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The importance of cholinergic tone in the release of pancreatic polypeptide by gut hormones in man
Life Sciences, Vol . 24, pp . 1989-1994 Printed in the U .S .A .
Pergamon Press
THE IMPORTANCE OF CHOLINERGIC TONE IN THE RELEASE OF PANCREATIC POLYPEPTIDE BY GUT HORMONS IN MAN T E Adrian, H S Bestarmen and S R Bloom Department of Medicine Royal Postgraduate Medical School London W12 OHS UK (Received in final form April 11, 1979) Summery The pancreatic polypeptide reeponee to both caerulein end Boots' aecretin wee greatly obtunded by prior administration of atropine . In addition basal plasma PP concentration fell by 54 ± 7,~ (mean t 5EM p < 0 .001) 3D minutes after etropinisation . Some degree of cholinergic tone thus appears essential both for maintaining basal PP levels end for the response to gut hormones . Pancreatic polypeptide (PP) is a 36 amino acid peptide produced by specific small grenuled endocrine cells in the pancreas (1) . Plasma levels rise dramatically after a meal rivalling the elevation of insulin (2,3,4) . Recent investigation of the effect of PP infusion in man, producing plasma concentretiona near to or within the physiological range, demonstrates a considerable inhibition both of bile Plow into the duodenum and pancreatic enzyme end juice output (5,6) . Abnormalities oP plasma PP have also been reported in various disease states for example, diabetes mellitus (3,7) end chronic pencreatitia (B) . The mechanisms controlling PP release have still not been fully elucidated . An entero-PP axis appears oP greater significance then changes in circulating nutriments in causing postprandial PP release (9) . Hormones that are known to enhance postprandial insulin release also appear to stimulate PP (10) . However, a recent report suggested that after vagotomy postprandial PP release was greatly reduced (4), though to e second study this finding was not confirmed (9) . To pursue this controversy we have therefore investigated the ePPect oP cholinergic blockade on the release oP PP by gut hormones . Methods Five healthy male volunteers, mean age 29 (range 26-33), mean weight 75 kg (range 65-88) were studied after en overnight Paet . Following e 30 minute basal period they received in random order and on different days one oP the following Pour regimes : 1)
A 40 minute intravenous infusion oP ceerulein ('caeruletide' Ltd) at a rate oP 100 ng kg -lh -1 .
2)
An intremuscular injection oP atropine (1 .2 mg) followed, after a further 30 minute period, by regime (1) .
3)
An intravenous infusion over 1 minute oP 2 Crick Herpsr Raper unita/kg Boots' aecretin (Boots !_td, Nottingham), a crude extract oP porcine intestine . 0024-3205/79/211989-0502 .00/0 Copyright (c) 1979 Pergamon Press Ltd
199 0
PP Release, Cholinergic Dependence
4)
Vol . 24, No . 21, 1979
1 .2 mg atropine IM followed after 30 minutes by regime (3) .
Plasma PP was measured by e previously described specific redioimmunoaesay sensitive to changes of 4 pmol/1 plasma (2) . Results Both ceerulein end Boots' aecretin caused e significant rise of plasma PP (Figure 1 end 2) . Atropine adminietretion, which caused a mean rise in pulse rate of 20 beats per minute (range 14-30) and e dry mouth in all subjacta, almost abolished the rise of plaeme PP induced both by ceerulein and Boots' aecretin . The mean PP peak with ceerulein infusion alone was 85 ± 14 pmol/1 (mean ± SEM) at 30 minutes end when the ceerulein was preceded by atropine it was reduced to 19 ± 6 pmol/1, a rise which is not etetietically significant . The mean PP peek following the injection of Boots' secretinwwae 165 t 22 pmol/1 at 6 minutes but this wee reduced to 14 ± 2 pmol/1, a rise which is not etetietically significant, with prior etropinieation . The magnitude of the rise with Boots' aecretin injection was similar to that seen with the ingestion of e small mixed meal in these subjacta (7) (a peak of 166 t 25 pmol/1 at 30 min) . It is also noteworthy that basal PP levels fell during the 30 minute period after adminietretion of atropine from a basal of 21 ± 4 pmol/1 to B t 2 pmol/1 (p C 0 .01) . Diecuseion In the isolated pancreas acetyl choline has been shown to release PP (10,11) . The release of PP during insulin-induced hypoglycaemia can be blocked by vagotomy or atropine (9,11) . Schwertz et et have also reported e greatly reduced poetprandiel PP release in patients studied shortly after e vagotomy (4), while Adrian et al found that in subjacta studied more then six months after successful truncel vagotomy, the poetprandiel PP release was not different from normal (9), e finding confirmed by Taylor (12) . These reaulte were difficult to reconcile . If e central vagal reflex was important then, as with acid secretion, it ought to have been blocked by a complete vagotomy . The long duration of PP elevation after a meal (in excess of 6 hours) perhaps made the central vegel reflex hypothesis somewhat unlikely . Our finding that the direct action of gut hormones on the PP cell can be blocked by atropine suggests e different mechanism . This may be considered to be more comparable to the influence on the acid secreting gastric parietal cell . Here it is well recognised that poetprandiel gastric acid secretion ie dependent on the close inter-reaction of acetyl cholina, geatrin end histamine becauee the removal of any single one of these influences very greatly diminiehee meal-stimulated acid production . Thus by analogy the effect of hormones on the PP cell may be strongly dependent on e facilitatory cholinergic tone . In these studies there was a significant fall in basal PP concentrations following the administration of atropine . However, Schwertz et al found no significant effect of atropine in sub~ecta with very low PP levels end this could perhaps reflect a different in basal cholinergic tone . In the dog, Taylor et al observed e reduction of approximately 40~ in the mean fasting plaeme PP concentration following atropinisation but this fall did not reach statistical significance (13) . In the dog atropine also reduced the poatprendiel PP response (13) . As no increase in PP concentrations wee seen during previous infusions with pure aecretin (6), it is possible that the releeae of PP by the crude gut extraôt "Boots' aecretin" reaulte from e contaminant . In other studies we have demonstrated that a low does of
Vol . 24, No . 21, 1979
100
PP Release, Cholinergic Dependence
1991
caerulein
50 -
Fio 1 Plaeme PP concentration during infusion of caerulein 100 ng/kg/hr in 5 healthy subjects with end Without prior intramuaculer administration of atropine 1 .2mg .
Fig 2 Plaéme PP concentration follo~ing en intravenous injection of Boots' aecretin 2 CHRU/kg in 5 healthy subjects with end without prior intremuecular edminietration of atropine 1 .2mg .
199 2
PP Release, Cholinergic Dependence
Vol . 24, No .
21, 1979
cholecystokinin (0 .5 IDU kgl h-1 GIH CCK, approx 10% pure) and aecretin (0 .1 CU kg-lh-1 GIH aecretin) elicits a PP response similar in magnitude to that seen with caerulein in this study (14) . Taylor et al observed only a small and statistically non-significant rise in PP levels during infusion of pure cholecyatokinin and gaetrin in man end the reason for the discrepancy is not immediately apparent . Experiments in an isolated perfuaed canine pancreas preparation have shown that PP increment produced by a low concentration UIP infusion was enhanced ten-fold when preceded by stimulation with acetyl choline (11) . Following vagotomy there is an initial depletion of local vagal fibres, but within a Pew months these have grown back (15,16,17,18) . However, the gland still remains unresponsive to central vagal signals, demonstrating the re-innervation to be e local phenomenon . This may thus explain the previous apparent discrepancy in poet-vagotomy postprandial PP release between early end late poet-vagotomy subjects . It appears that the PP cell may require a constant cholinergic tone to permit other substance to act in the physiological control Thus while central vagal of PP release, for example, postprandielly . connections are necessary for release in response to CNS stimulation, eg during hypoglycaemia, the postprandial release of PP only requires the Control of pancreatic presence of a function local cholinergic innervation . and alimentary hormone release is probably far more complex then previously considered and, if PP control is representative, seems likely to depend on complex substrate and neurohormonel interactions . Acknowledgmente Purified human pancreatic polypaptide and specific antiaera were generous gifts from Dr R E Chance (Lilly Research Laboratories, Indianapolis) . Generous support was received from The Wellcome Trust and the British Diabetic Association . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 .
L-I Larseon, F Sundler and R Hakaneon, Diabetologia _12 211-226 (1976) . T E Adrian, S R Bloom, ~ G Bryent, J M Polak, Ph Heitz and A J Barnes, Gut 17 940-944 (1976) . J C Floyd, S S Fajane and S Pek, Rec Prog Horm Rea 32 146-158 (1976) . T W Schwartz, S F Rehfeld, F Stadil, L-I Lersacn, R E Chance and N boon, Lancet I 1102-1105 (1976) . G R Greenberg, R F McCloy, T E Adrian, V S Chadwick, J H Baron and S R Bloom, Lancet _II 1280-1282 (1978) . T E Adrian, H S Beaterman, C N Mellinaon, G R Greenberg end S R Bloom, Gut 20 37-40 (1979) . T E Adrian, S R Bloom, H S Beaterman end M G Bry~nt, in Gut Hormonas (Ed S R Bloom) Churchill Livingstone, Edinburgh, 254-260 1978 . T E Adrian, H S Beaterman, C N Mallinson, C Garelotie end 5 R Bloom, Gut 20 37-4D (1979) . T E Adrian, 5 R Bloom, H S Bestarmen, A J Barnes, T J C Cooks, R C G Russell and R G Faber, Lancet I 161-163 (1977) . T E Adrian, S R Bloom, K Hermansen end J Iversen, Diabetologia 14 413-417 (1978) . T W Schwartz, J J Holst, J Fahrenkrug, S Lindkaer Jensen, 0 V Nielson, J F Rehfeld, 0 8 Schaffelitzky de ~uckadell end F Stadil, J clin Invest 61 781-789 (1978) . I L Taylor, M Felduran, C T Richardeon and J H Welsh, Geatroenterology 75 432-437 (1978) . I L Taylor, m Impicciatore, D C Carter and J H Walah, Amer J Physiol 235 E443-E447 (1978) .
Vol . 24, No . 21, 1979
14 . 15 . lfi . 17 . 18 .
PP Release, Cholinergic Dependence
T E Adrian, J clin Path (In Press) . 0 M Tiacornia, Amer J Gastroent 67 541-560 (1977) . R L Thambugala and J H Baron, Brit J 5urg 58 839-844 (1971) . N Uaysae, M J Bestie, J P Pascal, P Roux, Ch Martinel, A Lecroia and A Ribet, Geatroénterology 69 1269-1277 (1975) . M I Grosamen, in Cibe Foundation Svmaoaium London, Churchill, 208-220 (1962) .
1993
Pergamon Press
THE IMPORTANCE OF CHOLINERGIC TONE IN THE RELEASE OF PANCREATIC POLYPEPTIDE BY GUT HORMONS IN MAN T E Adrian, H S Bestarmen and S R Bloom Department of Medicine Royal Postgraduate Medical School London W12 OHS UK (Received in final form April 11, 1979) Summery The pancreatic polypeptide reeponee to both caerulein end Boots' aecretin wee greatly obtunded by prior administration of atropine . In addition basal plasma PP concentration fell by 54 ± 7,~ (mean t 5EM p < 0 .001) 3D minutes after etropinisation . Some degree of cholinergic tone thus appears essential both for maintaining basal PP levels end for the response to gut hormones . Pancreatic polypeptide (PP) is a 36 amino acid peptide produced by specific small grenuled endocrine cells in the pancreas (1) . Plasma levels rise dramatically after a meal rivalling the elevation of insulin (2,3,4) . Recent investigation of the effect of PP infusion in man, producing plasma concentretiona near to or within the physiological range, demonstrates a considerable inhibition both of bile Plow into the duodenum and pancreatic enzyme end juice output (5,6) . Abnormalities oP plasma PP have also been reported in various disease states for example, diabetes mellitus (3,7) end chronic pencreatitia (B) . The mechanisms controlling PP release have still not been fully elucidated . An entero-PP axis appears oP greater significance then changes in circulating nutriments in causing postprandial PP release (9) . Hormones that are known to enhance postprandial insulin release also appear to stimulate PP (10) . However, a recent report suggested that after vagotomy postprandial PP release was greatly reduced (4), though to e second study this finding was not confirmed (9) . To pursue this controversy we have therefore investigated the ePPect oP cholinergic blockade on the release oP PP by gut hormones . Methods Five healthy male volunteers, mean age 29 (range 26-33), mean weight 75 kg (range 65-88) were studied after en overnight Paet . Following e 30 minute basal period they received in random order and on different days one oP the following Pour regimes : 1)
A 40 minute intravenous infusion oP ceerulein ('caeruletide' Ltd) at a rate oP 100 ng kg -lh -1 .
2)
An intremuscular injection oP atropine (1 .2 mg) followed, after a further 30 minute period, by regime (1) .
3)
An intravenous infusion over 1 minute oP 2 Crick Herpsr Raper unita/kg Boots' aecretin (Boots !_td, Nottingham), a crude extract oP porcine intestine . 0024-3205/79/211989-0502 .00/0 Copyright (c) 1979 Pergamon Press Ltd
199 0
PP Release, Cholinergic Dependence
4)
Vol . 24, No . 21, 1979
1 .2 mg atropine IM followed after 30 minutes by regime (3) .
Plasma PP was measured by e previously described specific redioimmunoaesay sensitive to changes of 4 pmol/1 plasma (2) . Results Both ceerulein end Boots' aecretin caused e significant rise of plasma PP (Figure 1 end 2) . Atropine adminietretion, which caused a mean rise in pulse rate of 20 beats per minute (range 14-30) and e dry mouth in all subjacta, almost abolished the rise of plaeme PP induced both by ceerulein and Boots' aecretin . The mean PP peak with ceerulein infusion alone was 85 ± 14 pmol/1 (mean ± SEM) at 30 minutes end when the ceerulein was preceded by atropine it was reduced to 19 ± 6 pmol/1, a rise which is not etetietically significant . The mean PP peek following the injection of Boots' secretinwwae 165 t 22 pmol/1 at 6 minutes but this wee reduced to 14 ± 2 pmol/1, a rise which is not etetietically significant, with prior etropinieation . The magnitude of the rise with Boots' aecretin injection was similar to that seen with the ingestion of e small mixed meal in these subjacta (7) (a peak of 166 t 25 pmol/1 at 30 min) . It is also noteworthy that basal PP levels fell during the 30 minute period after adminietretion of atropine from a basal of 21 ± 4 pmol/1 to B t 2 pmol/1 (p C 0 .01) . Diecuseion In the isolated pancreas acetyl choline has been shown to release PP (10,11) . The release of PP during insulin-induced hypoglycaemia can be blocked by vagotomy or atropine (9,11) . Schwertz et et have also reported e greatly reduced poetprandiel PP release in patients studied shortly after e vagotomy (4), while Adrian et al found that in subjacta studied more then six months after successful truncel vagotomy, the poetprandiel PP release was not different from normal (9), e finding confirmed by Taylor (12) . These reaulte were difficult to reconcile . If e central vagal reflex was important then, as with acid secretion, it ought to have been blocked by a complete vagotomy . The long duration of PP elevation after a meal (in excess of 6 hours) perhaps made the central vegel reflex hypothesis somewhat unlikely . Our finding that the direct action of gut hormones on the PP cell can be blocked by atropine suggests e different mechanism . This may be considered to be more comparable to the influence on the acid secreting gastric parietal cell . Here it is well recognised that poetprandiel gastric acid secretion ie dependent on the close inter-reaction of acetyl cholina, geatrin end histamine becauee the removal of any single one of these influences very greatly diminiehee meal-stimulated acid production . Thus by analogy the effect of hormones on the PP cell may be strongly dependent on e facilitatory cholinergic tone . In these studies there was a significant fall in basal PP concentrations following the administration of atropine . However, Schwertz et al found no significant effect of atropine in sub~ecta with very low PP levels end this could perhaps reflect a different in basal cholinergic tone . In the dog, Taylor et al observed e reduction of approximately 40~ in the mean fasting plaeme PP concentration following atropinisation but this fall did not reach statistical significance (13) . In the dog atropine also reduced the poatprendiel PP response (13) . As no increase in PP concentrations wee seen during previous infusions with pure aecretin (6), it is possible that the releeae of PP by the crude gut extraôt "Boots' aecretin" reaulte from e contaminant . In other studies we have demonstrated that a low does of
Vol . 24, No . 21, 1979
100
PP Release, Cholinergic Dependence
1991
caerulein
50 -
Fio 1 Plaeme PP concentration during infusion of caerulein 100 ng/kg/hr in 5 healthy subjects with end Without prior intramuaculer administration of atropine 1 .2mg .
Fig 2 Plaéme PP concentration follo~ing en intravenous injection of Boots' aecretin 2 CHRU/kg in 5 healthy subjects with end without prior intremuecular edminietration of atropine 1 .2mg .
199 2
PP Release, Cholinergic Dependence
Vol . 24, No .
21, 1979
cholecystokinin (0 .5 IDU kgl h-1 GIH CCK, approx 10% pure) and aecretin (0 .1 CU kg-lh-1 GIH aecretin) elicits a PP response similar in magnitude to that seen with caerulein in this study (14) . Taylor et al observed only a small and statistically non-significant rise in PP levels during infusion of pure cholecyatokinin and gaetrin in man end the reason for the discrepancy is not immediately apparent . Experiments in an isolated perfuaed canine pancreas preparation have shown that PP increment produced by a low concentration UIP infusion was enhanced ten-fold when preceded by stimulation with acetyl choline (11) . Following vagotomy there is an initial depletion of local vagal fibres, but within a Pew months these have grown back (15,16,17,18) . However, the gland still remains unresponsive to central vagal signals, demonstrating the re-innervation to be e local phenomenon . This may thus explain the previous apparent discrepancy in poet-vagotomy postprandial PP release between early end late poet-vagotomy subjects . It appears that the PP cell may require a constant cholinergic tone to permit other substance to act in the physiological control Thus while central vagal of PP release, for example, postprandielly . connections are necessary for release in response to CNS stimulation, eg during hypoglycaemia, the postprandial release of PP only requires the Control of pancreatic presence of a function local cholinergic innervation . and alimentary hormone release is probably far more complex then previously considered and, if PP control is representative, seems likely to depend on complex substrate and neurohormonel interactions . Acknowledgmente Purified human pancreatic polypaptide and specific antiaera were generous gifts from Dr R E Chance (Lilly Research Laboratories, Indianapolis) . Generous support was received from The Wellcome Trust and the British Diabetic Association . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 .
L-I Larseon, F Sundler and R Hakaneon, Diabetologia _12 211-226 (1976) . T E Adrian, S R Bloom, ~ G Bryent, J M Polak, Ph Heitz and A J Barnes, Gut 17 940-944 (1976) . J C Floyd, S S Fajane and S Pek, Rec Prog Horm Rea 32 146-158 (1976) . T W Schwartz, S F Rehfeld, F Stadil, L-I Lersacn, R E Chance and N boon, Lancet I 1102-1105 (1976) . G R Greenberg, R F McCloy, T E Adrian, V S Chadwick, J H Baron and S R Bloom, Lancet _II 1280-1282 (1978) . T E Adrian, H S Beaterman, C N Mellinaon, G R Greenberg end S R Bloom, Gut 20 37-40 (1979) . T E Adrian, S R Bloom, H S Beaterman end M G Bry~nt, in Gut Hormonas (Ed S R Bloom) Churchill Livingstone, Edinburgh, 254-260 1978 . T E Adrian, H S Beaterman, C N Mallinson, C Garelotie end 5 R Bloom, Gut 20 37-4D (1979) . T E Adrian, 5 R Bloom, H S Bestarmen, A J Barnes, T J C Cooks, R C G Russell and R G Faber, Lancet I 161-163 (1977) . T E Adrian, S R Bloom, K Hermansen end J Iversen, Diabetologia 14 413-417 (1978) . T W Schwartz, J J Holst, J Fahrenkrug, S Lindkaer Jensen, 0 V Nielson, J F Rehfeld, 0 8 Schaffelitzky de ~uckadell end F Stadil, J clin Invest 61 781-789 (1978) . I L Taylor, M Felduran, C T Richardeon and J H Welsh, Geatroenterology 75 432-437 (1978) . I L Taylor, m Impicciatore, D C Carter and J H Walah, Amer J Physiol 235 E443-E447 (1978) .
Vol . 24, No . 21, 1979
14 . 15 . lfi . 17 . 18 .
PP Release, Cholinergic Dependence
T E Adrian, J clin Path (In Press) . 0 M Tiacornia, Amer J Gastroent 67 541-560 (1977) . R L Thambugala and J H Baron, Brit J 5urg 58 839-844 (1971) . N Uaysae, M J Bestie, J P Pascal, P Roux, Ch Martinel, A Lecroia and A Ribet, Geatroénterology 69 1269-1277 (1975) . M I Grosamen, in Cibe Foundation Svmaoaium London, Churchill, 208-220 (1962) .
1993