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Endocrinology Vol. 141, No. 7 2450-2457
Copyright © 2000 by The Endocrine Society

ARTICLES

Immunohistochemical Localization, Biochemical Characterization, and Biological Activity of Neurotensin in the Frog Adrenal Gland1

Flavie Sicard, Hubert Vaudry, Benedicte Braun, Nicolas Chartrel, Jerome Leprince, J. Michael Conlon and Catherine Delarue

European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, (INSERM U-413), Unité Affiliée au Centre National de la Recherche Scientifique (UA CNRS), University of Rouen, (F.S., H.V., B.B., N.C., J.L., C.D.), 76821 Mont-Saint-Aignan, France; and Regulatory Peptide Center, Department of Biomedical Sciences, Creighton University Medical School (J.M.C.), Omaha, Nebraska 68178

Address all correspondence and requests for reprints to: Dr. Hubert Vaudry, European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, UA CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry{at}univ-rouen.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The primary structure of neurotensin has been recently determined for the frog Rana ridibunda . In the present study, we have investigated the distribution and biochemical characterization of neurotensin-like immunoreactivity in the frog adrenal gland, using an antiserum directed against the conserved C-terminal region of the peptide. Neurotensin-like immunoreactivity was detected in two populations of nerve fibers: numerous varicose fibers coursing between adrenal cells, and a few processes located in the walls of blood vessels irrigating the gland. Reversed-phase HPLC analysis of frog adrenal gland extracts revealed the existence of a major peak of neurotensin-like immunoreactivity that exhibited the same retention time as synthetic frog neurotensin. The possible involvement of neurotensin in the regulation of steroid secretion was studied in vitro using perifused frog adrenal slices. For concentrations ranging from 10-10 to 10-5 M, synthetic frog neurotensin increased corticosterone and aldosterone production in a dose-dependent manner (EC50 = 1.2 x 10-9 M and 5.8 x 10-10 M, respectively). Repeated administration of neurotensin induced a reproducible stimulation of steroid output without any tachyphylaxis. Prolonged administration (3 h) of frog neurotensin caused a transient increase in corticosterone and aldosterone secretion followed by a decline of corticosteroid secretion. Neurotensin also produced a significant stimulation of corticosteroid secretion from dispersed frog adrenal cells. This study demonstrates that neurotensin is located in nerve processes innervating the adrenal gland of amphibians. The results also show that synthetic frog neurotensin exerts a direct stimulatory effect on corticosteroid output. Taken together, these data support the view that neurotensin, released by nerve fibers, may act as a local regulator of corticosteroid secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NEUROTENSIN (NT) is a tridecapeptide that was initially isolated from bovine hypothalami (1) and from bovine intestine (2). The primary structure of NT has been subsequently determined in various vertebrate species (Fig. 1Go). The amino acid sequence of NT is identical in all mammalian species yet studied except the opossum (3) and the guinea pig (4). The sequence of NT is also known in chicken (5), alligator (6), python (7), toad (8), and two species of frog (9, 10). Comparison of these various sequences indicates that the structure of the carboxyterminal hexapeptide has been totally conserved, whereas the sequence of the N-terminal heptapeptide has undergone a number of substitutions (Fig. 1Go). The NT precursor encompasses a hexapeptide called neuromedin N (NMN), which exhibits the same C-terminal sequence as NT (11, 12; Fig. 1Go).



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  		Figure 1. Comparison of the primary structures of neurotensin (NT) from different species and porcine neuromedin N (NMN). —, residue identity; pGlu, pyroglutamyl residue.

 
Biochemical and immunohistochemical studies have shown that NT is widely distributed in the brain, where it likely acts as a neurotransmitter and/or a neuromodulator (13, 14). NT is also abundant in the hypothalamo-pituitary complex (15) and in the gastrointestinal tract, where it is thought to function as a neurohormone (16, 17). Immunoreactive NT has been detected in the adrenal gland of various species including flat snake, rat, guinea pig, rabbit, bovine, and cat (18, 19). The distribution of NT in the adrenal gland of vertebrates exhibits marked species differences. For instance, NT-like immunoreactivity (NT-LI) has been detected in fibers innervating the hamster adrenal medulla (20), in epinephrine-producing cells in guinea pig (21) and rat (22), and in nerve fibers and norepinephrine-producing cells in cat (23) and snake (18) adrenal glands.

Studies aimed at investigating the effect of NT on corticosteroid secretion in mammals have led to contradictory results. In particular, NT was found to exert either an inhibitory (24) or a stimulatory effect (25, 26) on adrenal steroid secretion in rat. The possible role of NT in the regulation of adrenal steroidogenesis has never been investigated in nonmammalian vertebrates. In the present study, we have determined the localization of neurotensin in the adrenal gland of the frog Rana ridibunda. Biochemical characterization of the immunoreactive peptide was performed by combining HPLC analysis with RIA detection. Concurrently, the effect of synthetic frog NT (f NT) on corticosteroid secretion was studied in vitro on perifused frog adrenal slices.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male frogs (Rana ridibunda; body weight 30–40 g) originating from Albania were purchased from a commercial source (Couétard, St. Hilaire de Riez, France). Frogs were housed for at least 8 days in glass tanks with running water, at constant temperature (8 C) with a 12-h dark, 12-h light regimen (lights on from 0600–1800 h). Animal manipulations were performed according to the recommendations of the French Ethical Committee and under the supervision of authorized investigators.

Reagents and test substances
Triaminobenzoic acid ethyl ester (MS 222) and (N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid]) (HEPES), collagenase, protease and porcine NMN (pNMN) were purchased from Sigma (St. Louis, MO). Bio-Gel P-2 (200–400 mesh) was from Bio-Rad Laboratories, Inc. (Richmond, CA). (3-[125I]iodotyrosyl3)NT (2000 Ci/mmol), [1,2,6,7-3H]corticosterone (84 Ci/mmol) and [1,2,6,7-3H]aldosterone (82 Ci/mmol) were from Amersham International (Les Ulis, France). BSA was from Roche Molecular Biochemicals (Mannheim, Germany). The antiserum to bovine NT was raised in rabbit and the antibodies were directed against the conserved C-terminal region of the peptide (8). f NT, frog pituitary adenylate cyclase-activating polypeptide (f PACAP), frog galanin (f GAL), and frog calcitonin gene-related peptides (f CGRP) were synthesized by solid phase methodology as previously described (10). Fluorescein isothiocyanate-conjugated goat antirabbit {gamma}-globulins (GAR-FITC) were obtained from Nordic Immunological Laboratories (Tilburg, The Netherlands).

Immunofluorescence procedure
Frogs were anesthetized by immersion in 0.1% MS 222 for 15 min and perfused through the aortic bulb with 20 ml 0.1 M PBS, pH 7.3. The perfusion was carried on with 50 ml McLean’s fixative solution as previously described (27). The whole kidneys were quickly removed and immersed in the same fixative solution for 2 h. The tissues were rinsed overnight in PBS containing 15% sucrose and then transferred into a 30% sucrose solution for at least 24 h. Kidneys pieces were placed in an embedding medium (O.C.T. Tissue Tek, Reichert Jung, Nussloch, Germany) and frozen at -80 C. Adrenal sections were cut at 6 µm in a cryostat (Frigocut 2700, Leica Corp., Nussloch, Germany) and processed for indirect immunofluorescence as previously described (27). Briefly, tissue sections were incubated overnight at 4 C in a humid atmosphere with an antiserum directed against the C-terminal fragment of NT diluted 1:400 in PBS containing 1% BSA and 0.3% Triton X-100. The sections were rinsed in four baths of PBS and incubated for 90 min at room temperature with GAR-FITC (1:100). Finally, the sections were rinsed in PBS, mounted in PBS-glycerol (1:1) and coverslipped. The preparations were examined on a Leitz Orthoplan microscope equipped with a Vario-Orthomat photographic system (Leitz, Wetzlar, Germany). To study the specificity of the immunoreaction, the following controls were performed : 1) substitution of the NT antiserum with PBS; 2) incubation with nonimmune rabbit serum instead of the NT antiserum, and 3) preincubation of the NT antiserum (diluted 1:400) with f NT, pNMN, f PACAP, f GAL, f CGRP (10-6 M each).

Characterization of NT-like immunoreactivity in frog adrenal extracts
The adrenal glands from 50 animals were quickly dissected and kept frozen. The tissues were boiled in 0.5 M acetic acid (10 ml) for 15 min and then homogenized in a glass Potter homogenizer. The homogenate was centrifuged (4,000 x g; 15 min) and the pellet was used for the measurement of protein concentrations. The supernatant was submitted to partial purification on Sep-Pak C18 cartridges (Waters Associates, Milford, MA) as previously described (28). Bound material was eluted from the cartridges with 70% (vol/vol) acetonitrile/water and evaporated in a Speed-Vac concentrator (Savant Instruments, Hickville, NY). The Sep-Pak-prepurified adrenal extract was redissolved in 0.1% trifluoroacetic acid/water (1.5 ml) and injected directly onto a Vydac 218TP54 C18 reversed-phase HPLC column equilibrated with acetonitrile/water/trifluoroacetic acid (7.0 : 92.9 : 0.1, vol/vol/vol) at a flow rate of 1 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 35% (vol/vol) over 40 min. The fractions were collected every 1 min and NT-LI was determined by RIA. Synthetic f NT and pNMN, used as reference peptides, were chromatographed under the same conditions as the frog tissue extract.

The concentration of NT in the HPLC fractions was measured by RIA using (3-[125I]iodotyrosyl3)NT as a radioligand and the neurotensin antiserum at a dilution of 1:50,000. The IC50 of the assay was 1,000 pg/tube and the minimum detectable amount of peptide was 150 pg/tube.

Perifusion experiments
The effect of f NT on corticosteroid secretion by the frog adrenal gland was studied using a perifusion system technique previously described (29). For each perifusion chamber, adrenal glands from six frogs were dissected, sliced, and preincubated in 5 ml Ringer’s solution (15 mM HEPES, 100 mM NaCl, 2 mM KCl, and 15 mM NaHCO3, 2 mg/ml glucose, and 0.3 mg/ml BSA). The Ringer’s solution was gassed with O2/CO2 (95/5), and the pH was adjusted to 7.4. In some experiments, the effect of f NT was studied on acutely dispersed adrenal cells. For this purpose, adrenal cells were enzymatically dissociated using a 0.5% collagenase-1% protease solution, as previously described (27). The adrenal slices or the isolated cells were then transferred into a perifusion chamber (12 adrenal glands or 750,000 cells per chamber) and layered between several beds of Bio-Gel P-2. The adrenal slices or the dispersed cells were continuously perifused with gassed Ringer’s solution alone or with f NT freshly dissolved in Ringer’s solution, at a constant flow rate (200 µl/min) and temperature (24 C). Fractions of effluent perifusate were collected every 5 min and frozen until assay.

Corticosteroid RIA
Corticosterone and aldosterone concentrations were determined directly, without prior extraction, in 200–300 µl samples of effluent perifusate, as previously described (27). Direct measurements of corticosterone and aldosterone were validated by RIA quantification of corticosteroid after HPLC analysis of the effluent perifusate (30). The detection limits of the assays were 20 pg for corticosterone and 5 pg for aldosterone. For both assays, the intra and the interassay coefficients of reproducibility were 3% and 6%, respectively.

Calculations
Each perifusion pattern was established as the mean profile of corticosteroid production (± SEM) calculated over at least three independent experiments. The levels of corticosterone and aldosterone released were expressed as percentages of the basal values, calculated as the mean of eight samples (40 min), taken just before the infusion of the first pulse of f NT. ANOVA was performed to assess the dose-related stimulation induced by f NT. Paired t test was used after regression analysis for comparison between values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunohistochemical localization of NT in the frog adrenal gland
Immunofluorescence labeling of frog adrenal slices with an antiserum against NT revealed the presence of numerous immunopositive varicose nerve fibers coursing in the adrenal parenchyma (Fig. 2AGo). A few immunoreactive fibers were also observed in the walls of the blood vessels irrigating the adrenal gland (Fig. 2BGo). Preincubation of the NT antiserum with synthetic f NT or pNMN (10-6 M) resulted in complete extinction of the immunoreaction (Fig. 2CGo).In contrast, the immunofluorescence labeling was not affected after preincubation of the NT antiserum with 10-6 M f PACAP, f GAL, and f CGRP (data not shown). No fluorescence was observed when the NT antiserum was replaced with either nonimmune rabbit serum or PBS (data not shown).



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  		Figure 2. A and B, Immunofluorescence photomicrographs of frog adrenal slices labeled with an antiserum directed against the N-terminal portion of porcine neurotensin, showing the presence of varicose nerve fibers coursing in the adrenal parenchyma (A) and a few fibers within the walls of blood vessels (arrow) irrigating the gland (B). C, Control section incubated with NT antiserum preabsorbed with 10-6 M synthetic frog neurotensin. Scale bar, 50 µm.

 
Characterization of neurotensin-like immunoreactivity
The elution profile of a frog adrenal extract from the semipreparative Vydac C18 column is shown in Fig. 3Go. NT-like immunoreactivity eluted as a major peak that exhibited the same retention time as synthetic f NT. In contrast, no immunoreactive material co-eluted with synthetic pNMN.



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  		Figure 3. Reversed-phase HPLC analysis of a frog adrenal gland extract. Quantification of neurotensin in the elution fractions was performed by RIA using frog neurotensin (f NT) as a reference standard. The dashed line shows the concentration of acetonitrile in the eluting solvent. The retention times of synthetic f NT and porcine neuromedin N (pNMN) are indicated by arrows.

 
Effect of neurotensin on corticosteroid secretion
The effects of f NT on corticosteroid secretion by perifused frog adrenal slices are illustrated in Fig. 4Go. A 20-min pulse of f NT induced a transient increase in corticosterone (Fig. 4AGo) and aldosterone (Fig. 4BGo) output. Administration of graded doses of f NT (10-12 to 10-5 M) induced a dose-dependent stimulation of corticosterone and aldosterone [regression analysis, F (1, 30) = 78.39; P < 0.001 and F (1, 38) = 68.48; P < 0.001, respectively] (Fig. 4CGo). Half-maximum stimulation (EC50) of corticosterone and aldosterone was observed at concentrations of 1.2 x 10-9 M and 5.8 x 10-10 M, respectively, and maximum stimulation occurred at a concentration of 10-6 M.



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  		Figure 4. A and B, Effect of three graded concentrations of synthetic frog neurotensin (f NT) on the secretion of corticosterone (A) and aldosterone (B) by perifused frog adrenal slices. After a 120-min equilibration period, f NT was administered for 20 min (arrows), and the tissue was allowed to stabilize for another 90-min period before the next pulse of f NT was applied. The profiles represent the mean (± SEM) secretion pattern of three independent perifusion experiments. Each point is the mean corticosteroid production (expressed as a percentage of spontaneous steroid output) of eight consecutive fractions (40 min; open symbols) just preceding the infusion of f NT. C, Semilogarithmic plot comparing the effect of graded concentrations of f NT on corticosterone (•) and aldosterone ({blacksquare}) secretion. Experimental values were calculated from data similar to those shown in A and B. Each point represents the maximum amplitude of stimulation of corticosteroid secretion induced by f NT (peak height) compared with the mean corticosteroid levels observed just before the infusion of each dose of secretagogue (100% basal level). The mean basal levels of corticosterone and aldosterone secretion in these experiments were 0.73 + 0.1 and 1.24 + 0.36 ng/fraction, respectively.

 
Infusion of three equimolar doses of f NT (10-6 M; 20 min) induced a reproducible stimulation of corticosterone (Fig. 5AGo) and aldosterone (Fig. 5BGo) secretion without apparent tachyphylaxis. Figure 6Go shows the kinetics of the response of adrenal glands during a 3-h administration of f NT (10-6 M). The perifusion medium containing the neuropeptide was renewed every 20 min to minimize degradation of the diluted peptide. Prolonged exposure to f NT induced a transient stimulation of corticosterone (Fig. 6AGo) and aldosterone (Fig. 6BGo) secretion which reached a maximum within 40 min. Thereafter, despite continued perifusion with f NT, corticosteroid release declined, and two rebounds of the secretory activity were observed at approximately 60-min intervals.



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  		Figure 5. Effect of three equimolar concentrations of synthetic frog neurotensin (f NT ; 10-6 M; 20 min each) on the secretion of corticosterone (A) and aldosterone (B) by perifused frog adrenal slices. The pulses of f NT (arrows) were administered at 90-min intervals. The mean basal levels of corticosterone and aldosterone secretion in these experiments were 0.97 ± 0.23 and 0.50 ± 0.01 ng/fraction, respectively. See legend to Fig. 4Go for other designations.

 


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  		Figure 6. Effect of prolonged infusion of synthetic frog neurotensin (f NT ; 10-6 M; 3 h) on the secretion of corticosterone (A) and aldosterone (B) by perifused frog adrenal slices. The f NT solution was administered for 3 h (arrows) and the solution was renewed every 20 min (arrowheads) to minimize degradation of the peptide in the perifusion medium. The mean basal levels of corticosterone and aldosterone secretion in these experiments were 1.45 ± 0.23 and 0.32 ± 0.04 ng/fraction, respectively. See legend to Fig. 4Go for other designations.

 
The ability of f NT to stimulate steroid secretion from enzymatically dispersed adrenal cells was also tested (Fig. 7Go). Administration of f NT (10-6 M) for 20 min elicited a rapid and transient stimulation of corticosterone (Fig. 7AGo) and aldosterone (Fig. 7BGo) release. Application of a second dose of f NT (10-6 M), after a resting period of 120 min, induced a similar stimulation of steroid secretion without any desensitization phenomenon.



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  		Figure 7. Effect of two equimolar concentrations of synthetic frog neurotensin (f NT ; 10-6 M) on the secretion of corticosterone (A) and aldosterone (B) by perifused dispersed frog adrenal cells. The pulses of f NT were administered for 20 min (arrows) at 120-min interval. The mean basal levels of corticosterone and aldosterone in these experiments were 660 ± 48 and 6.36 ± 0.72 pg/fraction. See legend to Fig. 4Go for other designations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study has demonstrated the occurrence of a network of NT-containing fibers in the adrenal gland of the frog Rana ridibunda. This study has also shown that NT stimulates steroid secretion by dispersed frog adrenocortical cells.

Previous studies have shown that the frog adrenal gland is richly innervated by both extrinsic and intrinsic neurons (31). Extrinsic fibers originate from multiple types of nerves including the vagus nerve, the splanchnic nerve, sensitive nerves from dorsal root ganglia, and nerve branches associated with the walls of blood vessels. Intrinsic fibers mainly originate from subcapsular plexuses (32). Two types of NT-immunoreactive fibers were observed in the frog adrenal tissue, i.e. varicose fibers running between adrenal cells and a few fibers coursing along the walls of blood vessels. Several other regulatory peptides have previously been identified in fibers innervating the frog adrenal parenchyma : atrial natriuretic factor (33), tachykinins (32), PACAP (34), GAL (29), and CGRP (27). Most of these neuropeptides are generally found in fibers running between adrenal cells and along the walls of blood vessels irrigating the gland (27, 32, 34). The origin of these fibers, including NT-containing fibers is currently unknown. The occurrence of NT has previously been detected in the adrenal gland of various vertebrates. Depending on the species, NT is contained either in nerve fibers only (20), in chromaffin cells only (21, 22) or in both nerve fibers and chromaffin cells (18, 23).

The antiserum used in the present study was directed against the C-terminal region of NT (8). As expected, preincubation of the NT antiserum with pNMN, a peptide that possesses the same C-terminal sequence as NT (Fig. 1Go), completely abolished the immunofluorescence labeling. To determine whether the immunostained fibers innervating the frog adrenal gland contained NT, NMN, or both, we have characterized the immunoreactive peptide(s) by reversed phase HPLC analysis combined with RIA detection. The observation that a major immunoreactive peak coeluted with synthetic f NT indicates that the immunolabeled nerve fibers contain a mature form of NT.

Previous studies have shown that, in rat, NT can regulate the activity of the hypothalamo-pituitary-adrenal axis at different levels. In the paraventricular nucleus, NT stimulates the release of CRH (35, 36). At the pituitary level, NT triggers ACTH secretion (37, 38, 39). In the adrenal medulla, which contains a local CRH/ACTH system (40), NT can stimulate the release of both peptides (41). The occurrence of NT-immunoreactive fibers in the frog adrenal gland led us to examine the possible effect of the neuropeptide on corticosteroid secretion. The results presented herein show that synthetic f NT stimulates corticosterone and aldosterone output by perifused frog adrenal fragments in a dose-dependent manner.

In contrast to the mammalian adrenal gland, which is organized in cortical and medullary zones, the adrenal gland of amphibians is composed of adrenocortical cells tightly intermingled with chromaffin cells (42, 43). This peculiar anatomical organization favors paracrine communication between the two categories of cells (44). In fact, there is now clear evidence that frog chromaffin cells contain various biogenic amines (45, 46) and neuropeptides (47, 48, 49) that can modulate the secretion of corticosteroids. It was thus conceivable that f NT could stimulate the activity of adrenocortical cells indirectly, via an action on the release of regulatory factors from chromaffin cells. To determine whether NT acted directly on adrenocortical cells to enhance steroid secretion, we investigated the action of the peptide on enzymatically dispersed adrenal cells, a preparation in which the connections between the cells are disrupted. The fact that NT could stimulate corticosteroid secretion from isolated adrenal cells indicated that the effect of the peptide can be ascribed to a direct action on adrenocortical cells. However, nothing is known concerning the type of receptor mediating the effect of NT on corticosteroid production.

In mammals, studies designed to demonstrate a direct effect of NT on steroidogenesis have led to contradictory results. In vivo experiments have shown opposite effects of NT, either stimulatory in rabbit (50) or inhibitory in rat (51) on adrenal steroid secretion. Studies using in situ perfused rat adrenal glands indicate that NT causes a moderate increase in both corticosterone and aldosterone output (25, 26). In contrast, NT exerts an inhibitory effect on basal corticosterone secretion from dispersed fasciculata-reticularis cells (24) and on angiotensin II- or K+-stimulated aldosterone production from dispersed glomerulosa cells (52). Whether these divergent results can be ascribed to the different methodological approaches used, or actually reflect authentic species-specific responses, remains to be established.

The perifusion model provides valuable information regarding the kinetics of the response of the glands to secretagogues. Using this technique, we observed that prolonged administration of f NT (during 3 h) causes a rapid increase in steroid production which peaked at 30 min, followed by a rapid decline toward the baseline value. The decay of the response cannot be ascribed to degradation of f NT because the solution of the peptide was renewed every 20 min to avoid peptide damage. These data indicate that NT induced rapid desensitization of its own receptors. The occurrence of two rebounds during the sustained infusion of f NT suggests that the receptors are first internalized and then recycled at the cell surface. This desensitization phenomenon appears to be reversible as administration of repeated pulses of f NT at 90-min intervals induced a reproducible stimulation of corticosteroid secretion. A similar desensitization process has been described for several other corticotropic peptides including CGRP (27), ranakinin (32), and endothelins (53).

In conclusion, our results indicate that the adrenal gland of the frog Rana ridibunda is innervated by a network of neurotensinergic fibers. The immunoreactive peptide exhibits exactly the same retention time as synthetic f NT during HPLC analysis. Synthetic f NT directly stimulates in vitro corticosteroid secretion by perifused frog adrenal glands. These data support the view that endogenous NT, released by nerve endings in the vicinity of adrenocortical cells, can act locally as a modulator of corticosteroid secretion (54).


   Acknowledgments
 
We thank Dr. A. Fournier (INRS-Institut Armand Frappier, Montréal, Canada) for the generous gift of synthetic f PACAP, f galanin and f CGRP and Dr. D. Duterte-Boucher (CNRS UPRES-A 6036, Rouen, France) for valuable advice on statistical analysis.


   Footnotes
 
1 This work was supported by grants from INSERM (U-413), the National Science Foundation (IBN-980 6997), and the Conseil Régional de Haute-Normandie. Back

Received December 10, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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