Vagal, Cholinergic Regulation of Secretion

T. W. Schwartz, … , O. B. Schaffalitzky de Muckadell, F. Stadil

J Clin Invest. 1978;61(3):781-789. https://doi.org/10.1172/JCI108992.

Research Article

The effect of efferent, parasympathetic stimulation upon pancreatic polypeptide (PP) secretion was studied in three ways: (a) Plasma PP concentrations increased in response to -induced hypoglycemia in both normal subjects, from 11 pM (9.5-12.5) to 136 pM (118-147), n = 8 (median and interquartile range) and in duodenal ulcer patients, from 33 pM (21- 52) to 213 pM (157-233), n = 7. The PP response to hypoglycemia was diminished by atropine in normal subjects P( < 0.005) and completely abolished by vagotomy in the duodenal ulcer patients. (b) Electrical stimulation, 8 Hz, of the vagal nerves in anesthetized pigs induced an increase in portal PP concentrations within 30 s from 32 pM (28-39) to 285 pM (248-294), n = 12. Minimal stimulatory frequency was 0.5 Hz and maximal stimulatory frequency 8-12 Hz. Atropine inhibited the PP response to electrical stimulation. Median inhibition with 0.5 mg of atropine/kg body wt was 74%, range 31-90%, n = 6. The response was eliminated by hexamethonium. Adrenergic alpha and beta blockade did not influence the release of PP in response to vagal stimulation. (c) Acetylcholine stimulated, in a dose-dependent manner, the secretion of PP from the isolated perfused porcine , half-maximal effective dose being 0.19 μM; maximal PP output in response to 5 min stimulation was 228 pmol, range 140-342 pmol, n = 5. Atropine […]

Find the latest version: https://jci.me/108992/pdf Vagal, Cholinergic Regulation of Pancreatic Polypeptide Secretion

T. W. SCHWARTZ, J. J. HOLST, J. FAHRENKRUG, S. LINDKLER JENSEN, 0. V. NIELSEN, J. F. REHFELD, 0. B. SCHAFFALITZKY DE MUCKADELL, and F. STADIL, Institute of Medical Biochemistry, University of Aarhus, Arhus; Department of Clinical Chemistry, Bispebjerg Hospital, Copenhagen; Department of Surgical Gastroenterology C, Rigshospitalet, Copenhagen, Denmark

A B S R A C T The effect of efferent, parasympathetic INTRODUCTION stimulation upon pancreatic polypeptide (PP) secre- pancreatic polypeptide was isolated by Chance tion was studied in three ways: (a) Plasma PP concen- et al. as a side fraction during insulin purification (1). trations increased in response to insulin-induced hypo- It is a 36 straight chain polypeptide with no glycemia in both normal subjects, from 11 pM (9.5- similarity in sequence to any other known . 12.5) to 136 pM (118-147), n = 8 (median and inter- One or two aminoacids distinguish the known mam- quartile range) and in duodenal ulcer patients, from 33 malian pancreatic polypeptides (PP)l (1), whereas they pM (21-52) to 213 pM (157-233), n = 7. The PP re- have only 16 amino acids in common with the avian sponse to hypoglycemia was diminished by atropine in counterpart (2). normal subjects (P < 0.005) and completely abolished PP has been localized to secretory granules in a spe- by vagotomy in the duodenal ulcer patients. (b) Electri- cific endocrine type in the pancreas distinct from cal stimulation, 8 Hz, of the vagal nerves in anesthe- the A, B, D, and D, cells (3, 4). PP cells are found tized pigs induced an increase in portal PP concentra- both in the islets and in the acinar tissue and duct tions within 30 s from 32 pM (28-39) to 285 pM (248- epithelium. In some species PP cells are numerous 294), n = 12. Minimal stimulatory frequency was 0.5 in the extra-insular tissue and concentrated towards the Hz and maximal stimulatory frequency 8-12 Hz. Atro- duodenal part ofthe pancreas, whereas e.g., in the islets pine inhibited the PP response to electrical stimula- of the sheep the PP cells nearly outnumbers the insulin tion. Median inhibition with 0.5 mg of atropine/kg body cells (4). wt was 74%, range 31-90%, n = 6. The response was The action of PP has not yet been established al- eliminated by hexamethonium. Adrenergic alpha and though PP has been shown to affect many gastrointesti- beta blockade did not influence the release of PP in it PP response to vagal stimulation. (c) Acetylcholine stimu- nal functions (5). At present seems likely that acts lated, in a dose-dependent manner, the secretion of PP on the exocrine pancreatic tissue as a local regulator of from the isolated perfused porcine pancreas, half-maxi- secretion, in a way that may be conceived as an anti- mal effective dose being 0.19 ,uM; maximal PP output and -enhancing action (6, 7). in response to 5 min stimulation was 228 pmol, range It has recently been suggested that PP may also affect = satiety (8). 140-342 pmol, n 5. Atropine completely abolished During a meal PP concentrations in plasma increase this response. rapidly (9-13). The fool-mediated PP response consists The results of the present study together with the of a rapid primary phase (5-30 min) which is elimi- previously demonstrated poor PP response to food in nated by truncal vagotomy (10) and a prolonged sec- vagotomized patients, indicate that vagal, cholinergic ondary phase (0.5-6 h) which in man is reduced but stimulation is a major regulator of PP secretion. not abolished by vagotomy (10). The present study was This work was presented in part at the 11th Acta Endo- undertaken to investigate the effect of efferent para- crinologica Congress, 20-23 June 1977, Lausanne, Switzer- sympathetic stimulation on PP secretion. land, Abstract 161. 1977. Acta Endocritnol. 212 (Suppl.): 106. (Abstr.) ' Abbreviationis used in this paper: DU, duodenal ulcer; PP, Received for publication 18 Jtuly and in revised form 25 panereatic poly-; VIP, vasoactive intestinal polypep- October 1977. tide.

The Jouirnal of Clinical Investigation Volunme 61 March 1978 781-789 781 MIETHODS tweeni-assay coefficient of variation tested in 10 consecutive assays at concentrations of 30 and 75'pM was 12.8 and 12.1%. Radioimnmu noassay for paticreatic polypeptide Accuracy. Measurements of PP in plasma with added PP, and in dilutions of perfusate from the isolated panicreas (see Anitiserum. Anti-PP serumll (146-5), at generouis gift from below), porcine plasma, and human plasmla yielded results Drs. R. E. Chance and N. E. Moon (Eli Lilly & Co., Indianap- which deviated <20% from the expected values. The follow- olis, Ind.), was raised in a rabbit against subcutaneously in- ing specimens were exposed to gel permeation chromiiatog- jected highly purified bovine PP (BPP) coupled to albumin raphy: (a) perfusate from the isolated porcine pancreas during and mixed with complete Freund's adjuvant. The anltiserum stimulation with acetylcholinie; (b) porcine plasma obtained was used at a final dilution of 1/7.5 x 106. from the portal vein during electrical stimulation of the vagus Tracer. HighlyIpurified bovine PP was iodinlated by a nerve; and (c) human plasma obtained during in.sulini hypo- chloramine T method: 1 mCi 1251 (Behrinig-Werke AG, Mar- glycenmia. The gel permeation chromatography was performed burg/LLahi, West Germclany), 0.24 numlol (1 ,ug) highly pturified at 4°C on 10 x 1,000 mm Sephadex G-5( superfine columns BPP (a gift from Dr. Chance, Eli Lilly & Co.), and 18 nmol eluted with 1.0 M acetic acid with 2% equine serum and 0.2% (5 ,g) chloramine T was mixed in a total volume of 0.023 ml/ bovine serumii albumin. The fractions were lvophilize(d and 0.05 M sodium phosphate buffer, pH 7.4. The reactioni was reconstituted in barbitonie buffer 0.02 M, pH 8.4. In everv case terminated after 20 s by the addition of 26 nmol (5 ,ug) sodiun only one major immunoreactive peak with elution position metabisulfite in 0.010 ml sodium phosphate buffer. Theni 1.2 corresponding to purified PP was found, Fig. 1. Wheni plasma gnmol of potassium iodide in 0.1 nl wvas added followed by 0.2 ml of equine serum. The tracer was purifiedl on a 10 x 1,00( mm Sephadex G-50 superfine columni eluted with amlnmonium bicarbonate,(0.25 Xi, pH 8.2, at 4°C. The fractioins froml the 200- A ascending part of the 1251_PP peak gave the lowest unspecific binding without antibody (1-3%) and the highest binding in antibody surplus (90-95%). The specific radioactivity of the 150 - tracer was calculated to 200-250 /.Ci/4g (0.8-1.05 x 106 Ci/ m10ol). Standard. Highly purifiedl humclan PP (a gift from Dr. 100 - Chance, Eli Lilly & Co.) was usedl as standard. x in so- Assaly condition.s. Incubation was carriedl oUt 0.02 E dium barbitonie buffer, pH 8.4, with 0.2% bovine serumii al- 0C 50 - bumin (Ortho Pharmaceutical Corp., Raritani, N. J.) and 0.6 mnM thiomersal. The incubationi volume was 1.5 ml. In assays w 0- of plasma the saimple volume comprised 1/20 of the incubation UJQ- volume and in other assays at most 1/10 of the incubation CL volume. To obtain comparable incubation coniditions "hor- B mnone-free" plasma, twice treated with charcoal, was added to -J the standard and the same volume of buffer was added to the 0 50 - plasmiia samples. Incubation for 3 days was used in all assays a. except in assays on column eluates, where preincubation for 0 2 days was followed by 1 day of incuibation with tracer. 25 - Separation. Separation was performed with plasma- w Ir coated charcoal (Merck article 2186, Merck AG, Darmstadt, 0 West Germany). To each tube 0.5 ml of a preincubated char- z 0- coal slurry, 0.25 ml charcoal 40 g/liter and 0.25 ml outdated 0L plasma, was added. Centrifugation was performed 20 mni after addition of charcoal. Control samples were included in each 75- centrifugation. Specificity. No displacenment of the tracer from antiserum 50- could be demonstrated with up to 10( nM of: monocomponenit human insulin (Novo Research Institute, Bagsvaerd, Den- 25 - mark); monocomponent porcine pancreatic (Novo 0 - Research Institute); synthetic ovine (Beckman Instruments, Geneva, Switzerland); synthetic human - 6 25 50 75 100 17 (ICI Pharmaceutical Co., Alderly Park, Cheshire, England); highly purified porcine cholecystokinin (CCK 33); highly ELUTION VOLUME PERCENTAGE purified porcinie secretin; and highly purified porcine vasoactive intestinal polypeptide (VIP) all generous gifts FIGURE 1 Elution diagrams of PP immunoreactivity in: A from Dr. V. Mutt, Karolinska Institute, Stockholm, Sweden, perfusate from the isolated perfused porcine pancreas, B hu- and highly purified porcine gastric inhibitory polypeptide, a man plasma obtained during insulinhypoglycemia, C porcine generous gift from Dr. J. Browni, University of British plasma obtained during electrical stimulation of the vagus. Columbia, Vancouver, Canada. The samples were applied to Sephadex G-50 superfine col- Sensitivity and precision. The experimental detection umns (10 x 1,000 mm) eluted at 4°C with 1.0 M CH3COOH limit during equilibrium incubation was 2.8 pM and in pre- containing 2% equine serum and 0.2% bovine serum albumin. incubation assays 1.0 pM. The within-assay coefficient of The columns were all calibrated with 22NaCl and column A variation was 6.8, 7.1, 8.0, and 6.8% in buffer standards of also with '25I-albumin. Abscissa: percent of volume between 12, 25, 60, and 120 pM. The coefficient of variation was 9.7 the elution volume of 125I-albumin and 22NaCl. The arrow on and 12.7% in plasma samples of 15.7 and 49.7 pM. The be- top indicate the elution position of highly purified human PP.

782 Schwartz et al. wvas applicated without internal void voltume marker apparent andl eurrenit ais monitored on a dual-beam oscilloscope (type PP immunoreactivitv was determined in anid closely after the 502A, tektron ie, Guernsey Ltd., Englandl). void volume of the columiln (Fig. 1). A simiiilar apparent im- Itnlhibitioni. Atropine, 0.00.5, 0.05, 0.5-mg/kg, wvas injectedl munoreactivitv coulcI l)e detected in the void volume when the before repeated sull)maximal stimulationis in one pig, atropine hormone-free charcoal-treated plasnma that usually was added 0.5 mg/kg in six different pigs. Hexamiiethoniumt, 10( mg/kg was to the standards was applied. This apparent PP immuni oreac- usedl in four dlifferent pigs. In four other pigs submaximal tivitv could not be removed by specific PP immunoabsorption stimulcationis without inhibition wvere repeated. In five differ- (antibody from the same rabbit was used for assay). WVe there- eut pigs combined alpha and beta blockade was performed: fore conelude that the present assay meassures only a propraniolol (In{deral, ICI Pharmiiaceeuticatl Co.) was inljected which elutes in the samiie position as the highly purified PP. i.v. in a (lose of 1 mg/kg. Phenoxybenzarmine chloride, Fasting levels of PP in plasma from youing people (20-40 yr) (Alf'redl Benizon, Copenhllagein, Denmllark) in a dose of 1 mg/kg weere 13 pMI (range 2-30), it = 50, in agreenment with results (lillited in 100 ml isotoniic saline wvas inifused dutiring 1 h, after obtained by Floyd et al. (13) with another PP antiserun, with wvhiiel the vagal stimuilation wvas repeatedl. In these experi- iodinated highlx purified hulmian PP as tracer, and wvith another menits dextran, Macrodex 6%, Phlarnacia, Uppsala, Swveden, separation techniique. was infulsed to careftlly substitute blood anid fluiid losses.

Insulin hiypoglycenmia Isolated1, perfused, porcinie pa ucreas; ace tylcholihie Patientts. Eight normiial sul)jects, two femlales an(d six males, mediani age 22 yr, range 19-25, and seveni duodencal Preparatiotn. The isolationi anid preparation procedure has ulcer (DU) patients, one female and six miales, mediani age 52 I)een dlescribed in detail elsew.,here (16). Five pigs, Danishl vr, ranige 34-60( were studied after an overnight f:ast. Blood Landrace, weight: 26-3() kg, median 28 kg, were uised. The was taken from an aintecubital vein, cenitrifuged at 4°C acnd painereas was isolatedl together with their enitire vascular sup- serum was stored at -20°C before assayeed. Gastric juice was ply; the dllodeilnlim was nlot incluided in the preparation but aspirated continuously during the experimenit. Data concerni- the panereaticodniodealll vessels renmained intact. The pan- ing this acid secretioni anid gastrini release have been published creases were perflisedl It 380C with Krebs-Ringer bicarbonate (14, 15). solutioni enrieled with fumnarate, glutamiiate, and pyruvate all Stimiulatiotn. Hypoglycemia was achieved by i.v. injection 5 mM, Dextrani T 70 (Pharnmacia, Uppsala, Sweden) 4%, human of0.2 IU of instulin (Insulin LEO, Nordisk Insulinl Laboratory seruim albuminc (Trockeni Reinist, Behring Werke/Lhl}in, \Vest Gentofte, Denmark) per kilogramii body weight. Blood glucose Germany) 0.1%, aind 5 mM gluicose. The perfusate was oxy- conicentration, measured by the glucose oxidase method de- geniated with 95% oxygen and 5% CO2 anld perfused throuigh creased in all subjects l)elow 1.8 mM. the paciereats at a flo!v rate of 30( ml/min, thus the oxygen In1hibitioni. The normual subjects were studied on separate coinsuimiiptioni could be mlaintainiedl at 0.006-0.008 nml O2/I-in days with insulin alonie or after i.m. injection of 30 jig atropine/ per g tissue. Flow rate, pressure, temiiperatuire, Pco2, Po2 and( p1 kg body wt given 25 min before the insulini. The DU patients of the perfusate were measuiredl at 20 mni intervals. The ef- were studied before and 13 wk (8-26) after their operation, fluent was collected everv m-inutte and stored at -20(C unltil trunical vagotomv, and pyloroplasty. assaved. Stimutlationi. After a resting period of 30) miii 5mIistimulil- tion period(s with acetvlcholine 0.05, 0.1, 0.5, 2.5, and 100 Electrical stimulation of the vagel nerves /.M (final concenitrationi in perfusiate) were carried out sepa- rated by 15 min restinig periods. Preparationi. 28 female pigs, DaIIish Landrace, weight: 25 Inhibitioni. Atropine, 0.5 uiM (final concenitrationi in per- kg (20-32) were usedl. Food, but not water, was withdrawn 18 fusate), vas infuised 5 min before and( durinig sttimulition with h before experiments. Azaperone (4 mg/kg i.m.) was used for acetyleholine, 0.5 jNIM. premedicationi. Anesthesia was induced vith Halothane/N20) per 02 and maiintained with chloralose (.Merck AG, Darmstadt, West Germany) 70( mg/kg i.v. Polyethylene catheters were Statistics inserted into the portal vein and left femoral artery The results are, if not otherwise iildicated, preseinted as and vein. Intra-arteirial blood pressure and rectal temper- medlianii coneentrationi followed bw the iinter(luartile rainge in ature were continuously monitored (EMT 34 trans- brackets. The \Vilcoxon imiatched-pairs signed-ranks test or ducer, mingograph, E. Sch6nander, Stockholm, Sweden, wheni appropriate the Mann-Whitney test or the Friedmaunii and temperature recorder, Z8, ellab, Copenhagen, Denmark). tw%o-wN ay analysis of variance were used for statistical evaluia- A laparotomy was performed, the pylorus was ligated, and gas- tion (17). Differencees resultinig in P values <0.05 were con- tric content was continuously drained by suction via an oro- sidered signiificant. gastric tube. The splanichnic nerves were cut bilaterally. Through a thoracotomy the vagal trunks were identified above the diaphragm anid cut below the . The peripheral ends RESULTS of the nerves were passed through a bipolar platinum elec- trode. Blood was collected from the portal vein and the femoral Inisulin hypoglycemlia artery, in ice-chilled tubes, containing 500 kallikrein inactiva- tor un1its aprotinin (Trasylol, The Bayer Co., New York) and Durinig insulini hypoglycemia the plasmla concenitra- 50 IU heparin/ml blood. Samples were centrifuged at 40C and tions of PP in the eight normal subjects increased froml plasnma stored at -20°C until assayed. 11 pM (9.5-12.5) to a maximum of 136 pM (118-147) Stimulation. 5 miD stimulations with constant-current (8 mA) square-wave impulses (frequency 0.25-20 Hz, impulse at 45 min after the injection of insulin; PP levels were duration 5 ins) were carried out by means of an electronic still elevated 180 mmi after the iiujection, 46 pM (29- square-wave stimulator (Palmer, England) Applied voltage 55), Fig. 2. Atropine did not significanitly alter basal

Vagal, Cholinergic Regulation of Pancreatic Polypeptide Secretiotn 783 700 - 1000

- 600 - 900

200 - - 500 800

x 100 -to 'a L00 ) E x - CL 700

0L 300 CL 150 - 0- - 600

200 *_ -500 X 0. 100 0 100 - (L 0- -L00

-60 -30 0 30 60 90 120 150 180 - 300 TIME (Minutes) 50 - FIGURE 2 Plasma PP concentrations, median concentration - 200 and interquartile range (hatched area) in 8 normal subjects during insulin hypoglycemia (0.2 U x kg-' body weight) with- out (0) or with (0) atropine (0.03 mg/kg, i.m.) injected 25 - 100 min before insulin. 0 0 - 60 -30 0 30 60 90 120 PP concentrations, 10.1 pM (9.3-11.5) 5 min before and TIME (Minutes) 9.5 pM (9.0-14.0) 25 min after atropine. However, the FIGURE 3 Plasma PP concentrations, median concentration response to insulin hypoglycemia, from 9.5 pM (9.0- and interquartile range (hatched area) in seven duodenal ulcer 14.0) to a maximum of 31.5 pM (28.5-31.5) at 45 min patients during insulin hypoglycemia (0.2 U/kg body wt) be- after insulin, was diminished by atropine (P < 0.005) fore (0) and after (0) truncal vagotomy. at all points from 30 to 120 min after insulin injection. Basal PP concentrations in the DU patients before the operation were 33 pM (21-52), significantly higher 2.5 than those of the younger normal group. The PP re- sponse pattern to insulin hypoglycemia before the op- eration was similar to that of the normal group, Fig. 3. 2.0 However, the maximal PP concentrations were higher in the DU patients, 213 pM (157-233). After truncal vagotomy basal PP concentrations, 17.5 pMI (9.5-27.0), 7x were lower than preoperative levels (P < 0.05), and not E 0. different from the basal PP levels in the normal group. 0- No increase in PP concentrations was observed dur- 0L ing insulin hypoglycemia in the DU patients after trun- 1.5 cal vagotomy, Fig. 3 (P > 0.10, Friedmann two-way analysis). 0.5 Electrical stimulation Frequency-response studies were carried out in three pigs, Figs. 4 and 5. Half-maximal stimulatory fre- 0 60 120 quency was 2.5, 3.3, and 5.6 Hz, and maximal stimula- MINUTES tion was achieved with frequencies of 8, 12, and 12 Hz FIGURE 4 Portal plasm11a PP coieenitratiolis dutrinig graded respectively. Increase in portal plasma PP concentra- electrical stimulation with fre(uenecies from )0.25 to 20 Hz, in- tions could be detected during stimtilatioin with one dicated by bars, of' the vagal nerves in a pig.

784 Schwartz et al. 100 * 6 1500 z 0 I- . -J - D 300 75 (I) 0 -J x - 1000 7 I A x x E O 200 - x I 50 E cm 0. C) LiL 0 a- 0L 0 0 0L 0- 0 - 500 z 25 100- 0: wL A a-

0 0.3 1 3 10 -15 -10 -5 0 5 10 15 20 STIMULATIONS PER SECOND TIME (MINUTES) FIGURE 6 Portal plasim1a PP concenItration1s, m11ediaii conIeen1- FIGURE 5 Integriate(I PP responise in three indclividual pigs trationi anl interdlliartile rainge (hatchedI area) in responise to dutiring gradedl stimlltiation of the vagal nerves. The experimenlt 8 Hz inidicated 1v bar, of the vagal in 4 is hy electrical stimtilation, C1emoInstrated Fig. inidicatedl (0). The responises n = showvn have been callcilated b) initegrationi of PP conieentra- nerves in pigs, 12. tioin cturves dturing stimulclationi periods minutsi prestimulatory levels, if basal PP levels was niot reached before repeated stimulationi a caleuilated lbasal level was suhtracted. 0 0 impulse every other seconid, l)ut not with stimuitilationis 0 every 4th s. During submaximal stimuitilation, 8 Hz, PP conicenitra- 100 - 0 tions in portal plasmia increased within 30 s from 32 pMi /-6 (28-39) to 285 pNM (248-294), ni = 12, Fig. 6. PP con- / -: Z 75 - cenitrations in portal plasma, anid in arterial plasmiia (Fig. :y O 0 8), remainied elevated throughout the 5 min stinilulation 0- F- period. The PP response could be reproducedl in the LL saime pig duiring repeated stimultlationi 3 h after O D 50- the primary stimutlation. The integrated PP re- Z F- sponise durinig restimulationi averaged 91% of the pri- m-ar responise (rainge: 84-129%) ni = 5, Fig. 7. ) 25- w Atropinie iii cldose of 0.05 mi,g/kg was stufficienit to LLJ E inihihit the PP release in response to submaximal elec- 0 O trical stimilulation of the vagal nierves, Fig. 8. However, 0 - even with 0.5 mg/kg ofatropine the degree of inhibition varied; miiediani inihibition: 74% (ranige: 31-90%) n RES ATR HEX ADR = 6, Fig. 7. Inijectioni of' hexamiiethoniumlil 10 miig/kg le- FIGURE 7 The effect of (lifferent blocking agenit uipoin 8 Hz fore vagus stinudliationi elimliinatedl the PP responise, electricial, vagal stimlllliationi of PP secretioni. Ordiniate indi- Figs. 9 anid 7. Comiibined alpha (phenoxybenzaminiie) cates initegratted PP response cluirinig restimulaltioni as percent alnd beta (propraniolol) adreniergic blockade dicl niot sig- of initegratedl PP responise dutiring the primary stimutilationl. PP RES.-restimulation without anyx blocking agenits. ATR. Atro- nificaintly inifluienice the responise to vagal stimutla- pinie 0..5 miig/kg, HEX.-hexamethoniiumil, 10 Imlg/kg, ADR-com- tioll, n = 5, Fig. 7. bined atlpha, phenoxybenzamine 1 miig/kg, an(d beta, proprain- Cessationi of the electrical stimiutilation resuilted in a olol 1 miig/kg, adreniergic blockade.

Vagal, Clholitnergic Reguilationi of Patncreatic Poltlpeptide Secretion 7858 300 increased the secretion of PP from the isolated pancreas in a dose-dependent manner, Fig. 10 and 11. The lowest dose tested, 0.05 uM, stimulated the PP secre- tion in all experiments. Half-maximal effective dose of acetylcholine was 0.19 ,uM (range: 0.07-0.80 ,uM). 200 Maximal output of PP in response to 5 min stimulations 7x was 228 pmol (range: 140-342 pmol). In all experi- x E ments the stimulation of PP secretion was abolished by 0- 500 0- atropine (Fig. 10).

100 - DISCUSSION The present study shows that efferent, vagal stimula- tion releases PP, and that this is probably mainly medi- ated by acetylcholine. Together with the previously 60 demonstrated poor PP response to food in vagotomized MINUTES patients (10) these data indicate that vagal, cholinergic FIGURE 8 The effect of increasing doses of atropine on the stimulation is of major importance for the secretion plasma PP concentrations in the portal vein (*) and in the of PP. femoral artery (0) during repeated 8 Hz stimulations, indi- Insulin hypoglycemia. The increase in concentra- cated by bars, of the vagal nerves in a pig. tion of PP in plasma during insulin hypoglycemia is similar to the increase observed during a meal (11, rapid decline in PP concentrations, Fig. 6 and 8. After 13, 18). This response is closely correlated to the a more rapid decrement during the first five min, the hypoglycemia (11, 13). The fact that atropine strongly semilogarithmic disappearance curve for PP was linear inhibits the PP response (Fig. 2) indicates that vagal from 5 to 15 min after cessation of the stimulation, activity and not hypoglycemia per se stimulates the PP suggesting a half time of disappearance for endoge- cell. Accordingly low glucose concentrations did not nously released PP of 5.7 (4.5-6.9) minutes; ii = 12. enhance the release of PP from the isolated pancreas (unpublished observations). On the other hand the The isolated, perfused, porcine pancreas study with atropine dose not prove that the PP response Basal output of PP from the isolated pancreas was to hypoglycemia is mediated by a peripheral, choliner- 2.6 pmol/min (range: 1.4-3.6 pmol/min). Acetylcholine gic transmission, because atropine might also act on the central (19). 13 - The total elimination of the PP response to hypo- 12 - 11- 3 -

I - 8 10 z U - 0. 7 2 - 63- cE z 0- W 5- 0- z o 4 -5

2 - 1-1

1 -0 - 15 -10 - 5 0 5 10 15 20 25 30 TIME (MINUTES) 0.05 01 0.5 2.5 100 05 FIGURE 9 Portal plasma PP concentrations, median and 0 20 00 60 80 100 120 100 160 total ranige (hatched area), during electrical stimulation, in- TIME (minutes) dicated by bar, of the vagal nerves without (-) or with (0) pretreatment with hexamethonium, 10 mg/kg body wt, FIGURE 10 The effect of increasing concentrations of acetyl- n = 4. The ordinate indicates fractional PP concentration ob- choline (0.05-100 ,uM), indicated by closed bars, upon PP tained by division of actual concentration vith concentration concentrations in the perfusate from an isolated porcine pan- at time zero. creas. Addition of atropine, 0.5 ,tM, is indicated by open bar.

786 Schwartz et al. 100 - o sponse duiring vagal stimuitilation (Fig. 9) indicates that * a niicotinic isintercalated in the peripheral

0- vagal pathway distal to the diaphragm. This is in accord 4 . with the coincept of a classic parasympathetic pathway with a peripheral, postganglionaryneuirone activated I- bynicotiniic receptors. 4 The inhibitorx effect of atropine (Fig. 8)indicates so2 that the trainsillissio from the postganglionary neuronie to the PP cell is, at least in part, mediated by acetyl- z choliine acting on muscarinea receptor. In agreenment LLJ with the effect of atropine upon postganglionary, vagal cK Ll 11 of other functions (19), c stimulatioin gastrointestinal of are necessary toinhibit elec- , o large amiiounts atropine 01 10 loo trical, vagal PP release. However, even with a large ACETYLCHOLINE (MOLIl-Ix x 10-6) dose of atropiine, 0.5mg/kg, only 75% of the response FIGURE 11 Initegrated PPoutptutfrom0 the isolatedc poreinie couldl)eabolished (Fig. 7); thus other transmittersub- painereas in response to graded stimulationi with acetxlcholine stances apart from acetylcholine could be involved. It in fiveindividual . The ouitput was is likely that the cointaiining vagal integrationi of PP concentrationcurves from ti calecultedcl) nOt acetvlcholine started to PP levels retturnied top restimullastorvresfinfuionofv fibres (23) play anymajor role during electrical stimula- levels,minus this concentrationi,mutltiplied xwith theflO tion because combined alpha and beta adrenergic iate. blockade does not influence the PP response, Fig. 7. Furthermore electrical stimiiulation of the splanchniic nerves does not enhance PP secretion (unpublished gly cemia by truncal vagotomy (Fig. 3)indiicates, inac- observations). Peptidergic transmission might be in- cordance with findings by Adrian et al.(1E S), that all of volved in the neural stimulation of PPl)ecause e.g. theincrement in PP concentration duringi] nstslini hypo- VIP (24) aind gastrin (25) have been found in the vagus. glycemia is caused by vagal activity. OthermlechanllismslS Furthermore VIP containiing cell bodies are in soIm1e activated during hypoglycemia, e.g. the tdrenals;a anld species localized in pancreatic ganglia,2 aind VIP is re- the sympathetic nervous system, which miightpossil)ly leased by an atropine-resistantmechanism during elec- affect the secretion of PP, are entirely depe Zndento01 all trical, vagal stimulatioin (20). Finally, VIP andmembers intact vagal innervation. of the gastrin family of gastrointestinal horniones cain Electrical stimulation of the vagal 10 ervets The release PP (18,26). However, the possible involvement large increase in PP concentrations in p(ortal plasmiia of such substances in the electrical, vagal stimulation during electric stimulation of the vagal ner ves conifirmiis of PP secretion is presently difficult to delineate. that efferent vagal stimulation is a potent re leasemech- The isolated porcine pancreas. Low doses of acetyl- anism for PP. The increase in plasma concientrations;of choline stimulated the secretion of PP from the isolated PP during vagal stimulation does reflect iiocreasedPP pancreas. The half-maximal effective dose of acetylcho- secretion, since portal bloodflow was unch anged or in- line (0.2,uNl) for release of PP from the isolated porcine creased up to 40% during these stimulation s (20). MIore- pancreas is similar to the D50 of acetylcholine for stimu- over, a similar increase could be detectec I in the sys- latioin of the motor activity in the gastrointestinal mus- temic circulation, Fig. 8, which proves aIn acttial in- culature (27) and for stimulation of insulin release from creased splanchnic output. the isolated perfused rat pancreas (28). It is reasonable That the threshold frequency for PP rele ase is b)elow to assuime that acetylcholine acting on muscarinic re- one stimulation per second and that maxin- llal seretionl ceptors also is involved in the neural stimulation of PP is achieved with 8-12 Hz stimulation is iI n agreemilent secretioni, since atropine inhibited vagal stimulation, with the supposed physiological dischargl rate of' the Figs. 7 and 8, and because the effect of acetylcholine peripheral autonomic nervous system. B y/ recording oIn the isolated pancreas was completely abolished by from single or a few nerve fibres ofboth sympathetic an e(quimolar dose of atropine, Fig. 10. The amount of and parasympathetic nerves a continuous discharge of PP released during maximal stimulation with acetyl- impulses with frequencies at -1 Hz is fc )und during choline from the isolated porcine pancreas could very basal conditions, and during intense reflex stiimiulation well account for the increase in plasma PP concentra- the discharge rate is in the order of 10 H[z (21). Fur- thermore, similar frequency-response curvzes were ob- J. Holst, and 0. B. tained in studies on electrical, vagal stimul ation of acid Schaffalitzkv2 L-I. Larsson,de Muckadell.J. Fahrenkrug,InnervationJ. of the pancreas by secretion (22). vasoactive intestinal polypeptide inimunoreactive nerves. In The elimination by hexamethonium of F the PP re- preparation.

Vagal, Cholinergic Regulation of Pancreatic Polypeptide Secretiotn 787 tions during maximal, vagal stimulation in the same REFERENCES species. 1. Chance, R. E., F. M. Lin, M. G. Johnson, N. E. Moon, From these three studies taken together it can be D. E. Evans, W. E. Jones, and J. E. Koffenberger. 1975. argued: that efferent, vagal stimuilation is a potent Studies on a newly recognized pancreatic hormone with release mechanism for PP; that the vagal pathway gastrointestinal activities. 57th Annual Meetinig of the Eni- includes a peripheral neurone activated by nicotinic re- docrine Society. 1: 265/183A. (Abstr.) 2. Kimmel, J. R., L. J. Hayden, and H. G. Pollock. 1975. ceptors below the diaphragm; and that the final media- Isolation and characterization of a new pancreatic pol%- tor probably mainly is acetylcholine acting on mus- . J. Biol. Chemn. 250: 9369-9376. carinic receptors. 3. Larssoin, L.-I., F. Sundler, and R. Hakansoni. 1975. Im- The importance of efferent, vagal stimulation of the muniohistochemical localizationi of humani pancreatic polxpeptide (HPP) to a population of islet cells. Cell Tis- PP secretion in human physiology is illustrated by stud- sue Res. 156: 167-171. ies on vagotomized patients. In a prospective study 4. Larsson, L.-I., F. Sundler, and R. Hakanson. 1976. PaIn- with each patient being his own control we found that creatic polypeptide-a postulated new hormone: identifi- truncal vagotomy eliminated the first 15-25 min of the cation of its cellular storage site by light and electron otherwise rapid increase in PP concentrations microscopic immnunocytochemiistry. Diabetologia. 12: plasma 211-226. during a meal, and also significantly reduced the sec- 5. Lin, T. M., and R. E. Chance. 1974. Gastrointestinal ondary prolonged response.,10). These findings have actions of a new bovine pancreatic peptide (BPP). In En- partly been supported by the results of Adrian et al. docrinology of the gut. W. Y. Chey and F. P. Brooks, Edi- (18) and have been confirmed by Taylor et al. (29). tor. Charles B. Slack, Inc., Thorofare, New Jersey. 143- It has been suggested that the 145. elimination by 6. Lin, T. M., D. C. Evans, and R. E. Chance. 1974. Action vagotomy of the primary PP response might be of a bovine pancreatic polypeptide (BPP) on pancreatic due to impaired gastric emptying after the operation secretioni in dogs. Gastroeniterology. 66: 198-852 A (30). However, the afferent branch of the vago- (Abstr.) vagal reflex for PP release is activated by (pre- 7. Lin, T. M., D. C. Evans, R. E. Chance, and G. F. Spray. 1977. Bovine panicreatic peptide: Action on gastric and gastric) classic, cephalic stimulation (31), and by gastric pancreatic secretion in dogs. An. J. Physiol. 232: E311- food- ar d distention-receptors (26). We therefore con- E315. clude that vagal stimulation of the PP secretion is 8. Malaisse-Lagae, F., J-L. Carpentier, Y. C. Patel, W. J. responsible for the rapid, primary increase in plasma Malaisse, and L. Orci. 1977. Pancreatic polypeptide: A PP concentrations during a meal and that it also possible role in the regulation of food intake in the mouse. con- Hypothesis. Experientia (Basal). In press. tributes to the prolonged secondary PP response. 9. Floyd, J. C., R. E. Chance, M. Hayasha, N. Moon, and S. S. Vagal activity does not seem to influence the basal PP Fajans. 1975. Concentrations of a newly recognized pan- secretion in young normal subjects, because atropine creatic islet polypeptide in plasma of healthy subjects does not suppress their basal PP concentrations (Fig. 2). and in plasma and tumors of patients with insulin-se- PP creting islet cell tumors. Clin. Res. 23: 535A. (Abstr.) However, the elevated levels found in some DU pa- 10. Schwartz, T. W., J. F. Rehfeld, F. Stadil, L-I. Larsson, tients were normalized by vagotomy (10) (Fig. 3). The R. E. Chance, and N. Moon. 1976. Pancreatic-polypeptide difference between the DU patients and the normal response to food in duodenal-ulcer patients before and group may yet be due to difference in age because after vagotomy. Lantcet. 1: 1102-1105. the patients studied were and PP levels 11. Floyd, J. C., S. S. Fajans, and S. Pek. 1976. Regulation relatively old, in healthy subjects of the secretion of human pancreatic tend to increase with age (13). However, fluctuations polypeptide, a newly recognized pancreatic islet polypep- in PP concentrations and in the basic acid output in tide. Trans. Am. Physicians. 89: 146-158. DU patients are correlated, and moreover atropine nor- 12. Adrian, T. E., S. R. Bloom, M. G. Bryant, J. M. Polak, P. H. malizes the elevated PP (Schwartz, Olbe, and Sten- Heitz, and A. J. Barnes. 1976. Distribution and release of human pancreatic polypeptide. Gut. 17: 940-944. quist, unpublished observations). Thus, possibly some 13. Floyd, J. C., S. S. Fajans, S. Pek, and R. E. Chance. 1977. DU patients have elevated vagal activity, reflected in A newly recognized pancreatic polypeptide; plasma lev- increased basal PP concentrations. els in health and disease. Recent. Prog. Horm. Res. 33: 519-570. ACKNOWLEDGMENT 14. Stadil, F., J. Malmstrom, J. F. Rehfeld, and M. Miyata. 1974. Effect of atropine on hypoglycemic release of gas- Jetta Bach Haulrik, Evy D0rge, and Ulla S0gaard are gratefully trin in man. Acta Physiol. Scand. 92: 391-398. acknowledged for their skillful technical assistance. We are 15. Stadil, F., and J. F. Rehfeld. 1974. Gastrin response to also very grateful to Dr. R. E. Chance and Dr. Nancy E. Moon, insulin after selective, highly selective, and truncal vagot- Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, omy. Gastroenterology. 66: 7-15. Ind. U. S. A. for donating highly purified pancreatic polypep- 16. Lindkeer Jensen, S., C. Kuhl, 0. Vagn Nielsen, and J. J. tides and antisera. Holst. 1976. Isolation and perfusion of the porcine pan- This study was supported by grants from the Danish Medi- creas. Scand. J. Gastroent. 37 (Suppl.): 57-61. cal Research Council (J.nr. 512-7190) and from Landsforenin- 17. Siegel, S. 1956. Nonparametric statistics for the behavioral gen for Sukkersyge. sciences. McGraw-Hill Book Co., New York. 1.

788 Schwartz et al. 18. Adrian, T. E., S. R. Bloom, H. S. Besterman, A. J. Barnes, cell lines and normal nervous tissue. Scienice (WXVashi. T. J. C. Cooke, R. C. G. Russell, and R. G. Faber. 1977. D. C.). 192: 907-908. Mechanism of pancreatic polypeptide release in man. 25. Uvrn.s-WVallensten, K., J. F. Rehfeld, L.-I. Larsson, acndc Lancet. 1: 161-163. B. Uvnas. 1977. Heptadecapeptide gastrin in the vagal 19. Innes, I. R., and M. Nickerson. 1975. Atropine, scopol- nerve. Proc. Natl. Acad. Sci. In press. amine, and related antimuscarinic drugs. In The pharma- 26. Schwartz, T. W., and J. F. Rehfeld. 1977. NIechanism of cological basis of therapeutics. L. S. Goodman and A. pancreatic-polypeptide release. La icet I: 697-698. Gilman, Editors. Macmillan, Inc., New York. 516-522. 27. Clark, A. J. 1927. The reaction between acetylcholine and 20. Schaffalitzky de Muckadell, 0. B., J. Fahrenkrug and J. J. muscle cells. Part II.J. Physiol. 64: 123-143. Holst. 1977. Release of vasoactive intestinal polypeptide 28. Loubatieres-Mariani, NI. MI., J. Chapel, R. Alric, and A. (VIP) by electrical stimulation of the vagal nerves. Gas- Loubatieres. 1973. Studies of the cholinergic receptors in- troenterology. 72: 373-375. volved in the secretion of insulin using isolated perfused rat pancreas. Diabetologia. 9: 439-446. 21. Hillarp, N. A. 1960. Peripheral autonomic mechanisms. 29. Taylor, I. L., M. Impicciatore, and J. H. W7alsh. 1977. In Handbook of Physiology. Am. Physiol. Soc. Washing- Effect of atropine and vagotomy on the pancreatic poly- ton. Sec. 1. Vol. 2: 989-990. peptide response to a meal. Gastoenterology. 72: A-116- 22. Sjodin, L. 1975. Gastric acid responses to graded vagal 1139. stimulation in the anaesthetized cat. . 12: 17-24. 30. Bloom, S. R., and T. E. Adrian. 1977. Mechanism of pan- 23. Liedberg, G., K. C. Nielsen, C. H. Owman, and N.-O. creatic polypeptide release. Lancet. I: 698. Sjoberg. 1973. Adrenergic contribution to the abdominal 31. Schwartz, T. W. B. Steenquist, and L. Olbe. 1977. Vagal vagus nerves in the cat. Scand. J. Gastroent. 8: 177-180. regulation of PP secretion. In Proceeding from the gut hor- 24. Said, S. I., and R. N. Rosenberg, 1976. Vasoactive Intes- mone symposium, Lausanne, 18-19th June 1977. S. R. tinal Polypeptide: Abundant immunoreactivity in neural Bloom Editor. Churchill Livingston, Edinburgh. In press.

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