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Identification and Characterization in Rat Brain Membranes (Receptor Binding/Somatostatin Analogs/Neuropeptides/Neuropharmacology) COIMBATORE B

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Identification and Characterization in Rat Brain Membranes (Receptor Binding/Somatostatin Analogs/Neuropeptides/Neuropharmacology) COIMBATORE B Proc. Nati Acad. Sci. USA Vol. 78, No. 6, pp. 3930-3934, June 1981 Neurobiology Somatostatin receptors: Identification and characterization in rat brain membranes (receptor binding/somatostatin analogs/neuropeptides/neuropharmacology) COIMBATORE B. SRIKANT AND YOGESH C. PATEL* Fraser Laboratories, McGill University, Departments of Medicine, Neurology and Neurosurgery, Royal Victoria Hospital, and Montreal Neurological Institute, Montreal, Quebec H3A lAl, Canada Communicated by Brenda Milner, February 23, 1981 ABSTRACT We have identified and characterized specific sin, and bombesin were purchased from Bachem Fine Chem- receptors for tetradecapeptide somatostatin (SRIF; somatotropin icals (Torrance, CA). Other analogs of SRIF were provided by release-inhibiting factor) in rat brain using ['25I-Tyr"]SRIF as the D. Coy and C. Meyers (New Orleans, LA) and J. Rivier (La radioligand. These receptors are present in membranes obtained Jolla, CA). Synthetic 13H-endorphin was obtained from N. Ling from a subfraction of synaptosomes. Membranes derived from (La Jolla, CA), and [Met]enkephalin was from G. Tregear (Mel- cerebral cortex bind SRIF with high affinity (K. = 1.25 X 1010 bourne, Australia). Vasoactive intestinal polypeptide (VIP) was M-') andhave a maximum bindingcapacity (Bmw) of0. 155 X 10-12 a gift from S. I. Said (Dallas, TX). All other chemicals used were mol/mg. Neither opiates nor other neuropeptides appear to in- of analytical grade. fluence the binding of SRIF to brain membranes. Synthetic ana- Brain Membranes. Adult male logs with greater biological potency than SRIF-[D-Trp8]SRIF, Preparation of Sprague- [D-Cys14]SRIF, and [D-Trps, D-Cys'4]SRIF-bind to the recep- Dawley rats (150-200 g) were killed by decapitation. The cer- tors with greater avidity than SRIF, whereas inactive analogs ebral cortex was dissected and homogenized (10%, wt/vol) in [(2H)Ala3]SRIF and [Ala6]SRIF exhibit low binding. The ratio of 0.32 M sucrose/20 mM Tris-HCl, pH 7.5, with a Dounce ho- receptor density to endogenous somatostatin is high in the cortex, mogenizer, and membrane fractions were prepared by a mod- thalamus, and striatum, low in the hypothalamus, and extremely ification of the method of Cotman and Matthews (16). The ho- low in the brain stem and cerebellum. Thus, SRIF receptors in mogenate was centrifuged at 1000 X g for 5 min to remove the the brain appear to be a distinct, new class of receptors with a nuclear debris (fraction P1). The supernatant obtained was cen- regional distribution different from that ofendogenous somatostatin. trifuged at 10,000 x g for 45 min. The resulting pellet contain- ing the crude mitochondrial fraction (P2) was resuspended in The tetradecapeptide somatostatin (SRIF; somatotropin re- 0.32 M sucrose, applied to the top of a discontinuous Ficoll lease-inhibiting factor) originally discovered as a growth hor- (Pharmacia, Uppsala, Sweden) gradient (8-20%) in 0.32 M su- mone release-inhibiting factor in extracts ofsheep hypothalami crose, and sedimented at 63,580 X g for 45 min in a Beckman (1) has since been shown to be distributed throughout the ex- L5-65 ultracentrifuge. Subcellular fractions sedimenting in 20% trahypothalamic brain and in peripheral tissues and to exert a (vol/vol) Ficoll (P3) and at the different density interfaces 16- wide spectrum ofbiological actions (24). Although the precise 20% (P4), 12-16% (P5), 8-12% (P6), and 8% Ficoll/sucrose in- function of somatostatin in the central nervous system is un- terface (P7) were carefully collected and pelleted in 0.32 M su- known, the extensive distribution of somatostatin-containing crose at 10,000 X g. The synaptosomal fractions P5 and P6 were neurons in the brain (5), its synaptosomal localization (6), the hypoosmotically ruptured by swelling in 5 mM Tris-HCI (pH effects of microiontophoretically applied SRIF on the sponta- 7.5) twice. The final pellets obtained by centrifugation at 10,000 neous electrical activity of neurons (7), and the behavioral ef- x g were resuspended in 50 mM Hepes-KOH (pH 7.5). Ali- fects (8) of intracisternally injected SRIF (8) suggest that the quots of the membranes were resuspended in an identical vol- peptide may act as a neurotransmitter or neuromodulator (7, ume of 50 mM Tris-HCl (pH 7.5) for determination of protein 9). There is good evidence to suggest that the action of SRIF content (17) because Hepes interferes in this assay. Membrane on growth hormone secretion is mediated through binding to fractions from other areas ofthe brain were also prepared in this specific receptors which have been demonstrated in rat pitui- manner to determine the regional distribution ofSRIF receptors. tary tumor cells in culture (10) and in bovine pituitary plasma Histological Techniques. Membrane pellets isolated from membranes (11). Similar binding of SRIF to subcellular com- the cerebral cortex were fixed in 2.5% (vol/vol) glutaraldehyde ponents of neural tissue has not been reported. Therefore, the in 0.1 M sodium cacodylate for 2 hr, washed with 0.1 M sodium present study was designed to identify and characterize specific cacodylate/7% (wt/vol) sucrose, postfixed (2 hr) in Karnovsky's receptors for SRIF in rat brain synaptosomal membrane frac- reduced osmium (18), dehydrated, and embedded in epon; sec- tions that have been shown to possess specific binding sites for tions were prepared for electron microscopy. other neuropeptides (12-15). Preparation of Radioiodinated Peptide. Because SRIF does not contain a tyrosine or histidine residue, it is necessary to use MATERLALS AND METHODS suitable synthetic analogs for radioiodination. Three tyrosinated Synthetic cyclic SRIF was obtained from Ayerst Laboratories analogs ofSRIF, [Tyr']SRIF, Tyr-SRIF, and [Tyr"]SRIF, have (Montreal, Canada). Tyrosinated analogs of SRIF, thyrotropin- luteinizing hormone- Abbreviations: SRIF, synthetic tetradecapeptide somatostatin (soma- releasing hormone (TRH; thyroliberin), totropin release-inhibiting factor); VIP, vasoactive intestinal polypep- releasing hormone (LHRH; luliberin), substance P, neuroten- tide; TRH, thyrotropin-releasing hormone; LHRH, luteinizing hor- mone-releasing hormone (luliberin). The publication costs ofthis article were defrayed in part by page charge * To whom reprint requests should be addressed at: Room M3-10, payment. This article must therefore be hereby marked "advertise- Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. H3A lAl, Canada. 3930 Downloaded by guest on October 2, 2021 Neurobiology: Srikant and Patel Proc. Natl. Acad. Sci. USA 78 (1981) 3931 been developed for this purpose (19) and have been shown to Table 1. Integrity of tracer after incubation with synaptosomal possess similar biological activities to the native peptide (19, 20). cell membranes (fraction P5)* All three were selected for our SRIF binding experiments. They % intact were radioiodinated with Na1'2I (New England Nuclear) to high tracer specific activities (=1050 Ci/mmol; 1 Ci = 3.7 x 1010 becque- Tracer A B rels) by a modification of the chloramine-T technique as de- scribed (3). [125I-Tyr']SRIF 35 42 Stability of Radioiodinated SR-IF Analogs During Incuba- 125I-Tyr-SRIF 53 73 71 97 tion. Because SRIF and radioiodinated SRIF analogs are rapidly [125I-Tyr11]SRIF degraded by aminopeptidases and endopeptidases in plasma * In the absence (A) and presence (B) of antiproteolytic agents: Tra- and tissue extracts (21, 22), and because brain synaptosomal sylol, phenylmethylsulfonyl fluoride, and bacitracin. fractions are known to be associated with these enzymes (23), we first investigated the stability of the radioiodinated analogs teolytic agents were compared, [125I-Tyr']SRIF showed low during incubation with the synaptosomal membrane fractions specific binding (<1%) and high nonspecific binding (.14%). P5 and P6. Approximately 130,000 cpm (0.75 nM) of each 1I-Tyr-SRIF exhibited paradoxical binding in that more ra- analog was added to 0.50 ,ug of membrane protein and incu- dioligand was bound in the presence of 10 AuM SRIF than in its bated for 1 hr at 300C in the absence and presence of the fol- absence. Because of this phenomenon, it was not possible to lowing enzyme inhibitors: Trasylol (aprotinin; Bayer, Federal detect any specific binding with this radioligand. On the other Republic ofGermany) (500 kallikrein inhibitor units/ml), phen- hand, the best binding parameters were obtained with [125I- ylmethylsulphonyl fluoride (0.02 jig/ml), and bacitracin (0.02 Tyr"]SRIF. For these reasons, [125I-Tyrl]SRIF was the radioli- ,ug/ml). The final reaction volume was adjusted to 100 ,i1 with gand chosen for all subsequent binding experiments. 50 mM Hepes-KOH (pH 7.5) containing bovine serum albumin Identification of Membrane Fractions Containing Specific (10 mg/ml) and MgCl2 (5 mM). At the end of the incubation, Receptors for SRIF. The specific and nonspecific binding of the free radioligand was separated from bound radioligand by ['0I-Tyr"1]SRIF to the different subcellular fractions of the rat centrifugation and subjected to chromatoelectrophoresis (3) to cerebral cortex are compared in Table 2. Fraction P5 exhibited determine the percentage of intact tracer remaining. the highest specific and lowest nonspecific binding (9.67% and Determination of Specific Binding of Radioligand. About 4.12%, respectively). Morphologically this subfraction was en- 0.60 nM ofthe appropriate radioligand with 50 ,ug ofmembrane riched in synaptosomes (Fig. 1). Less than 0.5% specific binding protein were incubated with each antiproteolytic agent at 300C was observed in fraction P6. Both these fractions contained high until equilibrium had been reached. Radioligand bound to the concentrations of endogenous somatostatin, 9.7 and 11.0 ng/ membrane was separated by centrifugation (2500 x g), washed mg of protein, respectively, which could be released by hy- twice with 1 ml of 50 nM Hepes-KOH, pH 7.5/1% albumin, poosmotic lysis of the synaptosomes (Table 2). Removal of the and the radioactivity in the resulting pellet was assayed in a endogenous somatostatin in this manner resulted in a 7-fold in- Beckman L-4000 autogamma spectrometer.
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