Characterization of porcine gastric galanin

Peptides,Vol. 13, pp. 589-593, 1992

0196-9781/92 $5.00 + .00 Copyright© 1992PergamonPress Ltd.

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Characterization of Porcine Gastric Galanin T. J. M c D O N A L D , .1 A. KRANTIS,~ M. CLARKE,~ V. M U T T § A N D H. E. J ( ) R N V A L L ¶

*Departments of Medicine and Biochemistry and the Robarts Research Institute, London, Ontario, Canada, i'Department of Physiology, the University of Ottawa, Ottawa, Canada, ¢Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada, and Departments of§Biochemistry H and ~]ChemistryL Karolinska Institute, Stockholm, Sweden Received 24 December 1991 McDONALD, T. J., A. KRANTIS, M. CLARKE, V. MUTT AND H. E. JORNVALL. Characterization of porcine gastric galanin. PEPTIDES 13(3) 589-593, 1992.--The presence of a peptide capable of producing powerful contractions of rat small intestinal smooth muscle was detected in chromatographic fractions derived from porcine gastric corpus extracts. The pharmacological characteristics of this entity suggested that it might be galanin and on its purification to homogeneity, amino acid composition and sequence analysis demonstrated the identity of the gastric and intestinal forms of galanin. The presence of galanin in the gastric corpus tissue and its ability to affect gastric smooth muscle activity, gastrin release, and gastric acid secretion suggest potential important physiological roles for galanin in the stomach.

Galanin

Gastricacid secretion

HPLC

Neuropeptide

Gastrin

METHOD

THE gastric corpus is a functionally and anatomically distinct region of the GI tract, the unique nature of which is partially defined by the acid-secreting parietal cells present within its mucosa. Immunohistochemical studies have identified endocrinelike cells in the upper gastric mucosa possessing granules typical of those containing peptides but which do not stain with antibodies raised against known regulatory peptides (15). Antibodies to neuron-specific enolase, considered specific for neuronal structures and APUD cells, stain typical endocrine-like cells and neural structures in this part of the GI tract; certain of these structures do not stain with antibodies against known regulatory peptides (l 2). These studies suggest the presence of previously unrecognized bioactive peptides in this region. Similar considerations provided the stimulus for the initiation of studies on porcine nonantral gastric tissue extracts, which resulted in the isolation and characterization (9) of the bombesin-related gastrinreleasing polypeptide (GRP). During recent studies, we identified the presence of a factor in chromatographic fractions derived from porcine gastric corpus extracts, which potently stimulated contraction of rodent intestinal smooth muscle and which possessed characteristics resembling those ofgalanin, a peptide first isolated from upper small intestinal tissue extracts utilizing a chemical assay that detects the presence of the C-terminal alpha amide structure in peptides (l 7). Galanin possesses potent biological activities and is widely distributed in neural structures in mammalian central, peripheral, and enteral nervous systems (4,13). This report demonstrates an efficient isolation procedure for and the chemical characterization of porcine gastric galanin.

Initial Purification One thousand kg of freshly obtained porcine gastric corpus tissue was immediately heat-inactivated followed by extraction into dilute acetic acid and processed to yield 81.5 g ofa peptide concentrate by methods previously described in detail (10). This peptide concentrate was extracted with methanol as described (10) to yield a methanol insoluble fraction weighing 22.9 g, which was then applied to a carboxymethylcellulose column (8 × 18 cm) equilibrated with a 0.0225 M phosphate buffer, pH 6.4. On passage of this fraction through the column, the protein not retained was collected, and on desalting~ yielded 3.96 g of protein. This fraction was then chromatographed on a Sephadex G-25 (0 column (10 × 120 cm), equilibrated, and eluted with 0.2 M acetic acid. The peptide elution pattern was followed by measuring UV absorbance at 280 nm; fractions were pooled arbitrarily depending upon the absorbancy pattern and the peptides recovered by lyophilization. The peptide contained in the fraction eluting between 5460 and 5750 ml, designated fraction V, weighed 318 nag and contained an entity which produced potent stimulation of rodent smooth muscle contraction. A portion of this active Sephadex fraction (159 rag) was further purified on a preparative reverse-phase HPLC utilizing a Waters Delta-prep 3000 apparatus (Millipore, Toronto, Canada), equipped with a Model 481 variable wavelength detector and a Delta-prep C-18 column (19 × 300 ram). The solvent systems used were: A) 0.1% (v/v) trifluoroacetic acid (TFA) in water (Sequenal grade, Pierce Chemical Company, IL) and B) 0.12% (v/v) TFA in acetonitrile

Requests for reprints should be addressed to Dr. T. J. McDonald, Room 5-L2, P.O. Box 5339, University Hospital, London, Ontario, Canada N6A 5A5.

589

590 (HPLC Grade, Fischer Chemical Company, Toronto, Canada). The chromatogram was developed using a linear gradient between 18 to 80% solvent B over 135 rain. The flow rate was 15 ml/min and the protein elution pattern was monitored by UV absorption at 230 nm. The active fraction was recovered in a small peak at a retention time of 34 min. The bulk of the acetonitrile and TFA in the active fraction was removed via vacuum centrifugation (Heto, Emerston, Toronto, Ontario) and the peptides (690 #g) in this fraction were recovered by lyophilization.

Final Purification Procedures Semipreparative HPLC was performed using a Waters Associates apparatus consisting of two M6000A pumps, a 660 solvent programmer, a U6K injector, and a model 450 variable wavelength detector. Protein absorbancy patterns were monitored at 215 nm. Ion-exchange HPLC was performed on a TSKCM column (7.5 X 75 mm) (Millipore). The solvent systems employed were: C) 0.0225 M phosphate buffer, pH 6.4, and D) the same buffer containing 0.5 M sodium chloride. A portion (528 #g) of the active fraction recovered from preparative reversephase HPLC was dissolved in solvent C and applied to the TSK CM column; a linear gradient from 0 to 100% solvent D was developed over l h followed by isocratic elution at 100% D for 90 min. Ion-exchange HPLC eluates containing activity were pooled and frozen until performance of reverse-phase HPLC, when they were thawed and applied directly to reverse-phase columns. Final purification was performed by reverse-phase HPLC employing a 10 micron C-18 #BondaPak (3.9 × 300 ram) column (Millipore). The solvent systems employed were those described for the preparative HPLC system; the flow rate was l ml/min. The active ion-exchange eluate fraction was applied directly to the reverse-phase column and a linear gradient of 27 to 33% solvent B was developed over l h. The reversephase HPLC eluate fractions were subjected to vacuum centrifugation to remove the bulk of the organic phase and the peptides contained in each fraction were recovered by lyophilization.

Bioassays Fractions were tested for bioactivity on isolated rat small intestinal segments in gut-bath preparations. Segments (20 mm in length) of the proximal duodenum, proximal jejunum, and the proximal region of the distal ileum were obtained from Sprague-Dawley rats (male, 250-350 g) and positioned in Krebs buffer (37°C, pH 7.4) filled baths for recording mechanical activity in the longitudinal axis as described (6). Individual segments were placed under a resting tension of 1.5 g (maintained throughout the experiment) and allowed to equilibrate in the organ bath for 60 rain before drug treatment. To ensure that each tissue had sufficient tone for observation of relaxation and contraction responses, 5 X 10-8 M carbaehol (stimulates smooth muscle to contract) and 5 X 10-3 M o f t h e smooth muscle relaxant papaverine were applied. Only tissues displaying responses (i.e., contractions and relaxation to these agents) were subsequently tested with the chromatographic fractions and other drugs. Concentration-response data were derived using a noncumulative protocol, with a minimum of 10 rain between drug challenges. The lowest concentration was administered first and left in the bath until the maximal response had been achieved. In experiments where antagonist drugs were present in the bathing solution, the intestinal segments were allowed to equilibrate for a minimum of 15 rain or until the basal tone had

McDONALD ET AL. recovered to within 90% of resting level, before continuing the drug challenges.

Structural Analysis The amino acid composition of the purified gastric peptide was determined on a Beckman 121 M analyzer after hydrolysis at 110°C for 24 h in evacuated tubes containing 6 M HCI with 0.5% phenol. The purified gastric peptide (1.8 nmol) was degraded in an Applied Biosystems model 470A gas-phase sequencer and phenylthiohydantoin derivatives of the amino acids were analyzed by HPLC on a Nucleosil C-18 column with an acetonitrile gradient in sodium acetate as described (18). RESULTS

Isolation The purification of the gastric peptide was followed by its ability to produce dose-dependent contractions of rat small intestinal smooth muscle in organ-bath preparations. Figure 1 demonstrates the purification of the active preparative reversephase HPLC fraction on ion-exchange HPLC. The bioactivity was found to reside in a dominant symmetrical peak occurring at a retention time of 93 rain. Final purification was achieved by directly applying the active ion-exchange fraction to a C- 18 reverse-phase column (Fig. 2). The activity was found in a dominant symmetrical peak at retention time 27 rain; minimal impurities were removed by this final procedure. The final yield of purified peptide was 135 #g.

Structural Analysis Table 1 contains the amino acid analysis of the gastric galanin preparation compared with that of the known composition of intestinal galanin. The molar ratios were close to unity, in"du:ating a high degree of homogeneity of the preparation, and the amino acid composition of gastric galanin was identical to that of intestinal galanin, Sequence analysis of 1.8 nmol of the purified gastric peptide yielded the results shown in Table 2. All 29 residues, including the C-terminal alanine residue, were unequivocally identified. Although the presence or absence of an alpha amide structure at the C-terminal residue was not directly determined, a mixture of the gastric galanin preparation with a synthetic replicate of the intestinal peptide elutes as a single

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HG. 1. Purification of the preparative reverse-phase chromatographic active fraction on ion-exchange HPLC (see text for details). The activity was contained in the peak marked by the arrow.

STRUCTURE OF GASTRIC GALANIN

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TABLE 2 SEQUENCE ANALYSIS OF GASTRIC GALANIN

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FIG. 2. Final reverse-phase HPLC of the active ion-exchange HPLC fraction demonstrated in Fig. 1 (see text for details). The activity was contained solely within the dominant peak marked by the arrow.

symmetrical peak on high resolution reverse-phase HPLC (data not shown), providing strong evidence that the gastric peptide also contains the C-terminal alpha alanine amide structure.

Pharmacological Characteristics In untreated organ-bath preparations of rat duodenum taken 2-6 cm from the pylorus (n = 6), jejunum taken 12-16 cm from the pylorus (n = 20), and ileum taken 6-10 cm from the ileo-

Cycle

Residue

pmol Recovered

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His

408 262 140 410 297 38 273 185 224 230 301 171 162 59

15 16

Ala lie

196 137

17 18 19 20 21 22 23 24 25 26 27 28 29

Asp Asn His Arg Set Phe His Asp Lys Tyr Gly Leu Ala

75 112 38 40 8 56 24 29 83 38 33 20 16

TABLE 1 AMINO ACID ANALYSIS OF GASTRIC GALANIN: COMPARISON WITH INTESTINAL GALANIN

Residue Trp Lys His Arg Asx Thr Ser Glx Pro Giy Ala Val Met Ile Leu Tyr Phe Total

GastricGalanin 0.6 (1) 1.2 (1) 3.1 (3) 1.2 (1) 4.1 (4) 1.0 (1) 2.0 (2) 0.1 (0) 1.1 (1) 4.1 (4) 3.0 (3) 0 (0) 0 (0) 0.9 (I) 4.2 (4) 2.0 (2) 1.0 (1) (29)

IntestinalGalanin 1 1 3 1 4 1 2 0 1 4 3 0 0 1 4 2 1 29

The first values listed for gastric galanin indicate molar ratios of acid hydrolysates (values below 0.1 omitted), the values in parentheses are the sum from sequence analysis. The amino acid composition listed for intestinal galanin is taken from (17).

caecal junction (n = 20), the primary action of the gastric peptide was to induce concentration-dependent contractions (Fig. 3a,b). In all tissues tested, the responses were maximal within 20 s and readily reversible on washing the tissues with fresh Krebs solution. When compared with submaximal concentrations (5 × l0 -8 M) of the muscarinic agonist carbachol tested in the same segment, the amplitude of the response to the applied gastric peptide was 71 + 7% of that to the applied carbachol (Fig. 3a). The largest contractions induced by gastric peptide were obtained in jejunal segments; the duodenal segments were the least responsive. Contractions of the jejunal segments to applied gastric peptide were not prevented by treatment with tetrodotoxin (3 × l0 -7 M , n = 4), the muscarinic antagonist atropine 0 0 -7 M, n = 5), the serotonin (5-HT) receptor blocker methysergide (10 -6 M, n = 4) (Fig. 3a), or the E-receptor antagonist propranolol (3 × l0 -6 M, n = 3). The presence of the gastric peptide did not affect CCK-8-induced contractions and vice versa. These results are consistent with the gastric peptide having a direct effect upon rodent small intestinal muscularis via sites unrelated to functional receptors for acetylcholine, 5-HT, noradrenaline, or CCK-8. DISCUSSION

This study reports the isolation ofgalanin from porcine gastric corpus tissue extracts and demonstrates the identity of the gastric peptide with its intestinal counterpart. The presence of galanin

592

McDONALD ET AL.

0-5g

I

I

i

CARB

I p

5HT

I

I

I P

methy~Kglde r

I. I

I P

FIG. 3. (a) The effect of methysergide (10-6 M) on evoked contractions of the rat proximal jejunum. Serotonin (5-HT) (3 × 10-s M) and 100 ~1 of the active final reverse-phase eluate (Fig. 2) containing the gastric peptide (P) were tested before and 18 min after the appfication of methysergide (connected arrows). The contraction response to 5-HT was abolished by methysergide but the response to the gastric pepfide (100 t~l)was unaffected. The contraction response to 5 × 10-s M carbachol (CARB) can be used to compare the gastric peptide-evoked response. The duration of contact for the individual drugs is shown by the horizontal bars. The vertical bar corresponds to deflection generated by a force of 0.5 g. Washout artefacts are sometimes evident. (b) The effect of CCK-8 on the gastric peptide (P)-indueed contractions of the rat ileum. CCK-8 (5 × 10-9 M) Was al~lied and left in the bath (connected arrows), followed 3 rains later by another application of CCK-8 (5 × 10-9). In the presence of CCK-8 (final concentration 10-8 M), 100/tl of the active reverse-phase eluate containing the gastric peptide was re,applied. The gastric peptide-induced response was not blocked.

in the gastric corpus is not surprising, as galanin-like immunoreactivity in both the gastric antrum and corpus have been demonstrated in different species (1,3). Immunohistochemical studies have demonstrated the presence ofgalanin-posilive nerve cell bodies in the myenteric plexus of the gastric corpus of rat, mouse, and dog (3,5,11,13), but such nerve cell bodies were not readily demonstrable in porcine or guinea pig gastric myenteric plexuses (11). In all species studied, substantial numbers of galanin-positive nerve fibers are seen but the specific anatomical distribution varies somewhat between species. In all species, galanin-positive nerve fibers ramify among the intrinsic neurons of the gastric myenteric plexus. In rat, galam'n-posilive nerve fibers are more profuse in the submucosa and mucosa than in other species (13). In most species studied, ~ t ~ , i n ~ v e nerve fibers are relatively more abundant in the circular muscle layer (3,5,11,13) than in the longitudinal smooth muscle, the pig being an exception in that galanin-positive nerve fibers are relatively more abundant in the longitudinal smooth muscle layer (11). In the dog, galanin-posilive nerve fibers are particularly abundant in antral and pyloric circular smooth muscle; nerve ceil bodies are also particularly abundant in the myenteric plexus of this region (5). These anatomical locations are consistent with the

involvement of galanin neurons in the control of motility and secretory activity. In the dog, galanin directly inhibits ongoing circulatory smooth muscle contractions in both the antrum and corpus (5), while in other species, particularly rodents, galanin acts to stimulate GI smooth muscle contractility. In the present study, the pharmacology of the isolated gastric peplide in gut-i~th preparations of rat intestine was consistent with that of planin. In humans, galanin infusions significantly delay ~ c emptying and prolong intestinal transmit lime (2). In the isolat~ perfused rat stomach preparation, galanin inhibits basal and GRP(1827)-stimulated gastrin release by a t e t r o d o t o x i n - ~ n t mechanism (i.e., suggesting a nonneurai-mediated mechanism of action) without affecting somatostatin release (7,8). In an anes. thelized rat preparation, galanin did not afect ~ or bethanachol-slimulated acid secretion but potently inhibiled pentagastrin-stimulated acid secretion (14). Similarly, in conscious dogs, ~,,l=min potently inhibits bomb(~in- and 2 deoxyglmmsestimulated acid secretion without affecting basal, ~ n e or bethanachol-stimulated acid secretion (16). Hence, the occurrence of galanin, with a structure identical to its intestinal counterpart, in neural structures innervating the

S T R U C T U R E OF G A S T R I C G A L A N I N

593

smooth muscle, mucosa, and myenteric plexus neurons of the gastric corpus and antrum suggests that galanin may play important physiological roles in the stomach. In the corpus and antrum, it may play a role as a modulator of neuronal function in the myenteric plexus and may alter gastric motility to regulate gastric emptying. In the antrum, galanin may modulate the release of gastrin, whereas in the corpus, galanin may modulate the effect of certain gastric acid secretagogues.

ACKNOWLEDGEMENTS These studies were supported by operating grants from the Medical Research Council of Canada (T. J. McDonald, M. Clarke, and A. Krantis) and from the Swedish Medical Research Council (V. Mutt and H. E. JOrnvall). Excellent technical assistance by Ms. D. Feist and Nikos Vagelopoulos and excellent secretarial assistance by Ms. G. Kellett are gratefully acknowledged by the authors.

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