Synchronous Oscillations in the Basal Secretion of Pancreatic-Polypeptide and Gastric Acid

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1 Synchronos Oscillations in the Basal Secretion of Pancreatic-Polypeptide and Gastric Acid Depression by Cholinergic Blockade of Pancreatic-Polypeptide Concentrations in Plasma TH. SCHART, BO STNST, LARS GLB, and FLMMNG ST ADL nstitte of Medical Biochemistry, niversity of Aarhs, Aarhs, Denmark; Department of Srgical Gastroenterology D. Herlev Hospital, Copenhagen, Denmark; and Department of Srgery, Sahlgren Hospital, Giiteborg, Seden The effect of cholinergic blockade on the concentrations of pancreatic-polypeptide (PP) in plasma as stdied in 19 patients ith dodenal lcer (D) and 16 control sbjects. PP concentrations increased ith age both in control sbjects and in D patients. n the D patients atropine or benzilonim, an antimscarinic agent ith minimal cerebral actions, redced PP conc{jntrations from 47 (8-22) to 28 (7-53) pmol/liter, n = 18, median and total range. n the control sbjects atropine sppressed the PP concentrations from 17 (-257) to 11 (-41) pmol/liter, n = 15. Cholinergic blockade had only a minor effect in 1 patient and 1 control, both ith high PP concentrations. Both in the D patients and in the control grop the sppression by the cholinergic blockade as most marked in sbjects ith elevated PP concentrations. Spontaneos acid and PP secretion ere measred simltaneosly in 25 D patients. No correlation as fond beteen median acid secretion and median concentrations of PP. Hoever, in the individal patient a positive co variation as fond beteen flctations in spontaneos acid Received March 13, Accepted Jly 17, Address reqests for reprints to: Dr. T.. Schartz, nstitte of Medical Biochemistry, niversity of Aarhs, DK-8 Aarhs C, Denmark. This stdy as spported by grants from the Danish Medical Research Concil. no , , and ), The Sedish Medical Research Concil (project no. 17-x-76), and Landsforeningen for Skkersyge, Denmark. e thank vy D,lrge and Jetta Bach Halrik for technical assistance. e are very gratefl to Drs. R.. Chance and Nancy. Moon (Lilly Research Laboratory. ndiana) for donating highly prified pancreatic polypeptides and antisera. and to Parke, Davis & Co. (Seden) for spplying benzilonim bromide by the American Gastroenterological Association secretion and flctations in PP concentration. P <.5. e conclde that plasma concentrations of PP in the basal state are sppressible by cholinergic blockade and that PP concentrations flctate synchronosly ith the spontaneos secretion of gastric acid. These reslts sggest that PP concentrations in plasma before and after cholinergic blockade may possibly serve as indicator of abdominal vagal tone. Pancreatic-polypeptide (PP) is a polypeptide composed of 36 amino acids'" and originates from endocrine cells of the pancreas.3.4 PP is released in a biphasic manner dring a meal. S B 7 The initial, rapid PP response depends entirely on intact vagal innervation,s.8 hereas the secondary prolonged PP response is probably cased by combined interaction of hmoral and vagal stimlation. s. 7 - 'o n accordance ith the effect of vagotomy on the food mediated response, PP is released in a freqency dependent manner by electrical stimlation of the vagal nerves," and lo doses of acetylcholine stimlate the secretion of PP from the isolated perfsed porcine pancreas." Frthermore, the PP response to inslin hypoglycemia" is inhibited by atropine" and abolished by vagotomy.g 'l The present stdy as ndertaken in order to investigate hether PP secretion in the basal state as sppressible by cholinergic blockade and, hence, possibly correlated to gastric acid secretion. Patients and Methods Dodenal lcer Patients The effects of either 1 mg i.v. atropine (11 patients)

2 Janary 1979 SPONTANOS PANCRATC-POLYPPTD AND ACD SCRTON 15 or 1 mg i.v. benzilone (8 patients) on PP concentrations in plasma ere stdied after an overnight fast in 19 dodenal lcer (D) patients ith a median age of 48 (3-68) yr. Benzilonim bromide (Parke, Davis & Co., Detroit Mich.) is a qarternary antimscarinic agent ith minimal passage of the blood-brain barrier. On the control day blood samples ere collected every 15 min ithot any medication dring a 9-min period. On the test day, one of the anticholinergic drgs as injected after 45 min. The relationship beteen plasma concentrations of PP and basal secretion of gastric hydrochloric acid as stdied after an overnight fast in 25 D patients ith a median age of 48 (25-68) yr. Any anticholinergic medication as stopped 2 days before the stdy. Blood samples ere dran for PP determination from a peripheral vein every 15 min dring a 9-min stdy of spontaneos acid secretion. The diagnosis of chronic D disease as based on the history and verified by endoscopy and/or X-ray. Control Sbjects The effect of 1 mg i.v. atropine on plasma PP concentrations as stdied after an overnight fast in to grops of control sbjects: (a) 9 elderly sbjects, ith a median age of 63 (42-81) yr ithot pper gastrointestinal or renal diseases; and (b) 7 yong, normal sbjects ith a median age of 24 (2-32) yr. Radioimmnoassay The present PP radioimmnoassay has been described in detail elsehere." Antiserm: Anti-PP, 146-5, as sed in a final diltion of 1/7.5 X 1 6 Tracer: Highly prified bovine PP as iodinated by a chloramine-t method and prified on a 1 X 1, mm Sephadex G-5 SF colmn. Standard: Highly prified hman PP as sed as standard. Assay conditions: Sodim barbitone bffer,.2 mol/liter ph 8.4, ith.2% bovine serm albmin as sed as assay bffer. "Hormone-free" plasma, tice treated ith charcoal, as added to the standards. Separation: Free and bond tracer ere separated ith plasma-coated charcoal. Specificity: The assay did not cross-react ith a variety of pancreatic and gastrointestinal hormones." Frthermore, gel chromatography of hman plasma revealed only one immnoreactive peak ith eltion position corresponding to prified PP." An apparent immnoreactive peak is fond in the void volme of a Sephadex G-5 colmn applied ith plasma; hoever, this immnoreactivity cold not be removed by specific immnoabsorption in contrast to the immnoreactivity elting in the position of prified PP. A similar apparent immnoreactive peak is fond in the void volme hen the charcoal-treated plasma that sally is added to the standards is applied to the colmn. Ths, the nonspecific interference is not measred ith the present assay.l1 The variation in PP concentrations measred after atropine injection as de to variation in immnoextractable PP and not de to variation in nonspecific interference (T.. Schartz, npblished observation). Sensitivity and precision: The experimental detection limit dring eqilibrim incbation as 2.8 pmol/liter. The ithin assay coefficient of variation as in plasma samples at 15.7 and 49.7 pmol/liter, 9.7 and 12.7%, respectively. The beteen assay coefficient of variation tested in 1 consective assays at concentrations of 3 and 75 pmol/liter as 12.8% and 12.1 %, respectively. Accracy: Measrement of PP in plasma ith added hman PP and in diltions of plasma yielded reslts that deviated < 2% from the expected vales. Fasting levels of PP in plasma from yong people ere in agreement ith reslts obtained by Floyd et al. ith another PP antiserm, iodinated hman PP as tracer and ith another separation techniqe. '3 Determination of Acid Secretion A doble-lmen nasogastric tbe as placed in the antrm nder floroscopic control. Residal gastric contents ere discarded, and the gastric jice as collected by a sction pmp in 15-min periods. The amont of acid recovered as determined by potentiometric titration to ph 7 ith.1 mol/liter sodim hydroxide. The reslts ere corrected for losses of gastric contents to the dodenm by measrement of the recovery of phenol red continosly infsed into the stomach. Mean recovery as 91%. Calclations Reslts are presented as medians ith total range in brackets, becase PP concentrations are not normally distribted. PP concentration in the basal state in the individal sbject is calclated as the mean of the consective samples. Becase blood samples ere taken at the start of each acid collecting period, the mean concentration of PP for a period as sed for calclating correlation beteen PP and acid. hen patients ere tested more than one time, only the first observation day as sed for statistical analysis of relationship beteen PP and acid secretion. Spontaneos acid secretion is presented as the ratio of basal secretion/maximal acid secretion in response to pentagastrin 14 in order to correct the data for variation in parietal cell mass. '5 Statistical analyses ere performed by the Mann hitney -test, ilcoxon matched-pairs signed-ranks test, Friedman to-ay analysis of variance, and the Spearman rank correlation coefficient. '6 thics very sbject as informed of the prely scientific prpose and consented to the investigation. The project has been accepted by the thics Committee of the Faclty of Medicine, niversity of Goteborg (Goteborg, Seden). Reslts ffect of Anticholinergics D patients. Atropine redced plasma concentrations of pp from 49 (18-148) pmol/liter before the injection to 33 (8-53) pmol/liter 45 min after injection of atropine (P <.5, Figre 1). Benzilone sppressed plasma PP concentrations from 27 (8-16) pmol/liter to 21 (7-44) pmol/liter (P =.1, Figre 1). There as no difference beteen the effect of

3 16 SCHART T AL. GASTRONTROLOGY Vol. 76, No.1 2 A B x 5 2 x Ol, l- f:: a. a. a. c a. --' --' a. 8 a. - <{ ::: ::: <{ <{ a a o o Figre 1. The effect of anticholinergic medication on plasma PP concentrations in D patients. A. and B. Spontaneos secretion ithot medication. C. One milligram Lv., atropine, n = 11. D. One milligram Lv. benzilonim, n = 8. njection is indicated by arro. One patient ith relatively benzilone-resistant PP concentrations is indicated by dotted line. atropine and benzilone as determined by the fall in PP concentrations after injection of the drg (P >.1). n 1 patient, indicated by dotted line in Figre 1, benzilone had only a minor effect, sppression from 158 to 12 pmol/liter. A similar sppression as observed in this patient ith a doble dose of 2 mg i.v. benzilone. Basal PP concentrations did not fall significantly in the 19 D patients on the control day (see Figre 1). Basal PP concentrations increased ith age among the D patients (rs = +.37, P <.5). Control sbjects. Atropine decreased plasma PP concentrations in the grop of elderly controls from 37 (9-254) pmol/liter before injection to 18 (4-41) pmol/liter 45 min after injection of atropine (P <.5, Figre 2A). n onf! control sbject ith high PP concentrations, indicated by dotted line in Figre 2A, atropine had only a slight effect. Basal PP concentrations ere loer among the yong normal sbjects than among the elderly controls (P <.1). Considering all control sbjects as one grop PP concentrations also increased ith age, r. = +.8, P <.1. Atropine sppressed the PP levels in the yong normals from 9 (-22) to 4 (-12) pmol/liter (P <.5, Figre 2B). Generally, the sppressive effect of cholinergic blockade as most marked in sbjects, both D patients and normals, ith high PP concentrations in plasma (Figres 1 and 2). Relationship Beteen PP and Acid Secretion No correlation as fond beteen the median PP concentrations and the median spontaneos acid secretion in the D patients as a grop (rs =.14, P >.2, n = 25, Figre 3). Hoever, evidence of a positive covariation beteen basal PP concentrations and spontaneos acid secretion as fond in the individal patient (Figres 4 and 5). The coefficients of correlation (rs) beteen the six replicate determinations of acid secretion and PP concentrations in the individal patient ranged from -.39 to +.94 ith a median of +.54, n = 25 (Figre 5). The location of this distribtion as different from the location of a distribtion of rs vales ith the assmption that there is no relationship beteen PP and acid secretion in the individal patient (P <.5). 'T x l... 1 (71.) (67) i= 5 4: (63) (56) ::: 4: a TM (MNTS) B 'T x Ol. Figre 2. The effect of atropine injected at time, on plasma PP concentrations in control sbjects. A. lderly controls, n = 9. One sbject ith relatively atropine-resistant PP concentrations is indicated by dotted line. B. Yong controls, n = 7. Age is indicated in brackets. a l... 4: ::: 4: a..

4 Janary 1979 SPONTANOS PANCRATC-POLYPPTD AND ACD SCRTON n. i= 1.. -' c, 5. oc,. i= <l: \ o e c, a a..1 c, C,e.56 o -{> BAO/PAO a Figre 3. Scattergram of mean basal PP concentration and mean basal acid otpt as fraction of maximal pentagastrin stimlated acid otpt: r. =.14, P >.2, n = 25. The intraindividal correlation beteen concrrent PP and acid secretion is indicated (see text and Figre 5): (e) r. >.5; () < rs <.5; and (6) rs < O. Discssion The present stdy shos that plasma concentrations of PP in the basal state can be sppressed by peripheral, cholinergic blockade. Previosly e had fond a freqency-dependent release of PP by a i=!:d <l: electrical stimlation of the vagal nerves." The freqence-response crves corresponded to the physiological range of discharge rate in the atonomic nervos system. t7 Moreover, PP is released by lo doses of acetylcholine in a dose-dependent manner from the isolated pancreas." Taken together these reslts arge that the spontaneos secretion of PP is reglated mainly by tonic vagal activity. The depressive effect of atropine and benzilone on plasma PP concentrations accords ith or finding that trncal vagotomy normalized PP concentrations in those D patients ith highest PP concentrations in plasma before operation The elevated PP levels cold yet be de to nonvagal stimlatory mechanisms acting on a lo bt permissive vagal tone. This possibility is, hoever, less likely becase the vagally denervated PP cells can still be stimlated by hmoral agents. Ths, a secondary, prolonged PP response to food is fond after vagotomy in hmans. 5 Hoever, it cannot be dismissed that the vagal tone facilitates other stimlatory mechanisms, becase the secondary PP response to food is diminished after vagotomy.s The effect of vagotomy on the prolonged PP response in hmans is, hoever, so small that Adrian et a1. did not find any redction in a stdy here the patients ere not their on con ' '<' -l :: 1. <l:... ::(' DDS...1. fo" [" :5 en i i i i i i D i i i D 6 9. TM (MNTS) Figre 4. Spontaneos flctations in acid secretion (colmns) and concrrent plasma concentrations of PP (lines) in the 5 D patients ith highest PP levels. Different observation days are shon for patients K.A. and.n. The spontaneos acid secretion is expressed as a fraction of the maximal acid secretion dring a pentagastrin test.

5 18 SCHART T AL. GASTRONTROLOGY Vol. 76, No.1 "T X /. 'l r,.97 ra) r,.89ro) rs.51 (e) 125 a. rs.77 (e) rs.93 () 1 r! 2 KA ;Y' D rs /, D D N rs o,gt, rs.9[,../ 9 ::: / D... 1 B.P OJO a:: CO :: ::> ] COFFCNT OF BAO PAO CORRLATON ( rs Figre 5. Relationship beteen plasma PP concentrations and spontaneos acid secretion. pper part: Scattergrams of concrrent PP and acid secretion in the patients demonstrated in Figre 4. ach point represents one i5-min period. rs is Spearman's coefficient of correlation. Loer part: Histogram demonstrating the distribtion of Ts vales in 25 individal D patients. trols.9 Moreover, PP concentrations can be elevated oing to mechanisms that are independent of concrrent cholinergic stimlation; this is illstrated by the to sbjects in hom cholinergic blockade had only a minor effect (Figres 1 and 2). Ths, e believe that an elevated PP level that can be sppressed by cholinergic blockade is mainly governed by vagal activity. Vagal activity is assmed to participate in the control of the spontaneos acid secretion. '8 Determination of the basal acid secretion is distrbed by ambigos variations '8.'9 even ithin the same observation period!o.21 e fond a positive correlation beteen flctations in spontaneos acid secretion and flctations in plasma PP concentrations ithin a go-min period (Figres 4 and 5). This covariation is probably not de to mtal dependence of PP and acid secretion becase instillation of acid in the dodenm does not affect plasma PP concentrations'3; frthermore, maximal stimlation of acid secretion by pentagastrin infsion or netralization of the gastric content by alkaline perfsion does not change basal levels of PP (T.. Schartz,.-M. Schbon, and L. Olbe, npblished observations). Also, the flctations in PP concentrations per se cold not affect acid secretion, becase a dose of PP 4 times that hich affects exocrine pancreatic secretion is reqired to inflence the acid secretion. 22 The oscillations in PP and acid secretion have a periodicity compatible ith the ltradian rhythm fond in the atonomic nervos system, i.e. arond go min 23 (Figre 4). e therefore sggest that the synchronos oscillations in PP and acid secretion are de to concrrent alterations in vagal activity. t has not escaped the or notice that PP secretion cold possibly serve as an indicator of abdominal vagal tone. An independent measre of vagal activity old be sefl primarily to stdy the mechanism behind hypersecretion of acid in D patients.'5 On the above-stated gronds, e assme that the increment in PP concentrations above the level reached dring cholinergic blockade mainly is a reflection of vagal tone on the pancreas (specificity of the measre). Hoever, it is also essential that the PP secretion reflects the vagal tone on the parietal cell (accracy of the measre). The lack of correlation beteen median PP concentration and median acid secretion in the basal state does not invalidate the concept of PP as indicator of vagal tone, becase the acid secretion is inflenced by many synergistic mechanisms, most of hich do not affect PP secretion, e.g. gastrin!4 n the present stdy, it as fond that the PP and acid secretions are synchronos. Hoever, it cannot be decided hether the concrrent vagal activity is of eqal magnitde in the different branches of the vags. Althogh it is an intriging idea, the establishment of PP secretion as indicator of vagal tone aaits stdies of PP and acid secretion dring endogenos vagal stimlation, e.g., by sham feeding. The increased PP levels fond in some D patients 5 and in some healthy, elderly sbjects'3 can be sppressed by cholinergic blockade (Figres 1 and 2). This ptative age-dependent increase in vagal tone, i.e., PP levels, is in accord ith the prolongation of the P-R interval of the electrocardiogram ith age!5.26 n the present stdy the basal PP concentrations ere higher in the D patients than among the control sbjects, hich partly cold be de to the different age distribtion in the to grops, becase "normal" PP concentrations in D

6 Janary 1979 SPONTANOS PANCRATC-POLYPPTD AND ACD SCRTON 19 patients have been reported. 9 Ths it is still an open qestion hether D patients have higher PP levels (vagal tone) than trly age-matched control sbjects. References 1. Chance R. Jones : Polypeptides from bovine ovine. hman and porcine pancreas..s. Patent Office Kimmel JR, Hayden LJ. Pollock HG: solation and characterization of a ne pancreatic polypeptide hormone. J Bioi Chem 25: Larsson G-. Sndler F. Hl'kanson R: mmnohistochemical localization of hman pancreatic polypeptide (HPP) to a poplation of islet cells. Cell Tisse Res 156: Larsson L-. Sndler F. Hlkansson R: Pancreatic polypeptide-postlated hormone: identification of its celllar storage site by light and electron microscopic immnocytochemistry. Diabetologia 12: Schartz T. Rehfeld JF. Stadil F. et al: Pancreatic-polypeptide response to food in dodenal-lcer patients before and after vagotomy. Lancet : Adrian T. Bloom SR. Bryant MG. et al: Distribtion and release of hman pancreatic polypeptide. Gt 17: Schartz T. Stenqist B. Olbe L: Physiology of mammalian PP and the importance of vagal reglation. n Gt Hormones. dited by SR Bloom. dinbrgh. Chrchill Livingstone p Taylor L. mpicciatore M. alsh JH: ffect of atropine and vagotomy on the pancreatic polypeptide response to a meal (abstr). Gastroenterology 72:A-116/ Adrian T. Bloom SR. Besterman HS. et al: Mechanism of pancreatic polypeptide release in man. Lancet 1: Taylor L. Richardson CT. Feldman M. alsh JH: Mechanisms of hman pancreatic polypeptide (HPP) release (abstr). Clin Res 25:574A Schartz T. Holst JJ. Fahrenkrg J, et al: Vagal, cholinergic reglation of pancreatic polypeptide secretion. J Clin nvest 61: Floyd JC. Fajans SS. Pek S: Reglation in healthy sbjects of the secretion of hman pancreatic polypeptide. a nely recognized pancreatic islet polypeptide. Trans Am Physicians 89: Floyd JC. Fajans SS. Pek S. et al: A nely recognized pancreatic polypeptide; plasma levels in health and disease. Recent Prog Horm Res 33: Stenqist B. Kntson. Olbe L: Gastric acid responses to adeqate and modified sham feeding and to inslin hypoglycemia in dodenal lcer patients. Scand J Gastroenterol 13: Fordtran JC: Acid secretion in peptic lcer. n Gastrointestinal Disease: Pathophysiology. Diagnosis. Management. dited by JC Fordtran. M Schleisinger. Philadelphia. B Sanders Co p Siegel S: Nonparametric statistics for the behavioral sciences. Ne York. McGra-Hill Book Co Hillarp N A: Peripheral atonomic mechanisms. n Handbook of Physiology. Sect 1. Vol 2. ashington D.C.. American Physiological Society p alsh JH: Control of gastric secretion. n Gastrointestinal Disease: Pathophysiology. Diagnosis. Management. dited by JC Fordtran. M Schlesinger. Philadelphia. B Sanders Co p Baron JH: Stdies of basal and peak acid otpt ith an agmented histamine test. Gt 4: Gillespie G. lder JB. Smith S. et al: Analysis of basal acid secretion and its relation to the inslin response in normal and dodenal lcer sbjects. Gastroenterology 62: Faber RG. Hobsley M: Basal gastric secretion: reprodcibility and relationship ith dodenal lceration. Gt 18: Lin T-M. vans DC. Chance R. et al: Bovine pancreatic peptide: Action on gastric and pancreatic secretion in dogs. Am J Physiol 232: Levin B. Goldstein A. Natelson BH: ltradian rhythm of plasma noradrenalin in rhess monkeys. Natre (Lond) 272: Taylor L. Corazziari. alsh JH: Gastrin and CCK do not mediate the hman pancreatic polypeptide (HPP) response to a meal (abstr). Clin Res 25:113A Draper G. Brenn HG. Dpertis C: Changes in the electrocardiogram as criteria of individal constittion derived from its physiological panel. Am J Med Sci 194: Ostrander LD. Brandt RL. Kjelsberg MO. et al: lectrocardiographic findings among the adlt poplation of a total natral commnity. Tecmseh. Michigan. Circlation 31: