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Specific Binding to Rat Brain Membranes (Receptor Binding/Neuropeptide/Neuropharmacology) DUNCAN P Proc. Natl. Acad. Sci. USA Vol. 76, No. 2, pp. 660-664, February 1979 Biochemistry Vasoactive intestinal polypeptide: Specific binding to rat brain membranes (receptor binding/neuropeptide/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 Peptide. 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. Secretin 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 insulin (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 atropine-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.
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