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Effector mechanisms of peptides of the VIP family
Peptides. Vol. 7, Suppl. 1. pp. 101-107, 1986. Ankho International Inc. Printed in the U.S.A.
0196-9781/86 $3.00 + .00
Effector Mechanisms of Peptides of the VIP Family I JEAN CHRISTOPHE, MICHAL SVOBODA, MONIQUE LAMBERT, MAGALI WAELBROECK, JACQUES WINAND, JEAN-PAUL DEHAYE, MARIE-CLAIRE VANDERMEERS-PIRET, ANDRI~ VANDERMEERS AND PATRICK ROBBERECHT
Department o f Biochemistry and Nutrition, Medical School Unit'ersit6 Libre de Bruxelles, Brussels, B-IO00 Belgium
CHRISTOPHE, J., M, SVOBODA, M. LAMBERT, M. WAELBROECK, J. WINAND, J.-P. DEHAYE. M.-C. VANDERMEERS-PIRET, A. VANDERMEERS AND P. ROBBERECHT. EJI~,ctormechanisms ofpeptides q/'the VIP .[amily. PEPTIDES 7: Suppl. I, 101-107, 1986.--The present review is focused on the exocrine pancreas and liver where the only known effector mechanism of VIP is the activation of adenylate cyclase in plasma membranes. A two-state model of activation-deactivation of the enzyme visualizes the participation of VIP receptors and Ns, the guanyl nucleotide stimulatory protein of adenylate cyclase. In the rat pancreas, VIP and GRF receptors are indistinguishable and disulfide bridges influence their functional integrity. The antagonism of VIP and somatostatin perhaps requires, at the adenylate cyclase level, the contribution of Ni. the guanyl nucleotide inhibitory protein. The potentiation of VIP by various stimulants acting on Ca 2+ movements may rely on later events, e.g., on a concerted activation of protein kinases. When comparing quantitatively peptide binding to receptors with adenylate cyclase activation, cyclic AMP levels and amylase secretion, a tool is at hand to tailor synthetic agonists and antagonists of VIP, with appropriate changes in the N-terminal moiety of the peptide (a good agonist allows efficient coupling of receptors to the adenylate cyclase system). Apart from stimulussecretion coupling, VIP may influence protein synthesis in the rat pancreas, through the phosphorylation of ribosomal protein $6. and may alter the activity of the endoplasmic reticulum via the phosphorylation of Mr= 21 kDa and Mr 25 kDa proteins. In rat liver membranes, high affinity VIP receptors are specifically labelled with ~ZSl-helodermin and are coupled to adenylate cyclase (at variance with low affinity VIP receptors). These receptors are highly responsive to divalent cations and to guanyl nucleotides. VIP
GRF
Adenylate cyclase
Rat exocrine pancreas
T H E only well d o c u m e n t e d i n t e r m e d i a r y m e c h a n i s m w h e r e b y target tissues r e s p o n d to V I P is a d e n y l a t e c y c l a s e s t i m u l a t i o n . T h e r e results r e s p o n s e s as d i v e r s e as c o n t r a c tion, r e l a x a t i o n , electrical activity, h y p e r s e c r e t i o n o r h y p o s e c r e t i o n , d e p e n d i n g o n the tissue i n n e r v a t e d . T h e p r e s e n t o v e r v i e w will be c e n t e r e d o n the e x o c r i n e p a n c r e a s a n d liver. W e r e v i e w e d p r e v i o u s l y [5] the i n o t r o p i c a n d c h r o n o t r o p i c effects o f V I P o b s e r v e d o n h e a r t u p o n a d e n y late c y c l a s e stimulation.
Rat liver
ABBREVIATIONS VIP GRF CCK-PZ Ns (Gs) and Ni (Gi)
IBMX p(NH)ppG= Gpp(NH)p Kact = EC:,,,
THE EXOCRINE PANCREAS AND VIP
Two General Mechanisms of Response oJAcinar Cells VIP. a f t e r binding to specific r e c e p t o r s , s t i m u l a t e s the s e c r e t i o n o f h y d r o l a s e s from p a n c r e a t i c a c i n a r cells (in rat and e v e n m o r e so in the g u i n e a pig) (for r e v i e w , see [2,3]). It also e x e r t s a s e c r e t o r y effect o n ductal cells a n d c e n t r o a c i n a r cells a n d e n h a n c e s b l o o d flow within the gland. Figure 1 c o m p a r e s the p o s t u l a t e d a c t i o n o f V I P on a c i n a r cells with t h a t e x e r t e d by o t h e r s e c r e t a g o g u e s . In g e n e r a l , t h e s e a g e n t s are c a p a b l e to i n f l u e n c e cyclic A M P levels, (po-
I.A.
KD IC~.
vasoactive intestinal peptide human growth hormone-releasing factor cholecystokinin-pancreozymin the guanyl nucleotide regulatory proteins mediating, respectively, the activation and the inhibition of the catalytic subunit of adenylate cyclase isobutylmethylxanthine guanyl-5'-yl-imidodiphosphate concentration required to produce halfmaximal biological effect intrinsic activity=the relative efficacy of a given analog as compared to the maximal biological effect ligand concentration required to occupy 50% of the receptors ligand concentration required to inhibit half-maximally
~Paper presented at Second International Symposium on Vasoactive Intestinal Peptide (VIP) and Related Peptides (Cap d'Agde, June 18-22. 1985).
I01
CHRISTOPHE ET A L.
102
BASAL
FRONT
( B L O O D ANO NERVES1 Ca 2 +
ADENYLATE CYCLASE ~ . . . . . . FOASKOLIN
IP ~ I 3 P
RLIPASC,S,
[OA .....................................
...... D0
~ I]~RqL'I • . . . . . . . .
(a)
.... EGT~P
(b)
.... E ~(NH)ppG
(Hd)
.....
"
VIP t(lk+1
lGTPase
VIP
A!O
Ca2+ 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GUANYLATE CYCL~SE
A 2 3 IB 7
CaM
cGMP
// MELITTIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .~PLIPASE ... A2
FIG. 2. A two-state "on-off" cyclic model comparing the dynamic equilibrium of pancreatic adenylate cyclase between inactive (E0 and active (E,) states, in the presence of either (a) GTP or (b) p(NH) ppG (=Gpp(NH)p). Addition of VIP increases both k~j and k owing to the decrease of the activation energy for the "opening" of Ns. There results easier access as well as departure of the activating nucleotide.
POTENTIATION VIA FUSOGENS, PHOSPHORYLATIONS CYTDSKELETON, CHANNELS ETC --EMIOCYTOSIS TH~qOUGH SECRETORY POLE - c]-
FIG. 1. Postulated action of two groups of receptors on pancreatic acinar cells. The first group of receptors (R4 to R6) recognizes V1P (and helodermin Hd), secretin Sn, and cholecystokininpancreozymin PZ (when used at high concentration). The second group of receptors (R1 to R3) recognizes carbamylcholine Cb, bombesin Bo, and pancreozymin PZ (at low concentration). Adenylate cyclase, phospholipase(s) C. calcium, and guanylate cyclase may activate 4 protein kinases through, respectively, cyclic AMP (cAMP), diacylglycerol (DG) in the presence of phoshatidylserine (PS) and calcium, CaZ+-calmodulin(CAM), and cyclic GMP. IP, 13P, l: (poly)phosphatidylinositols, inositol 1,4,5-triphosphate and inositol. RER: endoplasmic reticulum with phosphoprotein Mr=21 kDa. Natural events may be duplicated with agents mentioned on both sides of the scheme such as TPA (12-O-tetradecanoylphorbol 13-acetate). The various effects of protein kinases and the action of phospholipase Ae may change the properties of phosphoproteins and produce fusogens such as diacylglycerol. Several of these steps are poorly defined (e.g., the activation and contribution of guanylate cyclase and phospholipase A0.
ly)phosphatidylinositol metabolism and/or calcium movements. VIpopreferring and secretin-preferring receptors belong to a first type of pancreatic receptors that, once occupied, provoke a sustained elevation of cyclic AMP in the presence of IBMX, and the activation of cyclic AMP-dependent protein kinase(s) A. Low affinity CCK-PZ receptors may act through a similar mechanism in the presence of supramaximal concentrations of CCK-PZ [34]. Muscarinic receptors for carbamylcholine, high-affinity CCK-PZ receptors (occupied at low CCK-PZ concentration), and bombesin receptors belong to the second type [2]. Any of these receptors, once occupied, eventually allows the activation of cyclic AMP-independent protein kinases, including calcium-activated, diacylglycerol and phospholipiddependent protein kinase C, Ca2+-calmodulin protein kinase, and probably also cyclic GMP-dependent protein kinase. Among the proximal steps activated after the occupancy of these receptors, the so-called phosphatidylinositol effect precedes Ca ~+ movements. Indeed, the stimulated phosphodiesterasic cleavage of polyphosphatidylinositols by phospholipase C liberates inositol-l,4,5-triphosphate, a messenger inducing the release of calcium from endoplasmic reticulum [33]. In addition, unsaturated diacylglycerol--the other product of phospholipase C action--activates protein kinase C in the presence of phospholipids (e.g., phosphatidylserine) even at Ca 2+ concentration as low as the resting concentration [20].
According to the scheme in Fig. 1, the exocrine pancreas is the site of convergence of several (neuro)hormones so that VIP may act simultaneously with another ligand. A potentiation of the secretory effect of VIP may occur provided that more than one mode of stimulus-effector coupling is implemented. Conceivably, this hypersecretory response is observed when the interplay of protein kinases (and protein phosphatases) leads to a phosphorylation pattern changing appropriately the properties of enzymes and structural proteins (e.g., those of the cytoskeleton [38] and the membrane of zymogen granules [13,14]). Furthermore, it is clear that acinar cells represent mostly a monodirectional system. An inhibition of the final secretory response is possible, however, and some evidence suggests that an inhibitory ligand, acting via N~, may be opposed to a stimulatory ligand (see below).
The VIP Activation o f Adenylate Cyclase by VIP, in Plasma Membranes From the Rat Pancreas, Proceeds Throul, h the Coupling o f VIP Receptors to Ns In rat pancreatic plasma membranes, a positive control of adenylate cyclase activity is exerted by several peptides including VIP. In the present case, the multicomponent adenylate cyclase system under consideration consists of VIP receptors lying at the outer membrane surface, and of Ns and the catalytic subunit present at the inner membrane surface [34]. The transducing component Ns(Gs) is a guanyl nucleotide regulatory stimulatory protein mediating the interaction between VIP receptors (as well as other stimulatory receptors, see Fig. 1) and the catalytic subunit, and activating this catalytic subunit when occupied with GTP or a nonhydrolyzable GTP analog such as Gpp(NH)p. Under physiological circumstances, the hydrolysis of GTP to GDP + Pi in Ns represents the major turn-off mechanism of adenylate cyclase activity. In previous work, we demonstrated the existence of such a hormone-dependent GTPase activity in rat pancreatic plasma membranes [ 15,16]. We will first examine the kinetic aspects of adenylate cyclase activation and deactivation and then the molecular aspects: (a) Figure 2 illustrates a model comparing the dynamic equilibrium of adenylate cyclase between inactive (E0 and active (E,) states, in the presence of either GTP or Gpp(NH)p. In this relatively simple two-state model [4, 18, 36] the degree of activation of the enzyme at steady state (E,)/(Et.0=k+J(k., + k - 0 and is accounted for with the three rate constants: k.~, k., and k , so that the concentration of E,
E F F E C T O R MECHANISMS OF V1P PEPTIDES ADENYLATE CYCLASE CYCLE
~
~)
103 VlP AND SOMATOSTATINCONTROLOF ADENYLATE CYCLASE THROUGHNS AND NI. RESPECTIVELY
ACTIVATION
VIP-~.~,
sSOMATOSTATIN
'~'-~VIP-R +NsGDP----DVIP-RW-NsGOP
W-NsGOP+GTP
:"COLLISION
-NsGTP-~,'NsGTP+VIP-RI
5. NsGTP+CATF-'ATP cAMP
COUPLING"
oVIP-RW-NsGTP+GDP:GTP/GDP EXCHANGE VI~+R__
AMPLIFICATION BY :
NS
NI
RECYCLINGOF R
: STIMULATION OF CAT
(~ DEACTIVATION 6. NsGTP---~NsGDP+P 7. C A T ~ I (FROM Ni)
ATP CAMP : GTPase ACTIVITY : N1
(~PHOSPHODIESTERASE ACTIVITY : cAMP
SUBUNITSOF NS AND Ni
*5'AMP-+H+
FIG. 3. Flowsheet of the molecular events stimulating the adenylate cyclase cycle in the presence of VIP and GTP.
Ns
•
increases when the activation rate k+~ increases, or when any of the deactivation rates k~ and k , decreases, or both. At rest, i.e., in the absence of added nucleotide, E~ predominates and adenylate cyclase activity is vanishingly low regardless of whether VIP is present or absent. When GTP is added, the determining step of activation is a pseudomonomolecular process, independent of nucleotide concentration, with rate constant k+~. Adenylate cyclase in the E,~ state can be deactivated to the E~ state by (I) the hydrolysis of GTP by the GTPase closely associated to Ns (rate constant k2) or (2) accessorily, by simple dissociation of the intact nucleotide GTP from Ns (rate constant k-0: the integrated rate of dissociation of the system k,,ff = k~ + k 1. When used alone GTP is a weak activator of adenylate cyclase (i.e., EflEt,,t remains low) because k+z is small compared with the rate of deactivation k,n. The addition of VIP, in the presence of GTP, facilitates the pseudomonomolecular " o p e n i n g " of Ns by decreasing the energy of activation of this process [35]. This allows easier access as well as departure of GTP, i.e., it increases both k+~ and k ~. When a stable nucleotide such as Gpp(NH)p (which cannot be hydrolyzed by the GTPase) is used, this nucleotide proves to be a better activator than GTP since k.,=0 and k+,>k ,: there results a persistently active state ofadenylate cyclase due to the formation of N's Gpp(NH) p. (b) There is a major difference between VIP activation of adenylate cyclase and the activation observed after a cholera toxin pretreatment of pancreatic plasma membranes [4,36]. For VIP stimulation of the catalytic system, small amounts of VIP receptors may suffice to generate optimal adenylate cyclase activity and stimulate GTPase in Ns. By contrast, the cholera toxin pretreatment, in the presence of NAD, provokes the ADP-ribosylation of Ns and this permanent chemical modification exerts an inhibitory effect on the GTPase activity so that k2 is then vanishingly low (Fig. 4). In several cell types [8, 18, 32], Ns is a heterotrimer made of 3 subunits: alphas, beta, and a small 8 kDa gamma subunit. Recently, it has become evident that in pancreatic acinar cells also, Ns contains a beta subunit of 35 kDa and a larger alphas subunit that may exist in 2 forms (of 50 and 42 kDa, respectively) [17,37]. It is the large alpha~ subunit that carries the GTP binding site and is able to hydrolyze GTP into GDP and Pi (Fig. 3). At rest, in the absence of a stimulating ligand such as VIP, GDP is tightly bound to alphas, making Ns unable to activate adenylate cyclase, and preventing GTP
N1
O(s 142 I< i 52 K
CHOLERA TOXIN
TREATMENT.
935 K (COMMON)
(X'l 35 K i
(9
..'
E)
cA!p I G
BORDETELLA PEATUSSIS
TREATMENT
I
FIG. 4. Possible molecular basis for opposite effects of VIP and somatostatin on adenylate cyclase regulation through, respectively, a stimulatory receptor Rs and Ns, and through an inhibitory receptor Ri and Ni.
binding. Under these conditions, the VIP receptor R, whose binding site is exposed, floats at the outer membrane surface, while a undefined portion of Ns [37] is more or less anchored to the catalytic subunit [18], at the inner membrane surface. In this state, there is clearly no coupling between the mobile VIP receptor and the rest of the system. VIP control starts with the binding of VIP to its receptor R (Fig. 4). A bimolecular collision then allows the floating VIP-R complex to interact with Ns loaded with GDP, resulting in the formation of a high-affinity ternary complex VIPR*-NsGDP. The subsequent first order "slow" conformational change accelerates the exchange of GDP with GTP in the alphas subunit of Ns. There follows: (1) a dissociation of the alpha~ and beta subunits of Ns, and (2) a low affinity state of R for VIP. This is why: (a) direct binding studies of VIP are not feasible in the presence of GTP, the rapid dissociation of 1251-VIP due to GTP provoking very low residual binding of the radioligand, and (b) only catalytic recycling amounts of R are required since a single receptor can activate several catalytic subunits, if enough VIP is still present (see below). The dissociation of the ternary complex is followed by "'coupling," i.e., the activation of adenylate cyclase by alpha~ remaining in contact with the enzyme. However, GTPase activity within alpha~ will hydrolyze the bound GTP and so turns off adenylate cyclase activation. The alphasbeta complex is then reconstituted into Ns and a new cycle of adenylate cyclase activation is initiated provided that VIP at the outer side of the membrane and fresh GTP (with ATP and Mg 2+) at the other side are available.
Possible Competition of Inhibitoo' (Neuro)Hormones With VIP at the Adenylate Cyclase Level in Pancreatic Acinar Cells In several cell types the hormonal control of adenylate
104 cyclase is dual and inhibitory hormone receptors Ri occupied by inhibitory (neuro)hormones may interact with Ni, a guanine nucleotide inhibitory protein mediating the inhibition of adenylate cyclase activity [8, 11, 12, 32]. Like Ns, Ni (Gi) is composed of at least two subunits: a small beta subunit (35 kDa) which is in fact identical to the beta component of Ns and a distinct alphai subunit (30 kDa) which can be ADP-ribosylated by Bordetella Pertussis toxin but not by cholera toxin (Fig. 3). This ADP-ribosylation inhibits the GTPase activity of alpha~ and therefore facilitates indirectly the activation of adenylate cyclase. Ns and Ni may, conceivably, implement a mutual antagonism in terms of physiological response, provided that these two regulatory proteins and appropriate receptors are present in adequate amounts in pancreatic plasma membranes [17,37]. A direct inhibitory effect of activated alpha~ on the catalytic subunit of adenylate cyclase could then intervene; moreover, free beta subunits liberated from N~ could associate with activated alpha~ subunits, lowering indirectly adenylate cyclase activation. We have documented the presence of N~ in rat pancreatic plasma membranes by identifying its 35 kDa subunit after ADP-ribosylation by Bordetella Pertussis toxin [17]. The presence of appropriate ligand and receptor, allowing GTP to dissociate Ni into the beta and alpha~ subunits, has been indirectly suggested (but not proven) in intact pancreatic acinar cells by Esteve et al. [11] who showed changes in cyclic AMP levels that might reflect a negative regulation of pancreatic adenylate cyclase activity by somatostatin. On the other hand, muscarinic receptors in the rat pancreas do not appear to be of a type whereby muscarinic agonists might antagonize the activation of adenylate cyclase by VIP.
Heterogeneity of VIP-Seeretin Receptors and Subheterogeneity of VIP-Preferring Receptors When comparing the ability of VIP and related peptides to interact with receptors and the accompanying adenylate cyclase system, a first distinction between "VIP-preferring" and "secretin-preferring" receptors can be established. The 2 types of receptors coexist in the pancreas from rat and guinea pig [3, 22, 23]. Fortunately, the C-terminal secretin fragments secretin-(5-27) and secretin-(7-27), that cannot stimulate adenylate cyclase, inhibit competitively and specifically adenylate cyclase activation through secretinpreferring receptors. These selective antagonists of secretin can, thus, be utilized as a tool to distinguish between "VIPpreferring" and "secretin-preferring" receptors. "'VIP-preferring" sites can in turn be subdivided into subtypes. Indeed, displacement curves of '2'H-VIP binding by VIP and VIP analogs are multiphasic: they develop on more than three logarithms, suggesting the presence of 2 or more subclasses of binding sites with distinct affinity for VIP and distinct ability to recognize VIP analogs. This conclusion is supported by multiphasic curves of adenylate cyclase activation in isolated pancreatic plasma membranes (see below). In practice, multiphasic dose-effect curves of inhibition of radioligand binding by VIP analogs can be analyzed by computer fitting to a model of two classes of binding sites [19]. Ideally, the demonstration of more than one receptor subtype should be based on the availability of highly selective agonists and antagonists for each subclass as well as on the chemical characterization of distinct receptor species. Such a demonstration could also lead to further pharmacological development. We will see later that 'z"I-
C H R I S T O P H E ET AL. helodermin can be used as a specific ligand for high-affinity VIP receptors in rat liver membranes [25,28]. Unfortunately the same radioligand cannot be utilized for the same purpose in the rat pancreas as helodermin binds to secretin-preferring receptors in this organ [6].
Kaet. Intrinsic Aetivity, and K~ Values as Parameters for ILvlimating the Re~alive E]fieuey ~f VIP Analogs and NonLinear Coupling Between VIP Binding and Biological Responses In pancreatic plasma membranes. KD values for the binding of VIP analogs to plasma membranes can be easily quantified even when the curves are multiphasic (see above). The potency K a c t = ECs, (i.e., the peptide concentration required for half-maximal adenylate cyclase activation) and intrinsic activio' I.A. (i.e., the relative efficacy of the analog as compared to that of 10/xM VIP) of each VIP agonist can be obtained by analysis of complete dose-effect curves of adenylate cyclase activation. In case of a multiphasic curve, a representation similar to that of Scatchard can be made: the ratio of cyclic A M P produced under peptidic stimulation over the peptide concentration is plotted against the percent of maximal adenylate cyclase stimulation and the saturation curve obtained can then be resolved graphically [41]. The relative effieaey of various VIP agonists to activate adenylate cyclase to the E,~ state (see above), in the presence of GTP, reflects how easily the preliminary ternary complex VIP-R-Ns(GDP) is formed. A good VIP agonist promotes an efficient interaction between VIP-R and Ns(GDP) and a rapid GDP-GTP exchange reaction so that Ns(GTP) is then available for adenylate cyclase activation. By contrast, an efficient VIP antagonist, such as (N-Ac-Tyr', DPheZ)-GRF(1-29)-NHe (see below), when binding to VIP receptors, is obviously unable to form an efficient ternary complex. The relative efficacy of coupling of a given V1P agonist relies not only on its structural pecularities but also on the membrane preparation utilized. Indeed, the activation rate k+, with the ligand under consideration depends linearly on the concentration of VIP receptors, as related to Ns and C concentration, since the recycling of each receptor allows the " o p e n i n g " of several Ns subunits (see above). In addition, collision coupling may also depend on the fluidity of the lipid bilayer of the membrane, i.e., on its phospholipid composition [18]. Differences in the efficacy of receptor-effector coupling among tissues may explain why a given VIP analog, with reduced potency, can only behave as a partial agonist in membranes from the rat pancreas while acting as a full agonist in rat liver (see below) and pituitary membranes (unpublished data). In dispersed intact rat pancreatic aeini. The KD value of VIP analogs can be determined in the presence of ~2"|-VIP. In the same preparation, the potency (Kact=EC~0 and intrinsic activity (I.A.) of all VIP agonists can be evaluated for cyclic AMP elevation and amylase secretion. In this integrated system, "high efficacy" for a full VIP agonist may signify that a low proportion of occupied receptors suffices to initiate a biological response, i.e., that the ligand produces already a maximum effect when occupying a limited proportion of receptors. The KD value for binding of this agonist is then higher thant he Kact values for biological effects. Two mechanisms may be responsible for the increased sensitivity to the ligand: (1) the abundance of " s p a r e " receptors; (2) a great metabolic sensitivity of intact
E F F E C T O R M E C H A N I S M S O F VIP PEPTIDES cells to low changes in the levels of messengers (such as cyclic A M P and cytosolic calcium).
Structural Requirements fi)r VIP Receptor Occupancy and Subsequent Adenylate Cyclase Activation in Rat Pancreatic Plasma Membranes Identity of VIP and GRF receptors and importance of disulfide bonds in VIP receptors. In rat pancreatic plasma membranes, GRF(1-29)-NH2 and GRF(1-40)-OH are partial agonists as compared to VIP and secretin, with relatively low intrinsic activity on adenylate cyclase [42]. This is not too surprising considering the considerable sequence homology of G R F with VIP (8 of 16 in the N-terminal moiety). The stimulatory effect of GRF can be d u e - - a priori----to the occupancy of either GRF-preferring receptors, VIP-preferring receptors or secretin-preferring receptors. We answered this question when discovering that (N-Ac-Tyr 1, D-Phe2) -, (His 1, D-Ala2, D-Sev~, NLeuZr) -, and (His ~, D-Ala 2, D-Thr 7, NLeu27)-GRF(1-29)-NH2 inhibit dose-dependently the VIP- as well as the GRFstimulated adenylate cyclase but not the secretin-stimulated adenylate cyclase [30,42], demonstrating that GRF acts through V1P-preferring receptors but not through distinct GRF-preferring receptors or through secretin-preferring receptors. Thus, VIP and GRF receptors are indistinguishable in the rat pancreas. VIP receptors are very susceptible towards 2-mercaptoethanol. Among VIP receptors, those with high affinity for VIP are already altered by simple exposure to 5 mM 2-mercaptoethanol, during pancreas homogeneization at 5°C. The importance of disulfide bonds for functional integrity is also evident when testing VIP receptors with 12'~I-VIP, after a preincubation of rat pancreatic plasma membranes with dithiothreitol: a low (1-2 mM) dithiothreitol concentration suffices already to reduce the number of VIP receptors; at higher dithiothreitol concentration, the affinity of the remaining VIP receptors is also reduced [26]. Structural requirements in the ligand. The free alpha-NH2 group of His 1in VIP is involved in adenylate cyclase activation as indicated by the low efficacy and potency of (Ac-Hism)-vIP. The position and integrity of the imidazole ring of His ~ is also important [27]. Our biological observations with a few VIP analogs and 31 G R F analogs [23, 27, 30, 42] fit adequately with the Chou-Fasman structural predictions established by D. Coy et al. for ligands of this type [9]: there is no preferred conformation for residues 1 to 3 in the native ligand, while residues 4 to 6 allow a beta sheet leading to a reverse turn between residues 6 and 10. There results that changes in position 2 increase or decrease the efficacy (depending on the amino acid utilized) while introducing a D-isomer in or immediately after the beta sheet (i.e., in position 4, 6, 7 or 8) markedly reduces the affinity. The hydrophobicity of Val in position 5 is also important for VIP receptor recognition [3, 22, 23]. VIP-Preferring Receptors and Stimulus-Secretion Coupling in Intact Rat Pancreatic Acini In dispersed rat pancreatic acini, VIP and the parent peptides PHI, secretin, and helodermin [24, 40] dosedependently increase cyclic A M P to comparable high values in the presence of 0.5 mM IBMX [6, 7, 39]. However, VIP and PHI provoke only a weak amylase release, whereas secretin and helodermin are secretagogues almost as efficacious
105 as CCK-PZ or carbamylcholine. In other terms, the low efficacy of VIP in stimulus-secretion coupling is due to the sollicitation of a relatively inefficient fraction of cyclic AMP pools rather than the total amount of cyclic AMP produced. This is of course compatible with the fact that VIP and secretin receptors, coupled to the adenylate cyclase system, represent different entities in the rat pancreas (see above). Concerning VIP-preferring receptors, dose-response curves for '"sI-VIP binding inhibition, when compared with secretory curves, suggest that only high-affinity VIP preferring receptors (Kact at 1 nM) are involved in amylase release. Dose-response curves for increased cyclic AMP levels are consistent with the presence of two subclasses of receptors for VIP whose occupancy produces a doubling and a trebling in cyclic A M P levels at, respectively, low and high VIP concentration. If high-affinity VIP receptors, that allow the first elevation in cyclic AMP, induce at best a modest secretion (+30%), they permit nevertheless an efficient synergistic action between VIP and secretatogues capable to increase Ca2+movements: for instance, when VIP is offered simultaneously with CCK-8 (the C-terminal octapeptide of CCK-PZ), the resulting amylase hypersecretion is greater than that obtained with one secretagogue only.
The Phosphorylation of Mr=33 kDa, 21 kDa, and 25 kDa Membrane-Bound Proteins in Rat Pancreatic Acini Submitted to VIP and Other Secretagogues [7. I0, 39] VIP, like other agents increasing cyclic AMP levels (secretin, helodermin, CCK-8 at high concentration, and forskolin) or agents increasing calcium movements (carbamylcholine and CCK-8) stimulates the phosphorylation of the Mr=33 kDa ribosomal protein $6. In addition, the first group of agents only (including VIP) enhances the phosphorylation of the Mr=21 kDa and Mr=25 kDa particulate proteins. These Mr=21 kDa and Mr=25 kDa phosphoproteins are mostly localized in large microsomal structures of intermediate R N A content. The first of these two proteins is reminiscent of an isoproterenol-responsive Mr=22 kDa phosphoprotein, recently documented in a fraction of endoplasmic reticulum from rat parotid endowed with high ATP-dependent calcium uptake activity [21]. None of the phosphorylation patterns of Mr=33 kDa, 25 kDa and 21 kDa proteins we observed in pancreatic acini appears to be directly involved in stimulus-secretion coupling. Rather, the systematic phosphorylation of the Mr=33 kDa ribosomal protein $6 could well influence protein synthesis whenever the gland is (neuro)hormonally stimulated, including by VIP. Besides, the more specific cyclic AMPdependent phosphorylation of the Mr=21 kDa and Mr=25 kDa proteins induced by agents stimulating adenylate cyclase (including VIP) might be concerned with the metabolic activity of endoplasmic reticulum but it is too early to suggest a relationship between cyclic AMP, the phosphorylation of these two endoplasmic proteins, and a stimulation of cytosolic Ca 2+ uptake. COVALENT CROSS-LINKINGOF VIP TO ITS RECEPTOR(S) IN PANCREATIC AND LIVER MEMBRANES FROM RAT After VIP binding to receptors, rat pancreatic and liver membranes can be treated with an appropriate bifunctional cross-linker, before irradiation with UV light. Among the cross-linking agents we used at a 1 mM concentration, N-hydroxysuccinimidylazidobenzoate appeared to be the
106
CHRISTOPHE ET AL.
most efficient (unpublished data of Svoboda et al.). Analysis after S D S - P A G E (under reducing conditions) and autoradiography demonstrated 2 bands of Mr 51 and 65 kDa in pancreatic plasma membranes and only one band of Mr 52 kDa in liver membranes, suggesting that differences in VIP receptor-effector coupling between the two tissues may already be concerned with the receptor itself. THE LIVER AND VIP |25, 28, 30, 311 In liver, a glycogenolytic action and increased biliary secretion range among the likely effects of VIP. In rat liver membranes, the mode of occupancy of VIP receptors, in the presence of ~zsI-VIP, reveals the presence of two subclasses of binding sites, one with a high affinity for VIP and a second one with low affinity for VIP. Competition curves develop, indeed, over a three logarithm concentration range. By contrast, competition curves between ~25I-helodermin and unlabelled VIP develop over a two logarithm concentration range, suggesting that '2'~l-helodermin recognizes only one class of receptors. Based on KD values, this subclass consists of high affinity VIP receptors. When compared with the dose-effect curve of VIP-stimulated adenylate cyclase activity in the same preparation of fresh rat liver membranes, it appears that VIP activates the enzyme after the occupancy of high-affinity VIP receptors. This conclusion can be generalized to VIP analogs, helodermin and helodermin fragments. The Vmax of enzymatic activity attained with these peptides (i.e., their intrinsic activity) is lower than that observed in the presence of glucagon. At variance with the situation prevailing in rat pancreatic plasma membranes, it thus appears that only high affinity VIP receptors can be coupled with adenylate cyclase in fresh rat liver membranes. This reflects the presence of a limited
amount of well coupled VIP receptors and not a general lack of Ns and/or catalytic subunits [1]. What is also atypical is the fact that C-terminal helodermin fragments lacking the first amino acid residues are still capable to activate the enzyme (whereas similar C-terminal VIP fragments are inactive) [31]. In addition, the absence of coupling between low affinity VIP receptors and adenylate cyclase leaves these receptors without a (known) effector system. Some of the properties of the high-affinity VIP receptors have been examined taking advantage of their specific labelling by '25I-helodermin [28]. Ca z+, Mg ~+ and Mn 2÷ increase ~2~l-helodermin binding whereas the reducing agent dithiothreitol decreases the binding. These effects being not accompanied by a modification of the KD of VIP (as established by competition curves) it appears that the divalent cations and dithiothreitol increase and reduce, respectively, the number of high-affinity VIP receptors. Besides, guanyl nucleotides accelerate the dissociation rate of both v'~Ihelodermin and ~2~I-VIP, bound to their receptors, but the IC~0 values of the nucleotides indicate that receptors occupied by '25I-helodermin are more sensitive to guanyl nucleotides than those occupied collectively by J2~I-VIP. To conclude, high affinity VIP receptors in the liver are perhaps waiting for mammalian helodermin [29] as a natural ligand and their coupling with Ns deserves further study.
ACKNOWLEDGEMENTS Aided by Grant 5 ROI-AM 17010-9 from the National Institutes of Health (USA), a "Concerted Action" from the Ministrre de la Politique Scientifique Beige (Belgium) and Grant 3.4504.81 from the Fonds de la Recherche Scientifique Medicale (Belgium). We thank Dr. D. H. Coy for the generous gift of GRF analogs.
REFERENCES
I. Amiranoff, B., M. Laburthe and G. Rosselin. Differential effects of guanine nucleotides on the first step of VIP and glucagon action in membranes from liver cells. Biochem Biophys Res Commun 96: 463-468, 1980. 2. Christophe, J., M. Svoboda, P. Calderon-Attas, M. Lambert, M. C. Vandermeers-Piret, A. Vandermeers, M. DeschodtLanckman and P. Robberecht. Gastrointestinal hormonereceptor interactions in the pancreas. In: Gastrointestinal Hormones, edited by G. B. J. Glass. New York: Raven Press, 1980, pp. 451-476. 3. Christophe, J. and P. Robberecht. Effect of VIP on the exocrine pancreas. In: Vasoactive Intestinal Peptide. edited by S. I. Said, in the series: Advances in Peptide Hormone Research. New York: Raven Press, 1982, pp. 235-252. 4. Christophe, J., M. Svoboda and M. Lambert. Determining steps in the regulatory GTPase cycle of rat pancreatic adenylate cyclase. In: The Control of Secretion. London: The Royal Society of London, 1982, pp. 13%150. 5. Christophe, J., M. Waelbroeck, P. Chatelain and P. Robberecht. Heart receptors for VIP, PHI and secretin are able to activate adenylate cyclase and to mediate inotropic and chronotropic effects. Species variations and physiopathology. Peptides 5: 341-353, 1984. 6. Christophe, J., M. Waelbroeck, J. P. Dehaye, J. Winand and P. Robberecht. Receptors for secretin, helodermin, VIP (vasoactive intestinal peptide), PHI, and GRF (growth hormone releasing factor) in the rat pancreas. In: Proceedings o f the 7th International Congress o f Endocrinology. edited by F. Labrie and L. Proulx. Amsterdam: Elsevier Science Publishers, 1984, pp. 773-778.
7. Christophe, J., M. C. Vandermeers-Piret, J. Rathe, J. P. Dehaye, N. D. Bui-Nguyen, J. Winand and A. Vandermeers. A comparison of the phosphorylation of Mr=33K, 21K, and 25K membranehound proteins in the rat pancreas, submitted to various secretagogues. In: Proceedings o f the IUPHAR 9th International Congress o f Pharmacology. vol 2, edited by W. Paton, J. Mitchell and P. Turner. London: Macmillan Press Ltd., 1984, pp. 147-156. 8. Codina, J., J. Hildebrandt, T. Sunyer, R. D. Sekura, C. R. Manclark, R. lyengar and L. Birnbaumer. Mechanisms in the vectorial receptor-adenylate cyclase signal transduction. In: Advances in Cyclic Nucleotide and Protein Phosphorylation Research, vol 17, edited by P. Greengard et al. New York:
Raven Press, 1984, pp. 111-125. 9. Coy, D. H., W. A. Murphy, J. Sueiras-Diaz, E. J. Coy and V. A. Lance. Structure-activity studies on the N-terminal region of growth hormone releasing factor. J Med Chem 28: 181-185, 1985. 10. Dehaye, J.-P., M. Gillard, P. Poloczek, M. Stievenart, J. Winand and J. Christophe. Effects of forskolin on adenylate cyclase activity and amylase secretion in the rat exocrine pancreas. J Cyclic Nucleotide Protein Phosphor Res 10: 269-280, 1985. 11. Esteve, J. P., N. Vaysse, C. Susini, J. M. Kunsch, D. Fourmy, L. Pradayrol, E. Wunsch, L. Moroder and A. Ribet. Bimodal regulation of pancreatic exocrine function in vitro by somatostatin-28. Am J Physiol 245. G208-G216, 1983. 12. Jakobs, K. H., K. Aktories and G. Schfiltz. Mechanisms and components involved in adenylate cyclase inhibition by hormones. In: Advam es in Cyclic Nucleotide and Protein Phosphorylation Research. vol 17, edited by P. Greengard et al. New York: Raven Press, 1984, pp. 135-143.
EFFECTOR
MECHANISMS
OF VIP PEPTIDES
13. Lambert, M., J. Camus and J. Christophe. Pancreozymin and caerulein stimulate in vitro protein phosphorylation in the rat pancreas. Biochem Biophys Res Cornmun 52: 935-942, 1973. 14. Lambert, M., J. C. Camus and J. Christophe. Phosphorylation in vitro of proteins in the pancreas and parotids of rats: effects of hormonal secretagogues and cyclic nucleotides. Biochem Pharmacol 24: 1755-1758, 1975. 15. Lambert, M., M. Svoboda and J. Christophe. Hormonestimulated GTPase activity in rat pancreatic plasma membranes. FEBS Lett 99: 303-307, 1979. 16. Lambert, M., M. Deschodt-Lanckman, J. Furnelle and J. Christophe. Similar characteristics of guanine nucleotide regulatory sites involved in adenylate cyclase activation, specific GTPase activity and cholecystokinin binding in rat pancreatic plasma membranes. J Cyclic Nueleotide Protein Phosphor Res 7 : 385397, 1981. 17. Lambert, M., M. Svoboda. J. Furnelle and J. Christophe. Solubilization from rat pancreatic plasma membranes of a cholecystokinin (CCK) agonist-receptor complex interacting with guanine nucleotide regulatory proteins coexisting in the same macromolecular system. Eur J Biochem 147: 611-617, 1985. 18. Levitzki, A. Reconstitution of membrane receptor systems. Biochim Biophys Acta 822: 127-153. 1985. 19. Minneman, K. P., L. R. Hegstrand and P. B. Molinoff. Simultaneous determination of beta I and beta 2 adrenergic receptors in tissues containing both receptor subtypes. Mol Pharmacol 16: 34-46, 1979. 20. Noguchi, M., H. Adachi, J. D. Gardner and R. T. Jensen. Calcium-activated, phospholipid-dependent protein kinase in pancreatic acinar cells. Am J Physiol 248: G692-G701, 1985. 21. Plewe, G., R. Jahn, A. Immelmann, C. Bode and H. D. Srling. Specific phosphorylation of a protein in calcium accumulating endoplasmic reticulum from rat parotid glands following stimulation by agonists involving cAMP as second messenger. FEBS Lett 166: 96-103, 1984. 22. Robberecht, P., P. Chatelain, M. Waelbroeck and J. Christophe. Heterogeneity of VIP-recognizing binding sites in rat tissues. In: Vasoaetive Intestinal Peptide, edited by S. I. Said, in the series: Advances in Peptide Hormone Research. New York: Raven Press, 1982, pp. 323-332. 23. Robberecht, P., M. Waelbroeck, M. Noyer, P. Chatelain, P. De Neef, W. Krnig and J. Christophe. Characterization of secretin and vasoactive intestinal peptide receptors in rat pancreatic plasma membranes using the native peptides, secretin-(7-27) and five secretin analogues. Digestion 23: 201-210, 1982. 24. Robberecht. P., M. Waelbroeck, J. P. Dehaye, J. Winand, A. Vandermeers, M. C. Vandermeers-Piret and J. Christophe. Evidence that helodermin, a newly extracted peptide from Gila monster venom, is a member of the secretin/VIP/PHI family of peptides with an original pattern of biological properties. FEBS Lett 166: 277-282, 1984. 25. Robberecht, P.. M. Waelbroeck, P. De Neef, J. C. Camus, A. Vandermeers, M.-C. Vandermeers-Piret and J. Christophe. Specific labelling by ~zSI-helodennin of high-affinity VIP receptors in rat liver membranes. FEBS Lett 172: 55-58, 1984. 26. Robberecht. P., M. Waelbroeck, J. Camus, P. De Neef and J. Christophe. Importance of disulfide bonds in receptors for vasoactive intestinal peptide (V1P) and secretin in rat pancreatic plasma membranes. Biochim Biophys Acta 773: 271-278, 1984. 27. Robberecht, P., M. Waelbroeck, J. C. Camus, P. De Neef, D. Coy and J. Christophe. Effects of His ~ modifications on the ability of vasoactive intestinal peptide to stimulate adenylate cyclase from rat and human tissues. Peptides 5: 877-881, 1984. 28. Robberecht, P., A. Vandermeers, M.-C. Vandermeers-Piret, P. De Neef, J. C. Camus, M. Waelbroeck, D. H. Coy and J. Christophe. Specific labelling of high affinity VIP receptors in rat liver and lung membranes by ~'-'5l-helodermin. In: Proceedings ~[" the INSERM Symposium on Regulatory Peptides. Gouvieux-Chantilly, France, May 9-11, in press, 1985.
107
29. Robberecht, P., J. De Graef, M.-C. Woussen, M.-C. Vandermeers-Piret, A. Vandermeers, P. De Neef, A. Cauvin, C. Yanaihara, N. Yanaihara and J. Christophe. Immunoreactive helodermin-like peptides in rat: a new class of mammalian neuropeptides related to secretin and VIP. Biochem Biophys Res Commun 130: 333-342, 1985. 30. Robberecht, P., M. Waelbroeck, D. Coy, P. De Neef, J.-C. Camus and J. Christophe. Comparative structural requirements of thirty GRF analogs for occupancy of GRF- and VIP-receptors and coupling to adenylate cyclase in rat adenopituitary, liver and pancreas. Peptides, submitted, 1985. 31. Robberecht, P., N. Yanaihara, C. Yanaihara, P. De Neef, J.-C. Camus, A. Vandermeers, M.-C. Vandermeers-Piret and J. Christophe. Interaction of synthetic N- and C-terminal fragments of helodermin with rat liver VIP receptors. Peptides, submitted, 1985. 32. Smigel, M., T. Katada, J. K. Northup, G. M. Bokoch, M. Ui and A. G. Gilman. Mechanisms of guanine nucleotide-mediated regulation of adenylate cyclase activity. In: Advances in ('yclic Nacleotide and Protein Phosphorylation Research, vol 17, edited by P. Greengard et al. New York: Raven Press, 1984, pp. 1-18. 33. Streb, H., R. F. lrvine, M. J. Berridge and I. Schulz. Release of Ca 2t from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-l,4,5-triphosphate. Nature 306: 67-69, 1983. 34. Svoboda, M., P. Robberecht, J. C. Camus, M. DeschodtLanckman and J. Christophe. Subcellular distribution and response to gastrointestinal hormones of adenylate cyclase in the rat pancreas. Partial purification of a stable plasma membrane preparation. Ear J Biochem 69: 185-193, 1976. 35. Svoboda M. and J. Christophe. Effect of hormone and guanyl nucleotide pretreatment on the activation energy of pancreatic adenylate cyclase. J Qvclic Nucleotide Protein Phosphor Res 5: 377-384, 1979. 36. Svoboda, M,, M. Lambert and J. Christophe. Distinct effects of the C-terminal octapeptide of cholecystokinin and of a cholera toxin pretreatment on the kinetics of rat pancreatic adenylate cyclase activity. Biochim Biophys Acta 675: 46-61, 1981. 37. Svoboda, M., J. Furnelle and J. Christophe. The differential detergent solubilization of adenylate cyclase and polypeptide ADP-ribosylated with cholera toxin suggests an excess of G/F protein relative to adenylate cyclase in rat pancreatic plasma membranes. FEBS Lett 135:207-211, 1981. 38. Vandermeers, A., M. C. Vandermeers-Piret, A. Hepburn, J. Rath6 and J. Christophe. Evidence for the existence of actomyosin ATPase in the rat pancreas. Isolation and biochemical characterization. Eur J Biochem 121: 295-299, 1982. 39. Vandermeers, A., M. C. Vandermeers-Piret, J. Rathr, J. P. Dehaye, J. Winand and J. Christophe. Phosphorylation of 3 particulate proteins in rat pancreatic acini in response to vasoactive intestinal peptide (VIP), secretin and cholecystokinin (CCK-8). Peptides 5: 359-365, 1984. 40. Vandermeers, A., M. C. Vandermeers-Piret, P. Robberecht, M. Waelbroeck, J. P. Dehaye, J. Winand and J. Christophe. Purification of a novel pancreatic secretory factor (PSF) and a novel peptide with VIP- and secretin-like properties (helodermin) from Gila monster venom. FEBS Lett 166: 273-276, 1984. 41. Waelbroeck, M., P. Robberecht, P. De Neef, P. Chatelain and J. Christophe. Binding of vasoactive intestinal peptide (VIP) and VIP-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: 83-90, 1981. 42. Waelbroeck, M., P. Robberecht, D. H. Coy, J. C. Camus, P. De Neef and J. Christophe. Interaction of growth hormonereleasing factor (GRF) and 14 GRF analogs with rat pancreatic VIP receptors. Discovery of (N-AC-TYR2, DPHE2)-GRF(1-29)-NH.., as a vasoactive intestinal peptide (VIP) antagonist. Endocrinology 116: 2643-2649, 1985.
0196-9781/86 $3.00 + .00
Effector Mechanisms of Peptides of the VIP Family I JEAN CHRISTOPHE, MICHAL SVOBODA, MONIQUE LAMBERT, MAGALI WAELBROECK, JACQUES WINAND, JEAN-PAUL DEHAYE, MARIE-CLAIRE VANDERMEERS-PIRET, ANDRI~ VANDERMEERS AND PATRICK ROBBERECHT
Department o f Biochemistry and Nutrition, Medical School Unit'ersit6 Libre de Bruxelles, Brussels, B-IO00 Belgium
CHRISTOPHE, J., M, SVOBODA, M. LAMBERT, M. WAELBROECK, J. WINAND, J.-P. DEHAYE. M.-C. VANDERMEERS-PIRET, A. VANDERMEERS AND P. ROBBERECHT. EJI~,ctormechanisms ofpeptides q/'the VIP .[amily. PEPTIDES 7: Suppl. I, 101-107, 1986.--The present review is focused on the exocrine pancreas and liver where the only known effector mechanism of VIP is the activation of adenylate cyclase in plasma membranes. A two-state model of activation-deactivation of the enzyme visualizes the participation of VIP receptors and Ns, the guanyl nucleotide stimulatory protein of adenylate cyclase. In the rat pancreas, VIP and GRF receptors are indistinguishable and disulfide bridges influence their functional integrity. The antagonism of VIP and somatostatin perhaps requires, at the adenylate cyclase level, the contribution of Ni. the guanyl nucleotide inhibitory protein. The potentiation of VIP by various stimulants acting on Ca 2+ movements may rely on later events, e.g., on a concerted activation of protein kinases. When comparing quantitatively peptide binding to receptors with adenylate cyclase activation, cyclic AMP levels and amylase secretion, a tool is at hand to tailor synthetic agonists and antagonists of VIP, with appropriate changes in the N-terminal moiety of the peptide (a good agonist allows efficient coupling of receptors to the adenylate cyclase system). Apart from stimulussecretion coupling, VIP may influence protein synthesis in the rat pancreas, through the phosphorylation of ribosomal protein $6. and may alter the activity of the endoplasmic reticulum via the phosphorylation of Mr= 21 kDa and Mr 25 kDa proteins. In rat liver membranes, high affinity VIP receptors are specifically labelled with ~ZSl-helodermin and are coupled to adenylate cyclase (at variance with low affinity VIP receptors). These receptors are highly responsive to divalent cations and to guanyl nucleotides. VIP
GRF
Adenylate cyclase
Rat exocrine pancreas
T H E only well d o c u m e n t e d i n t e r m e d i a r y m e c h a n i s m w h e r e b y target tissues r e s p o n d to V I P is a d e n y l a t e c y c l a s e s t i m u l a t i o n . T h e r e results r e s p o n s e s as d i v e r s e as c o n t r a c tion, r e l a x a t i o n , electrical activity, h y p e r s e c r e t i o n o r h y p o s e c r e t i o n , d e p e n d i n g o n the tissue i n n e r v a t e d . T h e p r e s e n t o v e r v i e w will be c e n t e r e d o n the e x o c r i n e p a n c r e a s a n d liver. W e r e v i e w e d p r e v i o u s l y [5] the i n o t r o p i c a n d c h r o n o t r o p i c effects o f V I P o b s e r v e d o n h e a r t u p o n a d e n y late c y c l a s e stimulation.
Rat liver
ABBREVIATIONS VIP GRF CCK-PZ Ns (Gs) and Ni (Gi)
IBMX p(NH)ppG= Gpp(NH)p Kact = EC:,,,
THE EXOCRINE PANCREAS AND VIP
Two General Mechanisms of Response oJAcinar Cells VIP. a f t e r binding to specific r e c e p t o r s , s t i m u l a t e s the s e c r e t i o n o f h y d r o l a s e s from p a n c r e a t i c a c i n a r cells (in rat and e v e n m o r e so in the g u i n e a pig) (for r e v i e w , see [2,3]). It also e x e r t s a s e c r e t o r y effect o n ductal cells a n d c e n t r o a c i n a r cells a n d e n h a n c e s b l o o d flow within the gland. Figure 1 c o m p a r e s the p o s t u l a t e d a c t i o n o f V I P on a c i n a r cells with t h a t e x e r t e d by o t h e r s e c r e t a g o g u e s . In g e n e r a l , t h e s e a g e n t s are c a p a b l e to i n f l u e n c e cyclic A M P levels, (po-
I.A.
KD IC~.
vasoactive intestinal peptide human growth hormone-releasing factor cholecystokinin-pancreozymin the guanyl nucleotide regulatory proteins mediating, respectively, the activation and the inhibition of the catalytic subunit of adenylate cyclase isobutylmethylxanthine guanyl-5'-yl-imidodiphosphate concentration required to produce halfmaximal biological effect intrinsic activity=the relative efficacy of a given analog as compared to the maximal biological effect ligand concentration required to occupy 50% of the receptors ligand concentration required to inhibit half-maximally
~Paper presented at Second International Symposium on Vasoactive Intestinal Peptide (VIP) and Related Peptides (Cap d'Agde, June 18-22. 1985).
I01
CHRISTOPHE ET A L.
102
BASAL
FRONT
( B L O O D ANO NERVES1 Ca 2 +
ADENYLATE CYCLASE ~ . . . . . . FOASKOLIN
IP ~ I 3 P
RLIPASC,S,
[OA .....................................
...... D0
~ I]~RqL'I • . . . . . . . .
(a)
.... EGT~P
(b)
.... E ~(NH)ppG
(Hd)
.....
"
VIP t(lk+1
lGTPase
VIP
A!O
Ca2+ 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GUANYLATE CYCL~SE
A 2 3 IB 7
CaM
cGMP
// MELITTIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .~PLIPASE ... A2
FIG. 2. A two-state "on-off" cyclic model comparing the dynamic equilibrium of pancreatic adenylate cyclase between inactive (E0 and active (E,) states, in the presence of either (a) GTP or (b) p(NH) ppG (=Gpp(NH)p). Addition of VIP increases both k~j and k owing to the decrease of the activation energy for the "opening" of Ns. There results easier access as well as departure of the activating nucleotide.
POTENTIATION VIA FUSOGENS, PHOSPHORYLATIONS CYTDSKELETON, CHANNELS ETC --EMIOCYTOSIS TH~qOUGH SECRETORY POLE - c]-
FIG. 1. Postulated action of two groups of receptors on pancreatic acinar cells. The first group of receptors (R4 to R6) recognizes V1P (and helodermin Hd), secretin Sn, and cholecystokininpancreozymin PZ (when used at high concentration). The second group of receptors (R1 to R3) recognizes carbamylcholine Cb, bombesin Bo, and pancreozymin PZ (at low concentration). Adenylate cyclase, phospholipase(s) C. calcium, and guanylate cyclase may activate 4 protein kinases through, respectively, cyclic AMP (cAMP), diacylglycerol (DG) in the presence of phoshatidylserine (PS) and calcium, CaZ+-calmodulin(CAM), and cyclic GMP. IP, 13P, l: (poly)phosphatidylinositols, inositol 1,4,5-triphosphate and inositol. RER: endoplasmic reticulum with phosphoprotein Mr=21 kDa. Natural events may be duplicated with agents mentioned on both sides of the scheme such as TPA (12-O-tetradecanoylphorbol 13-acetate). The various effects of protein kinases and the action of phospholipase Ae may change the properties of phosphoproteins and produce fusogens such as diacylglycerol. Several of these steps are poorly defined (e.g., the activation and contribution of guanylate cyclase and phospholipase A0.
ly)phosphatidylinositol metabolism and/or calcium movements. VIpopreferring and secretin-preferring receptors belong to a first type of pancreatic receptors that, once occupied, provoke a sustained elevation of cyclic AMP in the presence of IBMX, and the activation of cyclic AMP-dependent protein kinase(s) A. Low affinity CCK-PZ receptors may act through a similar mechanism in the presence of supramaximal concentrations of CCK-PZ [34]. Muscarinic receptors for carbamylcholine, high-affinity CCK-PZ receptors (occupied at low CCK-PZ concentration), and bombesin receptors belong to the second type [2]. Any of these receptors, once occupied, eventually allows the activation of cyclic AMP-independent protein kinases, including calcium-activated, diacylglycerol and phospholipiddependent protein kinase C, Ca2+-calmodulin protein kinase, and probably also cyclic GMP-dependent protein kinase. Among the proximal steps activated after the occupancy of these receptors, the so-called phosphatidylinositol effect precedes Ca ~+ movements. Indeed, the stimulated phosphodiesterasic cleavage of polyphosphatidylinositols by phospholipase C liberates inositol-l,4,5-triphosphate, a messenger inducing the release of calcium from endoplasmic reticulum [33]. In addition, unsaturated diacylglycerol--the other product of phospholipase C action--activates protein kinase C in the presence of phospholipids (e.g., phosphatidylserine) even at Ca 2+ concentration as low as the resting concentration [20].
According to the scheme in Fig. 1, the exocrine pancreas is the site of convergence of several (neuro)hormones so that VIP may act simultaneously with another ligand. A potentiation of the secretory effect of VIP may occur provided that more than one mode of stimulus-effector coupling is implemented. Conceivably, this hypersecretory response is observed when the interplay of protein kinases (and protein phosphatases) leads to a phosphorylation pattern changing appropriately the properties of enzymes and structural proteins (e.g., those of the cytoskeleton [38] and the membrane of zymogen granules [13,14]). Furthermore, it is clear that acinar cells represent mostly a monodirectional system. An inhibition of the final secretory response is possible, however, and some evidence suggests that an inhibitory ligand, acting via N~, may be opposed to a stimulatory ligand (see below).
The VIP Activation o f Adenylate Cyclase by VIP, in Plasma Membranes From the Rat Pancreas, Proceeds Throul, h the Coupling o f VIP Receptors to Ns In rat pancreatic plasma membranes, a positive control of adenylate cyclase activity is exerted by several peptides including VIP. In the present case, the multicomponent adenylate cyclase system under consideration consists of VIP receptors lying at the outer membrane surface, and of Ns and the catalytic subunit present at the inner membrane surface [34]. The transducing component Ns(Gs) is a guanyl nucleotide regulatory stimulatory protein mediating the interaction between VIP receptors (as well as other stimulatory receptors, see Fig. 1) and the catalytic subunit, and activating this catalytic subunit when occupied with GTP or a nonhydrolyzable GTP analog such as Gpp(NH)p. Under physiological circumstances, the hydrolysis of GTP to GDP + Pi in Ns represents the major turn-off mechanism of adenylate cyclase activity. In previous work, we demonstrated the existence of such a hormone-dependent GTPase activity in rat pancreatic plasma membranes [ 15,16]. We will first examine the kinetic aspects of adenylate cyclase activation and deactivation and then the molecular aspects: (a) Figure 2 illustrates a model comparing the dynamic equilibrium of adenylate cyclase between inactive (E0 and active (E,) states, in the presence of either GTP or Gpp(NH)p. In this relatively simple two-state model [4, 18, 36] the degree of activation of the enzyme at steady state (E,)/(Et.0=k+J(k., + k - 0 and is accounted for with the three rate constants: k.~, k., and k , so that the concentration of E,
E F F E C T O R MECHANISMS OF V1P PEPTIDES ADENYLATE CYCLASE CYCLE
~
~)
103 VlP AND SOMATOSTATINCONTROLOF ADENYLATE CYCLASE THROUGHNS AND NI. RESPECTIVELY
ACTIVATION
VIP-~.~,
sSOMATOSTATIN
'~'-~VIP-R +NsGDP----DVIP-RW-NsGOP
W-NsGOP+GTP
:"COLLISION
-NsGTP-~,'NsGTP+VIP-RI
5. NsGTP+CATF-'ATP cAMP
COUPLING"
oVIP-RW-NsGTP+GDP:GTP/GDP EXCHANGE VI~+R__
AMPLIFICATION BY :
NS
NI
RECYCLINGOF R
: STIMULATION OF CAT
(~ DEACTIVATION 6. NsGTP---~NsGDP+P 7. C A T ~ I (FROM Ni)
ATP CAMP : GTPase ACTIVITY : N1
(~PHOSPHODIESTERASE ACTIVITY : cAMP
SUBUNITSOF NS AND Ni
*5'AMP-+H+
FIG. 3. Flowsheet of the molecular events stimulating the adenylate cyclase cycle in the presence of VIP and GTP.
Ns
•
increases when the activation rate k+~ increases, or when any of the deactivation rates k~ and k , decreases, or both. At rest, i.e., in the absence of added nucleotide, E~ predominates and adenylate cyclase activity is vanishingly low regardless of whether VIP is present or absent. When GTP is added, the determining step of activation is a pseudomonomolecular process, independent of nucleotide concentration, with rate constant k+~. Adenylate cyclase in the E,~ state can be deactivated to the E~ state by (I) the hydrolysis of GTP by the GTPase closely associated to Ns (rate constant k2) or (2) accessorily, by simple dissociation of the intact nucleotide GTP from Ns (rate constant k-0: the integrated rate of dissociation of the system k,,ff = k~ + k 1. When used alone GTP is a weak activator of adenylate cyclase (i.e., EflEt,,t remains low) because k+z is small compared with the rate of deactivation k,n. The addition of VIP, in the presence of GTP, facilitates the pseudomonomolecular " o p e n i n g " of Ns by decreasing the energy of activation of this process [35]. This allows easier access as well as departure of GTP, i.e., it increases both k+~ and k ~. When a stable nucleotide such as Gpp(NH)p (which cannot be hydrolyzed by the GTPase) is used, this nucleotide proves to be a better activator than GTP since k.,=0 and k+,>k ,: there results a persistently active state ofadenylate cyclase due to the formation of N's Gpp(NH) p. (b) There is a major difference between VIP activation of adenylate cyclase and the activation observed after a cholera toxin pretreatment of pancreatic plasma membranes [4,36]. For VIP stimulation of the catalytic system, small amounts of VIP receptors may suffice to generate optimal adenylate cyclase activity and stimulate GTPase in Ns. By contrast, the cholera toxin pretreatment, in the presence of NAD, provokes the ADP-ribosylation of Ns and this permanent chemical modification exerts an inhibitory effect on the GTPase activity so that k2 is then vanishingly low (Fig. 4). In several cell types [8, 18, 32], Ns is a heterotrimer made of 3 subunits: alphas, beta, and a small 8 kDa gamma subunit. Recently, it has become evident that in pancreatic acinar cells also, Ns contains a beta subunit of 35 kDa and a larger alphas subunit that may exist in 2 forms (of 50 and 42 kDa, respectively) [17,37]. It is the large alpha~ subunit that carries the GTP binding site and is able to hydrolyze GTP into GDP and Pi (Fig. 3). At rest, in the absence of a stimulating ligand such as VIP, GDP is tightly bound to alphas, making Ns unable to activate adenylate cyclase, and preventing GTP
N1
O(s 142 I< i 52 K
CHOLERA TOXIN
TREATMENT.
935 K (COMMON)
(X'l 35 K i
(9
..'
E)
cA!p I G
BORDETELLA PEATUSSIS
TREATMENT
I
FIG. 4. Possible molecular basis for opposite effects of VIP and somatostatin on adenylate cyclase regulation through, respectively, a stimulatory receptor Rs and Ns, and through an inhibitory receptor Ri and Ni.
binding. Under these conditions, the VIP receptor R, whose binding site is exposed, floats at the outer membrane surface, while a undefined portion of Ns [37] is more or less anchored to the catalytic subunit [18], at the inner membrane surface. In this state, there is clearly no coupling between the mobile VIP receptor and the rest of the system. VIP control starts with the binding of VIP to its receptor R (Fig. 4). A bimolecular collision then allows the floating VIP-R complex to interact with Ns loaded with GDP, resulting in the formation of a high-affinity ternary complex VIPR*-NsGDP. The subsequent first order "slow" conformational change accelerates the exchange of GDP with GTP in the alphas subunit of Ns. There follows: (1) a dissociation of the alpha~ and beta subunits of Ns, and (2) a low affinity state of R for VIP. This is why: (a) direct binding studies of VIP are not feasible in the presence of GTP, the rapid dissociation of 1251-VIP due to GTP provoking very low residual binding of the radioligand, and (b) only catalytic recycling amounts of R are required since a single receptor can activate several catalytic subunits, if enough VIP is still present (see below). The dissociation of the ternary complex is followed by "'coupling," i.e., the activation of adenylate cyclase by alpha~ remaining in contact with the enzyme. However, GTPase activity within alpha~ will hydrolyze the bound GTP and so turns off adenylate cyclase activation. The alphasbeta complex is then reconstituted into Ns and a new cycle of adenylate cyclase activation is initiated provided that VIP at the outer side of the membrane and fresh GTP (with ATP and Mg 2+) at the other side are available.
Possible Competition of Inhibitoo' (Neuro)Hormones With VIP at the Adenylate Cyclase Level in Pancreatic Acinar Cells In several cell types the hormonal control of adenylate
104 cyclase is dual and inhibitory hormone receptors Ri occupied by inhibitory (neuro)hormones may interact with Ni, a guanine nucleotide inhibitory protein mediating the inhibition of adenylate cyclase activity [8, 11, 12, 32]. Like Ns, Ni (Gi) is composed of at least two subunits: a small beta subunit (35 kDa) which is in fact identical to the beta component of Ns and a distinct alphai subunit (30 kDa) which can be ADP-ribosylated by Bordetella Pertussis toxin but not by cholera toxin (Fig. 3). This ADP-ribosylation inhibits the GTPase activity of alpha~ and therefore facilitates indirectly the activation of adenylate cyclase. Ns and Ni may, conceivably, implement a mutual antagonism in terms of physiological response, provided that these two regulatory proteins and appropriate receptors are present in adequate amounts in pancreatic plasma membranes [17,37]. A direct inhibitory effect of activated alpha~ on the catalytic subunit of adenylate cyclase could then intervene; moreover, free beta subunits liberated from N~ could associate with activated alpha~ subunits, lowering indirectly adenylate cyclase activation. We have documented the presence of N~ in rat pancreatic plasma membranes by identifying its 35 kDa subunit after ADP-ribosylation by Bordetella Pertussis toxin [17]. The presence of appropriate ligand and receptor, allowing GTP to dissociate Ni into the beta and alpha~ subunits, has been indirectly suggested (but not proven) in intact pancreatic acinar cells by Esteve et al. [11] who showed changes in cyclic AMP levels that might reflect a negative regulation of pancreatic adenylate cyclase activity by somatostatin. On the other hand, muscarinic receptors in the rat pancreas do not appear to be of a type whereby muscarinic agonists might antagonize the activation of adenylate cyclase by VIP.
Heterogeneity of VIP-Seeretin Receptors and Subheterogeneity of VIP-Preferring Receptors When comparing the ability of VIP and related peptides to interact with receptors and the accompanying adenylate cyclase system, a first distinction between "VIP-preferring" and "secretin-preferring" receptors can be established. The 2 types of receptors coexist in the pancreas from rat and guinea pig [3, 22, 23]. Fortunately, the C-terminal secretin fragments secretin-(5-27) and secretin-(7-27), that cannot stimulate adenylate cyclase, inhibit competitively and specifically adenylate cyclase activation through secretinpreferring receptors. These selective antagonists of secretin can, thus, be utilized as a tool to distinguish between "VIPpreferring" and "secretin-preferring" receptors. "'VIP-preferring" sites can in turn be subdivided into subtypes. Indeed, displacement curves of '2'H-VIP binding by VIP and VIP analogs are multiphasic: they develop on more than three logarithms, suggesting the presence of 2 or more subclasses of binding sites with distinct affinity for VIP and distinct ability to recognize VIP analogs. This conclusion is supported by multiphasic curves of adenylate cyclase activation in isolated pancreatic plasma membranes (see below). In practice, multiphasic dose-effect curves of inhibition of radioligand binding by VIP analogs can be analyzed by computer fitting to a model of two classes of binding sites [19]. Ideally, the demonstration of more than one receptor subtype should be based on the availability of highly selective agonists and antagonists for each subclass as well as on the chemical characterization of distinct receptor species. Such a demonstration could also lead to further pharmacological development. We will see later that 'z"I-
C H R I S T O P H E ET AL. helodermin can be used as a specific ligand for high-affinity VIP receptors in rat liver membranes [25,28]. Unfortunately the same radioligand cannot be utilized for the same purpose in the rat pancreas as helodermin binds to secretin-preferring receptors in this organ [6].
Kaet. Intrinsic Aetivity, and K~ Values as Parameters for ILvlimating the Re~alive E]fieuey ~f VIP Analogs and NonLinear Coupling Between VIP Binding and Biological Responses In pancreatic plasma membranes. KD values for the binding of VIP analogs to plasma membranes can be easily quantified even when the curves are multiphasic (see above). The potency K a c t = ECs, (i.e., the peptide concentration required for half-maximal adenylate cyclase activation) and intrinsic activio' I.A. (i.e., the relative efficacy of the analog as compared to that of 10/xM VIP) of each VIP agonist can be obtained by analysis of complete dose-effect curves of adenylate cyclase activation. In case of a multiphasic curve, a representation similar to that of Scatchard can be made: the ratio of cyclic A M P produced under peptidic stimulation over the peptide concentration is plotted against the percent of maximal adenylate cyclase stimulation and the saturation curve obtained can then be resolved graphically [41]. The relative effieaey of various VIP agonists to activate adenylate cyclase to the E,~ state (see above), in the presence of GTP, reflects how easily the preliminary ternary complex VIP-R-Ns(GDP) is formed. A good VIP agonist promotes an efficient interaction between VIP-R and Ns(GDP) and a rapid GDP-GTP exchange reaction so that Ns(GTP) is then available for adenylate cyclase activation. By contrast, an efficient VIP antagonist, such as (N-Ac-Tyr', DPheZ)-GRF(1-29)-NHe (see below), when binding to VIP receptors, is obviously unable to form an efficient ternary complex. The relative efficacy of coupling of a given V1P agonist relies not only on its structural pecularities but also on the membrane preparation utilized. Indeed, the activation rate k+, with the ligand under consideration depends linearly on the concentration of VIP receptors, as related to Ns and C concentration, since the recycling of each receptor allows the " o p e n i n g " of several Ns subunits (see above). In addition, collision coupling may also depend on the fluidity of the lipid bilayer of the membrane, i.e., on its phospholipid composition [18]. Differences in the efficacy of receptor-effector coupling among tissues may explain why a given VIP analog, with reduced potency, can only behave as a partial agonist in membranes from the rat pancreas while acting as a full agonist in rat liver (see below) and pituitary membranes (unpublished data). In dispersed intact rat pancreatic aeini. The KD value of VIP analogs can be determined in the presence of ~2"|-VIP. In the same preparation, the potency (Kact=EC~0 and intrinsic activity (I.A.) of all VIP agonists can be evaluated for cyclic AMP elevation and amylase secretion. In this integrated system, "high efficacy" for a full VIP agonist may signify that a low proportion of occupied receptors suffices to initiate a biological response, i.e., that the ligand produces already a maximum effect when occupying a limited proportion of receptors. The KD value for binding of this agonist is then higher thant he Kact values for biological effects. Two mechanisms may be responsible for the increased sensitivity to the ligand: (1) the abundance of " s p a r e " receptors; (2) a great metabolic sensitivity of intact
E F F E C T O R M E C H A N I S M S O F VIP PEPTIDES cells to low changes in the levels of messengers (such as cyclic A M P and cytosolic calcium).
Structural Requirements fi)r VIP Receptor Occupancy and Subsequent Adenylate Cyclase Activation in Rat Pancreatic Plasma Membranes Identity of VIP and GRF receptors and importance of disulfide bonds in VIP receptors. In rat pancreatic plasma membranes, GRF(1-29)-NH2 and GRF(1-40)-OH are partial agonists as compared to VIP and secretin, with relatively low intrinsic activity on adenylate cyclase [42]. This is not too surprising considering the considerable sequence homology of G R F with VIP (8 of 16 in the N-terminal moiety). The stimulatory effect of GRF can be d u e - - a priori----to the occupancy of either GRF-preferring receptors, VIP-preferring receptors or secretin-preferring receptors. We answered this question when discovering that (N-Ac-Tyr 1, D-Phe2) -, (His 1, D-Ala2, D-Sev~, NLeuZr) -, and (His ~, D-Ala 2, D-Thr 7, NLeu27)-GRF(1-29)-NH2 inhibit dose-dependently the VIP- as well as the GRFstimulated adenylate cyclase but not the secretin-stimulated adenylate cyclase [30,42], demonstrating that GRF acts through V1P-preferring receptors but not through distinct GRF-preferring receptors or through secretin-preferring receptors. Thus, VIP and GRF receptors are indistinguishable in the rat pancreas. VIP receptors are very susceptible towards 2-mercaptoethanol. Among VIP receptors, those with high affinity for VIP are already altered by simple exposure to 5 mM 2-mercaptoethanol, during pancreas homogeneization at 5°C. The importance of disulfide bonds for functional integrity is also evident when testing VIP receptors with 12'~I-VIP, after a preincubation of rat pancreatic plasma membranes with dithiothreitol: a low (1-2 mM) dithiothreitol concentration suffices already to reduce the number of VIP receptors; at higher dithiothreitol concentration, the affinity of the remaining VIP receptors is also reduced [26]. Structural requirements in the ligand. The free alpha-NH2 group of His 1in VIP is involved in adenylate cyclase activation as indicated by the low efficacy and potency of (Ac-Hism)-vIP. The position and integrity of the imidazole ring of His ~ is also important [27]. Our biological observations with a few VIP analogs and 31 G R F analogs [23, 27, 30, 42] fit adequately with the Chou-Fasman structural predictions established by D. Coy et al. for ligands of this type [9]: there is no preferred conformation for residues 1 to 3 in the native ligand, while residues 4 to 6 allow a beta sheet leading to a reverse turn between residues 6 and 10. There results that changes in position 2 increase or decrease the efficacy (depending on the amino acid utilized) while introducing a D-isomer in or immediately after the beta sheet (i.e., in position 4, 6, 7 or 8) markedly reduces the affinity. The hydrophobicity of Val in position 5 is also important for VIP receptor recognition [3, 22, 23]. VIP-Preferring Receptors and Stimulus-Secretion Coupling in Intact Rat Pancreatic Acini In dispersed rat pancreatic acini, VIP and the parent peptides PHI, secretin, and helodermin [24, 40] dosedependently increase cyclic A M P to comparable high values in the presence of 0.5 mM IBMX [6, 7, 39]. However, VIP and PHI provoke only a weak amylase release, whereas secretin and helodermin are secretagogues almost as efficacious
105 as CCK-PZ or carbamylcholine. In other terms, the low efficacy of VIP in stimulus-secretion coupling is due to the sollicitation of a relatively inefficient fraction of cyclic AMP pools rather than the total amount of cyclic AMP produced. This is of course compatible with the fact that VIP and secretin receptors, coupled to the adenylate cyclase system, represent different entities in the rat pancreas (see above). Concerning VIP-preferring receptors, dose-response curves for '"sI-VIP binding inhibition, when compared with secretory curves, suggest that only high-affinity VIP preferring receptors (Kact at 1 nM) are involved in amylase release. Dose-response curves for increased cyclic AMP levels are consistent with the presence of two subclasses of receptors for VIP whose occupancy produces a doubling and a trebling in cyclic A M P levels at, respectively, low and high VIP concentration. If high-affinity VIP receptors, that allow the first elevation in cyclic AMP, induce at best a modest secretion (+30%), they permit nevertheless an efficient synergistic action between VIP and secretatogues capable to increase Ca2+movements: for instance, when VIP is offered simultaneously with CCK-8 (the C-terminal octapeptide of CCK-PZ), the resulting amylase hypersecretion is greater than that obtained with one secretagogue only.
The Phosphorylation of Mr=33 kDa, 21 kDa, and 25 kDa Membrane-Bound Proteins in Rat Pancreatic Acini Submitted to VIP and Other Secretagogues [7. I0, 39] VIP, like other agents increasing cyclic AMP levels (secretin, helodermin, CCK-8 at high concentration, and forskolin) or agents increasing calcium movements (carbamylcholine and CCK-8) stimulates the phosphorylation of the Mr=33 kDa ribosomal protein $6. In addition, the first group of agents only (including VIP) enhances the phosphorylation of the Mr=21 kDa and Mr=25 kDa particulate proteins. These Mr=21 kDa and Mr=25 kDa phosphoproteins are mostly localized in large microsomal structures of intermediate R N A content. The first of these two proteins is reminiscent of an isoproterenol-responsive Mr=22 kDa phosphoprotein, recently documented in a fraction of endoplasmic reticulum from rat parotid endowed with high ATP-dependent calcium uptake activity [21]. None of the phosphorylation patterns of Mr=33 kDa, 25 kDa and 21 kDa proteins we observed in pancreatic acini appears to be directly involved in stimulus-secretion coupling. Rather, the systematic phosphorylation of the Mr=33 kDa ribosomal protein $6 could well influence protein synthesis whenever the gland is (neuro)hormonally stimulated, including by VIP. Besides, the more specific cyclic AMPdependent phosphorylation of the Mr=21 kDa and Mr=25 kDa proteins induced by agents stimulating adenylate cyclase (including VIP) might be concerned with the metabolic activity of endoplasmic reticulum but it is too early to suggest a relationship between cyclic AMP, the phosphorylation of these two endoplasmic proteins, and a stimulation of cytosolic Ca 2+ uptake. COVALENT CROSS-LINKINGOF VIP TO ITS RECEPTOR(S) IN PANCREATIC AND LIVER MEMBRANES FROM RAT After VIP binding to receptors, rat pancreatic and liver membranes can be treated with an appropriate bifunctional cross-linker, before irradiation with UV light. Among the cross-linking agents we used at a 1 mM concentration, N-hydroxysuccinimidylazidobenzoate appeared to be the
106
CHRISTOPHE ET AL.
most efficient (unpublished data of Svoboda et al.). Analysis after S D S - P A G E (under reducing conditions) and autoradiography demonstrated 2 bands of Mr 51 and 65 kDa in pancreatic plasma membranes and only one band of Mr 52 kDa in liver membranes, suggesting that differences in VIP receptor-effector coupling between the two tissues may already be concerned with the receptor itself. THE LIVER AND VIP |25, 28, 30, 311 In liver, a glycogenolytic action and increased biliary secretion range among the likely effects of VIP. In rat liver membranes, the mode of occupancy of VIP receptors, in the presence of ~zsI-VIP, reveals the presence of two subclasses of binding sites, one with a high affinity for VIP and a second one with low affinity for VIP. Competition curves develop, indeed, over a three logarithm concentration range. By contrast, competition curves between ~25I-helodermin and unlabelled VIP develop over a two logarithm concentration range, suggesting that '2'~l-helodermin recognizes only one class of receptors. Based on KD values, this subclass consists of high affinity VIP receptors. When compared with the dose-effect curve of VIP-stimulated adenylate cyclase activity in the same preparation of fresh rat liver membranes, it appears that VIP activates the enzyme after the occupancy of high-affinity VIP receptors. This conclusion can be generalized to VIP analogs, helodermin and helodermin fragments. The Vmax of enzymatic activity attained with these peptides (i.e., their intrinsic activity) is lower than that observed in the presence of glucagon. At variance with the situation prevailing in rat pancreatic plasma membranes, it thus appears that only high affinity VIP receptors can be coupled with adenylate cyclase in fresh rat liver membranes. This reflects the presence of a limited
amount of well coupled VIP receptors and not a general lack of Ns and/or catalytic subunits [1]. What is also atypical is the fact that C-terminal helodermin fragments lacking the first amino acid residues are still capable to activate the enzyme (whereas similar C-terminal VIP fragments are inactive) [31]. In addition, the absence of coupling between low affinity VIP receptors and adenylate cyclase leaves these receptors without a (known) effector system. Some of the properties of the high-affinity VIP receptors have been examined taking advantage of their specific labelling by '25I-helodermin [28]. Ca z+, Mg ~+ and Mn 2÷ increase ~2~l-helodermin binding whereas the reducing agent dithiothreitol decreases the binding. These effects being not accompanied by a modification of the KD of VIP (as established by competition curves) it appears that the divalent cations and dithiothreitol increase and reduce, respectively, the number of high-affinity VIP receptors. Besides, guanyl nucleotides accelerate the dissociation rate of both v'~Ihelodermin and ~2~I-VIP, bound to their receptors, but the IC~0 values of the nucleotides indicate that receptors occupied by '25I-helodermin are more sensitive to guanyl nucleotides than those occupied collectively by J2~I-VIP. To conclude, high affinity VIP receptors in the liver are perhaps waiting for mammalian helodermin [29] as a natural ligand and their coupling with Ns deserves further study.
ACKNOWLEDGEMENTS Aided by Grant 5 ROI-AM 17010-9 from the National Institutes of Health (USA), a "Concerted Action" from the Ministrre de la Politique Scientifique Beige (Belgium) and Grant 3.4504.81 from the Fonds de la Recherche Scientifique Medicale (Belgium). We thank Dr. D. H. Coy for the generous gift of GRF analogs.
REFERENCES
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EFFECTOR
MECHANISMS
OF VIP PEPTIDES
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