Sodium Saccharin Inhibits Adenylyl Cyclase Activity in Non-Taste Cells

Cell. Signal. Vol. 9, No. 6, pp. 431–438, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0898-6568/97 $17.00 PII S0898-6568(97)00033-8

Sodium Saccharin Inhibits Adenylyl Cyclase Activity in Non-Taste Cells Karim Dib,† Francine Wrisez,‡ Amina El Jamali,† Bernard Lambert‡ and Claude Correze†* †Equipe INSERM, d’Endocrinologie Tour D1, Faculte´ de Pharmacie, 5, rue Jean-Baptiste Cle´ment 92296 Chaˆtenay-Malabry, France; and ‡Laboratoire de Biochimie, UPRES-A, UFR Sciences Exactes et Naturelles, Moulin de la Housse, BP 1039, 51687 Reims Cedex 2, France

ABSTRACT. We have studied the in vitro effect of sodium saccharin (NaSacch) on the rat adipocyte adenylyl cyclase complex. NaSacch (2.5–50 mM) inhibited significantly in a dose-dependent manner basal and isoproterenol-stimulated cAMP accumulation on isolated rat adipocytes. Similarly, NaSacch (2.5–50 mM) inhibited forskolin-stimulated adenylyl cyclase activity measured in the presence of Mg21-ATP on adipocyte, astrocyte and thyrocyte membrane fractions. In contrast, NaSacch did not inhibit but slightly increased the forskolin-stimulated adenylyl cyclase activity measured in the presence of Mn21-ATP and GDPbS, a stable GDP analogue. The effect of NaSacch was not mediated through either the A1-adenosine receptor (A1R) or the a2-adrenergic receptor (a2AR). The inhibitory effect of NaSacch was additive to that of A1R agonist and was not blocked by the addition of the a2AR antagonist RX 821002. Pretreatment of adipocytes with pertussis toxin slightly attenuated but did not abolish the inhibitory effect of NaSacch on forskolin-stimulated adenylyl cyclase activity on membrane fractions. These data suggest that the inhibitory effect of NaSacch on forskolin stimulated-adenylyl cyclase in adipocytes does not imply only Gi protein but also other direct or indirect inhibitory pathway(s) which remain to be determined. cell signal 9;6:431–438, 1997.  1997 Elsevier Science Inc. KEY WORDS. Sodium saccharin, G-proteins, Adenylyl cyclase, Pertussis toxin, a2-adrenergic receptor, A1-adenosine receptor, Adipocytes

INTRODUCTION Several studies have documented that sweet compounds elicit a G-protein-dependent cAMP response in taste cells. The artificial sweetener sodium saccharin (NaSacch) reproduces the sweet taste response in rats and therefore is commonly used as a sweet taste stimulus. NaSacch and sucrose have been found to increase adenylyl cyclase activity in membranes of pig circumvallate taste papillae [1], or rat tongue [2]. The latter effect was dependent on guanine nucleotides, suggesting that GTP-binding protein(s) was/were involved in the mechanism of NaSacch action [2]. The proposed model is that sweet molecules might act by binding to receptors coupled with effectors via GTP-binding protein(s). Intriguingly, NaSacch has also been shown to evoke a cAMP response in non-taste cells [3], indicating that at least in part identical signal transduction systems might be activated by sugars in taste and in non-taste cells. However, in non-taste cells, NaSacch has been found to be either activator or inhibitor of adenylyl cyclase. For instance, NaSacch stimulated adenylyl cyclase activity in membranes *Author to whom all correspondence should be addressed. Received 7 August 1996; accepted 18 December 1996.

prepared from skeletal muscle whereas NaSacch activated or inhibited adenylyl cyclase activity in liver membranes [3]. However, despite these studies, little is known of the mechanisms whereby sugars mediate taste transduction: sugar receptors have not yet been identified and the nature of the G-protein involved in NaSacch-induced sweet taste response remains unknown. Recently, a taste tissue-specific G-protein a-subunit (Ggust) has been cloned [4] and expressed in a baculovirus based system [5]. Ggust was shown to have homology with the retinal Gta and therefore one might expect that Ggust is involved in the termination of the cAMP signal by activating a phosphodiesterase rather than coupling sugar receptors with the catalytic subunits of adenylyl cyclase. In this report, in order to understand better the mechanism of NaSacch action in non-taste cells, we analysed the effect of NaSacch on adenylyl cyclase activity in rat adipocytes, rat brain astrocytes and pig thyrocytes. MATERIALS AND METHODS Materials [a-32P] ATP was purchased from Du Pont-New England Nuclear. [3H] cyclic AMP was provided from Amersham Corp.

432

Sodium Saccharin (NaSacch), N6(R-phenyl-isopropyl)-adenosine (PIA), 2-(2-methoxy-1,4-benzodioxan-2-yl) 2-imidazoline (RX 821002) were from Sigma. Collagenase (CLS; 207 IU/mg) was from Worthington Biochemicals. Pertussis toxin was from the Chemo-Sero-Therapeutic Research Institute (Tokyo); 4,3-(Butoxy)-4-methoxybenzyl imidazolidinone (RO 7-2956) was a gift of Hoffman-La Roche. 5-bromo-62-imidazoline-2-amino-quinoxaline (UK 14304) was kindly donated by Dr. Carpe´ne´ (U 317 INSERM, Toulouse). Male Wistar rats (180 g) were from Depre´ (Saint Doulchard, France). All reagents for cell cultures were obtained from Life-Technologies. Cell and Membrane Preparation White fat cells were isolated by collagenase digestion and incubated in Krebs-Ringer bicarbonate buffer 0.1 M, pH 7.4, containing Ca21 (1.3 mM) and 4% (w/v) fatty-acid-free albumin (Buffer A) [6]. Astroglial cells were prepared from the cerebral hemispheres of 2-day-old rats and cultured as described [7]. Thyrocytes were prepared by discontinuous trypsinisation of pig thyroid glands and cultured as described [8]. Crude membrane fractions from these cells were prepared as described [7]. Cyclic AMP Assay cAMP accumulation was measured on adipocytes (about 5 3 105 cells) by the radioimmunological method [9]. Briefly, adipocytes were incubated for 6 min at 378C in buffer A supplemented with 1 mM RO 7-2956 (a cAMP-phosphodiesterase inhibitor) and 2 mg/ml adenosine deaminase with or without isoproterenol (IPNE, 1 mM). cAMP was expressed versus the content of triacylglycerol (TAG) in the cells. TAG were extracted according to [10]. Pertussis Toxin Treatment of Cells For ADP-ribosylation, adipocytes were preincubated with or without pertussis toxin (10 mg/ml) in buffer A for 90 min. They were washed twice and membrane fractions were prepared. Adenylyl Cyclase Assay Aliquots of membranes (10–20 mg of protein) were incubated for 6 min at 308C in the presence of forskolin (10 mM) in 100 ml of buffer containing 40 mM Tris-HCl, (pH 7.4), 0.4 mM [a 32P] ATP (105 cpm/assay), 4 mM MgCl2 or 10 mM MnCl2, 1 mM RO 7-2956, 0.5 U/ml adenosine deaminase and an ATP-regenerating system. The [32P] cAMP produced was quantified as described [11]. Assays were carried out in triplicate and the protein content of the samples was estimated according to [12]. Phosphodiesterase Assay Phosphodiesterase activity was measured as described [13]. The reaction mixture contained, in 200 ml Tris-HCl 40

K. Dib et al.

mM, pH 7.4, 3.5 mM MgSO4, 0.25 mCi [3H] cAMP, 1 mM unlabelled cAMP and enzyme (soluble 105.000g or particulate fraction). Incubation was performed at 378C for 20 min and stopped by immersion in boiling water for 2 min. For the nucleotidase reaction, crotalus atrox venom (0.25 mg/ ml) were added to each assay. Adenosine was separated from the remaining cyclic nucleotides by chromatography on anionic exchange resin AG 1X2 and eluted with NaHCO3. Statistical Analysis The Student’s t-test for unpaired values was used to determine the levels of significance. Differences between means were considered significant when p was < 0.05. RESULTS Effect of NaSacch on cAMP Accumulation in Isolated Adipocytes Figure 1 depicts the dose-dependent effect of NaSacch (1–50 mM) on cAMP production in isolated adipocytes. In the presence of RO 7-2956, which inhibited completely the phosphodiesterase activity in fat cells and of adenosine deaminase which relieve the tonic inhibition of adenylyl cyclase mediated by Gi [14], the accumulation of cAMP in 6 min was stimulated 153 by the b-adrenergic agonist isoproterenol. Both basal and isoproterenol-stimulated cAMP accumulation were inhibited to the same extent by NaSacch. Significant inhibitory effects were observed when high concentrations of NaSacch were used in the assay. With 20 mM and 50 mM of NaSacch, basal cAMP production decreased from 2 to 1.5 and 1 nmol/mmol TAG/6 min, respectively (Fig. 1A) and from 30 to 20.5 and 15 nmol/ mmol TAG/6 min in isoproterenol-stimulated condition, respectively (Fig. 1B). It is unlikely that this phenomenon was due to sodium ions, since the Krebs-Ringer bicarbonate buffer used to incubate the fat cells contains 140 mM NaCl [6]. We also directly show that NaSacch exerted its action on the production and not on the degradation of cAMP since the sweetener affected neither the membrane bound nor the cytosolic low-Km cAMP phosphodiesterase (Table 1). Effects of PIA, UK and NaSacch on cAMP Accumulation in Isolated Adipocytes Firstly, we have compared the effect of NaSacch, on isoproterenol-stimulated cAMP accumulation in fat cells, with the very efficient inhibitory effects of phenyl-isopropyladenosine (PIA), a non-hydrolysable A1 adenosine receptor agonist [15]. PIA inhibited in a dose-dependent manner with maximum effects at 1–10 mM isoproterenol-stimulated cAMP accumulation in isolated adipocytes as previously shown [16]. NaSacch (50 mM), alone, as observed in Fig. 1, inhibited by about 43% the cAMP production and increased by about 2-fold the inhibitory effect of PIA at any of the tested concentrations. For example, 0.1 mM PIA inhibited by 33%

Sodium Saccharin Inhibits Adenylyl Cyclase Activity

433

pared the effects of RX 821002, an a2-adrenergic receptor antagonist which allowed the identification of a2-adrenergic receptor on intact fat cells [17], on the inhibitory effects of NaSacch and of UK 14304, a very selective and potent a2-adrenergic agonist [18]. As shown previously [19], UK 14304 inhibited significantly (by 66%) isoproterenol-stimulated cAMP accumulation and this effect was completely blocked by RX 821002. In contrast, RX 821002 failed to block the inhibitory effect of NaSacch (Fig. 3). All together, these results indicated that the inhibitory effects of NaS on b-adrenergic stimulation of cAMP production were neither related to A1 adenosine receptor nor to a2-adrenergic receptor stimulation.

Effect of NaSacch on ForskolinStimulated Adenylyl Cyclase Activity in Membrane Fractions from Different Non-Taste Cells

FIGURE 1. Concentration-dependent inhibition of cAMP accu-

mulation by NaSacch. Fat cells were incubated for 6 min with NaSacch at the indicated concentrations in the absence (A) or in the presence of isoproterenol (1 mM) (B) and cAMP levels were determined as described in Materials and Methods. Values are the mean 6 SE of 3 experiments carried out in triplicate. Statistical significance: *p , 0.05, **p , 0.001 (NaSacch versus control).

the isoproterenol-stimulated cAMP production in the absence of NaSacch, while in the presence of NaSacch a 77% inhibition was found. For higher concentrations of PIA (1–10 mM) less than 10.2% of cAMP are produced and the combination of NaSacch and PIA led to less than 5.8% of cAMP produced compared to the control (Fig. 2). In order to determine whether the inhibition of isoproterenol-stimulated cAMP accumulation by NaSacch could be due to stimulation of a2-adrenergic receptors, we com-

To have a better picture of the inhibitory effect of NaSacch, we utilized a membrane adenylyl cyclase assay which eliminates the possibility of endogenous inhibitory ligands generated by intact fat cells. The inhibitory effect of NaSacch on the stimulation of the adenylyl cyclase activity was measured in the presence of forskolin which activated the adenylyl cyclase activity with its usual substrate Mg21/ATP or with Mn21/ATP as substrate which uncoupled functional interactions between Gi proteins and the catalytic subunit [20, 21]. In the presence of Mg21/ATP, NaSacch (2.5–50 mM) inhibited in a dose-dependent manner the stimulation of adenylyl cyclase by forskolin (0.1 mM) in adipocyte membranes (Fig. 4A). Similar inhibitory effects of NaSacch were also observed in membranes prepared from other tissues, rat brain astroglial cells (Fig. 4B) and pig thyrocytes (Fig. 4C). Significant effects were observed with the lowest concentration of NaSacch (5 mM) on adipocyte membranes and with 10 mM on astroctye and thyrocyte membranes. The highest concentration of NaSacch (50 mM) inhibited by about 80% forskolin-stimulated adenylyl cyclase activity in the three types of membranes. The inhibitory effect of NaSacch was also observed when the adenylyl cyclase was stimulated by 10 mM GTP alone or in combination with forskolin (data not shown). To verify that the effect of NaSacch was not due to sodium salts, a parallel dose-dependent response of adenylyl cyclase to NaCl was carried out. It clearly demonstrated that NaCl (2.5–50 mM) had no inhibitory effect by itself and even had a slight stimulatory effect on adenylyl cyclase activity in adipocyte, astrocyte and thyroid membranes (Fig. 4A, B, C). In contrast, in the presence of Mn21/ATP as a substrate and with GDPbS to completely block GTP-dependent regulation of adenylyl cyclase, NaSacch failed to inhibit forskolin-stimulated adenylyl cyclase activity (Fig. 5). Even more, a slight stimulatory effect (1.4-fold) could be observed on each type of membranes whereas NaCl ions had no significant effect (Fig. 5A, B, C).

434

K. Dib et al. TABLE 1. Absence of effect of NaSacch on the cAMP phosphodiesterase activity expressed in (pmol adenosine/min/mg protein)

NaSacch (mM)

Particulate Soluble

0

5

10

20

35

40 6 10 29 6 8

45 6 6 36 6 7

45 6 7 44 6 10

44 6 14 30 6 7

47 6 1.3 —

Fat cells were incubated for 10 min at 378C in the presence of varying concentrations of NaSacch as indicated. At the end of the incubation period, cells were washed, homogenized and the homogenates centrifuged at 105.000 g. Aliquots (15–20 mg protein) of the particulate and soluble fractions were tested for phosphodiesterase activity as described under Materials and Methods. Values are the mean 6 SE of 4 experiments carried out in triplicate.

Consequences of the Treatment of Adipocytes with Pertussis Toxin on Adenylyl Cyclase Inhibition by NaSacch We examined the consequences of in vitro treatments of adipocytes with pertussis toxin which induced the ADP-ribosylation of Gia protein subunits and uncoupled inhibitory receptors from the catalyst. We found that the incubation of adipocytes with pertussis toxin resulted in a 1.5-fold increase in forskolin-stimulated adenylyl cyclase activity compatible with the removal of tonic inhibitory effect of Gi as previously reported [16]. In pertussis toxin-pretreated cells, the inhibitory effects observed for the low concentrations of NaSacch (5–20 mM) were partially decreased, whereas the inhibitory effects exerted by high concentrations of NaSacch (35–50 mM) were similar to control cells (Fig. 6). The inhibitory effects of NaSacch were also totally cholera toxin-insensitive (data not shown).

DISCUSSION The involvement of G-proteins in mediating sweet taste transduction has been proposed [2, 3] and permeable cAMP analogues have been shown to mimic the effects of sucrose and artificial sweeteners [22]. In this paper, we have studied the in vitro effect of NaSacch on adenylyl cyclase activity in rat adipocytes. We found that NaSacch (2.5–50 mM) inhibited significantly in a dose-dependent manner basal and isoproterenol-stimulated cAMP accumulation on isolated rat adipocytes (Figs. 1A and B). The inhibition induced by NaSacch is not mediated by activation of phosphodiesterase since the inhibition was observed in the presence of RO 7-2856, a potent phosphodiesterase inhibitor and since we

FIGURE 2. Concentration-dependent inhibition of cAMP accu-

FIGURE 3. Comparative inhibitory effects of NaSacch and UK

mulation by PIA. Fat cells were incubated for 6 min in the presence of isoproterenol (1 mM) with PIA at the indicated concentrations in the absence (s) or in the presence of NaS (50 mM) (d) and cAMP levels were determined as in Fig. 1. Values are the mean of 4 assays 6 SE. Statistical significance: *p , 0.05, **p , 0.001, PIA versus control without or with NaSacch.

14304 on the inhibition of cAMP accumulation. Fat cells were incubated for 6 min in the presence of isoproterenol (1 mM) alone or with UK (1 mM) or NaSacch (50 mM) supplemented with or without RX 821002 (100 mM) and cAMP levels were determined. Values are the mean of 6 assays 6 SE. Statistical significance: *p , 0.001 (treated samples versus control).

Sodium Saccharin Inhibits Adenylyl Cyclase Activity

FIGURE 4. Effect of NaSacch on adenylyl cyclase activity in

non-taste cell membranes. Adenylyl cyclase activities were measured in membranes from adipocytes (A), astrocytes (B) and thyrocytes (C) in the presence of forskolin (0.1 mM) and 4 mM Mg21 with NaSacch (d) or NaCl (s) at the indicated concentrations. Values are the mean 6 SE of 3–8 experiments carried out in triplicate for NaSacch. One typical experiment for NaCl is shown for each type of membrane. Statistical significance: *p , 0.05, **p , 0.02, ***p , 0.001 (NaSacch versus control).

435

show that NaSacch did not affect the particulate and cytosolic low-Km cAMP-phosphodiesterase (Table 1). Interestingly, the concentrations of NaSacch used in this work (5–50 mM) correlated with those needed to elicit taste sensation in humans [23–25]. The adenylyl cyclase family comprises at least eight different isoforms which are differentially regulated by protein kinases, Ca 21, the complex Ca21/calmodulin and the subunits of Gi/Go proteins [26]. Adipocyte cells expressed the three types of Gi proteins [27] and a G protein, immunologically related to Goa [28]. However, little is known about the isoforms expressed in fat cells. Polymerase chain reactions analyses have shown that white adipose tissue expressed mainly the isoform of type III and trace amounts for the isoform of type V [29]. It should be noted that the isoform III is stimulated by the complex Ca21/calmodulin [30] and inhibited by the subunits a of Gi proteins [31]. The negative control of adenylyl cyclase is mediated by Gi/Go proteins acting as coupler between inhibitory hormone receptors and the catalyst. Rat adipocytes possess mainly the A1 adenosine receptor class [32] and a2-adrenoceptors [33]. In order better to understand the mechanism whereby NaSacch induced inhibition of adenylyl cyclase activity, we investigated whether these two Gi-coupled receptors were involved in NaS-induced inhibition of cAMP production. We found that the inhibitory effect of NaSacch was additive to that of the A1 adenosine receptor agonist PIA (Fig. 2), and was not counteracted by the a2-adrenergic receptor antagonist RX 821002 (Fig. 3). These data clearly demonstrate that NaSacch action was mediated neither by a2-adrenergic receptor nor by A1-adenosine receptor and suggest that NaSacch uses a different route to inhibit adenylyl cyclase. NaSacch also inhibited in a dose-dependent manner forskolin-stimulated adenylyl cyclase measured in membrane fractions in the presence of Mg21/ATP as substrate (Fig. 4A). This inhibitory effect was observed in other non-taste membranes from astrocytes and thyrocytes (Fig. 4B, C), confirming that the action of NaSacch was not restricted to adipocytes and could be revealed on intact isolated cells as well as on membrane preparations. This situation was completely different when we substituted Mn21 for Mg21. In the presence of Mn21/ATP, which selectively abolished Gi proteins input [20, 21] and that of GDPbS, a competitive antagonist of Gs and Gi proteins, NaSacch failed to inhibit forskolin-stimulated adenylyl activity and even slightly increased it. Sodium salts have been shown to inhibit or stimulate membrane bound adenylyl cyclase depending on the system under scrutiny and in many cases affected the regulation of the enzyme by guanine nucleotides [34]. We clearly show in this work that the observed phenomena are not due to sodium ions since NaCl did not affect significantly forskolinstimulated adenylyl cyclase activity in the three types of membranes. (Figs. 4, 5). Cholera toxin which ADP-ribosylated Gs had no effect on the inhibition of adenylyl cyclase by NaSacch. This data

436

confirmed previous data showing that cholera toxin did not affect receptor-mediated inhibition of adenylyl cyclase [35]. In contrast, pertussis toxin which ADP-ribosylated Gi/Go proteins abolished the receptor-induced inhibition of adenylyl cyclase [36]. We show in this work, that the inhibitory effects of NaSacch on forskolin-stimulated adenylyl cyclase activity were slightly decreased for concentrations of this sweetener lower than 20 mM whereas those for the high concentrations up to 50 mM were not changed (Fig. 6). However, the functional integrity of the pertussis-toxin substrate in fat cells was already confirmed [16]. These results suggest that NaSacch should also inhibit adenylyl cyclase activity in a pertussis toxin-insensitive fashion. Up to now, we know of only one G protein (Gz) which mediated hormonal inhibition of cAMP accumulation in a pertussis toxin-insensitive fashion [37]. Gz has been shown to inhibit cAMP accumulation in response to the activation of a variety of inhibitory receptors, including the dopamine-D2, adenosine-A1, a2-adrenergic, lysophosphatidic acids and formyl peptide receptors [37, 38]. Recently, it has been shown that activated Gz was a more potent inhibitor of the type V adenylyl cyclase than Gia [39]. This type of adenylyl cyclase could be present in adipocytes. However, for the moment, the presence of Gz in these cells is still unknown. It has been proposed that the transduction of sweet and bitter taste involved specific membrane receptors coupled to heterotrimeric G proteins [4]. Gustducin, a taste receptor-specific G protein, has been proposed as a principal mediator of both bitter and sweet signal transduction. However, additional G proteins may also be involved including Gs, Gi, G14 [40]. To date, no taste receptor has been isolated and many observations supported the hypothesis that more than one sweet receptor is likely to exist [41]. Thus, more in-depth studies are required to examine whether the effect of NaSacch is due to specific receptors coupled to specific G proteins. On the other hand, it has been shown that a variety of cationic-amphiphilic neuropeptides (bradykinin, neurokinin P) and venom peptides (mastoparan) as well as polyamines such as 48/80 activated directly Gi/Go proteins, through a pertussis toxin-sensitive processus, by mimicking the role normally played by agonist-liganded receptors [42– 44]. Bradykinin has been also proposed to be one of the signal-transduction pathways for bitter sensation [45]. It has been shown that NaSacch and other sweeteners increased the GTPase activity of Gi/Go proteins reconstituted into phospholipid vesicles just like mastoparan did; thus it was proposed that non-sugar sweeteners could be direct activators of Gi/Go proteins [46]. Recently, it has been proposed that mastoparan may activate GTP hydrolysis by Gi proteins indirectly through interaction with nucleoside diphosphate kinase. This enzyme catalysed the phosphorylation of GDP to GTP, and thus could contribute to the stimulatory effect of venom on GTPase activity in HL-60 membranes

K. Dib et al.

FIGURE 5. Mn21 blocks the inhibitory effect of NaSacch on for-

skolin stimulated adenylyl cyclase activities. Adenylyl cyclase activities were measured in membranes from adipocytes (A), astrocytes (B) and thyrocytes (C) in the presence of forskolin (0.1 mM), 10 mM Mn21 and 200 mM of GDPbS with or without NaSacch (d) or NaCl (s) as described in Fig. 4. Values are the mean 6 SE of 2 experiments performed in triplicate.

Sodium Saccharin Inhibits Adenylyl Cyclase Activity

FIGURE 6. Pertussis toxin treatment does not abolish the inhibitory effect of NaSacch. Fat cells were preincubated for 90 min with (d) or without (s) 10 mg/ml of pertussis toxin and adenylyl cyclase activities were measured in membranes as described in Fig. 4. Results are expressed as % of control values without NaSacch and are the mean 6 SE of 3 experiments performed in triplicate. Statistical significance: *p , 0.05 (NaSacch versus control).

[47]. Moreover, the effect of mastoparan on the activation of GTP hydrolysis was partially pertussis toxin-sensitive and its effect on nucleoside diphosphate kinase was pertussis toxin-insensitive [48]. Thus by analogy to mastoparan, we cannot exclude the possibility that NaSacch could affect indirectly the activation of Gi like proteins. Such an hypothesis could explain, at least in part, the apparent discrepancies in its effectiveness to inhibit adenylyl cyclase and its partial pertussis toxin-insensitivity. Further studies are needed to address the question of whether the inhibitory effect of NaSacch reported herein can be explained by the existence of specific receptors for NaSacch or by a direct or indirect activation of G proteins. We would like to thank Dr. C. Carpe´ne´ for providing us with UK 14304 and Dr. L. Legendre for helpful advices.

References 1. Naim M., Ronen T., Striem B. J., Levinson M. and Zehavi U. (1991) Comp. Biochem. Physiol. 100, 455–458. 2. Striem B. J., Pace U., Zehavi U., Naim M. and Lancet D. (1989) Biochem. J. 260, 121–126. 3. Striem B. J., Naim M., Zehavi U. and Ronen T. (1990) Life Sciences 46, 803–810. 4. Margolskee R. F. (1993) Curr. Opin. Neurobiol. 3, 526–531. 5. Hoon M. A., Northup J. K., Margolskee R. F. and Ryba N. J. P. (1995) Biochem. J. 300, 629–636. 6. Rodbell M. (1967) J. Biol. Chem. 242, 5744–5750.

437 7. Dib K., El Jamali A., Jacquemin C. and Corre`ze C. (1994) Eur. J. Biochem. 219, 529–537. 8. Ge´rard C., Haye B., Jacquemin C. and Mauchamp J. (1982) Biochim. Biophys. Acta 710, 359–369. 9. Haye B., Aublin J. L., Champion S., Lambert B. and Jacquemin C. (1985) Mol. Cell. Endocrinol. 43, 41–50. 10. Folch J., Less M. and Stanley G. H. S. (1957) J. Biol. Chem. 226, 497–509. 11. Salomon Y., Londos C. and Rodbell M. (1974) Anal. Biochem. 58, 541–548. 12. Schaffner W. and Weissman C. (1973) Anal. Biochem. 56, 502–514. 13. Rutten W. J., Schoot B. M. and De Pont J. H. M. (1973) Biochim. Biophys. Acta 315, 378–383. 14. Lambert B. and Jacquemin C. (1983) FEBS Lett. 155, 31–34. 15. Green A. (1987) J. Biol. Chem. 262, 15702–15707. 16. Martiny L., Dib K., Haye B., Corre`ze C., Jacquemin C. and Lambert B. (1991) FEBS Lett. 286, 105–109. 17. Galitzky J., Larrouy D., Berlan M. and Lafontan M. (1990) J. Pharmacol. Exp. Ther. 252, 312–319. 18. Carpe´ne´ C., Galitzky J., Larrouy D., Langin D. and Lafontan M. (1990) Biochem. Pharmacol. 40, 437–445. 19. Rebourcet M. C., Carpe´ne´ C. and Lavau M. (1988) Biochem J. 252, 679–682. 20. Limbird L. E., Hickey A. R. and Lefkowitz R. J. (1979) J. Biol. Chem. 254, 2677–2683. 21. Jakobs K. H., Aktories K. and Schultz G. (1981) Adv. Cyclic Nucleotide Res. 14, 173–187. 22. Houpt T. A., Frankmann S. P. and Berlin R. (1996) Physiol. Behav. 59, 495–498. 23. Hellekant G., Roberts T., Segerstad C. H. and Van der Welt H. (1987) Ann. N. Y. Acad. Sci. 510, 356–358. 24. Naim M., Oukan E., Zenavi U. and Yaron L. (1985) Chem. Senses 11, 361–370. 25. Schiffman S. S., Sugarman D., Jakinovitch W. J., Paikin A. and Crofton V. (1987) Chem. Senses 12, 71–76. 26. Taussig R. and Gilman A. G. (1995) J. Biol. Chem. 270, 1–4. 27. Mitchell F. M., Griffiths S. L., Saggerson E. D. and Houslay M. D. (1989) Biochem. J. 262, 403–408. 28. Rouot B., Carrette J., Lafontan M., Lan Tran P., Fehrentz J. A., Bockaert J. and Toutant M. (1989) Biochem. J. 260, 307–310. 29. Manopoulos V. G., Liu J., Unsworth B. R. and Lelkes P. I. (1995) Biochem. Biophys. Res. Comm. 208, 323–331. 30. Choi E. J., Xia Z. and Storm D. R. (1992) Biochemistry 31, 6492–6498. 31. Chen J. and Iyengar R. (1993) J. Biol. Chem. 268, 12253– 12256. 32. Londos C., Cooper D. M. F., Schlegel W. and Rodbell M. (1978) Proc. Natl. Acad. Sci. USA 75, 5362–5366. 33. Lafontan M. and Berlan M. (1993) J. Lipid Res. 34, 1057–1090. 34. Joshi L. R. and Katz M. S. (1988) Second Messengers Phosphoprot. 12, 105–121. 35. Jakobs K. H., Aktories K. and Schultz G. (1984) Adv. Cyclic Nucleot. Prot. Phosphoryl. Res. 17, 135–143. 36. Katada T. and Ui M. (1981) J. Biol. Chem. 256, 8310–8317. 37. Wong Y. H., Conklin B. R. and Bourne H. R. (1992) Science 255, 339–342. 38. Tsu R. C., Lai H. W. L., Allen R. A. and Wong Y. H. (1995) Biochem. J. 309, 331–339. 39. Kozasa T. and Gilman A. G. (1995) J. Biol. Chem. 270, 1734–1741. 40. Wong G. T., Gannon K. S. and Margolskee R. F. (1996) Nature 381, 796–800. 41. Lindemann B. (1995) Am. J. Physiol. 10, 166–170. 42. Higashijima T., Uzu S., Nakajima T. and Ross E. M. (1988) J. Biol. Chem. 263, 6491–6494. 43. Mousli M., Bueb J. L., Bronner C., Rouot B. and Landry Y. (1990) Trends Pharmacol. Sci. 11, 358–362.

438 44. Higashijima T., Burnier J. and Ross E. M. (1990) J. Biol. Chem. 265, 14176–14186. 45. Spielman A. I., Huque T., Whitney G. and Brand J. C. (1992). In: Sensory Transduction (Corey D. P. and Roger S. D., Eds), pp. 308–324. The Rockfeller University Press, New York. 46. Naim M., Seifert R., Nu¨rnberg B., Gru¨nbaum L. and Schultz G. (1994) Biochem. J. 297, 451–454.

K. Dib et al. 47. Klinker J. F., Hagelu¨ken A., Gru¨nbaum L., Heilmann I., Nu¨rnberg B., Harhammer R., Offermans S., Schwaner I., Ervens J., Wenzel-Seifer K., Mu¨ller T. and Seifer R. (1994) Biochem. J. 304, 377–383. 48. Klinker J. F., Laugwitz K. L., Hagelu¨ken A. and Seifer R. (1996) Biochem. Pharmacol. 51, 217–223.