Characteriz in rat seminal v
estinal pepti
LUIS G. GUIJAR 0, M. SOL RODRIGUEZ-PENA, AND Departamento de Bioquimica y Biologia Molecular, Universidad 28871 AlcalcE de Henares-Madrid, Spain
GUIJARRO, LUIS G., M. SOL RODRIGUEZ-PENA, AND JUAN C. PRIETO. Characterization of vasoactive intestinal peptide receptors in rat seminal vesicle. Am. J. Physiol. 260 (Endocrinol. Metab. 23): E286-E291,1991.-Receptors for vasoactive intestinal peptide (VIP) in membranes from rat seminal vesicle were examined using 12”1-labeled VIP as ligand, The receptor binding was rapid, reversible, saturable, specific, and dependent on temperature and membrane concentration. At 15”C, the stoichiometric data suggested the presence of two classes of VIP receptors with & values of 0.54 and 44.4 nM and binding capacities of 73 and 1,065 fmol VIP/mg membrane protein, respectively. The interaction showed a high degree of specificity, as suggested by competition experiments with various peptides structurally related to VIP as follows: helodermin was 10 times, secretin 30 times, and rat growth hormone-releasing factor 300 times less potent than VIP, whereas glucagon did not recognize VIP receptors in concentrations of up to 10 PM. The binding of ‘“‘I-VIP to membranes was sensitive to the presence of GTP in the incubation medium in a dose-dependent manner. To characterize the molecular weight of these VIP receptors, ‘“‘I-VIP was covalently bound to membranes from rat seminal vesicle using dithiobis( succinimidyl propionate); sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized receptor revealed the presence of a specific component with a molecular mass of 47,000 Da as estimated in denaturing conditions. These findings, together with the known presence of VIP-containing nerves in the seminal vesicle, suggest a direct physiological role for this peptide in this accessory gland of the male genital tract. guanine linker
nucleotide
binding
proteins;
molecular
weight;
cross-
PEPTIDE (VIP)is ahighlybasic 2%amino acid peptide that was first isolated from porcine small intestine (25). VIP was originally considered to be a gut hormone but has later been seen to behave as a neurotransmitter or a neuromodulator (24). Immunohistochemical techniques have evidenced the presence of VIP-containing nerves in many organs, with the highest concentrations of the neuropeptide being localized in the digestive tract, central nervous system, and genitourinary tract (27,29). VIP affects a broad range of biological activities in many organs and tissues, including blood flow regulation, smooth muscle relaxation, exocrine secretion, and neuroendocrine activity (24, 29). The biological effects of VIP are thought to be mediated by its interaction with specific plasma membrane receptors in target cells, and this has been established in a whole range of tissues, the localization of which generally corVA~OACTIVEINTESTINAL
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0193~1849/91 $1.50 Copyright
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e receptors
JUAN C. PRIETO de Ahab,
relates with a VIP-like immunoreactivity (9, 23). The physiological and pharmacological characteristics of the VIP receptors have been extensively studied, but only recently has it been possible to investigate their molecular characteristics (9, 14). The seminal vesicle from humans, rats, and other species not only contains adrenergic and cholinergic nerve fibers but also those that store VIP and other peptides (5). VIP immunoreactive neurons seem to be closely associated with smooth muscle and subepithelial connective tissue close to the epithelium in the seminal vesicle (15, 20), suggesting a physiological role for VIP in this interesting glan.d of the male genital tract. Some recent results suggest VIP activity on secretory movements at this level (X,28). However, the involvement of VIP in motor activity, secretory mechanisms, metabolism, and cell proliferation in the seminal vesicle remains to be established. A recent preliminary report has shown the presence of specific binding sites for VIP in seminal vesicles from both rat and guinea pig when characterized by immunocytochemical techniques (20). However, there is no information on the pharmacological and molecular properties of VIP receptors in this gland, which can contribute to our knowledge of the direct action of VIP in the seminal vesicle. Using biochemical techniques we therefore studied the kinetics, stoichiometry, specificity, and dependence on gua.nyl nucleotides of VIP binding to plasma membranes from rat seminal vesicle. We also estimated the molecular size of these VIP receptors by using a covalent cross-linking procedure with the bifunctional reagent dithiobis( succinimidyl propionate) (DTSP). EXPERIMENTAL
PROCEDURES
Materials. Highly purified natural porcine VIP and secretin were supplied by Prof. V. Mutt (Karolinska Institute, Stockholm, Swedenj. Porcine glucagon was purchased from Novo. Helodermin and rat growth hormone-releasing factor (GRF) were obtained from Peninsula Laboratories. Bacitracin, phenylmethylsulfonyl fluoride (PMSF), bovine serum albumin, GTP and other nucleotides and nucleosides, DTSP, and electrophoresis reagents were purchased from Sigma Chemical. All other reagents were of the highest purity commercially available. ?-labeled VIP was prepared at a specific activity of -250 Ci/g and possesseda biological activity similar
1991 the American
Physiological
Society
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to that of native VIP (10, 21). Animals and membrane preparation. Sexually mature male Wistar rats (-250 g) were employed in this study. The animals were maintained in an environmentally controlled room (23°C; 12:12 h light-dark cycle), and food and water were provided ad libitum. Rats were killed by decapitation, and seminal vesicles were quickly removed, cleaned of the seminal fluid, dissected free from connective and adipose tissues, and then finely minced. For membrane preparation, the tissue was homogenized using a Polytron (setting 5, 3 min, Brinkmann Instruments) in 0.01 M triethanolamine-HCl buffer, pH 7.5, containing 0.25 M sucrose and 0.5 mM EDTA. After filtration through two layers of medical gauze, the homogenate was centrifuged at 1,500 g for 15 min at 4°C. The resulting supernatant was centrifuged at 30,000 g for 30 min at 4°C. The final pellet was washed twice in 20 mM tris(hydroxymethyl)aminomethane (Tris) HCl buffer, pH 7.5, and was immediately frozen at -70°C until used. Protein concentration was determined according to the method of Lowry et al. (13), using bovine serum albumin as a standard. VIP binding assay. In a standard binding assay, membranes (0.3 mg protein/ml) were incubated in 0.25 ml of 50 mM Tris. HCl buffer, pH 7.5, containing 1.4% bovine serum albumin, 1 mg/ml bacitracin, 0.05 mM PMSF, and 50 pM 12”1-VIP in the absence or presence of increasing amounts of unlabeled peptide (0.01-100 nM). Membrane-bound 1251-VIP was separated by filtration under vacuum using Whatman GB/F filters that had been pretreated with 1% polyethylenimine. Each filter was washed twice with 5 ml ice-cold Tris. HCl buffer, pH 7.5, and radioactivity was counted in a Kontron gamma counter at 70% efficiency. To determine nonspecific binding, parallel incubations were made in the presence of an excess (1 PM) of unlabeled VIP; nonspecific VIP binding averaged ~2% of the total radioactivity and was subtracted from the total binding to express the results as specific binding. The apparent equilibrium dissociation constants (&) and maximum numbers of binding sites expressed in terms of binding capacities (B,,,) were calculated from the stoichiometric data using the nonlinear iterative computer curve-fitting program (LIGAND) of Munson and Rodbard (17). The inactivation of 12’1VIP in the standard binding assay was estimated by talc adsorption, as previously described (7). Briefly, at the end of the incubation period, the reaction mixture was centrifuged at 30,000 g for 15 min. Then an aliquot of the supernatant (12”I-VIP) was mixed with an equal volume of 5% talc to adsorb intact tracer. After centrifugation for 5 min at 3,000 g, the radioactivity in the pellet was counted. Degradation was expressed as the percentage of unaltered tracer present in the control simultaneously incubated without membrane fraction. Cross-linking of 12’1-VIP to membranes. Membranes (-0.5 mg protein/ml) were incubated for 60 min at 15°C in 2.5 ml of the binding assay buffer containing 0.5 nM ‘““I-VIP. To determine nonspecific binding, parallel samples were incubated as above, but in the presence of excess (1 PM) unlabeled VIP. After incubation, the reaction mixture was diluted with 10 ml of ice-cold 60 mM l
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N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), pH 7.5, and centrifuged at 30,000 g for 15 min to remove unbound VIP. The pellet was then resuspended in 1 ml of the same buffer, but including 1 mM DTSP to initiate VIP-receptor cross-linking. After 15 min incubation at 4°C the reaction was stopped with 0.5 ml ice-cold 60 mM HEPES buffer, pH 7.5, containing 60 mM lysine as a reagent quench (8). The mixture was centrifuged for 15 min at 30,000 g, and the resulting pellet was resuspended by sonication (Dynatech sonic dismembrator; setting 35% of maximum, 1 min) in 0.1 ml of 60 mM Tris HCl buffer, pH 6.8, containing 10% glycerol, 0.001% bromophenol blue, and 3% sodium dodecyl sulfate (SDS). After 30 min at 60°C, the suspension was centrifuged at 30,000 g for 15 min, and the supernatant was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE and autoradiography. SDS-PAGE was carried out according to the method of Laemmli (11) in a 12% polyacrylamide slab gel (1.5 mm thickness) with a 3% polyacrylamide stacking gel. Molecular mass markers (triosephosphate isomerase 26,600; lactate dehydrogenase 36,500; fumarase 48,500; pyruvate kinase 58,000; fructose-6-phosphate kinase 84,000; fl-galactosidase 116,000; and cu2-macroglobulin 180,000) were run in parallel lanes. The gels were run, fixed, and dried as described previously (8). Then they were exposed for l-7 days at -70°C to a Du Pont Cronex-4 film with an intensifying screen (Du Pont Cronex Lightning Plus). RESULTS
Time and temperature studies on 12’I-VIP binding and degradation. Figure 1 illustrates the patterns of specific I”“1-VIP binding and degradation in membranes from rat seminal vesicle at three temperatures. Clearly, the binding (Fig. 1, top) was time- and temperature-dependent, with the maximal and stable binding obtained at 15°C between 30 and 120 min of incubation. This apparent equilibrium was accompanied by a very low level of peptide inactivation that did not reach 10% throughout the time interval studied (Fig. 1, bottom). At 25 and 37OC, the binding was rapid but transient, probably because of the higher degradation of ‘“51-VIP at these temperatures. It was therefore decided to undertake all subsequent incubations for 60 min at 15°C. Effect of membrane protein concentration. To determine whether the binding of ‘““I-VIP was proportional to the membrane concentration, the specific binding of 1251-VIP was studied at increasing membrane protein concentrations (Fig. 2). The binding of the neuropeptide was found to be linear at protein concentrations up to 0.6 mg/ml. The characteristics of binding were studied at a membrane protein concentration of 0.3 mg/ml. Dissociation of bound 12’1-VIP. The reversibility of the specific interaction between 12’I-VIP and membranes from rat seminal vesicle was assessed after a 60-min incubation at 15°C (Fig. 3). The addition of 0.1 PM unlabeled VIP resulted in a time-dependent displacement of the receptor-bound 12”1-VIP, with a 50% displacement of specifically bound tracer within 30 min at 15°C. The time course of dissociation of the complex
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10 20 30 40 50 Time (mid
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FIG. 3. Dissociation time course. Membranes from icle (0.3 mg membrane protein/ml) were preincubated VIP at steady state (60 min at 15”C), and dissociation adding 1 ,uM unlabeled VIP. Specific binding of tracer at indicated times. Results correspond to a representative of 2 performed in trip icate.
0.050
’ T
Time (hr).
rat seminal veswith 50 pM *“Iwas initiated by was determined experiment
IJIG. 1. Dependence of vasoactive intestinal peptide (VIP) binding and degradation on time and temperature. Fifty pM l’“I-labeled VIP were incubated with membranes from rat seminal vesicle (0.3 mg membrane protein/ml) at 15°C (o), 25°C (0), and 37°C (m). After indicated time intervals, specific binding (top) and degradation (bottom) of labeled neuropeptide were determined. Results correspond to representative experiments of 2 performed in triplicate at each temperature. . I//L 00
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FIG. 4. Competition binding of ‘““I-VIP at steady state. Membranes (0.3 mg membrane protein/ml) were incubated with 50 pM ‘““I-VIP for 60 min at 15OC in presence of increasing concentrations of unlabeled neuropeptide. Corresponding extent of specific ““I-VIP binding was expressed as a function of unlabeled VIP concentration (left; means t SE of 6 separate experiments performed in triplicate). Right: Scatchard analysis of same mean data.
/
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g
7
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(& = 44.4 t 4.1 nM) and high binding capacity t 150 fmol/mg protein; means t SE of 6 separate 0.3 0.6 0 experiments). Membrane Protein (mg/ml) Receptor specificity of 12,51VIP binding. The specificity of the VIP receptors in membranes from rat seminal FIG. 2. Dependence of VIP binding on membrane concentration. Specific receptor binding of 50 pM “‘I-VIP was determined at increasvesicle was investigated by determining the ability of ing concentrations of membrane protein after 60 min incubation at various peptides that are structurally related to VIP to 15°C. Results correspond to a representative experiment of 2 performed compete with the binding of 12”1-VIP (Fig. 5). The order in triplicate. of potency of the different peptides, as expressed by the tracer membranes followed multi-order kinetics, which concentration giving half-maximal inhibition of tracer binding, was as follows: VIP (I& = 1 nM) > helodermin suggested heterogeneity of VIP binding sites. Receptor binding of 1251-VIP at steady state. The addi- UC 50 = 10 nM) > secretin (ICso = 30 nM) >> rat GRF tion of unlabeled VIP caused a concentration-dependent UC 5. = 300 nM). In concentrations up to 10 PM, glucagon competition of the receptor-bound 12”1-VIP on mem- (another peptide structurally related to VIP) did not inhibit ““I-VIP binding at all. branes from rat seminal vesicle (Fig. 4, left), half-maximal inhibition (I&J being observed at -1 nM VIP. The Effect of guanyl nucleotides on 1251-VIP binding. The Scatchard analysis (26) of the stoichiometric data gave specific binding of 12”1-VIP to the membranes was moda curvilinear plot (Fig. 4, right) that was interpreted in ulated in a negative and dose-dependent manner by terms of a model of two classesof VIP receptors possess- guanyl nucleotides (Fig. 6). From 0.1 PM to 1 mM, GTP ing different affinities as follows: a first site of high and GDP inhibited the binding of the labeled peptide affinity (Kd = 0.54 2 0.04 nM) and low binding capacity with a similar potency, 50% inhibition of total specific (73 t 13 fmol VIP/ mg protein) and a second site of low binding measured at eauilibrium being obtained at -0.3 ’
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Kd 116.0 84.0 58.0 48.5
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26.6 :m [Peptide], -log M FIG. 5. Effect of VIP and related peptides on ‘251-VIP binding. Membranes from rat seminal vesicle (0.3 mg membrane protein/ml) were incubated with 50 pM “‘I-VIP in absence or presence of unlabeled VIP (o), helodermin (o), secretin (m), rat growth hormone-releasing factor (o), and glucagon (A). Specific binding of tracer was determined after 60 min incubation at 15°C and expressed as a percentage of maximal binding. Data are means + SE of 4 separate experiments performed in triplicate.
FIG. 7. Molecular mass determination of VIP receptors ,n membranes from rat seminal vesicle. Conditions for covalent labeling of using cross-linking reagent dimembrane protein by Y-VIP thiobis(succinimidy1 propionate) followed by SDS-polyacrylamide gel electrophoresis along with proteins of known relative molecular mass (M*) are described in EXPERIMENTAL PROCEDURES. Lanes of known M, are also described there. Lanes in autoradiography correspond to ‘251-VIP incubation in absence (A) or presence (B) of 1 PM unlabeled VIP. Reference protein size markers are indicated. Pattern is representative of 4 experiments.
mass) protein must represent the specific VIP receptor, since its labeling was greatly reduced when the crosslinking procedure was performed in the presence of 1 PM unlabeled VIP. Another widespread and weak band possessing an apparent molecular mass of 91,000 Da was also identified, indicating the existence of a second VIP binding protein of 88,000 Da. DISCUSSION
[Nucleotide],- log M FIG. 6. Dependence of VIP binding on guanyl nucleotides. ““I-VIP (50 PM) was incubated with membranes (0.3 mg membrane protein/ ml) in absence or presence of increasing concentrations of GTP (o), GDP (0), GMP (o), and ATP (A). Specific binding of ‘251-VIP was determined after 60 min incubation at 15°C. Results are means + SE of 3 separate experiments performed in triplicate.
mM GTP or GDP. Other nucleotides such as GMP and ATP did not affect the binding of ‘251-VIP to membranes even at a concentration as high as 1 mM. Molecular mass determination of VIP receptors by affinity labeling. VIP receptors in membranes from rat
seminal vesicle were identified at the molecular level by SDS-PAGE after cross-linking with the bifunctional agent DTSP as described in EXPERIMENTAL PROCEDURES. The conditions for the cross-linking reaction were optimized previously (data not shown). As indicated in Fig. 7, a major autoradiographic band was identified at 50,000 Da, which may correspond to a VIP binding protein with an apparent molecular mass of 47,000 Da plus the bound VIP. This 47,000-Da (relative molecular
The present study documents for the first time the properties of VIP receptors and their corresponding molecular size in membranes isolated from rat seminal vesicles. The basic characterization of the interaction of 1251-VIP with the receptors fulfilled the criteria of reversibility, saturability, specificity, and high affinity of a common hormone- or neurotransmitter-receptor binding reaction in agreement with that observed in other VIP binding systems like those in membranes isolated from gastric (6) or intestinal epithelium (l), lung (12), prostate (3), pancreas (22), liver (2), myometrial smooth muscle (19), and brain (30). The stoichiometric parameters of VIP receptors were obtained in optimal experimental conditions, since peptide degradation during the incubation period (60 min at 15°C) did not exceed 10%. Based on the results obtained from both the study of VIP binding at the steady state and the dissociation experiments, two classes of VIP receptors appear to be present in membranes from rat seminal vesicle; one with high affinity (& = 0.54 nM) that represented 6.4% of the total and another with low affinity (& = 44.4 nM). This heterogeneous pattern is common to all the VIP receptor systems so far described with very few exceptions, one being rat brain membranes, where a unique class of VIP receptors has been found (30). The dissociation constants observed in the present preparation are in the range of those tissues studied previously. It has
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been suggested that the existence of two classes of receptors for VIP can be due to the modulation of receptor affinity by the GTP-regulatory component, i.e., G proteins (9). In this context, it is to be noted that VIP binding to membranes from rat seminal vesicle was GTP sensitive, since the extent of VIP binding was inhibited in a dose-dependent manner. These data suggest that, in rat seminal vesicle, VIP receptors couple to some GTPbinding proteins. The competition experiments performed between 12’)1VIP and different unlabeled peptides that belong to the VIP family showed an order of efficacy common to other VIP binding systems (1, 3, 6, 12) as follows: VIP > helodermin > secretin > rat GRF and the lack of effect by glucagon. From this point of view it seems that the actions of VIP are mediated via similar receptors expressed in the different target tissues. The molecular characterization of VIP receptors was carried out by the covalent cross-linking of 12”1-VIP to membranes from rat seminal vesicle, using the bifunctional reagent DTSP. After protein solubilization and SDS-PAGE, a major 12”1-VIP-protein complex of M, 50,000 Da was identified. Assuming that one molecule of VIP linked to the receptor, the molecular mass of this component can be estimated to be ~47,000 Da. Another widespread and weak band was also observed, with an apparent molecular mass of 91,000 Da corresponding to a VIP binding component of 88,000 Da. Two alternatives could explain that the two classes of VIP receptors characterized by competitive displacement experiments were identified by SDS-PAGE primarily as a major component of -47,000 Da as follows: 1) two different classes of VIP receptors having similar molecular weight are present, and 2) the two classesof VIP receptors could be covalently attached to 1251-VIP by DTSP with different efficiency due to unequal localization and accessibility in the membrane. However, the 47,000- and 88,000-Da bands could correspond to the two classes of VIP receptors, as has been suggested for other VIP receptor systems (8). The major VIP binding proteins usually possess molecular massesranging from 46,000 to 53,000 Da depending on the systems studied and exhibit the high affinity and specificity of the VIP receptor (9, 14). It has been suggested that larger proteins may correspond to 1) complexes formed by the VIP receptor and a G, protein (9) or the VIP receptor and an cysprotein of 42,000 Da (4); 2) a precursor of the VIP receptor (18); and 3) a dimeric structure of the VIP-binding protein (16) In conclusion, the results presented here on the pharmacological and molecular weight identification of VIP receptors in rat seminal vesicle together with the known presence of VIP nerve fibers in this gland (5,20) strongly suggest that VIP may play a direct physiological role at this level. Some recent data support an effect of VIP on the secretory activity of seminal vesicle but not on either the basal tension or the neurogenic contraction of the corresponding smooth muscle in rat (15) and guinea pig (28). Nevertheless, the functional significance of VIP in the seminal vesicle remains to be clearly established, i.e, at levels as important as cell proliferation, hydroelectrolvtic movement, motor activitv, and metabolism.
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We thank Carol F. Warren from the Instituto de Ciencias de la Education de la Universidad de Alcala for linguistic assistance. This work was supported by Grants PM89-0096 and HF-100/1989 from Direction General de Investigation Cientifica y Tecnica, by the Consejo Superior de Investigaciones Cientificas (CSIC-INSERM, 1988), and by the Communidad Autonoma de Madrid. Address for reprint requests: J. C. Prieto, Departamento de Bioquimica y Biologia Molecular, Universidad de Alcala, 28871 Alcala de Henares-Madrid, Spain. Received
4 June
1990; accepted
in final
form
5 October
1990.
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cinema cells in culture (HTZ9). Eur. J. Biochem. 151: 411-417, 1985. 17. MUNSON, P. J., AND D. RODBARD. LIGAND: a versatile approach for characterization of ligand-binding systems. Anal. Biochem. 107: 220-239,198O. 18. NGUYEN, T. D., J. A. WILLIAMS, AND G. M. GRAY. Vasoactive intestinal peptide receptor on liver plasma membranes: characterization as a glycoprotein. Biochemistry 25: 361-368, 1986. 19. OTTESEN, B., P. STAUN-OLSEN, S. GAMMELTOFT, AND J. FAHRENKRUG. Receptors for vasoactive intestinal polypeptide on crude smooth muscle membranes from porcine uretus. Endocrinology 110: 2037-2043,1982.
20. POWER, R. F., A. E. BISHOP, J. WHARTON, C. 0. INYAMA, R. H. JACKSON, S. R. BLOOM, AND J. M. POLAK. Anatomical distribution of vasoactive intestinal peptide binding sites in peripheral tissues investigated by in vitro autoradiography. Ann. NY Acad. Sci. 527: 314-325,1988. 21.
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PRIETO, J. C., M. LABURTHE, AND G. ROSSELIN. Interaction of vasoactive intestinal peptide with isolated intestinal epithelial cells from rat. 1. Characterization, quantitative aspects and structural requirements of binding sites. Eur. J. Biochem. 96: 229-237, 1979. ROBBERECHT, P., M. WAELBROECK, M. NOYER, P. CHATELAIN, P. DE NEEF, W. K~NIG, AND J. CHRISTOPHE. Characterization of secretin and vasoactive intestinal peptide receptors in rat pancreatic plasma membranes using the native peptides, secretin (727) and five secretin analogues. Digestion 23: 201-210, 1982.
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23. ROSSELIN, G. The receptors of the VIP family peptides (VIP, secretin, GRF, PHI, PHM, GIP, glucagon, and oxyntomodulin). Specificities and identity. Peptides 7: 89-100, 1986. 24. SAID, S. I. Vasoactive intestinal peptide. J. Endocrinol. Inuest. 9: 191-200, 1986. 25. SAID, S. I., AND V. MUTT. Isolation from porcine-intestinal wall of a vasoactive octacosapeptide related to secretin and to glucagon. Eur. J. Biochem. 28: 199-204, 1972. z6 SCATCHARD, G. The attractions of proteins for small molecules ’ and ions. Ann. NY Acad. Sci. 51: 660-672,1949. 27 . SIMS, K. B., D. L. HOFFMAN, S. I. SAID, AND E. A. ZIMMERMAN. Vasoactive intestinal polypeptide (VIP) in mouse and rat brain: an immunocytochemical study. Brain Res. 186: 165-183, 1980. 28. STJERNQUIST, M., R. HAKANSON, S. LEANDER, C. OWMAN, F. SUNDLER, AND R. UDDMAN. Immunohistochemical localisation of substance P, vasoactive intestinal polypeptide and gastrin-releasing peptide in the vas deferens and seminal vesicle, and the effect of these and eight other neuropeptides on resting tension and neurally evoked contractile activity. Regul. Pept. 7: 67-86, 1983. F., E. EKBLAD, T. GRUNDITZ, R. HAKANSON, AND R. 29. SUNDLER, UDDMAN. Vasoactive intestinal peptide in the peripheral nervous system. Ann. N. Y. Acad. Sci. 527: 143-167, 1988. 30. TAYLOR, D. P., AND C. B. PERT. Vasoactive intestinal polypeptide: specific binding to rat brain membranes. Proc. Natl. Acad. Sci. USA. 76: 660-664, 1979.
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estinal pepti
LUIS G. GUIJAR 0, M. SOL RODRIGUEZ-PENA, AND Departamento de Bioquimica y Biologia Molecular, Universidad 28871 AlcalcE de Henares-Madrid, Spain
GUIJARRO, LUIS G., M. SOL RODRIGUEZ-PENA, AND JUAN C. PRIETO. Characterization of vasoactive intestinal peptide receptors in rat seminal vesicle. Am. J. Physiol. 260 (Endocrinol. Metab. 23): E286-E291,1991.-Receptors for vasoactive intestinal peptide (VIP) in membranes from rat seminal vesicle were examined using 12”1-labeled VIP as ligand, The receptor binding was rapid, reversible, saturable, specific, and dependent on temperature and membrane concentration. At 15”C, the stoichiometric data suggested the presence of two classes of VIP receptors with & values of 0.54 and 44.4 nM and binding capacities of 73 and 1,065 fmol VIP/mg membrane protein, respectively. The interaction showed a high degree of specificity, as suggested by competition experiments with various peptides structurally related to VIP as follows: helodermin was 10 times, secretin 30 times, and rat growth hormone-releasing factor 300 times less potent than VIP, whereas glucagon did not recognize VIP receptors in concentrations of up to 10 PM. The binding of ‘“‘I-VIP to membranes was sensitive to the presence of GTP in the incubation medium in a dose-dependent manner. To characterize the molecular weight of these VIP receptors, ‘“‘I-VIP was covalently bound to membranes from rat seminal vesicle using dithiobis( succinimidyl propionate); sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized receptor revealed the presence of a specific component with a molecular mass of 47,000 Da as estimated in denaturing conditions. These findings, together with the known presence of VIP-containing nerves in the seminal vesicle, suggest a direct physiological role for this peptide in this accessory gland of the male genital tract. guanine linker
nucleotide
binding
proteins;
molecular
weight;
cross-
PEPTIDE (VIP)is ahighlybasic 2%amino acid peptide that was first isolated from porcine small intestine (25). VIP was originally considered to be a gut hormone but has later been seen to behave as a neurotransmitter or a neuromodulator (24). Immunohistochemical techniques have evidenced the presence of VIP-containing nerves in many organs, with the highest concentrations of the neuropeptide being localized in the digestive tract, central nervous system, and genitourinary tract (27,29). VIP affects a broad range of biological activities in many organs and tissues, including blood flow regulation, smooth muscle relaxation, exocrine secretion, and neuroendocrine activity (24, 29). The biological effects of VIP are thought to be mediated by its interaction with specific plasma membrane receptors in target cells, and this has been established in a whole range of tissues, the localization of which generally corVA~OACTIVEINTESTINAL
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0193~1849/91 $1.50 Copyright
0
e receptors
JUAN C. PRIETO de Ahab,
relates with a VIP-like immunoreactivity (9, 23). The physiological and pharmacological characteristics of the VIP receptors have been extensively studied, but only recently has it been possible to investigate their molecular characteristics (9, 14). The seminal vesicle from humans, rats, and other species not only contains adrenergic and cholinergic nerve fibers but also those that store VIP and other peptides (5). VIP immunoreactive neurons seem to be closely associated with smooth muscle and subepithelial connective tissue close to the epithelium in the seminal vesicle (15, 20), suggesting a physiological role for VIP in this interesting glan.d of the male genital tract. Some recent results suggest VIP activity on secretory movements at this level (X,28). However, the involvement of VIP in motor activity, secretory mechanisms, metabolism, and cell proliferation in the seminal vesicle remains to be established. A recent preliminary report has shown the presence of specific binding sites for VIP in seminal vesicles from both rat and guinea pig when characterized by immunocytochemical techniques (20). However, there is no information on the pharmacological and molecular properties of VIP receptors in this gland, which can contribute to our knowledge of the direct action of VIP in the seminal vesicle. Using biochemical techniques we therefore studied the kinetics, stoichiometry, specificity, and dependence on gua.nyl nucleotides of VIP binding to plasma membranes from rat seminal vesicle. We also estimated the molecular size of these VIP receptors by using a covalent cross-linking procedure with the bifunctional reagent dithiobis( succinimidyl propionate) (DTSP). EXPERIMENTAL
PROCEDURES
Materials. Highly purified natural porcine VIP and secretin were supplied by Prof. V. Mutt (Karolinska Institute, Stockholm, Swedenj. Porcine glucagon was purchased from Novo. Helodermin and rat growth hormone-releasing factor (GRF) were obtained from Peninsula Laboratories. Bacitracin, phenylmethylsulfonyl fluoride (PMSF), bovine serum albumin, GTP and other nucleotides and nucleosides, DTSP, and electrophoresis reagents were purchased from Sigma Chemical. All other reagents were of the highest purity commercially available. ?-labeled VIP was prepared at a specific activity of -250 Ci/g and possesseda biological activity similar
1991 the American
Physiological
Society
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to that of native VIP (10, 21). Animals and membrane preparation. Sexually mature male Wistar rats (-250 g) were employed in this study. The animals were maintained in an environmentally controlled room (23°C; 12:12 h light-dark cycle), and food and water were provided ad libitum. Rats were killed by decapitation, and seminal vesicles were quickly removed, cleaned of the seminal fluid, dissected free from connective and adipose tissues, and then finely minced. For membrane preparation, the tissue was homogenized using a Polytron (setting 5, 3 min, Brinkmann Instruments) in 0.01 M triethanolamine-HCl buffer, pH 7.5, containing 0.25 M sucrose and 0.5 mM EDTA. After filtration through two layers of medical gauze, the homogenate was centrifuged at 1,500 g for 15 min at 4°C. The resulting supernatant was centrifuged at 30,000 g for 30 min at 4°C. The final pellet was washed twice in 20 mM tris(hydroxymethyl)aminomethane (Tris) HCl buffer, pH 7.5, and was immediately frozen at -70°C until used. Protein concentration was determined according to the method of Lowry et al. (13), using bovine serum albumin as a standard. VIP binding assay. In a standard binding assay, membranes (0.3 mg protein/ml) were incubated in 0.25 ml of 50 mM Tris. HCl buffer, pH 7.5, containing 1.4% bovine serum albumin, 1 mg/ml bacitracin, 0.05 mM PMSF, and 50 pM 12”1-VIP in the absence or presence of increasing amounts of unlabeled peptide (0.01-100 nM). Membrane-bound 1251-VIP was separated by filtration under vacuum using Whatman GB/F filters that had been pretreated with 1% polyethylenimine. Each filter was washed twice with 5 ml ice-cold Tris. HCl buffer, pH 7.5, and radioactivity was counted in a Kontron gamma counter at 70% efficiency. To determine nonspecific binding, parallel incubations were made in the presence of an excess (1 PM) of unlabeled VIP; nonspecific VIP binding averaged ~2% of the total radioactivity and was subtracted from the total binding to express the results as specific binding. The apparent equilibrium dissociation constants (&) and maximum numbers of binding sites expressed in terms of binding capacities (B,,,) were calculated from the stoichiometric data using the nonlinear iterative computer curve-fitting program (LIGAND) of Munson and Rodbard (17). The inactivation of 12’1VIP in the standard binding assay was estimated by talc adsorption, as previously described (7). Briefly, at the end of the incubation period, the reaction mixture was centrifuged at 30,000 g for 15 min. Then an aliquot of the supernatant (12”I-VIP) was mixed with an equal volume of 5% talc to adsorb intact tracer. After centrifugation for 5 min at 3,000 g, the radioactivity in the pellet was counted. Degradation was expressed as the percentage of unaltered tracer present in the control simultaneously incubated without membrane fraction. Cross-linking of 12’1-VIP to membranes. Membranes (-0.5 mg protein/ml) were incubated for 60 min at 15°C in 2.5 ml of the binding assay buffer containing 0.5 nM ‘““I-VIP. To determine nonspecific binding, parallel samples were incubated as above, but in the presence of excess (1 PM) unlabeled VIP. After incubation, the reaction mixture was diluted with 10 ml of ice-cold 60 mM l
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N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), pH 7.5, and centrifuged at 30,000 g for 15 min to remove unbound VIP. The pellet was then resuspended in 1 ml of the same buffer, but including 1 mM DTSP to initiate VIP-receptor cross-linking. After 15 min incubation at 4°C the reaction was stopped with 0.5 ml ice-cold 60 mM HEPES buffer, pH 7.5, containing 60 mM lysine as a reagent quench (8). The mixture was centrifuged for 15 min at 30,000 g, and the resulting pellet was resuspended by sonication (Dynatech sonic dismembrator; setting 35% of maximum, 1 min) in 0.1 ml of 60 mM Tris HCl buffer, pH 6.8, containing 10% glycerol, 0.001% bromophenol blue, and 3% sodium dodecyl sulfate (SDS). After 30 min at 60°C, the suspension was centrifuged at 30,000 g for 15 min, and the supernatant was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE and autoradiography. SDS-PAGE was carried out according to the method of Laemmli (11) in a 12% polyacrylamide slab gel (1.5 mm thickness) with a 3% polyacrylamide stacking gel. Molecular mass markers (triosephosphate isomerase 26,600; lactate dehydrogenase 36,500; fumarase 48,500; pyruvate kinase 58,000; fructose-6-phosphate kinase 84,000; fl-galactosidase 116,000; and cu2-macroglobulin 180,000) were run in parallel lanes. The gels were run, fixed, and dried as described previously (8). Then they were exposed for l-7 days at -70°C to a Du Pont Cronex-4 film with an intensifying screen (Du Pont Cronex Lightning Plus). RESULTS
Time and temperature studies on 12’I-VIP binding and degradation. Figure 1 illustrates the patterns of specific I”“1-VIP binding and degradation in membranes from rat seminal vesicle at three temperatures. Clearly, the binding (Fig. 1, top) was time- and temperature-dependent, with the maximal and stable binding obtained at 15°C between 30 and 120 min of incubation. This apparent equilibrium was accompanied by a very low level of peptide inactivation that did not reach 10% throughout the time interval studied (Fig. 1, bottom). At 25 and 37OC, the binding was rapid but transient, probably because of the higher degradation of ‘“51-VIP at these temperatures. It was therefore decided to undertake all subsequent incubations for 60 min at 15°C. Effect of membrane protein concentration. To determine whether the binding of ‘““I-VIP was proportional to the membrane concentration, the specific binding of 1251-VIP was studied at increasing membrane protein concentrations (Fig. 2). The binding of the neuropeptide was found to be linear at protein concentrations up to 0.6 mg/ml. The characteristics of binding were studied at a membrane protein concentration of 0.3 mg/ml. Dissociation of bound 12’1-VIP. The reversibility of the specific interaction between 12’I-VIP and membranes from rat seminal vesicle was assessed after a 60-min incubation at 15°C (Fig. 3). The addition of 0.1 PM unlabeled VIP resulted in a time-dependent displacement of the receptor-bound 12”1-VIP, with a 50% displacement of specifically bound tracer within 30 min at 15°C. The time course of dissociation of the complex
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1
0
1
I
1
0
1
2
1
’
’
’
I
10 20 30 40 50 Time (mid
60
FIG. 3. Dissociation time course. Membranes from icle (0.3 mg membrane protein/ml) were preincubated VIP at steady state (60 min at 15”C), and dissociation adding 1 ,uM unlabeled VIP. Specific binding of tracer at indicated times. Results correspond to a representative of 2 performed in trip icate.
0.050
’ T
Time (hr).
rat seminal veswith 50 pM *“Iwas initiated by was determined experiment
IJIG. 1. Dependence of vasoactive intestinal peptide (VIP) binding and degradation on time and temperature. Fifty pM l’“I-labeled VIP were incubated with membranes from rat seminal vesicle (0.3 mg membrane protein/ml) at 15°C (o), 25°C (0), and 37°C (m). After indicated time intervals, specific binding (top) and degradation (bottom) of labeled neuropeptide were determined. Results correspond to representative experiments of 2 performed in triplicate at each temperature. . I//L 00
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.
0
0
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1
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FIG. 4. Competition binding of ‘““I-VIP at steady state. Membranes (0.3 mg membrane protein/ml) were incubated with 50 pM ‘““I-VIP for 60 min at 15OC in presence of increasing concentrations of unlabeled neuropeptide. Corresponding extent of specific ““I-VIP binding was expressed as a function of unlabeled VIP concentration (left; means t SE of 6 separate experiments performed in triplicate). Right: Scatchard analysis of same mean data.
/
0
g
7
/
. /
(& = 44.4 t 4.1 nM) and high binding capacity t 150 fmol/mg protein; means t SE of 6 separate 0.3 0.6 0 experiments). Membrane Protein (mg/ml) Receptor specificity of 12,51VIP binding. The specificity of the VIP receptors in membranes from rat seminal FIG. 2. Dependence of VIP binding on membrane concentration. Specific receptor binding of 50 pM “‘I-VIP was determined at increasvesicle was investigated by determining the ability of ing concentrations of membrane protein after 60 min incubation at various peptides that are structurally related to VIP to 15°C. Results correspond to a representative experiment of 2 performed compete with the binding of 12”1-VIP (Fig. 5). The order in triplicate. of potency of the different peptides, as expressed by the tracer membranes followed multi-order kinetics, which concentration giving half-maximal inhibition of tracer binding, was as follows: VIP (I& = 1 nM) > helodermin suggested heterogeneity of VIP binding sites. Receptor binding of 1251-VIP at steady state. The addi- UC 50 = 10 nM) > secretin (ICso = 30 nM) >> rat GRF tion of unlabeled VIP caused a concentration-dependent UC 5. = 300 nM). In concentrations up to 10 PM, glucagon competition of the receptor-bound 12”1-VIP on mem- (another peptide structurally related to VIP) did not inhibit ““I-VIP binding at all. branes from rat seminal vesicle (Fig. 4, left), half-maximal inhibition (I&J being observed at -1 nM VIP. The Effect of guanyl nucleotides on 1251-VIP binding. The Scatchard analysis (26) of the stoichiometric data gave specific binding of 12”1-VIP to the membranes was moda curvilinear plot (Fig. 4, right) that was interpreted in ulated in a negative and dose-dependent manner by terms of a model of two classesof VIP receptors possess- guanyl nucleotides (Fig. 6). From 0.1 PM to 1 mM, GTP ing different affinities as follows: a first site of high and GDP inhibited the binding of the labeled peptide affinity (Kd = 0.54 2 0.04 nM) and low binding capacity with a similar potency, 50% inhibition of total specific (73 t 13 fmol VIP/ mg protein) and a second site of low binding measured at eauilibrium being obtained at -0.3 ’
JO
I
affinity
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I
(1,065
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Kd 116.0 84.0 58.0 48.5
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-50.0
26.6 :m [Peptide], -log M FIG. 5. Effect of VIP and related peptides on ‘251-VIP binding. Membranes from rat seminal vesicle (0.3 mg membrane protein/ml) were incubated with 50 pM “‘I-VIP in absence or presence of unlabeled VIP (o), helodermin (o), secretin (m), rat growth hormone-releasing factor (o), and glucagon (A). Specific binding of tracer was determined after 60 min incubation at 15°C and expressed as a percentage of maximal binding. Data are means + SE of 4 separate experiments performed in triplicate.
FIG. 7. Molecular mass determination of VIP receptors ,n membranes from rat seminal vesicle. Conditions for covalent labeling of using cross-linking reagent dimembrane protein by Y-VIP thiobis(succinimidy1 propionate) followed by SDS-polyacrylamide gel electrophoresis along with proteins of known relative molecular mass (M*) are described in EXPERIMENTAL PROCEDURES. Lanes of known M, are also described there. Lanes in autoradiography correspond to ‘251-VIP incubation in absence (A) or presence (B) of 1 PM unlabeled VIP. Reference protein size markers are indicated. Pattern is representative of 4 experiments.
mass) protein must represent the specific VIP receptor, since its labeling was greatly reduced when the crosslinking procedure was performed in the presence of 1 PM unlabeled VIP. Another widespread and weak band possessing an apparent molecular mass of 91,000 Da was also identified, indicating the existence of a second VIP binding protein of 88,000 Da. DISCUSSION
[Nucleotide],- log M FIG. 6. Dependence of VIP binding on guanyl nucleotides. ““I-VIP (50 PM) was incubated with membranes (0.3 mg membrane protein/ ml) in absence or presence of increasing concentrations of GTP (o), GDP (0), GMP (o), and ATP (A). Specific binding of ‘251-VIP was determined after 60 min incubation at 15°C. Results are means + SE of 3 separate experiments performed in triplicate.
mM GTP or GDP. Other nucleotides such as GMP and ATP did not affect the binding of ‘251-VIP to membranes even at a concentration as high as 1 mM. Molecular mass determination of VIP receptors by affinity labeling. VIP receptors in membranes from rat
seminal vesicle were identified at the molecular level by SDS-PAGE after cross-linking with the bifunctional agent DTSP as described in EXPERIMENTAL PROCEDURES. The conditions for the cross-linking reaction were optimized previously (data not shown). As indicated in Fig. 7, a major autoradiographic band was identified at 50,000 Da, which may correspond to a VIP binding protein with an apparent molecular mass of 47,000 Da plus the bound VIP. This 47,000-Da (relative molecular
The present study documents for the first time the properties of VIP receptors and their corresponding molecular size in membranes isolated from rat seminal vesicles. The basic characterization of the interaction of 1251-VIP with the receptors fulfilled the criteria of reversibility, saturability, specificity, and high affinity of a common hormone- or neurotransmitter-receptor binding reaction in agreement with that observed in other VIP binding systems like those in membranes isolated from gastric (6) or intestinal epithelium (l), lung (12), prostate (3), pancreas (22), liver (2), myometrial smooth muscle (19), and brain (30). The stoichiometric parameters of VIP receptors were obtained in optimal experimental conditions, since peptide degradation during the incubation period (60 min at 15°C) did not exceed 10%. Based on the results obtained from both the study of VIP binding at the steady state and the dissociation experiments, two classes of VIP receptors appear to be present in membranes from rat seminal vesicle; one with high affinity (& = 0.54 nM) that represented 6.4% of the total and another with low affinity (& = 44.4 nM). This heterogeneous pattern is common to all the VIP receptor systems so far described with very few exceptions, one being rat brain membranes, where a unique class of VIP receptors has been found (30). The dissociation constants observed in the present preparation are in the range of those tissues studied previously. It has
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been suggested that the existence of two classes of receptors for VIP can be due to the modulation of receptor affinity by the GTP-regulatory component, i.e., G proteins (9). In this context, it is to be noted that VIP binding to membranes from rat seminal vesicle was GTP sensitive, since the extent of VIP binding was inhibited in a dose-dependent manner. These data suggest that, in rat seminal vesicle, VIP receptors couple to some GTPbinding proteins. The competition experiments performed between 12’)1VIP and different unlabeled peptides that belong to the VIP family showed an order of efficacy common to other VIP binding systems (1, 3, 6, 12) as follows: VIP > helodermin > secretin > rat GRF and the lack of effect by glucagon. From this point of view it seems that the actions of VIP are mediated via similar receptors expressed in the different target tissues. The molecular characterization of VIP receptors was carried out by the covalent cross-linking of 12”1-VIP to membranes from rat seminal vesicle, using the bifunctional reagent DTSP. After protein solubilization and SDS-PAGE, a major 12”1-VIP-protein complex of M, 50,000 Da was identified. Assuming that one molecule of VIP linked to the receptor, the molecular mass of this component can be estimated to be ~47,000 Da. Another widespread and weak band was also observed, with an apparent molecular mass of 91,000 Da corresponding to a VIP binding component of 88,000 Da. Two alternatives could explain that the two classes of VIP receptors characterized by competitive displacement experiments were identified by SDS-PAGE primarily as a major component of -47,000 Da as follows: 1) two different classes of VIP receptors having similar molecular weight are present, and 2) the two classesof VIP receptors could be covalently attached to 1251-VIP by DTSP with different efficiency due to unequal localization and accessibility in the membrane. However, the 47,000- and 88,000-Da bands could correspond to the two classes of VIP receptors, as has been suggested for other VIP receptor systems (8). The major VIP binding proteins usually possess molecular massesranging from 46,000 to 53,000 Da depending on the systems studied and exhibit the high affinity and specificity of the VIP receptor (9, 14). It has been suggested that larger proteins may correspond to 1) complexes formed by the VIP receptor and a G, protein (9) or the VIP receptor and an cysprotein of 42,000 Da (4); 2) a precursor of the VIP receptor (18); and 3) a dimeric structure of the VIP-binding protein (16) In conclusion, the results presented here on the pharmacological and molecular weight identification of VIP receptors in rat seminal vesicle together with the known presence of VIP nerve fibers in this gland (5,20) strongly suggest that VIP may play a direct physiological role at this level. Some recent data support an effect of VIP on the secretory activity of seminal vesicle but not on either the basal tension or the neurogenic contraction of the corresponding smooth muscle in rat (15) and guinea pig (28). Nevertheless, the functional significance of VIP in the seminal vesicle remains to be clearly established, i.e, at levels as important as cell proliferation, hydroelectrolvtic movement, motor activitv, and metabolism.
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We thank Carol F. Warren from the Instituto de Ciencias de la Education de la Universidad de Alcala for linguistic assistance. This work was supported by Grants PM89-0096 and HF-100/1989 from Direction General de Investigation Cientifica y Tecnica, by the Consejo Superior de Investigaciones Cientificas (CSIC-INSERM, 1988), and by the Communidad Autonoma de Madrid. Address for reprint requests: J. C. Prieto, Departamento de Bioquimica y Biologia Molecular, Universidad de Alcala, 28871 Alcala de Henares-Madrid, Spain. Received
4 June
1990; accepted
in final
form
5 October
1990.
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cinema cells in culture (HTZ9). Eur. J. Biochem. 151: 411-417, 1985. 17. MUNSON, P. J., AND D. RODBARD. LIGAND: a versatile approach for characterization of ligand-binding systems. Anal. Biochem. 107: 220-239,198O. 18. NGUYEN, T. D., J. A. WILLIAMS, AND G. M. GRAY. Vasoactive intestinal peptide receptor on liver plasma membranes: characterization as a glycoprotein. Biochemistry 25: 361-368, 1986. 19. OTTESEN, B., P. STAUN-OLSEN, S. GAMMELTOFT, AND J. FAHRENKRUG. Receptors for vasoactive intestinal polypeptide on crude smooth muscle membranes from porcine uretus. Endocrinology 110: 2037-2043,1982.
20. POWER, R. F., A. E. BISHOP, J. WHARTON, C. 0. INYAMA, R. H. JACKSON, S. R. BLOOM, AND J. M. POLAK. Anatomical distribution of vasoactive intestinal peptide binding sites in peripheral tissues investigated by in vitro autoradiography. Ann. NY Acad. Sci. 527: 314-325,1988. 21.
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PRIETO, J. C., M. LABURTHE, AND G. ROSSELIN. Interaction of vasoactive intestinal peptide with isolated intestinal epithelial cells from rat. 1. Characterization, quantitative aspects and structural requirements of binding sites. Eur. J. Biochem. 96: 229-237, 1979. ROBBERECHT, P., M. WAELBROECK, M. NOYER, P. CHATELAIN, P. DE NEEF, W. K~NIG, AND J. CHRISTOPHE. Characterization of secretin and vasoactive intestinal peptide receptors in rat pancreatic plasma membranes using the native peptides, secretin (727) and five secretin analogues. Digestion 23: 201-210, 1982.
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23. ROSSELIN, G. The receptors of the VIP family peptides (VIP, secretin, GRF, PHI, PHM, GIP, glucagon, and oxyntomodulin). Specificities and identity. Peptides 7: 89-100, 1986. 24. SAID, S. I. Vasoactive intestinal peptide. J. Endocrinol. Inuest. 9: 191-200, 1986. 25. SAID, S. I., AND V. MUTT. Isolation from porcine-intestinal wall of a vasoactive octacosapeptide related to secretin and to glucagon. Eur. J. Biochem. 28: 199-204, 1972. z6 SCATCHARD, G. The attractions of proteins for small molecules ’ and ions. Ann. NY Acad. Sci. 51: 660-672,1949. 27 . SIMS, K. B., D. L. HOFFMAN, S. I. SAID, AND E. A. ZIMMERMAN. Vasoactive intestinal polypeptide (VIP) in mouse and rat brain: an immunocytochemical study. Brain Res. 186: 165-183, 1980. 28. STJERNQUIST, M., R. HAKANSON, S. LEANDER, C. OWMAN, F. SUNDLER, AND R. UDDMAN. Immunohistochemical localisation of substance P, vasoactive intestinal polypeptide and gastrin-releasing peptide in the vas deferens and seminal vesicle, and the effect of these and eight other neuropeptides on resting tension and neurally evoked contractile activity. Regul. Pept. 7: 67-86, 1983. F., E. EKBLAD, T. GRUNDITZ, R. HAKANSON, AND R. 29. SUNDLER, UDDMAN. Vasoactive intestinal peptide in the peripheral nervous system. Ann. N. Y. Acad. Sci. 527: 143-167, 1988. 30. TAYLOR, D. P., AND C. B. PERT. Vasoactive intestinal polypeptide: specific binding to rat brain membranes. Proc. Natl. Acad. Sci. USA. 76: 660-664, 1979.
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