[D-Arg1,D-Trp5,7,9,Leu11]Substance P coordinately and reversibly inhibits bombesin- and vasopressin-induced signal transduction pathways in Swiss 3T3 cells.

The novel substance P (SP) analogue, [D-Arg1,D-Trp5,7,9,Leu11]SP like [D-Arg1,D-Phe5,D-Trp7,9,Leu11]SP inhibited DNA synthesis induced by bombesin, vasopressin, and bradykinin, but did not interfere with the mitogenic response induced by other growth factors or pharmacological agents in Swiss 3T3 cells. [D-Arg1,D-Trp5,7,9,Leu11]SP reversibly inhibited bombesin-induced DNA synthesis, causing a 6-fold greater rightward shift in the bombesin dose response than [D-Arg1,D-Phe5,D-Trp7,9,Leu11]SP at identical concentrations (10 μM). We found that the new, more potent, SP analogue coordinately and reversibly inhibited bombesin-induced Ca2+ mobilization and protein kinase C (PKC) and mitogen-activated protein (MAP) kinase activation. The dose-response curves for bombesin-induced Ca2+ mobilization and MAP kinase activation were similarly displaced (51- and 40-fold, respectively) by [D-Arg1,D-Trp5,7,9,Leu11]SP. In addition, [D-Arg1,D-Trp5,7,9,Leu11]SP reversibly inhibited bombesin-induced tyrosine phosphorylation of Mr 110,000-130,000 and 70,000-80,000 bands as well as p125 focal adhesion kinase. [D-Arg1,D-Trp5,7,9,Leu11]SP also reversibly and coordinately inhibited vasopressin-induced Ca2+ mobilization, PKC stimulation, MAP kinase activation, tyrosine phosphorylation, and DNA synthesis in Swiss 3T3 cells. Surprisingly, deletion of the terminal Leu of [D-Arg1,D-Phe5,D-Trp7,9,Leu11]SP to yield [D-Arg1,D-Phe5,D-Trp7,9]SP1-10 resulted in a selective loss of inhibitory activity of this analogue against bombesin- but not vasopressin-stimulated DNA synthesis, Ca2+ mobilization, and MAP kinase activation. Collectively, these results suggest that SP analogues act at the receptor level to coordinately and reversibly antagonize bombesin- or vasopressin-induced signal transduction in Swiss 3T3 cells.

Interestingly, multiple neuropeptides, including bombesin and vasopressin, have also been implicated as autocrine and paracrine growth factors for small cell lung cancer (36), breast (37), and prostate cancer (38). Consequently, it may be useful to develop inhibitors of neuropeptide action, which have a broader spectrum than specific receptor antagonists (39).
SP analogues were initially synthesized to generate specific SP antagonists. Surprisingly, the synthetic SP analogues, [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP and [Arg 6 ,D-Trp 7,9 , MePhe 8 ]SP (6 -11) were found to inhibit the action of a broad range of neuropeptides structurally unrelated to SP, including bombesin-and vasopressin-stimulated DNA synthesis in Swiss 3T3 cells (40 -44). In contrast, they did not inhibit mitogenesis stimulated by either vasoactive intestinal peptide, which induces cAMP accumulation via G s , or platelet-derived growth factor, which signals through receptors with intrinsic tyrosine kinase activity (45). More recently, [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP and [Arg 6 ,D-Trp 7,9 ,MePhe 8 ]SP (6 -11) have been shown to inhibit small cell lung cancer cell proliferation in liquid culture, soft agar, and as xenografts in nude mice (46 -48). Despite their intriguing biological effects and potential importance as antiproliferative agents, the mechanism of action of SP analogues as broad spectrum inhibitors of neuropeptide-mediated signal transduction remains incompletely understood.
A recent report has proposed that [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP selectively uncouples PIP 2 -PLC-␤ from the bombesin receptor (49). It was therefore suggested that the inhibitory effect of the SP analogues on cell proliferation could be attributed to disruption of the coordinated regulation of bombesin-induced signal transduction pathways. This proposal was based on the fact that high concentrations of bombesin reversed the inhibition of MAP kinase but not PIP 2 -PLC-␤ activation caused by [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP in Swiss 3T3 cells (49). In contrast, we have recently demonstrated that high concentrations of either bombesin or vasopressin reverse the inhibitory effect of this SP analogue on inositol phosphate production in Swiss 3T3 cells (50). These discrepant results prompted us to examine the inhibitory effect of several novel SP analogues on the multiple signal transduction pathways induced by bombesin and vasopressin in Swiss 3T3 cells.

EXPERIMENTAL PROCEDURES
Cell Culture-Stock cultures of Swiss 3T3 fibroblasts were maintained in DMEM supplemented with 10% fetal bovine serum in a humidified atmosphere containing 10% CO 2 and 90% air at 37°C. For experimental purposes, cells were plated in 33-or 90-mm Nunc Petri dishes at 10 5 cells or 6 ϫ 10 5 cells/dish, respectively, in DMEM containing 10% fetal bovine serum and used after 6 -8 days when the cells were confluent and quiescent.

Assays of [ 3 H]Thymidine
Incorporation-Quiescent and confluent cells in 33-mm dishes were washed twice in DMEM and incubated at 37°C in 2 ml of a 1:1 mixture of DMEM and Waymouth medium containing 1 Ci/ml [ 3 H]thymidine with various additions as indicated. After 40 h, unless otherwise indicated, acid-precipitable material was measured as described previously (51).
Measurement of Intracellular Calcium-[Ca 2ϩ ] i was measured with the fluorescent Ca 2ϩ indicator fura-2/AME using a modification of the procedure previously described (52). Quiescent cells in 90-mm dishes were washed twice in DMEM and then incubated at 37°C for 10 min in 5 ml of DMEM with 1 M fura-2 tetraacetoxymethyl ester. The dishes were washed three times in phosphate-buffered saline at 37°C, and the cells were then suspended in 2 ml of electrolyte solution containing 120 mM NaCl, 5 mM KCl, 1.8 mM CaCl 2 , 0.9 mM MgCl 2 , 25 mM glucose, 16 mM Hepps, 6 mM Tris, and an amino acid mixture equivalent to DMEM (pH 7.2) by gentle scraping and transferred to a quartz cuvette. The suspension was stirred continuously and maintained at 37°C. Various factors were added as indicated in the figure legends. Fluorescence was monitored in a Perkin-Elmer LS-5 luminescence spectrophotometer with an excitation wavelength of 336 nm and emission wavelength of 510 nm. [Ca 2ϩ ] i was calculated using the formula [Ca 2ϩ ] i nM ϭ K(F Ϫ F min )/(F max Ϫ F), where F is the fluorescence at the unknown [Ca 2ϩ ] i , F max is the fluorescence after addition of 0.02% Triton X-100, and F min is the fluorescence after the Ca 2ϩ in the solution is chelated with 10 mM EGTA. The value of K was 220 nM for fura-2/AME (52). 32 P-Labeling of Cells and Analysis of 80K/MARCKS Phosphorylation-Quiescent and confluent cultures in 33-mm dishes were washed twice in phosphate-free DMEM and incubated at 37°C with this medium containing 50 Ci/ml of carrier-free [ 32 P]P i . After 18 h, various factors were added for the indicated times. The cells were then lysed in 500 l/dish of a solution containing 10 mM Tris/HCl, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 100 M Na 3 VO 4 , 50 mM phenylmethylsulfonyl fluoride, and 0.5% Triton X-100 (lysis buffer) supplemented with 3.5 g/ml aprotinin and 1 g/ml leupeptin and the lysates were clarified by centrifugation for at 15,000 ϫ g for 20 min at 4°C. The supernatants were then immunoprecipitated with specific anti-80K/MARCKS antibody (53) and the immunoprecipitates were further analyzed by SDS-PAGE prior to autoradiography.
Immunoprecipitation of Tyrosine Phosphorylated Proteins-Quiescent and confluent cultures of cells in 33-mm dishes were washed twice with DMEM, and then treated with factors as indicated in the figure legends prior to lysis at 4°C in 1 ml of lysis buffer. Lysates were centrifuged at 15,000 ϫ g for 20 min, and the supernatants were incubated for 4 h at 4°C with anti-mouse IgG agarose-linked mAbs directed against phosphotyrosine (Py72). The immunoprecipitates were washed three times with lysis buffer and further analyzed by SDS-PAGE and Western blotting (see below). Cells from parallel cultures treated in an identical fashion were suspended by trypsinization and counted using a Coulter counter to ensure equal numbers of cells per condition.
Immune Complex Assay of p42 MAPK Activity-Quiescent cultures of Swiss 3T3 cells were treated with factors as described in the figure legends and lysed at 4°C. Lysates were clarified by centrifugation at 15,000 ϫ g for 20 min at 4°C, and the supernatants were immunoprecipitated using the polyclonal anti-p42 MAPK antibody together with protein A-agarose beads (40 l, 1:1 slurry) for 2 h. Immune complexes were collected by centrifugation and washed once in lysis buffer and three times in kinase buffer (15 mM Tris-HCl, 15 mM MgCl 2 ). The kinase reaction was performed by resuspending the pellet in 25 l of kinase assay mixture containing kinase buffer, 1 mg/ml myelin basic protein-peptide (APRTPGGRR), 100 M ATP, 100 Ci/ml [␥-32 P]ATP, and 200 M microcystin LR. Incubations were performed for 10 min (linear assay conditions) at 30°C and terminated by spotting 20 l of the supernatant onto P81 chromatography paper (Whatman). Filters were washed four times, 5 min each, in 0.5% orthophosphoric acid, immersed in acetone, and dried before counting. The average radioactivity of two blank samples containing no immune complex was subtracted from the result of each sample. Results are expressed as a percentage of the maximum response obtained with either bombesin or vasopressin alone in the absence of inhibitors. The specific activity of [␥-32 P]ATP used was 900-1200 cpm/pmol. SDS-PAGE-Slab gel electrophoresis was performed essentially according to the method of Laemmli (54). Specifically, the slab gels were 1.5 mm thick with 1.5 cm of a 4% acrylamide stacking gel and 12 cm of 8 or 10% acrylamide resolving gel. Samples (100 ml) were electrophoresed at 20 V for 30 min, then run overnight at 50 V, and finally at 150 V for 30 min before terminating the run. Gels for 80K/MARCKS experiments were fixed in 25% methanol, 10% acetic acid (v/v) prior to drying under vacuum for 2 h at 80°C. Radioactivity was detected at Ϫ70°C using Fuji x-ray film with exposure times of 12-72 h.
Materials-Bombesin, vasopressin, EGF, and IgG-agarose were obtained from Sigma. Protein A-agarose was from Boehringer Mannheim. Anti-Tyr(P) mAb clone Py72 was obtained from the hybridoma development unit, Imperial Cancer Research fund, London, UK. PY20 anti-Tyr(P) mAb was from ICN. 4G10 anti-Tyr(P) mAb was from Upstate Biotechnology Inc., Lake Pladid, NY. The anti-p125 FAK mAb for Western blotting was obtained from AFFINITI Research Products Ltd., Nottingham, UK. The polyclonal anti-p42 MAPK (anti-ERK-2) antibody raised against a COOH-terminal peptide (EETARFQPGYRS) was a generous gift from Dr J. Van Lint (Katholieke Universiteit Leuven, Belgium). 125 11 ]SP resulted in a broad spectrum neuropeptide antagonist which was 5-fold more potent (44). We reasoned that further substitutions at this position may result in SP analogues with increased potency. We established that substitution of D-Phe at position 5 with D-Tyr did not significantly affect antagonistic activity against bombesin (data not shown). In contrast, when this residue was replaced by D-Trp to form [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP an increase in inhibitory activity was obtained (see below). In view of these results, [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP was selected for further investigation.  (Fig. 3, upper). In particular, the ability of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP to block MAP kinase activation was, just like Ca 2ϩ mobilization, reversed at high concentrations of bombesin. To further substantiate the results obtained with the immune complex MAP kinase assay, lysates of Swiss 3T3 cells stimulated with bombesin in the absence or presence of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP were subjected to SDS-PAGE followed by Western blotting with anti-MAP kinase antibody (55). The activation of MAP kinase induced by bombesin was inhibited by increasing concentrations of the SP analogue as judged by the disappearance of slower migrating forms in the mobility shift assay (Fig. 3, lower).
[D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ]SP 1-10 Reversibly Inhibits Vasopressin-but Not Bombesin-induced DNA Synthesis, Ca 2ϩ Mobilization, and MAP Kinase Activation-Recently, it has been shown that SP analogues are metabolized predominantly by oxidation of the amino acid at the COOH terminus (56). To test the effect of additional SP analogues on neuropeptide stimulated mitogenesis and signal transduction, we used synthetic peptide analogues which had substitutions or deletions of the terminal amino acid of [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP (shown in Fig.  6). Replacement of the terminal Leu of [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP with Val to produce [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Val 11 ]SP did not change the inhibitory activity of the SP analogue against either bombesin-or vasopressin-induced mitogenesis (Fig. 6), Ca 2ϩ mobilization, and MAP kinase activation (Fig. 7). In addition, the inhibitory effect of both SP analogues could be reversed by high concentrations of either neuropeptide (data not shown). In contrast, substitution of the terminal Leu with Gly resulted in a SP analogue with almost no inhibitory effect on mitogenesis, Ca 2ϩ mobilization, and MAP kinase activation stimulated by either neuropeptide (Figs. 6 and 7).
Deletion of the terminal Leu to form [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ]SP 1-10 produced an antagonist which potently inhibited vasopressin-induced mitogenesis (Fig. 6), Ca 2ϩ mobilization, and MAP kinase activation (Fig. 7). Surprisingly, [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ]SP 1-10 did not significantly block mitogenesis (Fig. 6) and only weakly inhibited Ca 2ϩ mobilization and MAP kinase activation (Fig. 7) 11 ]SP has been previously shown to block the action of multiple neuropeptides including bombesin and vasopressin in Swiss 3T3 cells (4, 40 -44) and to inhibit small cell lung cancer cell growth in vitro and as xenografts in vivo (46 -48). Here, we demonstrate that substitution of D-Phe at position 5 with D-Trp to form [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP produced a SP analogue with a further increase in potency against neuropeptide-induced mitogenesis. Importantly, this new SP analogue, like previously identified SP antagonists, inhibited DNA synthesis induced by bombesin, vasopressin, and bradykinin, but did not interfere with the mitogenic response induced by other growth factors or pharmacological agents.
A recent report has suggested that [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP can selectively inhibit the activation of PIP 2 -PLC-␤ but not MAP kinase at high bombesin concentrations in Swiss 3T3 cells (49). It was proposed that disruption of the coordinate regulation of bombesin-induced signaling pathways contributes to the growth inhibitory properties of [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP (49). This implies that the growth-inhibitory effects of the SP analogues should not be reversed by high concentrations of agonist. However, our results demonstrate that 1) the inhibition of DNA synthesis by [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP was reversed by increasing concentrations of bombesin, 2) although the inhibition curves for [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP on bombesin-induced Ca 2ϩ mobilization and MAP kinase activation were slightly different, the dose-response curves for bombesin-induced Ca 2ϩ mobilization and MAP kinase activation were similarly displaced by the SP analogue, and 3) importantly, the inhibitory effect of the SP analogue on both Ca 2ϩ mobilization and MAP kinase activation could be completely reversed at high bombesin concentrations. These results prompted us to perform additional experiments to test further the mechanism of action of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP and other related peptides.
It is well established that bombesin induces a rapid increase in the tyrosine phosphorylation of multiple substrates including p125 FAK (29 -35), through a signal transduction pathway that is mediated by p21 Rho (58,59). This pathway is not dependent on either PKC activation or Ca 2ϩ mobilization (32,34,60). Recently, we verified that the bombesin receptor transfected and stably expressed in Rat-1 cells mediates Ca 2ϩ mobilization, PKC activation as well as tyrosine phosphorylation of multiple substrates including p125 FAK (61). In the present study we demonstrate that [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP reversibly inhibited bombesin-induced tyrosine phosphorylation of multiple substrates including p125 FAK in Swiss 3T3 cells. These data support the proposition that this SP analogue coordinately inhibits the activation of the signal transduction pathways emanating from the bombesin receptor.
Vasopressin binds to a distinct G q -coupled receptor that also induces PIP 2 -PLC-mediated Ca 2ϩ mobilization, 80K/MARCKS phosphorylation, MAP kinase activation, and tyrosine phosphorylation of multiple substrates including p125 FAK in Swiss 3T3 cells (reviewed in Ref. 2). In order to substantiate our findings with bombesin, we also studied the effect of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP on vasopressin-stimulated signaling events. As previously seen with bombesin, we found that this SP ana- logue coordinately inhibited vasopressin-induced Ca 2ϩ mobilization, 80K/MARCKS phosphorylation, MAP kinase activation, tyrosine phosphorylation, and reinitiation of DNA synthesis. In particular, the dose responses for vasopressininduced Ca 2ϩ mobilization and MAP kinase activation were similarly displaced by [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP.
The coordinate inhibition of neuropeptide stimulated signal transduction pathways could be a feature specific to the new SP analogue, [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP, rather than a common property of all SP analogue antagonists. We verified that [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP could also coordinately block bombesin-and vasopressin-induced mitogenesis, Ca 2ϩ mobilization, and MAP kinase activation. Furthermore, we examined the effect of additional SP analogues generated by substitutions or deletion of the terminal amino acid of [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP. The conservative substitution of Leu 11 with Val yielded a peptide that behaved identically to [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP and [D-Arg 1 ,D-Trp 5 ,D-Trp 7,9 ,Leu 11 ]SP. Thus, three different SP analogues inhibit neuropeptide-induced mitogenesis, Ca 2ϩ mobilization, and MAP kinase activation in a reversible and coordinate fashion.
A model that accounts for the coordinate inhibition of bombesin or vasopressin stimulated signal transduction by [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP and related peptides is that these SP analogues interfere with agonist binding to their receptors. In fact, we have previously shown that SP analogues competitively inhibit ligand binding (50), but these findings could not rule out an indirect mechanism mediated by uncoupling of a G protein from the receptor. Surprisingly, deletion of the terminal Leu of [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP to form [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ]SP 1-10 has provided novel mechanistic insight into this problem. We found that [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ]SP 1-10 potently inhibited vasopressin-induced mitogenesis, Ca 2ϩ mobilization, and MAP kinase activation. In contrast, [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ]SP 1-10 did not significantly block bombesin-induced mitogenesis and only weakly inhibited Ca 2ϩ mobilization and MAP kinase activation, even at concentrations where these responses were completely inhibited by either [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP or [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Val 11 ]SP. Therefore, the SP analogue [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ]SP 1-10 coordinately inhibits vasopressin but not bombesin-induced signal transduction. This differential modulation strongly suggests that the truncated SP analogue acts as a potent vasopressin (but not bombesin) receptor antagonist. Our results imply that these inhibitory molecules block neuropeptide-mediated signal transduction at the receptor level.