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Demonstration of [125I]VIP binding sites and effects of VIP on cAMP-formation in rat insulinoma (RINm5F and RIN14B) cells
Regulator), Peptides, 40 (1992) 41-49
41
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-0115/92/$05.00
REGPEP 01192
Demonstration of [125I]VIP binding sites and effects of VIP on cAMP-formation in rat insulinoma (RINm5F and RIN14B) cells Mats Andersson a, R a n n a r Sillard a and Ake ROkaeus b Departments of Biochemistry ll~ and I b , Karolinska lnstitutet, Stockholm (Sweden)
(Received 21 January 1991; revised version received 25 February 1992; accepted 20 March 1992)
Key words: Vasoactive intestinal polypeptide; VIP-receptor; Beta-cell; Delta-cell; Insulin secretion; Adenylate cyclase
Summary Vasoactive intestinal polypeptide (VIP)-immunoreactive nerves have been demonstrated in close association with the islets of Langerhans, and VIP has been shown to stimulate insulin and somatostatin secretion. Using [125I]VIP and membranes prepared from rat insulinoma (RIN) cells, i.e., the subclones m5F (m5F; mainly insulinsecreting) and 14B (14B; mainly somatostatin-secreting), it was found that VIP (10 - 10_ 10 - 7 M) competitively inhibited the binding of[ 125I]VIP. A single c las s of high affinity binding sites with Kd values of 0.40 + 0.06 nM and 0.36 + 0.08 nM for m5F and 14B, respectively, with a corresponding number of binding sites (Bmax) of 163 + 20 and 254+ 51 fmol/mg protein was observed. The rank order of potency in inhibiting [125I]VIP binding was in both cell lines: VIP > helodermin > pituitary adenylate cyclase activating polypeptide 1-27 (PACAP27) > peptide histidine isoleucine (PHI) > secretin. VIP caused a dose-dependent increase in cAMP-formation in both m5F and 14B cell membranes with ECs0 values of 3.0 and 3.5 nM, respectively, but VIP (I" 1 0 - 9-3" 1 0 - 6 M) had no effect on insulin secretion (over 2 h) from the m5F cells. Thus, the data suggest that the VIP-receptors in these neoplastic rat cell lines, despite an apparent coupling to adenylate cyclase activity, seem to be functionally uncoupled to an effect on insulin secretion following an acute exposure to VIP.
Correspondence to: M. Andersson, Department of Biochemistry II, Karolinska Institutet, Box 60 400, S-104 01 Stockholm, Sweden.
42 Introduction
In man and rat, vasoactive intestinal polypeptide (VIP)-immunoreactive nerves have been demonstrated in intrapancreatic ganglia as well as in close association with the islets of Langerhans [1]. Both VIP and peptide with N-terminal histidine and Cterminal isoleucine amide (PHI) have been shown to potentiate glucose- and arginineinduced insulin release in anaesthetized rats [2], as well as insulin release from perifused newborn rat pancreas [3]. The degree of insulin, glucagon and somatostatin release from the rat pancreas is critically dependent on glucose and arginine levels [3,4] but in cultured fetal rat islets, which have a quantitative deficiency in their biphasic insulin response to glucose, acute VIP exposure did not effect insulin levels per se at any glucose concentration [5]. However, following chronic VIP treatment there was enhanced insulin response to glucose and an increased islet content of glucagon and somatostatin [5]. Using quantitative electron microscopic autoradiography [~25I]VIP binding sites have been demonstrated in rat pancreatic ~-,/3- and 6-cells [6]; in the case of fl-cells, labeling of both membrane surface and intracellular vesicles was observed, suggesting internalization of [125I]VIP binding sites [6,7]. The present study investigates whether the mainly insulin-secreting rat insulinoma (RIN), subclone m5F and the mainly somatostatin-secreting subclone 14B [9,10], could be used for characterizing [125I]VIP binding sites on fl- and b-cells and if so, whether or not they are coupled to adenylate cyclase activation and insulin secretion. Materials and Methods
Peptides and chemicals (3-[ l~-5I]iodotyrosyll°)VlP (2000 Ci/mmol) was obtained from Amersham (UK) and is referred to as [~25I]VIP. Synthetic VIP was obtained from Bachem (Switzerland), helodermin from (Peninsula) and PACAP27 was a generous gift from Dr. W. Schmidt, Department of Medicine, University of GOttingen, Germany. VIP, PHI and secretin were isolated from pig intestine as described [ 10]. Glucagon (for clinical use, NOVO Biolabs, Denmark) was purified by reverse-phase high-performance liquid chromatography (HPLC) before use. Tris-HC1, phosphoenolpyruvate, bacitracin, aprotinin, bovine serum albumin (BSA; fraction V), ATP, GTP and cyclic AMP (cAMP) were from Sigma (USA). Theophylline was from Fluka (Switzerland), EGTA and EDTA were from Merck (Germany), pyruvate kinase was from Boehringer-Mannheim (Germany), forskolin from Calbiochem (USA) and [2,8-3H]cAMP was obtained from New England Nuclear (USA). Cell culture and release experiments The cells, m5F and 14B, were grown at 37°C in 5~o C O 2 o n 24 well Costar plates (for secretion experiments) or in Costar flasks (for membrane preparations) containing 1 ml/well or 40 ml/flask of RPMI 1640 medium without glutamine (Gibco, UK). The medium was supplemented with 10~o fetal bovine serum (FBS; heat inactivated at 56°C for 30 min), 50 IU/ml penicillin and 50 #g/ml streptomycin. Media were changed 3 days after seeding the cells, and every second or third day thereafter as well
43 as on the day of the experiment when cells were nearly confluent (secretion experiments only). The confluent cells were then preincubated with 1 ml of fresh prewarmed media containing 1~o FBS for 2 h. Thereafter either of two protocols (A or B) were used. (A) At 2 h, media were replaced with 1 ml of fresh medium containing natural VIP (10 10 tO 10 - 6 M ) and incubated for 2 h under the conditions described above; a control plate receiving only fresh media was also incubated at the same time. At 4 h, plates were swirled and 50/A (m5F) of media was collected from each well, into prechilled Eppendorf-tubes, and stored at -20 °C until analysis by radioimmunoassay (RIA). (B) At 2 h, control samples (see above) were examined for interindividual variation of secretion in each well, i.e., as a measurement of the basal secretion. The amount of sample withdrawn was replaced by a 10 x concentrated synthetic VIP-solution to yield concentrations varying between 1" 10 9 M and 3.10- 6 M and incubated for an additional 2 h when another set of samples were examined for the possible effect ofVIP. Peptide degradation was examined under the same conditions by incubating cells with 3 . 1 0 - 6 M VIP (up to 48 h) and subsequently analyzing the remaining VIP in the medium, using reversed phase HPLC (Vydac 218TP54, 0.46 x 25 cm column). The amount of intact peptide eluting at the same retention time as VIP was calculated based on the absorbance at 2 214 nm.
Insulin radioimmunoassay RIA was performed with a rat insulin kit (NOVO Biolabs, Denmark); guinea-pig anti-insulin antibody (M8309, final dilution 1:350,000) was used in combination with 3000 cpm 125I-labeled porcine insulin (35.7 mCi/mg). Rat insulin (0.1-10 ng/ml) was used in standards (0.1 ml) and a suitably diluted aliquot of the sampled media was incubated in 0.5 ml of 40 mM phosphate-buffer (pH 7.4) with 0.1~o BSA, for 48 h at 4°C. Separation was carried out by the addition of 0.1 ml of outdated human bank plasma, 1 ml of charcoal/dextran T-70 suspension (1 ~o and 0.1 ~o, respectively) and centrifugation at 4°C.
Membrane preparations Cells were grown under the conditions described above. The medium was decanted and cells were rinsed twice with 10 ml of 50 mM Tris-HC1 (pH 7.4) at room temperature. Cells were then scraped off (5-10 ml of buffer/flask) and the resulting cellsuspension centrifugated (1200 g) for 15 rain at 4°C. The pellet was resuspended in 10 ml ice-cold buffer and homogenized using a Polytron (20,000 rpm, 20 s) followed by a 15 rain centrifugation (38,000 g) at 4°C and the resulting pellet rehomogenized (as above) in ice-cold buffer. Membranes for adenylate cyclase assays were prepared essentially as described above, but cells were rinsed and scraped off in ice-cold 130 mM phosphate-buffered saline (pH 7.2) and homogenized in 5 mM Tris-HC1 (pH 7.4). The final pellet was rehomogenized in 30 mM Tris-HC1 (pH 7.4) containing 1.5 mM theophylline, 8.25 mM MgCI2, 0.75 mM EGTA, 7.5 mM KC1 and 100 mM NaC1 (buffer A). Membranes for binding as well as adenylate cyclase assay were aliquoted into tubes on ice and stored at - 8 0 ° C until use. Protein content was determined using the BCA Protein Assay Reagent (Pierce, Europe B.V.) and BSA as standard.
44
Binding studies Cell membranes (0.43 mg/ml for m5F and 0.20 mg/ml for 14B) were incubated with 20-40 pM [125I]VIP in 50 mM Tris-HC1 (pH 7.4) containing 1.5 mM MgCI2, 300 #g/ ml bacitracin and 1~o BSA in the absence or presence of 10 l l - 1 0 - 6 M VIP, PACAP27, helodermin, PHI and secretin or 10- 6 M glucagon. Incubations were carried out in a total volume of 0.3 ml for 40 min at 30°C. Separation of free and membrane-bound ligands was achieved by centrifugation at 10,000 g for 5 min at 4 ° C. Radioactivity in the pellet was counted using a LKB 1282 gammacounter. The specific binding to membrane, calculated as the difference between the amount of bound [125I]VIP in absence and presence of 10 6M VIP, was about 5~o and nonspecific binding less than 2~o of the total radioactivity added to the membrane. Degradation of [125I]VIP (assayed after precipitation of the radioactive material remaining in the supernatant with 10~o trichloroacetic acid) was less than 20~o at the end of the incubation. In the kinetic experiments replicate aliquotes were removed from the incubation mixture after the appropriate time intervals and assayed for [125I]VIP binding.
Adenylate cyclase assay Adenylate cyclase was assayed essentially as described by Sutherland et al. [ 11 ] with some modifications. Briefly, membranes (0.01-0.03 mg/ml final concentrations) were incubated in buffer A (see above) also containing 100 #g/ml bacitracin, 20 #g/ml aprotinin, 0.05~o BSA, 10 mM ATP, 10 #M GTP and an ATP regenerating system consisting of 10 mM phosphoenolpyruvate and 30 #g/ml pyruvate kinase, in the absence of presence of synthetic or natural VIP (10-~1-10-5 M). Membrane preparation in buffer A (50/A) was added to the tubes containing all other components except GTP and ATP, which were added last to start the reaction. Incubations were carried out in a total volume of 150 gl at 30°C for 15 min in polypropylene tubes. The reaction was stopped by the addition of 50 #1 100 mM EDTA (pH 7.4), the tubes were subsequently boiled for 3 min and put on ice. cAMP-content in the tubes was determined according to Brown et al. [ 12] using a cAMP-binding protein prepared from bovine adrenal cortex and [2,8-3H]cAMP. Experiments performed with natural and synthetic VIP caused a similar adenylate cyclase activation in RIN cell membranes and thus results from both type of experiments have been combined.
Results
Specific binding of [125I]VIP to m5F and 14B cell membranes was saturable and time-dependent at 30°C reaching an apparent plateau between 30 and 120 min (Fig. 1A). Specific binding at 4°C was 5-fold lower than maximal binding at 30°C after 180 min in both cell membranes. Specific binding of [125I]VIP to membranes was dependent of membrane protein concentration, with an increase in specific binding up to at least 550/~g/ml for both m5F and 14B (Fig. 1B). Unlabeled VIP inhibited the binding of [ 125I]VIP to both m5F and 14B membranes in a concentration-dependent manner (Fig. 2A and B). Scatchard plot analysis [ 13] of
45
A
B
10
•
0
30
60
90
0
120
TIME (min)
•
•
a
200 400 600 MEMBRANE PROTEIN (p.g/ ml)
Fig. 1. Dependence of specific [125I]VIPbinding to RINm5F (O) and RIN14B ((3) cell membranes on time (A) and membrane protein concentration (B). Binding assays were performed as described under Materials and Methods in the absence and presence of 10 6 M VIP. The results are the mean of two experiments performed in duplicate with S.D. within 10~o.
the d a t a (n = 4) suggested the presence of a single class o f VIP-binding sites on membranes from both m 5 F and 14B cells. Analysis o f the d a t a with the ' L I G A N D ' program [ I4] resulted in dissociation constants (K d o f 0.40 + 0.06 n M a n d 0.36 + 0.08 n M , respectively. Competitive inhibition of [~25I]VIP binding to both cell membranes, showed a higher potency for VIP than for the related peptides helodermin, P A C A P , P H I , secretin and glucagon (Fig. 2A and B). Half-maximal inhibition, ICs0-values, obtained with m 5 F cells and 14B cells were: 0.45 + 0.04 and 0.4 _+0.05 for VIP, 0.81 + 0.2 and 0.7 + 0.1 for helodermin, 2.0 + 0.3 and 1.8 + 0.5 for P A C A P 2 7 , 10.4 + 0.1 and 11.8 + 2.9 n M for P H I , and 216 + 15 and 141 + 37 n M for secretin, respectively. G l u c a g o n (10 - 6 M ) had no effect on [L25I]VIP binding in either m 5 F or 14B cells. The estimated n u m b e r o f [125I]VIP binding-sites (Bmax) on m e m b r a n e s from m 5 F was found to be lower than in m e m b r a n e s from 14B, i.e., 163 _+20 and 254 + 51 fmol/mg protein, respectively, b a s e d on analysis with the ' L I G A N D ' program [ 14].
100
f
100f
i1
= "~60 "1
60
~.~4o ~
40
0u n -11
a -10 log
T -9
-S
[PEPTIDE](M)
n -7
-6
B~
n -11
i
I o10
n -9
!
I -us
log[PEPTIDE](M)
n -u7
-6
Fig. 2. Displacement of specific [ ~25I]VIPbinding to RINm5F (A) and RIN14B (B) cell membranes by VIP (O), helodermin (A), PACAP27 (if]), PHI (O) and secretin (A). Binding assays were carried out as described under Materials and Methods and the data are represented as the mean of three independent experiments performed in duplicate with less than 10~o S.D.
46
lOO .~ ~=
,.""
8o E
"""9
60
~E 40 [~-
Z
2o o i
I
I
I
i
I
I
11
10
9
8
7
6
5
-log [VIP] (M) Fig. 3. Effects of increasing concentrations of VIP on adenylate cyclase activity in RINm5F (O) and RIN 14B ( O ) cell membranes. The results are the mean _+ S.D. of two independent experiments performed in triplicate and are expressed as °~o of the difference between the basal activity and the activity obtained at 10 7 M VIP. The values for adenylate cyclase activity (pmol cAMP/min/mg protein) were: 284 + 23 and 256 _+ 13 (basal), 373 _+7 and 372 + 13 (at maximal stimulation by VIP) in m5F and 14B cell membranes, respectively.
Adenylate cyclase activation in m5F and 14B cell membranes by VIP was concentration-dependent, reaching maximal values between 10 7 and 10-6 M VIP as shown in Fig. 3. The dependence of adenylate cyclase activity upon VIP concentration seems to be similar within the error limits for m5F and 14B cell membranes and the estimated potencies, ECs0-values, were 3.0 and 3.5 nM for m5F and 14B, respectively (n = 2). The increase in adenylate cyclase activity caused by 10 7 M VIP was 13 + 23; of the maximum stimulation possible by 1 0 - 5 M forskolin in each cell line (1.11 + 0.04 nmol cAMP/min/mg protein in 14B cell membrane), suggesting that this fraction of adenylate cyclase activity is linked to VIP binding sites in both cell lines. Isolated natural VIP (10- 10 to 10- s M) had no significant effect (n = 2) on basal insulin secretion from m5F cells over 2 h (not shown). However, at higher concentrations (10- 7 to 10- 6 M) of isolated natural VIP, there was a tendency for an increased insulin secretion. On the other hand, synthetic VIP at concentrations of 1.10 '~ to 3 . 1 0 - 6 M (n = 2) had no effect. Under these conditions t,/: for degradation of VIP (3"10 6M) was found to be 10h.
Discussion
This study demonstrates the existence of VIP-preferring binding sites in cell membranes prepared from both RIN m5F and RIN 14B cells when using iodinated VIP as a ligand. Estimated affinity constants (Kd) from competitive binding experiments are of similar magnitude in both cell membranes (0.40 and 0.36 nM, respectively). The assignment of affinity and receptor number should be made with caution as experiments
47 were performed with only one concentration of [125I]VIP. The presence of only one population of high affinity sites in cell membranes from both cell lines studied here is similar to previous reports using different cell lines [15-17]. However, two classes of VIP binding sites have been reported in many cell and membrane preparations including rat liver [ 18]. The reason for this discrepancy remains unclear. The peptide potency in inhibiting [ 125I]VIP binding in both m5F and 14B membranes was: VIP > PHI > secretin and no glucagon binding, which is similar to that observed for VIP-receptors in other rat tissues such as intestine [19] and liver [18,20]. Both helodermin and PACAP27 interacts with [~25I]VIP binding sites with high affinity in accordance with previous findings in rat liver [21] and rat pancreatic acini [22]. In another report where intact m5F cells and [125I]PHI was used as ligand [23], rat PHI and VIP were equally potent in displacing the binding. This difference may be due to the use of different techniques employing [ 125I]PHI as ligand rather than [lzsI]VIP as has been observed in rat liver membranes [24]. Alternatively, the use of intact cells rather than crude membranes and synthetic rat PH1 rather than isolated natural porcine PHI could cause potency differences as observed in rat anterior pituitary gland [25]. The data from inhibition experiments show a somewhat higher number of binding sites in 14B membranes compared to m5F, in agreement with the study of Anteunis et al. [6]. They found more labeling of 6-cells than of ~cells after pancreatic perfusion with [~25I]VIP and quantitative electron microscopic autoradiography. VIP stimulated cAMP-formation in both type of cell membranes with a half-maximal stimulation occurring at a slightly higher concentration than half-maximal inhibition of [ 125I]VIP binding. These data suggest coupling of the VIP-binding sites to adenylate cyclase and may indicate the same mechanism of VIP mediated stimulation on adenylate cyclase in both cell lines. The somewhat higher value of adenylate cyclase basal activity obtained for m5F, compared to that reported before [26], can possibly be due to the differences in assay conditions and/or differences in cell passage number. Acute exposure of RIN m5F cells to VIP did not seem to significantly increase the level of basal insulin release over 2 h. Under identical conditions glucagon (10-6 M) produced a 59 + 11 ~o (n = 3) increase over basal levels and galanin (10- 6 M) a reduction by 34 + 3 ~o (n = 10) consistent with earlier studies (R0kaeus et al., unpublished observations). Since the increase in insulin secretion by VIP in vivo is apparently dependent on glucose, and the RIN-cells are not responsive to glucose elevation as the normal/?-cells [3,4], they may lack the proper response pattern to VIP observed in vivo. Hence, the neoplastic RIN-cells, at least in our hands, behave similarly to fetal rat islet cells which do not respond with an elevated insulin secretion to acute VIP exposure at any glucose concentration [5]. However, in a recent study [23], both synthetic rat PHI and synthetic VIP were stated to increase insulin secretion over 1 hour in a 'dose-independent' highly variable manner (between experiments). The reason for this difference, in terms of apparent 'dose independent' effect on insulin secretion and the lack of effect in our study, is not clear. In summary, we have found evidence for high affinity VIP binding sites that are positively coupled to adenylate cyclase but apparently not coupled to insulin secretion in the mainly insulin-secreting rat insulinoma cell-line.
48
Acknowledgements This study was supported by grants from The Swedish Medical Research Council (Projects No. 1010, 1101, 7906 and 8997), The Swedish Medical Society, Magnus Bergvall's Foundation, Karolinska Institute, The Nordic Insulin Fund, Skandigen AB, KabiGen AB, Swedish Hoechst Diabetes Foundation and Wenner-Gren Center Foundation. The authors wish to express their sincere gratitude to Professor Viktor Mutt for support and advice throughout this study and to Dr. Herbert Oie for providing the RINm5F and RIN14B cell lines. References 1 Bishop, A.E., Polak, J.M., Green, I.C., Bryant, M.G. and Bloom, S.R., The location of VIP in the pancreas of man and rat, Diabetologia, 18 (1980) 73-78. 2 Szec6wka, J., Lins, P. E., Tatemoto, K. and Efendic', S., Effects of porcine intestinal heptacosapeptide and vasoactive intestinal polypeptide on insulin and glucagon secretion in rats, Endocrinology, 112 (1983) 1469-1473. 3 Bataitle, D., Jarrousse, C., Vauclin, N., Gespach, C. and Rosselin, G., Effect of vasoactive intestinal peptide (VIP) and gastric inhibitory peptide (GIP) on insulin and glucagon release by perifused newborn rat pancreas. In P.P. Foa et al. (Eds.), Glucagon, its role in physiology and clinical medicine, SpringerVerlag, New York, 1977, pp. 255-269. 4 Szec6wka, J., Sandberg, E. and Efendic', S., The interaction of vasoactive intestinal polypeptide (VIP), glucose and arginine on the secretion of insulin, glucagon and somatostatin in the perfused rat pancreas, Diabetologia, 19 (1980) 137-142. 5 Rowley Ill, W. H., Fletcher, D.J., Dudek, R.W. and Brinn, J. E., Vasoactive intestinal polypeptide enhances hormone content and insulin release in cultured fetal rat islets, Proc. Soc. Exp. Biol. Med., 186 (1987) 165-169. 6 Anteunis, A., Astesano, A., Hejblum, G., Marie, J.-C., Portha, B. and Rosselin, G., Localization of vasoactive intestinal peptide binding sites in rat pancreatic islets, by computer-assisted analysis of electron microscopy video-autoradiographs, Ann. N.Y. Acad. Sci. USA, 527 (1988) 672-673. 7 Anteunis, A., Astesano, A., Portha, B., Hejblum, G. and Rosselin, G., Ultrastructural analysis of VIP internalization in rat fl- and acinar cells in situ, Am. J. Physiol., 256 (1989) G689-G697. 8 Bhathena, S.J., Awoke, S., Voyles, N.R., Wilkins, S., Recant, L., Oie, H.K. and Gazdar, A.F., Insulin, glucagon, and somatostatin secretion by cultured rat islet cell tumor and its clones, Proc. Soc. Exp. Biol. Med., 175 (1984) 35-38. 9 Gazdar, A.F., Chick, W.L., Oie, H.K., Sims, H.L., King, D.L., Weir G.C. and Lauris, V., Continuous, clonal, insulin-, and somatostatin-secreting cell lines established from a transplantable rat islet cell tumor, Proc. Natl. Acad. Sci. USA, 77 (1980) 3519-3523. 10 Said, S.I. and Mutt, V., Polypeptide with broad biological activity: isolation from small intestine, Science, 169 (1970) 1217-1218. 11 Sutherland, E. W., Rail, T. W. and Menon, T., Adenyl cyclase. I. Distribution, preparation and properties, J. Biol. Chem., 237 (1962) 1220-1227. 12 Brown, B.L., Ekins, R.P. and Albano, J.D.M., Saturation assay for cyclic AMP using endogenous binding protein, Adv. Cycl. Nucl. Res., 2 (1972) 25-40. 13 Scatchard, G., The attraction of proteins for small molecules and ions, Ann. N. Y. Acad. Sci., 51 (1949) 660-672. 14 Munson, P.J. and Rodbard, D., LIGAND: A versatile computerized approach for the characterization of ligand-binding systems, Anal. Biochem., 107 (1980) 220-239. 15 Couvineau, A., Rousset, M. and Laburthe, M., Molecular identification and structural requirement of vasoactive intestinal peptide (VIP) receptors in the human colon adenocarcinoma cell line, HT-29, Biochem. J., 231 (1985) 139-143.
49 16 Raymond, M. and Rosenzweig, S., Vasoactive intestinal peptide receptors on AR42J rat pancreatic acinar cells, Biochem. Biophys. Res. Commun., 179 (1) (1991) 176-182. 17 Wood, C., O'Dorisio, S., Vassalo, L., Malarkey, W. and O'Dorisio. T., Vasoactive intestinal peptide effects on GH3 pituitary tumor cells: high affinity binding, affinity labeling, and adenylate cyclase stimulation. Comparison with peptide histidine isoleucine and growth hormone-releasing factor, Regul. Pept., 12 (1985) 237-248. 18 Waelbroeck, M., Robberecht, P., De Neef, P., Chatelain, P. and Christophe, J., Binding of vasoactive intestinal peptide and its stimulation of adenylate cyclase through two classes of receptors in rat liver membranes. Effects of 12 secretin analogues and 2 secretin fragments, Biochim. Biophys. Acta, 678 (1981) 83-90. 19 Laburthe, M., The vasoactive intestinal peptide (VIP): an ubiquitous neuropeptide member of a structural family of regulatory peptides, Biochimie (Paris), 67 (1985) XI-XVII. 20 Bataille, D., Gespach, C., Laburthe, M., Amiranoff, B., Tatemoto, K., Vauclin, N., Mutt, V. and Rossetin, G., Porcine peptide having N-terminal histidine and C-terminal isoleucine amide (PHI). Vasoactive intestinal peptide (VIP) and secretin like effects in different tissues from the rat, FEBS Lett., 114 (1980) 240-242. 21 Dehaye, J.P., Winand, J., Damien, C., Gomez, F., Polocek, P., Robberecht, P., Vandermeers, A., Vandermeers-Piret, M.-C., Stievenart, M. and Christophe, J., Receptors involved in helodermin action on rat pancreatic acini, Am. J. Physiol., 251 (Gastrointest. Liver Physiol. 14) (1986) G602-G610. 22 Robberecht, P., Gourlet, P., Cauvin, A., Buscail, L., De Neef, P., Arimura, A. and Christophe, J., PACAP and VIP receptors in rat liver membranes, Am. J. Physiol., 260 (Gastrointest. Liver Physiol. 23) (1991) G97-G102. 23 G0ke, R. and Conlon, J.M., Binding sites for peptide-histidine-isoleucine (PHI) on rat insulinomaderived RINm5F cells, Mol. Cell. Endocrinol., 60 (1988) 211-215. 24 Paul, S., Chou, J. and Kubota. E., High affinity peptide histidine isolucine-preferring receptors in rat liver, Life Sci., 41 (1987) 2373-2380. 25 Wanke, I.E. and Rorstad, O.P., Receptors for vasoactive intestinal peptide in rat anterior pituitary glands: localization of binding to Lactotropes, Endocrinology, 126 (1990) 1981-1988. 26 Amiranoff, B., Lorinet, A.-M., Lagny-Pourmir, I. and Laburthe, M., Mechanism of galanin-inhibited insulin release. Occurrence of a pertussis toxin-sensitive inhibition of adenylate cyclase, Eur. J. Biochem., 177 (1988) 147-152.
41
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-0115/92/$05.00
REGPEP 01192
Demonstration of [125I]VIP binding sites and effects of VIP on cAMP-formation in rat insulinoma (RINm5F and RIN14B) cells Mats Andersson a, R a n n a r Sillard a and Ake ROkaeus b Departments of Biochemistry ll~ and I b , Karolinska lnstitutet, Stockholm (Sweden)
(Received 21 January 1991; revised version received 25 February 1992; accepted 20 March 1992)
Key words: Vasoactive intestinal polypeptide; VIP-receptor; Beta-cell; Delta-cell; Insulin secretion; Adenylate cyclase
Summary Vasoactive intestinal polypeptide (VIP)-immunoreactive nerves have been demonstrated in close association with the islets of Langerhans, and VIP has been shown to stimulate insulin and somatostatin secretion. Using [125I]VIP and membranes prepared from rat insulinoma (RIN) cells, i.e., the subclones m5F (m5F; mainly insulinsecreting) and 14B (14B; mainly somatostatin-secreting), it was found that VIP (10 - 10_ 10 - 7 M) competitively inhibited the binding of[ 125I]VIP. A single c las s of high affinity binding sites with Kd values of 0.40 + 0.06 nM and 0.36 + 0.08 nM for m5F and 14B, respectively, with a corresponding number of binding sites (Bmax) of 163 + 20 and 254+ 51 fmol/mg protein was observed. The rank order of potency in inhibiting [125I]VIP binding was in both cell lines: VIP > helodermin > pituitary adenylate cyclase activating polypeptide 1-27 (PACAP27) > peptide histidine isoleucine (PHI) > secretin. VIP caused a dose-dependent increase in cAMP-formation in both m5F and 14B cell membranes with ECs0 values of 3.0 and 3.5 nM, respectively, but VIP (I" 1 0 - 9-3" 1 0 - 6 M) had no effect on insulin secretion (over 2 h) from the m5F cells. Thus, the data suggest that the VIP-receptors in these neoplastic rat cell lines, despite an apparent coupling to adenylate cyclase activity, seem to be functionally uncoupled to an effect on insulin secretion following an acute exposure to VIP.
Correspondence to: M. Andersson, Department of Biochemistry II, Karolinska Institutet, Box 60 400, S-104 01 Stockholm, Sweden.
42 Introduction
In man and rat, vasoactive intestinal polypeptide (VIP)-immunoreactive nerves have been demonstrated in intrapancreatic ganglia as well as in close association with the islets of Langerhans [1]. Both VIP and peptide with N-terminal histidine and Cterminal isoleucine amide (PHI) have been shown to potentiate glucose- and arginineinduced insulin release in anaesthetized rats [2], as well as insulin release from perifused newborn rat pancreas [3]. The degree of insulin, glucagon and somatostatin release from the rat pancreas is critically dependent on glucose and arginine levels [3,4] but in cultured fetal rat islets, which have a quantitative deficiency in their biphasic insulin response to glucose, acute VIP exposure did not effect insulin levels per se at any glucose concentration [5]. However, following chronic VIP treatment there was enhanced insulin response to glucose and an increased islet content of glucagon and somatostatin [5]. Using quantitative electron microscopic autoradiography [~25I]VIP binding sites have been demonstrated in rat pancreatic ~-,/3- and 6-cells [6]; in the case of fl-cells, labeling of both membrane surface and intracellular vesicles was observed, suggesting internalization of [125I]VIP binding sites [6,7]. The present study investigates whether the mainly insulin-secreting rat insulinoma (RIN), subclone m5F and the mainly somatostatin-secreting subclone 14B [9,10], could be used for characterizing [125I]VIP binding sites on fl- and b-cells and if so, whether or not they are coupled to adenylate cyclase activation and insulin secretion. Materials and Methods
Peptides and chemicals (3-[ l~-5I]iodotyrosyll°)VlP (2000 Ci/mmol) was obtained from Amersham (UK) and is referred to as [~25I]VIP. Synthetic VIP was obtained from Bachem (Switzerland), helodermin from (Peninsula) and PACAP27 was a generous gift from Dr. W. Schmidt, Department of Medicine, University of GOttingen, Germany. VIP, PHI and secretin were isolated from pig intestine as described [ 10]. Glucagon (for clinical use, NOVO Biolabs, Denmark) was purified by reverse-phase high-performance liquid chromatography (HPLC) before use. Tris-HC1, phosphoenolpyruvate, bacitracin, aprotinin, bovine serum albumin (BSA; fraction V), ATP, GTP and cyclic AMP (cAMP) were from Sigma (USA). Theophylline was from Fluka (Switzerland), EGTA and EDTA were from Merck (Germany), pyruvate kinase was from Boehringer-Mannheim (Germany), forskolin from Calbiochem (USA) and [2,8-3H]cAMP was obtained from New England Nuclear (USA). Cell culture and release experiments The cells, m5F and 14B, were grown at 37°C in 5~o C O 2 o n 24 well Costar plates (for secretion experiments) or in Costar flasks (for membrane preparations) containing 1 ml/well or 40 ml/flask of RPMI 1640 medium without glutamine (Gibco, UK). The medium was supplemented with 10~o fetal bovine serum (FBS; heat inactivated at 56°C for 30 min), 50 IU/ml penicillin and 50 #g/ml streptomycin. Media were changed 3 days after seeding the cells, and every second or third day thereafter as well
43 as on the day of the experiment when cells were nearly confluent (secretion experiments only). The confluent cells were then preincubated with 1 ml of fresh prewarmed media containing 1~o FBS for 2 h. Thereafter either of two protocols (A or B) were used. (A) At 2 h, media were replaced with 1 ml of fresh medium containing natural VIP (10 10 tO 10 - 6 M ) and incubated for 2 h under the conditions described above; a control plate receiving only fresh media was also incubated at the same time. At 4 h, plates were swirled and 50/A (m5F) of media was collected from each well, into prechilled Eppendorf-tubes, and stored at -20 °C until analysis by radioimmunoassay (RIA). (B) At 2 h, control samples (see above) were examined for interindividual variation of secretion in each well, i.e., as a measurement of the basal secretion. The amount of sample withdrawn was replaced by a 10 x concentrated synthetic VIP-solution to yield concentrations varying between 1" 10 9 M and 3.10- 6 M and incubated for an additional 2 h when another set of samples were examined for the possible effect ofVIP. Peptide degradation was examined under the same conditions by incubating cells with 3 . 1 0 - 6 M VIP (up to 48 h) and subsequently analyzing the remaining VIP in the medium, using reversed phase HPLC (Vydac 218TP54, 0.46 x 25 cm column). The amount of intact peptide eluting at the same retention time as VIP was calculated based on the absorbance at 2 214 nm.
Insulin radioimmunoassay RIA was performed with a rat insulin kit (NOVO Biolabs, Denmark); guinea-pig anti-insulin antibody (M8309, final dilution 1:350,000) was used in combination with 3000 cpm 125I-labeled porcine insulin (35.7 mCi/mg). Rat insulin (0.1-10 ng/ml) was used in standards (0.1 ml) and a suitably diluted aliquot of the sampled media was incubated in 0.5 ml of 40 mM phosphate-buffer (pH 7.4) with 0.1~o BSA, for 48 h at 4°C. Separation was carried out by the addition of 0.1 ml of outdated human bank plasma, 1 ml of charcoal/dextran T-70 suspension (1 ~o and 0.1 ~o, respectively) and centrifugation at 4°C.
Membrane preparations Cells were grown under the conditions described above. The medium was decanted and cells were rinsed twice with 10 ml of 50 mM Tris-HC1 (pH 7.4) at room temperature. Cells were then scraped off (5-10 ml of buffer/flask) and the resulting cellsuspension centrifugated (1200 g) for 15 rain at 4°C. The pellet was resuspended in 10 ml ice-cold buffer and homogenized using a Polytron (20,000 rpm, 20 s) followed by a 15 rain centrifugation (38,000 g) at 4°C and the resulting pellet rehomogenized (as above) in ice-cold buffer. Membranes for adenylate cyclase assays were prepared essentially as described above, but cells were rinsed and scraped off in ice-cold 130 mM phosphate-buffered saline (pH 7.2) and homogenized in 5 mM Tris-HC1 (pH 7.4). The final pellet was rehomogenized in 30 mM Tris-HC1 (pH 7.4) containing 1.5 mM theophylline, 8.25 mM MgCI2, 0.75 mM EGTA, 7.5 mM KC1 and 100 mM NaC1 (buffer A). Membranes for binding as well as adenylate cyclase assay were aliquoted into tubes on ice and stored at - 8 0 ° C until use. Protein content was determined using the BCA Protein Assay Reagent (Pierce, Europe B.V.) and BSA as standard.
44
Binding studies Cell membranes (0.43 mg/ml for m5F and 0.20 mg/ml for 14B) were incubated with 20-40 pM [125I]VIP in 50 mM Tris-HC1 (pH 7.4) containing 1.5 mM MgCI2, 300 #g/ ml bacitracin and 1~o BSA in the absence or presence of 10 l l - 1 0 - 6 M VIP, PACAP27, helodermin, PHI and secretin or 10- 6 M glucagon. Incubations were carried out in a total volume of 0.3 ml for 40 min at 30°C. Separation of free and membrane-bound ligands was achieved by centrifugation at 10,000 g for 5 min at 4 ° C. Radioactivity in the pellet was counted using a LKB 1282 gammacounter. The specific binding to membrane, calculated as the difference between the amount of bound [125I]VIP in absence and presence of 10 6M VIP, was about 5~o and nonspecific binding less than 2~o of the total radioactivity added to the membrane. Degradation of [125I]VIP (assayed after precipitation of the radioactive material remaining in the supernatant with 10~o trichloroacetic acid) was less than 20~o at the end of the incubation. In the kinetic experiments replicate aliquotes were removed from the incubation mixture after the appropriate time intervals and assayed for [125I]VIP binding.
Adenylate cyclase assay Adenylate cyclase was assayed essentially as described by Sutherland et al. [ 11 ] with some modifications. Briefly, membranes (0.01-0.03 mg/ml final concentrations) were incubated in buffer A (see above) also containing 100 #g/ml bacitracin, 20 #g/ml aprotinin, 0.05~o BSA, 10 mM ATP, 10 #M GTP and an ATP regenerating system consisting of 10 mM phosphoenolpyruvate and 30 #g/ml pyruvate kinase, in the absence of presence of synthetic or natural VIP (10-~1-10-5 M). Membrane preparation in buffer A (50/A) was added to the tubes containing all other components except GTP and ATP, which were added last to start the reaction. Incubations were carried out in a total volume of 150 gl at 30°C for 15 min in polypropylene tubes. The reaction was stopped by the addition of 50 #1 100 mM EDTA (pH 7.4), the tubes were subsequently boiled for 3 min and put on ice. cAMP-content in the tubes was determined according to Brown et al. [ 12] using a cAMP-binding protein prepared from bovine adrenal cortex and [2,8-3H]cAMP. Experiments performed with natural and synthetic VIP caused a similar adenylate cyclase activation in RIN cell membranes and thus results from both type of experiments have been combined.
Results
Specific binding of [125I]VIP to m5F and 14B cell membranes was saturable and time-dependent at 30°C reaching an apparent plateau between 30 and 120 min (Fig. 1A). Specific binding at 4°C was 5-fold lower than maximal binding at 30°C after 180 min in both cell membranes. Specific binding of [125I]VIP to membranes was dependent of membrane protein concentration, with an increase in specific binding up to at least 550/~g/ml for both m5F and 14B (Fig. 1B). Unlabeled VIP inhibited the binding of [ 125I]VIP to both m5F and 14B membranes in a concentration-dependent manner (Fig. 2A and B). Scatchard plot analysis [ 13] of
45
A
B
10
•
0
30
60
90
0
120
TIME (min)
•
•
a
200 400 600 MEMBRANE PROTEIN (p.g/ ml)
Fig. 1. Dependence of specific [125I]VIPbinding to RINm5F (O) and RIN14B ((3) cell membranes on time (A) and membrane protein concentration (B). Binding assays were performed as described under Materials and Methods in the absence and presence of 10 6 M VIP. The results are the mean of two experiments performed in duplicate with S.D. within 10~o.
the d a t a (n = 4) suggested the presence of a single class o f VIP-binding sites on membranes from both m 5 F and 14B cells. Analysis o f the d a t a with the ' L I G A N D ' program [ I4] resulted in dissociation constants (K d o f 0.40 + 0.06 n M a n d 0.36 + 0.08 n M , respectively. Competitive inhibition of [~25I]VIP binding to both cell membranes, showed a higher potency for VIP than for the related peptides helodermin, P A C A P , P H I , secretin and glucagon (Fig. 2A and B). Half-maximal inhibition, ICs0-values, obtained with m 5 F cells and 14B cells were: 0.45 + 0.04 and 0.4 _+0.05 for VIP, 0.81 + 0.2 and 0.7 + 0.1 for helodermin, 2.0 + 0.3 and 1.8 + 0.5 for P A C A P 2 7 , 10.4 + 0.1 and 11.8 + 2.9 n M for P H I , and 216 + 15 and 141 + 37 n M for secretin, respectively. G l u c a g o n (10 - 6 M ) had no effect on [L25I]VIP binding in either m 5 F or 14B cells. The estimated n u m b e r o f [125I]VIP binding-sites (Bmax) on m e m b r a n e s from m 5 F was found to be lower than in m e m b r a n e s from 14B, i.e., 163 _+20 and 254 + 51 fmol/mg protein, respectively, b a s e d on analysis with the ' L I G A N D ' program [ 14].
100
f
100f
i1
= "~60 "1
60
~.~4o ~
40
0u n -11
a -10 log
T -9
-S
[PEPTIDE](M)
n -7
-6
B~
n -11
i
I o10
n -9
!
I -us
log[PEPTIDE](M)
n -u7
-6
Fig. 2. Displacement of specific [ ~25I]VIPbinding to RINm5F (A) and RIN14B (B) cell membranes by VIP (O), helodermin (A), PACAP27 (if]), PHI (O) and secretin (A). Binding assays were carried out as described under Materials and Methods and the data are represented as the mean of three independent experiments performed in duplicate with less than 10~o S.D.
46
lOO .~ ~=
,.""
8o E
"""9
60
~E 40 [~-
Z
2o o i
I
I
I
i
I
I
11
10
9
8
7
6
5
-log [VIP] (M) Fig. 3. Effects of increasing concentrations of VIP on adenylate cyclase activity in RINm5F (O) and RIN 14B ( O ) cell membranes. The results are the mean _+ S.D. of two independent experiments performed in triplicate and are expressed as °~o of the difference between the basal activity and the activity obtained at 10 7 M VIP. The values for adenylate cyclase activity (pmol cAMP/min/mg protein) were: 284 + 23 and 256 _+ 13 (basal), 373 _+7 and 372 + 13 (at maximal stimulation by VIP) in m5F and 14B cell membranes, respectively.
Adenylate cyclase activation in m5F and 14B cell membranes by VIP was concentration-dependent, reaching maximal values between 10 7 and 10-6 M VIP as shown in Fig. 3. The dependence of adenylate cyclase activity upon VIP concentration seems to be similar within the error limits for m5F and 14B cell membranes and the estimated potencies, ECs0-values, were 3.0 and 3.5 nM for m5F and 14B, respectively (n = 2). The increase in adenylate cyclase activity caused by 10 7 M VIP was 13 + 23; of the maximum stimulation possible by 1 0 - 5 M forskolin in each cell line (1.11 + 0.04 nmol cAMP/min/mg protein in 14B cell membrane), suggesting that this fraction of adenylate cyclase activity is linked to VIP binding sites in both cell lines. Isolated natural VIP (10- 10 to 10- s M) had no significant effect (n = 2) on basal insulin secretion from m5F cells over 2 h (not shown). However, at higher concentrations (10- 7 to 10- 6 M) of isolated natural VIP, there was a tendency for an increased insulin secretion. On the other hand, synthetic VIP at concentrations of 1.10 '~ to 3 . 1 0 - 6 M (n = 2) had no effect. Under these conditions t,/: for degradation of VIP (3"10 6M) was found to be 10h.
Discussion
This study demonstrates the existence of VIP-preferring binding sites in cell membranes prepared from both RIN m5F and RIN 14B cells when using iodinated VIP as a ligand. Estimated affinity constants (Kd) from competitive binding experiments are of similar magnitude in both cell membranes (0.40 and 0.36 nM, respectively). The assignment of affinity and receptor number should be made with caution as experiments
47 were performed with only one concentration of [125I]VIP. The presence of only one population of high affinity sites in cell membranes from both cell lines studied here is similar to previous reports using different cell lines [15-17]. However, two classes of VIP binding sites have been reported in many cell and membrane preparations including rat liver [ 18]. The reason for this discrepancy remains unclear. The peptide potency in inhibiting [ 125I]VIP binding in both m5F and 14B membranes was: VIP > PHI > secretin and no glucagon binding, which is similar to that observed for VIP-receptors in other rat tissues such as intestine [19] and liver [18,20]. Both helodermin and PACAP27 interacts with [~25I]VIP binding sites with high affinity in accordance with previous findings in rat liver [21] and rat pancreatic acini [22]. In another report where intact m5F cells and [125I]PHI was used as ligand [23], rat PHI and VIP were equally potent in displacing the binding. This difference may be due to the use of different techniques employing [ 125I]PHI as ligand rather than [lzsI]VIP as has been observed in rat liver membranes [24]. Alternatively, the use of intact cells rather than crude membranes and synthetic rat PH1 rather than isolated natural porcine PHI could cause potency differences as observed in rat anterior pituitary gland [25]. The data from inhibition experiments show a somewhat higher number of binding sites in 14B membranes compared to m5F, in agreement with the study of Anteunis et al. [6]. They found more labeling of 6-cells than of ~cells after pancreatic perfusion with [~25I]VIP and quantitative electron microscopic autoradiography. VIP stimulated cAMP-formation in both type of cell membranes with a half-maximal stimulation occurring at a slightly higher concentration than half-maximal inhibition of [ 125I]VIP binding. These data suggest coupling of the VIP-binding sites to adenylate cyclase and may indicate the same mechanism of VIP mediated stimulation on adenylate cyclase in both cell lines. The somewhat higher value of adenylate cyclase basal activity obtained for m5F, compared to that reported before [26], can possibly be due to the differences in assay conditions and/or differences in cell passage number. Acute exposure of RIN m5F cells to VIP did not seem to significantly increase the level of basal insulin release over 2 h. Under identical conditions glucagon (10-6 M) produced a 59 + 11 ~o (n = 3) increase over basal levels and galanin (10- 6 M) a reduction by 34 + 3 ~o (n = 10) consistent with earlier studies (R0kaeus et al., unpublished observations). Since the increase in insulin secretion by VIP in vivo is apparently dependent on glucose, and the RIN-cells are not responsive to glucose elevation as the normal/?-cells [3,4], they may lack the proper response pattern to VIP observed in vivo. Hence, the neoplastic RIN-cells, at least in our hands, behave similarly to fetal rat islet cells which do not respond with an elevated insulin secretion to acute VIP exposure at any glucose concentration [5]. However, in a recent study [23], both synthetic rat PHI and synthetic VIP were stated to increase insulin secretion over 1 hour in a 'dose-independent' highly variable manner (between experiments). The reason for this difference, in terms of apparent 'dose independent' effect on insulin secretion and the lack of effect in our study, is not clear. In summary, we have found evidence for high affinity VIP binding sites that are positively coupled to adenylate cyclase but apparently not coupled to insulin secretion in the mainly insulin-secreting rat insulinoma cell-line.
48
Acknowledgements This study was supported by grants from The Swedish Medical Research Council (Projects No. 1010, 1101, 7906 and 8997), The Swedish Medical Society, Magnus Bergvall's Foundation, Karolinska Institute, The Nordic Insulin Fund, Skandigen AB, KabiGen AB, Swedish Hoechst Diabetes Foundation and Wenner-Gren Center Foundation. The authors wish to express their sincere gratitude to Professor Viktor Mutt for support and advice throughout this study and to Dr. Herbert Oie for providing the RINm5F and RIN14B cell lines. References 1 Bishop, A.E., Polak, J.M., Green, I.C., Bryant, M.G. and Bloom, S.R., The location of VIP in the pancreas of man and rat, Diabetologia, 18 (1980) 73-78. 2 Szec6wka, J., Lins, P. E., Tatemoto, K. and Efendic', S., Effects of porcine intestinal heptacosapeptide and vasoactive intestinal polypeptide on insulin and glucagon secretion in rats, Endocrinology, 112 (1983) 1469-1473. 3 Bataitle, D., Jarrousse, C., Vauclin, N., Gespach, C. and Rosselin, G., Effect of vasoactive intestinal peptide (VIP) and gastric inhibitory peptide (GIP) on insulin and glucagon release by perifused newborn rat pancreas. In P.P. Foa et al. (Eds.), Glucagon, its role in physiology and clinical medicine, SpringerVerlag, New York, 1977, pp. 255-269. 4 Szec6wka, J., Sandberg, E. and Efendic', S., The interaction of vasoactive intestinal polypeptide (VIP), glucose and arginine on the secretion of insulin, glucagon and somatostatin in the perfused rat pancreas, Diabetologia, 19 (1980) 137-142. 5 Rowley Ill, W. H., Fletcher, D.J., Dudek, R.W. and Brinn, J. E., Vasoactive intestinal polypeptide enhances hormone content and insulin release in cultured fetal rat islets, Proc. Soc. Exp. Biol. Med., 186 (1987) 165-169. 6 Anteunis, A., Astesano, A., Hejblum, G., Marie, J.-C., Portha, B. and Rosselin, G., Localization of vasoactive intestinal peptide binding sites in rat pancreatic islets, by computer-assisted analysis of electron microscopy video-autoradiographs, Ann. N.Y. Acad. Sci. USA, 527 (1988) 672-673. 7 Anteunis, A., Astesano, A., Portha, B., Hejblum, G. and Rosselin, G., Ultrastructural analysis of VIP internalization in rat fl- and acinar cells in situ, Am. J. Physiol., 256 (1989) G689-G697. 8 Bhathena, S.J., Awoke, S., Voyles, N.R., Wilkins, S., Recant, L., Oie, H.K. and Gazdar, A.F., Insulin, glucagon, and somatostatin secretion by cultured rat islet cell tumor and its clones, Proc. Soc. Exp. Biol. Med., 175 (1984) 35-38. 9 Gazdar, A.F., Chick, W.L., Oie, H.K., Sims, H.L., King, D.L., Weir G.C. and Lauris, V., Continuous, clonal, insulin-, and somatostatin-secreting cell lines established from a transplantable rat islet cell tumor, Proc. Natl. Acad. Sci. USA, 77 (1980) 3519-3523. 10 Said, S.I. and Mutt, V., Polypeptide with broad biological activity: isolation from small intestine, Science, 169 (1970) 1217-1218. 11 Sutherland, E. W., Rail, T. W. and Menon, T., Adenyl cyclase. I. Distribution, preparation and properties, J. Biol. Chem., 237 (1962) 1220-1227. 12 Brown, B.L., Ekins, R.P. and Albano, J.D.M., Saturation assay for cyclic AMP using endogenous binding protein, Adv. Cycl. Nucl. Res., 2 (1972) 25-40. 13 Scatchard, G., The attraction of proteins for small molecules and ions, Ann. N. Y. Acad. Sci., 51 (1949) 660-672. 14 Munson, P.J. and Rodbard, D., LIGAND: A versatile computerized approach for the characterization of ligand-binding systems, Anal. Biochem., 107 (1980) 220-239. 15 Couvineau, A., Rousset, M. and Laburthe, M., Molecular identification and structural requirement of vasoactive intestinal peptide (VIP) receptors in the human colon adenocarcinoma cell line, HT-29, Biochem. J., 231 (1985) 139-143.
49 16 Raymond, M. and Rosenzweig, S., Vasoactive intestinal peptide receptors on AR42J rat pancreatic acinar cells, Biochem. Biophys. Res. Commun., 179 (1) (1991) 176-182. 17 Wood, C., O'Dorisio, S., Vassalo, L., Malarkey, W. and O'Dorisio. T., Vasoactive intestinal peptide effects on GH3 pituitary tumor cells: high affinity binding, affinity labeling, and adenylate cyclase stimulation. Comparison with peptide histidine isoleucine and growth hormone-releasing factor, Regul. Pept., 12 (1985) 237-248. 18 Waelbroeck, M., Robberecht, P., De Neef, P., Chatelain, P. and Christophe, J., Binding of vasoactive intestinal peptide and its stimulation of adenylate cyclase through two classes of receptors in rat liver membranes. Effects of 12 secretin analogues and 2 secretin fragments, Biochim. Biophys. Acta, 678 (1981) 83-90. 19 Laburthe, M., The vasoactive intestinal peptide (VIP): an ubiquitous neuropeptide member of a structural family of regulatory peptides, Biochimie (Paris), 67 (1985) XI-XVII. 20 Bataille, D., Gespach, C., Laburthe, M., Amiranoff, B., Tatemoto, K., Vauclin, N., Mutt, V. and Rossetin, G., Porcine peptide having N-terminal histidine and C-terminal isoleucine amide (PHI). Vasoactive intestinal peptide (VIP) and secretin like effects in different tissues from the rat, FEBS Lett., 114 (1980) 240-242. 21 Dehaye, J.P., Winand, J., Damien, C., Gomez, F., Polocek, P., Robberecht, P., Vandermeers, A., Vandermeers-Piret, M.-C., Stievenart, M. and Christophe, J., Receptors involved in helodermin action on rat pancreatic acini, Am. J. Physiol., 251 (Gastrointest. Liver Physiol. 14) (1986) G602-G610. 22 Robberecht, P., Gourlet, P., Cauvin, A., Buscail, L., De Neef, P., Arimura, A. and Christophe, J., PACAP and VIP receptors in rat liver membranes, Am. J. Physiol., 260 (Gastrointest. Liver Physiol. 23) (1991) G97-G102. 23 G0ke, R. and Conlon, J.M., Binding sites for peptide-histidine-isoleucine (PHI) on rat insulinomaderived RINm5F cells, Mol. Cell. Endocrinol., 60 (1988) 211-215. 24 Paul, S., Chou, J. and Kubota. E., High affinity peptide histidine isolucine-preferring receptors in rat liver, Life Sci., 41 (1987) 2373-2380. 25 Wanke, I.E. and Rorstad, O.P., Receptors for vasoactive intestinal peptide in rat anterior pituitary glands: localization of binding to Lactotropes, Endocrinology, 126 (1990) 1981-1988. 26 Amiranoff, B., Lorinet, A.-M., Lagny-Pourmir, I. and Laburthe, M., Mechanism of galanin-inhibited insulin release. Occurrence of a pertussis toxin-sensitive inhibition of adenylate cyclase, Eur. J. Biochem., 177 (1988) 147-152.