Gastrin release: Antrum microdialysis reveals a complex neural control

Regulatory Peptides 161 (2010) 22–32

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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / r e g p e p

Gastrin release: Antrum microdialysis reveals a complex neural control P. Ericsson a,⁎, R. Håkanson b, J.F. Rehfeld c, P. Norlén a a b c

Experimental and Clinical Pharmacology, Department of Laboratory Medicine, Lund University Hospital, S-221 85 Lund, Sweden Drug Target Discovery, Department of Experimental Medical Science, University of Lund BMC A 12, S-221 84 Lund, Sweden Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, DK-2100 Copenhagen, Denmark

a r t i c l e

i n f o

Article history: Received 9 May 2009 Received in revised form 6 October 2009 Accepted 11 January 2010 Available online 18 January 2010 Keywords: Endocrine cells Stomach Rat Vagus Tetrodotoxin Acid blockade Omeprazole

a b s t r a c t We used microdialysis to monitor local gastrin release in response to food, acid blockade and acute vagal excitation. For the first time, gastrin release has been monitored continuously in intact conscious rats in a physiologically relevant experimental setting in a fashion that minimizes confounding systemic effects. Microdialysis probes were placed in the submucosa on either side of the antrum, 3 days before the experiments. The concentration of gastrin in the antral submucosal compartment was about 20 times higher than in the microdialysate and estimated to be 5–10 times higher than in serum regardless of the prandial state. The rats were conscious during microdialysis except when subjected to electrical vagal stimulation. Acid blockade (omeprazole treatment of freely fed rats for 4 days), or bilateral sectioning of the abdominal vagal trunks (fasted, 3 days post-op.), raised the gastrin concentration in blood as well as microdialysate. The high gastrin concentration following omeprazole treatment was not affected by vagotomy. Vagal excitation stimulated the G cells: electrical vagal stimulation and pylorus ligation (fasted rats) raised the gastrin concentration transiently in both serum and microdialysate. Food intake induced a 2- to 3-fold increase in serum gastrin, while gastrin in antral microdialysate increased 10- to 15-fold. In unilaterally vagotomized rats (fasted, 3 days post-op.), food evoked a prompt peak gastrin release followed by a gradual decline on the intact side. On the vagotomized side of the antrum, the peak response seemed to be reduced while the microdialysate gastrin concentration remained elevated. Thus, unilateral vagotomy surprisingly raised the integrated gastrin response to food on the denervated side compared to the intact side, indicating that vagotomy suppresses an inhibitory as well as a stimulating effect on the G cells. While local infusion of atropine was without effect, infusion of the neuronal blocker tetrodotoxin (TTX) (which had no effect on basal gastrin) virtually abolished the food-evoked gastrin response and lowered the high microdialysate gastrin concentration in omeprazole-treated rats by 65%. We conclude that activated gastrin release, unlike basal gastrin release, is highly dependent on a neural input: 1) Vagal excitation has a transient stimulating effect on the G cells. The transient nature of the response suggests that the vagus has not only a prompt stimulatory but also a slow inhibitory effect on gastrin release. 2) Although vagal denervation did not affect the gastrin response to anacidity, the TTX experiments revealed that both food-evoked and anacidity-evoked gastrin release depends on neural input. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Gastrin from G cells in the antrum is the main stimulus of gastric acid secretion. Gastrin stimulates the ECL cells in the oxyntic mucosa [1,2] to mobilize histamine [3,4], which in turn stimulates the parietal cells to produce hydrochloric acid [4–8]. Gastrin is released in response to a variety of food-related stimuli, such as elevated luminal pH [9,10], intraluminal peptides, amino acids and amines [11,12], and distension of the stomach [13–15]. In addition to this, agents released from endocrine cells in the vicinity of the G cells (for example, somatostatin ⁎ Corresponding author. Unit of Diabetes & Celiac Diseases, Department of Clinical Sciences, CRC, Entrance 72, Bldg 91, Floor 10, Malmö University Hospital, S-205 02 Malmö, Sweden. Tel.: + 46 40 39 19 03; fax: + 46 40 39 19 19. E-mail address: [email protected] (P. Ericsson). 0167-0115/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2010.01.004

released from D cells) [16–18], as well as hormones reaching the antrum via the circulation [19–21], contribute to the control of gastrin release. Further, G-cell secretion is regulated by the enteric nervous system and the autonomic nervous system (via transmitters such as acetylcholine and gastrin-releasing peptide) [19,22–25]. Gastrin release has been the subject of numerous studies in the past using either in vivo or in vitro techniques. The drawback of the in vivo methods is that it is usually difficult to decide whether the G-cell response reflects a direct effect of the experimental intervention or occurs as a consequence of confounding systemic effects. In vitro methods, such as isolated stomachs [4,19,21,22], antral sheets [26] or isolated G cells [12,25,27,28] are generally thought to allow more direct studies of the G cell. However, in vitro methods are inherently “unphysiological” in that nervous and hormonal circuits that may affect the ability of the G cells to respond to stimuli are not operative.

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The method of microdialysis was first applied to the rat gastric submucosa by Bunnett et al. [29]. By using a similar approach, we have developed a protocol for the study of histamine mobilization from the ECL cells in the acid-producing part of the rat stomach with the use of microdialysis probes placed in the gastric submucosa [30–32]. The advantage of the microdialysis technique, as compared to measuring circulating concentrations of a substance, lies in the fact that the microdialysis probe operates as an artificial blood vessel. This allows the continuous monitoring of substances in the extracellular fluid in tissues of intact conscious animals [33,34]. Also, by reverse microdialysis agents can be delivered locally in the gastric submucosa via the probe, enabling stimulation or inhibition of the target cells with less risk of causing systemic effects [35]. Hence, the microdialysis technique has the advantage over in vitro methods and over other in vivo methods in that specific cell populations can be studied in whole animals under physiologically relevant experimental conditions. The usefulness of the microdialysis technique is limited mainly by the sensitivity of the monitoring assay and the ability of compounds to pass the dialysis membrane. Measurement of gastrin in serum following different kinds of experimental manipulations is a conventional approach to monitor gastrin release in vivo. However, these manipulations may interfere with the activity of the G cells. For instance, effects on acid secretion, on the central and peripheral nervous systems or on endocrine cells other than G cells may influence the G cells in an indirect manner, complicating the interpretation of the results. Another complicating factor in the in vivo situation is that following its release, gastrin is promptly being distributed, and diluted, in the blood stream, making it difficult to achieve precision in monitoring the process of gastrin release by measuring the serum gastrin concentration. The vascularly perfused rat stomach is an alternative experimental model, which reduces the impact of confounding systemic factors and makes it possible to monitor (even control) luminal acidity. However, there are problems: 1) The stomach is not in continuity with the remainder of the digestive tract. 2) Circulation is maintained artificially by perfusion with a salt solution. 3) All inputs from extra-gastric neurocrine and endocrine systems have been eliminated. The present study addresses the control of gastrin release from the G cells in the antrum. The primary purpose was to develop a protocol for studying gastrin release in intact, conscious rats in physiologically relevant experimental settings by the use of antral submucosal microdialysis and to compare measurement of gastrin in blood and microdialysate following a series of treatments assumed to influence gastrin release. The secondary purpose of the study was to explore the significance of nervous control of the G cells in relation to basal and stimulated gastrin release. 2. Materials and methods 2.1. Ethical approval The studies were approved by the local Animal Welfare Committee of Lund/Malmö. 2.2. Chemicals The proton pump inhibitor omeprazole was a gift from AstraZeneca (Mölndal, Sweden). Omeprazole was dissolved in 0.25% Methocel (methyl cellulose) (Dow Corning, Midland, MI, USA) and administered once daily (400 μmol kg− 1 day− 1) between 7:00 and 9:00 a.m. for 4 days by oral gavage. This treatment is known to block acid secretion [36]. The last dose was administered in the morning on the day of the experiment. Saline (0.9% NaCl) was used for perfusion via the microdialysis probes. Tetrodotoxin (TTX) (Alomone Labs, Jerusalem, Israel), a drug known to block nervous conductance [37], and atropine sulphate

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(Sigma, St. Louis, MO, USA), a muscarinic receptor blocker, were dissolved in saline for perfusion via the microdialysis probe. 2.3. Animals 130 male and 11 female (as specified) Sprague–Dawley rats (250– 300 g) were kept at a 12-h light and 12-h dark cycle in plastic cages (2– 3 in each cage) with free access to standard rat food pellets (B & K Universal, Sollentuna, Sweden) and tap water. When the rats were to be fasted, they were housed in individual wire mesh bottom cages with free access to water overnight for 24 h before the experiments. In experiments involving refeeding they were offered standard rat pellets and tap water for 3 h. Microdialysis experiments were performed on conscious animals, except those experiments that involved electrical vagal stimulation (see below). During sampling of microdialysate they were kept in Bollman-type restraining cages. Starting 1 week prior to the experiments the rats were familiarized with the Bollman cages by daily training for 1–2 h. Food and water was available during the training sessions. Blood samples for measurement of gastrin in serum were drawn from the tip of the tail, usually during the equilibration period (just before the start of microdialysate sampling) and at the termination of each experiment (after collecting the last microdialysate sample). Each rat was killed by exsanguination from the abdominal aorta following an overdose of chloral hydrate intraperitoneally. 2.4. Surgery 2.4.1. Anaesthesia If not otherwise stated, surgery, including implantation of the microdialysis probe (see below), was performed under chloral hydrate anaesthesia (300 mg kg− 1 intraperitoneally), 3 days prior to the microdialysis experiments. Surgery was performed on freely fed rats. Buprenorphine (Temgesic®, Schering-Plough, NJ, USA) was given subcutaneously (0.02 mg kg− 1) at the time of surgery to alleviate postoperative pain. No mortality was associated with the surgery. No antibiotics were used. Experiments involving electrical stimulation of the vagus (see Experimental design) were performed on rats anaesthetized with fluanisone/fentanyl/midazolam (15/0.5/ 7.5 mg kg− 1, intraperitoneally), since this anaesthesia has less inhibitory effect on gastric endocrine cells than chloral hydrate [31]. 2.4.2. Unilateral vagal denervation Unilateral vagotomy was performed by opening the abdominal cavity by a midline incision and by exposing the ventral vagus nerve along the oesophagus below the diaphragm before cutting it as close as possible to the stomach. The dorsal vagus nerve was left intact. The rats were fitted with microdialysis probes at the same time (see below). 2.4.3. Bilateral vagal denervation Total abdominal vagotomy was achieved by cutting both vagal trunks immediately below the diaphragm. A pyloroplasty was performed at the same time to prevent gastric dilation [38], and the rats were also fitted with microdialysis probes in the dorsal part of the antrum (see below). We have shown previously that pre- and postprandial serum gastrin concentrations in rats subjected to pyloroplasty do not differ from unoperated rats [39]. The effectiveness of the bilateral vagal denervation was verified by hypergastrinemia in the fasted state at the time of the experiment, usually 3 days after surgery (132 ± 36 pmol l− 1, n = 5, as compared to 15 ± 2 pmol l− 1, n = 16, in fasted intact rats). 2.4.4. Pylorus ligation The surgical procedure of acute pylorus ligation in conscious rats has been described in detail previously [40]. In short, each rat (freely fed) was fitted with a microdialysis probe on the dorsal side of the

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stomach (see below). A loose-fitting loop (noose) of silk thread was placed around the pylorus. The ends of the thread were passed through a plastic catheter that exited in the neck. By pulling the ends of the thread the loop tightens around the pylorus (no anaesthesia), ligating the pylorus without disturbing the rat. Ligation was applied 3 days after surgery (fasted male rats) and maintained until the animals were killed 4 h later. Gastric juice was collected at necropsy and its volume determined (11 ± 3 ml, n = 6), to verify that the ligation was successful. 2.5. Microdialysis 2.5.1. Implantation of the microdialysis probe Flexible microdialysis probes (MAB3.35.4, AgnTho's AB, Stockholm, length 4 mm, outer diameter 0.57 mm, 35 kDa cut-off) were used. The abdomen of the anaesthetized rat was opened by a midline incision. The serosa and the muscle layers of the dorsal or the ventral aspect (at times both) of the antrum were tangentially punctured by a needle (22 G) and a tunnel (5–8 mm long) was made in the submucosal layer. The orientation of the tunnel was from the border between corpus and antrum towards the pylorus, ending 1–2 mm proximally to the sphincter. In 6 rats, microdialysis probes were also implanted in the ventral aspect of the corpus. The orientation of the tunnel was from mid corpus towards the border between the corpus and the antrum, ending 5–10 mm proximally to the antrum. The microdialysis probe was then inserted into the tunnel and kept in place with sutures at the tunnel entrance. The inlet and outlet tubes were passed through the abdominal opening and tunnelled under the skin to a point at the nape of the neck where they were affixed with sutures. The time needed to implant probes was less than 20 min. Immediately upon recovery from the anaesthesia (less than 1 h), the rats were returned to their cage and given free access to food and water. The body weight was not affected by the surgery. In some cases food was withheld on the third night after surgery in preparation for experiments that involved fasted rats. 2.5.2. Sampling of microdialysate and blood Microdialysate was sampled 3 days after implantation of the microdialysis probe [32]. At this stage the rats had been fasted overnight (if not otherwise stated). All rats were conscious during the experiments except those that were subjected to electrical vagal stimulation. The inlet tube was connected to a microinfusion pump (Model 361, Sage Instrument, ATI Orion, Boston, USA) and the outlet was allowed to drain into 300 μl polyethylene vials. Perfusion of the microdialysis probes with 0.9% saline (1.2 μl min− 1) started at 7 a.m. After a 40 min equilibration period, collection of microdialysate commenced (see below). Basal samples were collected for 2 h before start of refeeding or stimulation (if not otherwise stated). Sampling started 3 min (the time needed for the perfusion medium to travel from the microdialysis membrane to the outlet of the probe) after start of food intake or stimulation. Food and water was made available in the Bollman cage. The amount of food ingested was measured in each case (food available at the start of the experiment minus the amount of food remaining at the end). In order to compare the gastrin concentration in serum with that of the microdialysate, blood was drawn (200 µl) from the tip of the tail, usually once during the equilibration period and once after collecting the last microdialysate sample, if not otherwise stated. Each rat and each probe was used once only. The position of the probe in the submucosa was verified at autopsy. Microdialysate and serum samples were stored at −20 °C until measurement of gastrin. 2.5.3. Determination of microdialysate equilibration period The length of the equilibration period was determined in an experiment in which microdialysate samples were collected every 30 min for 3 h after start of perfusion. In the 6 rats that were tested the baseline concentration of gastrin in the microdialysate was found to become stable within 30 min (not shown).

2.5.4. Reverse microdialysis The microdialysis technique allows local administration of bioactive agents in the gastric submucosa with reduced risk of inducing confounding systemic effects [35]. In short, by perfusing the probe with a solution containing the agent, the agent will diffuse into the tissue surrounding the microdialysis membrane. Tetrodotoxin (TTX) was administered by reverse microdialysis at concentrations from 1 to 100 µmol l− 1 [41,42]. Also, atropine was administered by reverse microdialysis (0.1 or 1 mmol l− 1). The concentration to be used was determined in a separate experiment where 4 rats were consecutively perfused with 0.01, 0.1, 1 or 10 mmol l− 1 (for 30 min at each concentration) of atropine via the microdialysis probes. Systemic effects of atropine are manifested by pupil dilation [43]. Pupil dilation (1 mm) was seen at 10 mmol l− 1 of atropine, but not at 0.01 to 1 mmol l− 1: Therefore, higher doses than 1 mmol l− 1 were not used in subsequent experiments.

2.6. Histological analysis of probe-carrying antral wall Specimens of the antral wall (5× 5 mm) were collected (3 days after implantation of microdialysis probe) from the area surrounding the microdialysis membrane and fixed by immersion in 4% formaldehyde (1 h). After rinsing in sucrose-enriched buffer (15%, w/v) for 72 h, the specimens were frozen in Tissue-Tek® O.C.T. (optimal cutting temperature) embedding medium and sectioned in a cryostat. Sections (10– 12 μm) were cut perpendicularly to the probe and thawed onto gelatinecoated glass slides. The sections were stained with haematoxylin and erythrosine before being mounted in Kaiser´s glycerol gelatine. The probe was invariably found to be positioned in the submucosa and the distance between the probe and the base of the mucosa was 255 ±20 µm (n = 5). A typical picture is shown in Fig. 1. Introduction of the microdialysis probe in the gastric submucosa is associated with some tissue damage and a reactive inflammatory response. The histological

Fig. 1. Histological analysis of antrum with microdialysis probe. Transverse section (12 µm thick) of the antral wall (stained with haematoxylin and erythrosin), showing mucosa (m), microdialysis probe (p) in the submucosa (sm), and muscularis externa (muscle). Bar = 200 µm. Implantation of the probe is known to cause tissue damage and an inflammatory response in the stomach wall [32]. Indeed, histological analysis revealed inflammatory cells around the probe and mild oedema in the submucosa. The mucosa was 360 ± 20 (SEM) µm in thickness (n = 10).The distance between the uppermost part of the probe and the basal part of the mucosa, where most of the G cells are located, was found to be 255 ± 20 µm. The probe (diameter 570 µm) resembles a large blood vessel. On the basis of the microanatomy, we suggest that the microdialysate gastrin concentration reflects the gastrin concentration in the extracellular compartment of the submucosa (and mucosa) of the antrum.

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analysis revealed invasion of inflammatory cells around and into the wall of the probe, and mild oedema in the submucosa. 2.7. Experimental design 2.7.1. Refeeding Rats fitted with microdialysis probes in the antrum were deprived of food for 24 h and then given free access to food (standard rat pellets) and water after collection of basal microdialysate for 2 h. Microdialysate samples were collected every 20 min during the first hour of refeeding and then every h. The effect of food intake was assessed: 1) in male rats fitted with microdialysis probes on both the ventral and the dorsal aspect of the antrum (8 rats), 2) in female rats fitted with a microdialysis probe on the dorsal aspect of the antrum (7 rats), 3) in male rats that were subjected to unilateral vagotomy (ventral side) fitted with probes on both the intact and the vagotomized side of the stomach (18 rats), 4) in male rats fitted with microdialysis probes on both the ventral and the dorsal aspect of the antrum, receiving TTX (1, 10 or 100 µmol l− 1) (17 rats) or atropine (0.1 or 1 mmol l− 1) (10 rats) via the ventral probe. In addition, the contribution of local neurons to gastrin release in response to elevated luminal pH was assessed in omeprazole-treated (4 days treatment) female rats fitted with microdialysis probes on both the ventral and the dorsal aspect of the antrum, receiving TTX (100 µmol l− 1) (5 rats) via the ventral probe. Perfusion with TTX or atropine started 1 h prior to start of refeeding and continued throughout the experiment. Blood samples were collected 1.5 h before and 3 h after start of refeeding. At termination of the experiments the stomach contents were collected and weighed. In order to monitor the serum gastrin response to food intake, serum was sampled repeatedly from two sets of male rats (equipped with single dummy microdialysis probes) (12 rats) during food intake: at 1 h before and 0, 20, 60 min, 2 h and 3 h after start of refeeding. 2.7.2. Recovery of gastrin In vitro recovery: The in vitro recovery of gastrin was calculated by placing microdialysis probes (n = 10) in vials containing serum (37 °C): 1) from omeprazole treated rats with a gastrin concentration of 480 pmol l− 1, or 2) from normal fasted rats, but with synthetic rat gastrin-17 added up to a final concentration of 35 nmol l− 1. The probes were perfused with 0.9% saline (1.2 μl min− 1). The gastrin concentration in the perfusate was determined and the recovery of gastrin (%) was calculated. The in vitro recovery of gastrin ranged from 4.8 ± 0.9% to 5.2 ± 0.7%. Recovery from corpus and antrum: In 6 omeprazole-treated rats with intact vagal innervations, microdialysis probes were implanted both in the antrum and the corpus. Three days later, blood samples (from the tail) and microdialysate samples (1 h) were collected. The gastrin concentrations in microdialysate from the corpus and antrum were 10 ± 1 pmol l− 1 and 136 ± 30 pmol l− 1, respectively. The corresponding serum gastrin concentration was 295 ± 30 pmol l− 1. Assuming that the microdialysis recovery of gastrin from the corpus and antrum is similar to the in vitro recovery (5%), it can be concluded that the gastrin concentration in the extracellular space of the submucosal compartment of the corpus is in the same range as in serum, while the gastrin concentration in the submucosal compartment of the antrum is about 10-fold higher than in serum. 2.7.3. Vagal activation by pylorus ligation and electrical vagal stimulation Pylorus ligation is known to cause vagal stimulation [44]. Basal microdialysate samples were collected for 2 h before the ligation (performed in 6 conscious fasted male rats as described under Surgery). Microdialysate samples were collected every 20 min during the first hour of stimulation and then every hour. Blood samples were collected 1.5 h before and 3 h after applying the ligature. Electrical vagal stimulation was performed in anaesthetized male rats (see Anaesthesia). The rats (fasted for 24 h) had a microdialysis probe implanted on

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the ventral side of the antrum and were kept on a warm surface (37 °C) during the experiment. The abdomen was opened and the ventral vagus gently exposed. Thirty minutes later collection of basal microdialysate samples commenced. The ventral vagus was stimulated electrically by means of a pair of platinum electrodes (diameter 0.25 mm, distance between electrodes 2 mm). A Grass stimulator (S48 stimulator, AstroMed. Inc., W Warwick, RI, USA) was used to generate 1 ms impulses of 5 V at 1 Hz (9 rats), 5 Hz (10 rats) or 20 Hz (7 rats). After 2 h of basal sampling of microdialysate, the electrical stimulation started. Samples were collected every 20 min during the electrical stimulation (1 h). Blood samples were collected 30 min before and 15 min after stimulation. In one experiment, rats were fitted with microdialysis probes on both sides of the antrum (n = 3). Three days later, the rats (fasted state) were anaesthetized and the abdomen was opened. The anterior vagus was stimulated (5 Hz, 1 h) and microdialysate was collected from both sides. 2.8. Measurement of gastrin We applied two different radioimmunoassays (RIAs) to measure gastrin, using two different antisera. The sensitivity of the conventional RIA used to measure serum gastrin was not high enough to permit the measurement of gastrin in the microdialysate in a reliable way. This made it necessary to develop an alternative, more sensitive RIA for this particular purpose. Details are given below. 2.8.1. Microdialysate gastrin Gastrin in the microdialysate was measured by a RIA using antiserum no. 92132/5 with rat gastrin-17 as standard and monoiodinated 125Igastrin-17 as tracer [45]. The antiserum was raised in a white Danish rabbit (no. 92132) against a gastrin analog corresponding to O-sulfated cholecystokinin (CCK)-10 extended at the N-terminus with a diglycine bridge directionally coupled to bovine serum albumin (BSA) [46]. The antiserum recognizes the bioactive C-terminal pentapeptide amide common to gastrin and CCK, but displays an affinity for sulfated gastrin17 which is 8.4 times higher than the affinity for sulfated CCK-8 [46]. The affinity (expressed by the “effective” equilibrium constant, K0eff [47]) for gastrin-17 (both sulphated and unsulphated) displayed by the antiserum was 32.6 × 1012 l mol− 1 [46]. Since CCK peptides so far have not been found in the mammalian antrum [48,49], the crossreactivity with CCK is without significance for the specificity of the gastrin measurements in the antral microdialysate. This premise was further corroborated in the present study by direct control measurements in rat antral extracts using an entirely specific CCK assay based on antiserum 92128 [46]. Gel chromatography on Sephadex G-50 superfine columns of rat antral extracts showed that the present gastrin-assay based on antiserum no. 92132 measured rat gastrin-34 and gastrin-17 with equimolar potency. Antiserum 92132 was diluted 1:700 000, resulting in 35% tracer binding. Barbital buffer (pH 8.5), containing 0.025% NaN3, 0.25% BSA and 0.25% EDTA, was used for all dilutions. For measurement of microdialysate gastrin the procedure was as follows: Standard samples (15 µl, containing different concentrations of gastrin) and microdialysate samples (15 µl) were incubated with the antiserum (200 µl) in polyethylene vials for 4 days (+4 °C). After adding tracer (50 µl), the samples were incubated for another 24 h (+4 °C). After the second incubation period goat anti rabbit γ-globulin (GAR) was added (250 µl, 2.5% GAR, 5% polyethylene glycol, PEG) together with normal rabbit serum (NRS) (50 µl, 1% NRS, 5% PEG) to separate bound tracer from free. After 2 h of incubation (+4 °C) with GAR and NRS, barbital buffer (150 µl) was added to each test tube and the samples were centrifuged. After decantation of the supernatant the radioactivity of the sedimented bound tracer was counted in a gamma counter. Gastrin concentrations in the microdialysate are expressed as picomole equivalents of rat gastrin-17 per liter. The gastrin assay was evaluated with respect to detection limit and intra- and interassay precision. The evaluation was performed as

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follows: Intraassay precision: The gastrin concentration was measured ten times within the same assay run in three microdialysate samples containing low, medium and high gastrin concentration, respectively. The coefficient of variation for the intraassay precision ranged from 6.2 to 7.7%. Interassay precision: The reproducibility of the estimations was assessed by five replicate measurements of ten different microdialysate samples during a period of two months. The coefficient of variation for the interassay precision ranged from 2.7 to 13.2%. Detection limit: The detection limit was defined as the gastrin concentration corresponding to two SD below the mean binding at a gastrin concentration of zero pmol/l. The calculated minimal detectable amount was 23 amol (i.e. 15 µl microdialysate with a concentration of 1.5 pmol l− 1) in the assay set-up of this study. The sensitivity of this assay was approximately 5 times higher than the sensitivity of the conventional RIA used to measure serum gastrin (see below). 2.8.2. Serum gastrin The concentration of gastrin in serum was measured by a RIA using antiserum no. 2604 [50] with rat gastrin-17 as standard and monoiodinated 125I-gastrin-17 as tracer [45]. Antiserum 2604 was raised against the 2-17 fragment of gastrin-17 and is specific for the bioactive C-terminal octapeptide amide. It binds gastrin-34 and gastrin17 with the same potency, irrespective of the degree of O-sulfation, and it displays no crossreactivity with CCK. The affinity (expressed by the “effective” equilibrium constant, K0eff [47]) for gastrin-17 (both sulphated and unsulphated) displayed by the antiserum was 1.1 × 1012 l mol− 1 [50]. Interestingly, the affinity for antiserum no. 92132 was 30 times higher than for antiserum no. 2604 (see Section 2.8.1). The serum gastrin concentration was measured in 50 µl of serum and expressed as picomole equivalents of rat gastrin-17 per liter. The reliability parameters, including the intraassay and interassay precisions have been reported before [51]. In our hands, the minimum detectable amount in the assay tubes was 115 amol. 2.9. Statistics Results are expressed as mean value ± SEM. The integrated gastrin responses are presented as the rise in microdialysate gastrin over basal concentrations during the period of activation (electrical stimulation and refeeding). Statistical significance was determined using Student's t test, or by one way analysis of variance (ANOVA) followed by Bonferroni's or Dunnett's multiple comparison test. p < 0.05 (*) was considered significant. Correlation between serum and microdialysate gastrin concentrations (Fig. 2) was assessed using Pearson correlation calculations. Statistics, concentration–response curves and graphs were calculated/constructed using the GraphPad PRISM program (version 3.00, GraphPad Software, San Diego, CA, USA). 3. Results 3.1. Gastrin in serum and microdialysate Gastrin was measured in serum and microdialysate collected from the same fasted or omeprazole-treated male rats with or without intact vagal innervation. The microdialysate gastrin concentration was well correlated to the serum gastrin concentration (Fig. 2) (r2 = 0.71, p < 0.001). The gastrin concentration in fasted rats with intact vagal innervation was 14 ± 1.6 pmol l− 1 and 5.2 ± 0.9 pmol l− 1 in serum and antral microdialysate, respectively (n = 8). It should be noted that the actual gastrin concentration in the antral submucosal compartment can be expected to be 20 times higher than the concentration in the microdialysate (the recovery of gastrin being 5%), i.e. the gastrin concentration in the antral submucosal compartment is estimated to be almost 10 times higher than the concentration of gastrin in serum. In fasted rats subjected to unilateral vagotomy the gastrin concentration was 12 ± 1.3 pmol l− 1 in serum and 3.5 ± 1.0 pmol l− 1 in antral

Fig. 2. Correlation between the gastrin concentration in serum and microdialysate. Basal gastrin concentration in serum and microdialysate in male rats (fasted for 24 h) with intact vagal innervation (○) (n = 8) or subjected to total abdominal vagotomy (VT) (●) (n = 5), and in omeprazole-treated male rats with intact vagal innervation (Δ) (n = 6) or subjected to VT (▲) (n = 5). The microdialysate gastrin concentration was well correlated to the serum gastrin concentration (r2 = 0.71, p < 0.001). Assuming that the in vivo recovery is similar to the in vitro recovery (5%), the gastrin concentration of the antral submucosal compartment is 5–10 times higher than the serum gastrin concentration. Blood was drawn from the tail immediately before sampling of microdialysate. Microdialysate was sampled for 1 h. Mean values ± SEM (vertical and horizontal bars). Correlation between serum and microdialysate gastrin concentrations was determined using Pearson correlation calculations.

microdialysate (denervated side) (n = 4). The gastrin concentration in both serum and microdialysate was raised by bilateral vagotomy (3 days before) to 132 ± 36 pmol l− 1 and 29±2.9 pmol l− 1 (n = 5), respectively, and by omeprazole treatment to 342±75 pmol l− 1 and 212 ± 69 pmol l− 1 (n = 6), respectively. When omeprazole treatment was combined with bilateral vagotomy, the serum and microdialysate gastrin concentrations were 331 ± 65 pmol l− 1 and 239 ± 40 pmol l− 1, respectively (n = 5), in fact quite similar to what was seen after omeprazole treatment alone in intact rats. 3.2. Gastrin release in response to refeeding The experiment was performed in fasted male and female rats (Fig. 3A) equipped with probes on either side of the antrum (Fig. 3B). All rats started to eat immediately after receiving food, resulting in a prompt increase in the microdialysate gastrin concentration. The response peaked during the first hour with a 15-fold increase in male rats (6.3 ± 2 pmol l− 1 versus 96 ± 19 pmol l− 1) and 8-fold in female rats (3 ± 0.9 pmol l− 1 versus 26 ± 4.5 pmol l− 1) (Fig. 3A, B), which was followed by a gradual decline. The gastrin response was similar on either side of the stomach (Fig. 3A). Upon termination of the experiments (3 h), the concentration of gastrin in the microdialysate remained 2 to 3-fold above the basal concentration in both male and female rats. At this time point, the corresponding gastrin concentrations in serum were elevated 2-fold in the male rats (17 ± 2 pmol l− 1 versus 38 ± 6 pmol l− 1) (n = 8) and by 50% in the female rats (19 ± 1 pmol l− 1 versus 30 ± 3 pmol l− 1) (n = 7). Male rats consumed more food than did female rats. At the end of the experiment the stomach content weighed 7.3 ± 0.8 g in male versus 4.7 ± 0.7 g in female rats (p < 0.05). The serum gastrin concentration was increased 3-fold 20 and 60 min after food intake in both groups of rats; 3 h after start of feeding the serum gastrin concentration was still twice that of the basal level (Fig. 3C). 3.3. Gastrin release in response to pylorus ligation Ligation of the pylorus in conscious fasted male rats raised the microdialysate gastrin concentration; it peaked within 20–40 min

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3.4. Gastrin release in response to electrical vagal stimulation Electrical stimulation (1 Hz, 5 Hz and 20 Hz for 1 h) of the ventral vagus nerve in fasted anaesthetized rats caused a prompt 3- to 10-fold increase in the microdialysate gastrin concentration on the stimulated side (Fig. 5A). At 15 min after start of stimulation, the serum gastrin concentration had increased 2- to 4-fold (Fig. 5B). In one pilot experiment of 3 rats, gastrin in the microdialysate was monitored on both sides following electrical stimulation (5 Hz, 1 h) of the ventral vagus. The gastrin concentration increased on both sides (Fig. 5C). 3.5. Gastrin release in response to refeeding in unilaterally vagotomized rats The experiments were performed on fasted male rats which had been subjected to unilateral ventral vagotomy 3 days earlier. Unilateral vagotomy per se had no effect on the basal microdialysate gastrin concentration on either side of the antrum. Intake of food raised the microdialysate gastrin concentration on the intact side of the antrum (10-fold increase within 20–40 min), after which it declined to reach a plateau at a level 4-fold above the basal concentration (Fig. 6A). The peak increase in microdialysate gastrin was similar on the vagotomized side of the antrum. After the peak (12-fold increase), the gastrin concentrations stayed at a plateau 10-fold above basal (Fig. 6A). As a consequence, the integrated gastrin response was more than 2-fold higher on the vagotomized side than on the intact side (p < 0.05) (Fig. 6B). Following food intake in unilaterally vagotomized rats, the serum gastrin concentration increased from 18 ± 2 pmol l− 1 (basal) to 45± 5 pmol l− 1 (3 h after start of feeding) (p < 0.001), an increase similar to that seen in intact rats (Fig. 3C). 3.6. The effect of atropine on food-evoked gastrin release The experiment was performed in fasted male rats fitted with one probe on the ventral and another on the dorsal side of the antrum. Infusion of atropine (0.1 or 1 mmol l− 1, starting 1 h prior to food intake) via the ventral probe did not affect the gastrin response to food intake, as compared to the dorsal, control side receiving saline (Fig. 7). The serum gastrin concentration (rats treated with 1 mmol l− 1) increased from 16 ± 2 pmol l− 1 (basal) to 28 ± 4 pmol l− 1 (3 h after start of feeding) (n = 5) (p < 0.05).

Fig. 3. Effects of food intake on gastrin mobilization. The gastrin concentration in microdialysate in response to food in fasted male (A) (n = 8) and female (B) (n = 6) rats equipped with microdialysis probes on the ventral (○) and dorsal (●) sides of the antrum. Access to food is indicated. Microdialysate samples were collected simultaneously from both sides of the antrum (male rats). There was no difference between the sides. The peak gastrin response was elevated 15 times over basal (male rats). Male and female rats differed in that the microdialysate gastrin concentration was higher in the male rats. The difference may reflect the fact that male rats eat more than female rats. At the end of the experiments the stomach content weighed 7 ± 0.8 g (male rats) and 5 ± 0.7 g (female rats) (p < 0.05). The serum gastrin response to food was assessed in fasted male rats (C) (n = 12) (these rats were equipped with dummy microdialysis probes). The blood samples were collected during food intake: at − 1 h, and 0, 20, 60 min, 2 h and 3 h after start of refeeding. The peak gastrin response was 3 times over basal. At the end of this experiment the stomach content weighed 8 ± 0.6 g. Mean values ± SEM. Statistical significance was assessed by ANOVA, followed by Dunnett's multiple comparison test (*p < 0.05, ***p < 0.001).

(6-fold increase) but started the return towards basal levels within 1 h (Fig. 4). Four hours after pylorus ligation the serum gastrin concentration was still higher than before ligation (28 ± 4 pmol l− 1 versus 18± 2 pmol l− 1, n = 6, p < 0.05).

Fig. 4. Gastrin mobilization in response to pylorus ligation. Gastrin was monitored in microdialysate in response to pylorus ligation (fasted male rats) as indicated. Microdialysis probes were placed on the dorsal side of the antrum 3 days before ligation (n = 6). The pylorus was ligated at time zero and the ligation was maintained for 4 h (no anaesthesia). Serum gastrin concentration was determined before (18 ± 2 pmol l-1) and after collection of the microdialysate samples (28 ± 4 pmol l-1). Mean values ± SEM. The rise in microdialysate gastrin 20 min after ligation (6-fold elevation) was statistically significant. Statistical significance was assessed by ANOVA, followed by Dunnett's multiple comparison test (*p < 0.05).

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Fig. 6. Gastrin mobilization in response to food intake in rats subjected to unilateral vagotomy. Gastrin was monitored in microdialysate (A) in response to food in fasted male rats, 3 days after unilateral (ventral) vagotomy; the rats were equipped with microdialysis probes on the ventral/vagotomized (○) as well as on the dorsal/intact (●) side of the antrum. Access to food is indicated. Microdialysate samples were collected from both sides of the antrum (n = 18). The serum gastrin concentration was determined once during the equilibration period (just before start of sampling of microdialysate) (18 ±2 pmol l− 1) and at termination of the experiment (after collection of the last microdialysate samples) (45±5 pmol l− 1) (n =12). At the end of the experiment the content of the stomach weighed 6±0.8 g. B shows the integrated microdialysate gastrin output on the intact side and on the vagotomized side during 3 h of refeeding. Mean values±SEM. Statistical significance was assessed by Student's t test (*p