INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

GASTRIN-RELEASING PEPTIDE (GRP), first isolated from an extract of porcine nonantral gastric tissue (16), belongs to a family of structurally related peptides whose members include bombesin from the skin of the frog Bombina bombina (1), alytensin from the skin of Alytes obstetricans (1), and neuromedin B (19) and neuromedin C (NMC) (20) from porcine spinal cord. NMC represents the COOH-terminal decapeptide of GRP, and structure-activity studies have shown that this peptide possesses the full biological potency and efficacy of GRP (14, 24). These peptides share the common sequence Trp-Ala-Xaa-Gly-His-Leu-Met.NH₂ at their COOH termini. GRP was originally regarded as the mammalian ortholog of bombesin, but subsequent work demonstrated that the stomach and brain of Bombina express a gene encoding a peptide structurally similar to GRP as well as the gene encoding bombesin (21). Similarly, both GRP and NMC were isolated from an extract of the brain of the frog Rana ridibunda, whereas bombesin was absent (4).

In mammals, GRP is localized exclusively to nerves distributed throughout the gastrointestinal tract, but immunohistochemical studies have demonstrated that in gastric tissues of the frogs, Rana pipiens, Rana catesbeiana, and Xenopus laevis, GRP is also produced in endocrine-like cells in the antral and fundic mucosa (12). Roles for GRP in the regulation of a wide range of mammalian physiological processes have been proposed. These include gastric motility, antral cell proliferation, feeding behavior, neuroendocrine secretion, pancreatic exocrine secretion, and thermoregulation (reviewed in Ref. 2). However, the biological actions of an amphibian GRP in an amphibian have not been investigated. This study involves the purification of two molecular forms of GRP and NMC from an extract of the intestine of the African clawed frog Xenopus laevis (Anura: Pipidae) and an investigation of the effects of Xenopus GRP on the motility of longitudinal and circular smooth muscle from the cardiac stomach of the animal.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Radioimmunoassay. GRP-like immunoreactivity (GRP-LI) was measured by radioimmunoassay using antiserum R354, raised against porcine GRP, as previously described (26). The antiserum shows full molar cross-reactivity with bombesin and NMC.

Tissue extraction. All experimental procedures were approved by University of Göteborg Ethical Committee. Adult specimens of X. laevis of both sexes (n = 60; body wt 60-110 g) were supplied by Horst Kähler, Bedarf für Forschung und Lehre, Hamburg, Germany. Animals were anesthetized by immersion in 0.1% 3-aminobenzoic acid ethyl ester in water buffered with NaHCO₃ to pH 7.4. The intestine and stomach were removed by dissection immediately after death and stored at 55°C until time of extraction. Tissue (92 g) was homogenized with ethanol-0.7 M HCl (3:1 by vol; 10 ml/g) using a Waring blender and stirred for 2 h at 0°C. After centrifugation (1,600 g, 30 min, 4°C), ethanol was removed from the supernatant under reduced pressure. After a further centrifugation (1,600 g, 30 min, 4°C), the extract was pumped at a flow rate of 2 ml/min through six Sep-Pak C₁₈ cartridges (Waters Associates, Milford, MA) connected in series. Bound material was eluted with acetonitrile-water-trifluoroacetic acid (70.0:29.9:0.1 by vol) and freeze-dried.

Purification of Xenopus GRP. The intestinal extract, after partial purification on Sep-Pak cartridges, was redissolved in 1 M acetic acid (5 ml) and chromatographed on a 100 × 2.5-cm column of Sephadex G-25 (Pharmacia, 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. GRP-LI in the fractions was measured at a dilution of 1:30. The fractions containing GRP-LI from gel permeation chromatography (denoted by the bar designated GRP in Fig. 1) were pooled and pumped at a flow rate of 2 ml/min onto a 25 × 1-cm Vydac 218TP510 (C₁₈) reverse-phase HPLC column (Separations Group, Hesperia, CA) equilibrated with 0.1% trifluoroacetic acid-water. The concentration of acetonitrile in the eluting solvent was increased to 21% over 10 min, followed by an increase to 49% over 60 min using linear gradients. Absorbance was measured at 214 and 280 nm, and fractions (1 min) were collected. The fraction containing GRP-LI (denoted by the bar in Fig. 2A) was rechromatographed on a 25 × 0.46-cm Vydac 214TP54 (C₄) column equilibrated with acetonitrile-water-trifluoroacetic acid (21.0:78.9:0.1 by vol) at a flow rate of 1.5 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 35% over 40 min using a linear gradient. Xenopus GRP-1 and GRP-2 were separately purified to apparent homogeneity by successive chromatographies at a flow rate of 1.5 ml/min on 25 × 0.46-cm Vydac 219TP54 phenyl and 25 × 0.46-cm Vydac 218TP54 (C₁₈) columns using the same elution conditions for the C₄ column.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Gel permeation chromatography on a Sephadex G-25 column of an extract of Xenopus intestine after partial purification on Sep-Pak cartridges. The fractions denoted by gastrin-releasing peptide (GRP) contain GRP-1 and GRP-2 and the fractions denoted by NMC contain neuromedin C. ABS280, absorbance at 280 nm.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Reverse-phase HPLC on a semi-preparative Vydac 218TP510 (C18) column of fractions from gel permeation chromatography containing GRP (A) and fractions from gel permeation chromatography containing NMC (B). The fractions (1 min) containing GRP-like immunoreactivity, denoted by the bars, were pooled for further purification. The dashed line shows the concentration of acetonitrile in the eluting solvent.

Purification of Xenopus NMC. The later-eluting fractions containing GRP-LI from gel permeation chromatography (denoted by the bar designated NMC in Fig. 1) were pooled and pumped at a flow rate of 2 ml/min onto a 25 × 1-cm Vydac 218TP510 (C₁₈) HPLC column equilibrated with 0.1% trifluoroacetic acid-water. The concentration of acetonitrile in the eluting solvent was increased to 35% over 60 min using linear gradients. Absorbance was measured at 214 and 280 nm, and fractions (1 min) were collected. The fraction containing GRP-LI (denoted by the bar in Fig. 2B) was rechromatographed on Vydac C₄, Vydac phenyl, and Vydac C₁₈ columns under the same conditions used for purification of Xenopus GRP.

Structural characterization. The primary structures of the peptides were determined by automated Edman degradation using a Perkin Elmer model 491A sequenator. Electrospray mass spectrometry was carried out using a Perkin Elmer Sciex API 150EX single quadrupole instrument. The accuracy of mass determinations was ±0.05%.

Peptide synthesis. Xenopus GRP-1 (APTSQQHTEQ¹⁰ LSRSNINTRG²⁰ SHWA VGHLM.NH₂) was synthesized on a 4-(2',4'-dimethoxy-phenyl-Fmoc-aminomethyl) phenoxyacetamidoethyl resin (Perkin Elmer, Foster City, CA) (0.025- mmol scale) using an Applied Biosystems peptide synthesizer (model 432A). Fmoc amino acid derivatives were activated with O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.075 mmol), 1-hydroxybenzotriazole hydrate (0.075 mmol), and diisopropylethylamine (0.150 mmol) as previously described (22). The peptide was cleaved from the resin with trifluoroacetic acid-water-thioanisole-1,2-ethanedithiol (99.0:0.50:0.25:0.25 by vol) at 25°C for 3 h.

Myotropic activity. Male and female specimens (55-110 g) of X. laevis were killed as previously described. The stomach was surgically removed and opened by cutting along the lesser curvature from the opening at the pylorus to the cardiac stomach. The cardiac stomach was dissected into strips (2.5 × 7 mm) oriented in the direction of either the longitudinal or circular smooth muscle. Each strip was mounted between the fittings of a Somedic Sales AB (Farsta, Sweden) differential pressure transducer by cotton strings tied at each end. The mounted strips were kept in glass organ baths containing 5 ml of modified McKenzie's toad Ringer solution (composition in g/l: 6.22 NaCl, 1.68 NaHCO₃, 1.30 HEPES, 0.24 KCl, 0.35 MgSO₄ · 7H₂O, 0.19 CaCl₂, 3.0 glucose). The McKenzie's solution was maintained at 20°C and aerated with 0.03% CO₂ in air to give a pH between 7.8 and 8.0. A Grass Model 7 Polygraph (Quincy, MA) was used to measure force. The strips were placed under an initial force of 10 mN, washed every 30 min with McKenzie's solution, and allowed to equilibrate at least 1 h.

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Statistical analysis. The data were analyzed by measuring the change in average force generated over a 3-min period at each concentration of GRP for each individual tissue preparation. Wilcoxon's matched-pairs signed-rank test was used to analyze the effects of drugs on the responses to Xenopus GRP-1 and differences between the responses to porcine GRP and Xenopus GRP-1. P values <0.05 were considered to indicate a significant difference.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Purification of Xenopus GRP. The GRP-LI in the extract of Xenopus intestine, after partial purification on Sep-Pak C₁₈ cartridges, was eluted from a Sephadex G-25 gel permeation column in two zones that were subsequently shown to contain GRP and NMC (Fig. 1). The fractions denoted by the GRP bar in Fig. 1 were pooled and chromatographed on a semipreparative Vydac C₁₈ column (Fig. 2A). GRP-LI was eluted in the single fraction denoted by the bar. Rechromatography of this material on an analytic Vydac C₄ column resulted in the separation of the GRP-LI into two discrete fractions associated with the well-defined peaks designated 1 and 2 (Fig. 3A). Peak 1 (containing GRP-1) and peak 2 (containing GRP-2) were each separately rechromatographed on an analytic Vydac phenyl column (Fig. 3, B and C). Both peptides were purified to near homogeneity, as assessed by a symmetrical peak shape and mass spectrometry, by a final chromatography on an analytic Vydac C₁₈ column. The final yields of the pure peptides were GRP-1 500 pmol and GRP-2 150 pmol.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Purification by reverse-phase HPLC of Xenopus GRP on an analytic Vydac 214TP54 (C4) column (A) and analytic Vydac 219TP54 (phenyl) column (B and C). Individual peaks were collected by hand, and the arrows indicate where peak collection began and ended. Peak 1 contains GRP-1 and peak 2 contains GRP-2.

Purification of Xenopus NMC. The later-eluting fractions from the Sephadex G-25 column containing GRP-LI (Fig. 1) were pooled and chromatographed on a semipreparative Vydac C₁₈ column (Fig. 2B). GRP-LI was eluted in the fractions denoted by the bar that were subsequently shown to contain NMC. The peptide was purified to near homogeneity by successive chromatographies on analytic Vydac C₄ and phenyl columns (chromatograms not shown), and the final yield of pure peptide was ~400 pmol.

Peptide characterization. The primary structures of Xenopus GRP-1 and GRP-2 were established by automated Edman degradation, and their amino acid sequences are shown in Fig. 4. The peptides are identical except for a single amino acid substitution at position 15 (AsnThr). The primary structure of Xenopus NMC was established as Gly-Ser-His-Trp-Ala-Val-Gly-His-Leu-Met, which corresponds to the amino acid sequence of the common COOH-terminal decapeptide of GRP-1 and GRP-2. The presence of a COOH terminally -amidated residue in the peptides was demonstrated by mass spectrometry [GRP-1 observed relative molecular mass (M_r) = 3,243.8 Da, calculated M_r = 3,243.6 Da; GRP-2 observed M_r = 3,230.5 Da, calculated M_r 3,230.6 Da; and NMC observed M_r = 1,092.8 Da, calculated M_r = 1,092.8 Da]. The calculated masses refer to the COOH terminally -amidated forms of the peptides.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   A comparison of the amino acid sequences of Xenopus GRP-1 and GRP-2 with the corresponding sequences of GRP-related peptides from species of other vertebrate taxa. , Residue identity. Residue deletions denoted by * are inserted into some sequences to maximize structural similarity, but the positions of these deletions are arbitrary.

Myotropic activity. Both Xenopus GRP-1 and porcine GRP produced a dose-dependent increase in the tension of longitudinal smooth muscle strips from the cardiac stomach of X. laevis (Fig. 5). The peptides produced an increase in the frequency and intensity of spontaneous rhythmic contractions in the strips so that the data points in Fig. 5 refer to the average force generated over a 3-min recording period. The potencies of both peptides were not significantly different (pD₂ = 8.93 ± 0.32 for Xenopus GRP-1 and pD₂ = 8.23 ± 0.32 for porcine GRP; n = 6). Similarly, the maximum increases in average tension induced by porcine GRP and Xenopus GRP-1 were not significantly different (29.2 ± 7.7 and 20.9 ± 6.8% of the response to 60 mM KCl, respectively; n = 6).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effects of increasing concentrations of synthetic Xenopus GRP-1 (xGRP) and porcine GRP (pGRP) on the contraction of isolated longitudinal smooth muscle strips from Xenopus cardiac stomach. Data are expressed as % of the maximum response produced by 60 mM KCl in the same strip (means ± SE; n = 6).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Effects of increasing concentrations of synthetic xGRP and pGRP on the relaxation of precontracted isolated circular smooth muscle strips from Xenopus cardiac stomach. Data points show means ± SE; n = 8. The 100% value represents baseline force before addition of carbachol (5 µM).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Effects of indomethacin (10 µM) on the ability of Xenopus GRP-1 to contract isolated longitudinal smooth muscle strips from Xenopus cardiac stomach. Data are expressed as % of the maximum response produced by the peptide (means ± SE; n = 6).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The African clawed frog Xenopus laevis is believed to have arisen following a tetraploidization event occurring in an ancestral species ~30 million years ago (8) and, as a result, expresses two nonallelic copies of many genes that encode neuroendocrine peptides. Examples of genes in X. laevis that have been identified in two copies include insulin, growth hormone, corticotropin-releasing hormone, proenkephalin A, cholecystokinin, proopiomelanocortin, and pancreatic polypeptide (reviewed in Ref. 11). Although gene duplication may lead to the evolution of a new gene having a new function, an alternative outcome following polyploidization is that one copy of the gene will eventually accumulate sufficient deleterious mutations that it is no longer expressed, i.e., becomes a pseudogene (29). The isolation of two forms of GRP from the intestine of X. laevis that differ only by a single amino acid substitution (Asn¹⁵Thr) demonstrates that both genes are expressed and encode functional peptides. The extent of the difference in sequence between the two isoforms of Xenopus regulatory peptides encoded by the duplicated genes is quite variable. For example, the two isoforms of corticotropin-releasing hormone are identical, neuropeptide Y and pancreatic polypeptide, similar to GRP, differ by one residue, whereas one of the isoforms of peptide tyrosine-tyrosine appears to have undergone an accelerated rate of mutation with the result that the two isoforms differ by six residues (11). There have been few studies comparing the biological activities of the two isoforms of the Xenopus peptides, but it has been reported that Xenopus prolactin-II is more potent than prolactin-I in suppressing the thyroid hormone-induced resorption of the larval tail, suggesting that the isoforms may be evolving toward specialized functions (31).

In Fig. 4, the amino acid sequences of Xenopus GRP-1 and GRP-2 are compared with the corresponding sequences of GRP from pig (16), human (27), chicken (15), the alligator Alligator mississipiensis (30), the amphibian Bombina orientalis (21), the rainbow trout Oncorhynchus mykiss (Teleostei) (10), and the spotted dogfish Scyliorhinus canicula (Elasmobranchii) (3). The data show that evolutionary pressure to conserve the structure of GRP has been very selective. The amino acid sequence at the NH₂-terminal region of the peptide has not been conserved at all during the evolution of vertebrates, whereas the COOH-terminal domain has been fully conserved except for the single conservative substitution (SerAsn) at the site corresponding to position 2 in NMC. The single arginine residue that constitutes the site of proteolytic cleavage in the generation of NMC has also been fully conserved. Our findings that myotropic activities of Xenopus GRP-1 and porcine GRP are indistinguishable demonstrate that evolutionary pressure has acted to conserve only the functionally important domain in GRP. Xenopus GRP-2 differs from Xenopus GRP-1 by a single amino acid substitution in a region of the peptide that is outside this domain, and so it is highly probable that Xenopus GRP-2 is equipotent with GRP-1. Consistent with previous work with R. ridibunda brain (4), bombesin was not identified in the extract of Xenopus gut, providing further support for the assertion that the bombesin gene is expressed only in tissue of frogs of the genus Bombina as a result of a localized gene duplication event within that lineage (21).

The inability of tetrodotoxin (a sodium channel blocker), atropine (a muscarinic receptor antagonist), and methysergide (an antagonist of 5-hydroxytryptamine) to modify the response to Xenopus longitudinal gastric smooth muscle to Xenopus GRP-1 suggests that the peptide interacts directly with receptors on the smooth muscle to produce contraction. In contrast, the significant attenuation of the response of the circular smooth muscle by indomethacin, an inhibitor of cyclooxygenase, indicates that the ability of GRP to relax this preparation is mediated, at least in part, by synthesis of prostanoids.

The dual contractile and relaxant effects of Xenopus GRP-1 on Xenopus gastric smooth muscle are appreciably different from the effects of porcine GRP (and/or bombesin) on gastrointestinal smooth muscle preparations from different species of mammal. Porcine GRP produced concentration-dependent contractions of both circular and longitudinal muscle strips isolated from different regions of human stomach and longitudinally cut strips from human duodenum (23). Circularly cut human duodenal strips showed no response to GRP (23). Longitudinal muscle strips from the human ileocecal region showed an increase in rhythmic activity in response to GRP, whereas circular muscle strips were unresponsive or showed a small decrease in tone (28). GRP and bombesin stimulated the spontaneously occurring contractions of both longitudinal and circular muscle from the antrum and corpus of the canine stomach (13, 14). The bombesin-stimulated contractions of longitudinal muscle involve the mediation of neuronally released acetylcholine, whereas the effects on contractions of circular muscle are myogenic. Circular smooth muscle strips from cat esophagus, fundus, and duodenum and longitudinal strips from the duodenum responded to NMC with tetrodotoxin- and atropine-resistant contractions (18). Direct contractile effects of GRP and bombesin have been demonstrated using freshly dispersed muscle cells from the guinea pig stomach (25) and from the longitudinal and circular muscle layers of human jejunum (17). In the latter case, cells from the circular and longitudinal muscle layers showed identical contractile responses.

Perspectives

The demonstration that Xenopus GRP shows potent effects on Xenopus gastric motility in vitro warrants further studies in vivo to demonstrate unequivocally that the peptide has a physiologically important role in the regulation of gastric function in this species. In particular, it is proposed to study the effects of intra-arterial infusions of synthetic Xenopus GRP on gastric motility and gastrointestinal blood flow in anesthetized Xenopus specimens using the same methodology used to measure these parameters in fish (9). Similarly, further insight in the physiological role of GRP in an amphibian will be gained by studying the effects of Xenopus GRP on the release of gastrin and the secretion of gastric acid in the in situ isolated perfused Xenopus stomach (6).