Localisation and neural control of the release of calcitonin gene-related peptide (CGRP) from the isolated perfused porcine ileum

Regulatory Peptides 98 (2001) 137–143 www.elsevier.com / locate / regpep

Localisation and neural control of the release of calcitonin gene-related peptide (CGRP) from the isolated perfused porcine ileum T.N. Rasmussen, P. Schmidt, S.S. Poulsen, J.J. Holst* Department of Surgical Gastroenterology C, Rigshospitalet and Departments of Medical Physiology and Anatomy B, The Panum Institute, University of Copenhagen, Copenhagen, Denmark Received 20 June 2000; received in revised form 30 November 2000; accepted 4 December 2000

Abstract By immunohistochemistry, CGRP-like immunoreactive (CGRP-LI) nerve fibres were found in the lamina propria along small vessels and in the lamina muscularis mucosae in the porcine ileum. Immunoreactive nerve cell bodies were found in the submucous and myenteric plexus. Upon HPLC-analysis of ileal extracts, CGRP-LI corresponded entirely to porcine CGRP plus smaller amounts of oxidised CGRP. Using isolated vascularly perfused segments of the ileum, we studied the release of CGRP-LI in response to electrical stimulation of the mixed extrinsic periarterial nerves and to infusion of different neuroblockers. In addition, the effect of infusion of capsaicin was studied. The basal output of CGRP-LI was 2.960.7 pmol / 5 min (mean6S.D.). Electrical nerve stimulation (8 Hz) significantly increased the release of CGRP-LI to 167616% (mean6S.E.M.) of the basal output (n 5 13). This response was unaffected by the addition of atropine (10 26 M). Nerve stimulation during infusion of phentolamine (10 25 M) with and without additional infusion of atropine resulted in a significant further increase in the release of CGRP-LI to 2616134% (n 5 5) and 240680% (n 5 9), respectively. This response was abolished by infusion of hexamethonium (3 3 10 25 M). Infusion of capsaicin (10 25 M) caused a significant increase in the release of CGRP-LI to 485682% of basal output (n 5 5). Our results suggest a dual origin of CGRP innervation of the porcine ileum (intrinsic and extrinsic). The intrinsic CGRP neurons receive excitatory input by parasympathetic, possibly vagal, preganglionic fibres, via release of acetylcholine acting on nicotinic receptors. The stimulatory effect of capsaicin suggests that CGRP is also released from extrinsic sensory neurons.  2001 Elsevier Science B.V. All rights reserved. Keywords: Enteric nervous system; Sympathetic nerves; Cholinergic nerves; Neuropeptides; Immunohistochemistry; Pig

1. Introduction Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide produced by tissue-specific alternative processing of the primary transcript of the calcitonin gene [1,2]. In addition to its presence in the central and peripheral nervous system, CGRP has been identified throughout the enteric nervous system by immunohistochemistry [3] and radioimmunoassay [4]. CGRP-immunoreactivity in the mammalian small intestine is thought to *Corresponding author. Department of Medical Physiology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark. Tel.: 1 45-35-327-518; fax: 1 45-35-327-537. E-mail address: [email protected] (J.J. Holst).

originate from extrinsic (primary afferent) sources and from intrinsic neurons of the myenteric and submucosal plexuses [5–8]. The localisation of CGRP in the small intestine suggests a regulatory role for CGRP in small intestinal function. Indeed, administration of CGRP has been found to cause a transient inhibition of field-stimulation induced contractions of longitudinal muscle strips from the human jejunum and ileum and of circular muscle strips from human ileum [9–11]. In circular muscle preparations of the guinea pig ileum, CGRP was found to have both excitatory and inhibitory motor effects [12]. Phasic contractions of the unstimulated muscle were evoked resulting from activation of nerves (sensitive to tetrodotoxin, TTX), and were apparently mediated by cholinergic motor neurons and neuroeffector transmission

0167-0115 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 00 )00242-1

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via muscarinic acetylcholine receptors. In contrast, CGRP was found to have a moderate inhibitory effect on distension-induced peristalsis [12]. In the isolated perfused porcine ileum, CGRP has been demonstrated to cause dose-dependent contractions, as measured by manometry [13]. The response was abolished by atropine suggesting involvement of cholinergic motorneurons. A neural release of CGRP was reported by Mason et al. [14] studying cultured trigeminal ganglion cells, and other studies have demonstrated a basal and evoked release from enteric nerves of isolated rat ileum [15,16]. However, little information is available about the extrinsic nervous control of CGRP release from the small intestine. In order to gain more insight into the relationship between CGRPergic nerves in the intestine and the extrinsic and intrinsic parts of the enteric nervous system, we investigated both the immunohistochemical localisation of CGRP in the porcine ileum and mechanisms for nervous release of CGRP, as measured by radioimmunoassay in the effluent from isolated, perfused preparations of porcine ileum with intact innervation.

pigs and specimens of porcine spinal cord was obtained from a local slaughterhouse. The samples was frozen immediately and extracted with acid ethanol as previously described (method II in [17]). The extracts were concentrated by reverse-phase low pressure liquid chromatography on a 3 3 15 cm glass column packed with Techoprep C18 40–63 mm (HPLC Technology, Macclesfield, Cheshire, UK) and eluted with a linear gradient of 10–90% ethanol containing 0.1% trifluoroacetic acid (TFA) (Merck, Darmstadt, Germany). The CGRP-like immunoreactive (CGRP-LI) fractions were pooled and subjected to reversephase high pressure liquid chromatography (RP-HPLC) using LKB equipment (LKB, Broma, Sweden) with absorbance monitoring at 226 nm. The samples were loaded onto MN Nucleosil cartridges with 300-5 C18 as packing material (Macherey-Nagel, Duren, Germany) and eluted at 1 ml / min with a gradient of Acetonitrile (Rathburn, Walkerburn, UK) / water / TFA(Pierce Chemical Co., Rockford, IL, USA) (phase A 0:99.9:0.1 and phase B 99.9:0:0.1). Fractions of 1 ml were collected and analysed for CGRP-LI.

2.3. Perfusion experiments 2. Material and methods

2.1. Immunohistochemistry Pig ileum tissue for immunohistochemical investigation was obtained by perfusion of an isolated preparation of the ileum, first with saline for a few minutes to remove blood and thereafter with ice-cold 4% paraformaldehyde (No. 4005; Merck, Darmstadt, Germany) in 0.1 M sodium phosphate, pH 7.4, for 10 min. Specimens were excised and postfixed in the same solution for 24 h and transferred to 20% sucrose in 0.1 M phosphate buffer for cryoprotection. After a further 24 h, tissue blocks were frozen in carbon dioxide and cut into sections of | 10 mm using a cryostat. For immunohistochemistry, the sections were preincubated for 30 min with normal swine serum diluted 1:10 and then incubated for 20 h at 48C with CGRP antiserum (code 352) (see below) diluted 1:400, 1:1600, and 1:2400 in 0.1 M phosphate buffer with 0.3% Triton X and 0.1% human serum albumin. For visualisation of the immunoreaction, biotin-conjugated swine anti-rabbit IgG (E353; DAKO, Denmark) diluted 1:40 was used as the second layer (60 min), and streptavidin conjugated with Texas Red (RPN 1233; Amersham International, Buckinghamshire, UK) diluted 1:50 as the third layer (30 min). The sections were examined in a Zeiss fluorescence microscope fitted with an appropriate filter (Zeiss No. 00). Photographs were taken with Kodak Tri-X Pan, 400 ASA film.

2.2. Chromatography Specimens of ileum were excised from anaesthetised

Seventeen pigs, strain LYY, weighing 14–16 kg were fasted overnight with free access to water, and were then anaesthetised without premedication with 2.5% halothane in N 2 O / O 2 for induction, followed by i.v. infusion of chloralose (100 mg / kg, Merck, Darmstadt, Germany). A 60–90 cm segment of mid-ileum was isolated together with the supplying mesenteric artery and vein. During the isolation, great care was taken to preserve all visible nerve fibres to the segment; most fibres formed a dense network around the supplying artery. After positioning of catheters in the artery and vein, the preparation was excised and placed in a previously described, single pass perfusion system [18] and perfused at a constant rate of 0.5 ml g 21 min 21 . Changes in the effluent concentration of the neuropeptides, therefore, accurately reflect changes in their output. The perfusion pressure was recorded continuously. A bipolar platinum electrode, shaped like a hook and embedded in a frame of acryl was carefully positioned around the artery (and the periarterial nerves) and kept in place by means of a loose ligature. The nerves were stimulated electrically with square wave impulses of 10 mA for a duration of 4 ms at a frequency of 8 Hz. The perfusion medium consisted of Krebs–Ringer bicarbonate solution containing, in addition, 0.1% human serum albumin, 5% dextran (Dextran T-70, Pharmacia, Uppsala, Sweden), 5 mmol / l glucose, 100,000 kallikrein inhibiting units / l of aprotinin (Trasylol  , Behringwerke, Marburg, Germany) and a mixture of amino acids (5 mmol / l, Amorex Asa  , Pharmacia, Sweden). Freshly washed bovine erythrocytes were added to the medium immediately prior to use, to give a haematocrit of 20%. Indomethacin (5 mg / l, Confortid  , Dumex, Copenhagen,

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Denmark) was added to prevent generation of prostaglandins in the red blood cells of the perfusate. At regular intervals, PO 2 , PCO 2 , haemoglobin saturation, pH, and standard bicarbonate were measured using ABL-II automatic equipment (Radiometer, Copenhagen, Denmark). It was thus possible to calculate oxygen consumption, which averaged 18.161.9 ml g 21 min 21 (mean6S.D.) in the unstimulated state. The venous effluent was collected for 1-min periods in polyethylene tubes (Minisorb  , Nunc, Roskilde, Denmark) and immediately centrifuged at 4 o C. The supernatants were decanted and kept at 2 208C until assayed for CGRPimmunoreactivity.

2.4. Experimental protocol Electrical stimulation of the periarterial nerves was performed in 17 perfusion experiments (several nerve stimulations were carried out in each experiment). After a 30-min equilibration period, the nerves were stimulated at a frequency of 8 Hz, which invariably resulted in an increase in perfusion pressure from 4063 to 6165 mmHg, n 5 11. In seven perfusion experiments, nerve stimulation was repeated in the presence of atropine 10 26 M (DAK Laboratories, Copenhagen, Denmark) and in five experiments, in the presence of phentolamine 10 25 M (Regitin  , Ciba-Geigy AG, Basel, Switzerland); in nine experiments the nerves were stimulated in the presence of both atropine and phentolamine, and in four experiments in the presence of hexamethonium 3 3 10 25 M (Sigma, St Louis, MO, USA) in addition to atropine and phentolamine. The agents were infused for at least 10 min before nerve stimulation, atropine at least 20 min before. In five perfusion experiments, the effect of capsaicin 10 25 M (Sigma) on the release of CGRP was investigated. Capsaicin and the nerve blockers atropine, phentolamine and hexamethonium were infused by means of precision pumps attached to the arterial line, in amounts calculated to yield final perfusate concentrations as indicated above. In two perfusion experiments, nerve stimulations were repeated four times during the course of 160 min without infusion of any pharmacological agents. In other two experiments, the basal release of CGRP-LI was measured over a period 45 min without manipulation or stimulation.

2.5. Radioimmunoassay CGRP-LI was measured by radioimmunoassay using antisera produced against synthetic human a-CGRP [12– 24] raised in rabbits [19]. Antisera code no. 302 was used. Iodinated human a-[Tyr 0 ]CGRP (Peninsula Laboratories, Belmont, CA) was prepared according to the stoichiometric chloramine-T method as previously described [19]. In short, 300 ml of unknowns and standards (human a-CGRP from Peninsula Laboratories) were preincubated for 72 h at 48C with 100 ml of antiserum (final dilution

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1:125,000) and 100 ml of tracer (4000 c.p.m.). Separation of bound and free peptide was performed by mixing the samples with 1.5 ml of plasma coated activated charcoal. Intra-assay variation was less than 5% and the inter-assay variation was less than 10% at a CGRP concentration of 64 pmol / l. Detection limits were , 5 pmol / l. The antiserum showed no cross-reaction with neurokinin A, substance P, galanin (all from Bachem, Bubendorf, Switzerland), somatostatin-14, gastrin-17, gastrin-releasing peptide and vasoactive intestinal polypeptide (all from Peninsula Laboratories) in concentrations up to 10,000 pmol / l. There was 100% cross-reactivity with human a- and human b-CGRP (Peninsula Laboratories) and with the single known form of porcine CGRP [19]. The recovery of CGRP added to venous effluent deviated less than 610% from the expected values.

2.6. Statistical evaluation The effects of the different stimuli were evaluated by comparison of the accumulated peptide output obtained during the last 3 min of stimulation plus 2 min after stimulation (this period was chosen because of the latency of the response), and outputs accumulated in the 5-min period immediately preceding the start of stimulation, using Student’s t-test for paired data. Differences resulting in P-values less than 0.05 were considered significant. All results are given as means6S.E.M., except where otherwise indicated. The output data are presented in percent of basal output due to some variation in the length of the intestine included in the preparation.

3. Results

3.1. Immunohistochemistry In the mucosa, rather few CGRP-LI varicose nerve fibres were observed in the lamina propria — often along small vessels or in the lamina muscularis mucosae. In the submucosa, immunoreactive ganglionic cells were observed in the submucous plexus and varicose nerve fibres were localised along vessels and in the ganglia around nerve cell bodies of the submucous nerve plexus. In the external muscle coat, there were immunoreactive ganglionic cells in the myenteric plexus and nerve fibres in the plexus and in the muscle layers (Fig. 1).

3.2. Chromatography Upon HPLC the majority of CGRP-LI in extracts of ileum and spinal cord eluted at same positions (Fig. 2). CGRP-LI from the porcine spinal cord has previously been purified and sequenced [19] and, as in the present investigation, three immunoreactive peaks were identified (Fig. 2A). The first of the three peaks was found to be a

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Fig. 1. (A) A few varicose CGRP-immunoreactive nerve fibres in the lamina propria of the ileum. (B) Minor magnification of the mucosa also demonstrating the submucous plexus with an immunoreactive ganglionic cell (lower arrow). (C) The myenteric plexus showing immunoreactive nerve fibres and a few ganglionic cells. (D) Larger magnification showing the myenteric plexus with varicose nerve fibres (arrow) and a single immunoreactive ganglionic cell. (E) Varicose nerve fibre in the external muscle layer. Magnifications: A, D and E: 3 360; B and C: 3 190.

metabolite of CGRP, which was not found in the ileum (maybe because the amount was too low to be detected). Peak no. 2 was found to be porcine CGRP, and no. 3 to be the oxidised form of the peptide [19]. The intestinal CGRP-LI comprised two peaks eluting with identical retention time as peak 2 and 3 from the spinal cord (Fig. 2B). We conclude,therefore, that the two peaks represent porcine CGRP 1–37 and oxidised CGRP.

3.3. Perfusion experiment The basal output of CGRP-LI measured during the control periods was 2.960.7 pmol / 5 min (mean6S.D.). Electrical stimulation of the mixed periarterial nerves significantly increased the release of CGRP-LI from the isolated, perfused, porcine ileum (Fig. 3). The average output increased to 167616% of the basal output (P 5 0.0016, n 5 13). The effect of nerve stimulation was not affected by infusion of atropine (10 26 M) (152636% of

basal value (P 5 0.02, n 5 7)) (Fig. 4A). Electrical stimulation of the nerves during infusion of the a-blocking agent, phentolamine (10 25 M) with (Fig. 4B) and without additional infusion of atropine (Fig. 4C) resulted in a significant increase in the release of CGRP-LI to 2616134% (P 5 0.03, n 5 5) and 240680% (P 5 0.02, n 5 9) respectively. After further addition of the cholinergic, nicotinic blocker, hexamethonium, (3 3 10 25 M) (Fig. 4D), the response was lost. The basal release of CGRP-LI was not significantly affected by the infusion of these three agents (2.660.8 pmol / 5 min before and 2.860.7 pmol / 5 min after the addition of the agents). Infusion of capsaicin (10 25 M) (Fig. 5) significantly increased the release of CGRP-LI to an average of 485682% of the basal output (P 5 0.01). Electrical nerve stimulation invariably resulted in an increase in perfusion pressure from 4063 to 6165 mmHg (P 5 0.01). This increase was nearly abolished when nerve stimulation was performed during infusion of phen-

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Fig. 4. Release of CGRP into the venous effluent of isolated, perfused, porcine ileum in response to electrical stimulation of the periarterial nerves (8 Hz, 4 ms) during ongoing infusion of atropine (10 26 M) (A), phentolamine (10 25 M) (B), atropine (10 26 M) 1 phentolamine (10 25 M) (C) and atropine (10 26 M) 1 phentolamine (10 25 M) 1 hexamethonium (3 3 10 25 M) (D). The data are expressed as mean changes from basal secretion in percent and presented as mean6S.E.M., n 5 7 (A), n 5 5 (B), n 5 9 (C), n 5 4 (D). Asterisks mark values that differ significantly (P , 0.05) from the values in the preceding basal period.

Fig. 2. HPLC of CGRP-LI from extracts of porcine spinal cord A and porcine ileum B. The extracts were first concentrated by reverse-phase low pressure liquid chromatography (Techoprep C18 40–63 mm). CGRPimmunoreactive fractions were pooled and subjected to reverse-phase high pressure liquid chromatography (A and B) and eluted with a gradient of acetonitrile (ACN, left ordinate scale) in water / trifluoroacetic acid (TFA). Eluted immunoreactivity (right ordinate scale) was measured by radioimmunoassay.

Fig. 5. Release of CGRP into the venous effluent of the isolated, perfused, porcine ileum in response to infusion of capsaicin (10 25 M). The data are expressed as changes from basal secretion in percent and presented as mean6S.E.M., (n 5 5).

tolamine (4363 mmHg). Atropine had no effect on the perfusion pressure observed during nerve stimulation. During infusion of capsaicin, the perfusion pressure decreased from 4163 to 3663 mmHg (P 5 0.02). Repeated nerve stimulation without infusion of any pharmacological agents showed no significant differences in the CGRP-LI output (coefficient of variation was 16%)(not shown). In the two perfusion experiments where the release of CGRP-LI was followed without manipulation or stimulation for 80 min, the coefficients of variation for the min to min release was 16 and 23%.

Fig. 3. Release of CGRP into the venous effluent of isolated, perfused, porcine ileum in response to electrical stimulation of the periarterial nerves (8 Hz, 4 ms). The data are expressed as changes from basal secretion in percent and presented as mean6S.E.M., (n 5 13).

4. Discussion In the present study, we examined the nervous control of the release of CGRP-LI using the isolated perfused porcine

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ileum as an experimental model. With this preparation, which is perfused in a single-pass system, it is possible to control the composition of the perfusion medium and thereby avoid metabolic, nervous, endocrine or luminal signals from extra-intestinal tissues that might influence the intrinsic nerves of the intestine; in addition, it is possible to infuse pharmacological agents in concentrations that could not have been used in the intact animal. The preparation is well defined and there is no doubt about the origin of the peptide release. In addition, changes in blood flow subsequent to nerve stimulation or agonist infusion that might influence neuropeptide release are avoided, because the blood flow is kept constant. The chosen parameters for electrical stimulation are derived from previous experimentation with determination of other neuropeptides and transmitters, as well as observations of motility and perfusion pressure responses.Thus, we have established that release of neuropeptides is best observed when a long impulse duration (from 1 ms and above) is employed. The impulse current was chosen to guarantee activation of all fibres in the periarterial network of nerve fibres. Both peptide release and motor and pressure responses are frequency dependent. Eight Hz was chosen because this frequency provides a reproducible response that does not show tachyphylaxis and does not impede perfusate flow. Finally, the architecture and the functions of the porcine small intestine are very similar to those of the human intestine, and it is believed that results obtained in a pig model may be particularly relevant to human physiology [20]. An intestinal release of CGRP has previously been described only in a few studies. Belai and Burnstock [16] found that electrical field stimulation of the muscular coat of the rat ileum increased the release of CGRP in a Ca 21 -dependent manner. Further pharmacological characterisation of the release was not attempted. In the present study, we found that electrical stimulation of the mixed periarterial nerves to the ileum significantly increased the release of CGRP-LI. This response was not affected by the addition of the cholinergic, muscarinic blocker, atropine, but was enhanced after the administration of the a-adrenergic blocking agent, phentolamine, alone or in combination with atropine. However, these responses were abolished by infusion of the cholinergic, nicotinic blocker, hexamethonium. Our results therefore indicate that the CGRP neurons receive excitatory input from parasympathetic, possibly vagal, preganglionic fibres, via release of acetylcholine acting on nicotinic receptors. In addition the intrinsic CGRP neurons seem to receive sympathetic inhibitory fibres. In agreement with these findings and in agreement with studies in dogs and rats [3,8], we found CGRP-LI containing nerve cell bodies in the submucous and myenteric plexus in the porcine ileum. This localisation appears to be unique for the small intestine, since CGRP-containing

nerve cell bodies are not seen in other regions of the digestive system, at least in dogs [3]. In rats, CGRP has been reported to be present not only in intrinsic nerves of the small intestine but also in sensory nerves [21]. Indeed, in our study, capsaicin significantly released CGRP-LI from the porcine ileum. Capsaicin acts selectively on populations of peptide-containing thin primary afferent neurons, making this drug an important tool in the investigation of the functions of these neurons [22], and the results therefore suggest that also in the porcine ileum, CGRP is present in afferent, sensory nerve fibres. The intrinsic origin of a portion of CGRP-immunoreactive fibres innervating the porcine ileum is indicated by the presence of CGRP-LI in cells bodies of both the myenteric and submucosal plexus. In agreement with this, other studies have shown that neonatal treatment of rats with capsaicin (which causes a permanent degradation of most of the small-diameter sensory neurons) only reduces the CGRP content in the ileum by approximately 50% [8]. Together with the marked release of CGRP-LI after capsaicin, thought to activate extrinsic sensory nerve fibres, these findings therefore support the hypothesis of a dual origin of the CGRP innervation (intrinsic and extrinsic). Regarding the functions of afferent neuropeptides in the gut, it is important to consider that peptide-containing sensory nerves at the visceral levels not only transmit information to the CNS, but may also exert a local ‘effect’ function by transmitter release from their peripheral endings [23,24]. Electrical stimulation of the mixed periarterial nerves to the ileum elevated perfusion pressure. This elevation probably reflects arteriolar vasoconstriction, and suggests that the electrical stimulation activated sympathetic fibres to the blood vessels of the preparation. Indeed, a-adrenergic blockade abolished the pressure increase, supporting this hypothesis. Other studies have shown that infusion of noradrenaline resulted in a pressure response similar to the pressure response to nerve stimulation [25]. The CGRP release is unlikely to contribute to this rise in perfusion pressure, since studies on the isolated perfused porcine ileum have shown that infusion of CGRP (10 28 M) decreases the perfusion pressure significantly [13]. Rather, the observed decrease in perfusion pressure during infusion of capsaicin could be the result of the released CGRP. Previous motility studies in our laboratory, using the same experiment model, have shown that electrical stimulation of the mixed extrinsic nerves had no significant motor effect, alone or with atropine or phentolamine, but increased motility during co-infusion of both blockers. This effect was abolished by hexamethonium. Infusion of capsaicin weakly stimulated small intestinal motility [26]. To what extent CGRP contribute to the motility observed in these experiments will require further investigations. In conclusion, our results suggest a dual origin of the CGRP innervation of the porcine ileum (intrinsic and

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extrinsic). The intrinsic CGRP neurons receive excitatory input by parasympathetic, possibly vagal, preganglionic fibres, via release of acetylcholine acting on nicotinic receptors, whereas thin extrinsic sensory fibres were activated by capsaicin.

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