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Endocrinology Vol. 139, No. 10 4140-4146
Copyright © 1998 by The Endocrine Society

ARTICLES

Isolation, Primary Structure, and Effects on {alpha}-Melanocyte-Stimulating Hormone Release of Frog Neurotensin1

Laurence Desrues, Marie-Christine Tonon, Jerome Leprince, Hubert Vaudry and J. Michael Conlon

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, University of Rouen (L.D., M.-C.T., J.L., H.V.), 76821 Mont-Saint-Aignan, France; and Regulatory Peptide Center, Department of Biomedical Sciences, Creighton University School of Medicine (J.M.C.), Omaha, Nebraska 68178-0405

Address all correspondence and requests for reprints to: Dr. J. M. Conlon, Department of Biomedical Sciences, Creighton University Medical School, Omaha, Nebraska 68178-0405.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neurotensin (NT) was isolated in pure form from the small intestine of the European green frog, Rana ridibunda, and its primary structure was established as pGlu-Ala-His-Ile-Ser-Lys-Ala-Arg-Arg-Pro-Tyr-Ile-Leu. This sequence contains five amino acid substitutions (Leu2->Ala, Tyr3->His, Glu4->Ile, Asn5->Ser, and Pro7->Ala) compared with human NT. A peptide with identical chromatographic properties was identified in an extract of frog brain. Synthetic frog NT produced a concentration-dependent increase in {alpha}MSH release from perifused frog pars intermedia cells, with an ED50 of 5 x 10-9 M. A maximum response (276.3 ± 45.5% above basal release) was produced by a 10-8-M concentration. Repeated administration of NT to melanotrope cells revealed the occurrence of a rapid and pronounced desensitization mechanism. The data are consistent with a possible role for the peptide as a hypophysiotropic factor in amphibians.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NEUROTENSIN (NT) is a tridecapeptide that was first isolated from an extract of bovine hypothalami (1) and subsequently, in an identical molecular form, from bovine intestine (2). Studies using antisera raised against bovine NT in immunohistochemistry and RIA have shown that NT, or a structurally related antigen, is present in nervous and/or intestinal tissue from all classes of vertebrate studied, including Agnatha (lampreys and hagfish), as well as in a wide range of invertebrate species (3). In nonmammalian species, much higher concentrations of NT-like immunoreactivity (NT-LI) were measured using antisera directed against the C-terminal region of NT compared with N-terminally directed antisera, suggesting that the C-terminal region of the molecule has been more strongly conserved during evolution (3). Our knowledge of the structures, localization, and biological properties of nonmammalian NTs is fragmentary. The amino acid sequence of NT is known only for the chicken (4), alligator (5), python (6), and one species of frog, Rana temporaria (7). The distribution of the peptide in the central nervous system has been studied in detail for the quail, Coturnix coturnix japonica (8); the lizard, Gallotia galloti (9); two species of amphibian, the tree frog, Hyla meridionalis (9), and the Northern leopard frog, Rana pipiens (10); and two species of teleost fish, the goldfish, Carassius auratus (9), and the green molly, Poecilia latipinna (11).

In the tree frog, a number of nuclei containing NT-immunoreactive fibers projecting to the median eminence were identified, particularly in the preoptic region and the ventral hypothalamic nucleus (9). Such an innervation has suggested that NT may exert hypophysiotropic functions in this amphibian. NT-immunoreactive cells were also identified in the anterior lobe of the pituitary, although the nature of these cells, characterized in rats as gonadotrophs and thyrotrophs (12), is not known. The presence of NT-immunoreactive fibers and cells in the optic tectum of both H. meridionalis (9) and R. pipiens (10) has suggested that NT may be involved in the processing of visual information. At this time, the biological effects of NT on pituitary function in amphibia are unknown; therefore, the aim of the present study was to purify and characterize NT from the European green frog, Rana ridibunda, and to study its effects on the release of {alpha}MSH from the melanotrope cells of the pars intermedia of the same species.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue extraction
Adult specimens of the frog R. ridibunda were obtained from a commercial source (Couétard, St. Hilaire de Riez, France). Animal manipulations were performed according to the recommendations of the French ethical committee and under the supervision of authorized investigators. Small intestines were collected from 428 animals, and whole brains were from 267 animals; the tissues were immediately frozen on dry ice. Frozen intestinal tissue (220 g wet weight) was homogenized with acetone-1 M HCl (97:3, vol/vol; 4 ml/g tissue) using a Waring blender (Fisher Scientific, Pittsburgh, PA). After centrifugation (1600 x g, 30 min, 4 C), the precipitate was rehomogenized with acetone-0.01 M HCl (80:20, vol/vol; 2 ml/g tissue). After a further centrifugation (1600 x g, 30 min, 4 C), acetone was removed from the supernatant under reduced pressure. The frozen brain tissue (24.9 g wet weight) was homogenized in ethanol-0.7 M HCl (3:1, vol/vol; 10 ml/g tissue) using a Waring blender. After centrifugation (1600 x g, 30 min, 4 C), ethanol was removed from the supernatant under reduced pressure. Peptide material was isolated from the supernatants of the intestinal and brain extracts by passage through 8 Sep-Pak C18 cartridges (Waters Associates, Milford, MA) as previously described (6). Bound material was eluted from the cartridges with 70% (vol/vol) acetonitrile-water and lyophilized.

NT RIA
NT-LI was measured using antiserum 2/7 directed against a site in the C-terminal to central region of pig NT as previously described (5). The antiserum cross-reacts with chicken NT, but shows negligible reactivity with xenopsin and neuromedin N.

Gel permeation chromatography
The intestinal extract, after partial purification on Sep-Pak cartridges, was redissolved in 1 M acetic acid (5 ml) and chromatographed on a 2.5 x 100-cm column of Sephadex G-25 (Pharmacia Biotech, Uppsala, Sweden) equilibrated with 1 M acetic acid at a flow rate of 48 ml/h. Absorbance was measured at 280 nm, and fractions (8 ml) were collected. NT-LI in the fractions was measured by RIA at appropriate dilution.

Purification of frog NT by HPLC
The fractions containing NT-LI from gel permeation chromatography of the intestinal extract were pooled and pumped at a flow rate of 2 ml/min onto a 1 x 25-cm Vydac 218TP510 C18 reverse phase HPLC column (Separations Group, Hesperia, CA) equilibrated with 0.1% (vol/vol) trifluoroacetic acid-water. The concentration of acetonitrile in the eluting solvent was raised to 21% over 10 min, maintained at this concentration for 30 min, and raised to 49% over 60 min using linear gradients. Absorbance was measured at 214 and 280 nm, and fractions (1 min) were collected. Frog NT was purified to apparent homogeneity by successive chromatographies on a 0.46 x 25-cm Vydac 214TP54 C4 column, a 0.46 x 25-cm Vydac 219TP54 phenyl column, and a 0.46 x 25-cm Vydac 218TP54 C18 column, using the elution conditions summarized in Fig. 2Go.



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  		Figure 2. Purification of frog NT on analytical Vydac C4 (A), Vydac phenyl (B), and Vydac C18 (C) reverse phase columns. The columns were eluted at a flow rate of 1.5 ml/min, with linear gradients of increasing acetonitrile concentration, denoted by the dashed lines. The fractions containing NT-LI are denoted by NT. The upward-pointing arrows show where peak collection began and ended. In C, the downward-pointing arrow shows the retention time of synthetic frog NT.

 
The lyophilized extract of frog brain, after partial purification on Sep-Pak cartridges, was redissolved in 0.1% trifluoroacetic acid-water (4 ml) and injected directly onto a 1 x 25-cm Vydac 218TP510 C18 reverse phase HPLC column equilibrated with 0.1% trifluoroacetic acid-water. The column was eluted under the same conditions as those used for the chromatography of the intestinal extract (Fig. 1Go), and fractions (1 min) were collected.



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  		Figure 1. Reverse phase HPLC on a semipreparative Vydac C18 column of an extract of frog intestine after partial purification by gel permeation chromatography. The fractions denoted by the bar contained NT-LI. The dashed line shows the concentration of acetonitrile in the eluting solvent.

 
Peptide synthesis
Frog NT was synthesized by the solid phase methodology on a 0.025-mmol scale using an Applied Biosystems model 432 synthesizer (St. Quentin en Yvelines, France). Fluorenylmethoxycarbonyl (F-moc)-labeled amino acids were coupled as their hydroxybenzotriazole active esters following the manufacturer’s standard protocols. The peptide was cleaved from the resin using trifluoroacetic acid-water-ethanedithiol-thioanisol (900:30:30:40, vol/vol/vol/vol) and purified to homogeneity by reverse phase HPLC. The identity of the peptide was verified by analysis of the amino acid composition and automated Edman degradation.

Structural analysis
Amino acid compositions were determined by precolumn derivatization with phenylisothiocyanate using an Applied Biosystems model 420A derivatizer and model 130A separation system. The detection limit for phenylthiocarbamyl amino acids was 1 pmol. Hydrolysis (24 h at 110 C in 5.7 M HCl) of approximately 500 pmol sample was performed. Automated Edman degradation was performed using an Applied Biosystems model 471A Sequenator modified for on-line detection of phenylthiohydantoin amino acids under gradient elution conditions. The detection limit was 1 pmol.

The purified frog NT (~1 nmol) was incubated for 18 h at 4 C with 1 µg pyroglutamyl aminopeptidase (Calbiochem, Torrance, CA) in 0.1 M sodium phosphate buffer, pH 8.0, containing 5% (vol/vol) glycerol, 10 mM EDTA, and 5 mM dithiothreitol (total volume, 100 µl). The reaction mixture was chromatographed on a 0.46 x 25-cm Vydac 218TP54 C18 using the same elution conditions as those shown in Fig. 2Go.

Dissociation of frog melanotrope cells
Adult male frogs were decapitated, and the neurointermediate lobes were collected in a frog Ringer’s solution consisting of 112 mM NaCl, 2 mM KCl, 2 mM CaCl2, 15 mM HEPES, 2 g/liter glucose, and 0.3 g/liter BSA (pH 7.4). The neurointermediate lobes were rinsed in calcium-free Ringer’s medium and gently agitated for 30 min at 22 C in a dissociation solution composed of 2 mg/ml collagenase type IA (Sigma Chemical Co., St. Louis, MO) freshly dissolved in calcium-free Ringer’s solution. After removal of the neural lobes by sedimentation, dispersed intermediate lobe cells were centrifuged (50 x g, 22 C, 3 min), rinsed three times with Ringer’s medium containing 1% kanamycin (Sigma Chemical Co.) and 1% of an antibiotic-antimycotic solution (Bioproducts, Gagny, France), pH 7.4, and then transferred into the perifusion chambers.

Perifusion of dispersed melanotrope cells
The perifusion system used in this study has been previously described in detail (13). Briefly, dispersed melanotrope cells (equivalent to 12 intermediate lobes/chamber) were mixed with Bio-Gel-P2 beads (200–400 mesh; Bio-Rad Laboratories, Richmond, CA) and layered in perifusion chambers. The cells were continuously supplied either with Ringer’s solution alone or with synthetic frog NT (10-9-10-6 M) freshly dissolved in Ringer’s solution at a constant flow rate (0.3 ml/min) and temperature (24 C). After a 2-h stabilization period, the perifusion effluent from each column was collected as 7.5-min fractions during the stabilization periods and as 1- or 2.5-min fractions during administration of NT. The fractions collected were immediately chilled on ice until {alpha}MSH RIA.

{alpha}MSH RIA
The {alpha}MSH concentration was measured in each fraction on the same day as the perifusion experiment using a double antibody RIA procedure (14). The perifusion profiles were expressed as percentages of the basal secretory level. The basal values were calculated as the mean of four consecutive fractions (7.5-min each) collected just before the administration of synthetic NT. Each figure represents the mean profile of {alpha}MSH release (±SEM) established from at least four independent experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Purification of frog NT
The NT-LI in the extract of frog small intestine, after partial purification on Sep-Pak cartridges, was eluted from a Sephadex G-25 column as a single peak with the same elution volume as synthetic human NT (Kav = 0.65–0.85). The fractions containing immunoreactive material were pooled and chromatographed on a semipreparative C18 HPLC column, as shown in Fig. 1Go. The NT-LI was eluted in the single fraction, denoted by the bar in Fig. 1Go. After rechromatography on an analytical Vydac C4 column (Fig. 2AGo), NT-LI was associated with the sharp peak, delineated by the arrows. Frog NT was purified to apparent homogeneity, as assessed by a symmetrical peak shape, by chromatography on analytical Vydac phenyl (Fig. 2BGo) and C18 (Fig. 2CGo) columns. The final yield of pure peptide was 3 nmol.

As shown in Fig. 3Go, the NT-LI in an extract of whole frog brain, after partial purification on Sep-Pak cartridges, was eluted from a semipreparative Vydac C18 as a single peak whose retention time was the same as the NT-LI in the intestinal extract and the same as that of synthetic frog NT.



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  		Figure 3. A comparison of the retention times on a semipreparative Vydac C18 column of the NT-LI in extracts of frog brain (A) and intestine (B). The column was eluted under the same conditions as those described in Fig. 1Go. The arrow shows the retention time of synthetic frog NT.

 
Structural characterization
Attempts to determine the amino acid sequence of frog NT by automated Edman degradation were unsuccessful, indicating that the peptide did not contain a free {alpha}-amino group. However, sequence analysis was accomplished after removal of the N-terminal pyroglutamyl residue by incubation with pyroglutamyl aminopeptidase. The amino acid sequence of frog des-pGlu1 NT was established as: Ala(109)-His(48)-Ile(55)-Ser(11)-Lys(33)-Ala(25)-Arg(23)-Arg(28)-Pro(34)-Tyr(27)-Ile(19)-Leu(9). The values in parentheses show the yields of amino acid phenylthiohydantoins in picomoles. The proposed structure of frog NT, including the presence of an N-terminal pyroglutamic acid residue, was confirmed by determination of its amino acid composition [found: Glx 0.9 (1), His 1.0 (1), Arg 2.2 (2), Ala 2.2 (2), Pro 1.1 (1), Tyr 0.8 (1), Ile 1.9 (2), Leu 1.2 (1), Lys 1.0 (1) mol residue/mol peptide]. The values in parentheses show the numbers of residues predicted from the proposed structure. The retention time of synthetic frog NT on an analytical Vydac C18 column, under the conditions of chromatography shown in Fig. 2CGo, was identical to that of the endogenous peptide.

Effect of NT on {alpha}MSH release
Pilot experiments revealed that repeated pulses of NT to perifused frog neurointermediate lobes caused stimulation of {alpha}MSH release, with marked attenuation of the response of the tissue to the peptide. To avoid the bias of tachyphylaxis, a single pulse of NT was administered to each perifusion chamber. Exposure of dispersed frog melanotrope cells to a 15-min pulse of NT caused a concentration-related stimulation of {alpha}MSH release (Fig. 4Go). The maximum increase in {alpha}MSH secretion (+276.3 ± 45.5%) occurred at a concentration of NT of 10-8 M, and the apparent half-maximum response was observed at a dose of approximately 5 x 10-9 M. At higher concentrations (3.16 x 10-8 and 10-7 M), the effect of NT on {alpha}MSH release gradually declined. For each dose of NT tested, the maximum stimulatory effect was observed within 3 min after the onset of peptide administration. Thereafter, despite continued NT infusion, {alpha}MSH secretion gradually decreased, and some rebound was consistently observed 8–13 min after the initial transient peak. Administration of three consecutive pulses of NT (10-8 M each) at 60-min intervals led to a substantial reduction of the amplitude of the response (Fig. 5Go). In contrast, administration of TRH (10-8 M) after two pulses of NT (10-8 M) induced a much stronger response than the third pulse of NT (10-8 M; inset to Fig. 5Go). The increase in total {alpha}MSH release over basal after TRH was 307 ± 36% compared with an increase of 154 ± 8% after NT (P < 0.01).



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  		Figure 4. Effects of graded doses of NT on {alpha}MSH secretion by dispersed frog melanotrope cells. Perifused cells were exposed for 15 min to NT (10-9-10-7 M) as shown by the arrows. The profiles represent the mean secretion pattern (±SEM) of at least four independent perfusion experiments. The spontaneous level of {alpha}MSH release (100% basal level) was calculated as the mean hormone concentration in the four consecutive fractions (30 min) collected before the onset of NT administration ({circ}). The mean basal secretion rate of {alpha}MSH in these experiments was 46.2 ± 7.3 pg/min·450,000 cells.

 


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  		Figure 5. Effects of three consecutive pulses of NT (10-8 M) on {alpha}MSH secretion from perifused frog melanotrope cells. Perifused cells were exposed for 15 min to NT at 60-min intervals. The profile represents the mean secretion pattern (±SEM) of four independent perifusion experiments. The spontaneous level of {alpha}MSH release (100% basal level) was calculated as the mean hormone concentration in the four consecutive fractions (30 min) collected before the onset of the first pulse of NT ({circ}). Inset, Comparison of the effects of a third pulse of NT or TRH, administered after two consecutive pulses of NT, on {alpha}MSH secretion from perifused frog melanotrope cells. Perifused cells were exposed twice for 15 min to NT (10-8 M). The cells were subsequently exposed for 15 min to NT or TRH (10-8 M each). The effects of NT and TRH were calculated as the total amounts of {alpha}MSH released during a 12.5-min period starting after the onset of NT and TRH infusion (solid bars) and compared with the amounts of {alpha}MSH released during the 12.5 min just preceding NT and TRH administration (open bars). Values are the mean ± SEM of three independent perifusion experiments. The mean basal secretion rate of {alpha}MSH in these experiments was 79.5 ± 14.8 pg/min·450,000 cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The primary structure of NT isolated from the extract of Rana ridibunda intestine is compared with that of NTs already characterized in representative species from various vertebrate taxa in Fig. 6Go. The amino acid sequence of NT has been more strongly conserved among the reptiles than among amphibia. The sequence is identical in chicken (4), alligator (5), and python (6). In contrast, NT from the European grass frog R. temporaria (7) differs from the peptide isolated from R. ridibunda by the substitution (Ser2->Ala) and from the peptide isolated from the cane toad Bufo marinus by two additional substitutions (His3->Ile and Ile4->Val; Warner, F., E. Burcher, and J. M. Conlon, unpublished data). The sequence of frog NT contains five amino acid substitutions compared with that of human NT (15). All of these substitutions are located in the N-terminal region of the molecule, whereas the amino acid sequence of the C-terminal domain is identical in all vertebrate species investigated to date. Interestingly, structure-activity relationships have shown that the biologically active domain of the NT molecule is located in the C-terminal 8–13 moiety of the peptide (3, 16, 17) whose sequence has been fully preserved from frog to man. This observation indicates that strong evolutionary pressure has acted to conserve the sequence of the active determinant of NT. Although the primary structure of NT from frog brain was not established, the fact that the NT-LI detected after reverse phase HPLC of the brain extract had the same retention time strongly suggests that the sequences of NT from brain and intestine are identical.



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  		Figure 6. A comparison of the primary structures of NT from species of different vertebrate taxa. -, Residue identity; <E, pyroglutamyl residue.

 
Nucleotide sequence analysis of cloned complementary DNAs encoding prepro-NT from mammals has shown that the NT sequence is flanked at its N-terminus by a Lys-Arg processing site and the sequence of neuromedin N (18). Our attempts to detect by RIA neuromedin N-like immunoreactivity in the extracts of R. ridibunda intestine and brain using antisera raised against either neuromedin N or LANT-6 [the homolog of neuromedin N isolated from the chicken (19)] were unsuccessful (our unpublished data). This suggests that the neuromedin N sequence is absent from the amphibian prepro-NT or that the amino acid sequence of the peptide is sufficiently different from those of the mammalian and chicken analogs as to be unrecognizable by the available antisera.

NT can modulate pituitary hormone secretion via an indirect effect on hypothalamic neurons (20). In particular, NT inhibits PRL and {alpha}MSH secretion through a stimulatory action on tuberoinfundibular dopaminergic neurons (21). In contrast, NT has been shown to exert a direct stimulatory action on PRL release from cultured rat anterior pituitary cells (22, 23). However, despite the striking similarities between the neuroendocrine mechanisms regulating PRL and {alpha}MSH secretion (24), a possible direct effect of NT on melanotrope cell activity has never been investigated. The present study has demonstrated that synthetic frog NT is a potent stimulator of {alpha}MSH secretion from frog pars intermedia cells. As a matter of fact, frog NT exhibited the same potency as TRH, a well recognized {alpha}MSH-releasing hormone in amphibia (13, 25, 26, 27).

The source of the NT that may regulate melanotrope cell activity in frog is currently unknown. It has been observed previously that the median eminence of the frog H. meridionalis is innervated by a dense network of NT-positive fibers (9). The occurrence of a few NT-immunoreactive cells has also been found in the pars distalis of H. meridionalis (9). It is thus conceivable that NT, transported by portal blood vessels and/or secreted locally within the pars distalis, may diffuse toward the pars intermedia. Alternatively, NT produced by peripheral organs, notably by the gastrointestinal tract, may be transported by the blood and thus may act as a hormone stimulating melanotrope cell activity.

The perifusion technique provides valuable information concerning the kinetics of the response of endocrine cells. The transient effect evoked by a 15-min administration of NT and the attenuation of the response observed after repeated administration of NT pulses are indicative of a rapid and sustained tachyphylaxis. This desensitization mechanism probably accounts for the weakened responses induced by high concentrations of NT. In any event, the attenuation of the secretory activity cannot be ascribed to exhaustion of the releasable pool of {alpha}MSH, inasmuch as other stimulatory factors, including TRH and acetylcholine, do not induce similar tachyphylaxis (27, 28). Consistent with these findings, we found that after two consecutive administrations of NT, a pulse of TRH induces a much higher response than a third pulse of NT.


   Acknowledgments
 
The authors thank Dr. Robert Carraway, University of Massachusetts (Worcester, MA), for valuable advice regarding tissue extraction.


   Footnotes
 
1 This work was supported by the NSF, INSERM, and the Conseil Régional de Haute-Normandie. Back

Received February 10, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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J H Li, F Sicard, M A Salam, M Baek, J LePrince, H Vaudry, K Kim, H B Kwon, and J Y Seong
Molecular cloning and functional characterization of a type-I neurotensin receptor (NTR) and a novel NTR from the bullfrog brain
J. Mol. Endocrinol., June 1, 2005; 34(3): 793 - 807.
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A. Belmeguenai, L. Desrues, J. Leprince, H. Vaudry, M.-C. Tonon, and E. Louiset
Neurotensin Stimulates Both Calcium Mobilization from Inositol Trisphosphate-Sensitive Intracellular Stores and Calcium Influx through Membrane Channels in Frog Pituitary Melanotrophs
Endocrinology, December 1, 2003; 144(12): 5556 - 5567.
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F. Sicard, H. Vaudry, B. Braun, N. Chartrel, J. Leprince, J. M. Conlon, and C. Delarue
Immunohistochemical Localization, Biochemical Characterization, and Biological Activity of Neurotensin in the Frog Adrenal Gland
Endocrinology, July 1, 2000; 141(7): 2450 - 2457.
[Abstract] [Full Text] [PDF]

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