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Proc. Natl. Acad. Sci. USA Vol. 76, No. 2, pp. 660-664, February 1979 Biochemistry Vasoactive intestinal polypeptide: Specific binding to rat brain membranes ( binding//neuropharmacology) DUNCAN P. TAYLOR AND CANDACE B. PERT* Section on Biochemistry and Pharmacology, Biological Psychiatry Branch, National Institute of Mental Health, Bethesda, Maryland 20014 Communicated by Elizabeth F. Neufeld, November 9, 1978

ABSTRACT The binding of radiolabeled vasoactive intes- (Cleveland, OH) and J. Gardner. VIP18-28 was also received tinal polypeptide (VIP) to rat brain membranes was investigated. from G. Makhlouf (Richmond, VA). Other analogs were ob- Specific binding of 125I-labeled VIP was reversible and saturable (Bmax = 2.2 pmol/g of wet tissue). Brain membranes exhibited tained courtesy of J. Gardner. a high affinity for 1251-labeled VIP (KD = 1 nM) at a single class Preparation of Radiolabeled . Natural porcine VIP of noninteracting sites. Binding of 125I-labeled VIP paralleled was radiolabeled by the chloramine-T procedure as described its immunohistochemical localization, being enriched in cere- (26). Na'251 was from New England Nuclear; chloramine-T bral cortex, hippocampus, striatum, and thalamus, with the from Eastman; sodium metabisulfite from Fisher. The specific notable exception of the hypothalamus, which had low levels activity of the iodinated peptide was 700-900 Ci/mmol. of binding. The density of sites was greater in synaptosomal Preparation of Membranes. Adult male Sprague-Dawley fractions relative to mitochondrial or nuclear fractions. and partial sequences of it and VIP inhibited binding to brain rats (175-200 g) were decapitated, and the brains were dis- membranes with an order of potency similar to that found in sected. The medulla, pons, cerebellum, and midbrain, which other systems. The findings suggest the existence of a unique contained a minimum of receptor sites, were routinely re- new class of brain receptors. moved. The remaining brain was weighed, homogenized in 100 vol of 50 mM Tris-HCI (pH 7.5) at 4°C with a Brinkmann Vasoactive intestinal polypeptide (VIP), an octacosapeptide Polytron (setting 5 for 15 sec), held on ice for 20 min, and isolated from porcine intestine (1), induces vasodilation in centrifuged at 39,000 X g for 20 min. The resulting pellet was various vascular beds (for a review, see ref. 2) and relaxes resuspended in 10 vol of 50 mM Hepes-KOH (pH 7.4), con- smooth muscle (3). VIP has mixed effects on secretory processes, taining 10 mg of bovine serum albumin per ml (Cohn Fraction inhibiting gastric acid secretion (4) while stimulating small V, Sigma). In assays in which protein levels were to be deter- intestinal secretion (5), biliary secretion (6), pancreatic release mined, by the method of Lowry et al. (27), membranes were of (7), and colonic ion transport (8). The immunohisto- resuspended in 50 mM Tris-HCl (pH 7.5) at 4°C, and bovine chemical localization of VIP in peripheral and central nervous serum albumin was used as a standard. tissues (9-21) suggests a possible role for VIP in neuro- Subcellular fractions were prepared as described (28). transmission which is supported by findings that VIP is localized Fractions from the crude mitochondrial pellet (P2) were pel- in the synaptosomal fractions of rat and dog brains (15, 19). leted in 0.32 M sucrose/50 mM Tris-HCl, pH 7.5, at 39,000 X Moreover, treatment of crude synaptosomes from cerebral g for 20 min. All pellets were resuspended in 50 mM Tris-HCl cortex or synaptic vesicles from hypothalamus with 50 mM (pH 7.5) at 4°C, as above. potassium results in the release of VIP immunoreactivity (15, Binding Assay. Routinely, 100 Al of freshly prepared 19). Further, electrical stimulation of vagal nerves in pigs results membranes (0.6 mg of protein) was incubated with 30-90 fmol in an elevation in plasma levels of VIP that is -resistant of 1251-VIP at 370C for 20 min in the presence or absence of (22). Finally, VIP is capable of exciting neurons from the rat competitor. The buffer was 5 mM MgCl2 in 50 mM Hepes- sensory motor cerebral cortex (23). The latency of excitation, KOH, pH 7.4. Ten milligrams of bovine serum albumin, 0.2 ,ug from 10 sec to more than a minute, is compatible with the of bacitracin (Sigma), and 3270 kallikrein inhibitor units of possible mediation of VIP's effect by adenosine 3',5'-cyclic aprotinin (Sigma) were added per ml to prevent sticking of the monophosphate. VIP stimulates accumulation of this nucleotide peptide to vessel walls and to inhibit proteases. The total volume in rat brain slices (24) and stimulates adenylate cyclase activity was 0.3 ml. in membrane preparations from guinea pig brain (25). Membrane-bound 125I-VIP was separated from free peptide We have undertaken an investigation of the VIP receptor in by centrifugation. During incubation, aliquots were removed the brain. Thus, the data presented here indicate that 125I- from the incubation mixture and layered over 1.3 ml of a labeled VIP (125I-VIP) binds with high affinity to a site on rat warmed solution of 0.32 M sucrose, 50 mM Hepes-KOH (pH brain membranes. 7.4), 5 mM MgCl2, and 10 mg of bovine serum albumin per ml. At the termination of incubation, the aliquots were centrifuged at 6500 X g for 1 min in an Eppendorf microcentrifuge (model MATERIALS AND METHODS 5411). The supernatants were rapidly aspirated and discarded, VIP and Analogs. Purified porcine VIP was graciously and the pellets were rinsed with an additional 1 ml of warm provided by J. Gardner (Bethesda, MD) and V. Mutt (Stock- sucrose/Hepes/bovine serum albumin/MgCl2 and centrifuged holm, Sweden). Synthetic porcine VIP and VIP1028 were gifts for another minute. The second supernatant was aspirated and from J.-K. Chang (Peninsula Laboratories, Palo Alto, CA). discarded, and the washed pellets were assayed for radioactivity VIP18-28 and secretin15 27 were donated by M. Bodanszky in a Searle gamma counter.

The publication costs of this article were defrayed in part by page Abbreviations: VIP, vasoactive intestinal polypeptide; 125I-VIP, charge payment. This article must therefore be hereby marked "ad- 125I-labeled VIP; IC5o, concentration required for half-maximal in- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate hibition of binding. this fact. *To whom reprint requests should be addressed. 660 Downloaded by guest on September 29, 2021 Biochemistry: Taylor and Pert Proc. Natl. Acad. Sci. USA 76 (1979) 661 in Fig. 2 A and C. From the experiment depicted in Fig. 2A, double-reciprocal plots of total binding and that which occurred in the presence of 0.1 1iM unlabeled VIP (nonspecific binding) 0 gave apparent maximal amounts of binding of 0.68 and 0.28 x fmol of VIP per mg of tissue, respectively. In addition, such plots gave half-lives (t1/2) of 1.5 and 1.0 min, respectively. (Coefficients of determination were 0.95 for total binding and E 0.97 for nonspecific binding.) The curves shown in Fig. 2A have 11 been plotted from these values according to the equation b = C A ; (Bmax-t)/(t + t1/2). Thus, plateaus are reached in total and 0 nonspecific binding by 10 and 5 min, respectively. In this ex- 0. periment the apparent Bmix for specific binding was 0.40 fmol of VIP per mg of tissue. A plot of ln(Bmax/Bmax - B) against time gave a straight line (Fig. 2B), in accordance with a pseudo-first-order reaction (29), with a slope of 0.16 (coefficient of determination = 0.99). The curve for specific binding in Fig. 50 100 150 2A was obtained by plotting the constants from Fig. 2B ac- Membranes, pl per assay cording to the equation b = Bmax (1 - ekt). Specific binding FIG. 1. Binding of 1251-VIP as a function of membrane concen- reached a plateau at 15 min (Fig. 2A). No specific binding was tration in the absence (0) or presence (@) of 0.1 yM VIP. The dif- observed at periods up to 1 hr at 40C (data not shown). ference between the two curves represents the membrane dependence The addition of 0.1 tM unlabeled VIP reversed specific of specific VIP binding (A&). Four percent of the total cpm added ad- binding (Fig. 2C). A plot of In B/Bo against time gave a straight hered to the walls of the vessel in the absence of membranes. When line (Fig. 2D), in accordance with a zero-order reaction (29), 100 pl of membranes was added, the membrane-associated binding with a slope of -0.12 minI (coefficient of determination = of labeled peptide was 30% of the total cpm added, of which 65-70% was displaced in the presence of 0.1 pM unlabeled VIP. Each assay 0.97) and an apparent half-life of 7 min. The curve for the was performed in duplicate; the mean value is indicated. The range dissociation (Fig. 2C) was obtained by using the constant ob- of values was within -5% of the mean; the results of this experiment tained in Fig. 2D according to the equation b = Boe-kt. are representative of two other experiments. By the procedure of Kitabgi et al. (29) the association (k+1 = 2.2 X 106 M-1 sec') and dissociation rate constants (k-1 = RESULTS 2.0 X 10-3 sec-1) were calculated. Based on these rate constants, Binding of Radiolabeled Peptide to Membranes. Fig. 1 the equilibrium dissociation constant (Kd = k-llk+l = 0.9 nM) illustrates that 125I-VIP binds to rat brain membranes as a linear was calculated. function of membrane concentration for total and nonspecific Equilibrium Binding Studies. The concentration depen- binding (binding in the presence of 0.1 tM VIP). In addition, dence of 125I-VIP binding to rat brain membranes was inves- Fig. 1 shows that in the absence of membranes, 250 cpm (4% tigated. Fig. 3 illustrates that specific binding appeared to be of the total cpm added) were adsorbed to the walls of the cen- saturable. Fig. 3, lower inset, shows that a double-reciprocal trifuge tube in the presence or absence of 0.1 AM VIP. We plot of the specific binding data yields a straight line (coefficient routinely used 100 AI of membranes per assay since the ratio of determination = 0.99), suggesting that binding occurs at a of specific binding (total minus nonspecific binding) relative single class of sites. From this plot the equilibrium constant (Kd to tube background was enhanced. Specific binding represented = 1.0 nM) and the maximal number of binding sites (Bmax = 65-70% of total membrane-dependent binding in these ex- 2.2 pmol/g of wet tissue) were calculated. The curve in Fig. 3 periments. (center) was plotted from these values according to the equation Kinetic Binding Data. The time course of 125I-VIP associ- b = Bmax-[LI/[L] + Kd, in which [L] is the ligand concentration. ation to and dissociation from rat brain membranes is shown In addition, VIP appeared to bind to noninteracting sites be-

, 0.3 0 .05D ED _0 l

X,# ECM 0.1. - b

M--00~~~~ ol vN-A 10 15 20 0 5 10 15 5 10 15 Time, min Time, min Time, min Time, min FIG. 2. Time course of 125I-VIP association and dissociation. (A) Binding of0.3 nM 125I-VIP as a function of time in the absence (0) or presence (-) of 0.1 pM unlabeled VIP. The difference between the two represents the time course of specific VIP binding (A). Each assay was performed in triplicate; the mean value is indicated. SEM was less than 3%. The results of this experiment are representative of three others. (B) Replot of specific 1251-VIP binding data obtained in A, with value of 0.40 fmol of VIP per mg of wet tissue as Bmax. The straight line was obtained by linear regression analysis (r2 = 0.99). (C) 125I-VIP (0.1 nM) was incubated with membranes for 20 min. Then 0.1 ,M unlabeled VIP was added, and the amount of 125I-labeled peptide-receptor complex was determined as a function of time. Each assay was performed in duplicate; the mean value is indicated. The range of values was less than 5%. The results of this experiment are representative of three others. Essentially all of the specifically bound 125I-VIP was dissociated by 40 min (not shown). Curve generation is described in the text. (D) Replot of specific 125I-VIP binding obtained in C. The straight line was obtained by linear regression (r2 = 0.97). Downloaded by guest on September 29, 2021 662 Biochemistry: Taylor and Pert Proc. Natl. Acad. Sci. USA 76 (1979)

Table 1. Agents with no effect on '251-VIP binding Receptor agonists Biogenic amines and antagonists A(THI-IO Acetylcholine Atropine o ED I -y-Amino- E E Angiotensin II butyric acid (+)Butaclamol .1.0 oo-1.O D-Aspartic acid Carbamylcholine [D-Ala5l1ombesin 1,-Aspartic acid

0.8 -_ CCK-OP Haloperidol U Dopamine Harmine 0.6 4 0 DALA Epinephrine Hexamethonium 0 ~~~~~~~~~~-~ C * 3 [Leu5JEnkephalin L- Isoproterenol :3 0 00.4 ~~~~~~E2 [Met5 Levallorphan n ~~~~~~~~~~C6 Glucagon Histamine >0.2 Insulin 5-Hydroxy- >0.2 ~~~~~~2~~4 6 8 10 1/F, 1/nM Litorin tryptamine a-MSH Norepinephrine 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Phenoxybenzamine [VIP], nM Phentolamine Fi(t. 3. Binding of 1251-VIP as a function of radiolabeled peptide concentration. Points are the means of triplicate assays; the results Ranatensin Pilocarpine of the experiment are representative of two other experiments. Curve Secretin16 (50 MM) Pimozide generation is described in the text. (Upper Inset) Hill plot of the same Procaine data by using the Bmax of 2.2 fmol/mg of wet tissue obtained in the double-reciprocal plot. The line was generated by linear regression Propranolol analysis (rO = 0.98). (Lower Inset) Double-reciprocal plot of the same TRF data. The line was generated by linear regression analysis (r2 = 0.99). VIPs--(, (50 MM) d-Tubocurarine F = [free ligand]; B = bound ligand. VIP18E28 (50 MM) Substances exhibited no displacement of 1251-VIP at 10 PM. Some cause a Hill plot of the specific binding data (Fig. 3, upper peptides were tested at higher concentrations (given in parentheses). inset) yielded a line with a slope of 0.98 (coefficient of deter- ACTH, adrenocorticotropic hormone; CCK-OP, COOH-terminal mination = 0.98). octapeptide of cholecystokin; DALA, [I)-Ala2,Met5lenkephalinamide; Inhibition of 125I-VIP Binding. The ability of a wide variety (Y-MSH, aY-melanocyte-stimulating hormone (melanotropin); TRF, of , putative , and brain-re- thyrotropin-releasing factor (thyroliberin). ceptor antagonists to inhibit specific 125I-VIP binding was in- vestigated. Inhibition of 125I-VIP binding by native or synthetic VIP was half-maximal (IC50) at 4 nM and was abolished at 1 AtM probit analysis). The synthetic COOH-terminal sequence (Fig. 4). Natural secretin inhibited specific VIP binding 1/100th VIPIo-28 inhibited 125I-VIP binding only 1/1000th as effectively as effectively as native VIP (IC50 = 600 nM). The synthetic as the intact peptide (IC50 = 45 ,uM). The other partial se- COOH-terminal sequences of secretin inhibited 125I-VIP quences of VIP, either the NH2-terminal VIPI-10 or the binding at very high concentrations. The most potent of these COOH-terminal VIP18-28, as well as secretinl 6, glucagon, the was [Gln9, Asnl5]secretin5-27 (IC50 = 25 AtM), while the un- COOH-terminal octapeptide of , other neuro- substituted fragment, secretin5-27, had a potency similar to the peptides, putative neurotransmitters, and brain-receptor agents, shorter secretin sequences, secretin1427 and secretinl527 (IC50 do not inhibit binding of the high-affinity '25I-VIP binding site values of 0.2 mM, 0.3 mM, and 0.8 mM, respectively, by log- (see Table 1). Regional Distribution of VIP Receptor. The regional dis- o tribution of 1251-VIP binding sites was investigated. Table 2 100 CD C -C Table 2. Regional distribution of specific 1251-VIP binding fl 75 in rat brain IL activity 1251-VIP specifically bound, o 500 Region fmol/mg protein U Striatum 10.0 ± 1.2 ± O 25 Hippocampus 8.6 1.6 Ole~~~~~~~ Cortex 7.5 ± 0.8 Thalamus 6.7 ± 1.0 Midbrain 3.2 ± 0.6 10 9 8 7 6 5 4 Hypothalamus 1.1 i 0.6 -log [peptidel, M Cerebellum <0.7 <0.7 FiG. 4. Inhibition of 1251-VIP binding by various peptides. The Medulla/pons percent of '251-VIP bound specifically is plotted as a function of un- Regions from fresh rat brains were dissected on ice and weighed labeled peptide concentration for VIP (0), secretin (0), [Gln9, and membranes were prepared. Binding assays were performed in Asnl5lsecretin5-27 (A), VIPIO 28 (v), secretin5g27 (A), secretin1427 triplicate with membranes from each brain region and 0.3 nM 1251- (*), and secretinl5-27 (0°)- VIP. Means ± SEM of four separate experiments are indicated. Downloaded by guest on September 29, 2021 Biochemistry: Taylor and Pert Proc. Natl. Acad. Sci. USA 76 (1979) 663 librium binding studies (Kd = 1.0 nM), and competition studies (Kd = 4.0 nM) agree. Nanomolar dissociation constants for specific VIP binding have been obtained with rat fat-cell plasma membranes (31), liver plasma membranes (31-33), enterocytes from rat small intestine (34), a cell line from a human colon carcinoma (35), and cat pancreatic membranes (33). In addition, VIP at nanomolar levels effects half-maximal stimulation of adenosine 3',5'-cyclic monophosphate accu- mulation in rat fat-cell plasma membranes (31) and stimulates adenylate cyclase half-maximally in rat fat-cell plasma mem- branes (36), in membranes from murine islets of Langerhans (37), and in membranes from human colon adenocarcinoma (38). Monovalent cations have no effect at physiological concen- trations on the affinity of the receptor for 1251-VIP, whereas divalent cations increase binding affinity. The addition of or- ganic solvents to aqueous solutions of VIP, which possibly dis- rupts the intrachain linkages, resulted in an increase of helical character, as determined by optical rotatory dispersion (39). It may be that divalent cations alter the conformation of the [Salt], mM peptide and/or the receptor or alter the electrostatic interactions between them. FIG. 5. Binding of 125IVIP as a function of salt concentration. VIP and secretin are similar in terms of chemical structure Membranes were incubated with 1251-VIP (0.3 nM) for 20 min in the presence of various concentrations of MgCl2 (0). MnCl2 (0), CaCI2 (40) and spectrum of biological activity (41). In our hands se- (A), NaCi (3), and KCI (-). Incubations were terminated by cen- cretin is bound with less than 1% the affinity of VIP, in agree- trifugation of aliquots through 1.3 ml of a buffer containing 50 mM ment with studies by others of binding sites in pancreatic acinar Tris-HCI (pH 7.5), 0.32 M sucrose, and 10 mg of bovine serum albumin cells (26) and liver and fat-cell membranes (31, 36). It has been per ml. The pellets were washed once with the same buffer and ra- suggested that VIP may contain two independent "command dioactivity was determined. sequences," one located in the NH2-terminal sequence and the other in the COOH-terminal sequence (42). We were unable shows that the highest density of sites was found in striatum. to obtain any inhibition of 12-I-VIP binding by concentrations The medulla/pons and cerebellum had no significant level of of VIPI-10 or VIP182s as high as 50 ,4M, and similar results were specific binding. Intermediate receptor densities were observed found in pancreatic acinar cell (26) and liver plasma mem- in the hippocampus, cortex, thalamus, midbrain, and hypo- branes (6). These fragments have biological activities at least thalamus. Values represent determinations at a single ligand 1/5000th that of the complete peptide in several systems (4, 6, concentration (0.3 nM) rather than Bmax; they are proportional 40, 42-45). Partial sequences of secretin were much less effi- to total receptor number only if the Kd values are very similar cient as inhibitors of 125I-VIP binding compared to the intact in all regions. peptide. A rank order of potency for secretin fragments similar Miscellaneous Binding Data. The subcellular distribution to ours (secretin5-27 > secretin14 27 > secretin15-27 > secretin1-6) of '251-VIP binding sites was investigated. The total number was found in studies of VIP binding and adenosine 3',5'-cyclic and density of specific 125I-VIP binding sites was greater in the monophosphate accumulation in pancreatic acinar cells (26, P2 than P1 fraction as well as the B than C fraction (data not 44). The replacement of the negatively charged glutamyl-9 and shown). Gray and Whittaker (28) observed that the P2 fraction aspartyl-15 residues with their neutral amides in the secretin5 27 contains crude mitochondria and fraction B is composed of fragment resulted in a 7-fold increase in inhibitory potency synaptosomes, whereas the PI fraction contains nuclear debris compared to the unsubstituted fragment. A similar increase was and the C fraction is composed of mitochondria. This subcel- observed in studies on pancreatic acinar cells (46). The helical lular localization is consistent with the possible association of structure of the disubstituted secretins527 fragment, as revealed receptors with membranes derived from nerve terminals. by circular dichroism, is comparable to that of VIP (47), while The effects of various cations were investigated. Fig. 5 shows those of shorter VIP sequences are not (39). Therefore, the in- that specific 125I-VIP binding was doubled by millimolar creased potency of the disubstituted fragment indicates that concentration of CaCl2 and MnCI2 and almost tripled by 5 mM charge as well as conformation may be important in the VIP- MgCl2. In contrast, NaCl and KCI had no significant effect on receptor interaction. specific binding at concentrations up to 100 mM. Other neuropeptides, putative neurotransmitters, and brain-receptor agents have low affinity for the VIP receptors DISCUSSION studied here (Table 1). Similarly, the regional distribution of This study indicates that rat brain membranes bind radiolabeled the VIP receptor (Table 2) differs from that of the opiate (48), VIP with high affinity. The binding is specific, saturable (2.2 muscarinic cholinergic (49), fl-adrenergic (50), pmol of sites per g of wet tissue), and reversible. I25I-VIP binds (51), neurotensin (52), bombesin (30), and carnosine (53) re- with high affinity to a single class of noninteracting sites. The ceptors. These data suggest that 125I-VIP binds to a unique class rate constants for association and dissociation (k+l = 2.2 X 106 of brain receptors. M-1 sec-1, k-1 = 2.0 X 10-3 sec') of 125I-VIP to rat brain The density of receptor sites does not always parallel the membranes are similar to those obtained for the neuropeptides distribution of VIP immunoreactivity in the brain (12). Whereas bombesin (k+l = J.8 X 106 M-' sec1, k-1 = 1.7 X 10-3 sec-1; pons, medulla, cerebellum, and midbrain are low in both VIP ref. 30) and neurotensin (k+l = 1.8 X 106 M-' sec1, kI = 0.8 content and receptor density and hippocampus and cortex are X 10-3 sec1; ref. 29). The equilibrium dissociation constants enriched in both, the hypothalamus has a relatively high ratio calculated from kinetic binding studies (Kd = 0.9 nM), equi- of VIP to receptor. One possible explanation is that neurons with Downloaded by guest on September 29, 2021 664 Biochemistry: Taylor and Pert Proc. Natl. Acad. Sci. USA 76 (1979)

cell bodies containing VIP in the hypothalamus terminate in 25. Deschodt-Lanckman, M., Robberecht, P. & Christophe, J. (1977) other regions of the brain. The possibility that layer I of the FEBS Lett. 83, 76-80. neocortex, which is devoid of cell bodies and highly enriched 26. Christophe, J. P., Conlon, T. P. & Gardner, J. D. (1976) J. Biol. in VIP receptor sites (unpublished data), represents the terminal Chem. 251,4629-4634. field of a VIPergic hypothalamic projection is under investi- 27. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. gation. The behavioral significance of VIP receptors clustered 28. Gray, E. G. & Whittaker, V. P. (1962) J. Anat. 96,79-86. in the various brain loci has barely begun to be explored 29. Kitabgi, P., Carraway, R., van Rietschoten, J., Granier, C., (54). Morgat, J. L., Menez, A., Leeman, S. & Freychet, P. (1977) Proc. Natl. Acad. Sci. USA 74, 1846-1850. Note Added in Proof. After this paper was submitted, a report of 30. Moody, T. W., Pert, C. B., Rivier, J. & Brown, M. R. (1978) Proc. specific binding of VIP to synaptosomal preparations of guinea pig Natl. Acad. Sci. USA 75,5372-5376. forebrain appeared (55). 31. Bataille, D., Freychet, P. & Rosselin, G. (1974) Endocrinology 95,713-721. We are indebted to Dr. J. Gardner for his helpful comments. D.P.T. 32. Laburthe, M., Bataille, D. & Rosselin, G. (1977) Acta Endocrinol. is the recipient of a National Institute on Drug Abuse National Research 84,588-599. Service Award. 33. Bataille, D., Besson, J., Bastard, C., Laburthe, M. & Rosselin, G. (1977) in First International Symposium on Hormonal Recep- 1. Said, S. I. & Mutt, V. (1970) Science 169, 1217-1218. tors in Digestive Tract Physiology, eds. Bonfils, S., Fromageot, 2. Said, S. I. (1975) in Gastrointestinal Hormones, ed. Thompson, P. & Rosselin, G. (Elsevier, Amsterdam), pp. 113-125. J. C. (University of Texas Press, Austin, TX), pp. 591-597. 34. Laburthe, M., Besson, J., Hui Bon Hoa, D. & Rosselin, G. (1977) 3. Piper, P. J., Said, S. I. & Vane, J. R. (1970) Nature (London) 225, C. R. Hebd. Seances Acad. Sci. Ser. D 284, 2139-2142. 1144-1146. 35. Laburthe, M., Rousset, M., Boissard, C., Chevalier, G., Zweibaum, 4. Makhlouf, G. M., Zfass, A. M., Said, S. I. & Schebalin, M. (1977) A. & Rosselin, G. (1978) Proc. Natl. Acad. Sci. USA 75, 2772- Proc. Soc. Exp. Biol. Med. 157,565-568. 2775. 5. Barbezat, G. 0. & Grossman, M. I. (1971) Science 174, 422- 36. Desbuquois, B., Laudat, M. H. & Laudat, P. (1973) Biochem. 424. Biophys. Res. Commun. 53,1187-1194. 37. Frandsen, E. K. & Moody, A. J. (1977) in First International 6. Makhlouf, G. M., Said, S. I. & Yau, W. M. (1974) Castroenter- Symposium on Hormonal Receptors in Digestive Tract Physi- ology 66,737 (abstr.). ology, eds. Bonfils, S., Fromageot, P. & Rosselin, G. (Elsevier, 7. Schebalin, M., Said, S. I. & Makhlouf, G. M. (1977) Am. J. Physiol. 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