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Vasoactive intestinal polypeptide (VIP) in the pig pancreas: role of VIPergic nerves in control of fluid and bicarbonate secretion
Regulatory Peptides, 8 (1984) 245-259 Elsevier
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RPT 00279
Vasoactive intestinal polypeptide (VIP) in the pig pancreas: role of VIPergic nerves in control of fluid and bicarbonate secretion Jens J. Hoist ~, Jan Fahrenkrug 2, Svend Knuhtsen (, Steen L. Jensen 3, S. Seier Poulsen 4 and O. Vagn Nielsen 3 I Institute of Medical Physiology C and 4 Department of Anatomy B, The Panum Institute, University of Copenhagen, 2 Department of Clinical Chemistry, Bispebjerg Hospital, and 3 Department of Surgery C, Rigshospitalet, Copenhagen, Denmark (Received 3 January 1984; revised manuscript received 1 February 1984; accepted for publication 16 March 1984)
Summa~ Vasoactive intestinal polypeptide (VIP) in the pig pancreas is localized to nerves, many of which travel along the pancreatic ducts. VIP stimulates pancreatic fluid and bicarbonate secretion like secretin. Electrical vagal stimulation in the pig causes an atropine-resistant profuse secretion of bicarbonate-rich pancreatic juice. In an isolated perfused preparation of the pig pancreas with intact vagal nerve supply, electrical vagal stimulation caused an atropine-resistant release of VIP, which accurately parallelled the exocrine secretion of juice and bicarbonate. Perfusion of the pancreas with a potent VIP-antiserum inhibited the effect of vagal stimulation on the exocrine secretion. It is concluded, that VIP is responsible for (at least part of) the neurally controlled fluid and bicarbonate secretion from the pig pancreas. peptidergic nerves; perfused pancreas; antiserum treatment
Introduction In the pig, electrical stimulation of the vagus nerves causes an atropine-resistant, profuse secretion from the pancreas of fluid and bicarbonate [1,2], which is indepenAddress all correspondence to: Jens J. Hoist, MD, Institute of Medical Physiology C, The Panum Institute, University of Copenhagen, Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark. Telephone: (01) 35 79 00 ext. 2541. 0167-0115/84/$03.00 © 1984 Elsevier Science Publishers B.V.
246 dent of increases in the concentration of secretin in plasma [2]. Under the same conditions a splanchnic release of the neuropeptide, vasoactive intestinal polypeptide (VIP), can be demonstrated [3], and we have shown that part of the splanchnic VIP output is derived from the pancreas [4]. Furthermore, in the pancreas VIP is exclusively localized to nerves [5-8], and (porcine) VIP is a full secretin-like agonist on the pig pancreas [9]. We therefore hypothesized that neuronally released pancreatic VIP was responsible for the atropine-resistant fluid and bicarbonate secretion in response to vagal stimulation in this species [2,4]. To examine this hypothesis in detail, we developed an experimental model, the isolated perfused pig pancreas with intact vagal nerve supply. This model allowed us to study in a truly isolated system concomitant changes in the pancreatic exocrine secretion and changes in the release into the perfusate of this neuropeptide during parasympathetic nerve stimulation with and without addition of relevant pharmacological blockers. Furthermore, it was feasible to study the effect of the only specific VIP-antagonist available, namely VIP-antiserum. In addition, we studied the chromatographic profile of VIP in extracts of pig pancreas as well as in perfusate obtained during nerve stimulation, and we studied in detail the localization of intrapancreatic VIP-containing nerves using immunohistochemistry.
Materials and Methods
22 pigs, Danish Landrace, weighing 14-16 kg, were used as donor animals. They were fasted for 12 h but had free access to water. Premedication was not given. Anaesthesia was induced with 2.5% halothane and after the onset of surgical anaesthesia, halothane was substituted by intravenous infusion of chloralose (No. 2420, Merck, Darmstadt, F.R.G., 100 mg/kg). The operative procedure and the perfusion system are related to our previously described method for isolation and perfusion of the (denervated) pig pancreas [10-13]. The abdomen was opened through a long mid-line incision. The spleen was removed. An 8-10 cm long segment of the aorta comprising both the coeliac and the superior mesenteric arteries was dissected free after careful removal of the left adrenal and division of the lumbar arteries. The pancreas was dissected free from the colon and the duodenum at the root of the mesentery, and the vessels of the mesentery isolated and divided, taking care not to injure ganglionic tissue around the superior mesenteric artery. The vagus nerves were then identified around the oesophagus and the lesser omentum in which the vagal branches to the pancreas run was dissected free as close to the lesser curvature of the stomach as possible. The portal vein was isolated at the porta hepatis and the remaining structures in the hepatoduodenal ligament divided. A feeding tube (Argyll No. 5) was positioned in the pancreatic duct. The pancreas was isolated from the proximal duodenum and from the inferior caval vein, and after excision of the aortic segment and division of the portal vein and the vagus nerves the preparation could be lifted out. The aorta was catheterized immediately after isolation and perfusion started. The duration of ischemia was a matter of a few seconds. The preparation was placed in the
247
previously described perfusion system [10], and perfused 'once-through' with a synthetic medium, supplemented with 12-15% washed human or bovine erythrocytes as previously described [11,12]. The perfusion medium consisted of a Krebs-Ringer bicarbonate solution containing in addition 0.1% human albumin, 5% Dextran T-70, 5 mmol/1 glucose, aprotinine and a mixture of amino acids (total concentration 5 mmol/1) [12]. Perfusion flow was kept constant at approx. 0.4 m l / m i n per g whereby an adequate oxygen supply to the preparation was ensured [11]. Oxygen consumption and acid-base status of the perfusion medium were monitored at regular intervals using the ABL-2 automatic equipment (Radiometer, Copenhagen, Denmark). In six perfusion experiments, the 'once-through' perfusion system was changed for 20-30-rain periods to a recirculation system by leading the venous effluent to a 40 ml reservoir to which the arterial line was also connected. The reservoir was connected to a Radiometer T T T 60 titrator and ABU 13 autoburette, by which a pH at approx. 7.4 could be maintained. The glucose concentration of the reservoir perfusate was monitored every 10 min using an 'Eytone' Reflectance Meter * (Ames), and small amounts of glucose added if necessary. At the start of recirculation 1-2 ml of either non-immune rabbit serum or VIP-antiserum 5600-9 was added to the reservoir. This antiserum, which recognizes the C-terminal part of the VIP molecule, was raised against natural porcine VIP coupled to bovine serum albumin [14]. It had a binding capacity of 0.7 nmol V I P / m l and a binding energy of 1.3 x 1011 l / m o l . The vagus nerves were threaded through a bipolar tunnel electrode [2], and electrical stimulations were carried out using 8 mA square wave impulses of 4 ms duration at 1-32 Hz for 5 min. The perfusate from the portal vein was collected for 1-min periods in chilled tubes (Minisorp, NUNC, Roskilde, Denmark) and centrifuged within a few minutes at 4°C. The supernatant was decanted and frozen immediately, pending radioimmunoassay for VIP [14]. The recovery of VIP added to perfusate was linear and within + 10% of the expected values. Pancreatic juice was led to a drop-counter connected to a microprocessor capable of calculating flow rate from a fixed drop size and determination of the time interval between the drop which just fell and the preceding drop. Flow, calculated this way, was continuously recorded. Furthermore the secretion was collected for 5- or 10-min periods, and analysed for volume, and bicarbonate and protein concentration as previously described [10]. Student's t-test for paired data and analysis of variance (for evaluation of minute to minute changes) were used for statistical evaluation of the results. Differences resulting in P-values less than 0.05 were considered significant.
Immunohistochemistry Catheters were introduced into the major pancreatic arteries of anaesthetized pigs and the pancreas perfused with ice-cold saline followed by a 4% solution of paraformaldehyde in 0.1 m o l / l sodium phosphate at pH 7.4. The tissues were postfixed for 24 h and transferred to sucrose (20% in 0.1 mol/1 sodium phosphate,
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p H 7.4) overnight. They were then frozen in melting freon-22 and sections of 10/~m cut on a cryostat. For immunohistochemistry the unlabelled antibody peroxidase-antiperoxidase (PAP) technique was applied as described by Sternberger [15]. VIP antiserum code nr 5600-9 was diluted 1 : 400 and 1 : 1600. Chromatography
Fresh pancreatic tissue was obtained from anaesthetized pigs and extracted using an acid/ethanol technique (Method II, [16]), and the extract subjected to Sephadex G-50 gel filtration in 0.5 m o l / l acetic acid [16]. The column effluent was freeze-dried and subjected to VIP radioimmunoassay [14]. Effluent from the perfused pancreas obtained during vagal stimulation was applied directly to 1000 × 16 mm Sephadex G-50 (fine grade) columns eluted with a 0.125 mol/1 ammonium bicarbonate solution containing 0.1 mol/1 NaC1 and 0.1% human serum albumin (Behringwerke, M a r b u r g / L a h n , F.R.G.). Both columns were calibrated with VIP, and 22NaC1 and ~25I-labelled albumin were added to all samples for internal calibration. Elution position is referred to by the coefficient of distribution K,~ = ( V e - V o ) / V i, where Ve is the elution volume of the substance in question, Vo the excluded volume and V~ the available inner volume, determined as the difference between the elution volumes of 22Na+ and 125I-labelled albumin.
Results Fig. 1 shows the results of 12 Hz electrical stimulations of the vagus nerves in 15 perfusion experiments. Vagal stimulation caused a 24-fold increase in the average flow of juice (5 min volume expressed as m l / m i n ) and a 38-fold and a 70-fold increase in bicarbonate and protein output, respectively. At the same time VIP output increased from mean values between 0.10 and 0.13 p m o l / m i n to maximally 1.4 p m o l / m i n . The effluent concentrations of VIP in the basal state ranged between 0 and 25 pmol/1, whereas the peak values ranged between 26 and 210 pmol/1. The magnitude of the response was frequency-dependent as shown in Fig. 2, which illustrates the parallel changes of exocrine secretion and VIP output with varying frequency of stimulation. Significant increases in VIP output and exocrine secretion were noted already at 1 Hz (not shown) whereas maximum responses were seen between 12 and 16 Hz. The VIP and exocrine responses were highly reproducible. Thus the coefficients of variation of the magnitude of the VIP output and the exocrine responses were below 10% in an experiment in which six identical repeated stimulations were carried out. Simultaneous continuous recording of flow rate of pancreatic juice and measurement of VIP output during vagal stimulation were carried out in 8 perfusion experiments. The results showed a remarkable parallelism between the time course of pancreatic flow of juice and VIP output (Fig. 3). The effect of vagal stimulation with or without the addition to the arterial line of atropine to a calculated concentration of 1 / t m o l / 1 (during the stimulation as well as for 10 min before and after) was studied in seven perfusion experiments (Fig. 4). The
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Fig. 1. Effect of electrical vagal stimulation (12 Hz, 4 ms, 8 mA) on VIP output and exocrine secretion from the isolated perfused pig pancreas. Data are presented as mean +S.E.M. from 15 perfusion experiments. Open circles and vertical asterisks indicated changes which are significantly different from values in the prestimulation period. 0,3 0,2
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Fig. 3. Effect of electrical vagal stimulation on VIP output and flow of juice from the isolated perfused pig pancreas. Flow was recorded continuously, and venous effluent sampled for 1-min periods for VIP determination. The flow rate corresponding to the mid of these 1-min periods was read off the recordings for construction of the mean curve shown in the top panel. Mean _+S.E.M. of 8 perfusion experiments. Open circles denote values which are significantly different from baseline values.
p r o t e i n secretion was greatly r e d u c e d b y a t r o p i n e in the b a s a l state as well as d u r i n g s t i m u l a t i o n s (to 10 a n d 14%, respectively) whereas b i c a r b o n a t e o u t p u t increased (by 21%). A l s o flow rate a n d V I P o u t p u t increased after a d d i t i o n of atropine, particularly d u r i n g the last m i n u t e s of stimulation. In five e x p e r i m e n t s the effects of vagal s t i m u l a t i o n were studied before a n d after a d d i t i o n to the arterial line of h e x a m e t h o n i u m to a c a l c u l a t e d c o n c e n t r a t i o n of 30 / ~ m o l / l . This a b o l i s h e d in all cases c o m p l e t e l y the increases in V I P o u t p u t a n d exocrine secretion ( d a t a not shown). In five p e r f u s i o n e x p e r i m e n t s the following p r o t o c o l was followed: first 4 H z vagal stimulation, then a d d i t i o n of 1 /~mol/1 a t r o p i n e to the p e r f u s a t e a n d a r e p e a t e d 4 H z stimulation; then recirculation for 2 0 - 3 0 min with 1 - 2 ml n o n - i m m u n e r a b b i t s e r u m a d d e d to the reservoir followed b y a n o t h e r 4 H z stimulation; then recirculation for 2 0 - 3 0 min with 1 ml of a n t i s e r u m 5600-9 a d d e d to the
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Fig. 4. Effect of 10 6 mol/1 atropine on V1P output and exocrine secretion during electrical vagal stimulation of the isolated perfused pig pancreas. Results are presented as mean _+S.E.M. of 7 perfusion experiments. Frequencies of stimulation between 4 and 12 Hz. Flow of juice presented as in Fig. 3. Open circles denote values which are significantly different from prestimulation values. Asterisks denote significant differences between the paired experiments with (upper panel) or without (lower panel) atropine. reservoir and a subsequent 4 H z stimulation; and finally 30 min once-through perfusion without serum addition followed by a 4 H z stimulation. The results of these experiments are shown in Fig. 5. Preceding recirculation with VIP antiserum inhibited the exocrine response to vagal stimulation to 58 + 14% of the values obtained after recirculation with n o n - i m m u n e serum. After the prolonged 'wash-out' period without recirculation or antiserum addition the exocrine response to vagal stimulation increased again to 77 + 10% of the control value.
Immunohistochemistry Fig. 6 shows five sections of a pig pancreas stained for VIP using the imm u n o p e r o x i d a s e technique. There is a dense innervation around intercalated ducts (part e) as well as larger ducts (parts b and d); heavily stained intrapancreatic
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Fig. 5. (A) Exocrine response to vagal stimulation of the isolated perfused pig pancreas after control serum or VIP-antiserum. Results of a single representative experiment. A direct reproduction of continuous recording of flow of juice. See text for details of protocol. Note the direction of the time axis from right to left. (B) Exocrine response to electrical vagal stimulation of the isolated perfused pig pancreas after control serum or VIP-antiserum. Exocrine secretion was collected for 5 min periods, and flow expressed as m l / m i n . Mean ± S.E.M. of 5 perfusion experiments. See text for details of protocol.
Fig. 6. Immunoperoxidase staining of VIP-nerves in the pig pancreas. (a) Intrapancreatic ganglion with some VIP immunoreactive nerve cell bodies and many VIP fibres; x 140. (b) Interlobular, excretory duct with VIP-fibres (large arrows) and fibres around an interlobular duct (small arrow), x 160. (c) Large nerve trunk with VIP fibres; x 215. (d) VIP-fibres along minor intralobular duct; x 215. (e) VIP-fibres along an intercalated duct; x 240.
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Fig. 7. Immunoperoxidase staining for rabbit gamma globulin in the perfused pancreas after perfusion for 30 min with V1P-antiserum. Stained fibres encircling a larger duct. Magnification, x 200.
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Fig. 8. Gel filtration profiles of VIP. (A) and (B) Synthetic VIP. (C) Ethanol extract of pig pancreas. (D) Effluent from the isolated perfused pig pancreas obtained during vagal stimulation. (A) and (C) 50 x 1000 mm columns eluted with 0.5 m o l / l acetic acid. (B) and (D) 16x 1000 mm columns eluted with 0.125 mol/1 ammonium bicarbonate, containing also NaC1 (0.1 mol/1), human serum albumin (0.1%), and thiomersal (0.6 mmol/l). Both columns operated at 4 o C at a flow rate of 0.03 × bed volume/h.
255 ganglia with large nerve trunks are also seen (parts a and c). In two recirculation experiments biopsies were cut out of the pancreas 10 rain after a 30 min recirculating perfusion with VIP-antiserum as above. The biopsies were fixed in paraformaldehyde, and processed for immunoperoxidase staining as above but without incubation with VIP-antiserum. One of the sections is shown in Fig. 7. There was a clear immunostaining of beaded nerve fibres in relation to pancreatic ducts with an appearance very similar to that shown in Fig. 6. Pancreatic biopsies obtained from the same pancreases before antiserum perfusion and subjected to the same immunoperoxidase procedure (again without application of VIP antiserum) did not show such structures.
Chromatographic profile The gel filtration profile of immunoreactive VIP in the pig pancreas is shown in Fig. 8, which shows that the vast majority of the immunoreactivity co-eluted with synthetic or pure natural VIP. Also immunoreactive VIP present in the effluent from the perfused pancreas during vagal stimulation co-eluted with synthetic VIP.
Discussion
The following lines of evidence support the conclusion that VIP is directly involved in the neuroeffector transmission of the atropine-resistant pancreatic exocrine response to electrical stimulation of the vagus nerves: (1) VIP-containing nerve fibres are found in close relation to the ducts in the pig pancreas. Earlier reports have documented the presence of VIP-containing nerve fibres in pancreas from a number of species [5-8] including pigs, but their particular relation to the exocrine tissue was not reported. An association of VIP-nerves with the main pancreatic duct in cats was noted by Sundler et al. [7]. In this report we can show that in the pig pancreas VIP fibres are typically seen in association with intercalated ducts as well as larger ducts. The fibres had a characteristic beaded appearance suggesting that the fibres might release VIP from the beads 'en passant'. Since the majority of the fluid and bicarbonate is believed to be produced by the duct cells [17], the association between the VIP nerves and the ducts would be a prerequisite for VIP to be responsible for this secretion. The gel filtration experiments show that the pancreatic VIP in all probability is identical to intestinal or central nervous VIP, indicating that in the pancreas, the VIP precursor is being processed in the same way as in other tissues where this has been examined [18]. (2) In the pig, VIP stimulates pancreatic bicarbonate and fluid secretion with the same efficacy as secretin [9] whereas its potency is about 1/100 of that of secretin. Thus the effluent concentrations determined in this study (maximally 270 pmol) would not be expected to be high enough to influence exocrine secretion. However, it is unlikely that the initial space of distribution of newly neuronally released VIP is as large as that of VIP administered intravascularly. Considering the close association between VIP-containing nerve fibres and ducts demonstrated here, it can be safely
256 assumed, that the local concentrations of VIP may be several orders of magnitude higher than those reached in venous effluent from the preparation. VIP, the half-maximally effective concentration of which for pancreatic exocrine secretion is about 1 nmol/1 in the pig [9], is, therefore, sufficiently potent to be responsible for this secretion after vagal stimulation. (3) A peptide, which by gel filtration is indistinguishable from VIP, is being released from pancreatic nerves during electrical stimulations, a fact which is evident from the present investigation. There were initial reports on a location of immunoreactive VIP to pancreatic endocrine cells [19], but later investigations have shown, that with antisera directed against the C-terminal part of the VIP molecule, only nerve fibres can be detected in the normal pancreas [6]. We have used such an antiserum in the present investigation, and it is therefore likely, that all of the VIP determined in the effluent is of neuronal origin. (4) Pancreatic secretion of fluid and bicarbonate and VIP release were closely parallel under all circumstances tested. This particularly concerns the atropine resistance of both, the main reason for suspecting a non-cholinergic, peptidergic transmission in the vagal effects on. fluid and bicarbonate secretion, but is also illustrated by the similar effect on both of hexamethonium~ the parallelism of the time course, and the frequency dependence. (5) The exocrine response in the isolated preparation is similar to the response obtained in vivo. The exocrine responses to vagal stimulation obtained with this preparation were similar to those obtained with electrical vagal stimulations in anaesthesized pigs [2]. This proves that the vagal effect is independent of secretin or other circulating hormones, which may be present in plasma but not in the synthetic perfusate. In addition, the effect was independent of changes in pancreatic blood flow [20], since our perfusion flow was kept constant throughout. (6) Finally, VIP-antiserum inhibited the effect of vagal stimulation on the fluid secretion. Since a potent VIP-antagonist is not yet available, we tried to block the supposed action of VIP with a potent high-titre VIP-antiserum [14]. Preliminary experiments showed that single-pass perfusion of the pancreas with VIP antiserum diluted 50-100 times with perfusion medium was without effect on the vagus effect on secretion. We therefore designed the recirculation experiments to increase the probability that anti-VIP ),-globulins could extravasate and reach the interstitial tissue around the ducts and VIP containing nerves. That such extravasation is probable is evident from the finding that pancreatic capillaries are highly fenestrated [21]. Fig. 7 shows immunohistochemical evidence that the antibodies were actually present in the interstitial tissues. The fact that the exocrine response to vagal stimulation was suppressed by addition of VIP antiserum and increased again after prolonged once-through perfusion supports the conclusion that the inhibitory effect of antiserum addition was indeed due to a specific blocking of neuronally released VIP. Thus our results strongly suggest that VIP functions as a final transmitter of the vagal effect on pancreatic secretion of fluid and bicarbonate. The atropine resistance of the exocrine response excludes participation of acetylcholine acting on muscarinic receptors, although such interaction is likely to occur when atropine is not present.
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Possible co-existence of VIP and acetylcholine in the same nerve fibres [22] points to the importance of this interaction. Pancreatic sympathetic fibres would not be expected to participate, since electrical stimulation of the splanchnic nerves innervating the pig pancreas is not associated with stimulated exocrine secretion [13]; on the contrary, simultaneous stimulation of both the vagus nerves and the splanchnic nerves Strongly inhibits the exocrine response to vagal stimulation [2]. The possible participation of other pancreatic neuropeptides should be considered, however. Nerves containing cholecystokinin-related peptides are present in the pig pancreas, but are mainly found to innervate the islets [23]; furthermore, we have never been able to detect CCK-like immunoreactivity in the effluent from the isolated perfused pancreas during vagal stimulation (Rehfeld and Hoist, unpublished results). We therefore find CCK-like peptides unlikely candidates. Substance P-immunoreactive nerves are not found in the exocrine tissue [6]; substance P is not an agonist for the secretion of fluid and bicarbonate in the pig pancreas, and it is not released during vagal stimulation (Schaffalitzky de Muckadell and Hoist, unpublished results). The only other neuropeptide with a significant effect on pancreatic exocrine secretion is gastrin-releasing polypeptide (GRP) which is found in intrapancreatic nerves, and which is released during electrical vagal stimulation [24]. GRP particularly potently stimulates enzyme secretion which is strongly inhibited by atropine. This, and the 50% reduction of the fluid response caused by VIP antiserum, support the notion of a predominant role of VIP in the transmission of the vagal effect. Again, however, it is likely that the two systems interact in the intact organism. It was recently reported, that the human VIP precursor contains another peptide, PHM-27, the structure of which greatly resembles the porcine neuropeptide PHI and shows extensive homology with VIP itself [25]. Possibly the PHI of the pig corresponds to PHM-27 of the human precursor, and PHI might therefore be released concomitantly with VIP during nerve stimulation, if the precursor is indeed being processed to release these two peptides. In three perfusion experiments, however, we studied the effect of synthetic PHI obtained from Peninsula Laboratories (Belmont, California 94002) in concentrations up to 10 nmol/l and found it to be 100-fold less potent than VIP on pancreatic exocrine secretion. We think that the present series of experiments have brought us as far as it is presently possible in trying to establish the role of VIP in pancreatic neuro-effector transmission. Our results corroborate further the spreading acceptance of peptides as potential transmitters of neuronal activity.
Acknowledgements This study was supported by grants from the Danish Medical Research Council and P. Carl Petersens Fond. Vibeke Jerris, Anita Hansen, Sorn Haagen Nielsen and Letty Klarskow performed excellent technical assistance. Professor L.-I. Larsson assisted at the immunohistochemical procedures.
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References 1 Hickson, J.C.D., The secretion of pancreatic juice in response to stimulation of the vagus nerves in the pig, J. Physiol., 206 (1970) 257-297. 2 Holst, J.J., Schaffalitzky de Muckadell, O.B. and Fahrenkrug, J., Nervous control of pancreatic exocrine secretion in pigs, Acta Physiol. Scand., 105 (1979) 33-51. 3 Fahrenkrug, J., Galbo, H., Hoist, J.J. and Schaffalitzky de Muckadell, O.B., The influence of the autonomic nervous system on the release of Vasoactive Intestinal Polypeptide from the gastrointestinal tract in pigs, J. Physiol., 280 (1978) 405-422. 4 Fahrenkrug, J., Schaffalitzky de Muckadell, O.B., Hoist, J.J. and Jensen, S.L., Vasoactive intestinal polypeptide in vagally mediated pancreatic secretion of fluid and HCO 3, Am. J. Physiol., 237 (1979) E535-E540. 5 Larsson, L.-I., Fahrenkrug, J., Hoist, J.J. and Schaffalitzky de Muckadell, O.B., Innervation of the pancreas by vasoactive intestinal polypeptide (VIP) immunoreactive nerves, Life Sci., 22 (1978) 773-780. 6 Larsson, L.-I., Polak, J.M., Buffa, R., Sundler, F. and Solcia, E., On the immunocytochemical localization of the vasoactive intestinal polypeptide, J. Histochem. Cytochem., 27 (1979) 936-938. 7 Sundler, F., Alumets, J., Hhkanson, R., Fahrenkrug, J. and Schaffalitzky de Muckadell, O.B., Peptidergic nerves in the pancreas, Histoche.mistry, 55 (1978) 173-176. 8 Bishop, A.E., Polak, J.M., Green, I.C., Bryant, M.G. and Bloom, S.R., The location of VIP in the pancreas of man and rat, Diabetologia, 18 (1980) 73-78. 9 Jensen, S.L., Fahrenkrug, J., Holst, J.J., Nielsen, O.V. and Schaffalitzky de Muckadell, O.B., Secretory effects of VIP on isolated perfused porcine pancreas, Am. J. Physiol., 235 (1978) E387-E391. 10 Jensen, S.L., Fahrenkrug, J., Holst, J.J., Kuhl, C., Nielsen, O.V. and Schaffalitzky de Muckadell, O.B., Secretory effects of secretin on isolated perfused porcine pancreas, Am. J. Physiol., 235 (1978) E381-E386. 11 Hoist, J.J., Jensen, S.L., Nielsen, O.V. and Schwartz, T.W., Oxygen supply, oxygen consumption, and endocrine and exocrine secretions of the isolated, perfused, porcine pancreas, Acta Physiol. Scand., 109 (1980) 7-13. 12 Holst, J.J., Lauritsen, K.B., Jensen, S.L., Schaffalitzky de Muckadell, O.B. and Nielsen, O.V., Secretin release from the isolated, vascularly perfused pig duodenum, J. Physiol., 318 (1981) 327-337. 13 Hoist, J.J., Jensen, S.L., Knuhtsen, S. and Nielsen, O.V., Autonomic nervous control of pancreatic somatostatin secretion, Am. J. Physiol., 245 (1983) E542-E548. 14 Fahrenkrug, J. and Schaffalitzky de Muckadell, O.B., Radioimmunoassay of vasoactive intestinal polypeptide (VIP) in plasma, J. Lab. Clin. Med., 89 (1977) 1379-1388. 15 Sternberger, L., Immunohistochemistry. Prentice Hall, Englewood Cliffs, NY, 1974, pp. 129-171. 16 Newgard, C.B. and Hoist, J.J., Heterogeneity of somatostatin like immunoreactivity in extracts of porcine, canine and human pancreas, Acta Endocrinol. (Kbh.), 98 (1981) 564-572. 17 Scratcherd, T., Hutson, D. and Case, R.M., The secretion of fluid and electrolytes from the pancreas, Phil. Trans. R. Soc. Lond. B, 296 (1981) 167-178. 18 Fahrenkrug, J., Vasoactive intestinal polypeptide: measurement, distribution, and putative neurotransmitter function, Digestion, 19 (1979) 149-169. 19 Buffa, R., Capella, C., Solcia, E., Frigerio, B. and Said, S.I., Vasoactive Intestinal Peptide (VIP) cells in the pancreas and gastro-intestinal mucosa, Histochemistry, 50 (1977) 217-227. 20 Lenninger, S., Effects of acetylcholine and papaverine on the secretion and blood flow from the pancreas of the cat, Acta Physiol. Scand., 89 (1973) 260-268. 21 Haraldsson, B., Rippe, B., Moxham, B.J. and Folkow, B., Permeability of fenestrated capillaries in the isolated pig pancreas with effects of bradykinin and histamine, as studied by simultaneous registration of filtration and diffusion capacities, Acta Physiol. Scand., 114, 67-74. 22 Lundberg, J.M., Evidence for the co-existence vasoactive intestinal polypeptide (V1P) and acetylcholine in neurons of cat exocrine glands. Morphological, biochemical and functional studies, Acta Physiol. Scand., suppl., 496 (1981) 1-57. 23 Rehfeld, J.F., Larsson, L.-I., Goltermann, N.R., Schwartz, T.W., Hoist, J.J., Jensen, S.L and Morley,
259 J.S., Neural regulation of pancreatic hormone secretion by the C-terminal tetrapeptide of CCK, Nature, 284 (1980) 33-38. 24 Hoist, J.J., Knuhtsen, S., Knigge, U., Jensen, S.L., Nielsen, O.V. and Larssom L.-I., Gastrin Releasing Polypeptide (GRP-Mammalian Bombesin) - a new peptide transmitter in the pancreas, Digestion, 25 (1982) 37-38. 25 Itoh, N., Obata, K., Yanaihara, N. and Okamoto, H., Human preprovasoactive intestinal polypeptide contains a novel PH1-27-1ike peptide, PHM-27~ Nature, 304 (1983) 547-549.
245
RPT 00279
Vasoactive intestinal polypeptide (VIP) in the pig pancreas: role of VIPergic nerves in control of fluid and bicarbonate secretion Jens J. Hoist ~, Jan Fahrenkrug 2, Svend Knuhtsen (, Steen L. Jensen 3, S. Seier Poulsen 4 and O. Vagn Nielsen 3 I Institute of Medical Physiology C and 4 Department of Anatomy B, The Panum Institute, University of Copenhagen, 2 Department of Clinical Chemistry, Bispebjerg Hospital, and 3 Department of Surgery C, Rigshospitalet, Copenhagen, Denmark (Received 3 January 1984; revised manuscript received 1 February 1984; accepted for publication 16 March 1984)
Summa~ Vasoactive intestinal polypeptide (VIP) in the pig pancreas is localized to nerves, many of which travel along the pancreatic ducts. VIP stimulates pancreatic fluid and bicarbonate secretion like secretin. Electrical vagal stimulation in the pig causes an atropine-resistant profuse secretion of bicarbonate-rich pancreatic juice. In an isolated perfused preparation of the pig pancreas with intact vagal nerve supply, electrical vagal stimulation caused an atropine-resistant release of VIP, which accurately parallelled the exocrine secretion of juice and bicarbonate. Perfusion of the pancreas with a potent VIP-antiserum inhibited the effect of vagal stimulation on the exocrine secretion. It is concluded, that VIP is responsible for (at least part of) the neurally controlled fluid and bicarbonate secretion from the pig pancreas. peptidergic nerves; perfused pancreas; antiserum treatment
Introduction In the pig, electrical stimulation of the vagus nerves causes an atropine-resistant, profuse secretion from the pancreas of fluid and bicarbonate [1,2], which is indepenAddress all correspondence to: Jens J. Hoist, MD, Institute of Medical Physiology C, The Panum Institute, University of Copenhagen, Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark. Telephone: (01) 35 79 00 ext. 2541. 0167-0115/84/$03.00 © 1984 Elsevier Science Publishers B.V.
246 dent of increases in the concentration of secretin in plasma [2]. Under the same conditions a splanchnic release of the neuropeptide, vasoactive intestinal polypeptide (VIP), can be demonstrated [3], and we have shown that part of the splanchnic VIP output is derived from the pancreas [4]. Furthermore, in the pancreas VIP is exclusively localized to nerves [5-8], and (porcine) VIP is a full secretin-like agonist on the pig pancreas [9]. We therefore hypothesized that neuronally released pancreatic VIP was responsible for the atropine-resistant fluid and bicarbonate secretion in response to vagal stimulation in this species [2,4]. To examine this hypothesis in detail, we developed an experimental model, the isolated perfused pig pancreas with intact vagal nerve supply. This model allowed us to study in a truly isolated system concomitant changes in the pancreatic exocrine secretion and changes in the release into the perfusate of this neuropeptide during parasympathetic nerve stimulation with and without addition of relevant pharmacological blockers. Furthermore, it was feasible to study the effect of the only specific VIP-antagonist available, namely VIP-antiserum. In addition, we studied the chromatographic profile of VIP in extracts of pig pancreas as well as in perfusate obtained during nerve stimulation, and we studied in detail the localization of intrapancreatic VIP-containing nerves using immunohistochemistry.
Materials and Methods
22 pigs, Danish Landrace, weighing 14-16 kg, were used as donor animals. They were fasted for 12 h but had free access to water. Premedication was not given. Anaesthesia was induced with 2.5% halothane and after the onset of surgical anaesthesia, halothane was substituted by intravenous infusion of chloralose (No. 2420, Merck, Darmstadt, F.R.G., 100 mg/kg). The operative procedure and the perfusion system are related to our previously described method for isolation and perfusion of the (denervated) pig pancreas [10-13]. The abdomen was opened through a long mid-line incision. The spleen was removed. An 8-10 cm long segment of the aorta comprising both the coeliac and the superior mesenteric arteries was dissected free after careful removal of the left adrenal and division of the lumbar arteries. The pancreas was dissected free from the colon and the duodenum at the root of the mesentery, and the vessels of the mesentery isolated and divided, taking care not to injure ganglionic tissue around the superior mesenteric artery. The vagus nerves were then identified around the oesophagus and the lesser omentum in which the vagal branches to the pancreas run was dissected free as close to the lesser curvature of the stomach as possible. The portal vein was isolated at the porta hepatis and the remaining structures in the hepatoduodenal ligament divided. A feeding tube (Argyll No. 5) was positioned in the pancreatic duct. The pancreas was isolated from the proximal duodenum and from the inferior caval vein, and after excision of the aortic segment and division of the portal vein and the vagus nerves the preparation could be lifted out. The aorta was catheterized immediately after isolation and perfusion started. The duration of ischemia was a matter of a few seconds. The preparation was placed in the
247
previously described perfusion system [10], and perfused 'once-through' with a synthetic medium, supplemented with 12-15% washed human or bovine erythrocytes as previously described [11,12]. The perfusion medium consisted of a Krebs-Ringer bicarbonate solution containing in addition 0.1% human albumin, 5% Dextran T-70, 5 mmol/1 glucose, aprotinine and a mixture of amino acids (total concentration 5 mmol/1) [12]. Perfusion flow was kept constant at approx. 0.4 m l / m i n per g whereby an adequate oxygen supply to the preparation was ensured [11]. Oxygen consumption and acid-base status of the perfusion medium were monitored at regular intervals using the ABL-2 automatic equipment (Radiometer, Copenhagen, Denmark). In six perfusion experiments, the 'once-through' perfusion system was changed for 20-30-rain periods to a recirculation system by leading the venous effluent to a 40 ml reservoir to which the arterial line was also connected. The reservoir was connected to a Radiometer T T T 60 titrator and ABU 13 autoburette, by which a pH at approx. 7.4 could be maintained. The glucose concentration of the reservoir perfusate was monitored every 10 min using an 'Eytone' Reflectance Meter * (Ames), and small amounts of glucose added if necessary. At the start of recirculation 1-2 ml of either non-immune rabbit serum or VIP-antiserum 5600-9 was added to the reservoir. This antiserum, which recognizes the C-terminal part of the VIP molecule, was raised against natural porcine VIP coupled to bovine serum albumin [14]. It had a binding capacity of 0.7 nmol V I P / m l and a binding energy of 1.3 x 1011 l / m o l . The vagus nerves were threaded through a bipolar tunnel electrode [2], and electrical stimulations were carried out using 8 mA square wave impulses of 4 ms duration at 1-32 Hz for 5 min. The perfusate from the portal vein was collected for 1-min periods in chilled tubes (Minisorp, NUNC, Roskilde, Denmark) and centrifuged within a few minutes at 4°C. The supernatant was decanted and frozen immediately, pending radioimmunoassay for VIP [14]. The recovery of VIP added to perfusate was linear and within + 10% of the expected values. Pancreatic juice was led to a drop-counter connected to a microprocessor capable of calculating flow rate from a fixed drop size and determination of the time interval between the drop which just fell and the preceding drop. Flow, calculated this way, was continuously recorded. Furthermore the secretion was collected for 5- or 10-min periods, and analysed for volume, and bicarbonate and protein concentration as previously described [10]. Student's t-test for paired data and analysis of variance (for evaluation of minute to minute changes) were used for statistical evaluation of the results. Differences resulting in P-values less than 0.05 were considered significant.
Immunohistochemistry Catheters were introduced into the major pancreatic arteries of anaesthetized pigs and the pancreas perfused with ice-cold saline followed by a 4% solution of paraformaldehyde in 0.1 m o l / l sodium phosphate at pH 7.4. The tissues were postfixed for 24 h and transferred to sucrose (20% in 0.1 mol/1 sodium phosphate,
248
p H 7.4) overnight. They were then frozen in melting freon-22 and sections of 10/~m cut on a cryostat. For immunohistochemistry the unlabelled antibody peroxidase-antiperoxidase (PAP) technique was applied as described by Sternberger [15]. VIP antiserum code nr 5600-9 was diluted 1 : 400 and 1 : 1600. Chromatography
Fresh pancreatic tissue was obtained from anaesthetized pigs and extracted using an acid/ethanol technique (Method II, [16]), and the extract subjected to Sephadex G-50 gel filtration in 0.5 m o l / l acetic acid [16]. The column effluent was freeze-dried and subjected to VIP radioimmunoassay [14]. Effluent from the perfused pancreas obtained during vagal stimulation was applied directly to 1000 × 16 mm Sephadex G-50 (fine grade) columns eluted with a 0.125 mol/1 ammonium bicarbonate solution containing 0.1 mol/1 NaC1 and 0.1% human serum albumin (Behringwerke, M a r b u r g / L a h n , F.R.G.). Both columns were calibrated with VIP, and 22NaC1 and ~25I-labelled albumin were added to all samples for internal calibration. Elution position is referred to by the coefficient of distribution K,~ = ( V e - V o ) / V i, where Ve is the elution volume of the substance in question, Vo the excluded volume and V~ the available inner volume, determined as the difference between the elution volumes of 22Na+ and 125I-labelled albumin.
Results Fig. 1 shows the results of 12 Hz electrical stimulations of the vagus nerves in 15 perfusion experiments. Vagal stimulation caused a 24-fold increase in the average flow of juice (5 min volume expressed as m l / m i n ) and a 38-fold and a 70-fold increase in bicarbonate and protein output, respectively. At the same time VIP output increased from mean values between 0.10 and 0.13 p m o l / m i n to maximally 1.4 p m o l / m i n . The effluent concentrations of VIP in the basal state ranged between 0 and 25 pmol/1, whereas the peak values ranged between 26 and 210 pmol/1. The magnitude of the response was frequency-dependent as shown in Fig. 2, which illustrates the parallel changes of exocrine secretion and VIP output with varying frequency of stimulation. Significant increases in VIP output and exocrine secretion were noted already at 1 Hz (not shown) whereas maximum responses were seen between 12 and 16 Hz. The VIP and exocrine responses were highly reproducible. Thus the coefficients of variation of the magnitude of the VIP output and the exocrine responses were below 10% in an experiment in which six identical repeated stimulations were carried out. Simultaneous continuous recording of flow rate of pancreatic juice and measurement of VIP output during vagal stimulation were carried out in 8 perfusion experiments. The results showed a remarkable parallelism between the time course of pancreatic flow of juice and VIP output (Fig. 3). The effect of vagal stimulation with or without the addition to the arterial line of atropine to a calculated concentration of 1 / t m o l / 1 (during the stimulation as well as for 10 min before and after) was studied in seven perfusion experiments (Fig. 4). The
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p r o t e i n secretion was greatly r e d u c e d b y a t r o p i n e in the b a s a l state as well as d u r i n g s t i m u l a t i o n s (to 10 a n d 14%, respectively) whereas b i c a r b o n a t e o u t p u t increased (by 21%). A l s o flow rate a n d V I P o u t p u t increased after a d d i t i o n of atropine, particularly d u r i n g the last m i n u t e s of stimulation. In five e x p e r i m e n t s the effects of vagal s t i m u l a t i o n were studied before a n d after a d d i t i o n to the arterial line of h e x a m e t h o n i u m to a c a l c u l a t e d c o n c e n t r a t i o n of 30 / ~ m o l / l . This a b o l i s h e d in all cases c o m p l e t e l y the increases in V I P o u t p u t a n d exocrine secretion ( d a t a not shown). In five p e r f u s i o n e x p e r i m e n t s the following p r o t o c o l was followed: first 4 H z vagal stimulation, then a d d i t i o n of 1 /~mol/1 a t r o p i n e to the p e r f u s a t e a n d a r e p e a t e d 4 H z stimulation; then recirculation for 2 0 - 3 0 min with 1 - 2 ml n o n - i m m u n e r a b b i t s e r u m a d d e d to the reservoir followed b y a n o t h e r 4 H z stimulation; then recirculation for 2 0 - 3 0 min with 1 ml of a n t i s e r u m 5600-9 a d d e d to the
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Immunohistochemistry Fig. 6 shows five sections of a pig pancreas stained for VIP using the imm u n o p e r o x i d a s e technique. There is a dense innervation around intercalated ducts (part e) as well as larger ducts (parts b and d); heavily stained intrapancreatic
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Fig. 5. (A) Exocrine response to vagal stimulation of the isolated perfused pig pancreas after control serum or VIP-antiserum. Results of a single representative experiment. A direct reproduction of continuous recording of flow of juice. See text for details of protocol. Note the direction of the time axis from right to left. (B) Exocrine response to electrical vagal stimulation of the isolated perfused pig pancreas after control serum or VIP-antiserum. Exocrine secretion was collected for 5 min periods, and flow expressed as m l / m i n . Mean ± S.E.M. of 5 perfusion experiments. See text for details of protocol.
Fig. 6. Immunoperoxidase staining of VIP-nerves in the pig pancreas. (a) Intrapancreatic ganglion with some VIP immunoreactive nerve cell bodies and many VIP fibres; x 140. (b) Interlobular, excretory duct with VIP-fibres (large arrows) and fibres around an interlobular duct (small arrow), x 160. (c) Large nerve trunk with VIP fibres; x 215. (d) VIP-fibres along minor intralobular duct; x 215. (e) VIP-fibres along an intercalated duct; x 240.
254
Fig. 7. Immunoperoxidase staining for rabbit gamma globulin in the perfused pancreas after perfusion for 30 min with V1P-antiserum. Stained fibres encircling a larger duct. Magnification, x 200.
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255 ganglia with large nerve trunks are also seen (parts a and c). In two recirculation experiments biopsies were cut out of the pancreas 10 rain after a 30 min recirculating perfusion with VIP-antiserum as above. The biopsies were fixed in paraformaldehyde, and processed for immunoperoxidase staining as above but without incubation with VIP-antiserum. One of the sections is shown in Fig. 7. There was a clear immunostaining of beaded nerve fibres in relation to pancreatic ducts with an appearance very similar to that shown in Fig. 6. Pancreatic biopsies obtained from the same pancreases before antiserum perfusion and subjected to the same immunoperoxidase procedure (again without application of VIP antiserum) did not show such structures.
Chromatographic profile The gel filtration profile of immunoreactive VIP in the pig pancreas is shown in Fig. 8, which shows that the vast majority of the immunoreactivity co-eluted with synthetic or pure natural VIP. Also immunoreactive VIP present in the effluent from the perfused pancreas during vagal stimulation co-eluted with synthetic VIP.
Discussion
The following lines of evidence support the conclusion that VIP is directly involved in the neuroeffector transmission of the atropine-resistant pancreatic exocrine response to electrical stimulation of the vagus nerves: (1) VIP-containing nerve fibres are found in close relation to the ducts in the pig pancreas. Earlier reports have documented the presence of VIP-containing nerve fibres in pancreas from a number of species [5-8] including pigs, but their particular relation to the exocrine tissue was not reported. An association of VIP-nerves with the main pancreatic duct in cats was noted by Sundler et al. [7]. In this report we can show that in the pig pancreas VIP fibres are typically seen in association with intercalated ducts as well as larger ducts. The fibres had a characteristic beaded appearance suggesting that the fibres might release VIP from the beads 'en passant'. Since the majority of the fluid and bicarbonate is believed to be produced by the duct cells [17], the association between the VIP nerves and the ducts would be a prerequisite for VIP to be responsible for this secretion. The gel filtration experiments show that the pancreatic VIP in all probability is identical to intestinal or central nervous VIP, indicating that in the pancreas, the VIP precursor is being processed in the same way as in other tissues where this has been examined [18]. (2) In the pig, VIP stimulates pancreatic bicarbonate and fluid secretion with the same efficacy as secretin [9] whereas its potency is about 1/100 of that of secretin. Thus the effluent concentrations determined in this study (maximally 270 pmol) would not be expected to be high enough to influence exocrine secretion. However, it is unlikely that the initial space of distribution of newly neuronally released VIP is as large as that of VIP administered intravascularly. Considering the close association between VIP-containing nerve fibres and ducts demonstrated here, it can be safely
256 assumed, that the local concentrations of VIP may be several orders of magnitude higher than those reached in venous effluent from the preparation. VIP, the half-maximally effective concentration of which for pancreatic exocrine secretion is about 1 nmol/1 in the pig [9], is, therefore, sufficiently potent to be responsible for this secretion after vagal stimulation. (3) A peptide, which by gel filtration is indistinguishable from VIP, is being released from pancreatic nerves during electrical stimulations, a fact which is evident from the present investigation. There were initial reports on a location of immunoreactive VIP to pancreatic endocrine cells [19], but later investigations have shown, that with antisera directed against the C-terminal part of the VIP molecule, only nerve fibres can be detected in the normal pancreas [6]. We have used such an antiserum in the present investigation, and it is therefore likely, that all of the VIP determined in the effluent is of neuronal origin. (4) Pancreatic secretion of fluid and bicarbonate and VIP release were closely parallel under all circumstances tested. This particularly concerns the atropine resistance of both, the main reason for suspecting a non-cholinergic, peptidergic transmission in the vagal effects on. fluid and bicarbonate secretion, but is also illustrated by the similar effect on both of hexamethonium~ the parallelism of the time course, and the frequency dependence. (5) The exocrine response in the isolated preparation is similar to the response obtained in vivo. The exocrine responses to vagal stimulation obtained with this preparation were similar to those obtained with electrical vagal stimulations in anaesthesized pigs [2]. This proves that the vagal effect is independent of secretin or other circulating hormones, which may be present in plasma but not in the synthetic perfusate. In addition, the effect was independent of changes in pancreatic blood flow [20], since our perfusion flow was kept constant throughout. (6) Finally, VIP-antiserum inhibited the effect of vagal stimulation on the fluid secretion. Since a potent VIP-antagonist is not yet available, we tried to block the supposed action of VIP with a potent high-titre VIP-antiserum [14]. Preliminary experiments showed that single-pass perfusion of the pancreas with VIP antiserum diluted 50-100 times with perfusion medium was without effect on the vagus effect on secretion. We therefore designed the recirculation experiments to increase the probability that anti-VIP ),-globulins could extravasate and reach the interstitial tissue around the ducts and VIP containing nerves. That such extravasation is probable is evident from the finding that pancreatic capillaries are highly fenestrated [21]. Fig. 7 shows immunohistochemical evidence that the antibodies were actually present in the interstitial tissues. The fact that the exocrine response to vagal stimulation was suppressed by addition of VIP antiserum and increased again after prolonged once-through perfusion supports the conclusion that the inhibitory effect of antiserum addition was indeed due to a specific blocking of neuronally released VIP. Thus our results strongly suggest that VIP functions as a final transmitter of the vagal effect on pancreatic secretion of fluid and bicarbonate. The atropine resistance of the exocrine response excludes participation of acetylcholine acting on muscarinic receptors, although such interaction is likely to occur when atropine is not present.
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Possible co-existence of VIP and acetylcholine in the same nerve fibres [22] points to the importance of this interaction. Pancreatic sympathetic fibres would not be expected to participate, since electrical stimulation of the splanchnic nerves innervating the pig pancreas is not associated with stimulated exocrine secretion [13]; on the contrary, simultaneous stimulation of both the vagus nerves and the splanchnic nerves Strongly inhibits the exocrine response to vagal stimulation [2]. The possible participation of other pancreatic neuropeptides should be considered, however. Nerves containing cholecystokinin-related peptides are present in the pig pancreas, but are mainly found to innervate the islets [23]; furthermore, we have never been able to detect CCK-like immunoreactivity in the effluent from the isolated perfused pancreas during vagal stimulation (Rehfeld and Hoist, unpublished results). We therefore find CCK-like peptides unlikely candidates. Substance P-immunoreactive nerves are not found in the exocrine tissue [6]; substance P is not an agonist for the secretion of fluid and bicarbonate in the pig pancreas, and it is not released during vagal stimulation (Schaffalitzky de Muckadell and Hoist, unpublished results). The only other neuropeptide with a significant effect on pancreatic exocrine secretion is gastrin-releasing polypeptide (GRP) which is found in intrapancreatic nerves, and which is released during electrical vagal stimulation [24]. GRP particularly potently stimulates enzyme secretion which is strongly inhibited by atropine. This, and the 50% reduction of the fluid response caused by VIP antiserum, support the notion of a predominant role of VIP in the transmission of the vagal effect. Again, however, it is likely that the two systems interact in the intact organism. It was recently reported, that the human VIP precursor contains another peptide, PHM-27, the structure of which greatly resembles the porcine neuropeptide PHI and shows extensive homology with VIP itself [25]. Possibly the PHI of the pig corresponds to PHM-27 of the human precursor, and PHI might therefore be released concomitantly with VIP during nerve stimulation, if the precursor is indeed being processed to release these two peptides. In three perfusion experiments, however, we studied the effect of synthetic PHI obtained from Peninsula Laboratories (Belmont, California 94002) in concentrations up to 10 nmol/l and found it to be 100-fold less potent than VIP on pancreatic exocrine secretion. We think that the present series of experiments have brought us as far as it is presently possible in trying to establish the role of VIP in pancreatic neuro-effector transmission. Our results corroborate further the spreading acceptance of peptides as potential transmitters of neuronal activity.
Acknowledgements This study was supported by grants from the Danish Medical Research Council and P. Carl Petersens Fond. Vibeke Jerris, Anita Hansen, Sorn Haagen Nielsen and Letty Klarskow performed excellent technical assistance. Professor L.-I. Larsson assisted at the immunohistochemical procedures.
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