Proc. Nati Acad. Sci. USA Vol. 79, pp. 2379-2383, April 1982 Medical Sciences

Human lung small-cell carcinoma contains bombesin (radioimmunoassay/immunohistochemistry/peptide hormones/high-performance liquid chromatography) MICHAEL D. ERISMAN*t, R. ILONA LINNOILA*t, OSCAR HERNANDEZ§, RICHARD P. DiAUGUSTINE*, AND LAWRENCE H. LAZARUS*¶ *Laboratory of Pulmonary Function and Toxicology, and §Laboratory of Environmental Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Communicated by George H. Hitchings, December 18, 1981

ABSTRACT The presence of immunoreactive bombesin in a tide in the tumor, (ii) its quantitation in extracts by radioim- human lung small-cell carcinoma grown in nude mice was estab- munoassay, and (iii) analysis by gel-filtration chromatography lished by several criteria: (i) Radioimmunoassay of tissue extracts and reverse-phase HPLC. for bombesin revealed approximately 6.5 pmol/g of tissue; (ii) bombesin was found in 12-14% of the tumor cells by immuno- histochemical localization; (iii) gel filtration ofsmall-cell carcinoma MATERIALS AND METHODS extract on Sephadex G-75 and Bio-Gel P-4 gave only a single peak Tumor. A SCC resected from a 51-year-old man with few ofimmunoreactivity, which occurred at the elution volume ofbom- apparent clinical endocrinopathies was propagated subcutane- besin; and (iv) reverse-phase HPLC of acid-solubilized extracts ously in athymic (nude) mice by Reid et al. (22) and generously separated the immunoreactive material into three discrete peaks, supplied by G. Sato (University of California, San Diego). Tu- one of which eluted with a retention time identical to that of syn- mors from two separate passages were analyzed separately. The thetic bombesin. The presence of bombesin may represent the tumors, 8.71 and 6.36 g wet weight, were removed and sec- ectopic expression of this peptide in small-cell carcinoma, because tioned for histology, and the remainders of each were minced immunoreactive bombesin was found in human fetal and neonatal at 10C. They were homogenized in 5 ml of ice-cold 0.01 M so- lung but apparently not in adult lung tissue [Wharton, J., Polak, dium phosphate buffer, pH 6.8, containing 1 mM phenylmeth- J. M., Bloom, S. R., Ghatei, M. A., Solcia, E., Brown, M. R. & ylsulfonyl fluoride. The homogenate was slowly added to 25 ml Pearse, A. G. E. (1978) Nature (London) 273, 769-770]. The im- munoreactive bombesin previously found in mammalian tissues ofboiling water, boiled 30 min, and cooled on ice; then formic is considerably larger than amphibian bombesin; these data sub- acid was added to a final concentration of 1.0 M (23, 24) and the stantiate the presence of a mammalian form of bombesin in a hu- mixture was stirred for 60 min. The extract was clarified by cen- man tumor that may have a structure similar to that of the am- trifugation (37,000 X g for 30 min at 40C), and the supernatant phibian peptide. was freeze-dried. The yield was approximately 33 mg of dry extract per g of tissue. Small oat-cell carcinoma accounts for 10-25% of identifiable Immunohistochemistry. Sections of tumor (0.1 cm3) were human lung cancers (1). This neoplasm is characterized by a frozen to the temperature of liquid nitrogen, lyophilized, and rapid growth rate and a propensity to metastasize (1) and is fre- fixed with either diethylpyrocarbonate or parabenzoquinone quently a source for "ectopic" hormone production (1-7). Many vapors (25, 26); other sections (0.03 cm3) were fixed either in ofthe clinical symptoms manifested by patients with small-cell 10% buffered formalin or Bouin's fluid and embedded in par- carcinoma (SCC) can be attributed to increased levels ofectopic affin. Samples for electron microscopy (0.03 cm3) were fixed in hormones (5-7). For example, the syndrome of inappropriate 2.6% glutaraldehyde/2.0% formaldehyde (wt/vol) and embed- secretion of antidiuretic hormone is apparently due to ded in Epon. Immunohistochemistry used an improved im- [Arg]vasopressin (5, 6, 8), whereas Cushing syndrome is con- munoglobulin-enzyme bridge technique (27) with antisera sidered to be caused by ectopic adrenocorticotropin (ACTH) against the following peptides at 1:1,000 dilution: ,B subunit of formation and secretion, which is nonsuppressible by dexa- human chorionic gonadotropin (hCGB; batch 2, Pituitary Hor- methasone (3, 5, 7). Other symptoms may be due to the pres- mone Distribution Program, National Institutes of Health), ence ofpresently unknown or undiscovered peptide hormones. bombesin (see below), physalaemin (PS-1) (23, 24), ACTH (A7, In this regard, bombesin, a tetradecapeptide from anuran skin, midregion specific) (28), Mr 16,000 region of pro-opiomelano- elicits numerous physiological and pharmacological responses cortin (B. Eipper, University of Colorado) and human /3-en- (9, 10) that resemble some of the clinical symptoms observed dorphin (V. Fang, University of Chicago). Sheep anti-rabbit for SCC.(5, 11). Bombesin-like immunoreactivity was detected gamma globulin was purchased from Antibodies, Inc. (Davis, in numerous mammalian tissues (12-20), including human fetal CA), and rabbit anti-peroxidase serum and sheep anti-rabbit and neonatal lung, but is apparently absent from adult lung (21). antiserum were from Cappel Laboratories (Cochranville, PA). Because many immunological and enzymological changes that Controls for the specificity of the interaction included incuba- appear in tumors resemble those normally associated with fetal tion ofserial sections oftissues with antigen-inactivated antisera tissues, it seemed reasonable to evaluate the presence ofbom- besin in a lung neoplasm noted for ectopic hormone production. Abbreviations: SCC, small-cell carcinoma; ACTH, adrenocorticotropin; Bombesin in a human lung SCC grown in nude mice was dem- RIA, radioimmunoassay; GH, somatotropin (growth hormone), hCG', ,B subunit of human chorionic gonadotropin. onstrated by (i) immunohistochemical localization of this pep- t Present address: Institute for Cancer Research, Fox Chase, Phila- delphia, PA 19111. The publication costs ofthis article were defrayed in part by page charge t Present address: Laboratory ofPathology, NCI, NIH, Bethesda, MD payment. This article must therefore be hereby marked "advertise- 20205. nent" in accordance with 18 U. S. C. §1734 solely to indicate this fact. ¶ To whom reprint requests should be addressed. 2379 Downloaded by guest on September 24, 2021 2380 Medical Sciences: Erisman et al. Proc. Natl. Acad. Sci. USA 79 (1982)

FIG. 1. Immunohistochemical localization of bombesin in SCC. (A) Hematoxylin/eosin stain of a diethylpyrocarbonate vapor-fixed (26) sample. (x 120.) (B-F) Immunostaining (black areas denoted by arrows) on tissue fixed with parabenzoquinone vapor (25), which givesthe photomicrographs their grayish tone. (B-D) Consecutive serial sections (6 am) immunostained with hCGB antiserum (B), physalaemin antiserum (C), and ACTH antiserum (D) and counterstained with toluidine blue. (x 290.) (E andF) Serial sections (12 gim) immunostained with bombesin (E) andphysalaemin (F) antisera. (x290.)

(100-250 ug of peptide per ml) or normal rabbit serum. Ar- Radioimmunoassays (RIAs). [Tyr8]Bombesin (donated by J. gyrophilia was determined by using the silver impregnation E. Rivier, the Salk Institute, La Jolla, CA) was iodinated and method of Grimelius (29) on Bouin's fixed paraffin sections. purified according to published procedures (30). Antiserum to Downloaded by guest on September 24, 2021 Medical Sciences: Erisman et al. Proc. Natl. Acad. Sci. USA 79 (1982) 2381 bombesin was raised in rabbits by injecting TPCK-trypsin Immunohistochemistry. Previous studies showed that vapor- (Worthington)-digested bombesin (des-calcitonin and gastrin and J. van study positive immunostaining was observed after incubation Wyk for somatomedin C (both at the University of North Car- with antisera to several peptides and hormones: (i) hCGB was olina, Chapel Hill). The hCG/3 level was determined by En- found in approximately 9-13% of the tumor cells in several docrine Sciences (Tarzana, CA), and human somatotropin fields ofview (Fig. 1B); (ii) physalaemin was observed in 9-11% (hGH, human growth hormone) was measured with RIA kits of the cells (Fig. 1 C and F); and (iii) bombesin occurred in supplied by CIS Radiopharmaceuticals (Bedford, MA). 12-14% of the cells (Fig. 1E). The specific immunoprecipitate The bombesin RIA mixture contained 50 mM sodium phos- formed by these antisera was located in either the same single phate at pH 7.6, 0.025% bovine serum albumin, 0.085% gamma cells or groups ofcells, on the basis oftheir localization in serial globulins, 1% Trasylol, 30 mM 2-mercaptoethanol, 25 mM sections. Groups of 2-15 immunopositive cells were scattered NaCl, 5,000 cpm of "WI-labeled [Tyr8]bombesin, antiserum unevenly throughout the specimen. Cells containing ACTH- (BM-XII-165-4; 1:4,000 final dilution), and either bombesin like immunoreactivity were infrequent and did not occur in the standards (Bachem Fine Chemicals, Torrance, CA) or extract same region as cells immunopositive for bombesin, physalae- in a final volume of 100 ,1. Incubation ofassay mixtures in poly- min, or hCG3 (Fig. 1C). No immunostaining was detected by propylene tubes was complete after 22 hr at 40C and was ter- using antisera to the Mr 16,000 region ofpro-opiomelanocortin minated by adding 0.05 ml of outdated human plasma and 1 ml or to ,3-endorphin, or by using controls of nonimmune serum of 15.6% polyethylene glycol 6000. After centrifugation of the or antisera previously adsorbed with excess peptide. samples and aspiration of the supernatants, the radioactivities RIA of Peptides. Further characterization of the tumor in- in the pellets were measured. The background in the absence cluded RIAs for ectopic products normally associated with this of antiserum or antiserum in the presence of excess bombesin neoplasm (2, 5, 7, 8, 35, 36); e.g., the levels ofimmunoassayable (60 pmol) was 3.61 ± 1.54% ofthe total cpm (mean ± SD). Our ACTH, calcitonin, and hCGJ3 were 10.5, 0.18, and 410 pmol/ bombesin antiserum recognized the region between residues g of tissue, respectively. The amount of physalaemin-like im- Leu4 and Gln-7 due to the slight crossreactivity with litorin munoreactivity was low, about 0.8 pmol/g; analyses of hGH, (0.14%) and substance P (0.00027%). Other peptides (physa- gastrin, and somatomedin C were consistently negative, in laemin, neurotensin, and bradykinin) were ineffective in com- agreement with an earlier report (3). The level of immuno- petition in amounts as high as 10 nmol. The sensitivity of the reactive bombesin, 5.81 and 7.13 pmol/g in the SCC samples, assay (50% displacement) was 21.1 ± 9.6 pg (12.5 ± 5.7 fmol). on the other hand, was similar to that ofACTH. Bombesin im- Chromatography. The lyophilized extract of SCC was redis- munoreactivity from both the initial acid extract and partially solved in 0.1 M acetic acid/i mM EDTA and clarified by cen- purified material produced displacement curves parallel to the trifugation; 52% of the extract remained insoluble and was dis- curve for bombesin (Fig. 2). carded. Aliquots of that solution were fractionated on a Chromatographic Characterization of Bombesin Immuno- Sephadex G-75 column (1.5 X 90 cm) equilibrated in acid/ reactivity. An aliquot of resolubilized SCC was initially frac- EDTA at4°C. Other samples were resolubilized in 1.0 M formic tionated on Sephadex G-75 in order to detect high molecular acid at 450C and clarified by centrifugation, and 150 ,ul of this weight forms of bombesin, as described for ovine hypothalami solution was chromatographed on Bio-Gel P-4 (100-200 mesh; (14), rat stomach, intestine (13, 17), and plasma (13), porcine 0.7 x 49cm; Bio-Rad) (23, 24). Aliquots ofthe 0.25-ml fractions nonantral gastric mucosa (18), and chicken proventriculus (20); were immunoassayed directly. Samples were injected into we found only a single species of immunoreactive material that either a Spherisorb octadecylsilica (ODS) 5-,um or a Whatman had the same elution volume as bombesin (data not shown). ODS 5-gm column, using Laboratory Data Control HPLC units Further analysis on Bio-Gel P-4 confirmed the presence of an fitted with Rheodyne injectors, and were eluted with linear immunoreactive peptide that coincided with the elution volume gradients of one of the following solvent systems: (a) 20-75% (vol/vol) methanol in 0.01 M Tris HCl, pH 7.0, (b) 0-80% (vol/ Volume, ,4 vol) acetonitrile in 0.01 M heptafluorobutyric acid (32), or (c) 0 0.1 1.0 10 0-60% (vol/vol) acetonitrile in 0.02 M sodium phosphate, pH 6.97. After a 5-min delay, fractions were collected at 20-sec to 90 1-min intervals at a flow rate of 0.8-1.0 ml/min. A blank col- umn run was performed before each analysis of tumor extract; 80 70 these controls established a true background for the RIA and o 60 ruled out any possible contamination of the injector, precol- IrD- 50 umn, or metal/Teflon surfaces by either the extract or the syn- x 40 thetic bombesin standard. 1- 30 20 RESULTS Electron microscopy of this human lung SCC propagated in athymic mice revealed the high nucleus-to-cytoplasm ratio typ- ical ofthese tumors (11, 33, 34). Very few, if any, small dense- cored vesicles were identified in the cells, and the cytoplasm was essentially free ofrough endoplasmic reticulum. At the light Bombesin, pg microscope level, SCC was composed of small fusiform cells (Fig. IA). Silver impregnation indicated scattered groups of FIG. 2. RIA for bombesin. The initial SCC extract (A), column frac- 2-10 argyrophilic cells that account for about 10% of the total tion (0), and a bombesin standard (e) were assayed. B, labeled bom- cell population in the tumor. besin bound; Bo, labeled bombesin bound in the absence of competitor. Downloaded by guest on September 24, 2021 2382 Medical Sciences: Erisman et al. Proc. Natl. Acad. Sci. USA 79 (1982)

O 150 0 0 '-

e0 100 0 I-- Un C.)x

- a0 50 4.) C-t C.)

'4-4 ._ C.) 50 ax ctJ tU 'U r. 20 40 60 80 0 Fraction

FIG. 3. Molecular sieving of SCC extract. A 150-,ul aliquot (5-24 mg) was chromatographed on Bio-Gel P-4 (23, 24). The numbered ar- rows denote markers: void volume V., bovine serum albumin; 1, 125I- labeled [Tyr5]bombesin; 2, 125I-labeled physalaemin; 3, 125I-labeled physalaemin-(1-8)-octapeptide. The bar graph is iodinated bombesin and o denotes immunoreactivity. The total volume Vt corresponds to fraction 142.

ofbombesin (Fig. 3). Reverse-phase HPLC, on the other hand, 35 40 45 separated the bombesin immunoreactive material into three Retention time, min discrete components. In solvent systems a and b with 1-min fractions, the retention times were 43, 45, and 49-50 min, and FIG. 4. Reverse-phase HPLC of SCC extract and synthetic bom- 30, 33, and 36 min, respectively. In solvent c (Fig. 4) with 20- besin. An aliquot (50 .D) of formic acid-solubilized extract was injected sec the three immunoreactive at onto a Spherisorb octadecylsilica 5-/Am column in 0.02 M sodium phos- resolution, peaks emerged phate, pH 6.97, and eluted with a linear gradient of 0-60% acetonitrile 36:20-36:40, 39:00-39:40, and 43:20-43:40 min, respectively. in buffer at a flow rate of 1 ml/min, and fractions were collected at 20- The last peak coincided with the appearance of the synthetic sec intervals. Immunoreactivity in the blank column, between 25- and amphibian bombesin in the three solvents used. 50-min retention time, averaged 17.7 pg per fraction, with no peaks corresponding to those observed in eitherthe tumor extract or the bom- DISCUSSION besin standard peptide. The major conclusions that can be drawn from our data are that (i) immunoreactive bombesin was present in a SCC resected account for the immunopositive staining ofbombesin in a small from a male patient at a level comparable to ACTH (refs. 2-4, percentage of the tumor cells. Alternately, the absence of im- our data), which is a common ectopic hormone in these tumors munostaining may indicate that a peptide is either below the (1, 2, 5, 7) and (ii) this molecule appeared to be chromatograph- level of the threshold for detection by immunohistochemical ically similar to the amphibian peptide. To verify these conclu- techniques or absent altogether. sions, we applied several of the criteria required for the as- Molecular sieving confirmed that the immunoreactivity in sessment of ectopic tumor products (7): RIA, immunohis- the tumor extracts resided in a relatively small peptide (Fig. 3). tochemistry, gel filtration chromatography, and reverse-phase HPLC analyses demonstrated that these extracts contained a HPLC. peptide that appeared to be very similar to amphibian bom- RIAs detected nanogram levels ofboth bombesin and ACTH besin. The superposition of the retention profiles on HPLC of in extracts of the tumor. This was confirmed by immunohis- one peak ofimmunoreactive bombesin from the tumor with that tochemical localization of the peptides in serial sections of the of synthetic bombesin argues against any major differences in tissue, which suggested that the same cell, or group of cells, structure because HPLC offers a high degree of resolution of contain bombesin as well as hCGf3 and physalaemin-like im- limited differences in a peptide, such as the separation ofpep- munoreactivity, whereas ACTH was found in different cells tides differing by only one D amino acid isomer replacement (Fig. 1). The presence of multiple peptide hormones in the tu- (40) or its sulfoxide derivative (41). The heterogeneity of im- mor could be accounted for by the apparent heterogeneity of munoreactivity in the tumor extracts revealed by HPLC may SCC of the lung (37), which contains numerous foci with the be due to the presence of an oxidized methionine residue (un- appearance ofcells at different stages ofdifferentiation (33). This published data). diversity in biochemical (8) and chromosomal variability (34), It is also interesting from an evolutionary viewpoint that a also observed during the establishment ofdistinct cell lines from mammalian tissue would contain a peptide resembling an am- the tumor (34, 38, 39), may account for the appearance ofectopic phibian peptide and that this peptide, expressed only in fetal/ peptides. Because the expression ofhormones may also change neonatal lung tissue (21), would reappear in an adult lung neo- during passage ofthe tumor in nude mice (4), this could possibly plasia. The reappearance of fetal isozymes in cancerous adult Downloaded by guest on September 24, 2021 Medical Sciences: Erisman et aL Proc. Natl. Acad. Sci. USA 79 (1982) 2383

tissues is a well-established observation. The tumor might rep- 18. McDonald, T. J., Jornvall, H., Nilsson, G., Vagne, M., Ghatei, resent a reversion to a biochemical state present in prenatal M., Bloom, S. R. & Mutt, V. (1979) Biochem. Biophys. Res. Com- tissue one of several mechanisms (7), including the process- mun. 90, 227-233. by 19. Moody, T. M., Thoa, N. B., O'Donohue, T. L. & Pert, C. B. ing of an inactive precursor, thereby enabling the reexpression (1980) Life Sci. 26, 1707-1712. of a peptide hormone. 20. Melchiorri, P. (1980) in Gastrointestinal Hormones, ed. Glass, In mammals, bombesin elicits numerous pharmacological G. B. J. (Raven, New York), pp. 717-725. and physiological effects (9, 10), several of which resemble the 21. Wharton, J., Polak, J. M., Bloom, S. R., Ghatei, M. A., Solcia, clinical symptoms reported for patients with SCC (1, 2, 5, 11). E., Brown, M. R. & Pearse, A. G. E. (1978) Nature (London) For example, the marked reduction in the absorption of water 273, 769-770. 22. Reid, L. M., Holland, J., Jones, C., Wolf, B., Niwayama, G., and electrolytes in the intestine due to bombesin (10) could Williams, R., Kaplan, N. 0. & Sato, G. (1978) in Proceedings of contribute to hyponatremia (5, 11), generally assumed to be the Symposium on the Use of Athymic (Nude) Mice in Cancer caused by inappropriate secretion ofantidiuretic hormone. Sus- Research, eds. Houchens, D. P. & Ovejera, A. A. (Fischer, New tained clinical effects could then result from the constant low- York), pp. 107-121. level secretion of a bioactive form of bombesin, such as found 23. Lazarus, L. H. & DiAugustine, R. P. (1980) AnaL Biochem. 107, infusion studies in human subjects (42, 43), as well as 350-357. during 24. Lazarus, L. H., Linnoila, R. I., Hernandez, 0. & DiAugustine, other peptide hormones from the tumor. Once in plasma, bom- R. P. (1980) Nature (London) 287, 555-558. besin could interact with tissue receptor sites (44), thereby 25. Pearse, A. G. E., Polak, M. M., Adams, C. & Kendall, P. A. mediating intracellular changes that stimulate secretion of en- (1974) Histochem. J. 6, 347-352. zymes (44-46), or affect electrical potentials (10, 46), or bring 26. Pearse, A. G. E. & Polak, J. M. (1975) Histochem.J. 7, 179-186. about the release of other peptide hormones, such as insulin 27. Petrusz, P., DiMeo, P., Ordronneau, P., Weaver, C. & Keefer, 48), glucagon (48), gastrin (9, 10, 42, 43, 49), and prolactin D. A. (1975) Histochemistry 46, 9-26. (47, 28. Lazarus, L. H., DiAugustine, R. P., Khan, M. N., Jahnke, G. D. and GH (50). With the growing evidence of ectopic secretion & Erisman, M. D. (1981) Clin. Chem. 27, 549-552. of peptides by SCC and other neoplasms (6, 7, 11), we suggest 29. Grimelius, L. (1968) Acta Soc. Med. Ups. 73, 243-270. that bombesin should be considered among the various peptides 30. Lazarus, L. H., Perrin, M. H. & Brown, M. R. (1977) J. Biol causing endocrinologic abnormalities in patients with lung tu- Chem. 252, 7174-7179. mors. Furthermore, bombesin might then serve as a potential 31. Goodfriend, T., Levine, L. & Fasman, G. D. (1964) Science 144, marker in the screening of patients with SCC. 1344-1356. peptide 32. Bennett, H. P. J., Browne, C. A. & Solomon, S. (1981)J. Liquid After submission of this article, Moody et al. (51) and Wood Chromatogr. 3, 1353-1365. et aL (52) presented data that cell lines derived from SCC and 33. Mackay, B., Osborne, B. M. & Wilson, R. A. (1977) in Lung Can- tumor samples from patients, respectively, contain bombesin- cer, ed. Straus, M. J. (Harcourt, Brace, Jovanovich, New York), like immunoreactivity. pp. 71-84. 34. O'Hara, H. & Okamoto, T. (1977) Cancer Res. 37, 3088-3095. 35. Roos, B. A., Lindall, A. W., Baylin, S. B., O'Neil, J., Frelinger, We acknowledge and appreciate the skilled technical assistance of A. L., Birnbaum, R. S. & Lambert, P. W. (1979) Endocr. Res. J. Frushstick, G. D. Jahnke, C. M. Soldato, and F. Talley, and the in- Commun. 6, 169-190. valuable comments of the reviewers. M.D.E. was supported by the 36. Rosen, S. W., Weintraub, B. D., Vaitukaitis, J. L., Sussman, H., American Lung Association. Hershman, J. M. & Muggia, F. M. (1975) Ann. Intern. Med. 82, 71-83. 37. Abeloff, M. D., Eggleston, J. C., Mendelsohn, G., Ettinger, D. 1. Greco, F. A. & Oldham, R. K. (1979) N. Engl. J. Med. 301, & Baylin, S. B. (1979) Am. J. Med. 66, 757-764. 355-358. 38. Baylin, S. B., Abeloff, M. D., Goodwin, G., Carney, D. N. & 2. Rees, L. H., Bloomfield, G. A., Rees, G. M., Corrin, B., Gazdar, A. F. (1980) Cancer Res. 40, 1990-1994. Franks, L. M. & Ratcliffe, J. G. (1974)J. Clin. Endocrinol Metab. 39. Lieblich, J. M., Weintraub, B. D., Krauth, G. H., Kohler, P. 38, 1090-1097. O., Rabson, A. S. & Rosen, S. W. (1976) J. Nati Cancer Inst. 56, 3. Gewirtz, G. & Yalow, R. S. (1974)J. Clin. Invest. 53, 1022-1032. 911-917. 4. Shimosato, Y., Kameya, T., Nagai, K., Hirohashi, S., Koide, T., 40. Rivier, J. & Burgus, R. (1979) in Biological/Biomedical Applica- Hayashi, H. & Nomura, T. (1976) J. Natl Cancer Inst. 56, tions ofLiquid Chromatography, ed. Hawk, G. L. (Dekker, New 1251-1260. York), pp. 147-161. 5. Jones, A. W. (1978) Chest, Heart, Stroke J. 3, 16-24. 41. Floor, E. & Leeman, S. E. (1980) AnaL Biochem. 101, 498-503. 6. Sherwood, L. M. (1979) in Contemporary Endocrinology, ed. 42. Melchiorri, P. (1978) in Gut Hormones, ed. Bloom, S. R. Ingbar, S. H. (Plenum, New York), Vol. 1, pp. 341-386. (Churchill-Livingstone, Edinburgh), pp. 534-540. 7. Baylin, S. B. & Mendelsohn, G. (1980) Endocr. Rev. 1, 45-77. 43. Varner, A. A., Modlin, I. M. & Walsh, J. H. (1981) Regul Pept. 8. Rees, L. H. (1976) Clin. Endocrinoi 5, 363s-372s. 1, 289-296. 9. Erspamer, V. & Melchiorri, P. (1973) Pure Appl Chem. 35, 44. Jensen, R. T., Moody, T., Pert, C., Rivier, J. E. & Gardner, J. 463-494. D. (1978) Proc. Natl Acad. Sci. USA 75, 6139-6143. 10. Erspamer, V. (1980) in Gastrointestinal Hormones, ed. Glass, G. 45. Deschodt-Lanckman, M., Robberecht, P., de Neef, P., Lam- B. J. (Raven, New York), pp. 343-361. mens, M. & Christophe, J. (1976) J. Clin. Invest. 58, 891-898. 11. Shields, T. W. & Ritts, R. E. (1974) Bronchial Carcinoma 46. Iwatsuki, K. & Petersen, 0. H. (1978) J. Clin. Invest. 61, 41-46. (Thomas, Springfield, IL). 47. Kaneto, A., Kaneto, T., Nakaya, S., Kajinuma, H. & Kosaka, K. 12. Polak, J. M., Hobbs, S., Bloom, S. R., Solcia, E. G. & Pearse, (1978) Metabolism 27, 549-553. A. G. E. (1976) Lancet i, 1109-1110. 48. Ipp, E. & Unger, R. H. (1979) Endocr. Res. Commun. 6, 37-42. 13. Brown, M., Allen, R., Villarreal, J., Rivier, J. & Vale, W. (1978) 49. Delle Fave, G., Kohn, A., de Magistris, L., Mancuso, M. & Life Sci. 23, 2721-2728. Sparvoli, C. (1980) Life Sci. 27, 993-999. 14. Villarreal, J. A. & Brown, M. R. (1978) Life Sci. 23, 2729-2734. 50. Rivier, C., Rivier, J. & Vale, W. (1978) Endocrinology 102, 15. Dockray, G. J., Vaillant, C. & Walsh, J. H. (1979) Neuroscience 519-522. 4, 1561-1568. 51. Moody, T. W., Pert, C. S., Gazdar, A. F., Carney, D. N. & 16. Moody, T. W. & Pert, C. B. (1979) Biochem. Biophys. Res. Com- Minna, J. D. (1981) Science 214, 1246-1248. mun. 90, 227-233. 52. Wood, S. M., Wood, J. R., Ghatei, M. A., Lee, Y. C., 17. Walsh, J. H., Wong, H. C. & Dockray, G. J. (1979) Fed. Proc. O'Shaughnessy, D. & Bloom, S. R. (1981) J. Clin. EndocrinoL Fed. Am. Soc. Exp. Biot 38, 2315-2319. Metab. 53, 1310-1312. Downloaded by guest on September 24, 2021