- Email: [email protected]
Effect of freezing on the coupling of VIP receptors to adenylate cyclase in rat liver membranes
Life Sciences, Vol. 42, pp. 505-510 Printed in the U.S.A.
EFFECT
Pergamon Journals
OF FREEZING ON THE COUPLING OF VIP RECEPTORS ADENYLATE CYCLASE IN RAT LIVER MEMBRANES.
TO
Patrick ROBBERECHT,Magali WAELBROECK,Philippe DE NEEFf Jean-ClaudeCAMUS, Philippe GOURLET and Jean CHRISTOPHE . Department of Biochemistryand Nutrition,School of Medicine, Universit Libre de Bruxelles,Boulevard de Waterloo 115, B-1000 Brussels, Belgium. (Received in final form November 24, 1987)
Summary In fresh rat liver plasma membranesi2$igh affinity VIP receptors were specifically labelled with [ I] helodermin and were well coupled to adenylate cyclase while low affinity VIP receptors were not. After freezing and thawing low affinity VIP receptors were also coupled to adenylate cyclase. This modification of adenylate cyclase activation was specific for the VIP response as freezing and thawing did not modify Gpp(NH)p, NaF and glucagon stimulations. High and low affinity binding sites for Vasoactive Intestinal Peptide (VIP) are present in rat liver membranes [1,2]. These sites present the usual characteristics of hormone receptors : reversible binding, sensitivity to guanyl nucleotides, and high specificity [1,3]. They resemble similar VIP receptors in other tissues when consideringthe structural ligand requirements for receptor occupancy end their potential coupling with adenylate cyclsse as a membrane effector system [4,5,6]. In rat liver this last characteristic presents, however, some particularities: a) the occupancy of VIP receptors in intact hepatocytes is followed, even at high ligand concentration,by only a modest increase in cyclic AMP when compared to that obtained with glucagon [ 7,8]; b) VIP stimulation of adenylate cyclase in highly purified rat liver plasma membranes is obvious in the absence of added GTP whereas glucagon stimulation requires the addition of the nucleotide [ 91 ; c) despite a high density of VIP receptors and the high sensitivityof adenylate cyclaae towards VIP, the efficacy of enz me stimulation (i.e. the amount of cyclic AMP produced) is low ]5,6,9 r . The metabolic effects of VIP on liver are qualitativelyidentical to the biochemical events triggered by other hormones acting through adenylate cyclase activation [ 7,8,10] but are evident at high peptide concentrationonly so that it has been hypothesizedthat high affinity VIP receptors are in fact mainly concerned with the initiation of the hepatic degradationof VIP [ll]. As there wss no explanationavailable for the unique profile of coupling of VIP receptors to adenylate cyclase in rat liver membranes, we inferred from new data that this coupling depended, at least partly, on the physical state of membranes : freezing and thawing of membranes greatly increasedthe capacity of VIP to stimulate cyclic AMP production.
* To whom correspondenceshould be addressed. 0024-3205188$3.00 +.OO Copyright (c) 1988 Pergamon Journals Ltd.
506
VIP Receptors
and Adenylate
Cyclase
Materials
Vol.
Coupling
42, No. 5,
1988
and methods
1. Preparation of hepatic membranes : Crude hepatic
membranes were prepared The membranes were resuspendedin 1 mM sodium bicarbonate to a protein concentrationof 5 mg/ml. These membranes were either used immediately or tested after freezing during from
fresh
rat
liver
as
previously
described
111 .
30 min in liquid nitrogen and rapid thawing. 2. Determinationof adenylatecyclase activity : Adenylate cyclase activity was determinedwith minor modificationsof the orocedure of Salomon et al. 1121 as previouslydescribed[l]. It. was verified that cyclic AMP production was linear for at least 8 min under all conditionstested. 3. Binding of [1251]heloderminand [lz5 I]VIP to hgatic membranes. a) radioiodination of VIP and helodermin : VIP and helodermin were radioiodinatedsimilarly with a chloramin-T technique as previously reported 11,131. [lz51]VIP was separated from Free iodine by cellulose adsorption and elution with bovine serum albumin [ 14 1. [ lz511 helodermin was seoarated from free 1251 by adsorption on a Sep Pack C 18 cartridge then eluted with 60 % acetonitrile. [1251]helodermin was further purified by reverse phase chromatography on a C 18 t] bondapak colu n eluted with a linear 35-45 ?A gradient of acetonitrile.The monoiodinated[ T 251]heldderminused was separated from the unlabelledpeptide (in preparation). b) Binding conditions : standard binding of [1251]VIP and ['25~]helodermin was carried out in a 50 mM Tris-maleatebuffer (pH 7.4) containing 0.5 mg/ml bacitracin,2 mM MgC12, serum albumin, 45,000 cpm membrane protein in a final The tubes were incubated at I] VIP binding and during 3D min for 37 PC during 15 min for [1251]heloderminbinding i.e. until binding equilibriumwas attained. Tracer degradation represented no more than 15 X of the free ligand [1,13]. The radioactivity bound to membranes was separated from Free radioactivity by Filtrationthrough glass-Fiberfilters GF/C (Whatman, Maidstone, UK) presoaked overnight in 0.1 X polyethyleneimine according to Schachter et al [15]. Non specific binding was determined in the presence of 1 FM unlabelled VIP and accounted For less than 20 % of total binding. Total binding always represented less than 15 % of the total radioactivityoffered. 4. Heloderminwas purified from the Gila monster venom From Siqma Chemical Co (St. Louis, MO, USA) as previouslydescribed[16]. Synthetic VIP was a generous (it_aj-~j? University,New Orleans, LA USA). Carrier-free P] ATP (10 Ci/mmol) and [R-'HIcyclic AMP (2030 Ci/mmol) were pur&ased from Amersham International (Bucks, UK). Bovine serum albumin (FractionV), bacitracin, ATP, cyclic AMP, GTP, pyruvate kinase and phospho(enol)pyruvate were From Sigma Chemical Co. (St. Louis, MO, USA), Gpp(NH) was from Boehringer (Mannheim,FRG.). 5. Dose-effect curves of adenylate cyclase activation and binding data were analyzed by computer using the "Ligand" programme developed by Richardson and Humrich [17]. Statistical analysis was performed using the Student's test on paired values. RESULTS
Adenylate cyclase activity in Freshly prepared crude rat liver membranes as well as in the same frozen and defrosted membranes was stimulated by GPP(NH)P, sodium fluoride, glucagon and VIP (Figs. 1 and 2). Cyclic AMP production was identical in both types of membranes under basal conditions and in the presence of Gpp(NH)p, NaF or in the combined presence of glucagon and 10 IJM GTP. The concentrationsof Gpp(NH)p, NaF and glucagon required For half-
Vol.
42, No. 5,
1988
VIP Receptors
and Adenylate
Cyclase
Coupling
507
maximum enzyme stimulation were also identical. The capacity of VIP to stimulate the enzyme increased markedly in defrosted membranes as compared to freshly prepared membranes (Fig. 1). The higher efficacy of VIP observed after freezing and thawing was accompanied by an apparent decrease in peptide potency. This resulted, however, from a biphasic pattern of the dose-effect curve of VIP (Fig. 1 and Table I) that was compatible (best-fitted) with the coexistence of two functional (sub)classes of : a high affinitylow capacity class, identical to the only VIP receptors active component found in fresh membranes, and a new low affinityhigher capacity class revealed only in defrosted membranes. TABLE I. Cyclase by VIP,
Best Fit of Dose-Effect Curves of VIP Adenylate [ lz51] VIP and [ lz511 Helodermin Binding Inhibition Existence of one or two Functionally Distinct Sites.
-~
DEFROSTED Results
[ lZ511 VIP
[ “3
helodermin 9 Max. (X1
Max. activity*
Yd (nM)
“(q”’ /D
0.3 + 0.1
16 f. 3
0.3 + 0.1 10 -+3
30 70
0.1 10
+ 0.1 -+2
05 15
-0.2 + 0.1 10.0 z 4
16 + 4 26 T - 5
0.3 + 0.2 10 T3 -
35 65
0.15 10
+ 0.05 T3 -
80 20
Kact (nM) FRESH
and
th e
RECEPTOROCCUPANCY
ADENYLATECYCLASE ACTIVATION
MEMBRANES
Activation Considering
from data presented in Fig. I (left panel) and Fig. 3 4 experiments. Maximal adenylate cyclase activation in the presence of 10 pM VIP was expressed as pm01 cyclic AMP produced.min-l.mg protein-l over basal activity. The Kd of receptor occupancy and the % of sites occupied were calculated using a “ligand” program [ 171 . All curves were best fitted bv a two-receotors model. FIG. 1.’ Effects of increasing concentrations of VIP (left RAT LIVER MEMBRANES * panel) and glucagon (right panel) on cyclic AMP production 165by crude rat hepatic membranes. Experiments were performed, in the presence of 10 IJM GTP, on freshly prepared membranes kept until use over ice (open symbols) or on the same membranes previously frozen during 15 min in liquid nitrogen and thawed immediately before testing (closed symbols). The results were the Teans 2 SEM of 6 experiments. Indicates where values on frozen membranes differed dh 4-M -10 -9. -6 -7 -6 -5 -5 significantly (p < 0.05) from [VIP CONCENTRATION1 llog I41 IGLUCAGON CONCENTRATION] (log HI corresponding data on fresh membranes. +and were
were calculated
Kd (nM)
the
means + SEM of
1
.
.
I
508
VIP Receptors
and Adenylate
Cyclase
Coupling
42, No. 5,
1988
FIG. 2 Effects of increasing concentrations of Gpp(NH)p (left panel) and WaF (right panel) on cyclic AMP production by crude rat hepatic membranes. Enzyme determinations were performed in the absence GTP. 0 f S a me representation an d svmbols as in Fio. 1. a
I
[Gpp(NH1 p CONCENTRATION] (log Ml
Vol.
2 4 6 8 10 [NaFCONCENTRATION] ImMJ
Binding sites in fresh and defrosted membranes were studied by competition curves, using [ 1251]VIP and [ 1251] helodermin as tracers and unlabelled IP as competitor (Fig. 3). As detailed in Table I, the inhibition of [ l2 ! I] VIP binding was best fitted considering the recognition of two classes of receptors, one with a low (0.3 nM) and a second one with a high (10 nM) Kd. The total binding capac’ty of [ 195 proportions of the twoT~~2~~V,I~~~~~~~~~~~~~~~~] ~{o~~~~~tee~ ~~~~~~~ defrosted membranes. more specifically the high affinity VIP binding sites) confirmed the coexistence of two classes of VIP receptors and their stability after freezing and thawing. As shown in Table I, there was after thawing an excellent correlation between the two Kact of VIP stimulation of adenylate cyclase and the two Kd values for VIP binding sites. FIG. DEFROSTEO(n=4)
[VIP CONCENTRATION1
absence of added peptide. and defrosted membranes.
Ilog Ml
The amounts of
tracer
3.
Inhibition of 11251] VIP (left pane-l) -and [ 1251 ] helodermin (righ t panel) binding to fresh or defrosted (I) (0) hepatic membranes by increasing concentrations of unlabelled VIP. The results were the means of 4 experiments made in duplicate a nd w er e expressed in X of tracer specifically bound in bound were identical in fresh
Discussion Despite a high density of VIP receptors in fresh rat hepatic membranes, VIP-stimulated adenylate cyclase activity remained low as compared to the glucagon response (present results and ref. 18). The potency of VIP was, however, remarkably high, making rat hepatic adenylate cyclase one of the most sensitive model for VIP stimulation [4]. In 1980, Amiranoff et al [9] suggested that the discrepancy between the high number of VIP receptors and the low efficacy of the neuropeptide was due to a defect in the GTP binding protein Ns
Vol.
42, No. 5,
1988
VIP Receptors and Adenylate Cyclase Coupling
509
coupling VIP receptors to the catalytical unit. This hypothesis was not supported, however, by recent data from the same group showing that most of the liver VIP receptors still interact with Ns after membrane solubilizationt~l. It remains t at VIP binding characterized by [ l&l ]VIp cross_linki~~,tes’differ in rat membranes’ from liver VIP receptors in ra: intestinal epithelial membranes, a system in which receptors are efficiently and potently coupled to adenylate cyclase [19,201. The Mr values of 51 and 89 kDa found for VIP binding proteins in liver contrast with Mr of 73 and 33 kDa found in intestinal cells. There is at present no argument suggesting that such structuraldifferencesare associatedwith functionaldifferences. The present binding studies confirmed that two classes of VIP binding sites can be identified in rat li membranes, their precise analysis being greatly facilitatedby the use of [1% Ilheloderminthat selectively labels high affinity VIP receptors [1,13 1 The high affinity VIP receptorswere coupled to adenylate cyclase (Table I and refs. 6 and 13) in freshly prepared membranes. After freezing and thawing both high and low affinity receptorswere coupled to the enzyme. This situation was not further modified by repeated freezing and thawing (data not shown). The interpretation of this unique observation, made so far on rat liver membranes only and for VIP receptors only, is highly speculative : It is conceivable that freezing and thawing modify the motility of 1. allowing a better association between receptors, Ns and membrane components, the catalytical unit. This hypothesis relies on the general dynamic properties
of biological membranes [211 and on the non permanent coupling of hormonal the absence of coupling receptors with adenylate cyclase 1221 . Of course, between low affinity VIP receptors and adenylate cyclase observed before freezing leaves this subclass of receptors without a known effector system in the "fresh" state. Considering thst the physical state of membranes influences adenylate 2. cyclsse activation [23-251 it is also possible that freezing induced discrete modifications of lipids and/or proteins in membrane structures that were sufficient to modify the efficscy of coupling between membrane proteins. all hormonal as well as Gpp(NH)p and fluoride responses are affected. Usually, The highly specific alteration of the VIP response in the present study, in the face of a general perturbation of membranes, is not a completely isolated observation as we showed recently that aliphatic alcohols potentiate the effect of secretin but not those of glucsgon and isoproterenol in rat heart membranes
[261.
the
Whatever the explanation, the present VIP receptor and the Gs-catalytical
data indicate that coupling between unit system can be selectively
modulated by membrane freezing . Acknowledgements. This work was supported Research (Belgium).
by Grant 3.4571.85
from the Fund for
Medical
Scientific
References 1. 2. 3. 4. 5. 6.
M. WAELBROECK, P. ROBBERECHT, P. DE NEEF, P. CHATELAIN P. and J. CHRISTOPHE,Biochim. Biophys. Acta 678 83-90 (1981). A. COUVINEAU, B. AMIRANOFF and M. L4BmHE, J. Biol. Chem. 261, 1448214489 (1986). B. DESBUQUOIS,Eur. J. Biochem. 46 439-450 (1974). P. ROBBERECHT, M. WAELBROECK ,J.C. CAMUS, P. DE NEEF, D. COY and J. CHRISTOPHE,Peptides 5 877-881 (1984). P. ROBBERECHT, M. WAEFBROECK, D. COY, P. DE NEEF, J.C. CAMUS and J. CHRISTOPHE,Peptides 7, Suppl. 1 53-59 (1986). P. ROBBERECHT,D.H. COY, P. DE NEEF, J.C. CAMUS, A. CAUVIN, M. WAELBROECK and J. CHRISTOPHE,Eur. J. Biochem.-159 45-49 (1986).
510
7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18.
19. 20. 21. 22. 23. 24. 25. 26.
VIP Receptors
and Adenylate
Cyclase
Coupling
Vol.
42,
No.
5,
1988
J.C. SOUQUET, J.P. RIOU, M. BEYLOT, J.A. CYAYVIALLE and R. MORNEX, FEES Lett. gLlU15-120 (1982). J.E. SILVESTRE, L. MONGE and J. MARCO, Endocrinologb, ?l2",",",',%25";~983). El. AMIRANOFF, MTABURTHE and G. ROSSELIN, Biochem. Biophys. Res. Commun. 96 463-468 (1980). r LEISER and J.J. BLUM, FEBS Lett. 173 407-413 (1984). S. GAMMELTOFT, P. STAUN-OLSEN, B. OmSEN and J. FAHRENKRUG, Peptides 2 367-370 (1984). Y. SALOMON, C. LONDOS and M. RODBELL, Anal. Biochem. 58 541-548 (1974). P. ROBBERECHT, M. WAELBROECK, P. DE NEEF, J.C. CAMUS,4. VANDERMEERS, M.-C. VANDERMEERS-PIRET and J. CHRISTOPHE, FEBS Lett. 172 55-58 (1984). 3. CHRISTOPHE, T.P. CONLON and J.D. GARDNER, J. Biol. Chem. 251 4629-4634 (1976). DICKINSON, C.M. MILES and P.S. SEVER, FEBS Lett. M. SCHACHTER, K.E.J., 199 125-129 (1986). A.VANDERMEERS, M.C. VANDERMEERS-PIRET, P. ROBBERECHT, M. WAELBROECK, J.P. DEHAYE, J. WINAND and J. CHRISTOPHE, FEBS Lett. 166 273-276 (1984). A. RICHARDSON and A. HUMRICH, Trends Pharmacol. Sci. 5 47-49 (1984). A. LABURTHE, D. BATAILLE, M. ROUSSET, J. BESSON, Y.-i3ROERS, A. ZWEIBAUM and G. ROSSELIN, in Proceedings of the membrane proteins action of the 11th FEBS Meeting, Copenhagen 1977, pp. 271-290, Pergamon Press, Oxford (19781. .-- -, A. COUVINEAU and M. LABURTHE, Biochem. J. 225 473-479 (1985). M. LABURTHE, B. BREANT and C. ROUYER-FESSARD, Eur. J. Biochem. 139 181187 (1984). M.J. KARNOVSKY, A.M. KLEINFELD, R.L. HOOVER and R.D. KLAUSNER, J. Cell. Biol. 94 l-6 (1982). G. RIMm, E. HANSKI, S. BRAUN and A. LEVITZKI, Nature, 276 394-396 (1978). L.M. GORDON, R.D. SAURHEBER, J.A. ESGATE, I. DIPPLE, R.J. MARCHMONT and M.D. HOUSLAY, J. Biol. Chem. 255 4519-4527 (1980). J. GARNIER PXETERRIER, D. DAVELOOSE and J. VIRET, kocELm%,'; 21 1581-1586'(1982) and D.' DAVELOOSE, Biochemistry R. SALESSE, -5: GARNIER -21 1587-1590, (1982). P. CHATELAIN, P. ROBBERECHT, M. WAELBROECK, J.C. CAMUS and J. CHRISTOPHE, J. Membrane Biol. -93 23-32 (1986).
EFFECT
Pergamon Journals
OF FREEZING ON THE COUPLING OF VIP RECEPTORS ADENYLATE CYCLASE IN RAT LIVER MEMBRANES.
TO
Patrick ROBBERECHT,Magali WAELBROECK,Philippe DE NEEFf Jean-ClaudeCAMUS, Philippe GOURLET and Jean CHRISTOPHE . Department of Biochemistryand Nutrition,School of Medicine, Universit Libre de Bruxelles,Boulevard de Waterloo 115, B-1000 Brussels, Belgium. (Received in final form November 24, 1987)
Summary In fresh rat liver plasma membranesi2$igh affinity VIP receptors were specifically labelled with [ I] helodermin and were well coupled to adenylate cyclase while low affinity VIP receptors were not. After freezing and thawing low affinity VIP receptors were also coupled to adenylate cyclase. This modification of adenylate cyclase activation was specific for the VIP response as freezing and thawing did not modify Gpp(NH)p, NaF and glucagon stimulations. High and low affinity binding sites for Vasoactive Intestinal Peptide (VIP) are present in rat liver membranes [1,2]. These sites present the usual characteristics of hormone receptors : reversible binding, sensitivity to guanyl nucleotides, and high specificity [1,3]. They resemble similar VIP receptors in other tissues when consideringthe structural ligand requirements for receptor occupancy end their potential coupling with adenylate cyclsse as a membrane effector system [4,5,6]. In rat liver this last characteristic presents, however, some particularities: a) the occupancy of VIP receptors in intact hepatocytes is followed, even at high ligand concentration,by only a modest increase in cyclic AMP when compared to that obtained with glucagon [ 7,8]; b) VIP stimulation of adenylate cyclase in highly purified rat liver plasma membranes is obvious in the absence of added GTP whereas glucagon stimulation requires the addition of the nucleotide [ 91 ; c) despite a high density of VIP receptors and the high sensitivityof adenylate cyclaae towards VIP, the efficacy of enz me stimulation (i.e. the amount of cyclic AMP produced) is low ]5,6,9 r . The metabolic effects of VIP on liver are qualitativelyidentical to the biochemical events triggered by other hormones acting through adenylate cyclase activation [ 7,8,10] but are evident at high peptide concentrationonly so that it has been hypothesizedthat high affinity VIP receptors are in fact mainly concerned with the initiation of the hepatic degradationof VIP [ll]. As there wss no explanationavailable for the unique profile of coupling of VIP receptors to adenylate cyclase in rat liver membranes, we inferred from new data that this coupling depended, at least partly, on the physical state of membranes : freezing and thawing of membranes greatly increasedthe capacity of VIP to stimulate cyclic AMP production.
* To whom correspondenceshould be addressed. 0024-3205188$3.00 +.OO Copyright (c) 1988 Pergamon Journals Ltd.
506
VIP Receptors
and Adenylate
Cyclase
Materials
Vol.
Coupling
42, No. 5,
1988
and methods
1. Preparation of hepatic membranes : Crude hepatic
membranes were prepared The membranes were resuspendedin 1 mM sodium bicarbonate to a protein concentrationof 5 mg/ml. These membranes were either used immediately or tested after freezing during from
fresh
rat
liver
as
previously
described
111 .
30 min in liquid nitrogen and rapid thawing. 2. Determinationof adenylatecyclase activity : Adenylate cyclase activity was determinedwith minor modificationsof the orocedure of Salomon et al. 1121 as previouslydescribed[l]. It. was verified that cyclic AMP production was linear for at least 8 min under all conditionstested. 3. Binding of [1251]heloderminand [lz5 I]VIP to hgatic membranes. a) radioiodination of VIP and helodermin : VIP and helodermin were radioiodinatedsimilarly with a chloramin-T technique as previously reported 11,131. [lz51]VIP was separated from Free iodine by cellulose adsorption and elution with bovine serum albumin [ 14 1. [ lz511 helodermin was seoarated from free 1251 by adsorption on a Sep Pack C 18 cartridge then eluted with 60 % acetonitrile. [1251]helodermin was further purified by reverse phase chromatography on a C 18 t] bondapak colu n eluted with a linear 35-45 ?A gradient of acetonitrile.The monoiodinated[ T 251]heldderminused was separated from the unlabelledpeptide (in preparation). b) Binding conditions : standard binding of [1251]VIP and ['25~]helodermin was carried out in a 50 mM Tris-maleatebuffer (pH 7.4) containing 0.5 mg/ml bacitracin,2 mM MgC12, serum albumin, 45,000 cpm membrane protein in a final The tubes were incubated at I] VIP binding and during 3D min for 37 PC during 15 min for [1251]heloderminbinding i.e. until binding equilibriumwas attained. Tracer degradation represented no more than 15 X of the free ligand [1,13]. The radioactivity bound to membranes was separated from Free radioactivity by Filtrationthrough glass-Fiberfilters GF/C (Whatman, Maidstone, UK) presoaked overnight in 0.1 X polyethyleneimine according to Schachter et al [15]. Non specific binding was determined in the presence of 1 FM unlabelled VIP and accounted For less than 20 % of total binding. Total binding always represented less than 15 % of the total radioactivityoffered. 4. Heloderminwas purified from the Gila monster venom From Siqma Chemical Co (St. Louis, MO, USA) as previouslydescribed[16]. Synthetic VIP was a generous (it_aj-~j? University,New Orleans, LA USA). Carrier-free P] ATP (10 Ci/mmol) and [R-'HIcyclic AMP (2030 Ci/mmol) were pur&ased from Amersham International (Bucks, UK). Bovine serum albumin (FractionV), bacitracin, ATP, cyclic AMP, GTP, pyruvate kinase and phospho(enol)pyruvate were From Sigma Chemical Co. (St. Louis, MO, USA), Gpp(NH) was from Boehringer (Mannheim,FRG.). 5. Dose-effect curves of adenylate cyclase activation and binding data were analyzed by computer using the "Ligand" programme developed by Richardson and Humrich [17]. Statistical analysis was performed using the Student's test on paired values. RESULTS
Adenylate cyclase activity in Freshly prepared crude rat liver membranes as well as in the same frozen and defrosted membranes was stimulated by GPP(NH)P, sodium fluoride, glucagon and VIP (Figs. 1 and 2). Cyclic AMP production was identical in both types of membranes under basal conditions and in the presence of Gpp(NH)p, NaF or in the combined presence of glucagon and 10 IJM GTP. The concentrationsof Gpp(NH)p, NaF and glucagon required For half-
Vol.
42, No. 5,
1988
VIP Receptors
and Adenylate
Cyclase
Coupling
507
maximum enzyme stimulation were also identical. The capacity of VIP to stimulate the enzyme increased markedly in defrosted membranes as compared to freshly prepared membranes (Fig. 1). The higher efficacy of VIP observed after freezing and thawing was accompanied by an apparent decrease in peptide potency. This resulted, however, from a biphasic pattern of the dose-effect curve of VIP (Fig. 1 and Table I) that was compatible (best-fitted) with the coexistence of two functional (sub)classes of : a high affinitylow capacity class, identical to the only VIP receptors active component found in fresh membranes, and a new low affinityhigher capacity class revealed only in defrosted membranes. TABLE I. Cyclase by VIP,
Best Fit of Dose-Effect Curves of VIP Adenylate [ lz51] VIP and [ lz511 Helodermin Binding Inhibition Existence of one or two Functionally Distinct Sites.
-~
DEFROSTED Results
[ lZ511 VIP
[ “3
helodermin 9 Max. (X1
Max. activity*
Yd (nM)
“(q”’ /D
0.3 + 0.1
16 f. 3
0.3 + 0.1 10 -+3
30 70
0.1 10
+ 0.1 -+2
05 15
-0.2 + 0.1 10.0 z 4
16 + 4 26 T - 5
0.3 + 0.2 10 T3 -
35 65
0.15 10
+ 0.05 T3 -
80 20
Kact (nM) FRESH
and
th e
RECEPTOROCCUPANCY
ADENYLATECYCLASE ACTIVATION
MEMBRANES
Activation Considering
from data presented in Fig. I (left panel) and Fig. 3 4 experiments. Maximal adenylate cyclase activation in the presence of 10 pM VIP was expressed as pm01 cyclic AMP produced.min-l.mg protein-l over basal activity. The Kd of receptor occupancy and the % of sites occupied were calculated using a “ligand” program [ 171 . All curves were best fitted bv a two-receotors model. FIG. 1.’ Effects of increasing concentrations of VIP (left RAT LIVER MEMBRANES * panel) and glucagon (right panel) on cyclic AMP production 165by crude rat hepatic membranes. Experiments were performed, in the presence of 10 IJM GTP, on freshly prepared membranes kept until use over ice (open symbols) or on the same membranes previously frozen during 15 min in liquid nitrogen and thawed immediately before testing (closed symbols). The results were the Teans 2 SEM of 6 experiments. Indicates where values on frozen membranes differed dh 4-M -10 -9. -6 -7 -6 -5 -5 significantly (p < 0.05) from [VIP CONCENTRATION1 llog I41 IGLUCAGON CONCENTRATION] (log HI corresponding data on fresh membranes. +and were
were calculated
Kd (nM)
the
means + SEM of
1
.
.
I
508
VIP Receptors
and Adenylate
Cyclase
Coupling
42, No. 5,
1988
FIG. 2 Effects of increasing concentrations of Gpp(NH)p (left panel) and WaF (right panel) on cyclic AMP production by crude rat hepatic membranes. Enzyme determinations were performed in the absence GTP. 0 f S a me representation an d svmbols as in Fio. 1. a
I
[Gpp(NH1 p CONCENTRATION] (log Ml
Vol.
2 4 6 8 10 [NaFCONCENTRATION] ImMJ
Binding sites in fresh and defrosted membranes were studied by competition curves, using [ 1251]VIP and [ 1251] helodermin as tracers and unlabelled IP as competitor (Fig. 3). As detailed in Table I, the inhibition of [ l2 ! I] VIP binding was best fitted considering the recognition of two classes of receptors, one with a low (0.3 nM) and a second one with a high (10 nM) Kd. The total binding capac’ty of [ 195 proportions of the twoT~~2~~V,I~~~~~~~~~~~~~~~~] ~{o~~~~~tee~ ~~~~~~~ defrosted membranes. more specifically the high affinity VIP binding sites) confirmed the coexistence of two classes of VIP receptors and their stability after freezing and thawing. As shown in Table I, there was after thawing an excellent correlation between the two Kact of VIP stimulation of adenylate cyclase and the two Kd values for VIP binding sites. FIG. DEFROSTEO(n=4)
[VIP CONCENTRATION1
absence of added peptide. and defrosted membranes.
Ilog Ml
The amounts of
tracer
3.
Inhibition of 11251] VIP (left pane-l) -and [ 1251 ] helodermin (righ t panel) binding to fresh or defrosted (I) (0) hepatic membranes by increasing concentrations of unlabelled VIP. The results were the means of 4 experiments made in duplicate a nd w er e expressed in X of tracer specifically bound in bound were identical in fresh
Discussion Despite a high density of VIP receptors in fresh rat hepatic membranes, VIP-stimulated adenylate cyclase activity remained low as compared to the glucagon response (present results and ref. 18). The potency of VIP was, however, remarkably high, making rat hepatic adenylate cyclase one of the most sensitive model for VIP stimulation [4]. In 1980, Amiranoff et al [9] suggested that the discrepancy between the high number of VIP receptors and the low efficacy of the neuropeptide was due to a defect in the GTP binding protein Ns
Vol.
42, No. 5,
1988
VIP Receptors and Adenylate Cyclase Coupling
509
coupling VIP receptors to the catalytical unit. This hypothesis was not supported, however, by recent data from the same group showing that most of the liver VIP receptors still interact with Ns after membrane solubilizationt~l. It remains t at VIP binding characterized by [ l&l ]VIp cross_linki~~,tes’differ in rat membranes’ from liver VIP receptors in ra: intestinal epithelial membranes, a system in which receptors are efficiently and potently coupled to adenylate cyclase [19,201. The Mr values of 51 and 89 kDa found for VIP binding proteins in liver contrast with Mr of 73 and 33 kDa found in intestinal cells. There is at present no argument suggesting that such structuraldifferencesare associatedwith functionaldifferences. The present binding studies confirmed that two classes of VIP binding sites can be identified in rat li membranes, their precise analysis being greatly facilitatedby the use of [1% Ilheloderminthat selectively labels high affinity VIP receptors [1,13 1 The high affinity VIP receptorswere coupled to adenylate cyclase (Table I and refs. 6 and 13) in freshly prepared membranes. After freezing and thawing both high and low affinity receptorswere coupled to the enzyme. This situation was not further modified by repeated freezing and thawing (data not shown). The interpretation of this unique observation, made so far on rat liver membranes only and for VIP receptors only, is highly speculative : It is conceivable that freezing and thawing modify the motility of 1. allowing a better association between receptors, Ns and membrane components, the catalytical unit. This hypothesis relies on the general dynamic properties
of biological membranes [211 and on the non permanent coupling of hormonal the absence of coupling receptors with adenylate cyclase 1221 . Of course, between low affinity VIP receptors and adenylate cyclase observed before freezing leaves this subclass of receptors without a known effector system in the "fresh" state. Considering thst the physical state of membranes influences adenylate 2. cyclsse activation [23-251 it is also possible that freezing induced discrete modifications of lipids and/or proteins in membrane structures that were sufficient to modify the efficscy of coupling between membrane proteins. all hormonal as well as Gpp(NH)p and fluoride responses are affected. Usually, The highly specific alteration of the VIP response in the present study, in the face of a general perturbation of membranes, is not a completely isolated observation as we showed recently that aliphatic alcohols potentiate the effect of secretin but not those of glucsgon and isoproterenol in rat heart membranes
[261.
the
Whatever the explanation, the present VIP receptor and the Gs-catalytical
data indicate that coupling between unit system can be selectively
modulated by membrane freezing . Acknowledgements. This work was supported Research (Belgium).
by Grant 3.4571.85
from the Fund for
Medical
Scientific
References 1. 2. 3. 4. 5. 6.
M. WAELBROECK, P. ROBBERECHT, P. DE NEEF, P. CHATELAIN P. and J. CHRISTOPHE,Biochim. Biophys. Acta 678 83-90 (1981). A. COUVINEAU, B. AMIRANOFF and M. L4BmHE, J. Biol. Chem. 261, 1448214489 (1986). B. DESBUQUOIS,Eur. J. Biochem. 46 439-450 (1974). P. ROBBERECHT, M. WAELBROECK ,J.C. CAMUS, P. DE NEEF, D. COY and J. CHRISTOPHE,Peptides 5 877-881 (1984). P. ROBBERECHT, M. WAEFBROECK, D. COY, P. DE NEEF, J.C. CAMUS and J. CHRISTOPHE,Peptides 7, Suppl. 1 53-59 (1986). P. ROBBERECHT,D.H. COY, P. DE NEEF, J.C. CAMUS, A. CAUVIN, M. WAELBROECK and J. CHRISTOPHE,Eur. J. Biochem.-159 45-49 (1986).
510
7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18.
19. 20. 21. 22. 23. 24. 25. 26.
VIP Receptors
and Adenylate
Cyclase
Coupling
Vol.
42,
No.
5,
1988
J.C. SOUQUET, J.P. RIOU, M. BEYLOT, J.A. CYAYVIALLE and R. MORNEX, FEES Lett. gLlU15-120 (1982). J.E. SILVESTRE, L. MONGE and J. MARCO, Endocrinologb, ?l2",",",',%25";~983). El. AMIRANOFF, MTABURTHE and G. ROSSELIN, Biochem. Biophys. Res. Commun. 96 463-468 (1980). r LEISER and J.J. BLUM, FEBS Lett. 173 407-413 (1984). S. GAMMELTOFT, P. STAUN-OLSEN, B. OmSEN and J. FAHRENKRUG, Peptides 2 367-370 (1984). Y. SALOMON, C. LONDOS and M. RODBELL, Anal. Biochem. 58 541-548 (1974). P. ROBBERECHT, M. WAELBROECK, P. DE NEEF, J.C. CAMUS,4. VANDERMEERS, M.-C. VANDERMEERS-PIRET and J. CHRISTOPHE, FEBS Lett. 172 55-58 (1984). 3. CHRISTOPHE, T.P. CONLON and J.D. GARDNER, J. Biol. Chem. 251 4629-4634 (1976). DICKINSON, C.M. MILES and P.S. SEVER, FEBS Lett. M. SCHACHTER, K.E.J., 199 125-129 (1986). A.VANDERMEERS, M.C. VANDERMEERS-PIRET, P. ROBBERECHT, M. WAELBROECK, J.P. DEHAYE, J. WINAND and J. CHRISTOPHE, FEBS Lett. 166 273-276 (1984). A. RICHARDSON and A. HUMRICH, Trends Pharmacol. Sci. 5 47-49 (1984). A. LABURTHE, D. BATAILLE, M. ROUSSET, J. BESSON, Y.-i3ROERS, A. ZWEIBAUM and G. ROSSELIN, in Proceedings of the membrane proteins action of the 11th FEBS Meeting, Copenhagen 1977, pp. 271-290, Pergamon Press, Oxford (19781. .-- -, A. COUVINEAU and M. LABURTHE, Biochem. J. 225 473-479 (1985). M. LABURTHE, B. BREANT and C. ROUYER-FESSARD, Eur. J. Biochem. 139 181187 (1984). M.J. KARNOVSKY, A.M. KLEINFELD, R.L. HOOVER and R.D. KLAUSNER, J. Cell. Biol. 94 l-6 (1982). G. RIMm, E. HANSKI, S. BRAUN and A. LEVITZKI, Nature, 276 394-396 (1978). L.M. GORDON, R.D. SAURHEBER, J.A. ESGATE, I. DIPPLE, R.J. MARCHMONT and M.D. HOUSLAY, J. Biol. Chem. 255 4519-4527 (1980). J. GARNIER PXETERRIER, D. DAVELOOSE and J. VIRET, kocELm%,'; 21 1581-1586'(1982) and D.' DAVELOOSE, Biochemistry R. SALESSE, -5: GARNIER -21 1587-1590, (1982). P. CHATELAIN, P. ROBBERECHT, M. WAELBROECK, J.C. CAMUS and J. CHRISTOPHE, J. Membrane Biol. -93 23-32 (1986).