Expression of prepro-VIP derived peptides in the gastrointestinal tract of normal, hypothyroid and hyperthyroid rats

Neuropeptides (1996) 30 (3), 237-247 © PearsonProfessionalLtd 1996

Expression of prepro-VIP derived peptides in the gastrointestinal tract of normal, hypothyroid and hyperthyroid rats 1". BuhP, C. Nilsson 2, E. Ekblad 3, A. H. Johnsen 4, J. Fahrenkrug ~ 1University Department of Clinical Biochemistry, Bispebjerg Hospital,Denmark 2Wallenberg Laboratory, Division of Molecular Neurobiology, University of Lund, Sweden 3Department of Medical Cell Research, University of Lurid, Sweden 4Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Denmark

Summary Vasoactive intestinal polypeptide (VIP) is a widespread neuropeptide involved in the autonomic nervous control of smooth muscle activity, blood flow and secretion. To study the biosynthetic processing of the VIP precursor in the gut of normal, hypo- and hyperthyroid rats we used antisera against the five functional domains of the precursor molecule, prepro-VIP 22-79, peptide histidine isoleucine (PHI), prepro-VIP 111-122, VIP and prepro-VIP 156-170, to quantify and characterize VIP precursor peptides by radioimmunoassay and chromatography and examine their cellular localization and co-localization by immunohistochemistry. All five peptides were expressed in the gut but not in equimolar amounts as expected from the structure of the VIP precursor. A high concentration of PHV, the C-terminally extended form of PHI which includes prepro-VIP 111-122, was found in the small intestine. Immunohistochemically the prepro-VIP derived peptides were shown to coexist in neuronal elements. Changes in thyroid hormone status induced moderate changes in peptide expression in the gut, the most prominent being a 2-fold increase in all prepro-VIP derived peptides in the gastric fundus of hypothyroid rats. The findings indicate that differences in the post-translational processing of prepro-VIP exist in neurons of the rat gut and that hypo- and hyperthyroidism induce differential changes in peptide expression.

INTRODUCTION

Vasoactive intestinal polypetide (VIP) is a 28 amino acid peptide located in neurons of both the central and peripheral nervous system? ,2 VIP is established as a neurotransmitter involved in nervous control of smooth muscle activity, blood flow, and exo- and endocrine secretion) VIP is derived from a 170 amino acid precursor (prepro-VIP) 4,5 which by post-translational proteolytic cleavage gives rise to the following peptide sequences (Fig. 1): a signal peptide, prepro-VIP 22-79 (the N-terminal flanking peptide), peptide with N-terminal histidine and Received 13 December 1995 Accepted 14 February 1996 Correspondence to: Thora Buhl MD, University Department of Clinical Biochemistry, Bispebjerg Hospital, Bispebjerg Bakke 23, DK-2400 Copenhagen NV, Denmark. Fax: +45 35 31 39 55.

C-terminal isoleucine/methionine (PHI/PHM; rat/human), prepro-VIP 111-122 (the bridging peptide), VIP itself, and prepro-VIP 157-170 (the C-terminal flanking peptide). In some instances the dibasic cleavage site C-terminally to PHI/PHM is left uncleared resulting in a C-terminally extended form designated PHV (peptide with N-terminal histidine and C-terminal valine).6 Besides VIP, PHI/PHM and PHV have been shown to be biologically active. 7,s Although all peptides are derived from the same precursor, equimolar amounts of the different peptide fragments are often not found, as has been reported for several different tissues, 9,I° indicating that the post-translational processing of prepro-VIP can be regulated meeting the different demands of various tissues. The aim of this study was to examine the distribution and tissue-specific expression of the various prepro-VIP derived sequences and VIP mRNA in the gastrointestinal 237

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Fig.1 Schematic representation of the structure of rat VIP precursor and its processing products. The putative signal peptide is from 1-21, after that follows: prepro-VIP 22-79 (the N-terminal flanking peptide), PHI, prepro-VIP 111-122 (the bridging peptide), VIP and prepro-VIP 156-170 (the C-terminal flanking peptide). At the bottom is shown the C-terminally extended form of PHI, designated PHV (prepro-VIP 81-122). The amino acid residues at putative posttranslational processing sites, as well as the detected sequences of the various antisera used, are indicated.

tract of normal rats by radioimmunoassay and immunohistochemistry. Hypothyroidism is known to increase VIP expression in the anterior pituitary of rats. n-14 It is well known that the clinical states of hypo- and hyperthyroidism change the activity of the gastrointestinal tract, and a decreased responsiveness of intestinal epithelial cells to VIP in hypothyroid rats has been reported. 15 Since no information on the influence of thyroid status on VIP expression in the gastrointestinal tract exists, we also included tissue from the gut of hyper- and hypothyroid aduk rats.

trois. Hyperthyroidism was induced by injection of 20 gg L-thyroxine (Sigma, St Louis, MO; in saline)/100 g body weight per day subcutaneously for 10 days, and control animals were injected with 0.9% saline solution only. Finally, the animals were weighed and anaesthetized with sodium pentobarbital (50-100 mg/kg body weight) i.p. Blood samples were collected and the thyroid status of the animals was measured (Amerlite TT4 Assay, Amersham, UK). Tissue specimens from the gastric fundus, gastric antrum, small intestine and colon were rapidly removed and processed for either radioimmunoassay (RIA) or immunohistochemistry.

MATERIALS AND METHODS Animals and treatments

Random-bred male Sprague-Dawley rats (Mollegaard Breeding Centre Ltd, Denmark) weighing 220 g were used. They were kept at room temperature under controlled lighting (on at 06:00, off at 18:00) with free access to food and water. Hypothyroidism was induced by adding propylthiouracil (PTU, Sigma) (0.05%) to the drinking water for 25 days. Animals given water without PTU served as conNeuropeptides (1996) 30(1), 237-247

Extraction, radioimmunoassays and chromatography Tissue extraction

The specimens, n = 10 for all samples, were immediately frozen in liquid nitrogen and stored at -20°C. The frozen specimens were weighed and submitted to boiling water/acetic acid extraction, a6 The average extraction recoveries (n = 4) of the various prepro-VIP derived peptides, determined by addition of known amounts of the peptides prior to extraction, were as follows: prepro-VIP © Pearson Professional Ltd 1996

VlP and thyroid hormones

22-79: 82%; PHI: 92%; prepro-VIP 111-122: 94%; PHV: 90%; VIP: 82%; and prepro-VIP 156-170: 96%. The data are presented without corrections for extraction recoveries. RIA of prepro-VlP 22-79 Antisera to the N-terminal region of the VIP precursor (prepro-VIP 22-79) were obtained by immunization with synthetic rat prepro-VIP 22-79 (custom synthesis Cambridge Research Biochemicals, Cambridge, UK) coupled to bovine serum albumin (BSA; Sigma, St Louis, MO) with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (CDI; Sigma). Conjugates were prepared by adding 0.2 gmol of prepro-VIP 22-79 to 0.03 gmol of BSA, 52 ~mol of CDI, and 2.6 mmol (0.2 ml) of N,N-dimethylformamide (E. Merck, Darmstadt, Germany) in 1.5 ml of 0.04 mol/L phosphate buffer, pH 7.4. The conjugation mixture was stirred for 20 h at room temperature. Eight random-bred white Danish rabbits were immunized and 5 animals produced suitable antisera after the fifth immunization. Before immunization, the antigen was emulsified with a double volume of Freund's complete adjuvant (The State Serum Institute (SSI), Copenhagen, Denmark) for the initial immunization and with a double volume of incomplete Freund's adjuvant (SSI) for booster injections. Each rabbit received a dose of the antigen mixture equal to 30 nmol of peptide for initial immunization and 15 nmol for boosters subcutaneously at multiple sites on the back. The animals were boosted once every 8 weeks and bled 10 days after each immunization by ear vein puncture. The antisera were stored at -20°C and used for both radioimmunoassays and for immunohistochemistry. Prepro-VIP 22-79 was radiolabelled with 123Iusing chloramine T (E. Merck) as previously described 17 to a specific radioactivity of 60 Bq/fmol. The selected antiserum (code no. 4Y06-7) was used in a final titre of 1 x 103. It displayed no cross-reactivity with the other prepro-VIP derived sequences or related peptides. The ICs0value (the concentration of prepro-VIP 22-79 giving 50% displacement of labelled peptide) was 33 pmol/L and the detection limit of the assay was 5 pmol/L. To characterize the region specificity of the selected antiserum fragments of synthetic rat prepro-VIP 22-79 were produced by cleavage of 30 nmol aliquots with 5 gg trypsin, 5 gg endoproteinase Glu-C and 0.8 ~g endoproteinase Asp-N (all sequence grade from Boehringer Mannheim), the incubation conditions were as described by the manufacturer. Following incubation, the digestion mixture was applied to a reverse phase HPLC column (Vydac C18, 5g, 4.6 x 250 ram). Peaks of UV-absorbing material (monitored at 214 nm) were collected manually. The isolated fragments were identified by mass spectrometry © Pearson Professional Ltd 1996

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using a plasma desorption mass spectrometer (No-Ion 20, Applied Biosystems). Ambiguous identities were unravelled by sequence analyses using a 475A protein sequencer (Applied Biosystems). The following fragments were used for characterization of the antiserum: trypsin treatment: 22-36, 37-55, 41-55 and 56-77, Glu-C treatment: 22-44, 45-79 and 68-79, Asp-N treatment: 38-56 and 59-68. The concentrations of the peptides were determined by amino acid analysis employing an Aminoquant Analyzer (Hewlett-Packard). Based on radioimmunochemical specificity studies, the antiserum used was found to recognize the region around residues 17-35 of the prepro-VIP 22-79 molecule. RIA of PHI (prepro-VlP 81-107) and PHV (prepro-VlP 81-122)

Details on production of antiserum and radioimmunoassay of PHI (code no. 3692-3, critically dependent on the C-terminal isoleucine amide) and the N-terminally directed antiserum (code no. R8403) recognizing both PHI and PHV with equimolar potency have previously been described) 8,21 The concentrations of PHV were calculated by subtracting the results of measurements with antiserum 3692-3 from the results obtained with antiserum R8403. Both antisera were used in a final titre of 25 × 103, the IC50value was 21 pmol/L and the detection limit was 10 pmol/L for both assays. RIA of prepro- VIP 111-122 The selected antiserum against the bridging peptide of the VIP precursor (code no. 185B-9) was raised against human prepro-VIP 111-122 and cross-reacts fully with rat prepro-VIP 111-122 and PHV. Details of antisera production and assay procedures have been reported earlier.9,10 The concentration of prepro-VIP 111-122 was calculated by subtraction of the calculated PHV from the results obtained with antiserum 185B-9. The antiserum was used in a final titre of 4 × 103, ICs0 value was 20 pmol/L and the detection limit was 5 pmol/L. RIA of VIP (prepro-VIP 125-152) Antibody production (code no. 5603-7) and VIP assay procedure have been published. 17,22 The antiserum is critically dependent on the C-terminal amide since it does not react with VIP free acid or VIP C-terminally extended with glycine. It was used in a final titre of 1.25 × 105, ICs0 was 14 pmol/L and the detection limit was 2 pmol/L. RIA of prepro-VIP 156-170 Antisera to the C-terminal flanking peptide was raised against synthetic rat prepro-VIP 156-170. The peptide was N-terminally extended with a cysteine residue (custom synthesis, Cambridge Research Biochemicals, Neuropeptides (1996) 30(1), 237-247

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Cambridge, Ut0 and coupled to BSA using m-maleimidobenzoyl-N-hydroxy-succinimide esterY Eight randombred white Danish rabbits were immunized with the prepro-VIP 156-170 conjugate and 6 rabbits produced adequate antisera after the fourth immunization. The immunization procedure was as described above for prepro-VIP 22-79. Prior to radiolabelling prepro-VIP 156-170 was tyrosylated at the amino terminus (due to absence of tyrosine residues). The peptide was radiolabelled with 12~Iby chloramine T (Sigma) as previously described, lz The specific radioactivity was 55 Bq/fmol. The selected antiserum (code no. C957-5) was used in a final titre of 10 × 103 with a ICs0 value of 17 pmol/L, and showed no cross-reactivity with other prepro-VIP derived sequences or related peptides. The detection limit of the assay was 5 pmol/L. Assay procedure The procedures for VIP, PHI, PHV, and prepro-VIP 111-122 have previously been published, 9,1z18Assay conditions for prepro-VIP 22-79 and prepro-VIP 156-170 were as follows: 500 btl of extracted and reconstituted sample or standard was incubated with 200 ~tl of diluted antiserum for 48 h at 4°C. All assay incubations were performed in 0.04 mol/L phosphate buffer containing 0.4% HSA, pH 7.4. After addition of 3-5 fmol of labelled peptides (100 btl) and incubation for another 48 h, bound and free peptides were separated at 4°C by absorption to plasma-coated activated charcoal (Sigma). After centrifugation at 4°C, the supernatant and charcoal precipitate were counted. Synthetic rat prepro-VIP 22-79 and rat prepro-VIP 156-170 were used as standards. Serial dilutions of various tissue extracts (treated as well as untreated animals) were parallel to standard curves. The within-assay variations were 4.5 and 6.3%, respectively, and the corresponding between-assay variations were 12.4 and 10.2%. Gel filtration and reverse-phase high performance liquid chromatography Reconstituted tissue extract of gastric fundus and small intestine (control rats) was subjected to both gel permeation chromatography on a Sephadex G-50 superfine column (11 × 1000 re_m, Pharmacia Fine Chemicals AB, Uppsala, Sweden) and to reverse-phase high performance liquid chromatography (HPLC) using a Cls column (Lichrocart 7 ~tm 125 mm × 4 ram, Merck, Germany). Details on the chromatographic systems have previously been published, s, 16 Both columns were calibrated in a separate run with a mixture containing 10 nmol of each of the following synthetic peptides: VIP, rat PHI, rat PHV, rat prepro-VIP 22-79, rat prepro-VIP 111-122, and rat prepro-VIP 156-170. The column recoveries of the various peptides were on average 70% for gel chromatography and 85% for HPLC. Neuropeptides (1996)30(1), 237-247

Immunohistochemistry. The specimens were immediately immersed in ice-cold Stefanini fixative (2% formaldehyde and 0.2% picric acid in 0.1 mol/L phosphate buffer, pH Z2) for 24-48 h. They were then thoroughly rinsed in Tyrode's solution containing 10% sucrose overnight, frozen on dry ice and sectioned in a cryostat at 5-10 ~tm thickness. The sections were processed for immunocytochemical demonstration of prepro-VIP 22-79 (code no. 4Y06; 1:320), PHI (3692; 1:320), prepro-VIP 111-122 (5Y89; 1:320), VIP (5306; 1:1280), and prepro-VIP 156-170 (C957; 1:320) using indirect immunofluorescence,a4 The antisera used were raised as described above. The sections were rinsed 3 x 10 min in phosphate buffered saline (PBS, x 1) containing 0.25% triton X-100, then preincubated with normal swine serum (DAKO, code no. 901, Glostrup, Denmark) for 20 min at room temperature in order to prevent unspecific binding, followed by incubation with the primary antiserum in appropriate dilutions for 20 h at 4°C in a moist chamber. The site of the antigen-antibody reaction was revealed by application of fluorescein isothiocyanate (FITC-)-conjugated swine antirabbit immunoglobulins (affinity-isolated, DAKO, code no. F 205; 1:40) for 45 min at room temperature. The sections were subsequently rinsed and mounted in a mixture of glycerol and buffer (1:1). Specificity of the immunoreactivity was tested by preabsorption of the antisera with synthetic peptides, all in a concentration of 20 gg/ml diluted antiserum. Simultaneous double immunostaining was carried out using a guinea pig VIP antiserum (Milab, Maim6, Sweden; code no. B-GP 340-1; 1:320) together with a secondary antibody labelled with Texas red (affiniPure donkey anti-guinea pig; Jackson ImmunoResearch Laboratories, Inc., West Baltimore, USA; code no. 706-075-148; 1:100) and each of the above-mentioned rabbit antisera with a secondary antibody labelled with FITC (DAKO; code no. F205; 1:40) in order to reveal coexistence of VIP and the other prepro-VIP derived peptides. Statistical analysis

The results are given as mean + SEM. The Mann-Whimey U-test for non-paired observation was used to analyse the significance of differences between means. Differences resulting in P ( 0.05 were considered significant. RESULTS

Both control groups (normal and saline-injected) and the T4-treated rats showed normal gain in body weight, while the PTU-treated rats had a limited weight gain (not shown). The total T 4 level in serum decreased by 850/0 in the hypothyroid rats compared to controls, while in the © Pearson ProfessionalLtd 1996

VlP and thyroid hormones

hyperthyroid rats the total T 4 level increased by 112% compared to controls given saline injections. R a d i o i m m u n o a s s a y s and chromatography

The concentrations of prepro-VIP 22-79, PHI, PHV, prepro-VIP 111-122, VIP and prepro-VIP 156-170 in the different regions of the gastrointestinal tract are illustrated in Figure 2 for normal and hypothyroid rats, and in Figure 3 for normal saline-injected control and hyperthyroid rats. Of the regions examined, the gastric fundus exhibited the lowest concentration of prepro-VIP derived peptides. In the gastric fundus of normal rats all six prepro-VIP derived sequences were expressed (Fig. 2A), though the concentration of prepro-VIP 111-122 was barely above the detection limit. The concentration of the other sequences ranged from 7 to 52 pmol/g. In the gastric antrum of normal rats, the highest concentrations of prepro-VIP 22-79, PHI, prepro-VIP 111-122 and VIP were found, being 183, 79, 125 and 212 300

pmol/g, respectively (Fig. 2B). The concentrations of PHV and prepro-VIP 156-170 were considerably lower, being 42 and 47 pmol/g, respectively. In normal small intestine no prepro-VIP 111-122 was detected and accordingly PI-IV (PHI extended with prepro-VIP 111-122) was found in high concentration (144 pmol/g) (Fig. 2C). Similar concentrations were found for prepro-VIP 22-79 and VIP (122 and 153 pmol/g, respectively), whereas PHI and prepro-VIP 1.56-170 were low (32 and 38 pmol/g, respectively). All six prepro-VIP derived peptides were expressed in the colon of normal rats though the concentration of prepro-VIP 111-122 was fairly low (16 pmol/g) (Fig. 2D). Prepro-VIP 22-79 was the peptide which occurred in the highest concentration in this region (107 pmol/g) followed by VIP (94 pmol/g). The concentration of PHI, PHV and prepro-VIP 156-170 ranged from 36 to 53 pmol/g. Changes in the thyroid hormone status induced tissuespecific alterations in the expression of prepro-VIP derived peptides. Hypothyroidism caused a significant 300

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Fig. 2 Concentration of prepro-VIP derived peptides (mean _+SEM) in extracts from rat gastric fundus (A), gastric antrum (B), small intestine (C) and colon (D). Controls shown in solid bars and hypothyroids in hatched bars; n = 10. The P values: *** P < 0.005, ** P_< 0.01, * P < 0.02.

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Fig. 3 Concentration of prepro-VlP derived peptides (mean _+SEM) in extracts from rat gastric fundus (A), gastric antrum (B), small intestine (C) and colon (D). Saline-injected controls (NaCI controls) shown in open bars and hyperthyroid in cross-hatched bars; n = 10. The P values: *** P_< 0.005, ** P < 0.01, * P<_ 0.02.

increase in peptide content only in the gastric fundus (Fig. 2A). In the gastric antrum neither hypo- nor hyperthyroidism led to any significant changes in the expression of the various peptide sequences (Figs 2B & 3B). In the small intestine, hyperthyroidism selectively raised the concentration of prepro-VIP 156-170 (Fig. 3C) whereas all other peptides were unchanged in the small intestine of hypothyroid animals compared to controls. In the colon, prepro-VIP 22-79 and PHI increased significantly in hypothyroid animals (Fig. 2D), whereas hyperthyroidism caused a significant increase of prepro-VIP 156-170 only (Fig. 3D). When extracts of small intestine from normal male rats were fractionated on a Sephadex G-50 superfine column and on a reverse-phase HPLC C18 column, immunoreactive components corresponding to synthetic prepro-VIP 22-79, PHI, PHV, prepro-VIP 111-122, VIP and preproVIP 156-170 were identified by the respective antisera (Figs 4 & 5). Neuropeptides (1996) 30(1), 237-247

Immunohistochemistry

In normal rats, all the prepro-VIP derived peptides were visualized in neuronal elements of the gastrointestinal tract. Numerous immunoreactive nerve fibres were demonstrated with all the antisera in all layers including intramural ganglia throughout the gastrointestinal tract, and immunoreactive nerve fibres were often seen close to the blood vessels. The immunostaining was intense with exception of the prepro-VIP 156-170 antiserum which was slightly weaker. Immunoreactive cell bodies were seen in both the submucous and myenteric ganglia. In the stomach, immunoreactive nerve fibres within the mucosalsubmucosa and the muscle layers were numerous, whereas fibres in the submucous plexus were less numerous. In the myenteric ganglia of the stomach, intensely immunoreactive cell bodies were detected. In the small intestine, numerous immunoreactive nerve fibres were demonstrated in the internal circular muscle © Pearson Professional Ltd 1996

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layer and in the mucosa/submucosa with extensions to the tips of the villi, and to a lesser extent in the external muscle layer. A strong immunostaining of nerve cell bodies particularly in the submucous but also in myenteric ganglia of the small intestine was observed (Fig. 6). Also in the colon, numerous immunoreactive nerve fibres were found in the internal circular muscle layer and in the mucosa/submucosa with many fibres running close to the epithelium. In the submucous ganglia of the colon, nerve cell bodies were numerous and intensely immunostained whereas in the myenteric ganglia they were fewer. Double immunostaining for VIP and the other preproVIP derived peptides in the different regions of the gastrointestinal tract revealed a total coexistence of VIP

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Fig. 5 Panels showing the elution profiles of extracts from normal male rat small intestine (controls) after fractionation by reversephase HPLC on a C18column. Aliquots of identical fractions were assayed by radioimmunoassaysfor the prepro-VIP derived peptides using the antisera indicated. Arrowheadsindicate elution position of the synthetic peptides. The ethanol gradient is indicated by the full line.

with the other peptides in the same nerve fibres and nerve cell bodies (Fig. 6). Neither hypo- nor hyperthyroidism caused any observable changes in the frequency, distribution or staining intensity of the various preproVIP derived peptides in any of the regions studied. No staining was seen after preabsorption of the antisera with the respective synthetic peptides.

DISCUSSION In the present study we have shown, using sequencespecific radioimmunoassays against the functional domains of the VIP precursor, that all prepro-VIP derived Neuropeptides (1996) 30(1), 237-247

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Neuropeptides (1996) 30(1), 237-247

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Fig. 6 Photomicrographs of sections of normal male rat small intestine (controls) double immunostained with guinea pig VIP antiserum (code no. B-GP 340-1): (B, D, F, H); and with following antisera: prepro-VIP 22-79 (code no. 4Y06) (A), PHI (code no. 3692) (C), prepro-VIP 111-122, which also detects the extended form of PHI named PHV (code no. 185B) (E), and prepro-VIP 156-170 (code no. C957) (G). Scale bars: 100 tam (A, B); 50 tam (C-H).

peptides are expressed in the rat gastrointestinal tract, the concentration being highest in the gastric antrum and small intestine, and lowest in the gastric fundus. By immunohistochemistry the VIP precursor peptides were visualized in neuronal cell bodies and nerve fibres innervating smooth muscle, blood vessels and the mucosa throughout the gastrointestinal tract. The prepro-viP derived peptides had an identical distribution, suggesting that they were co-localized, a suggestion which was supported by double-staining experiments We were unable to demonstrate any distinct differences in the pattern of co-localization of VIP precursor peptides as reported by Raybould & Dimaline, 25 who found a subpopulation of VIP-containing neurons in the myenteric plexus of rat stomach, which did not stain with their antiserum against the N-terminal flanking peptide. From the structure of the VIP precursor, one would expect that the prepro-viP derived peptides were expressed in equimolar amounts, although tissue- or cellspecific post-translational processing could give rise to different peptide profiles. Our radioimmunochemical data showed that there were regional differences in the tissue concentration of the various peptides throughout the gastrointestinal tract of normal rat. An interesting finding was the high level of PHV (the C-terminally extended form of PHI, which includes prepro-VIP 111-122) in the rat small intestine and accordingly prepro-VIP 111-122 was virtually absent in this region. The processing pattern, however, seems to vary between species, since PHV constituted only a small proportion in human small intestine, while the PHV concentration is © Pearson Professional Ltd 1996

high in the human gastric antrum. 1° Our results on prepro-VIP 22-79 (N-terminal flanking peptide) and preproVIP 156-170 (C-terminal flanking peptide) do not fully agree with the data reported by Dimaline & Vowles,26 who used antisera raised against synthetic peptides corresponding to short sequences of the N-terminal and C-terminal flanking peptides. The concentrations of the N-terminal peptide were similar to what was found for prepro-VIP 22-79 in the present study. They reported, however, that in the stomach and colon of the rat, the N-terminal peptide concentrations were 2-fold higher than VIE while we found almost equimolar concentrations of prepro-VIP 22-79 and VIP throughout the gastrointestinal tract. We were unable to confirm their suggestion that the N-terminal peptide in the rat stomach and intestine existed in different molecular forms. Since the antiserum used by Dimaline & Vowles26 was raised against prepro-viP 165-169 and reacted poorly with prepro-viP 156-170, they detected low concentrations of the C-terminal peptide throughout the rat gut. Our data in man 1° and in the present study in rat show that the C-terminal flanking peptide is expressed as prepro-viP 156-170 and that the C-terminal lysine-residue of prepro-VIP is not normally removed during processing. Accordingly, Dimaline & Vowlesa6 could detect higher concentrations of immunoreactive C-terminal flanking peptide by removal of the lysine with carboxypeptidase B. At present it is unknown why and how the VIP precursor is differentially processed. PHI and PHV are both biologically active with a spectrum of action similar to that of VIP. Interestingly PHV, the extended form of PHI, is in Neuropeptides (1996) 30(1), 237-247

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m a n y systems more potent than PHI itself, a, s The functions of the remaining prepro-VIP derived peptides, ff any, remain to be clarified. It is likely that the alternative processing pathways of the VIP precursor could be explained by differences in the composition and activity of the enzymes responsible for post-translational processing. Search for enzymes responsible for the endoproteolytic cleavage of peptide precursors has so far resulted in cloning of cDNAs for two prohormone convertases, PC2 and PC1/3 found in neuroendocrine tissues. 2z 28 It remains to be clarified if these enzymes are involved in the processing of the VIP precursor. Thyroid hormones are essential to normal development and function in most tissues of the body. So far, limited information is available on the influence of thyroid hormone status of gastrointestinal neuropeptides and their receptors. A decreased responsiveness of intestinal epithelial cells to VIP in hypothyroid rats has been reported, 15 suggesting a role for VIP in the pathophysiology of the myxedematous intestine. In hypothyroid neonatal rats exposed to antithyroid medication from day 16 of gestation to 8 weeks postnatally, an increase in VIP content in the d u o d e n u m and jejunum, but a decrease in the ileum and colon, were reported. 29 However, in the present study on a d u k rats, changes in thyroid hormone status induced no or only moderate changes in the expression of prepro-VIP derived peptide concentrations in the rat gastrointestinal tract, the most prominent change being a 2-fold increase in all preproVIP derived peptides in the gastric fundus after induction of hypothyroidism. VIP is an important neuropeptide in the enteric nervous system and there is evidence that the peptide is an inhibitory neurotransmitter, responsible for the vagally mediated receptive relaxation of the stomach, and for the descending inhibition of the peristaltic reflex. 3 Patients with thyroid hormone diseases often suffer from gastrointestinal disturbances, obstipation in hypothyroidism and diarrhoea in hyperthyroidism. Whether VIP or VIPrelated peptides play a pathophysiological role in these gastrointestinal disorders in m a n remains to be elucidated, but the observed alterations in the concentrations or prepro-VIP derived peptides in hypo-and hyperthyroid rats make a role for VIP less likely. ACKNOWLEDGEMENTS

The skilful technical assistance of Anita Hansen, Lea Larsen, Juliano Olsen, Anna Hlin Schram and Doris Persson is gratefully acknowledged. The help from the staff of the Department of Pathology, Bispebjerg hospital, is acknowledged. Antiserum R 8403 was a kind gift from Professor N. Neuropeptides (1996) 30(1), 237-247

Yanaihara, Laboratory of Bioorganic Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan. This study was supported by the Danish Biotechnology Centre for Signal Peptide Research, 11o Henriksen Foundation, The Danish Medical Research Council (no. 12-0819-1), The Danish Foundation for the Advancement of Medical Science, Gerda and Aage Haensch's Foundation, NOVO Foundation, Dr Johan Boserup and Lise Boserup Foundation (no. 204-1EVR), The Danish Hospital Foundation for Medical Research, Region of Copenhagen, The Faroe Islands and Greenland (no. 14/91 and no. 8/92), the Medical Faculty of Lund University, Sweden, the Swedish Medical Research Council (no. 14X-05680-16 and no. 4499) and Magnus Bergwalls Foundation, Sweden. REFERENCES

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