Retraction

NEUROSCIENCE. For the article ‘‘Functional characterization of anonclassical nicotine receptor associated with inositolphospho-lipid breakdown and mobilization of intracellular calciumpools,’’ by Marianne Garnier, Marek Lamacz, Marie ChristineTonon, and Hubert Vaudry, which appeared in number 24,November 22, 1994, of Proc. Natl. Acad. Sci. USA (91, 11743–11747), the authors note that ‘‘A regrettable mistake has beenmade in the preparation of the nicotine solutions used through-out this study. One microliter of nicotine was erroneouslyconsidered to be equivalent to 1 �g of nicotine; however, it wasequal to 1 mg. Thus, all the concentrations of nicotine used inour study were 1,000 times higher than reported. The authorsapologize for the erroneous information that may have misledother investigators.’’

Marianne GarnierMarek LamaczMarie Christine TononHubert Vaudry

www.pnas.org�cgi�doi�10.1073�pnas.0234540100

382 � PNAS � January 7, 2003 � vol. 100 � no. 1 www.pnas.org

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Proc. Nati. Acad. Sci. USAVol. 91, pp. 11743-11747, November 1994Neurobiology

Functional characterization of a nonclassical nicotine receptorassociated with inositolphospholipid breakdown andmobilization of intracellular calcium pools

(nicodnic receptor/cytosolic calcdum/phosphatddylinositol turnover/a-melanocyte-stimulatlng horMone/melanotrope cells)

MARIANNE GARNIER, MAREK LAMACZ, MARIE CHRISTINE TONON, AND HUBERT VAUDRY*European Institute for Peptide Research, Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Sant6 et de la Recherche MddicaleUnite 413, Unite Associde Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France

Communicated by Aaron B. Lerner, August 10, 1994

ABSTRACT Classical nicotinic receptors are neurotrans-mitter-gated channels that, upon activation by acetylcholine,induce the opening of an intrinsic cationic channel. We haverecently observed that, in frog pituitary melanotrophs, nicotinestimulates a-melanocyte-stimulating hormone (a-MSH) re-lease through a noncholinergic mechanism. In the study re-ported here, we investigated the intracellular events thatmediate the response offtog melanotrophs to nicotine. Nicotinewas capable of stimulating a-MSH release in the absence ofCa2+ and/or Na+ in the extracellular medium. A short pulseof nicotine induced a rapid and transient increase of cytosolicfree Ca2+ concentration ([Ca2+i). The effect of nicotine onCa2+ mobilization was not affected in the absence of Na+ andCa2+ in the extracellular medium, indicating that the nicotine-evoked increase in [Ca2+]J did not result from Na+ or Ca2+influx. Nicotine induced both an increase in inositol trisphos-phate and a reduction in phosphaditylinositol bisphosphateconcentrations but did not affect cAMP production. The pres-ent results indicate that nicotine-induced stimulation ofa-MSHrelease in frog melanotrophs can be explained by activation ofinositolphospholipid breakdown and mobilization of inositoltrisphosphate-dependent intracellular Ca2e pools. These dataprovide evidence for the existence of an unusual type ofnoncholinergic nicotine receptor positively coupled to phos-pholipase C.

The nicotinic acetylcholine receptor (nAChR) superfamilyconsists of two categories of neurotransmitter-gated ionchannels, the muscle-type nAChR and the neuronal-typenAChR (1, 2). Both receptor types are pentameric complexesformed through the association of several distinct subunits(i.e., a, f3, y or e, and 8) (3). The subunit stoichiometry ofmuscle-type nAChRs is thought to be a2/3yS, while neuronaltype nAChRs are composed of only a and 3 subunits (4, 5).Besides these two categories ofnAChR, Abood et al. (6) havereported the existence of noncholinergic nicotinic bindingsites in the rat brain. However, the functional properties ofthis nonclassical nicotinic receptor have never been investi-gated.The intermediate lobe of the pituitary, which is composed

of a single population of endocrine cells, is an appropriatemodel in which to investigate the mechanism of action ofneurohormones and neurotransmitters (7). In the pig, ace-tylcholine stimulates the electrical activity of melanotropecells through activation of typical neuronal-type nAChR (8).In the frog Rana ridibunda, acetylcholine stimulates hor-monal secretion and electrical activity of melanotrope cellsthrough activation of Ml muscarinic receptors (9). In thislatter species, nicotine also appears to be a potent stimulator

of a-melanocyte-stimulating hormone (a-MSH) release andspiking activity (10), but the effect of nicotine is neithermimicked by nicotinic cholinergic agonists nor blocked bynicotinic antagonists (10), suggesting that the action of nic-otine on frog melanotrope cells is mediated by a nonclassicalnicotine receptor, which might be related to the noncholin-ergic nicotinic binding site characterized by Abood et al. (6)in the rat brain.The aim of the present study was to investigate the mech-

anism(s) of coupling of this nonclassical type of nicotinereceptor in frog melanotrophs. The results show that the actionof nicotine is not mediated through activation of plasmamembrane Na+ or Ca2+ channels and does not depend onadenylate cyclase activity. In contrast, the effect ofnicotine onfrog melanotrope cells is associated with activation of inosi-tolphospholipid breakdown and mobilization of inositoltrisphosphate-dependent intracellular Ca2+ stores.

MATERIALS AND METHODSAnimals. Adult male frogs (Rana ridibunda) of about 30-g

body weight were purchased from a commercial supplier(Couttard, St. Hilaire de Riez, France). The animals weremaintained under artificial illumination (light on from hour0600 to 1800) in a temperature-controlled room (8 ± 0.50C).They were kept under running water for at least 1 week beforesacrifice. The frogs were killed by decapitation betweenhours 0800 and 0900, and the neurointermediate lobes (NILs)were dissected under a microscope. Then the NILs werepreincubated for 15 min in Ringer's solution (15 mM Hepesbuffer/112 mM NaCl/2mM KCI/2mM CaCl2) supplementedwith 2 g ofglucose and 0.3 g ofbovine serum albumin per liter.The solution was gassed for 15 min with 02/CO2, 95:5(vol/vol), before use, and the pH was adjusted to 7.35.

Reagents. Nicotine and Hepes were obtained from Merck.Collagenase, protease, ionomycin, EGTA, thapsigargin, andLeibovitz culture medium were purchased from Sigma. Ka-namycin, the antimycotic-antibiotic solution, and bovineserum albumin (fraction V) were purchased from BoehringerMannheim. Indo 1-AM (acetoxymethyl ester) was from Mo-lecular Probes. Anion-exchange resin AG 1-X8 (100-200mesh; formate form) and Bio-Gel P-2 were purchased fromBio-Rad. Silica gel 60 F254 high-performance thin-layer chro-matography (TLC) plates were from Merck.

Perifusion Technique. The perifusion system used in thisstudy has been described (11). Intact NILs were mixed withBio-Gel P-2 beads and transferred into plastic columns(0.9-cm i.d.; three NILs per column). The tissues were

Abbreviations: [Ca2+]i, intracellular calcium concentration; a-MSH,a-melanocyte-stimulating hormone; nAChR, nicotinic acetylcholinereceptor; NIL, neurointermediate lobe.*To whom reprint requests should be addressed.

11743

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

11744 Neurobiology: Gamier et al.

perifused with the Ringer's solution at constant flow rate (0.3ml/min) and temperature (280C) throughout the experiment.The effluent medium was collected as 7.5-min fractionsduring the stabilization period and 2.5-min fractions duringinfusion of the secretagogues. The collected samples wereimmediately chilled at 40C, and the concentration of a-MSHwas measured in each fraction on the same day as theperifusion experiment by using a double-antibody radioim-munoassay procedure (12).The perifusion profiles were calculated and expressed as

percentages of the basal secretory level. All experimentswere carried out at least three times. The basal level wascalculated as the mean of four consecutive fractions (7.5 mineach) collected just before the infusion of the secretagogues.Student's t test was used to compare the mean secretoryactivities within the same set of experiments.

Cell Culture. Melanotrope cells were enzymatically dis-persed as described (13). Briefly, eight NILs were incubatedfor 30 min at 260C in culture medium [Leibovitz mediumsupplemented with 200 mg of glucose and 63 mg of CaCl2 perliter, 1% kanamycin, and 1% antimycotic-antibiotic solution(pH 7.35)] containing 0.5% collagenase type IA and 1%protease type IX. The NILs were then dispersed by gentleaspiration with a siliconized Pasteur pipette with a flame-polished tip. The cell suspension was centrifuged (50 x g for5 min) and rinsed three times with culture medium supple-mented with 10%o heat-inactivated fetal calf serum. The dis-persed cells were suspended at a density of approximately15,000 cells per dish. The cells were cultured on coverslips thathad previously been coated with gelatin (250 ttg/ml) andpoly(L-lysine) (10 ,g/ml) (13). The cells were allowed to attachto the coverslips for 24 hr at 26°C in a humidified atmosphere.Then, the culture medium was renewed every 48 hr.Calcium Measurement. For microfluorimetric studies, the

cells were cultured for 3-5 days. Before each experiment, thecells were incubated in the dark for 30 min at 22°C with 5 ,uMindo 1-AM in the culture medium solution. At the end of theincubation period, the cells were washed twice with 1 ml offresh medium. The cytosolic (intracellular) calcium concen-tration ([Ca2+]1) was monitored by a dual emission microflu-orimetric system constructed from a Nikon Diaphot invertedmicroscope equipped for epifluorescence with an oil immer-sion objective (x 100 CF Fluor series; numerical aperture,1.3). The fluorescence emission of indo 1-AM, induced byexcitation at 355 nm, was recorded at two wavelengths (405nm, corresponding to the Ca2+-complexed form, and 480 nm,corresponding to the free form) by separate photometers (P1;Nikon). The 405/480-nm ratio was determined by using ananalogical divider (constructed by B. Dufy, Centre Nationalde la Recherche Scientifique, Unitd de Recherche Associde1200, Bordeaux, France) after conversion of single-photon

currents to voltage signals. All three signals (405 nm, 480 nm,and the 405/480-nm ratio) were continuously recorded witha three-channel voltage recorder (BD 100/101; Kipp &Zonen, Delft, The Netherlands). [Ca2+]i was calculated fromthe following formula established by Grynkiewicz et al. (14):[Ca2+]i = Kd X (3 (R - Rmin)/(Rmax - R), where R is the405/480-nm ratio, Rin represents the minimum fluorescenceratio obtained after incubation of cells with 10mM EGTA and10 ,uM ionomycin for 3 hr, R. represents the maximumfluorescence ratio obtained after incubation of cells with 10mM CaCl2 and 10 ,uM ionomycin for 3 hr, and P is the ratioof fluorescence yield from the Ca2+n/CaW2+ indicator at 480nm. The average values of Rmin, Rm., and 8 were 0.164 ±0.03 (n = 55), 1.82 ± 0.06 (n = 50), and 1.62 (n = 50),respectively. Kd = 250 nM is the dissociation constant forindo 1-AM (15). All secretagogues were injected in thevicinity of the cells by a pressure ejection system.

Analysis of Inositolphosphoipid Turnover. Measurement ofmembrane phospholipid breakdown was performed as de-scribed (16). The NILs were prelabeled in Leibovitz mediumwith myO_[3H]inositol (100 ,uCi/ml; 1 uCi = 37 kBq) for 18 hrat 21°C. The pulse medium was discarded, and the NILs werewashed six times and incubated for 10 min in Krebs-Ringersolution containing 10 mM LiCl. Then, the tissues -wereexposed for 30 s or 1 min to 10 ,tM nicotine, and the reactionwas stopped by addition of 250 A of ice-cold 20% trichloro-acetic acid. The NILs were homogenized, and precipitatedproteins were removed by centrifugation (13,000 X g for 10min). The acid-soluble fractions were stored at -20°C untilanalysis of [3H]inositol phosphates. Phospholipids were ex-tracted from protein pellets with 200 /1 of chloroform/methanol, 2:1 (vol/vol).

Inositol phosphates were separated after NaOH neutral-ization by anion-exchange chromatography on AG 1-X8 resin(formate form) columns (1 x 2 cm) as described (16). Free[3H]inositol was eluted by water; inositol monophosphate,inositol bisphosphate, and inositol trisphosphate were elutedby a step gradient of ammonium formate (0.2 M, 0.45 M, and0.8 M, respectively) in 0.1 M formic acid. For each AG 1-X8column, 38 fractions (4 ml each) were collected. Ten millili-ters of scintillation fluid was added to each fraction, and theradioactivity was determined in a 1217 Rackbeta spectrom-eter (LKB-Pharmacia).

[3H]Inositol-containing phospholipids were separated byTLC on silica gel 60 F2M TLC plates. The dried samples werereconstituted with 20 Al of chloroform/methanol, 2:1 (vol/vol), and applied to the TLC plates. The samples werechromatographed with a mixture of chloroform/methanol/25% ammonia solution/water, 50:39:2:9 (vol/vol). After a2-hr migration, the radioactivity incorporated in the differentinositolphospholipid fractions was measured by using an

Na+/Ca2+-free medium

B 500-

0c

4.*i 300-0

-

(A 100 -

nicotine Ca2+-free medium500

300

100*

60 180Time, min

60 180Time, min

500

300

100

300 60 180Time, m

300

FIG. 1. Effect of 10 ,uM nicotine on a-MSH secretion by perifused frog NILs in normal conditions (Left), in Ca2+-free medium (Center) orin Ca2+- and Na+-free medium (Right). Each profile represents the mean value of a-MSH release (+SEM) from 20 (Left) or 6 (Center and Right)independent experiments. The spontaneous level of a-MSH release (100% basal level) was calculated as the mean a-MSH secretion rate during30 min just preceding the administration of the first secretagogue.

Proc. Natl. Acad Sci. USA 91 (1994)

Proc. Natl. Acad. Sci. USA 91 (1994) 11745

N N N N N N

V V V 9 VI200-

g 120.

.

N10U-. 30-

10-

1 min

FIG. 2. Effects of repeated pulses (5 s each) of 10 ,uM nicotine on[Ca2+]i in melanotrope cells. The profile represents the ratio ofemission fluorescence signals (405/480 nm). The arrows indicate theonset of nicotine application.

automatic TLC linear analyzer (Tracemaster 20; Berthold,Elancourt, France) operated by an Epson PCAX computer.To identify the radioactive bands, a series of phospholipidstandards was chromatographed in the same conditions andvisualized by exposure to iodine vapors.

Quantification of cAMP. Whole NILs were preincubatedfor 30 min at 22°C in a Krebs-Ringer solution containing 0.1mM isobutylmethylxanthine. The NILs were then incubatedfor 10 min with 10 ALM nicotine. Incubation was stopped byaddition of ice-cold 20% trichloroacetic acid. The NILs werehomogenized and centrifuged (13,000 x g for 10 min), andtrichloroacetic acid was eliminated from the supernatant bythree successive rinses with 1 ml of water-saturated diethylether. After evaporation of the ether phase, the supernatantwas lyophilized, and the cAMP content in the dried ex-tractwas measured with a RIA kit (Amersham).

RESULTSEffect of Nicotine on a-MSH Release. A single pulse of 10

,gM nicotine for 10 min induced a marked stimulation of

Na+- and Ca2+-free medium

N

N200 -l

160 -

.

+

30 -

+_F

10-

1 min

a-MSH release from perifused frog NILs (Fig. 1 Left). Theresponse to nicotine developed very rapidly and reached amaximum within 7.5 min. Thereafter, the secretion ofa-MSHdeclined despite the continuous infusion of the secretagogue.As previously reported (17), suppression of Ca2+ in the

incubation medium caused a dramatic inhibition of sponta-neous a-MSH release (Fig. 1 Center). However, in Ca2+-freeconditions, nicotine retained its a-MSH-releasing activity.Similarly, suppression of both Ca2+ and Na+ from theincubation medium did not affect the stimulative action ofnicotine (Fig. 1 Right).

Effect of Nicotine on [Ca2+]i. The effects of nicotine on[Ca2+]i were studied by monitoring the fluorescence signal ina total of 346 cells. Ejection of 10 ,uM nicotine in the vicinityof cultured melanotrophs provoked a rapid (10 s) and massiveincrease of [Ca2+]i, which increased from 50 ± 15 nM to 190± 40nM (n = 25). Sequential administration ofnicotine to thesame cells provoked a substantial decrease of the amplitudeofthe Ca2+ response (Fig. 2). After the fourth pulse, the effectof nicotine on [Ca2+]i virtually disappeared.

Origin of Ca2+ MobUized by Nicotine. Suppression of bothNa+ and Ca2+ from the incubation medium did not impair thestimulative effect of nicotine on [Ca2i]i (n = 16) (Fig. 3 Left).In a Ca2+-free medium supplemented with 2 mM Mn2 ,nicotine still induced a massive increase in [Ca2+]i (n = 76)(Fig. 3 Right). In the same culture medium, a depolarizingpulse of KCl caused a decrease of both 405- and 480-nmfluorescence signals, indicating that an influx of Mn2+ actu-ally occurred into the cells (Inset of Fig. 3 Right). Thapsi-gargin, an antagonist of reticulum Ca2+-dependent ATPases(18) was used to block mobilization of the intracellularinositol trisphosphate-regulated Ca2+ pool (n = 32). Thapsi-gargin induced a gradual increase of [Ca2+J1i associated witha marked decrease of nicotine-induced elevation of [Ca2W](Fig. 4 Left). After 30 min of exposure to thapsigargin,nicotine was totally devoid of effect on [Ca2+]i (Fig. 4 Right).

Effect of Nicotine on Inositolphospholipid Turnover andcAMP Production. The effect of nicotine on phosphatidyl-inositol metabolism was studied by using myo-[3H]inositol-prelabeled whole-frog NILs (Fig. 5). The tissues were incu-bated with 10 mM LiCl during the 10 min preceding theadministration of nicotine in order to block the activity of

[Ca2+] = 0

[Mn2+] 2 mM

1 min

FIG. 3. Effect of 10 pM nicotine pulse for 5 s on [Ca2+]i in the absence of Ca2+ and Na+ and in the presence of 2 mM EGTA and 116 mMcholine (Left) or in the absence of Ca2+ and in the presence of 2 mM Mn2+ (Right) in the culture medium.

KCI

1.7i ~4n0.49.31 -t483.1:

-1 min

Neurobiology: Gamier et al.

11746 Neurobiology: Gamier et al.

thapsigargin

N

thapsigargin1

N

165-3

r-" ~ W Z } r-S/ Z_ I %

30

1 5 35Time, min

250

40-1// -r---1 62 64Time, min

FIG. 4. Effect of 10 ,AM nicotine pulse for 5 s on [Ca2+]1 in the presence of 10 aM thapsigargin. (Left) After a first pulse of nicotine, the cellwas perifused with 10 j.M thapsigargin for 30 min; then the cell was exposed to a second pulse of nicotine. (Right) A pulse of nicotine was given60 min after the onset of thapsigargin infusion.

inositol-monophosphate phosphatase and inositol-polyphos-phate 1-phosphatase. Exposure of the NILs to 10 AM nico-tine caused within the first 30 s a significant increase of EP3inositol trisphosphate in the cytosolic fraction (P < 0.01)associated with a marked reduction of phosphatidylinositolbisphosphate in the membrane fraction (P < 0.005). There-after, levels of inositol trisphosphate and phosphatidylinosi-tol bisphosphate returned to control levels within 1 min. Incontrast, incubation of frog NILs with 10 ,uM nicotine had noeffect on cAMP production (data not shown).

DISCUSSIONThe effects ofacetylcholine are mediated through two classesof membrane receptors-namely, the ionotropic nicotinic

0.4

0.3

0.2

0.1

5u 0.0

< 6 -0

5-

4-

4 -3-

2.-

30s 60s

C 30S 60s

FIG. 5. Effect of nicotine on levels of [3H]inositol trisphosphate(Upper) and phosphatidylinositol bisphosphate (Lower) in frogNILs. The tissues were prelabeled with myo-[3H]inositol for 18 hr at21'C and then incubated for 10 min in the presence of 10 mM LiCl.Finally, the lobes were exposed for 30 or 60 s to 10 ,uM nicotine. Eachcolumn represents the mean SEM of 10 determinations. **, P <0.001; ***, P < 0.005 (Student's t test).

receptors and the metabotropic muscarinic receptors. Twofamilies of nAChR-i.e., the muscle-type and neuronal-typenAChR-have previously been identified on the basis oftheirtissue distribution, pharmacological profile, and molecularstructure (see ref. 19 for a review). Both of these nAChRtypes are transmembrane cationic channels that activateunder cholinergic stimulation. We have recently reportedthat nicotine stimulates the electrical and secretory activity offrog pituitary melanotrophs through a nonclassical type ofreceptor that is not sensitive to acetylcholine and that is notblocked by either neuronal-type or muscle-type nicotinicantagonists (10). In the present study, we have investigatedthe transduction pathways that are responsible for the actionof nicotine on this previously unreported type of receptor.We first observed that the mechanism of action of nicotine

on melanotrope cells does not involve cation influx. Inparticular, the effect of nicotine on a-MSH release andintracellular Ca2W mobilization did not depend on the pres-ence of Ca2+ or Na+, or both, in the extracellular medium. Inaddition, the Mn2+-quenching technique (20) confirmed thatthe increase in [Ca2l] could not be accounted for by an influxof Ca2+. These observations strongly suggested that, in ourmodel, nicotine-evoked hormone secretion is not mediatedthrough activation of a conventional ionotropic nicotinicreceptor.The fact that nicotine caused an increase in [Ca2+], that

could not be ascribed to Ca2+ influx led us to investigate thepossible effect of nicotine on intracellular Ca2+ stores. Thenicotine-induced increase of [Ca2+]i was abolished by thap-sigargin, a drug known to deplete the inositol trisphosphate-sensitive Ca2+ pool (18, 21-23), suggesting that the effect ofnicotine could be ascribed to Ca2+ release from the endo-plasmic reticulum. Concurrently, nicotine was found to stim-ulate inositolphospholipid turnover in frog neurointermediatelobes. In contrast, nicotine did not affect adenylate cyclaseactivity. These data support the concept that nicotine stim-ulates a novel type of receptor positively coupled to phos-pholipase C. Nicotine-evoked activation of this receptorwould be responsible for generation of inositol trisphosphateand subsequently to Ca2+ release from the endoplasmicreticulum store. Grassi et al. (24) have reported that nicotineincreases inositolphospholipid turnover and induces mobili-zation of cytosolic Ca2+ in mouse C2C12 myotubes. On theone hand, the "metabotropic nicotinic receptor" investi-gated by these authors exhibits striking functional similaritieswith the nicotine receptor presently described in frog mel-anotrophs. On the other hand, however, the pharmacologicalprofiles of the two receptor models appear to be fundamen-tally different, inasmuch as the mouse myotube nicotinicreceptor can be activated by acetylcholine (24).

Proc. Natl. Acad. Sci. USA 91 (1994)

Proc. Natl. Acad. Sci. USA 91 (1994) 11747

Interestingly, the effect of nicotine on both a-MSH secre-tion and Ca2+ mobilization exhibited strong and rapid desen-sitization. This observation shows that the response to nic-otine is strictly controlled by a down-regulation process.Although the cellular mechanism involved in this process hasnot been investigated, such a desensitization phenomenonstrongly suggests that nicotine actually mimics the effect ofsome endogenous ligand. The nature of this putative ligand iscurrently a matter of speculation. Since the pars intermediaof the frog pituitary is richly supplied by peptidergic axonterminals (25), it is conceivable that nicotine mimics thestimulatory action of a novel-peptide involved in the regula-tion of pituitary melanotrophs. In support of this hypothesis,it has previously been shown that several neuropeptidesmodulate the activity of frog melanotrope cells (26-29).

In summary, our data indicate that the stimulatory effect ofnicotine on frog melanotrope cells is mediated through anon-cholinergic metabotropic receptor. The action of nico-tine is associated with stimulation of phosphatidylinositolbreakdown and mobilization of inositol trisphosphate-dependent intracellular Ca2+ stores, suggesting that thisnonclassical receptor type belongs to the G protein-linkedreceptor superfamily with seven hydrophobic transmem-brane domains and is positively coupled to phospholipase C.

This work was supported by the Centre National de la RechercheScientifique (Unite de Recherche Associde 650), the Institut Nationalde la Recherche Mddicale (Contrat de Recherche Externe 89-4015),the Commission of the European Communities (ERBCHRXCT-920017), and the Conseil Regional de Haute-Normandie. M.G. wasthe recipient of a doctoral fellowship from the Ministtre del'Enseignement Supdrieur et de la Recherche.

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Neurobiology: Gamier et al.