NEUROPEPTIDE Y RECEPTORS FROM CALF BRAIN: EFFECT OF CRUDE CONUS VENOM PREPARATIONS ON [3H]NPY BINDING

Pergamon

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PII: S0197-0186(96)00066-6

Newochem.Int. Vol.29,No. 6, pp. 669476, 1996 Copyright01996 ElsevierScienceLtd Printedin Great Britain.All rightsreserved 0197+186/96$15.00+0.00

NEUROPEPTIDE Y RECEPTORS FROM CALF BRAIN: EFFECT OF CRUDE CONUS VENOM PREPARATIONS ON [3H]NPYBINDING EVA CZERWIEC,* JEAN-PAUL De BACKER, GEORGES VAUQUELINt and PATRICK M. L. VANDERHEYDEN Department of Protein Chemistry,Institute for Molecular Biologyand Biotechnology,Free Universityof Brussels(V.U.B.), Paardenstraat 65, B-1640St GenesiusRode, Belgium (Received 12 February 1996;accepted20 June 1996) Abstract—NPY receptors are identifiedin calf frontal cortex and hippocampusmembrane preparations

by bindingof N-[propionyl-3H]neuropeptideY. Saturation and competitionbindingdata withPYY,NPY(18-36)and NPY itselffit with a singleclassof sites:for the radiofigandKD= 1.4~0.5 nM, B~aX=434+180 fmol/mg protein in frontal cortex, &= O.7+0.2 nM, B~.X=267~ 50 fmol/mg protein in hippocampus. bipfmsic in both membrane Competitioncurves of the Y,-subtypeselectiveagonist [Leu31proqd]Npyare , preparations:highaffinitysites(i.e.Y,-subtype)amount to 80V0in frontal cortexand 23’?40 in hippocampus. The remainingsites are of the Y,-subtype.Out of 23 Conus venompreparations, 17inhibit the binding of [3H]NPYin both membranepreparations, but onlytwo of them (from Conus ardicus and C. pennaceus) do so with high potency (IC50<5 ~g protein/ml). Only one venom preparation (from C. rnercator) had weak discriminatory properties (IC,0Y2/IC,0Y,=6). Venom from C. anemone increased the [3H]NPY binding 5fold and with an IC,Oof 15-18 flg protein/ml. This binding occurred to the venom itself and was unrelated to the NPY receptors since it was equaIIy potent when displaced by [Leu 31, prosd]Npy, NPY-(18–36), pyy and NPY. Copyright ~ 1996 Elsevier Science Ltd

The neurotransmitter neuropeptide Y (NPY) is released both by central and peripheral neurons (Lundberg et al., 1982; O’Donohue et al., 1985; Stanley and Leibowitz, 1985). It is part of a family of homologous regulatory peptides, including peptide YY (PYY) and pancreatic polypeptide (PP) (Tatemoto et al., 1982; Tatemoto and Mutt, 1980), all of which are 36 amino acids long and characterized by a common and highly conserved tertiary structure: the PP-fold (Schwartz et al., 1990). NPY is highly abundant in the central nervous system (CNS) where it participates in the control of a wide variety of functions including psychomotor activities, cognitive functions, sexual behaviour, food intake, blood pressure regulation, circadian rhythmicity and neuroendocrine regulation (0’ Donohue et al., 1985; Stanley and Leibowitz, 1985). In the peripheral nervous system NPY is associated with sympathetic vascular control and release of cate*Current address: Marine Biological laboratory, 7 MBL Street, Wood’s Hole, MA 02543-1015, U.S.A. TTo whom all correspondence should be addressed.

cholamines (Westfall et al., 1990). NPY receptors are members of the G-protein-coupled receptor family and can be investigated directly by binding studies with radiolabelled NPY or PYY (Dumont et al., 1993; Widdowson and Halaris, 1990). Based on the ranking order of potency of NPY and certain peptide analogues, these receptors are currently classified into three subtypes: Y,, Y2, and Yj receptors. Whereas NPY does not discriminate between the three subtypes, PYY is only recognised with high affinity by the Y, and Yz receptors. C-terminal fragments of NPY such as NPY-(13–36) and NPY-(18–36) display poor affinity for the YI receptors, whereas NPY-analogues in which Ile at position 31 and Gly at position 34 are substituted by Leu and Pro, respectively (i.e. [Pro34]NPY and [Leu31,Pro34]NPY), possess greatly reduced affinity for the Y2- receptors (Boublik et al., 1989; Fuhlendorff et al., 1990; Grundemar and Hikanson, 1994; Sheikh et al., 1989). Synthetic non-peptide antagonists with high selectivity for the Y, receptors have recently been developed (Rudolf et al., 1994; Serradeil-Le Gal et al., 1995). NPY receptors have been divided into subtypes

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only recently and just one of them, the YI receptor, has been cloned unambiguously (Jorgensen et al., 1990). Because of the very limited range of existing NPY receptor subtype selective ligands and the similar chemical structure of most of them, it is plausible that certain NPY receptor subtypes have escaped identification until now. Venoms and natural toxins from various origins— bacteria, plants and animals—have often played a key role in the identification and classification process of membrane-bound receptors and ion channels. They have proven to be particularly successful discriminatory tools for the division of hormone and neurotransmitter receptors into different subtypes. In certain instances, their discriminatory power may well exceed that of other natural or synthetic ligands. This is well illustrated by the case of muscarinic receptors for which synthetic ligands such as pirenzepine provide a much poorer distinction between the Ml- and M2-subtypes than the venom of the marine snail Conus tessulatus (Czerwiec et al., 1993). The above considerations have prompted us to explore the ability of venom preparations from various Conus species to interact with NPY receptors. In the present study, we show that venom preparations from several CoruIs species inhibit [3H]NPY binding to NPY receptors in calf frontal cortex and hippocampus membrane preparations. These regions are particularly rich in Y, and Y2 receptors, respectively. No discrimination between these receptor subtypes was seen for 20 venoms and only a weak discrimination was noticed for the venom of C. mercator. To our surprise, we found that the venom of C. anemone increases the binding of [3H]NPY and that this increase is related to the ability of one or more of the venom’s components to bind the radioligand by itself. Although Conus venoms have already been shown to affect a variety of cellular targets (Fainzilber et al., 1995; Olivera et al., 1990, 1991; Shon et al., 1994), the present data constitute the first evidence that such venoms contain components that are capabable of interacting with peptide neurotransmitters. MATERIALSAND METHODS Chemicals N-[propionyl-3H] neuropeptide Y ([3H]NPY) (80 Ci/mmol) was obtained from Amersham (Little Chalfont, U.K.). Neuropeptide Y (NPY, porcine), polypeptide Y (PYY, porcine) and the analogs [Leu31,Pro34]NPY(porcine) and NPY(18-36) (porcine) were from Serva (Heidelberg, Germany). Bovine serum albumin (BSA) (Fraction V) was from Sigma (St Louis, MO, U.S.A.). All other chemicals were of the highest grade commercially available.

Conus venom preparations Specimens were taken live from the Philippines, near Cebu (C. arenatus, C. aulicus, C. canonicus, C. eburneus (var. polyglotta), C. furvus, C. geographus, C. lizreratus, C. liuidus, C. magus, C. marmoreus, C. mercator, C. miles, C. mustellinus, C. namocanus, C. pennaceus, C. rattus, C. vexi[lum, C. vitulinus and C. uirgo), from the Seychelles (C. tessulatus), from near Dakkar, Senegal (C. pulcher), and from South-West Australia (C. anemone). The gastropod were frozen, shipped by air to Brussels in dry ice, and stored at –20”C until use. At &l°C, the venom ducts of C. anemone, C.prdcher and C. tessulatus were dissected out of the animals, the venom was squeezed out of the duct and homogenised in 10 VOIS30 mM ammonium acetate (w/v) with a Polytron mixer and sonicated three times for 10 s in a Soniprep 150 sonicator. Whole ducts were homogenised and sonicated for the other species. Suspensions were centrifugated at 9,000 g for 10 min and the resulting supernatants were stored at –20”C. Membrane preparation Calf brains were obtained from a local slaughterhouse within 2 h post mortem and kept on ice until dissection. Frontal cortex and hippocampusweredissectedat 4“C,rapidlyfrozenin liquidnitrogenand kept at —80°Cuntil further preparation. All subsequentsteps werecarried out at l&l°C. The brain samples were homogenisedwith an Ultraturrax and Potter–Elvejhemhomogeniserin Krebs–Ringer buffer (137mM NaCl, 2.68mM KC1,2.05 mM MgCl,, 1,80mM CaClz,20 mM HEPES (pH 7.4)).The homogenatewas centrifugedat 30,000g for 20min and pelletswere resuspended in the same buffer. This procedure was repeated twice and the finalpellet was resuspendedin Krebs–Ringerbuffercontaining 10?4oglycerol (v/v). The obtained suspension was divided in 1 ml batches and stored in liquid nitrogen until use. Protein concentration determination Protein concentrations were determined using a modification of the Sopachem Ultra Sensitive Total Protein Assay, based on the Pyrogallol Red–Molybdate complex method (Watanabe et al., 1986),,with BSA as a standard. Radioligandbinding Assays were performed in 200 @ Krebs–Ringer buffer containing 0. 1‘7. w/v BSA in plastic 96-well plates. Frontal cortex and hippocampus membrane suspensions (100 ,agprotein/assay) were incubated for 60 min at 30°C with N-[propionyl-3H] neuropeptide Y ([3H]NPY, 0.2–5 nM in saturation experiments and 0.5 nM in competition experiments). Competitor concentrations ranged from 0.01 nM to 1 PM for NPY and analogues and (typically) from 1–100 #g/ml for venoms. Non specific binding was measured in the presence of 0.1 PM NPY and ranged between 30 and 40°/0 of total binding in all experiments. After incubation, the samples were rapidly filtered through glass fibre filters (Whatman GF/C, incubated in an aqueous solution of 0.3% (v/v) polyethyleneimine 15 min prior to filtration and prewashed with ice-cold Krebs–Ringer buffer) using a Skatron Cell Harvester. Filters were then washed four times with ice-cold Krebs–Ringer buffer, first for 2 s and then for 1 s in the subsequent steps. Filters were dried for 10 s, removed from the harvester and placed in polyethylene scintillation vials

Neuropeptide Y receptors from calf brain with 250 Y10.1 N NaOH and 3.5 ml scintillation fluid (Optiphase II, LKB). The amount ofradioligand on the filters was counted in a liquid scintillation counter.

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100 80-

Data analysis Binding experiments were done in triplicate (unless stated otherwise), Ki and IC50values of competitors and venom preparations were calculated by non-linear curve-fitting (best-fitted to a 1- or 2-site model, with binding parameters obtained by iteration with the Solver add-in function of MS Excel 3), Values are expressed as means ~S.E.M, (standard error of means).

601 40200

-i] RESULTS

-io

-9

Competitor

.8

.7

-6

CONCENTRATION (LOGM)

and characterization of NPYreceptors in calf frontal cortex and hippocampus membrane preparations

Identl$cation

Saturation binding experiments with [propionyl-3H] neuropeptide Y ([3H]NPY) reveal that calf hippocampus and frontal cortex membranes contain an apparently homogeneous class of high affinity sites for this radioligand. The affinity is equal for both regions (KD = 1.4 nM ~0.5 nM, nH= 1.02+ 0.03, n = 3) in cortex and (KD =0.7 ~ 0.2 nM, nH =0.97+0.06, n =3) in hippocampus but the density of sites is higher in frontal cortex (B~.X=434* 180 fmol/mg protein) than in hippocampus (B~,X= 267 ~ 50 fmol/mg protein). In cortex and hippocampus, competition binding experiments with NPY, PYY and NPY-(18–36) result in steep curves and are best analysed in terms of a one-site model (Fig. 1(A) and (B)). The Ki values and, hence, the ranking order of potency of these competitors is identical for both tissues: Ki NPY= Ki PYY < Ki NPY-(18–36) (Table 1). On the other hand, competition curves for the NPY analogue [Leu31, Pro34]NPY are biphasic in both preparations. [Leu3’,Pro34] displays high affinity (Ki< 1 nM) for 80+ IYo of the sites in frontal cortex membranes and for 23 ~ 3Y0 of the sites in hippocampus membranes (Table 1). The remaining sites display about 1000-fold lower affinity for this competitor in both tissues. Ejfects of crude Conus [3~NPY binding

venom preparations

on

[3H]NPY binding to calf hippocampus and frontal cortex membranes was tested in the presence of increasing concentrations (typically 1–100 pg protein/ml) of crude venom from 22 Conus species. The venoms of C. pennaceus and C. aulicus inhibited the binding with high potency (IC,O<5 yg protein/ml) but did not discriminate between the two membrane preparations (Table 2). Several venoms inhibited the binding with low potency (IC50ranging between 10 and 100

2L-J!G2L -il

.io

-9

.i

-7

-6

COMPETITORCONCENTRATION (LOG M)

Fig. 1.NPY, NPY-(18–36), [Leu31,Pro34]NPYand PYY competition binding to NPY receptors in calf cortex and hippocampus. (A) Calf cortex and (B) hippocampus membrane preparations were incubated with 0.5 nM [3H]NPY and increasing concentrations of the agonists NPY (A), NPY(18-36) (~), [Leu31,Pro34]NPY(.) and PYY (t). Binding is expressed in percent control binding, i.e. binding in the presence of buffer only. Table 1 lists the K, values from the different curves. Vahres are means and bars are S.E.M. from three experiments,

#g protein/ml) and did not discriminate between the two membrane preparations (IC50-rati0 <2, Table 2): C. tessulatus, C. marmoreus, C. lividus, C. mercatur, C. pulcher, C. eburneus, C. geografus, C. planorbis, C. magus, C. canonicus, C. vexillum. A third class of venoms inhibited [3H]NPY binding with low potency in calf frontal cortex and showed even at least 2-fold lower potency in calf hippocampus: C. mercator, C. vitulinus, C. rattus (Table 2). The ICSOof the venom of C. mercator for hippocampus membranes was found to be six times lower than for cortex membranes but the two other venoms were too weak to allow the determination of their IC50 for hippocampus membranes. A fourth class of venoms were poor inhibitors (IC50>100 pg protein/ml) for both membrane preparations: C. arenatus, C.furvus, C litteratus,

672

E. Czerwiec et al. Table 1.Parametersfor NPY-, PYY-,[Leu3’,Pro3’]NPYand NPY-(18-36)forcompetition with [3H]NPY bindingincalffrontalcortexandhippocampus membranepreparations,TheKi values and relativeamountsof high (HA) and low(LA) affinitysitesrefer to the competitioncurvesin Fig. l(A) and (B).Valuesare meansand S.E,M. of threeexperiments Cortex Competitor NPY PYY NPY-(18-36) [Leu3’,Pro34] NPY (HA) [Leu]’,Pro”] NPY (LA)

Hippocampus R (%)

Ki (nM)

R (%)

Ki (nM)

1.05+0.05 1.3io.l 29,5~0.5 0.7+0.2 326f27

100 100 100 80~ 1 2011

o.7fo,l 0.7+0,3 25+0,5 o.4fo,l 590+20

100 100 100 23*2 77*2

Table 2. Parametersfor crude Conus venompreparationsfor competingwith [3H]NPYbindingin calf frontal cortex(80% Y,, 20% YJ and hippncampus(23% Y,, 77% Y,) and for [3H]idazoxanbindingin retina membranepreparations(a,-adrenergicreceptors(Conventset rd., 1987)).Icj, valuesare givenin #g protein/ml.Y2/Y,,cc,/Y,and aJY, refersto the ratio of IC,Ovaluesobtainedfor the respectivereceptors. (–) IG, > 100pg protein/ml.Valuesare meansand S.E.M. of threeexperiments Species C.pennaceus C. ardicus C tessulatus C. marmoreus C. liuidus C. mercator C. prdcher C. eburneus C. oitulinus

C. geografu.r C. magus C. canonicus C. rattus C. uexillum C. arenatus C. furvus C. Iitteratus C. miles C. mustellinus C. namocanus C. uirga

CortexY, (80%)Ic~O 1.3+0.2 3.4* 1 loi4 17t2.5 18*2 24*9 31+11 34*4.5 38*4 43~8 55* 1 56* 14 56k20 62*3 —

HippocampusYg(770/.)IC50 1.4-LO.6 3.4+0.9 21i5 11*1 24* 1.5 150+24 19+6 64t 19 — 31*4 51+3 110+6 — 79*4 —

— — — —

C. miles, C mustelinus, C. namocanus and C. virgo (Table 2). The behaviour of the venom of C. anemone was completely unexpected; it increased the binding of [3H]NPY up to five times (Fig. 2(A) and (B)). This increase was dose-dependent with a half-maximal effect at 15+ 1 Kg protein/ml and 18tO.5 pg protein/ml in the presence of cortex and hippocampus membranes, respectively. Competition binding studies with NPY, PYY and the analogues, [Leu3i,Pro34]NPY and NPY-(1 8–36), were carried out to investigate whether the increased binding involved YI receptors, Yz receptors or even unrelated sites. For this purpose venom from C. anemone (in a final concentration of 25 pg protein/ml, increasing the binding up to 3-fold) was included in the competition assay in cortex or hippocampus membrane suspensions. Characteristics of the increased [3H]NPY binding clearly differed from those of the [3H]NPY binding in control membranes

RetinaIc~O

YJY, 1

— 102+14

—

18t2.3 41+6 18+0.7 29+6 57* 1.5 — 52*9 5+1 —

— —

— —

1 2.1 0.6 1,3 6 0.6 1,9 >2 0.7 0.9 2 >2 1.3 — — — — — — —

~21y1 >77 5.3 1,8 >6 >6 4.2 >3 0.5 1 0.4 0.5 1 >2 0.8 <0,05 — — — — — —

>72 5.3 0.8 >9 >4 0.7 >5 0.3 <0.4 0.5 0.5 0.5 — 0.6 <0.05 — — — — — —

(Figs 1 and 2 inset). [Leu3’,Pro34]NPY,NPY-(18-36), PYY and NPY inhibition curves were all steep and were analysed according to a one-site model; IC50 values were comparable and the potency ranking order was identical for both membrane preparations: PYY< [Leu31,Pro34]NPY%NPY-(18-36) ZZNPY (Table 3). Control experiments, in which membranes were omitted, revealed that the venom increased the binding of [3H]NPY to the same extent as in the presence of membranes. DISCUSSION

NPY receptors have already been studied in the CNS of rat, pig and man. From these studies, it appears that the three decribed subtypes are present in the CNS and that their abundance varies from one region to another and, for the same brain region, from one species to another (Busch-Sarensen et al., 1989;

Neuropeptide Y receptors from calf brain

673

(Grundemar et al., 1991a,b). In the present study, we found that calf frontal cortex and hippocampus membranes contain NPY receptors with high (nanomolar) affinity for the radioligand [3H]NPY. Their densities are higher than those reported for human and rat cortex (Widdowson and Halaris, 1990), comparable to that reported for pig cortex and lower than for pig hippocampus (Busch-%rensen et al., 1989). Competition binding studies indicate that PYY disA places the binding of [3H]NPY to calf cortex and hippocampus membranes to the same extent and with the same potency as NPY itself (Fig. 1). Since Y3receptors VENOMCONCENTRATION(Logmg proteiidml) are characterised by their very low affinity for PYY (Grundemar et al., 1991a, 1991b) our data indicate 3aoo that Y3 receptors are not present at detectable levels 2500 in these brain regions. The Y, and Y2 receptor subtypes are usually discriminated from each other on basis of their difference in affinity for synthetic NPY analogues such as [Leu3’,Pro34]NPY, which is Y11500 selective, and C-terminal fragments (e.g. NPY-(18– 36)), which act preferentially on Y2 receptors. The competition binding data with [Leu31, Pro34]NPY are clearly biphasic in both membrane preparations. The high affinity sites represent about 113-1 80Y0 of the labelled sites in cortex membranes and VENOMCONCENTRATION(Logmg prdeinlml) about 23°A in hippocampus membranes (Fig. 1). Fig. 2. Increase of [3H]NPY binding mediated by C. anemone These high affinity sites have, in both tissues, a ranking venom: competition binding with NPY, NPY-(18–36), order of potency that fits with a Y,-subtype: NPY = [Leu3’,Pro34]NPY and PYY. Membrane preparations form PYY=[Leu3’,Pro34] NPY> NPY-(18–36). The phar(A) calf cortex (80% Y,, 20% Y,) and (B) hippocampus macological profile of the low affinity sites is con(23% Y,, 77% Y,) were incubated with O.5nM [3H]NPY and increasing concentrations of C. anemone venompreparation. sistent with that of a Y2-subtype: NPY =PYY > NPYBindingis givenas total cpm counted.Valuesare means and (18-36) >[Leu31,Pro34]NPY. It can thus be concluded bars are S.E.M from three experiments.Competition with that both membrane preparations contain Y, as well the agonistsNPY (A), NPY-(18–36)(~), [Leu31,Pro34]NPY as Y2 receptors and that their ratio is about 4: 1 in calf (o) and PYY (+) on increased[3H]NPYbindingin the presence of C. anemone venom is shown in the insets. (A) Calf frontal cortex membranes and 1:3in calfhippocampus membranes. These relative amounts are comparable cortex and (B) hippocampus membrane preparations were incubated with 0.5 nM [3H]NPY, C. anemone venom prepto those reported for rat cortex and hippocampus aration (final concentration 25 pg protein/ml, increasing the (Dumont et al., 1993). binding up to 3-fold) and increasing concentrations of comCompetition curves with the C-terminal fragment petitors. Increased specific binding is given in cpm and was NPY-(18–36) are steep and the potency of this peptide calculated by subtracting non specific binding (measured in is about 30 times lower than for NPY in both memthe presence of 0.1 PM NPY) from control binding (measured in the presence of C. anemone venom and buffer alone). brane preparations. These findings suggest that, at Table 3 lists the ICWvalues and Hill coefficients from the least in calf membranes, NPY-(18–36) does not disdifferent curves. Values are means and S.D. from two experitinguish between the Y, and Yz receptor subtypes on ments. its own and is therefore not suitable for discriminatory purposes when used as the only competitor. The second part of this study, dealing with the effect Dumont et al., 1990, 1993; Widdowson and Halaris, 1990). Y2 receptors clearly predominate in the hip- of Conus venoms on [3H]NPY binding to calf cortex pocampus of all investigated species. Y, receptors, on and hippocampus membranes, provided two noteworthy observations. Firstly, of the 23 venoms tested, the other hand, predominate in frontal cortex of rat and pig (Busch-S@rensen et a[., 1989; Dumont et al., 17 inhibited the binding of [3H]NPY (by at least soy. at 100 pg protein/ml) to at least one of the membrane 1993). Less is known about the Y, receptor subtype, preparations (Table 2). Yet these venoms showed which has been identified in the brainstem from rat

674

E. Czenviec et al. Table 3. Parametersfor NPY-, PYY-, [Leu31,Pro34]NPYand NPY-(18–36)for competingwith [3H]NPYbindingto a mixtureof venomfrom C. anemone (finalconcentration2Spg proteinlml) and frontalcortexor hippocampusmembranepreparations.IcjOvaluesand Hillcoefficientsrefer to the competitioncurvesin Fig. 2(A)and (B).Valuesare meansand S.D, fromtwo experiments Competitor

Cortex IC50 (nM)

NPY PYY NPY-(18-36) [Leu3’,Pro’4]NPY

13*5 1.8+0.1 5.9+0.5 5.4+0.9

marked differences in potency and only two (C. pennaceus and C. aulicus) could inhibit the binding with IC50values below 5 ,ug protein/ml. Both venom preparations were equipotent in calf cortex and hippocampus membranes and hence did not present NPY receptor subtype selectivity. From the other active venoms, none produced preferential inhibition in hippocampus membranes. For calf cortex membranes, only the venoms of C. mercator, C. vitulinus and C. rattus showed any, albeit limited, selectivity: 6-fold for the venom of C. mercator (no values could be calculated for the other venoms because ICSO values in hippocampus were too low). Although the tested Conus venoms display no or only limited NPY receptor subtype selectivity, some are quite effective at differentiating the NPY receptors from other G-protein-coupled receptors such as the az-adrenergic receptor. This is clearly illustrated for the venoms of C. pennaceus and C. aulicus, which inhibit the binding of the uz-adrenergic antagonist [3H]RX781094 to its receptors in calf retina membranes with appreciably lower potency than the binding of [3H]NPY in cortex and hippocampus; about five times for the venom of C. aulicus and over 70 times for the venom of C. pennaceus. Alternatively, the venom of C. arenatus is a quite potent inhibitor of the binding to ~J-adrenergic receptors and more than 20 times less potent for the NPY receptors in cortex and hippocampus (Table 2). Since certain of the tested venoms can readily differentiate between u2-adrenergic and NPY receptors, it could be deduced that the limited Y, versus Yz receptor selectivity of these toxins is due to the structural similarity between these receptor subtypes. The validity of this deduction may be verified by comparing the amino acid sequences of the two receptor subtypes but, since only the Y, receptor has been cloned without ambiguity (Blomqvist et al., 1995), such verification will have to await cloning data for the Y, receptor. Secondly, it was found that the venom from C. anemone contains one or more components capable of increasing the binding of [3H]NPY as assessed by

‘H

0.96~0.05 0.81tO.005 o.88io.08 1,1*0.9

Hippocampus IC,O(nM) ‘H 11*3 0.85~0.05 7.0+0.1 5.3+ 1

0.84+0.09 0.82+0.001 0.7210.05 1.09fo.7

our filtration binding method. Since this increase occurs independently from the presence of membranes, it is likely that these venom components interact with [3H]NPY itself and then get trapped by the glass fibre filters. An alternative explanation such as the precipitation of [3H]NPY is, in our opinion, much less likely because of the very low concentration of this radioligand (0.5 nM) in the assay. Interestingly, these venom components do not have a pharmacological profile that would classify them as one of the known NPY receptor subtypes (Table 3); thus, they seem to be unrelated to any of the known NPY receptor subtypes. The high affinity of these components for NPY-(18–36) further indicates that they bind the NPY molecule at its carboxyl terminal. Isolation of the venom components responsible for decreasing the binding of [3H]NPY to Y, and Y2receptors in the venoms of C. pennaceus and C. aulicus, and for the direct interaction with the radioligand receptors in the venom of C. anemone, will allow us to gain further insight in their molecular structure and properties. In this context, most of the bioactive components from Conus venoms have been shown to be peptides. These “conopeptides” were shown to interact with a variety of cellular targets, especially with voltage-controlled and ligand-gated ion channels: a-conotoxins bind to nicotinic acetylcholine receptors, p-conotoxins target sodium channels in muscle, b-conotoxins bind to molluscan sodium channels, conantokins interact with the glutamate NMDA receptor and co-conotoxins block presynaptic calcium channels (Fainzilber et al., 1995; Olivera et al., 1991; Shon et al., 1994).Conotoxins have also been reported to interact with several species of monoamine receptors belonging to the superfamily of G-proteincoupled receptors, and have been used to discriminate between cq-adrenergic and 5-HTl~ serotonergic receptors in the human CNS and between Ml- and M2muscarinic receptor subtypes in calf retina (Czerwiec et al., 1993, 1989; De Vos et al., 1991). The ability of such venom components to interact with large peptides such as NPY, and their receptors, constitute

Neuropeptide Y receptors from calf brain

novel additions to the above list of conotoxin actions. Since both actions are intended to interfere with the function of NPY in the circulatory system (i.e. vasoconstriction (Eason et al., 1994)) of the animals on which cone snails prey, the ensuing vasodilatation could result in a faster distribution of other, more lethal conotoxins in their organism. In this context, these toxins could be related to the vasopressin-like peptides isolated from C. geograjius and C. magus (Cruz et al., 1987). Acknowledgements—We are very grateful to Mr G, Van Geel for C. tessulatus specimens, Mr M. Gabelish for C. anemone specimens and to the slaughterhouses of Anderlecht and Gee] for the facilities offered in obtaining calf brains. We are most obliged to Astra–Hissle (Sweden) Astra (Germany) and Astra (Belgium) for their kind support. This text presents research results of the Belgian programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming. The scientific responsibility is assumed by its authors. G. Vauquelin is Research Director of the National Fund for Scientific Research, Belgium. The Department of Protein Chemistry is recognised as a Prescribed Scientific Organisation by the Wildlife Protection Authority, Australia.

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