AgRP(83–132) Acts as an Inverse Agonist on the Human-Melanocortin-4 Receptor

Wouter A. J. Nijenhuis, Julia Oosterom and Roger A. H. Adan

Molecular Neuroscience Rudolf Magnus Institute for Neurosciences University Medical Center Utrecht Utrecht, the Netherlands 3584 CG


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The central melanocortin (MC) system has been demonstrated to act downstream of leptin in the regulation of body weight. The system comprises {alpha}-MSH, which acts as agonist, and agouti-related protein (AgRP), which acts as antagonist at the MC3 and MC4 receptors (MC3R and MC4R). This property suggests that MCR activity is tightly regulated and that opposing signals are integrated at the receptor level. We here propose another level of regulation within the melanocortin system by showing that the human (h) MC4R displays constitutive activity in vitro as assayed by adenylyl cyclase (AC) activity. Furthermore, human AgRP(83–132) acts as an inverse agonist for the hMC4R since it was able to suppress constitutive activity of the hMC4R both in intact B16/G4F melanoma cells and membrane preparations. The effect of AgRP(83–132) on the hMC4R was blocked by the MC4R ligand SHU9119. Also the hMC3R and the mouse(m)MC5R were shown to be constitutively active. AgRP(83–132) acted as an inverse agonist on the hMC3R but not on the mMC5R. Thus, AgRP is able to regulate MCR activity independently of {alpha}-MSH. These findings form a basis to further investigate the relevance of constitutive activity of the MC4R and of inverse agonism of AgRP for the regulation of body weight.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Melanocortin receptors (MCRs) are G-protein coupled receptors (GPCRs) that are positively coupled to the cAMP pathway. Of the five MCRs that have been cloned, the MC1R is expressed on melanocytes in the skin where it is involved in regulation of pigmentation. The MC4R is expressed in the brain where it was shown to be involved in regulating metabolism and food intake both in rodents (1, 2, 3, 4) and humans (5, 6). The MC3R is expressed in the brain, placenta, and gut. The exact role of the MC3R is still unclear, but it has been proposed that in the brain the MC3R could have a regulatory role in metabolism and food intake upstream of the MC4R (7).

In addition to the melanocortins (e.g. ACTH and {alpha}- and {gamma}-MSH), which act as agonists on MCRs, endogenous high-affinity peptide ligands for MCRs have been found that act as antagonists, namely agouti protein (agouti) (8, 9, 10) and agouti-related protein (AgRP) (11, 12). The existence of different ligands with opposing actions on the same receptor provides a mechanism to tightly regulate MCR activity and to integrate opposing signals at the receptor level.

Agouti is a paracrine factor expressed in dermal papilla in the hair follicle, where it regulates the switch from eumelanin to phaeomelanin synthesis by melanocytes in mice (13). In vitro, agouti is an antagonist for the MC1R and MC4R (10), and it has been suggested that agouti regulates pigmentation by blocking {alpha}-MSH binding to the MC1R (14). However, several effects of agouti cannot be explained by competition with {alpha}-MSH only. For instance, it has been reported that incubation of cells expressing the MC1R with agouti in the absence of {alpha}-MSH resulted in lower cAMP formation (15, 16), decreased melanogenesis (16, 17, 18) and cell growth (19), and lower tyrosinase expression (16). Furthermore, agouti can inhibit the response to cholera toxin (17, 20). These effects could be explained by inverse agonism of agouti.

AgRP, which shares sequence homology with agouti, is expressed in the arcuate nucleus of the hypothalamus, subthalamic nucleus, and the adrenal gland (11) and is a potent antagonist for the MC3R and MC4R, and a weak antagonist for the MC5R (12, 21, 22). Considerable evidence exists suggesting that AgRP is involved in body weight regulation (11, 12, 23, 24) by acting downstream of the adipose tissue-derived satiety factor leptin (25, 26). AgRP reduces the maximal response of the human-MC4R (hMC4R) to {alpha}-MSH, and evidence was provided that AgRP reduces activation of the cAMP pathway in the absence of agonist, as assayed by measurement of cAMP levels and reporter gene activity (12, 27).

Understanding the mechanisms by which melanocortin ligands regulate their receptors is essential for unraveling the physiological role of the melanocortin system. Therefore, we tested directly whether the human (h) MC4R displays constitutive activity and whether AgRP is able to suppress this constitutive activity. We used synthetic AgRP(83–132) (28) and B16/G4F (29) cell lines stably expressing different levels of the hMC4R and assayed for adenylyl cyclase activity.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Constitutive Activity of the hMC4R
No specific [125I]NDP-MSH ([Nle4-D-Phe7]-{alpha}-MSH) binding was detected in wild-type B16/G4F cells (data not shown). Also, incubation of this cell line with up to 1 µM {alpha}-MSH did not induce adenylyl cyclase (AC) activity (Table 1Go), indicating that there is no expression of endogenous MC receptors in this cell line.


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Table 1. AC Activities of Wild-Type B16/G4F Cells and B16/G4F Cells Expressing the Human MC3R or Mouse MC5R

 
Four clones of B16/G4F cells were generated, each with a different expression level of functional hMC4R as measured by [125I]NDP-MSH binding and response to {alpha}-MSH (Fig. 1Go). The cell lines were tested for basal (unstimulated) and forskolin-induced AC activity. The response to forskolin was determined because it has been shown to correlate with the amount of constitutive activity of receptors expressed in cell lines (30). Basal AC activity was not significantly different (0.54, 0.51, and 0.41% cAMP) in three clones expressing 1.6 x104, 5.0 x104, and 5.6 x 104 receptors per cell (clones MC4–1, MC4–2, and MC4–3, respectively). However, significantly higher (1.26% cAMP, P < 0.05) AC activity was detected in clone MC4–4, which expresses 23 x 104 receptors per cell (Fig. 1Go). The forskolin-induced AC activity correlated with the expression level of the clones (Fig. 1Go, r = 0.97 and r = 0.93 respectively, P < 0.001).



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Figure 1. Spontaneous Activity of the hMC4R

Basal ({diamondsuit}) AC activity and the response to 1 µM forskolin ({blacksquare}) and 1 µM {alpha}-MSH ({square}) of four cell lines expressing the hMC4R. AC activity expressed as % of [3H]ATP converted to [3H]cAMP is plotted against the receptor expression level of the cell lines. Data are given as mean ± SEM (n = 3). Replicated four times.

 
Suppression of Constitutive Activity of the hMC4R by AgRP(83–132)
The effect of 100 nM human AgRP(83–132) on basal and forskolin-induced AC activity of hMC4R-expressing cells was determined (Fig. 2Go). AgRP(83–132) suppressed basal AC activity up to 60% in cells expressing the hMC4R. This effect, however, was not seen in clone MC4–1, which displayed the lowest level of hMC4R expression. Similarly, AgRP(83–132) suppressed forskolin-induced AC activity only in the three cell lines with the highest expression of the hMC4R. Suppression of both basal and forskolin-induced AC activity was most profound in cells with high expression levels of the hMC4R. AgRP(83–132) did not alter basal and forskolin-induced AC activity in wild-type B16/G4F cells (Fig. 2Go).



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Figure 2. Suppression of AC Activity by AgRP(83–132) in Cells Expressing the hMC4R

Effect of 100 nM AgRP(83–132) on basal (A) and forskolin (1 µM)-induced (B) AC activity of wild-type (wt) B16/G4F cells and B16/G4F cells with different expression levels of the hMC4R. AC activities of untreated (black bars) cells were set at 100% for each cell line. Relative AC activities of cells treated with AgRP(83–132) are given in gray bars. Data are shown as mean ± SEM (n = 3 or 4). *, Statistically significant different (P < 0.01). Replicated four times.

 
AgRP(83–132) suppressed basal and forskolin-induced AC activity in a dose-dependent manner (Fig. 3Go). The EC50 values were 21 and 37 nM, respectively; 1 µM SHU9119, originally identified as antagonist for the hMC4R (31), blocked the effect of {alpha}-MSH on AC activity in a cell line expressing the hMC4R at 3.6 x 104 receptors per cell (Fig. 4AGo). In this cell line, SHU9119 also blocked the effect of AgRP(83–132) on both basal and forskolin-induced AC activity when coadministered (Fig. 4Go, B and C). SHU9119 by itself did not influence AC activity in these cells. Also in clone MC4–4, which expresses 23 x 104 receptors per cell and shows a larger response to AgRP(83–132), SHU9119 was able to block the effect of AgRP(83–132) on basal AC activity (Fig. 4DGo). However, in this clone SHU9119 displayed weak partial agonism.



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Figure 3. Dose-Dependent Suppression of hMC4R Activity by AgRP(83–132)

Effect of AgRP(83–132) on basal (A) and forskolin (1 µM)-induced (B) AC activity in cells expressing the hMC4R (23 x 104 receptors per cell). Data are shown as mean ± SEM (n = 3). Replicated three times.

 


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Figure 4. SHU9119 Blocks the Effect of AgRP(83–132) and {alpha}-MSH on the hMC4R

A, Effect of 1 µM SHU9119 on 1 µM {alpha}-MSH-induced AC activity in cells expressing the hMC4R at 3.9 x 104 receptors per cell. B, Effect of 1 µM SHU9119 on inhibition of AC activity by 200 nM AgRP(83–132) in the same cell line as in panel A. C, Effect of 1 µM SHU9119 on the inhibition of forskolin-induced AC activity by 200 nM AgRP(83–132) in the cells mentioned in panel A. D, Effect of 1 µM SHU9119 on the inhibition of AC activity by AgRP(83–132) in clone MC4–4. Basal (A, B, and D) and forskolin-treated (C) samples were set at 100%. Where multiple compounds were used in one sample, compounds were added simultaneously. Abbreviations: MSH, {alpha}-MSH; S, SHU9119; F, forskolin; and A, AgRP(83–132). Data are expressed as mean ± SEM (n = 3 for A, C, and D; n = 4 for B). *, Statistically significant different from basal (A, B, and D) or forskolin-treated (C) (P < 0.05). Replicated two (A, C, and D) and three (B) times.

 
To investigate the influence of receptor internalization, AgRP(83–132) was also tested in a cell-free AC activity assay using membrane preparations instead of intact cells. Incubation of membranes prepared from clone MC4–4 with AgRP(83–132) reduced basal and forskolin-induced AC activity, but did not affect AC activity in membranes from B16/G4F cells (Fig. 5Go).



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Figure 5. AgRP(83–132) on hMC4R-Containing Membranes

Effect of 200 nM AgRP(83–132) on basal (A) and forskolin (1 µM)-induced (B) AC activity in membranes from clone MC4–4 and B16/G4F cells. *, Statistically significant different (P < 0.05). Replicated three times.

 
The Effect of AgRP(83–132) on the hMC3R and the mMC5R
Cells expressing the hMC3R (190 x 104 receptors per cell) and the mouse (mMC5R, 54 x104 receptors per cell) showed increased basal and forskolin-induced AC activity compared with wild-type cells (Table 1Go). Both cell lines responded to {alpha}-MSH (Table 1Go). The hMC3R-expressing cells and membrane preparations from these cells showed 20–35% reduction in basal and forskolin-induced AC activity upon incubation with 200 nM AgRP(83–132) (Fig. 6Go). There was no effect of 200 nM AgRP(83–132) on basal and forskolin-induced AC activity in cells expressing the mMC5R (data not shown).



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Figure 6. Effect of AgRP(83–132) on the hMC3R

Effect of 200 nM AgRP(83–132) treatment (gray bars) on basal (A), and forskolin (1 µM)-induced (B) AC activity of cells expressing the hMC3R and membranes of these cells. Bars indicate mean ± SEM (n = 4). *, Statistically significant different (P < 0.01). Replicated two (membranes) and three (cells) times.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study, we show that the hMC4R displays constitutive activity in vitro and that AgRP(83–132) acts as an inverse agonist for this receptor. These findings represent a new mechanism by which MCR activity may be regulated in vivo.

In cells expressing the hMC4R, AgRP(83–132) lowered both basal and forskolin-induced AC activity in a dose-dependent manner. The data suggest that the effect of AgRP(83–132) occurs via direct interaction with the hMC4R since this effect was not seen in wild-type B16/G4F cells and was blocked by 1 µM of the MC4R antagonist SHU9119. Although at this high dose of SHU9119 there was a partial agonistic effect of SHU9119 on cells expressing the highest number of hMC4R (clone MC4–4), this was only a small effect compared with the effect of AgRP(83–132). In any case, SHU9119 was able to fully block the effect of AgRP(83–132) on this clone. This is most likely via competition for binding to the hMC4R since SHU9119 has nanomolar affinity for the hMC4R (31) and at 1 µM it is thus expected that all receptors are occupied.

The effect of AgRP(83–132) on AC activity was most profound in the cell line with the highest hMC4R expression level. The effect was less in cells expressing an intermediate level of the hMC4R and not detectable in cells expressing very low amounts of hMC4R. Thus, the efficacy of AgRP(83–132) to suppress basal and forskolin-induced AC activity correlates with the expression level of the receptor.

Inverse agonism is defined as the ability of a ligand to stabilize the inactive conformation of a receptor (32). The results discussed above suggest that AgRP(83–132) is an inverse agonist for the hMC4R. However, the decreases in AC activity observed here could also be due to hMC4R internalization induced by AgRP(83–132), since AC activity correlates with receptor density of a constitutively active receptor (33). To investigate the role of endocytosis in the suppression of AC activity by AgRP(83–132), AC activity was measured in cell membranes. During homogenization, the actin cytoskeleton, which is necessary for endocytosis (34), is disrupted mechanically. Additionally, endocytosis needs cytosolic components, which are washed away during membrane preparation. Therefore it is expected that endocytosis is severely impaired or absent in isolated membranes. Because AgRP(83–132) suppressed constitutive activity of the hMC4R in membrane preparations to the same extent as in intact cells, it is reasonable to conclude that internalization is not the underlying mechanism for the effects of AgRP(83–132) on the hMC4R. Therefore, based on the results that 1) AgRP(83–132) suppressed AC activity via direct interaction with the hMC4R, 2) this suppression depended on the expression level of the hMC4R, and 3) this suppression occurred in intact cells as well as in membrane preparations, we conclude that AgRP(83–132) is an inverse agonist for the hMC4R.

Because inverse agonism can only be measured in the presence of constitutive activity, it is to be expected that in our test system the hMC4R displays constitutive activity. Indeed, the response to 1 µM forskolin correlated with the expression levels of the hMC4R. This was shown before to be characteristic for constitutively active receptors (30). Also, compared with the other hMC4R-expressing clones, the clone with the highest hMC4R expression level showed elevated basal AC activity. These data strongly support the notion that the hMC4R is constitutively active. However, basal AC activity in the clones with lower hMC4R expression did not correlate with the expression level of the receptor. This is not due to limited sensitivity of the assay because, upon incubation with AgRP(83–132), lower AC activity could be measured in these clones. One obvious explanation is that intracellular compensatory mechanisms exist that are able to counteract basal constitutive activity only at low expression levels of the receptor. At higher expression levels of the receptor (as in clone MC4–4) or in the presence of forskolin, these mechanisms may not be able to compensate for constitutive activity. Thus, in these experiments the use of forskolin allows a more sensitive detection of constitutive activity, as has been described previously (30).

Expressing the hMC3R or the mMC5R in B16/G4F cells increased basal and forskolin-induced AC activity as compared with wild-type cells. This indicates that both receptors are constitutively active. As is shown in Fig. 6Go, AgRP(83–132) is an inverse agonist for the hMC3R. In contrast, although the mMC5R has constitutive activity, AgRP(83–132) did not affect AC activity in cells with this receptor. This is in agreement with the low affinity of AgRP for the MC5R (12, 21, 22).

Several lines of evidence suggest that activation of central MCR inhibits food intake (1, 3). If the hMC4R exhibits constitutive activity in vivo, this would contribute to the tonic inhibition of the melanocortin system on feeding. The observed haploinsufficiency of obese human subjects with hMC4R gene variants reported in several studies (5, 6, 35) and the intermediate obese phenotype of mice heterozygous for MC4R deletion (1) may therefore be the result of a gene dosage effect resulting in decreased constitutive activity. However, it cannot be excluded that an impaired response to {alpha}-MSH in the heterozygous mutants causes the haploinsufficiency.

Most likely, full-length AgRP also acts as an inverse agonist because the C terminus of AgRP has been shown to possess the same pharmacological properties in vitro and in vivo as full-length AgRP (22, 24, 27). Furthermore, other studies already showed effects of nearly full-length AgRP that do not fit with neutral antagonism (12, 27).

This indicates that AgRP may reduce the activity of the melanocortin system independently of {alpha}-MSH in the brain. POMC and AgRP are expressed in different neurons (36, 37). If AgRP(83–132) only blocks {alpha}-MSH-induced activation, the presence of {alpha}-MSH (i.e. activation of POMC neurons) is necessary for AgRP to function. However, if AgRP acts independently of {alpha}-MSH, AgRP-containing neurons can act independently of POMC-containing neurons, thereby adding a new level of regulation to the melanocortin system. Indeed, in rat brain, AgRP and {alpha}-MSH production seem to be counterregulated since there is increased AgRP mRNA and decreased POMC mRNA expression in response to fasting (38, 39). The opposite effect is seen in response to leptin (26, 36, 38, 39, 40). Interestingly, the changes in AgRP mRNA levels are larger than those measured for POMC (25). Thus, for the regulation of body weight the melanocortin system may be controlled more by AgRP than {alpha}-MSH. Similarly, for the MC1R it is the expression level of agouti rather than that of {alpha}-MSH that determines the coat color (13).

The data presented here form a basis to further investigate the relevance of constitutive activity of the MC4R and of inverse agonism of AgRP for the regulation of body weight.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals and Ligands
Forskolin was purchased from Sigma (Steinheim, Germany), and synthetic human AgRP(83–132) was obtained from Phoenix Pharmaceuticals, Inc. (Mountain View, CA). {alpha}-MSH and NDP-MSH were purchased from Bachem (Bubendorf, Switzerland). SHU9119 was synthesized as described previously (41).

Generation of Cell Lines
B16/G4F cells (29) were grown in RPMI 1640 medium (Life Technologies, Inc., Paisley, U.K.) supplemented with 10% FCS (Integro, Zaandam, The Netherlands) and 15 mM sodium hydrogen carbonate (NaHCO3). Cells were transfected with hMC3R (42), hMC4R (43), or mMC5R (44) cDNA cloned into pcDNA3 (Invitrogen, Carlsbad, CA) using calcium phosphate precipitation. Clones, stably expressing MCRs, were selected in medium containing the neomycin analog G418 (800 µg/ml, Life Technologies, Inc.).

AC Activity
AC activity was determined using a modified method of Salomon (45).

Intact-Cell Assay
Cells were grown in 24-well plates and incubated for 2 h with 2 µCi/ml [3,8-3H]-adenine (21.7 Ci/mmol, NEN Life Science Products, Boston, NA) in DMEM (Life Technologies, Inc.) containing 0.2% BSAe (BSA, ICN Biomedicals, Inc., Aurora, OH), 2 mM L-glutamine (Life Technologies, Inc.) and nonessential amino acids. Subsequently, the cells were washed with PBS (Life Technologies, Inc.) containing 0.25 mM isobutylmethylxanthine (IBMX, Sigma) (PBS/IBMX) and incubated for 20 min with compounds diluted in PBS/IBMX. Then 1 ml of cold stop solution (5% trichloric acid (Merck & Co. Inc., West Point, PA), 1 mM cAMP (Roche Molecular Biochemicals, Indianapolis, IN), 1 mM ATP (Roche Molecular Biochemicals)] per well was added and the plates were centrifuged at 250 x g. Finally, ATP and cAMP fractions were separated on Dowex (AG-50W-X4, Bio-Rad Laboratories, Inc. Hercules, CA) and alumina (WN-3, Sigma) columns, respectively. ATP and cAMP fractions were dissolved in scintillation cocktail (Ultima Gold, Packard, Meriden, CT) and counted in a ß-counter.

AC activity was calculated as the percentage of [3H]ATP that is converted to [3H]cAMP using the equation [3H]cAMP/([3H]cAMP + [3H]-ATP). In a typical experiment, from one well containing approximately 2 x·105 cells, 1.2 pmol (equivalent to 60,000 dpm) of tritiated cAMP and ATP were counted.

Membrane Assay
Two days after plating into 10-cm dishes, approximately 1.5 x 109 cells were scraped and suspended in PBS. After centrifugation (15 min; 500 x g) the cells were homogenized in 10 ml solution containing 1 mM NaHCO3, 1 mM dithiothreitol (DTT, Sigma), 0,2 mM magnesium acetate (MgAc), 200 µg/ml DNaseI (Roche Molecular Biochemicals), and protease inhibitors (Complete, EDTA-free, Roche Molecular Biochemicals) at 4 C, using a Teflon on glass homogenizer. Homogenates were centrifuged for 15 min at 1,500 x g to remove intact cells and cell debris. The supernatant was centrifuged for 45 min at 40,000 x g, and the final pellet was resuspended in 1 mM NaHCO3 + 1 mM DTT. Total protein content was determined using Bradford reagents with BSA as standard. Assay mixtures contained 25 mM Tris acetate (pH 7.5), 5 mM MgAc, 0.5 mM ATP, 1 mM DTT, 0.1 mM IBMX, 0.1 g/l BSA, 10 µM GTP (Roche Molecular Biochemicals), 50 µM cAMP, 1 µCi/ml [2,8-3H]-ATP (34.5 Ci/mmol, NEN Life Science Products, Boston, NA), 5 mM phosphocreatine (Sigma) and 50 U/ml creatine kinase (Sigma). The assay was started by adding 20 µg of total membrane protein (contained in 60 µl) to 40 µl of assay mixtures and compounds at the appropriate concentrations. Incubations were performed at 30 C for 30 min. After incubation, samples were treated exactly as described above for the intact cell assay, except that 0.9 ml of stop solution was added. Typically, from one sample a total amount of 2.3 pmol (equivalent to 180,000 dpm) of [3H]cAMP and [3H]ATP were counted.

Receptor Expression
Bmax of the cell lines was determined in saturation experiments with [125I]NDP-MSH as tracer. NDP-MSH was iodinated using bovine lactoperoxidase (Calbiochem, La Jolla, CA) and [125I]Na (ICN Biochemicals, Inc.) according to Oosterom et al. (46) and subsequently HPLC purified on a C18 column (µBondapak 3.9 x 300 mm, Waters Corp., Milford, MA). Cells were washed with Tris-buffered saline (TBS) supplemented with 2.5 mM calcium chloride and incubated for 30 min at room temperature with tracer diluted in Ham’s F10 medium (Life Technologies, Inc.) supplemented with 2.5 mM calcium chloride, 0.25% BSA, and 200 KIU/ml aprotinin (Sigma). After two washes with ice-cold TBS (+ 2.5 mM calcium chloride) the cells were lysed in 1 M sodium hydroxide and samples were counted in a {gamma}-counter.

Data Analysis
The Spearman correlation coefficient was determined to assess correlation between receptor expression level and AC activity response to forskolin or {alpha}-MSH. Differences in basal AC activity between the hMC4R-expressing cell lines were evaluated using the Student-Newman-Keuls test. Student’s t test was used to analyze the effect of AgRP(83–132) treatment on basal and forskolin-induced AC activity. EC50 values were calculated with curve fitting (nonlinear, variable slope) using Prism software (GraphPad Software, Inc., San Diego, CA).


    ACKNOWLEDGMENTS
 
We thank D. H. Vrinten M.D. for her help with the statistical analysis of the data.


    FOOTNOTES
 
Address requests for reprints to: Dr. R. A. H Adan, Rudolf Magnus Institute for Neurosciences, Utrecht University, Universiteitsweg 100, 3584 CG Utrecht, the Netherlands. E-mail: A.H.Adan{at}med.uu.nl

J. Oosterom was supported by Netherlands Organisation for Scientific Research (NWO) Grant 903–49-162.

Received for publication April 11, 2000. Revision received August 14, 2000. Accepted for publication October 3, 2000.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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