Proc. Nati. Acad. Sci. USA Vol. 74, No. 8, pp. 3424-3428, August 1977 Cell Biology Vasoactive intestinal polypeptide in brain: Localization in and release from isolated nerve terminals (synaptosomes/neurotransmitters/hypothalamic, cortical peptides/gastrointestinal hormones/K+) ANTONIO GIACHETTI*, SAMI I. SAID*tf, ROLLAND C. REYNOLDS§, AND FRANK C. KONIGESt Departments of * Pharmacology, * Internal Medicine, and § Pathology, University of Texas Health Science Center, Dallas, Texas 75235; and tVeterans Administration Hospital, Dallas, Texas 75216 Communicated by Berta Scharrer, May 19, 1977 ABSTRACT The vasoactive intestinal polypeptide was Labeled VIP was obtained from Nuclear International Corp. present in synaptosomal (nerve ending) preparations from ce- (Waltham, MA); DL-[3H]-NE (12 mCi/mmol) was from New rebral cortex, hypothalamus, and striatum of rat brain in higher concentrations than in these tissues as a whole. The total content England Nuclear Corp. (Boston, MA); a-endorphin, somatos- and relative specific activity of the peptide increased with tatin, Met- and Leu-enkephalin, and neurotensin were pur- progressive purification of the synaptosomal fractions and chased from Peninsula Laboratories, Inc. (San Carlos, CA). All generally followed the distribution of known synaptosomal other chemicals were obtained from Sigma Chemical Co. (St. constituents-dopamine, norepinephrine, and lactate dehy- Louis, MO). Pargyline (N-methyl-N-2-propynylben- drogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27). The zylamine) was a gift of Abbot Labs (Chicago, IL). peptide was also released from synaptosomal pellets with in- creased K+ concentration, and this release was Ca2+-dependent. Preparation of Synaptosomes. Male rats (Sprague-Dawley, The findings suggest a role for vasoactive intestinal polypeptide 350 g) were killed by decapitation. Brains were removed, and as a transmitter or modulator of synaptic function. hypothalamic, striatal, and cortical areas were dissected ac- cording to the method of Glowinsky and Iversen (13). All pro- Vasoactive intestinal polypeptide (VIP), a 28-residue peptide cedures were carried out at 40. Brain areas were homogenized that is structurally related to secretin and glucagon, has been in 10 volumes of cold 0.32 M sucrose containing pargyline (0.1 isolated from porcine duodenum (1, 2). Until recently, its mM) in a glass homogenizer with a Teflon pestle (Kontes) for normal presence was thought to be limited to the gastrointes- 30 sec at 600 rpm. Subsequent centrifugations, collection, and tinal tract, but the discovery of high levels of VIP immuno- designation of fractions were essentially as described by reactivity in neural cell lines and in mammalian brain (3-5) has Whittaker (14) and DeRobertis (15), with the following mod- established its presence in the nervous system as well. VIP thus ification. The nuclear fraction (P1) was washed once and re- joins a number of other polypeptides-including somatostatin, centrifuged at 1000 X g for 10 min. The combined supernatants substance P. neurotensin, and the enkaphalins-that have been were centrifigued at 12,000 X g for 20 min; the pellet was re- shown to occur both in gut and in brain (6-11). suspended once in the original volume of 0.32 M sucrose and A possible functional role for VIP in the central nervous recentrifuged as above. The new pellet, P2, was subfractionated system is suggested by its selective localization in brain, being by centrifugation in sucrose gradients. at highest concentration in cerebral cortex and hypothalamus For centrifugation on discontinuous density gradients, the and virtually absent in cerebellum (3). The recent demonstra- P2 pellet was resuspended in half the original volume of 0.32 tion, by immunofluorescence, of VIP in cell bodies and nerve M sucrose, and 1 ml of this suspension was layered on a dis- terminals in cortex and in hypothalamic and other nuclei (12) continuous density gradient consisting of equal amounts of 0.8 supports this possibility. M and 1.2 M sucrose and centrifuged at 51,000 X g for 2 hr We have investigated the subcellular distribution of VIP in (rotor SW39, Beckman model L ultracentrifuge). Six fractions selected areas of rat brain, on the basis that nerve-cell constit- were collected by aspiration: (i) 0.5-0.6 ml of 0.32 M sucrose; uents are localized in compartments that correspond to their (Hi) 1 ml, at the interface with 0.8 M sucrose, containing white function and that the subcellular localization of the peptide fluffy material (myelin); (iii) 1 ml of 0.8 M sucrose; (iv) 1 ml, could thus provide an indication of its function. VIP was con- at the interface between 0.8 M and 1.2 M sucrose, showing two centrated in synaptosomal (nerve ending) preparations, which distinct bands-a light, faint one on top and a heavy one un- were also enriched in dopamine and norepinephrine (NE). The derneath (no attempt was made to separate these two bands, peptide also could be released from synaptosomes by depolar- which contained the bulk of the synaptosomes); (v) 1-1.2 ml, ization with high [K+], and the release was Ca2+-dependent. containing the remainder of the 1.2 M sucrose; and (v) pellet, These findings provide evidence for a physiologic role for VIP containing mitochondria. in synaptic function, possibly as a neurotransmitter or modu- Data are expressed for three final fractions: 1, comprising lator. fractions (i) and (ii) (lighter than 0.8 M sucrose); 2, combining (iii-v) (between 0.8 and 1.2 M); and 3, fraction (vi) (heavier MATERIALS AND METHODS than 1.2 M). VIP for iodination and for standard solutions in the radioim- Continuous sucrose gradients were formed in SW39 tubes munoassay was the natural porcine peptide (2), prepared in the with a standard gradient apparatus, by mixing equal amounts laboratory of V. Mutt, Karolinska Institute, Stockholm. 125I- of 1.6 M and 0.8 M sucrose. The resuspended P2 fraction (1 ml = 10 mg of protein) was layered on top of the gradient, which The costs of publication of this article were defrayed in part by the was then centrifuged at 51,000 X g for 120 min. By puncturing payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore Abbreviations: VIP, vasoactive intestinal polypeptide; NE, norepi- be hereby marked "advertisement" in accordance with 18 U. S. C. nephrine; SDH, succinate dehydrogenase; LDH, lactate dehydroge- §1734 solely to indicate this fact. nase. 3424 Downloaded by guest on September 26, 2021 Cell Biology: Giachetti et al. Proc. Natl. Acad. Sci. USA 74 (1977) 3425 Table 1. Distribution of VIP immunoreactivty in subeelhlalr 80- fractions of rat brain VIP content, ng/g* Cru Hypothalamus Striatum Cerebral 0 Fraction (4) (4) cortex (3) + 60- 0 Homogenate 12.10 + 0.70 17.60 + 4.50 26.40 : 4.60 P1 2.60 + 0.34 3.05 + 0.55 5.51 d 0.31 S2 5.50 1 0.75 4.80 1 0.55 13.64 ± 2.44 40- P2 9.54 I 0.61 13.60 b 2.90 19.76 1 1.94 0 * Values are means + SEM. Numbers in parentheses are the numbers of determinations. L' 20- the tubes at the bottom and counting drops, 18 fractions of approximately 0.3 ml each were collected. Because this volume F-L 4' was too small to permit assays in duplicate, fractions from two gradients were usually combined and the volume of each 0 _ ..r _ fraction was brought up to 1.0 ml with distilled water. Pi S2 P2 When hypothalamic or cortical tissues were labeled in vitro FIG. 1. Subcellular distribution of immunoreactive VIP, protein, with DL-[3H]NE, blocks of tissue were dissected with a razor SDH, and NE in rat hypothalamus. Distribution is expressed as blade. Each block, weighing 25-30 mg, was incubated in 2 ml percentage of total recovered content or activity in the subcellular of Krebs-Henseleit buffer containing [3H]NE (0.2 MAM) for 20 fractions PI, S2, and P2. Values are means + SEM from four experi- min at 370 in an atmosphere at 95% 02/5% CO2. At the end of ments. o, VIP; a, protein; X, SDH; al, NE. the incubation, blocks were rinsed in Krebs-Henseleit buffer, VIP was measured by a sensitive radioimmunoassay as de- weighed and homogenized in 0.32 M sucrose, and fractionated scribed (20), with the following modifications: a high-titer as outlined above. (>1:150,000), specific antiserum was produced by immuni- Release Experiments. Experiments testing the release of the zation with VIP-bovine serum albumin conjugate, and 125I- peptide were carried out on P2 pellets prepared from cerebral labeled VIP was prepared by the lactoperoxidase method. The cortex. The pellets were resuspended in Krebs solution to obtain lower limit of sensitivity of the assay was 20 pg. The assay a protein concentration of 4 mg/ml (11.25 ml of Krebs solution showed negligible crossreactivity with these peptides: secretin, per g of original tissue). One-half milliliter of the P2 suspension glucagon, cholecystokinin-pancreozymin, porcine pancreatic was mixed in a centrifuge tube with 0.5 ml of each of four polypeptide, substance P, somatostatin, a-endorphin [fl-lipo- variants of Krebs solution: (i) normal (5.9 mM) [K+], absent tropin-(61-76)], neurotensin, Leu-enkephalin, Met-enkephalin,, Ca2+; (i) high (55 mM) [K+], normal (1.3 mM) [Ca2+]; (iii) high bradykinin, and angiotensin II. [K+], absent Ca2+; and (iv) normal [K+], normal [Ca2+]. The Samples for electron microscopic examination of synapto- mixtures were incubated at 30° for 30 min, following which somal fractions were prepared as described (21). they were centrifuged at 40,000 X g for 10 min. The superna- Statistical analysis was performed by Student's t test, using tant solution was pipetted off and assayed for VIP immuno- paired or unpaired comparisons.