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Valosin Stimulates Gastric and Exocrine Pancreatic Secretion and Inhibits Fasting Small Intestinal Myoelectric Activity in the Dog
GASTROENTEROLOGY 1987;92:1181-6
Valosin Stimulates Gastric and Exocrine Pancreatic Secretion and Inhibits Fasting Small Intestinal Myoelectric Activity in the Dog STANISLAW J. KONTUREK, WOLFGANG E. SCHMIDT, VIKTOR MUTT, JAN W. KONTUREK, and WERNER CREUTZFELDT Institute of Physiology, Medical Academy, University of Krakow, Krakow, Poland; Division of Gastroenterology and Endocrinology, Department of Medicine, University of G6ttingen, G6ttingen, Federal Republic of Germany; and Department of Biochemistry II, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden
Valosin, a novel 25-amino acid gastrointestinal peptide with N-terminal valine and C-terminal tyrosine, has recently been isolated from porcine upper gut extracts. Its physiologic role is unknown and it does not belong to one of the structurally related gut peptide families. Assuming that valosin may influence gastrointestinal functions, we investigated the effect of high-performance liquid chromafographypure valosin on gastric and exocrine pancreatic secretion and on the intestinal myoelectric activity in conscious dogs. Intravenous injection of valosin (0.125-1 J-Lg/kg) dose-dependently increased gastric acid secretion 80-fold over basal, corresponding to 18% of the maximal pentagastrin-induced effect. Pepsin output increased 10-fold over basal (30% of the pentagastrin-stimulated secretion). Half-maximal stimulation by pentagastrin could be further increased dose-dependently ~y simultaneous administration of valosin. Pancreatic bicarbonate secretion was stimulated ll-fold over basal at 1.0 J-Lg/kg, reaching about 6% of the secretin-induced maximal output, whereas protein secretion increased 12-fold over basal, corresponding to about 55% of the cholecystokinin-induced maximal output. In fasted dogs, spontaneously occurring migrating myoelectric complexes were substantially delayed during infusion of valosin at a dose of 0.2 J-Lg/kg. These experiments indicate that valosin may Received July 10, 1986. Accepted December 8, 1986. Address requests for reprints to: Prof. Dr. Werner Creutzfeldt, Division of Gastroenterology and Endocrinology, Department of Medicine, Georg-August-University of G6ttingen, Robert-Koch Strasse 40, D-3400 G6ttingen, Federal Republic of Germany. © 1987 by the American Gastroenterological Association 0016-5085/87/$3.50
represent a novel member of the regulatory gastrointestinal peptides. During recent years, an increasing number of novel gastrointestinal peptides have been purified from extracts of upper porcine intestine using as isolation criterion either structural characteristics like the C-terminal amide group (1) or a biological activity defined by a bioassay system. Recently, we have developed a screening strategy for the characterization of unknown peptide fractions depending on high-performance liquid chromatography and Nterminal sequence determination (2,3). Using this method, a novel peptide comprising 25 amino acid residues from a porcine upper gut extract has been isolated and the complete primary structure has been elucidated (2). As this peptide, which was designated "valosin" from the N-terminal residue valine and the C-terminal residue tyrosine, does not show convincing sequence homology to any peptide sequence reported so far, whether determined directly or deduced from the corresponding nucleotide sequence, it may represent a member of an as yet unidentified family of gastrointestinal peptides. Preliminary biological experiments on dogs revealing that the peptide exhibits several activities within the gastrointestinal tract have been reported in abstract form (4), where valosin was provisionally denoted as "peptide VQY." This study deals with the effect of valosin on gastric and exocrine pancreatic secretion and intestinal myoelectric activity in conscious dogs when administered intravenously. Abbreviations used in this paper: CCK, cholecystokinin; MMC, migrating motor complex.
1182
KONTUREK ET AL.
Materials and Methods Preparation of Valosin Valosin was pur~fieQ from upper porcine intestine as described recently (2). The pure peptide eluting as a single peak from a reverse-phase C-18 high-performance liquid chromatography-column (Vydac RP-201 TPB, 10 f.Lm, 300 A wide pore) in a trifluoroacetic acid/acetonitrile solvent system was used for the biological studies. Lyophilized peptide was dissolved in physiologic saline containing 1 gldl canine serum albumin immediately before the experiment and administered intravenously as a bolus or as an infusion as described below.
Secretory Studies Secretory studies were carried out on 4 conscious dogs equipped with gastric fistulas (5) and Herrera-type pancreatic fistulas (6) as described previously (7). The studies were performed after an 18-h fasting period. Gastric and pancreatic secretions were collected at 15-min intervals. The volume of each sample was measured. The output of HCl into the gastric juice was determined by titration of the samples to pH 7.0 by means of an autotitrator (Radiometer, Copenhagen, Denmark). Pepsin activity was measured by the method of Anson (8). Bicarbonate and protein in the pancreatic juice were determined as described (7). Several series of tests were performed in each animal. Basal secretions of gastric and pancreatic juices were collected for 60 min. Then various doses of valosin were injected intravenously as a single bolus; no more than two doses were given on anyone test day. After each injection of valosin, the secretions were collected for 90 min until the secretory rate completely returned to the basal level. For comparison, the maximal gastric acid output and the maximal pancreatic bicarbonate and protein secretion were determined in response to a 90-min intravenous infusion of a constant dose of pentagastrin (8 f.Lglkg . h) (9), secretin (4 U/kg . h) (10), or cholecystokinin (CCK-33) (4 Ulkg. h) (11), respectively. In tests with stimulated gastric secretion, pentagastrin (4 f.Lglkg . h) was infused at a constant rate to induce near maximal gastric acid output. When the secretion reilched il sustained plateau, usually after 60 min, valosin was added to the intravenous infusion as a single bolus injection at a dose qf 0.125, 0.25, or 0.5 f.Lg/kg, no more than two doses being administered on anyone test day. In tests with stimulated pancreatic secretion, secretin (2 U/kg· h) or CCK-33 (2 U/kg. h) was infused at a constant rate to induce near maximal pancreatic bicarbonate or protein secretion, respectively. When the stimulated secretion reached a constant plateau, valosirt was added to the infusion as an intravenous injection at a dose of 0.125, 0.25, or 0.5 f.Lglkg. Pancreatic juice was collected for at least 90 min after each dose of valosin; no more than two doses were given on anyone test day. Blood samples were taken from a peripheral vein twice during the period of basal secretion and at 15-30-min intervals after injection of valosin for the determination of
GASTROENTEROLOGY Vol. 92, No.5, Part 1
plasma gastrin (12,13) and pancreatic polypeptide (14,15) using specific radioimmunoassays.
Motility Studies Studies investigating the myoelectric activity of the small intestine were performed in conscious dogs equipped with monopolar silver electrodes implanted in the duodenum (10 j:;m distal to the pylorus), jejunum (20 cm distal to the ligament of TreitzJ, and ileum (20 cm oral to the ileocecal junction). The intestinal myoelectric activity was examined either in the fasted or in the postprandial state after ingestiOIi of a meat meal (25 g/kg). After a fasting period of 18 h, two spontaneously occurring migrating motor complexes (MMCs) were recorded. When phase III was passing the upper jejunum, valosin dissolved in saline containing 1 g/dl canine serum albumin was added to an intravenous saline infusion either as a single bolus injection at a dose of 0.2 f.Lglkg or as a constant infusion at a dose of 0.1-0.4 f.Lg/kg. h over a 2.5-h period. The different phases of MMC were identified using the criteria of Code and Marlett (16). The MMC interval was measured as the time period between two consecutive phase III patterns at the level of midjejunum. In the postprandial state, valosin was injected according to the same protocol; in control experiments, injections or infusions of canine serum albumin (1 gldl) in physiologic saline were administered.
Statistics Results were analyzed by the two-tailed Student's t-test for unpaired data adapted for multiple comparisons according to Holm (17); p < 0.05 was considered to be significant.
Results Effects of Valosin on Gastric and Exocrine Pancreatic Secretion
The effect of various doses of valosin on gastric acid and pepsin secretion is shown in Figure 1. Single bolus injections of valosin dose-dependently increased gastric acid secretion 8o-fold over basal levels and pepsin output lo-fold over basal levels. At the highest dose tested (1 J.Lg/kg). the stimulation of acid secretion reached ~18% and the increase in pepsin output reached ~30% of the effect induced by pentagastrin at a dose of 8 J.Lg/kg. h, which represents a maximal stimulus for gastric acid secretion in the dog. The rise in acid output was accompanied by a small, but dose-related increase in plasma gastrin levels reaching a peak 15 min after injection of valosin, as illustrated in Figure 2. When valosin was added to a half-maximal background stimulation by pentagastrin (4 J.Lg/kg. h), gastric acid secretion was further stimulated dosedependently. The response to the combination of pentagastrin plus valosin was higher than the sum of
BIOLOGICAL EFFECTS OF VALOSIN
May 1987
13{ 3
Ii!!
i
H+
o Pepaln
'E a
M
0
Table 1. Effect of Valosin Added as Bolus Injection in Various Doses to an Intravenous Infusion of Pentagastrin on Gastric Acid Secretion a
40
C
Gastric acid secretion (mmol H+ 130 min)
30 C
2
'E
E E
20
..I
a
M
en ..sc: .iii
10 a. Ql a.. 1.()
Ya60aln
8.0
0
1~-~"lr~
Figure 1. Effect of valosin injected intravenously as a single bolus on basal gastric acid and pepsin secretion in fasted conscious dogs equipped with gastric fistulas. Nearmaximal secretion was achieved with infusion of pentagastrin (8 JLg/kg. h). Mean ± SEM of four tests in 4 animals .• p < 0.05, •• p < 0.005.
the separate responses to pentagastrin and valosin alone as shown in Table 1. The effect of valosin on pancreatic protein and bicarbonate secretion is shown in Figure 3. Intravenous administration of the peptide resulted in a dose-dependent increase in both parameters of exocrine pancreatic secretion. At 1.0 JLglkg, the protein secretion was stimulated 12-fold over basal, which corresponded to about 55% of the CCK-induced maximal output and bicarbonate secretion increased ii-fold over basal, reaching about 6% of the secretininduced maximal response. This increase in pancreatic secretion was accompanied by a dose-dependent
•
50
· /I I
• : pp
J.. Q. Q.
30
.,Is
20
i; to
III
to
E
"'to
9.45 ± 1.08 1.08 ± 0.18 11.24 ± 0.79 11.70 ± 1.24 12.73 ± 0.81b
Mean ± SEM of four experiments in 4 animals. b p < 0.05, compared with pentagastrin alone.
g kg/h
(JI!Vkg]
~ 40
Pentagastrin alone (4 JLg/kg· h) Valosin alone (0.5 JLg/kg) Pentagastrin + valosin 4 JLg/kg. h + 0.125 JLg/kg 4 JLg/kg . h + 0.25 JLg/kg 4 JLg/kg· h + 0.50 JLg/kg a
0
o : gastrin
1/ /1
I::::::~.JL ~/.r y I
•
increment in plasma pancreatic polypeptide levels, with a peak at 15 min after injection (Figure 2). In combination with a CCK background stimulation (2 U/kg . h), valosin failed to significantly affect the pancreatic bicarbonate or protein secretion, as summarized in Table 2. When valosin was combined with a constant background stimulation by secretin (2 Ulkg. h), it resulted in a small but significant increase in both pancreatic bicarbonate and protein output above the level obtained by secretin alone. Effects of Valosin on Intestinal Myoelectric Activity In fasted dogs, spontaneously recycling MMCs were recorded with a mean MMC interval of
•• 2000
I!ll1I
o T
••
0
11000
0.125
0.25
0.5
1,0
Figure 2. Peak plasma gastrin and plasma pancreatic polypeptide (PP) levels 15 min after intravenous bolus injection of valosin at various doses in fasted dogs .• p < 0.05, compared to basal levels. Mean ± SEM of four experiments in 4 animals.
5T
••
4
•
!e
~ 0
l
0.125
f
..E..
.
3
)
2
;l!
iii
Q.
500
7
6
Bicarbonate
""l Q25
Valo"", ~kgJ
'l5.
••
Protein
1500
0
10
o
1183
h h
Q5
1.0
"/
I
4.0
CCK ~/kg/hj
rn; 4.0
0
Secretin
[LVkgjh]
Figure 3. Pancreatic protein and bicarbonate secretion in fasted dogs equipped with pancreatic fistulas after intravenous bolus injection of valosin at various doses. Nearmaximal stimulation of bicarbonate output by secretin (4 U/kg . h) and of protein secretion by CCK (4 U/kg . h) are included for comparison. Mean ± SEM of four tests in 4 animals .• p < 0.05, •• p < 0.005, compared with basal secretion.
1184
KONTUREK ET AL.
GASTROENTEROLOGY Vol. 92, No.5, Part 1
Table 2. Effect of Valosin Added as Bolus Injection in Various Doses to an Intravenous Infusion of Secretin or Cholecystokinin on Pancreatic Bicarbonate and Protein Secretion U
J
:~ ~ /bi\4J .. .,
Pancreatic secretion
Secretin Alone (2 U/kg' h) Plus valosin 0.125 JLg/kg 0.25 JLg/kg 0.50 JLg/kg
Cholecystokinin Alone (2 U/kg' h) Plus valosin 0.125 JLglkg 0.25 JLglkg
0.50 JLg/kg a
Bicarbonate (mmol!30 min)
Protein (mg/30 min)
4.32 ± 0.63
253 ± 74
4.48 ± 0.72 5.65 ± 0.51 b 5.94 ± 0.71 b
297 ± 84 340 ± 74 b 390 ± 60 b
0.81 ± 0.09
1180 ± 176
0.92 ± 0.11 0.69 ± 0.12 0.71 ± 0.10
1042 ± 198 1210 ± 177 1360 ± 224
Mean ± SEM of four experiments in 4 animals; no more than two different doses were administered to 1 animal on anyone test day. b p < 0.05, compared with secretin or cholecystokinin alone.
about 78 ± 12 min. After intravenous injection or infusion of valosin, a dose-dependent prolongation of the MMC interval and an increase in the overall spike activity were recorded as depicted in Figure 4. After ingestion of a meat meal, the MMCs were interrupted and replaced by a more uniform spike activity that was characterized by a larger percentage of slow waves accompanied by spikes (Table 3). Valosin administered during such postprandial periods did not affect the pattern of myoelectric activity occurring after feeding.
Discussion This study demonstrates that in conscious dogs valosin, a novel gastroint~stinal peptide isolated from porcine small intestine (2), stimulates both gastric and exocrine pancreatic secretion. In addition, it seems to modulate the pattern of myoelectric activity in the small bowel in the fasting state. Although the results have been obtained with essentially pure natural peptide isolated from porcine gut extracts by high-performance liquid chromatography, it has to be shown that all effects can be reproduced with synthetic valosin in order to exclude the theoretical possibility that an as yet not identified contaminant is responsible for the biological activity. The effect of valosin on gastric secretion is characterized by a dose-dependent increase in basal acid and pepsin secretion reaching, at a dose of 1 /Lg/kg, 18% and 30%, respectively, of the pentagastrin effect at 8 /Lg/kg . h. Valosin added to a dose of pentagastrin
Valosin (O,2J1g/k9/h)
Valosin (O.2J1g/kg)
DUODENUM
.=
.~
Cf)
.l:
.,iJ
~
~
..
'E I:!
~
'=lJ ,~ ~ ~ ~
d
~
':l~ ~j~ ~ ~ 1
2
I
5
4
3
6
JEJUNUM
~ ILEUM I
7
Time (hours)
Figure 4. Effect of intravenously injected valosin on small bowel motility: percentage of slow waves with spike potentials as equivalents of the slow MMCs, recorded in the duodenum, jejunum, and ileum of a fasted dog before and after bolus injection of valosin (0.2 JLglkg) at 2.5 h and after infusion of valosin (0.2 JLg/kg. h) for 2.5 h starting at 4.0 h. The representative record from 1 animal is shown; similiar results were obtained from 3 other animals. The time interval between two successive MMCs is markedly prolonged after injection, and even more markedly prolonged after infusion of peptide valosin (see also Table 3).
that caused near-maximal acid secretion resulted in a further augmentation of acid output. This effect could be mediated via a gastrin-independent mechanism, although the small concomitant release of gastrin that was observed after injection of different doses of valosin could contribute to the overall stimulation of gastric secretion. A bombesinlike acTable 3. Effect of Intravenously Administered Valosin on Small Bowel Motilityu Slow waves with spikes Fasted Fasted 0.10 0.20 0.40
+ valosin JLglkg· h JLg/kg . h JLglkg· h
Food alone Food + valosin (0.40 JLg/kg· h) a
(%)
MMC interval (min)
10.3 ± 1.4
78 ± 12
11.8 ± 1.9 16.3 ± 2.4 b 24.5 ± 3.7 b
92 ± 11 124 ± 16 b 136 ± 14b
43.8 ± 5.2
Absent
49.1 ± 8.6
Absent
Percentage of slow waves with spike potentials and time interval between successive migrating myoelectric complexes (MMCs) in the midjejunum of fasted and fed conscious dogs with and without infusion of valosin at various doses. Mean ± SEM of four recordings in 4 animals; one dose was given to 1 animal per test day. b p < 0.05, compared to the fasted state.
May 1987
tion of valosin, however, seems unlikely because bombesin does not significantly augment gastric acid secretion in response to pentagastrin (18), but increases serum gastrin levels sevenfold. Valosin, however, raises serum gastrin levels just twofold at much higher dosages, but is able to further stimulate maximal gastric acid secretion induced by pentagastrin. In this respect and concerning the localization of the peptide, valosin resembles enterooxyntin (9,19), a candidate hormone of the small intestine that has been reported to augment maximal pentagastrin-induced gastric acid secretion after being released from the upper gut in response to food. Whether or not valosin is identical or, at least, present in the partially purified peptide fractions from porcine gut containing the enterooxyntinlike bioactivity (20) remains to be investigated. Valosin stimulated basal pancreatic secretion of bicarbonate and protein, reaching about 6% of the secretin maximum and 55% of the CCK maximum at a dose of 1 J,Lg/kg, respectively. Thus valosin seems to exhibit a preferential pancreozyminlike activity. When combined with a half-maximal secretin stimulation, valosin augmented the bicarbonate and protein response to this hormone. The release of gastrin that was observed during the intravenous administration of valosin could contribute to the stimulation of the exocrine pancreas, as it is known that, at least in the dog, gastrin is able to stimulate pancreatic secretion (21). However, the increase in serum gastrin levels was small and occurred only at valosin doses that were several fold higher than the threshold dose for stimulation of pancreatic protein secretion. Whether the peptide itself in addition possesses an intrinsic stimulatory activity on the exocrine pancreas or is capable of releasing CCK has to be demonstrated in further experiments. During intravenous administration of valosin, a dose-dependent release of pancreatic polypeptide was noticed. This peptide, which is a well-established inhibitor of exocrine pancreatic secretion (22), may limit the stimulatory effect of valosin. The inhibitory action of valosin on intestinal motility was strictly confined to the fasting state. The propagation of the interdigestive MMC seems to be delayed, while an increase in spike activity by prolonging the phase II pattern in the small bowel was observed. Similiar effects on intestinal myoelectric motility were reported after the administration of exogenous pancreatic polypeptide (23). It is unlikely, however, that this action of valosin is mediated through the release of endogenous pancreatic polypeptide, because valosin influenced motility at a dose that did not yet increase serum pancreatic polypeptide levels. It has to be elucidated whether
BIOLOGICAL EFFECTS OF VALOSIN
1185
an intrinsic activity of valosin on neuronal or muscular elements is involved. Further evaluation of the physiologic role of valosin will require immunocytochemical localization of the peptide and the development of a radioimmunoassay to address the question whether the peptide is released into the circulation to affect gastric and pancreatic secretion and intestinal myoelectric activity or whether it acts as a local neuroendocrine modulator. Molecular cloning of a cDNA encoding the valosin precursor is needed to solve another problem. Since the natural peptide, as isolated from porcine gut, does not possess an amino acid a-amide as C-terminus, it still has to be excluded that valosin represents a fragment of a hitherto unknown protein in the small gut that coincidentally exhibits certain biological effects.
References 1. Tatemoto K, Mutt V. Chemical determination of polypeptide hormones. Proc Natl Acad Sci USA 1978;75:4115-9. 2. Schmidt WE, Mutt V, Carlquist M, Kratzin H, Conlon JM, Creutzfeldt W. Valosin: isolation and characterization of a novel peptide from porcine intestine. FEBS Lett 1985; 191:264-8. 3. Schmidt WE, Mutt V, Kratzin H, Carlquist M, Conlon JM, Creutzfeldt W. Isolation and characterization of ProSSl_32, a peptide derived from the N-terminal region of preprosomatostatin. FEBS Lett 1985;192:141-6. 4. Schmidt WE, Mutt V, Konturek SJ, Creutzfeldt W. Peptide VQY: isolation and characterization of a new biologically active gastrointestinal peptide. Dig Dis Sci 1985;29:75S. 5. Thomas JE. An improved cannula for gastric and intestinal fistulas. Proc Soc Exp BioI Med 1941;46:260-1. 6. Herrera F, Kemp DR, Tsukamoto M, Woodward ER, Dragstedt LR. A new cannula for the study of pancreatic function. J Appl PhysioI1968;25:207-9. 7. Konturek SJ, Pucher A, Radecki T. Comparision of vasoactive intestinal peptide and secretin in stimulation of pancreatic secretion. J Physiol (Lond) 1976;255:497-509. 8. Anson ML. The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J Gen Physiol 1948;22:78-89. 9. Debas HT, Slaff GF, Grossman MI. Intestinal phase of gastric acid secretion: augmentation of maximal response of Heidenhain pouch to gastrin and histamine. Gastroenterology 1975;68:691-8. 10. Konturek SJ, Thor P, Dembinski A, Krol R. Comparison of secretin and vasoactive intestinal peptide on pancreatic secretion in dogs. Gastroenterology 1975;68:1527-35. 11. Debas HT, Grossman MI. Pure cholecystokinins: pancreatic and bicarbonate response. Digestion 1969;9:469-81. 12. Yalow RS, Berson SA. Radioimmunoassay of gastrin. Gastroenterology 1970;58:1-4. 13. Fokina A, Konturek SJ, Kwiecien N, Radecki T. Role of gastric antrum in gastric and intestinal phases of gastric secretion. J Physiol (Lon d) 1979;295:229-39. 14. Floyd JC, Fajans SS, Pek S, Chance RE. A newly recognized pancreatic polypeptide: plasma levels in health and disease. Recent Prog Horm Res 1977;33:519-70. 15. Swierczek JS, Konturek SJ, Tasler J, Jaworek J, Cieczkowski M. Pancreatic polypeptide and vagal stimulation of gastric and pancreatic secretion in dogs. Hepatogastroenterology 1981;28:206-9.
1186 KONTUREK ET AL.
16. Code CF, Marlett JA. The inter digestive myoelectric complex of the stomach and small bowel of dog. J Physiol (Land) 1975;246:298-309. 17. Holm S. A simple sequentially rejective multiple test procedure. Scand J Statist 6:65-70. 18. Pappas D, Hamel D, Debas R, Walsh JR, Tache Y. Cerebroventricular bombesin inhibits gastric acid secretion in dogs. Gastroenterology 1985;89:43-8. 19. Grossman MI. Candidate hormones of the gut. Gastroenterology 1974;67:730-55. 20. Vagne M, Mutt V. Entero-oxyntin: a stimulant of gastric acid
GASTROENTEROLOGY Vol. 92, No.5, Part 1
secretion extracted from porcine intestine. Scand J GastroenteroI1980;15:17-22. 21. Valenzuela JE, Bugat R, Grossman MI. Effect of big and little gastrins on pancreatic and gastric secretion. Proc Soc Exp BioI Med 1978;159:237-8. 22. Chance RE, Cieszkowski M, Jaworek J, Konturek SJ, Swierczek JS, Tasler J. Effect of pancreatic polypeptide and its C-terminal hexapeptide on meal and secretin-induced pancreatic secretion in dogs. J Physiol (Land) 1981;314:1-9. 23. Hall KE, Diamant NE, EI-Sharkawy TY, Greenberg RG. Effect of pancreatic polypeptide on canine migrating motor complex and plasma motilin. Am J PhysioI1983;245:178-85.
Valosin Stimulates Gastric and Exocrine Pancreatic Secretion and Inhibits Fasting Small Intestinal Myoelectric Activity in the Dog STANISLAW J. KONTUREK, WOLFGANG E. SCHMIDT, VIKTOR MUTT, JAN W. KONTUREK, and WERNER CREUTZFELDT Institute of Physiology, Medical Academy, University of Krakow, Krakow, Poland; Division of Gastroenterology and Endocrinology, Department of Medicine, University of G6ttingen, G6ttingen, Federal Republic of Germany; and Department of Biochemistry II, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden
Valosin, a novel 25-amino acid gastrointestinal peptide with N-terminal valine and C-terminal tyrosine, has recently been isolated from porcine upper gut extracts. Its physiologic role is unknown and it does not belong to one of the structurally related gut peptide families. Assuming that valosin may influence gastrointestinal functions, we investigated the effect of high-performance liquid chromafographypure valosin on gastric and exocrine pancreatic secretion and on the intestinal myoelectric activity in conscious dogs. Intravenous injection of valosin (0.125-1 J-Lg/kg) dose-dependently increased gastric acid secretion 80-fold over basal, corresponding to 18% of the maximal pentagastrin-induced effect. Pepsin output increased 10-fold over basal (30% of the pentagastrin-stimulated secretion). Half-maximal stimulation by pentagastrin could be further increased dose-dependently ~y simultaneous administration of valosin. Pancreatic bicarbonate secretion was stimulated ll-fold over basal at 1.0 J-Lg/kg, reaching about 6% of the secretin-induced maximal output, whereas protein secretion increased 12-fold over basal, corresponding to about 55% of the cholecystokinin-induced maximal output. In fasted dogs, spontaneously occurring migrating myoelectric complexes were substantially delayed during infusion of valosin at a dose of 0.2 J-Lg/kg. These experiments indicate that valosin may Received July 10, 1986. Accepted December 8, 1986. Address requests for reprints to: Prof. Dr. Werner Creutzfeldt, Division of Gastroenterology and Endocrinology, Department of Medicine, Georg-August-University of G6ttingen, Robert-Koch Strasse 40, D-3400 G6ttingen, Federal Republic of Germany. © 1987 by the American Gastroenterological Association 0016-5085/87/$3.50
represent a novel member of the regulatory gastrointestinal peptides. During recent years, an increasing number of novel gastrointestinal peptides have been purified from extracts of upper porcine intestine using as isolation criterion either structural characteristics like the C-terminal amide group (1) or a biological activity defined by a bioassay system. Recently, we have developed a screening strategy for the characterization of unknown peptide fractions depending on high-performance liquid chromatography and Nterminal sequence determination (2,3). Using this method, a novel peptide comprising 25 amino acid residues from a porcine upper gut extract has been isolated and the complete primary structure has been elucidated (2). As this peptide, which was designated "valosin" from the N-terminal residue valine and the C-terminal residue tyrosine, does not show convincing sequence homology to any peptide sequence reported so far, whether determined directly or deduced from the corresponding nucleotide sequence, it may represent a member of an as yet unidentified family of gastrointestinal peptides. Preliminary biological experiments on dogs revealing that the peptide exhibits several activities within the gastrointestinal tract have been reported in abstract form (4), where valosin was provisionally denoted as "peptide VQY." This study deals with the effect of valosin on gastric and exocrine pancreatic secretion and intestinal myoelectric activity in conscious dogs when administered intravenously. Abbreviations used in this paper: CCK, cholecystokinin; MMC, migrating motor complex.
1182
KONTUREK ET AL.
Materials and Methods Preparation of Valosin Valosin was pur~fieQ from upper porcine intestine as described recently (2). The pure peptide eluting as a single peak from a reverse-phase C-18 high-performance liquid chromatography-column (Vydac RP-201 TPB, 10 f.Lm, 300 A wide pore) in a trifluoroacetic acid/acetonitrile solvent system was used for the biological studies. Lyophilized peptide was dissolved in physiologic saline containing 1 gldl canine serum albumin immediately before the experiment and administered intravenously as a bolus or as an infusion as described below.
Secretory Studies Secretory studies were carried out on 4 conscious dogs equipped with gastric fistulas (5) and Herrera-type pancreatic fistulas (6) as described previously (7). The studies were performed after an 18-h fasting period. Gastric and pancreatic secretions were collected at 15-min intervals. The volume of each sample was measured. The output of HCl into the gastric juice was determined by titration of the samples to pH 7.0 by means of an autotitrator (Radiometer, Copenhagen, Denmark). Pepsin activity was measured by the method of Anson (8). Bicarbonate and protein in the pancreatic juice were determined as described (7). Several series of tests were performed in each animal. Basal secretions of gastric and pancreatic juices were collected for 60 min. Then various doses of valosin were injected intravenously as a single bolus; no more than two doses were given on anyone test day. After each injection of valosin, the secretions were collected for 90 min until the secretory rate completely returned to the basal level. For comparison, the maximal gastric acid output and the maximal pancreatic bicarbonate and protein secretion were determined in response to a 90-min intravenous infusion of a constant dose of pentagastrin (8 f.Lglkg . h) (9), secretin (4 U/kg . h) (10), or cholecystokinin (CCK-33) (4 Ulkg. h) (11), respectively. In tests with stimulated gastric secretion, pentagastrin (4 f.Lglkg . h) was infused at a constant rate to induce near maximal gastric acid output. When the secretion reilched il sustained plateau, usually after 60 min, valosin was added to the intravenous infusion as a single bolus injection at a dose qf 0.125, 0.25, or 0.5 f.Lg/kg, no more than two doses being administered on anyone test day. In tests with stimulated pancreatic secretion, secretin (2 U/kg· h) or CCK-33 (2 U/kg. h) was infused at a constant rate to induce near maximal pancreatic bicarbonate or protein secretion, respectively. When the stimulated secretion reached a constant plateau, valosirt was added to the infusion as an intravenous injection at a dose of 0.125, 0.25, or 0.5 f.Lglkg. Pancreatic juice was collected for at least 90 min after each dose of valosin; no more than two doses were given on anyone test day. Blood samples were taken from a peripheral vein twice during the period of basal secretion and at 15-30-min intervals after injection of valosin for the determination of
GASTROENTEROLOGY Vol. 92, No.5, Part 1
plasma gastrin (12,13) and pancreatic polypeptide (14,15) using specific radioimmunoassays.
Motility Studies Studies investigating the myoelectric activity of the small intestine were performed in conscious dogs equipped with monopolar silver electrodes implanted in the duodenum (10 j:;m distal to the pylorus), jejunum (20 cm distal to the ligament of TreitzJ, and ileum (20 cm oral to the ileocecal junction). The intestinal myoelectric activity was examined either in the fasted or in the postprandial state after ingestiOIi of a meat meal (25 g/kg). After a fasting period of 18 h, two spontaneously occurring migrating motor complexes (MMCs) were recorded. When phase III was passing the upper jejunum, valosin dissolved in saline containing 1 g/dl canine serum albumin was added to an intravenous saline infusion either as a single bolus injection at a dose of 0.2 f.Lglkg or as a constant infusion at a dose of 0.1-0.4 f.Lg/kg. h over a 2.5-h period. The different phases of MMC were identified using the criteria of Code and Marlett (16). The MMC interval was measured as the time period between two consecutive phase III patterns at the level of midjejunum. In the postprandial state, valosin was injected according to the same protocol; in control experiments, injections or infusions of canine serum albumin (1 gldl) in physiologic saline were administered.
Statistics Results were analyzed by the two-tailed Student's t-test for unpaired data adapted for multiple comparisons according to Holm (17); p < 0.05 was considered to be significant.
Results Effects of Valosin on Gastric and Exocrine Pancreatic Secretion
The effect of various doses of valosin on gastric acid and pepsin secretion is shown in Figure 1. Single bolus injections of valosin dose-dependently increased gastric acid secretion 8o-fold over basal levels and pepsin output lo-fold over basal levels. At the highest dose tested (1 J.Lg/kg). the stimulation of acid secretion reached ~18% and the increase in pepsin output reached ~30% of the effect induced by pentagastrin at a dose of 8 J.Lg/kg. h, which represents a maximal stimulus for gastric acid secretion in the dog. The rise in acid output was accompanied by a small, but dose-related increase in plasma gastrin levels reaching a peak 15 min after injection of valosin, as illustrated in Figure 2. When valosin was added to a half-maximal background stimulation by pentagastrin (4 J.Lg/kg. h), gastric acid secretion was further stimulated dosedependently. The response to the combination of pentagastrin plus valosin was higher than the sum of
BIOLOGICAL EFFECTS OF VALOSIN
May 1987
13{ 3
Ii!!
i
H+
o Pepaln
'E a
M
0
Table 1. Effect of Valosin Added as Bolus Injection in Various Doses to an Intravenous Infusion of Pentagastrin on Gastric Acid Secretion a
40
C
Gastric acid secretion (mmol H+ 130 min)
30 C
2
'E
E E
20
..I
a
M
en ..sc: .iii
10 a. Ql a.. 1.()
Ya60aln
8.0
0
1~-~"lr~
Figure 1. Effect of valosin injected intravenously as a single bolus on basal gastric acid and pepsin secretion in fasted conscious dogs equipped with gastric fistulas. Nearmaximal secretion was achieved with infusion of pentagastrin (8 JLg/kg. h). Mean ± SEM of four tests in 4 animals .• p < 0.05, •• p < 0.005.
the separate responses to pentagastrin and valosin alone as shown in Table 1. The effect of valosin on pancreatic protein and bicarbonate secretion is shown in Figure 3. Intravenous administration of the peptide resulted in a dose-dependent increase in both parameters of exocrine pancreatic secretion. At 1.0 JLglkg, the protein secretion was stimulated 12-fold over basal, which corresponded to about 55% of the CCK-induced maximal output and bicarbonate secretion increased ii-fold over basal, reaching about 6% of the secretininduced maximal response. This increase in pancreatic secretion was accompanied by a dose-dependent
•
50
· /I I
• : pp
J.. Q. Q.
30
.,Is
20
i; to
III
to
E
"'to
9.45 ± 1.08 1.08 ± 0.18 11.24 ± 0.79 11.70 ± 1.24 12.73 ± 0.81b
Mean ± SEM of four experiments in 4 animals. b p < 0.05, compared with pentagastrin alone.
g kg/h
(JI!Vkg]
~ 40
Pentagastrin alone (4 JLg/kg· h) Valosin alone (0.5 JLg/kg) Pentagastrin + valosin 4 JLg/kg. h + 0.125 JLg/kg 4 JLg/kg . h + 0.25 JLg/kg 4 JLg/kg· h + 0.50 JLg/kg a
0
o : gastrin
1/ /1
I::::::~.JL ~/.r y I
•
increment in plasma pancreatic polypeptide levels, with a peak at 15 min after injection (Figure 2). In combination with a CCK background stimulation (2 U/kg . h), valosin failed to significantly affect the pancreatic bicarbonate or protein secretion, as summarized in Table 2. When valosin was combined with a constant background stimulation by secretin (2 Ulkg. h), it resulted in a small but significant increase in both pancreatic bicarbonate and protein output above the level obtained by secretin alone. Effects of Valosin on Intestinal Myoelectric Activity In fasted dogs, spontaneously recycling MMCs were recorded with a mean MMC interval of
•• 2000
I!ll1I
o T
••
0
11000
0.125
0.25
0.5
1,0
Figure 2. Peak plasma gastrin and plasma pancreatic polypeptide (PP) levels 15 min after intravenous bolus injection of valosin at various doses in fasted dogs .• p < 0.05, compared to basal levels. Mean ± SEM of four experiments in 4 animals.
5T
••
4
•
!e
~ 0
l
0.125
f
..E..
.
3
)
2
;l!
iii
Q.
500
7
6
Bicarbonate
""l Q25
Valo"", ~kgJ
'l5.
••
Protein
1500
0
10
o
1183
h h
Q5
1.0
"/
I
4.0
CCK ~/kg/hj
rn; 4.0
0
Secretin
[LVkgjh]
Figure 3. Pancreatic protein and bicarbonate secretion in fasted dogs equipped with pancreatic fistulas after intravenous bolus injection of valosin at various doses. Nearmaximal stimulation of bicarbonate output by secretin (4 U/kg . h) and of protein secretion by CCK (4 U/kg . h) are included for comparison. Mean ± SEM of four tests in 4 animals .• p < 0.05, •• p < 0.005, compared with basal secretion.
1184
KONTUREK ET AL.
GASTROENTEROLOGY Vol. 92, No.5, Part 1
Table 2. Effect of Valosin Added as Bolus Injection in Various Doses to an Intravenous Infusion of Secretin or Cholecystokinin on Pancreatic Bicarbonate and Protein Secretion U
J
:~ ~ /bi\4J .. .,
Pancreatic secretion
Secretin Alone (2 U/kg' h) Plus valosin 0.125 JLg/kg 0.25 JLg/kg 0.50 JLg/kg
Cholecystokinin Alone (2 U/kg' h) Plus valosin 0.125 JLglkg 0.25 JLglkg
0.50 JLg/kg a
Bicarbonate (mmol!30 min)
Protein (mg/30 min)
4.32 ± 0.63
253 ± 74
4.48 ± 0.72 5.65 ± 0.51 b 5.94 ± 0.71 b
297 ± 84 340 ± 74 b 390 ± 60 b
0.81 ± 0.09
1180 ± 176
0.92 ± 0.11 0.69 ± 0.12 0.71 ± 0.10
1042 ± 198 1210 ± 177 1360 ± 224
Mean ± SEM of four experiments in 4 animals; no more than two different doses were administered to 1 animal on anyone test day. b p < 0.05, compared with secretin or cholecystokinin alone.
about 78 ± 12 min. After intravenous injection or infusion of valosin, a dose-dependent prolongation of the MMC interval and an increase in the overall spike activity were recorded as depicted in Figure 4. After ingestion of a meat meal, the MMCs were interrupted and replaced by a more uniform spike activity that was characterized by a larger percentage of slow waves accompanied by spikes (Table 3). Valosin administered during such postprandial periods did not affect the pattern of myoelectric activity occurring after feeding.
Discussion This study demonstrates that in conscious dogs valosin, a novel gastroint~stinal peptide isolated from porcine small intestine (2), stimulates both gastric and exocrine pancreatic secretion. In addition, it seems to modulate the pattern of myoelectric activity in the small bowel in the fasting state. Although the results have been obtained with essentially pure natural peptide isolated from porcine gut extracts by high-performance liquid chromatography, it has to be shown that all effects can be reproduced with synthetic valosin in order to exclude the theoretical possibility that an as yet not identified contaminant is responsible for the biological activity. The effect of valosin on gastric secretion is characterized by a dose-dependent increase in basal acid and pepsin secretion reaching, at a dose of 1 /Lg/kg, 18% and 30%, respectively, of the pentagastrin effect at 8 /Lg/kg . h. Valosin added to a dose of pentagastrin
Valosin (O,2J1g/k9/h)
Valosin (O.2J1g/kg)
DUODENUM
.=
.~
Cf)
.l:
.,iJ
~
~
..
'E I:!
~
'=lJ ,~ ~ ~ ~
d
~
':l~ ~j~ ~ ~ 1
2
I
5
4
3
6
JEJUNUM
~ ILEUM I
7
Time (hours)
Figure 4. Effect of intravenously injected valosin on small bowel motility: percentage of slow waves with spike potentials as equivalents of the slow MMCs, recorded in the duodenum, jejunum, and ileum of a fasted dog before and after bolus injection of valosin (0.2 JLglkg) at 2.5 h and after infusion of valosin (0.2 JLg/kg. h) for 2.5 h starting at 4.0 h. The representative record from 1 animal is shown; similiar results were obtained from 3 other animals. The time interval between two successive MMCs is markedly prolonged after injection, and even more markedly prolonged after infusion of peptide valosin (see also Table 3).
that caused near-maximal acid secretion resulted in a further augmentation of acid output. This effect could be mediated via a gastrin-independent mechanism, although the small concomitant release of gastrin that was observed after injection of different doses of valosin could contribute to the overall stimulation of gastric secretion. A bombesinlike acTable 3. Effect of Intravenously Administered Valosin on Small Bowel Motilityu Slow waves with spikes Fasted Fasted 0.10 0.20 0.40
+ valosin JLglkg· h JLg/kg . h JLglkg· h
Food alone Food + valosin (0.40 JLg/kg· h) a
(%)
MMC interval (min)
10.3 ± 1.4
78 ± 12
11.8 ± 1.9 16.3 ± 2.4 b 24.5 ± 3.7 b
92 ± 11 124 ± 16 b 136 ± 14b
43.8 ± 5.2
Absent
49.1 ± 8.6
Absent
Percentage of slow waves with spike potentials and time interval between successive migrating myoelectric complexes (MMCs) in the midjejunum of fasted and fed conscious dogs with and without infusion of valosin at various doses. Mean ± SEM of four recordings in 4 animals; one dose was given to 1 animal per test day. b p < 0.05, compared to the fasted state.
May 1987
tion of valosin, however, seems unlikely because bombesin does not significantly augment gastric acid secretion in response to pentagastrin (18), but increases serum gastrin levels sevenfold. Valosin, however, raises serum gastrin levels just twofold at much higher dosages, but is able to further stimulate maximal gastric acid secretion induced by pentagastrin. In this respect and concerning the localization of the peptide, valosin resembles enterooxyntin (9,19), a candidate hormone of the small intestine that has been reported to augment maximal pentagastrin-induced gastric acid secretion after being released from the upper gut in response to food. Whether or not valosin is identical or, at least, present in the partially purified peptide fractions from porcine gut containing the enterooxyntinlike bioactivity (20) remains to be investigated. Valosin stimulated basal pancreatic secretion of bicarbonate and protein, reaching about 6% of the secretin maximum and 55% of the CCK maximum at a dose of 1 J,Lg/kg, respectively. Thus valosin seems to exhibit a preferential pancreozyminlike activity. When combined with a half-maximal secretin stimulation, valosin augmented the bicarbonate and protein response to this hormone. The release of gastrin that was observed during the intravenous administration of valosin could contribute to the stimulation of the exocrine pancreas, as it is known that, at least in the dog, gastrin is able to stimulate pancreatic secretion (21). However, the increase in serum gastrin levels was small and occurred only at valosin doses that were several fold higher than the threshold dose for stimulation of pancreatic protein secretion. Whether the peptide itself in addition possesses an intrinsic stimulatory activity on the exocrine pancreas or is capable of releasing CCK has to be demonstrated in further experiments. During intravenous administration of valosin, a dose-dependent release of pancreatic polypeptide was noticed. This peptide, which is a well-established inhibitor of exocrine pancreatic secretion (22), may limit the stimulatory effect of valosin. The inhibitory action of valosin on intestinal motility was strictly confined to the fasting state. The propagation of the interdigestive MMC seems to be delayed, while an increase in spike activity by prolonging the phase II pattern in the small bowel was observed. Similiar effects on intestinal myoelectric motility were reported after the administration of exogenous pancreatic polypeptide (23). It is unlikely, however, that this action of valosin is mediated through the release of endogenous pancreatic polypeptide, because valosin influenced motility at a dose that did not yet increase serum pancreatic polypeptide levels. It has to be elucidated whether
BIOLOGICAL EFFECTS OF VALOSIN
1185
an intrinsic activity of valosin on neuronal or muscular elements is involved. Further evaluation of the physiologic role of valosin will require immunocytochemical localization of the peptide and the development of a radioimmunoassay to address the question whether the peptide is released into the circulation to affect gastric and pancreatic secretion and intestinal myoelectric activity or whether it acts as a local neuroendocrine modulator. Molecular cloning of a cDNA encoding the valosin precursor is needed to solve another problem. Since the natural peptide, as isolated from porcine gut, does not possess an amino acid a-amide as C-terminus, it still has to be excluded that valosin represents a fragment of a hitherto unknown protein in the small gut that coincidentally exhibits certain biological effects.
References 1. Tatemoto K, Mutt V. Chemical determination of polypeptide hormones. Proc Natl Acad Sci USA 1978;75:4115-9. 2. Schmidt WE, Mutt V, Carlquist M, Kratzin H, Conlon JM, Creutzfeldt W. Valosin: isolation and characterization of a novel peptide from porcine intestine. FEBS Lett 1985; 191:264-8. 3. Schmidt WE, Mutt V, Kratzin H, Carlquist M, Conlon JM, Creutzfeldt W. Isolation and characterization of ProSSl_32, a peptide derived from the N-terminal region of preprosomatostatin. FEBS Lett 1985;192:141-6. 4. Schmidt WE, Mutt V, Konturek SJ, Creutzfeldt W. Peptide VQY: isolation and characterization of a new biologically active gastrointestinal peptide. Dig Dis Sci 1985;29:75S. 5. Thomas JE. An improved cannula for gastric and intestinal fistulas. Proc Soc Exp BioI Med 1941;46:260-1. 6. Herrera F, Kemp DR, Tsukamoto M, Woodward ER, Dragstedt LR. A new cannula for the study of pancreatic function. J Appl PhysioI1968;25:207-9. 7. Konturek SJ, Pucher A, Radecki T. Comparision of vasoactive intestinal peptide and secretin in stimulation of pancreatic secretion. J Physiol (Lond) 1976;255:497-509. 8. Anson ML. The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J Gen Physiol 1948;22:78-89. 9. Debas HT, Slaff GF, Grossman MI. Intestinal phase of gastric acid secretion: augmentation of maximal response of Heidenhain pouch to gastrin and histamine. Gastroenterology 1975;68:691-8. 10. Konturek SJ, Thor P, Dembinski A, Krol R. Comparison of secretin and vasoactive intestinal peptide on pancreatic secretion in dogs. Gastroenterology 1975;68:1527-35. 11. Debas HT, Grossman MI. Pure cholecystokinins: pancreatic and bicarbonate response. Digestion 1969;9:469-81. 12. Yalow RS, Berson SA. Radioimmunoassay of gastrin. Gastroenterology 1970;58:1-4. 13. Fokina A, Konturek SJ, Kwiecien N, Radecki T. Role of gastric antrum in gastric and intestinal phases of gastric secretion. J Physiol (Lon d) 1979;295:229-39. 14. Floyd JC, Fajans SS, Pek S, Chance RE. A newly recognized pancreatic polypeptide: plasma levels in health and disease. Recent Prog Horm Res 1977;33:519-70. 15. Swierczek JS, Konturek SJ, Tasler J, Jaworek J, Cieczkowski M. Pancreatic polypeptide and vagal stimulation of gastric and pancreatic secretion in dogs. Hepatogastroenterology 1981;28:206-9.
1186 KONTUREK ET AL.
16. Code CF, Marlett JA. The inter digestive myoelectric complex of the stomach and small bowel of dog. J Physiol (Land) 1975;246:298-309. 17. Holm S. A simple sequentially rejective multiple test procedure. Scand J Statist 6:65-70. 18. Pappas D, Hamel D, Debas R, Walsh JR, Tache Y. Cerebroventricular bombesin inhibits gastric acid secretion in dogs. Gastroenterology 1985;89:43-8. 19. Grossman MI. Candidate hormones of the gut. Gastroenterology 1974;67:730-55. 20. Vagne M, Mutt V. Entero-oxyntin: a stimulant of gastric acid
GASTROENTEROLOGY Vol. 92, No.5, Part 1
secretion extracted from porcine intestine. Scand J GastroenteroI1980;15:17-22. 21. Valenzuela JE, Bugat R, Grossman MI. Effect of big and little gastrins on pancreatic and gastric secretion. Proc Soc Exp BioI Med 1978;159:237-8. 22. Chance RE, Cieszkowski M, Jaworek J, Konturek SJ, Swierczek JS, Tasler J. Effect of pancreatic polypeptide and its C-terminal hexapeptide on meal and secretin-induced pancreatic secretion in dogs. J Physiol (Land) 1981;314:1-9. 23. Hall KE, Diamant NE, EI-Sharkawy TY, Greenberg RG. Effect of pancreatic polypeptide on canine migrating motor complex and plasma motilin. Am J PhysioI1983;245:178-85.