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The neuropeptide head activator stimulates amylase release from rat pancreas in vitro
Letters, 38 0983) 287-289 Elsevier Scientific Publishers k d a n d Ltd.
287
THE N E U R O P ~ E HEAD ACTIVATOR STIMULATES AMYLASE RELEASE FROM RAT P A N C R E ~ ~ VITRO
G.E. FEURLE, H. BODENMOLLER and I. BA(~A
MediTj~ische Poliklinik, University o f Heidelbe.~g, Hospitalstr. 3 und Max-Planck-lnstitut f~r Med~nische Forschung, A bteilung Biophysik, 6900 Heidelberg (F.R.G.) (Received March 17th, 1983; Revised version received April 21st, 1983; Accepted April 27th, 1983)
Key N~rds: neuropeptide - head activator - exocrine pancreas - amylase release - gastrointestinal hormones - hydra - rat
The neurol~'ptide head activator previously located in neural cells of the freshwater hydra was found to stimulate release of amylase from rat pancreatic Iobules in vitro. It is unknown whether this effect may have physiological implications.
Recently an undecapeptide isolated from the freshwater coelenterate Hydra atwnuafa controlling henri-specific growth and differentiation in this coelenterate [2] was also found in the mammalian upper gut [I ]. Whereas the function of this peptide in the hydra is well known - hence the designation: head activator - no function has so far been found in mammals. As this peptide is present in high concentrations in the upper intestine of the rat we reasoned that it misht affect exocrine pancreatic secretion similar to the peptides secretin and pancreozymin (CCK), which also originate in the upper small bowel. Pancreatic Iobules were isolated from mongrel albino rats according to the method of Scheele and Palade 131. Four of these Iobules were incubated in 4 ml medium 199 containing 25 mM HEPES, 0.1 g/I g-glutamine, 2.2 g/I bicarbonate and Earle's salts (Gibeo) for 120 rain and gassed intermittently with 95% Oz and 5% COz as described [3]. Aliquots of 0.5 ml of supernatant medium were removed and replaced by fresh medium at 30 min intervals. At the end of the incubation period the tissue was homogenized in a Potter system (teflon grinder in glass vessel) and centrifuged at 10,000 g for 30 min. Amylase in the medium samples and the extract supernatant was determined with the Phadebas method. Synthetic head activator (Bachem, Switzerland) was added in final concentrations of 10- 7 - - 1 0 - It M , each concentration in at least 4 experiments. Caerulein (Takus, Farmitalia) in a final concentration of 10-~o M and incubation medium without additives served as controls.
OJ0,~J940/S3/$ 03.00 © 1983 Elsevier Scientific Publishers Ireland LM,
We found that the head activator produced a time (Fig. I) and dose (Fig. 2) dependent release o f amylase. Optimal concentration was 10 - m M, which stimulated pancreatic amylase release to approximately the same degree as 10- m M caemlein. The structure of the coelenterate head activator has no d ~ b l e homology with any of the known peptides regulating exocrine pancreatic secretion. Its action as a pancreatic secretalgogue may therefore represent a new principle of pancreatic stimulation. In vivo, however, we failed to find a reproducible stimulatory effect o f the peptide head activator from the same batch on exocrine pancreatic secretion in experiments in conscious dogs equipped with a Thomas fistula (unpublished).
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Vi~. I. C,mulativc relea~ of amy!a~ calculated as propo~-d pr~'iously 131 from rat pancreatic Iobulc~ it, vilro duri.8 a 2 h incubation with the neuropeptide head activator, the CCK analogue cacrulcin, and in a control experiment without additions. At 90 and 120 min there was a significant (P
(P
289
Even high doses (2 x 10-s mol) injected intravenously failed to elicit pancreatic secretion, yet produced some retching and irritation in our animals. In principle pancreatic enzyme release may take place either via the luminal cell surface into the pancreatic duct system in the form of exocrine secretion, or via the interstiti~ sides of the a c i ~ cells into the i n t e r c e H ~ space or the capill~es, One explanation for the discrepancy of the in vitro and in vivo effects of the head activator could be that this neuropeptide preferentially releases amylase through the interstitial sides of the acinar cells. Serum amylase concentration, however, remained unchanged when head activator was infused in our animals. Further studies, therefore, will be necessary to elucidate fully the mode and way of action of this neuropeptide upon the exocrine pancreas. In preliminary experiments we found that 24 h in vitro incubation of tritiated head activator with 5 ml canine blood resulted in the recovery of only 20°70 of the peptide as determined by high pressure liquid chromatography; and radioimmunoassay demonstrated only 25070of the expected plasma concentrations 5 min after intravenous injection of 2 × I0- -~ mol head activator in a dog. From these studies a rapid loss (degradation?) of the peptide in canine blood seems evident. The stimulator)' action of the coelenterate head activator on the exocrine pancreas of the rat in vitro therefore cannot be generalized at this point of knowledge. if physiological inferences can be drawn from our study at all, non-endocrine modes of action of the head activator on exoerine function of the pancreas, i.e. paracrine or neuroerine mechanisms, have to be considered besides species differences.
This work was supported by the Deutsche Forschungsgemeinschaft Fe 127/5-5 and Scha 253/8. I Bodenmuller, H. and Schaller, H.C., Conserved amino acid sequence of a neuropeptide, the head activator, from coelenterates to humans, Nature (Lond.), 293 (1981) $79-580. 2 Schaller, H.C. and Bodenmiiller, H., Isolation and amino acid sequence of a morphoseneti¢ peptide from hydra, Proc. nat. Acad. Sci. U.S.A., 78 (1981) 7000~7004. 3 Schccle, G.A. and Palade, G.E., Studies on the guinea pi8 pancreas, J. biol. Chem., 250 (1975) 2660-2.670.
287
THE N E U R O P ~ E HEAD ACTIVATOR STIMULATES AMYLASE RELEASE FROM RAT P A N C R E ~ ~ VITRO
G.E. FEURLE, H. BODENMOLLER and I. BA(~A
MediTj~ische Poliklinik, University o f Heidelbe.~g, Hospitalstr. 3 und Max-Planck-lnstitut f~r Med~nische Forschung, A bteilung Biophysik, 6900 Heidelberg (F.R.G.) (Received March 17th, 1983; Revised version received April 21st, 1983; Accepted April 27th, 1983)
Key N~rds: neuropeptide - head activator - exocrine pancreas - amylase release - gastrointestinal hormones - hydra - rat
The neurol~'ptide head activator previously located in neural cells of the freshwater hydra was found to stimulate release of amylase from rat pancreatic Iobules in vitro. It is unknown whether this effect may have physiological implications.
Recently an undecapeptide isolated from the freshwater coelenterate Hydra atwnuafa controlling henri-specific growth and differentiation in this coelenterate [2] was also found in the mammalian upper gut [I ]. Whereas the function of this peptide in the hydra is well known - hence the designation: head activator - no function has so far been found in mammals. As this peptide is present in high concentrations in the upper intestine of the rat we reasoned that it misht affect exocrine pancreatic secretion similar to the peptides secretin and pancreozymin (CCK), which also originate in the upper small bowel. Pancreatic Iobules were isolated from mongrel albino rats according to the method of Scheele and Palade 131. Four of these Iobules were incubated in 4 ml medium 199 containing 25 mM HEPES, 0.1 g/I g-glutamine, 2.2 g/I bicarbonate and Earle's salts (Gibeo) for 120 rain and gassed intermittently with 95% Oz and 5% COz as described [3]. Aliquots of 0.5 ml of supernatant medium were removed and replaced by fresh medium at 30 min intervals. At the end of the incubation period the tissue was homogenized in a Potter system (teflon grinder in glass vessel) and centrifuged at 10,000 g for 30 min. Amylase in the medium samples and the extract supernatant was determined with the Phadebas method. Synthetic head activator (Bachem, Switzerland) was added in final concentrations of 10- 7 - - 1 0 - It M , each concentration in at least 4 experiments. Caerulein (Takus, Farmitalia) in a final concentration of 10-~o M and incubation medium without additives served as controls.
OJ0,~J940/S3/$ 03.00 © 1983 Elsevier Scientific Publishers Ireland LM,
We found that the head activator produced a time (Fig. I) and dose (Fig. 2) dependent release o f amylase. Optimal concentration was 10 - m M, which stimulated pancreatic amylase release to approximately the same degree as 10- m M caemlein. The structure of the coelenterate head activator has no d ~ b l e homology with any of the known peptides regulating exocrine pancreatic secretion. Its action as a pancreatic secretalgogue may therefore represent a new principle of pancreatic stimulation. In vivo, however, we failed to find a reproducible stimulatory effect o f the peptide head activator from the same batch on exocrine pancreatic secretion in experiments in conscious dogs equipped with a Thomas fistula (unpublished).
I
coe~ul~n
a~1ose
*,6
/
lcj
/i /
l
~i
,
.
,
/
: ~!/II/ //
/
i/
,
,
"
.
~,9
0 9
,
.
M
Vi~. I. C,mulativc relea~ of amy!a~ calculated as propo~-d pr~'iously 131 from rat pancreatic Iobulc~ it, vilro duri.8 a 2 h incubation with the neuropeptide head activator, the CCK analogue cacrulcin, and in a control experiment without additions. At 90 and 120 min there was a significant (P
(P
289
Even high doses (2 x 10-s mol) injected intravenously failed to elicit pancreatic secretion, yet produced some retching and irritation in our animals. In principle pancreatic enzyme release may take place either via the luminal cell surface into the pancreatic duct system in the form of exocrine secretion, or via the interstiti~ sides of the a c i ~ cells into the i n t e r c e H ~ space or the capill~es, One explanation for the discrepancy of the in vitro and in vivo effects of the head activator could be that this neuropeptide preferentially releases amylase through the interstitial sides of the acinar cells. Serum amylase concentration, however, remained unchanged when head activator was infused in our animals. Further studies, therefore, will be necessary to elucidate fully the mode and way of action of this neuropeptide upon the exocrine pancreas. In preliminary experiments we found that 24 h in vitro incubation of tritiated head activator with 5 ml canine blood resulted in the recovery of only 20°70 of the peptide as determined by high pressure liquid chromatography; and radioimmunoassay demonstrated only 25070of the expected plasma concentrations 5 min after intravenous injection of 2 × I0- -~ mol head activator in a dog. From these studies a rapid loss (degradation?) of the peptide in canine blood seems evident. The stimulator)' action of the coelenterate head activator on the exocrine pancreas of the rat in vitro therefore cannot be generalized at this point of knowledge. if physiological inferences can be drawn from our study at all, non-endocrine modes of action of the head activator on exoerine function of the pancreas, i.e. paracrine or neuroerine mechanisms, have to be considered besides species differences.
This work was supported by the Deutsche Forschungsgemeinschaft Fe 127/5-5 and Scha 253/8. I Bodenmuller, H. and Schaller, H.C., Conserved amino acid sequence of a neuropeptide, the head activator, from coelenterates to humans, Nature (Lond.), 293 (1981) $79-580. 2 Schaller, H.C. and Bodenmiiller, H., Isolation and amino acid sequence of a morphoseneti¢ peptide from hydra, Proc. nat. Acad. Sci. U.S.A., 78 (1981) 7000~7004. 3 Schccle, G.A. and Palade, G.E., Studies on the guinea pi8 pancreas, J. biol. Chem., 250 (1975) 2660-2.670.