytosolic Ca2+ levels but antagonized the increase in cytosolic Ca2+ caused by BN. (Psi13,14, Leu14)BN antagonized the clonal growth of SCLC cells caused by BN. These data indicate that reduced peptide bond analogues may disrupt the autocrine growth cycle of SCLC cells by functioning as BN receptor antagonists.     

Arachidonic acid release by bombesin. A novel postreceptor target for heterologous mitogenic desensitization

Prolonged exposure of Swiss 3T3 cells to vasopressin causes heterologous mitogenic desensitization to bombesin and structurally related peptides including gastrin-releasing peptide (GRP) without down-regulation of the bombesin receptor. The number and affinity of bombesin/GRP receptor sites and modulation of 125I-GRP binding by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) are unaffected in membrane preparations from vasopressin-treated cultures. Stimulation of inositol phosphate accumulation, mobilization of intracellular calcium, production of diacylglycerol, and transmodulation of the epidermal growth factor receptor by bombesin are similarly unaffected. Thus, the heterologous mitogenic desensitization is not due to uncoupling of bombesin receptor from transducing G protein(s) or to an inability to activate phospholipase C. Bombesin, unlike vasopressin, causes a rapid dose-dependent release of [3H]arachidonic acid and prostaglandin E2 from Swiss 3T3 cells (EC50 approximately 4 nM), which is inhibited by the specific bombesin receptor antagonist [Leu13-psi(CH2NH)-Leu14]bombesin. Crucially, release of [3H]arachidonic acid and prostaglandin E2 by bombesin is completely suppressed by prolonged pretreatment with vasopressin (EC50 = 0.6 nM). The mitogenic action of bombesin is restored by adding arachidonic acid to vasopressin-treated cells. We conclude first that arachidonic acid release is an early signal in the mitogenic response to bombesin and second that pretreatment with vasopressin induces heterologous mitogenic desensitization to bombesin by a novel mechanism: inhibition of arachidonic acid release.     

Comparison of dopamine uptake and release in vitro in sheep and rat striatum.

Cocaine inhibits tritium-labeled dopamine ([3H]DA) uptake in rat (IC50 approximately 400 nM) and sheep (IC50 approximately 1 microM) striatum. GBR 12909, a selective DA uptake inhibitor, potently inhibits [3H]DA uptake in rat (IC50 less than 10 nM), but is less effective (only 60% of the uptake is inhibited at a concentration of 10 microM) and less potent (IC50 approximately 300 nM) in sheep. [3H]DA release from slices of rat or sheep striatum is stimulated by potassium (15-50 mM). In the presence of nomifensine (10 microM), cocaine (10 microM) had no effect on potassium-stimulated [3H]DA release in either species. [3H]DA release is increased by N-methyl-D-aspartate (NMDA) (10-1000 microM) in rat striatum but NMDA did not stimulate [3H]DA release in sheep striatum. These findings suggest that NMDA receptors either are absent from or do not regulate release of preloaded [3H]DA in sheep striatum.     

Chronic desensitization to bombesin by progressive down-regulation of bombesin receptors in Swiss 3T3 cells. Distinction from acute desensitization

Prolonged exposure (40 h) of Swiss 3T3 cells to bombesin induced homologous desensitization to bombesin and structurally related peptides including mammalian gastrin releasing peptide (GRP). The ability of bombesin to mobilize intracellular Ca2+, inhibit epidermal growth factor binding, and stimulate DNA synthesis was profoundly and selectively inhibited. In contrast, Ca2+ mobilization by either vasopressin or bradykinin was unaffected, indicating that chronic desensitization is mechanistically distinct from acute desensitization of Ca2+ mobilization. Prolonged (24 or 40 h) pretreatment with bombesin also induced a 78 +/- 5% loss of bombesin receptor binding sites in both intact and plasma membrane preparations of Swiss 3T3 cells without an apparent change in receptor affinity (Kd = 1.9 +/- 0.1 x 10(-9) M and Kd = 1.8 +/- 0.2 x 10(-9) M for control and pretreated cells, respectively). Loss of 125I-GRP binding was slow and progressive with half-maximal loss of binding occurring after 7 h and maximal after approximately 14 h. Cross-linking of 125I-GRP to intact cultures and membrane preparations revealed an identical time-dependent loss of the Mr = 75,000-85,000 cross-linked band, previously identified as the bombesin receptor. Prolonged exposure of the cells to phorbol 12,13-dibutyrate, epidermal growth factor, cholera toxin, or mitogenic combinations of these agents did not alter 125I-GRP binding. Receptor down-regulation and loss of mitogenic responsiveness to bombesin were: (a) induced in a parallel dose-dependent manner by bombesin (ED50 = 1 nM), GRP (ED50 = 2 nM), and neuromedin B (ED50 = 20 nM), but not by the biologically inactive fragment GRP (1-16); (b) inhibited by the specific bombesin antagonist [Leu13-psi(CH2NH)-Leu14] bombesin, and (c) reversed upon removal of bombesin with a similar time course (full recovery after 15 h). On the basis of these observations, we propose that prolonged pretreatment of Swiss 3T3 cells with bombesin induces homologous desensitization to peptides of the bombesin family by down-regulation of cell surface bombesin receptors.     

Somatostatin and lanthanum discriminate two Ca2+ mobilizing mechanisms regulated by bombesin receptors in peptic cells

Bombesin (BB)-stimulated pepsinogen secretion from frog esophageal peptic acini was inhibited 40% by somatostatin (SS) (IC50 = 1 nM), and by 30-50% by low concentrations (10(-7)-10(-4)M) of lanthanum chloride. SS inhibited basal secretion as well as both early (0-2 min) and late (2-30 min) BB-stimulated secretory phases. By contrast, LaCl3 selectively inhibited the late secretory phase and was without effect on basal secretion. SS (100 nM) and LaCl3 (30 microM) attenuated BB-stimulated 45Ca2+ uptake, and the combination resulted in additive inhibition. High K+ media decreased basal secretion, abolished SS but not LaCl3 inhibition of BB-stimulated secretion, and blocked SS inhibition of BB-mediated 45Ca2+ uptake. These findings suggest the existence on peptic cells of distinct La3+-sensitive, and somatostatin-sensitive, K+ dependent, Ca2+ mobilizing mechanisms which contribute to BB receptor-mediated secretion.     

Development of a potent bombesin receptor antagonist with prolonged in vivo inhibitory activity on bombesin-stimulated amylase and protein release in the rat.

Of the various types of potent bombesin(Bn)/gastrin releasing peptide receptor antagonists that have been discovered, the desMet14-methyl ester peptides are devoid of residual agonist activity and are among the most potent in terms of in vitro receptor blockade and also in terms of their prolonged inhibition of bombesin-stimulated amylase and protein release in the rat. We have now examined the in vitro and in vivo properties of a new series of methyl ester analogues, [D-Phe6]Bn(6-13)OMe, [D-Phe6,D-Ala11]Bn(6-13)OMe, N alpha-propionyl-[D-Ala24]GRP(20-26)OMe, and [D-pentafluoro-Phe6,D-Ala11]Bn(6-13)OMe, which have an additional D-amino acid substituent and some highly lipophilic moieties at the N-terminus. All analogues were able to potently antagonize the ability of Bn to stimulate amylase release from rat acinar cells, with IC50 values of 2.4, 2.5, 0.6, and 1.3 nM, respectively. The four peptides were found to have binding affinities for these cells comparable to Bn itself, with K(i)s of 10.3, 2.8, 5.5, and 3.6 nM, respectively, but all had little or no affinity for neuromedin B receptors on murine C6 cells. Single bolus IV injections of these peptides were found to potently inhibit amylase and protein release caused by IV infusion of bombesin into the rat. Generally the peptides containing the D-Ala substituent were longer acting than [D-Phe6]Bn(6-13)OMe, so that [D-Phe6,D-Ala11]Bn(6-13)OMe and N alpha-propionyl-[D-Ala24]GRP(20-26)OMe displayed significant inhibitory effects for up to 1.5 h after administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leu13-psi(CH2NH)Leu14]bombesin is a specific bombesin receptor antagonist in Swiss 3T3 cells

The pseudopeptide [Leu13-psi(CH2NH)Leu14]bombesin blocks bombesin-stimulated mitogenesis in Swiss 3T3 cells in a competitive and reversible manner, but not that of other mitogens. It inhibits the mobilization of intracellular Ca2+ and activation of protein kinase C by bombesin-like peptides. It acts at receptor level, as shown by inhibition of [125I]GRP binding and reduction in cross-linking of the Mr 75-85,000 receptor-associated protein. Thus [Leu13-psi(CH2NH)Leu14]bombesin is a specific bombesin receptor antagonist in Swiss 3T3 cells which blocks long-term growth promoting effects of bombesin-like peptides.     

Leu31-Pro34] neuropeptide Y identifies a subtype of 125I-labeled peptide YY binding sites in the rat brain.

Subtypes of the neuropeptide Y (NPY) receptor in the rat brain were identified by the use of the selective Y-1 analog, [Leu34-Pro34] NPY. In rat brain homogenate binding studies, [Leu31-Pro34] NPY was found to produce a partial inhibition of 100 pM 125I-labeled peptide YY (PYY) binding with a plateau at 50-1000 nM [Leu31-Pro34] NPY resulting in a 70% inhibition of binding. The C-terminal fragment NPY 13-36, a putative Y-2 agonist, exhibited very little selectivity in rat brain homogenates. Scatchard analysis of 125I-labeled PYY binding to rat brain homogenate yielded biphasic plots with Kd values of 40 and 610 pM. Inclusion of 100 nM [Leu31-Pro34] NPY was found to eliminate the low affinity component of 125I-labeled PYY binding leaving a single, high affinity binding site with a Kd of 68 pM. In autoradiographic studies, displacement curves indicated that [Leu31-Pro34] NPY completely inhibited binding in the cerebral cortex with little effect on the binding in the hypothalamus. On the other hand NPY 13-36 inhibited binding in the hypothalamus at low concentrations but required higher concentrations to inhibit binding in the cerebral cortex. Other brain regions such as the hippocampus, appeared to contain both subtypes. Subsequent to these studies, a quantitative autoradiographic map was conducted using 50-100 pM 125I-labeled PYY in the presence and absence of [Leu31-Pro34] NPY which produced a selective displacement of binding in certain distinct brain regions. These areas included the cerebral cortex, certain thalamic nuclei and brainstem while ligand binding was retained in other brain regions including the zona lateralis of the substantia nigra, lateral septum, nucleus of the solitary tract and the hippocampus. Numerous brain regions appeared to contain both receptor subtypes. Therefore, the Y-1 and Y-2 receptor subtypes exhibited a somewhat distinct distribution in the brain. In addition, 125I-labeled PYY appears to label the Y-2 receptor with relatively higher affinity when compared to the Y-1 receptor.     

Inhibition by mepacrine and amylase secretion from intact and permeabilized rat pancreatic acini.

In intact rat pancreatic acini, the phospholipase A2 inhibitor mepacrine did not affect basal amylase release but dose-dependently inhibited the carbachol (IC50 65 microM) and CCK-8 (IC50 210 microM)-stimulated amylase release. In permeabilized acini, mepacrine shifted the dose-response curve for calcium to the right by a factor 2 and inhibited the release of amylase stimulated by GTPrS. From these results we conclude that carbachol, CCK-8 and GTPrS probably activate a phospholipase A2 closely coupled to exocytosis.     

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.     

Functional and molecular characterization of CCK receptors in the rat pancreatic acinar cell line AR 4-2J.

Competitive inhibition binding studies on membranes from the rat pancreatic AR 4-2J cell line revealed the predominance (80%) of low selectivity CCK receptors (KD of 1 nM and 4 nM for, respectively, CCK-8 and gastrin-17I (G-17I] over selective receptors (20% with a KD of 1 nM and 1 microM for, respectively, CCK-8 and G-17I). Amylase secretion was stimulated by low concentrations of CCK-8, G-17I and CCK-4. G-17I-induced amylase secretion was unaffected by 100 nM of the selective peripheral CCK-A receptor antagonist L-364,718, suggesting that amylase hypersecretion followed non-selective CCK receptor activation, a function normally assumed by selective CCK-A receptors in rat pancreatic acini. Direct ultraviolet irradiation of AR 4-2J cell membranes preloaded with 125I-BH-CCK-33 or 125I(Leu)G(2-17)I resulted in covalent cross-linking with, respectively, a 90 kDa protein and a 106 kDa protein, both distinct from the 81 kDa CCK binding species revealed in normal rat pancreatic membranes. Gpp[NH]p increased the dissociation rate of CCK-8 and G-17I from AR 4-2J cell membranes, indicating a coupling of receptors with guanyl nucleotide regulatory protein(s) G. [32P]ADP-ribosylation of AR 4-2J cell membranes allowed to detect the presence of two Gs alpha (the 50 kDa form predominating over the 45 kDa form) and one Gi alpha (41 kDa). However, Gi and Gs may not be involved in gastrin stimulation of amylase secretion, as Bordetella pertussis toxin and cholera toxin pretreatment of cells did not suppress G-17I-dependent amylase secretion.