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Characterization of rat intestinal immunoreactive motilin (IR-M) Vogel, Lee 1987

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CHARACTERIZATION OF RAT INTESTINAL IMMUNOREACTIVE MOTILIN (IR-M) BY Lee Vogel B.Sc. University of British Columbia 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT OF PHYSIOLOGY UNIVERSITY OF BRITISH COLUMBIA We accept this thesis as conforming to the required standards. THE UNIVERSITY OF BRITISH COLUMBIA APRIL,1987 © L e e B. V o g e l , 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) I 1 1 SUMMARY Interdigestive myoelectric a c t i v i t y i n rat i n t e s t i n e has been recorded and characterized. The i n t e r d i g e s t i v e pattern of a c t i v i t y can be disrupted by o r a l glucose and high doses of the duodenal ulcerogen cysteamine. Intravenous glucose had no e f f e c t on the i n t e r d i g e s t i v e myoelectric pattern, nor did high doses of porcine m o t i l i n . Attempts were made to develop a hybridoma c e l l l i n e secreting antibodies that would recognize rat I n t e s t i n a l immunoreactive m o t i l i n (IR-M). The murine myeloma c e l l l i n e NS1 was fused with murine B - c e l l s primed against porcine m o t i l i n . One hundred of the monoclonal c e l l l i n e s produced secreted monoclonal antibodies that recognized porcine m o t i l i n . Attempts to i d e n t i f y a c e l l l i n e secreting antibodies with the a b i l i t y to s t a i n rat i n t e s t i n a l t i s s u e , however, produced only negative r e s u l t s . Rat i n t e s t i n a l IR-M has been characterized with respect to immunocytochemistry (ICC), radioimmunoassay (RIA), and chromatographic properties. The b i o l o g i c a l a c t i v i t y of p a r t i a l l y p u r i f i e d rat i n t e s t i n a l IR-M has also been evaluated u t i l i z i n g a rabbit i s o l a t e d duodenal muscle s t r i p preparation. Five d i f f e r e n t antisera and one monoclonal antibody directed against natural porcine m o t i l i n were u t i l i z e d i n an e f f o r t to detect IR-M containing c e l l s i n rat i n t e s t i n a l t i s s u e s . A v a r i e t y of techniques were employed including tissue f i x a t i o n with either Bouins, paraformaldehyde, or benzoquinone. In a d d i t i o n a v a r i e t y of s t a i n i n g methods including, f l u o r e s c e i n conjugated second antibody, peroxidase-antiperoxidase or peroxidase conjugated second antibody techniques i i i were used. A l l methods using these antibodies f a i l e d to detect IR-H i n the rat small i n t e s t i n e . 125 Porcine m o t i l i n was able to displace I - m o t i l i n from antisera 13-3, 72X and M03. These antisera were u t i l i z e d i n a m o t i l i n RIA to evaluate acid extracts of rat i n t e s t i n a l tissue for IR-M. Only antisera 13-3 and 72X were capable of detecting IR-M i n gut extracts, and these antisera gave d i f f e r e n t d i s t r i b u t i o n s of IR-M In the proximal small bowel. Rat i n t e s t i n a l tissue was extracted i n t o 2% t r i f l u o r o a c e t i c acid and the soluble f r a c t i o n c l a r i f i e d by c e n t r i f u g a t i o n . This acid extracted material was p r e c i p i t a t e d with sodium chloride then dissolved i n methanol at pH 6.0. Methanol soluble material was p r e c i p i t a t e d with ether and the ether p r e c i p i t a t e then dissolved i n water and chromatographed on Sep-Pak cartridges (Waters). Sep-Pak cartridges were eluted with 50% a c e t o n i t r i l e : 0.1% TFA. The 50% eluate was then fractionated further using cation exchange, gel f i l t r a t i o n and reverse phase high pressure l i q u i d chromatography (HPLC). Rat i n t e s t i n a l IR-M peaks from cation exchange chromatography on SP-Sephadex-C25 (Pharmacia) were concentrated and examined for c o n t r a c t i l i t y i n a rabbit duodenal muscle s t r i p preparation. P u r i f i c a t i o n a f t e r SP-Sephadex-C25 was approximately 20 f o l d . Desensitization of rabbit duodenum to porcine m o t i l i n could be demonstrated by pre-treatment with m o t i l i n . C o n t r a c t i l e a c t i v i t y of p a r t i a l l y p u r i f i e d rat i n t e s t i n a l IR-M was not i n h i b i t e d by pre-treatment with m o t i l i n . Chromatography on Bio-Gel P-10 (Biorad) eluted with 0.2M acetic acid yielded an IR-M peak co-eluting with natural porcine m o t i l i n . On HPLC, using a l i n e a r gradient of w a t e r / a c e t o n i t r i l e (10-45% a c e t o n i t r i l e i n 30 min), rat i n t e s t i n a l IR-M did not co-elute with natural porcine m o t i l i n . In conclusion, the molecular weight of rat i n t e s t i n a l IR-M appeared to be s i m i l a r to porci ne m o t i l i n as these two substances demonstrated co-elution on gel permeation chromatography. The lack of co-elution with porcine m o t i l i n on HPLC i n d i c a t e s that other molecular c h a r a c t e r i s t i c s of rat i n t e s t i n a l IR-M, such as hydrophobicity, are not s i m i l a r to porcine m o t i l i n . Furthermore, p a r t i a l l y p u r i f i e d rat i n t e s t i n a l IR-M did induce a c o n t r a c t i l e response i n rabbit duodenal muscle s t r i p s but porcine m o t i l i n did not de s e n s i t i z e t h i s preparation to the c o n t r a c t i l e a c t i v i t y of rat i n t e s t i n a l e x t r a c t s . This suggests that the c o n t r a c t i l e a c t i v i t y of these two compounds i s induced v i a d i f f e r e n t receptor mechanisms. I t i s concluded that the immunoreactive m o t i l i n found i n rat i n t e s t i n a l extracts does not resemble natural porcine m o t i l i n i n structure or b i o l o g i c a l a c t i v i t y . CONTENTS PAGE INTRODUCTION 1 METHODS MIGRATING MYOELECTRIC COMPLEX (MMC) A) ELECTRODE IMPLANT 13 B) ELECTRICAL RECORDING 14 MONOCLONAL ANTIBODY PRODUCTION A) IMMUNIZATION 15 B) PREPARATION OF MYELOMA CELL LINE 15 C) FUSION AND HAT SELECTION 16 D) CLONAL EXPANSION OF HYBRIDOMAS 17 RADIOIMMUNOASSAY i ) IODINATION 17 i i ) ASSAY CONDITIONS 18 i i i ) SEPARATION 18 CYANOGEN BROMIDE CLEAVAGE 19 HIGH PRESSURE LIQUID CHROMATOGRAPHY A) HPLC EQUIPMENT AND BUFFERS 20 B) ELUTION WITH ACETONITRILE 20 IDENTIFICATION OF MOTILIN FRAGMENTS A) SPECTROPHOTOMETRY ANALYSIS 21 B) N-TERMINAL RESIDUE DETERMINATION 22 ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) 23 IMMUNOCYTOCHEMISTRY i ) TISSUE FIXATION A) BOUINS 24 B) BENZOQUINONE FIXATION 25 v i page C) PARAFORMALDEHYDE 25 i i ) STAINING 25 TISSUE EXTRACTION 27 CHROMATOGRAPHIC CHARACTERIZATION OF RAT INTESTINAL IR-M A) ION EXCHANGE CHROMATOGRAPHY B) GEL FILTRATION CHROMATOGRAPHY C) HPLC SMOOTH MUSCLE CONTRACTILE ACTIVITY RESULTS MIGRATING MYOELECTRIC COMPLEX 31 HYBRIDOMA PRODUCTION 32 SEPARATION OF CNBr CLEAVED MOTILIN FRAGMENTS 38 IDENTIFICATION OF MOTILIN FRAGMENTS 39 CHARACTERIZATION OF MOTILIN ANTISERA 45 RADIOIMMUNOASSAY 45 TISSUE EXTRACTION 50 CHROMATOGRAPHIC CHARACTERIZATION OF RAT INTESTINAL IR-M 50 CONTRACTILE ACTIVITY 51 IMMUNOCYTOCHEMISTRY 52 DISTRIBUTION OF IR-M 52 DISCUSSION i) INTERDIGESTIVE INTESTINAL MYOELECTRIC 61 ACTIVITY IN THE RAT i i ) PORCINE-MOTILIN MONOCLONALS DO NOT 65 DETECT IR-M IN THE RAT i i i ) CHARACTERIZATION OF RAT INTESTINAL IR-M 66 REFERENCES 78 28 28 28 29 v i i LIST OF TABLES TABLE PAGE I LIST OF ANTISERA AND THEIR CHARACTERISTICS 49 II EXTRACTION OF RAT INTESTINAL IR-M 59 III DISTRIBUTION OF RAT INTESTINAL IR-M 60 v i i i LIST OF FIGURES FIGURE PAGE 1. ELECTRODE IMPLANT AND MYOELECTRIC RECORDING 33 2. INTEGRATED MYOELECTRIC ACTIVITY; FED RAT AND FASTED RAT PLUS CYSTEAMINE 34 3. INTEGRATED MYOELECTIC ACTIVITY; FASTED RAT, IV GLUCOSE AND ORAL GLUCOSE 35 4. INTEGRATED MYOELECTRIC ACTIVITY; FASTED RAT, PORCINE MOTILIN AND MOTILIN ANTISERA 36 5. ANTI-MOTILIN TITRE OF SERUM FROM MOTILIN IMMUNIZED MICE PRIOR TO FUSION 37 6. CHROMATOGRAMS OF CNBr CLEAVED MOTILIN 41 7. MOTILIN FRAGMENTS RE-CHROMATOGRAPHED 42 8. ULTRA VIOLET SPECTRA OF MOTILIN FRAGMENTS 43 9. POSITION OF DANSYL-AMINO ACIDS AFTER CHROMATOGRAPHY 44 10. REGIONAL SPECIFICITY OF ANTISERA 13-3 AND 72X 46 11. REGIONAL SPECIFICITY OF ANTISERA 74 AND M03 47 12. SERIAL DILUTIONS OF RAT INTESTINAL EXTRACT IN RIA 48 13. ION EXCHANGE AND GEL FILTRATION PROFILES OF IR-M 53 14. HPLC PROFILE OF IR-M 54 15. CONTRACTILE ACTIVITY OF RAT INTESTINAL EXTRACT 55 16. MOTILIN INDUCED DESENSITIZATION OF RABBIT DUODENAL MUSCLE 56 17. CROSS DESENSITIZATION; PORCINE-MOTILIN—RAT INTESTINAL IR-M 57 18. STAINING OF PORCINE DUODENUM WITH ANTISERUM 13-3 58 -1-INTRODUCTION The e x t r i n s i c and i n t r i n s i c neural c i r c u i t r y involved i n i n t e s t i n a l m o t i l i t y Is now r e l a t i v e l y w ell established. The three types of autonomic nerves involved i n the control of g a s t r o i n t e s t i n a l m o t i l i t y are the parasympathetic nerves, the noradrenergic nerves and the non-adrenergic non-cholinergic (NANC) i n h i b i t o r y and excitatory nerves (100). In general a c e t y l c h o l i n e acts as an excitatory neuromuscular transmitter within the GI t r a c t , whereas postganglionic sympathetic a c t i v i t y p r i m a r i l y suppresses m o t i l i t y by i n h i b i t i n g acetylcholine release (100). Descending i n h i b i t i o n was f i r s t described by Bayliss and S t a r l i n g i n 1899 (2,3). I t i s now generally accepted that i n the stomach and small i n t e s t i n e a wave of re l a x a t i o n propagates aborally ahead of the zone of contraction. Receptive r e l a x a t i o n of the stomach f a c i l i t a t e s the entry of food from the oesophagus. In the i n t e s t i n e descending i n h i b i t i o n i s an i n t e g r a l part of the p e r i s t a l t i c r e f l e x pathway that f a c i l i t a t e s the aboral movement of chyme. I t has been evident for several years that the neural pathways involved i n descending i n h i b i t i o n are NANC i n nature. Recently, considerable attention has been given to vasoactive i n t e s t i n a l peptide (VIP) containing neurons and t h e i r r o l e i n the e n t e r i c nervous system as possible mediators of descending i n h i b i t i o n . VIP containing neurons i n the guinea pig have been shown to project i n an aboral d i r e c t i o n at v i r t u a l l y a l l l e v e l s of the GI t r a c t (35,36). Furthermore, VIP and i t s co-synthesized -2-analogue PHI appear to be the only neural peptides that cause direct relaxation of smooth muscle, and are contained In neurons projecting from the myenteric plexus to the smooth muscle layers (39,57). These observations are part of a growing body of evidence suggesting that VIP i s the major i n h i b i t o r y transmitter i n r e f l e x i n h i b i t o r y pathways of the g a s t r o i n t e s t i n a l t r a c t . Other e n t e r i c neuropeptides such as neurotensin, and somatostatin, when administered exogenously i n small doses, also produce an i n h i b i t o r y e f f e c t on g a s t r o i n t e s t i n a l smooth muscle a c t i v i t y (100). Myenteric somatostatin neurons, however, do not project beyond t h i s plexus (52). Thus, i t appears as though somatostatin may have an i n d i r e c t r o l e i n regulating smooth muscle function possibly by i n h i b i t i n g a c e t y l c h o l i n e release from myenteric c h o l i n e r g i c neurons as well as by modulating the release of i n h i b i t o r y peptides (57). Studies u t i l i z i n g i s o l a t e d cross-perfused stomach preparations have provided evidence that m o t i l i t y patterns are also dependent on hormonal mechanisms (73). When the i s o l a t e d stomach was perfused with blood from a fed animal the g a s t r i c m o t i l i t y pattern resembled that seen i n the i n vivo fed state. Conversely, the fed pattern of m o t i l i t y was abolished when the i s o l a t e d stomach was perfused with blood from a fasted animal. Several known g a s t r o i n t e s t i n a l hormones may play a role In the humoral mediation of m o t i l i t y patterns. Two such hormones, g a s t r i n and cholecystokinin (CCK), are known to be released following ingestion of a meal and can delay g a s t r i c emptying as well as disrupt the f a s t i n g pattern of motor a c t i v i t y i n the proximal small bowel. The only i n t e s t i n a l peptide that i s both -3-inhibited by ingestion of a meal and shows enhanced release during the fasted state is motilin. The physiological role of this peptide remains controversial, but i t appears to be associated with the regulation of interdigestive gastrointestinal motility. The original isolation of motilin resulted from attempts to identify the stimulus responsible for increased gastric motility seen with alkalinization of the duodenum (14). Early experiments in man had demonstrated that duodenal perfusion with a 1% sodium bicarbonate solution induced rapid gastric emptying (86,87). During the same period Thomas et. a l . (93) demonstrated an increase in gastric motor activity and a concomitant decrease in gastric emptying time when gastric contents were diverted from the duodenum. In dog experiments Instillation of alkaline buffer or porcine pancreatic juice into the duodenum was found to el icit motor activity in the extrinsically denervated stomach (12). These results clearly indicated that a humoral mechanism was involved but the distinction between release of a stimulatory agent or inhibition of an inhibitor was not apparent. Several commercially available duodenal extracts were assayed for gastric motor-stimulating activity (9). A pancreozymin preparation (Boots Pure Drug Co. U.K.) demonstrated a powerful motor stimulatory activity. It was subsequently shown that the motor stimulating ability could be separated from the pancreozymin activity (14), thus demonstrating that this crude material contained a separate motor stimulating factor. The isolation and characterization of the motor stimulating factor from this Pancreozymin preparation proved unfeasible. However, side fractions produced during the isolation of -4-8 e c r e t i n from hog duodenal mucosa (62) were extracted and the purified extract sequenced (10). This revealed a 22 amino acid polypeptide that was named motilin on the basis of its stimulatory activity in fundic pouches (10,11,13). Although i t was not known at the time the term motilin had been introduced previously (28,79) for a hypothetical agent with the ability to induce intestinal peristalsis (64). Once the complete amino acid sequence of motilin was known* (11), i t was synthesized (103) and both the synthetic and natural forms have been used to study the actions of motilin and to develop antibodies for radioimmunoassay (RIA) and immunocytochemical (ICC) studies. Motilin-like immunoreactivity (IR-M) has been demonstrated in a variety of mammalian species. However, the only other species in which this peptide has been isolated and sequenced is the dog. Canine motilin differs from porcine motilin in 5 of its 22 amino acids (75,80). This represents a relatively large species variation in the amino acid composition of motilin as other intestinal peptides such as VIP, somatostatin and secretin show no variation between mammalian species characterized to date. The fact that porcine motilin has been utilized to generate antisera to investigate the physiology of motilin In other animal models may underly much of the controversy relating to the origin, action, and release of this peptide. Immunocytochemical studies have demonstrated motilin positive cells in the upper small intestine of several species, but the specific cell type has been the subject of debate. One group, using antisera raised against natural porcine motilin, has described *The question of whether the identification of glutamic acid at position 14 is an artefact or represents heterogeneity of the natural peptide remains unresolved (88 b). -5-m o t i l i n p o s i t i v e c e l l s which represent a subpopulation of enterochromaffin (EC) c e l l s d i s t i n c t from substance P containing EC c e l l s (43,71,78). More recently, s u b c e l l u l a r f r a c t i o n a t i o n of human jejunal c e l l homogenates has confirmed the existence of two subtypes of EC c e l l s (26): EC-1 c e l l s containing substance P, and EC-2 c e l l s containing m o t i l i n . Another set of studies u t i l i z i n g antisera raised against synthetic porcine m o t i l i n f a i l e d to detect m o t i l i n i n EC c e l l s of any type (30,44). These l a t t e r observations led to the r e v i s i o n of the Lausanne c l a s s i f i c a t i o n of gastro-enteropancreatic endocrine c e l l s to include the designation Mo for s p e c i f i c m o t i l i n c e l l s (89,90). Controversy also surrounds the reported l o c a l i z a t i o n of m o t i l i n i n CNS t i s s u e s . Radioimmunoassay and immunocytochemical data have described r e l a t i v e l y large concentrations of m o t i l i n i n c e r e b e l l a r tis s u e (19,20,21,66). A more recent report, employing several an t i s e r a , f a i l e d to f i n d any IR-M peaks when screening chromatographed extracts of r a t cerebellum (54). In addition, the same work indi c a t e d that only one of the eight antisera used showed ICC staini n g of the cerebellum. This s t a i n i n g , however, was not blocked by pre-incubation with m o t i l i n . These r e s u l t s suggest that ICC l o c a l i z a t i o n of m o t i l i n i n the cerebellum may r e s u l t from non-s p e c i f i c binding of an uncharacterized f r a c t i o n of the a n t i s e r a . In support of t h i s view i t has been observed that several monoclonal antibodies raised against porcine m o t i l i n do not s t a i n CNS ti s s u e i n a va r i e t y of species i n c l u d i n g the rat (S. Vincent personal communication). -6-The stimulus for the release of motilin and its physiological role in the gastrointestinal tract are two areas that are also poorly understood as the two primary models for study of this peptide (man and dog) have yielded only equivocal data. Oral or intravenous fat stimulated motilin release in man (23), but had no effect on motilin release in dogs (61). Mixtures of amino acids inhibit motilin release in both man and dog (23,61). However, a mixed meal produces either minor increases or no change in motilin levels in man and pig (22,60). Similarly, motilin infusion in dogs speeds gastric emptying of liquid but not solid meals, and has the opposite effect in man, where gastric emptying of solids but not liquids is enhanced (23,27,82). Motilin appears to act in the gut by modulating the release of acetylcholine as well as an unknown non-adrenerglc non-cholinergic excitatory transmitter. The actions of motilin in the gut have been most thoroughly studied in vivo in association with the migrating motor complex (MMC) (see below). This naturally occurring complex appears to be induced by motilin and is blocked by atropine and tetrodotoxin (68,83), suggesting that motilin acts to release acetylcholine and/or other neurotransmitters. This action may also vary at different levels of the gastrointestinal tract. Motilin induced activity in the canine stomach in vivo (31,32) or ex vivo (25) was reduced by hexamethonium and totally blocked by tetrodotoxin or atropine. In the small Intestine atropine alone produces only a partial blockade of motilin induced activity. Tetrodotoxin or a combination of atropine and hexamethonium are required to abolish the -7-response (31,32). These r e s u l t s suggest that the action of m o t i l i n i n the stomach i s mediated e n t i r e l y by a c h o l i n e r g i c mechanism, whereas i n the small i n t e s t i n e i t i s v i a both cholinergic and non-cholinergic pathways. It has been suggested that the non-cholinergic excitatory transmitter acting i n the small i n t e s t i n e i s opioid i n character and possibly met-enkephalin (30). A more recent report u t i l i z i n g the i s o l a t e d perfused canine small i n t e s t i n e has demonstrated that motilin-induced c o n t r a c t i l e a c t i v i t y i s e f f e c t i v e l y antagonized by both tetrodotoxin and atropine (45). Furthermore, perfusion of the bowel segment with naloxone did not affect the response to m o t i l i n . The c o n f l i c t i n g r e s u l t s reported for either i n vivo, i n s i t u , or ex vivo preparations appear to r e f l e c t the degree to which external neural elements are l e f t i n t a c t . There are some data i n d i c a t i n g that m o t i l i n has a d i r e c t action on i n t e s t i n a l smooth muscle. These data have come from i n v i t r o experiments, and are almost e x c l u s i v e l y derived from studies with i s o l a t e d s t r i p s of rabbit i n t e s t i n a l muscle. Tissues from a l l other species except man are r e f r a c t o r y to m o t i l i n (1,85,91). In the r a b b i t , c o n t r a c t i l e responses of muscle s t r i p s i n v i t r o were not affected by neural blocking agents or antagonists to several transmitter substances. Calcium antagonists or incubation i n a calcium free medium abolished the response to m o t i l i n (1). These r e s u l t s suggest that i n the rabbit m o t i l i n acts d i r e c t l y on smooth muscle, presumably by enhancing calcium' i n f l u x . A recent study has reported the s p e c i f i c binding of m o t i l i n to homogenates of rabbit i n t e s t i n a l t i s s u e (15). Subcellular f r a c t i o n a t i o n indicated that -8-binding occurs at the plasma membrane. The relative distribution of motilin receptors to neural elements or other tissue types within the homogenate was not examined. It was suggested, however, that because of the very high amount of binding some of the receptors must be localized to the smooth muscle cells (15). The effect of motilin on rabbit tissue in vitro may reflect a species difference. However, the isolated rabbit duodenal muscle strip exhibits a high degree of basal contractility in comparison to other species. Consequently i t has been suggested that increased neuronal activity associated with a high level of basal contraction, as seen in the rabbit, may be required to observe an effect with motilin in other species (58). The focus of attention concerning the physiology of motilin has been its association with phasic contractile activity during the fasted state. The first detailed investigation of myoelectric activity associated with interdigestive phasic contractions was by Szurszewski in 1969 (92). This pattern of activity originates in the stomach or upper small bowel and propagates distally to the ileum, thus the name migrating myoelectric complex. These myoelectric events associated with the interdigestive period in man have since been characterized into four recurring phases. Phase I corresponds to a period of quiescence lasting 60-70 minutes. Phase II is a period of intermittent contractions of 15-25 minutes. Phase III is a 4 to 6 minute period of regular strong contractions, followed by phase IV, a brief transition phase back to quiescence, the overall period of the MMC cycle being 90-100 minutes (100). The variations in the -9-myoelectric a c t i v i t y occurring from phase II though phase IV have also been termed a c t i v i t y f r o n t s . In man and i n dog several groups found c y c l i c v a r i a t i o n s i n plasma m o t i l i n l e v e l s that peaked i n a s s o c i a t i o n with phase III of the MMC (49,50,95,96). This c o r r e l a t i o n between m o t i l i n peaks and i n t e r d i g e s t i v e myoelectric a c t i v i t y has stimulated considerable research i n t o a possible r o l e for m o t i l i n i n the regulation of MMC's. The primary research model i n t h i s case i s the dog, i n which the following relationships have been observed. Peaks of IR-M are associated with passage of an a c t i v i t y front through the duodenum (76). This association i s independent of vagal a c t i v i t y (42). A c t i v i t y fronts i n i t i a t e d i n the jejunum show no c o r r e l a t i o n with peaks i n plasma IR-M (70). Intravenous Infusion of m o t i l i n i n fasted dogs w i l l i n i t i a t e c o n t r a c t i l e a c t i v i t y resembling premature a c t i v i t y fronts (48,101). Phasic a c t i v i t y remains associated with endogenous IR-M peaks i n denervated fundic pouches (94), but not i n autotransplanted j e j u n a l loops (84). F i n a l l y , i t has been demonstrated that low doses of m o t i l i n antisera w i l l temporarily i n h i b i t the MMC i n the antrum and small bowel, while larger doses induce prolonged i n h i b i t i o n (55,74). The conclusion that must be drawn from a l l these data i s that m o t i l i n does play an a c t i v e r o l e i n generating or modulating a c t i v i t y fronts i n the stomach, at least i n the dog. M o t i l i n , however, does not appear to be involved i n the propagation of a c t i v i t y fronts a b o r a l l y from the duodenum to the terminal ileum. Evidence for a s i m i l a r a c t i o n of m o t i l i n i n other species i s less conclusive except, s u r p r i s i n g l y , i n the pig where -10-motilin does not appear to have any association with interdigestive phasic activity (17). An important question that has received l i t t le attention to date Is: what factors influence the cyclic pattern of motilin release? It is apparent that the major site for release of motilin in the dog is the upper small intestine and the only motilin dependent segment of the MMC is the gastric component. Consequently, i t is most likely that the motilin released from the upper small intestine is having an endocrine effect at the level of the stomach. One group has suggested that motilin release is secondary to smooth muscle contraction (30). However, this does not explain how plasma IR-M peaks coincide with gastric phase III activity when the stomach has been shown to contain few i f any motilin cells (6,71). Plasma IR-M concentrations were increased by bilateral vagal blockade in conscious animals (42), and decreased by vagal stimulation (sham feeding) (56). These observations suggest that cyclic patterns of release may be regulated primarily by vagal inhibitory pathways. As outlined above, virtually every aspect of motilin physiology remains controversial. Most of the differences probably result from the use of a number of different antisera which undoubtedly display widely varying specificities for porcine motilin. Interspecies differences (5 amino acid difference between porcine and canine motilin) may account for variability in detectable levels of lmmunoreactive motilin (IR-M). Similarly, infusion of porcine motilin ln non-porcine models may produce effects different from those caused by the endogenous hormone. Furthermore, the most prominent feature -11-associated with m o t i l i n release, the MMC, i s only observed l n vivo. Presently, the only adequately characterized i n vivo model for m o t i l i n i s i n the dog. I t i s apparent that a well characterized rodent model i s needed to help solve the enigma that t h i s peptide has presented. The i n t e r d i g e s t i v e migrating myoelectric complex has been observed i n the rat (72,81,99). In addition i t has been observed that intraduodenal administration of glucose and amino acids disrupt the MMC i n rats (81). Subcutaneous administration of morphine sulfate (99) and intravenous administration of duodenal ulcerogens, cysteamine and p r o p r i o n i t r i l e (72) have also been shown to disrupt the MMC pattern i n r a t s . Although porcine m o t i l i n reportedly increases g a s t r i c emptying i n rats (65) the e f f e c t s of porcine m o t i l i n on the rat MMC have not been documented. Furthermore, there i s presently no data Indicating plasma m o t i l i n l e v e l s or d i s t r i b u t i o n i n the rat using e i t h e r RIA or ICC. I t i s evident from several studies that r e l a t i v e l y few porcine-motilin antisera cross-react with m o t i l i n peptide(s) i n rat i n t e s t i n a l e x t r acts. This coupled with the r e l a t i v e l y large species d i f f e r e n c e between canine and porcine m o t i l i n , suggests rat m o t i l i n may be very d i f f e r e n t from the porcine peptide. The o v e r a l l objective of t h i s work was to further characterize the rat with respect to several aspects concerning the physiology of m o t i l i n . Three strategies were applied i n working toward t h i s objective. F i r s t , porcine m o t i l i n was employed i n e f f o r t s to produce hybridoma c e l l l i n e s that would secrete monoclonal antibodies with the a b i l i t y to recognize Immunoreactive m o t i l i n (IR-M) i n rat -12-i n t e 8 t i n a l tissue. Second, bipolar electrodes chronically Implanted within the rat small intestine were used to record and characterize intestinal interdigestive migrating myoelectric complexes. Third, a combination of the original and more recent techniques for the isolation of motilin were applied to the extraction of motilin like peptide(s) from the rat digestive system. The rat intestinal IR-M obtained was characterized with respect to its chromatographic properties and contractile activity. -13-METHODS AND MATERIALS MIGRATING MYOELECTRIC COMPLEX (MMC)  A. ELECTRODE IMPLANT WIstar rats of either sex weighing between 250-300g were anaesthetized with sodium pentabarbitol, 100/il/100g (Somnotol, M.T.C. Pharmaceuticals; Mississauga, Ontario). A 3cm incision was made in the mid-line of the abdominal wall and the stomach and proximal small intestine exposed. From one to three pairs of bipolar electrodes were attached to the smooth muscle layer at three locations; the antrum, duodenum, and jejunum. Electrodes were constructed from teflon coated stainless steel wire, uninsulated diameter 75 ;im. (Mediwire Corp., New York). Teflon insulation was removed from a small segment of the ends to be attached to the smooth muscle. Using a 27 gauge needle as a trocar, electrodes were inserted through the serosa and tied off close to the intestinal wall (Figure IA). Two such electrodes were Inserted approximately 2mm apart to form a bipolar set. Each wire from the three bipolar sets were then bundled together and passed through a puncture wound in the abdominal wall, then tunnelled subcutaneously to the mid-scapular region. Each set . was then soldered to a bipolar electrode plug (Plastic Products Company; Roanoke, Virginia) and mounted in dental acrylic. The dental acrylic was moulded to form a flange that could be anchored subcutaneously leaving the electrode plugs exposed. After surgery animals were allowed to recover for 5 days before electrical recordings were made. There was sufficient serosal proliferation around the electrodes during this period to effectively insulate each wire in a bipolar set. In some animals a jugular cannula was also i n s e r t e d . This cannula was tunnelled subcutaneously, exposed i n the mid-scapular region and anchored to the dental a c r y l i c flange. Glucose was administered o r a l l y at a dose of lg«kg \ and intravenously at a dose of 10 mg.kg \ min ^ . Intravenous glucose was administered v i a a p e r i s t a l t i c pump (Holter, model 921; Pasadena, C a l i f o r n i a ) as a s t e r i l e s o l u t i o n of 180 mM glucose i n 1/3 normal s a l i n e (Abbott; Montreal, Quebec) at an i n f u s i o n rate of approximately 0.1 ml«mln Cysteamine was administered v i a the jugular cannula at doses of 6 mg and 11 mg In a 0.25 ml vehicle of s t e r i l e s a l i n e . M o t i l i n at doses of 5 jig and 35 ug i n 0.25 ml s t e r i l e s a l i n e was also administered v i a the jugular cannula. M o t i l i n antiserum M03 was d i l u t e d 1:1 i n s t e r i l e s aline and 0.50 ml injec t e d i n t o the jugular cannula. B. ELECTRICAL RECORDING Following an overnight f a s t , animals were attached to the recording equipment v i a the three electrode plugs at the mid-scapular region. Rats were then l e f t unrestrained for the recording period with no access to food or water. E l e c t r i c a l signals from rat smooth muscle were amplified v i a a Tektronics type 122 low-level preamplifier with a high frequency f i l t r a t i o n of 50 Hz, and a time constant of 0.2 sec (Tektronics Inc. Portland, Oregon), and recorded on a Gould model 2200 chart recorder (Gould Inc. Cleveland, Ohio). -15-MONOCLONAL ANTIBODY PRODUCTION A. IMMUNIZATION Natural porcine m o t i l i n was conjugated to keyhole lympet haemocyanin (KLH) by the carbodiimide method of Goodfriend (37). The motilin-KLH conjugate was emulsified In complete Freunds adjuvant, and an aliquot equivalent to 50 ug m o t i l i n was administered subcutaneously to Balb/c mice. Twenty-one days a f t e r the primary I n j e c t i o n the mice were bled from a t a i l vein and tested f o r antibody production. A l l animals were then given a secondary i n j e c t i o n of motilin-KLH conjugate equivalent to 50 pg m o t i l i n . Twelve days a f t e r the secondary immunization animals were tested f o r antibody production. Those animals responding adequately were then rested f o r t h i r t y days before receiving the f i n a l antigen boost p r i o r to fusion. On days 5, 4, and 3 p r i o r to the planned fusion date, immune mice were given a further intravenous i n j e c t i o n of 10 pg unconjugated m o t i l i n i n 0.1 ml s a l i n e . B. PREPARATION OF THE MYELOMA CELL LINE The NS1 c e l l l i n e (p3-NSl-Ag4-l; Salk I n s t i t u t e ; San Diego, C a l i f o r n i a ) was rendered 8-azaguanine r e s i s t a n t by continuous culture i n the presence of t h i s drug. The 8-azaguanine r e s i s t a n t c e l l s selected f o r fusion were stored at -70^ C i n 10% dimethylsulphoxide (DMSO), 20% f o e t a l c a l f serum (FCS), and 70% Delbecco's Modified Eagle Medium (DME). Several days p r i o r to fusion the NS1 c e l l s were thawed and cultured i n DME + 10% FCS. Only NS1 c e l l s i n the logarithmic growth phase and at a c e l l density of approximately 1-2 x 10~* ce l l s / m l were used f o r fusion. -16-C. FUSION AND HAT SELECTION A m o t i l i n immunized Balb/c mouse was s a c r i f i c e d by c e r v i c a l d i s l o c a t i o n , i t s spleen was then excised and dispersed into single c e l l suspension by gentle s i e v i n g . Both the spleen and NS1 c e l l s were washed 3 times i n warm phosphate-buffered saline (PBS) and resuspended i n warm DME. An aliquot of spleen c e l l s were then d i l u t e d with a fixed volume of tris-NH^Cl s o l u t i o n (pH 7.2) to f a c i l i t a t e the l y s i s of red blood c e l l s . The density of both the NSl and spleen c e l l s was calc u l a t e d . They were then mixed at a r a t i o of 2:1 (spleen:NSl) and spun together at 400 x g f o r 10 min. The supernatant was removed and the p e l l e t l e f t to e q u i l i b r a t e i n a 37^C water bath for 2-3 min. Following e q u i l i b r a t i o n , 1.0 ml of 50% polyethylene g l y c o l (PEG) (Serva, 33136, M.W. 4000, Heidelburg, W. Germany), i n warm DME was added over a period of 1 min with gentle s t i r r i n g . This was followed by an a d d i t i o n a l minute of s t i r r i n g , and the addition of 9.0 ml of warm DME, again with gentle s t i r r i n g , over a period of 4 min. The fusion mixture was then centrifuged at 400 x g for 10 min and the supernatant discarded. DME was added and the c e l l s incubated at 37^C for 30 min. During t h i s period a fresh thymocyte-HAT medium was prepared as follows: 20 ml FCS, 1.0 ml lOOx hypoxanthine thymidine (HT) buffer plus 0.1 ml lOOOx amlnopterine (59), si n g l e c e l l suspension or 4 thymuses from 2-4 wk old mice, and DME to 100 ml. The hybrid c e l l s were then gently resuspended i n 100 ml thymocyte-HAT o medium for every 1 x 10 spleen c e l l s used i n the fusion. F i n a l l y , the c e l l s were gently dispersed i n t o m i c r o t i t r e plates at 0.2 ml per well (2 x 10 5 c e l l s / w e l l ) , and incubated at 37°C with 10% C0 2 for approximately 14 days. -17-D. CLONAL EXPANSION OF HYBRIDOMAS Hybrldoma cultures that exhibited a strong positive reaction on the ELISA were cloned by limiting dilution. Hybrid cultures to be cloned were counted for viable cells using a Speirs-Levy Eosinyl counting chamber. A thymocyte medium was prepared (1 juvenile murine thymus per 25 ml DME + 20% FCS) and used to dilute the desired cultures to a density of 400 cells/ml. Hybridoma cells (at 400 cells/ml) were then serially diluted in six steps, on a 96 well microtitre plate, from 20 cells/well to less than 1 cell/well. Cells were incubated for 8-12 days at 37°C, 10% C02 humidified. When cell cultures reached an appropriate size (individual populations approximately 2mm in diameter; approximately 1-5 x 10^  cells) their supematants were tested for motilin antibodies using the ELISA and immunocytochemistry. For ease of handling, hybridomas that tested positive for anti-motilin activity by ELISA were pooled into groups of 2 or 3, then further cultured in 24 well plates for approximately 7 days. Using this method the supematants from 101 "ELISA positive" cell populations were collected and immunocytochemically screened for anti-motilin activity on rat intestinal tissue. RADIOIMMUNOASSAY i ) Iodination Motilin was iodinated following the chloramine-T method of Hunter 125 125 and Greenwood (47). The I-motilin was separated from free I on a Sephadex G-25 column (10 x 1 cm) eluted with 0.2M acetic acid, 0.5% bovine serum albumin (BSA), 0.1% trifluoroacetic acid, 1.0% trasylol. 125 Possible fragmentation of I-motilin by the iodination procedure was -18-125 assessed by the a b i l i t y of I-moti l i n to bind to dextran coated charcoal. The f r a c t i o n with the highest percentage of counts ( greater than 95%) bound to the charcoal p e l l e t was retained for use i n RIA. This f r a c t i o n was then d i l u t e d to approximately 2x10^ cpm i n a c i d i f i e d ethanol and stored at -20° C. When stored i n t h i s manner 1 2 5 I - m o t i l i n could be used f o r up to 10 weeks. Before use i n an assay each new 125 batch of I - m o t i l i n was tested i n a standard curve f o r i t s a b i l i t y to be displaced by the addition of natural porcine m o t i l i n . i l ) Assay Conditions Antiserum 13-3 (rabbit; f i n a l d i l u t i o n 1:500,000) or 72-X (guinea pig; f i n a l d i l u t i o n 1:40,000) was incubated together with 125 I-motil i n (2000 cpm), sample or standard i n assay buffer (0.04M phosphate pH. 6.5, 5% charcoal extracted plasma, 0.25% t r a s y l o l ) i n a f i n a l volume of 1.0 ml for 48 h at 4^ C. Intra-assay and inter-assay v a r i a b i l i t y were obtained from control samples of 2.0 ng/ml which were included i n each assay. Assays were accepted when these control values assayed between 1.90 to 2.10 ng/ml m o t i l i n . i i i ) Separation 125 Following Incubation, bound and free I - m o t i l i n were separated by adsorption of the free component on dextran coated charcoal. Two hundred m i c r o l i t e r s of separation buffer prepared as follows: 0.04M phosphate pH 6.5, 5% charcoal extracted plasma, 1.25% charcoal (Fisher S c i e n t i f i c , Vancouver, B.C.), 0.25% dextran M.W. 70,000 (Pharmacia; ^ r . William Chey Dr. John C. Brown -19-Dorval, Quebec), was added to each tube. Tubes were then vortexed and centrifuged at 2000 x g f o r 30 min. Supematants were discarded and the tubes allowed to dry inverted for 6 h before counting the p e l l e t s . P e l l e t s were counted f o r 3 min on an automatic gamma counter (Model 1285:Searle A n a l y t i c Inc.; Des P l a i n s , I l l i n o i s ) CYANOGEN BROMIDE CLEAVAGE M o t i l i n antisera were characterized with respect to t h e i r a b i l i t y to bind either N-termlnal or C-terminal fragments of natural porcine m o t i l i n . Amino and carboxyl terminal fragments of m o t i l i n were created by cleavage at the methionyl residue with cyanogen bromide (CNBr) (40,41). Cyanogen bromide (Eastman Kodak; Rochester, N.Y.) was dissolved i n 70% (v/v) formic acid to a f i n a l concentration of 10 mg/ml. Cleavage was performed at 22^C f o r 6 h i n the dark at a peptide concentration of 2mg/ml. Following the 6 h incubation the reaction mixture was d i l u t e d 20x with d i s t i l l e d water and l y o p h i l i z e d . The CNBr cleavage fragments were separated by high pressure l i q u i d chromatography, and i d e n t i f i e d by both absorption spectra and N-termlnal residue determination. -20-HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC). A. HPLC EQUIPMENT AND BUFFERS A l l HPLC equipment was from Waters Associates Inc. . Reverse phase chromatography was carried out using a u-Bondapak column (Waters) (3.9mm x 30 cm) consisting of a si l ica matrix with attached C-18 chains. The solvent system utilized was water and acetonitrile (CH^CN). Prior to use, each solvent was degassed under vacuum for 10 min., then trifluoroacetic acid added to a final concentration of 0.1%. To prevent reabsorption of atmospheric gases, solvents were also maintained in an atmosphere of helium during their use. Samples were applied via a Waters U6K injection system, and mixing of the solvents to form gradients of decreasing polarity was accomplished by using a Waters 660 solvent programmer. Effluent was monitored for absorbance at 220 nm. B. ELUTION WITH ACETONITRILE A semi-empirical approach was applied to the elution of compounds from the u-Bondapak column with acetonitrile. Only linear gradients were used and generally a wide gradient (5%-75% acetonitrile) was applied over 30 min in a preliminary run to gain an estimation of the concentration range in which peptides in the mixture eluted. Upon determining the acetonitrile concentration range in which the sample eluted i t was possible to progressively raise the in i t ia l acetonitrile concentrations and lower the final acetonitrile concentrations of the gradient in order to optimise resolution and elution time. The time -21-over which the gradient was run was also progressively shortened so that an optimal gradient slope was maintained (generally less than a 3% rise i n acetonitrile concentration per min). Sensitivity of the detection apparatus also required adjustment according to the size and concentration of the sample being chromatographed. Monitoring the effluent for absorbance at a wavelengh of 220 nm provided the greatest sensitivity for peak detection. When concentrated samples of a relatively crude extract were chromatographed the sensitivity of the spectrophotometer was reduced to 2.0 Arbitrary Units Ful l Scale (AUFS). As well, the chart recorder could be adjusted to attenuate peaks i n order that relatively large amounts of protein did not exceed the limit of detection. Similarly the sensitivity of the spectrophotometer was increased (to a maximum of 0.01 AUFS) and attenuation of recorded peaks reduced when small relatively pure samples were chromatographed. Spectrophotometric detection and recording of protein peaks was simultaneous with column outflow. This facilitated the separation and collection of peaks by pooling of the outflow during each spectrophotometry peak and discarding the effluent between peaks. In order to,check the homogeneity of collected peaks, they were f i r s t lyophilized, reconstituted i n dH20: 0.1% TFA, then rechromatographed on HPLC. IDENTIFICATION OF MOTILIN FRAGMENTS  A) Spectrophotometric analysis Peaks identified from the HPLC chromatographs of CNBr cleaved material were subjected to spectrophotometric analyses i n the -22-ultravlolet range using a Pye Unicam Sp8-100 spectrophotometer. As motilin contains a single tyrosine residue on the N-tertrdnal side of the CNBr cleavage site, only uncleaved motilin and the N-terminal fragment are expected to exhibit an absorption peak at 280 nm. B) N-terminal residue determination Amino terminal residues of CNBr cleaved motilin fragments were determined using the dansyl (DNS) method of Gray (38). Dansyl-amino acids were detected by the method of Woods and Wang (102) in which 2 thin layer chromatography on 5 cm polyamide plates is employed. Approximately 5nMol of uncleaved motilin or CNBr cleaved motilin fragments were aliquoted into 6 x 50 mm glass culture tubes and lyophilized. Samples were then dissolved in 2.0 ul of 1% sodium bicarbonate, centrifuged and relyophilized. Samples were redlssolved in 2.5ul distilled water, and 2.5ul of dansyl-chloride (5-dimethylaminonapthalene-l-sulphonyl chloride, 2.5mg/ml in acetone; Sigma, St Louis, Missouri.) added. This reaction mixture was then centrifuged, sealed and incubated for 20 min at 45 ^ C , following which the tubes were again centrifuged and relyophilized. Dansyl-peptides were then acid hydrolized by adding 50 ul of 5.7M HCl, centrifuging, sealing the tube in a flame and heating at 100^  C. for 18 h. Following acid hydrolysis the tubes were centrifuged, opened, diluted 50% with distilled water and lyophilized over sodium hydroxide pellets. Thin layer chromatography on polyamide plates (Chen Chin trading Co. Taipal, Taiwan) was employed to identify dansyl-amino acids. To each tube 2.5 ul of 50% pyridine was added, and 0.25 ul -23-2 spotted on each side of a 5 cm polyamide plate. A standard solution containing DNS-glutamic acid, DNS-phenylalanine, DNS-E-lysine, DNS-tyrosine and DNS-amide each at approximately 0.5mg/ml was also spotted onto one side of the plate. Ascending chromatography was performed i n two dimensions with the following solvents: Dimension Solvent 1 I water/90% formic acid (200/3 v/v) 2 II benzene/acetic acid (9/1 v/v) 2 I I I hexane/butanol/acetic acid (3/3/1 v/v/v) Plates were viewed under a short wave u l t r a v i o l e t source, with i d e n t i f i c a t i o n being made by comparison to the standards on one side of the pla t e . ENZYME-LINKED-IMMUNOSORBENT-ASSAY (ELISA) Carboxy-terminal (1-13) and amide-terminal (14-22) m o t i l i n fragments generated by CNBr cleavage were u t i l i z e d i n the ELISA as a means of i d e n t i f y i n g the binding epitopes of m o t i l i n a n t i s e r a . A 96 well m i c r o t i t r e plate (Falcon) was i n i t i a l l y coated with m o t i l i n . The coating procedure consisted of d i s s o l v i n g 0.5 jig m o t i l i n per ml carbonate buffer (15mM sodium carbonate, 35mM sodium bicarbonate, 3mM sodium azide, pH 9.6) and dispensing lOOul to each w e l l . Plates were then incubated at 4^ C f o r 24 h. The m o t i l i n antisera were applied a f t e r washing the plate 3X with PBS-Tween. After a p p l i c a t i o n of the f i r s t antibody layer, plates were incubated at 22^ C for 2 h. The plates were again washed with PBS-Tween and the second antibody -24-(rabbit anti-mouse at 1:3000 or goat anti-rabbit at 1:2000 or goat anti-guinea pig at 1:2000) covalently linked to alkaline phosphatase was added (100ul) to each well. The plates were again incubated at room temp, for 2 h, and subsequently washed 3X with PBS-tween. The assay was developed by adding 100 ji l of lmg para-nitro-phenyl-phosphate(Sigma; St Louis, Missouri) in 1ml of diethanolamine buffer (10% diethanolamine, 0.5mM magnesium chloride, 3mM sodium azide) and the phosphatase reaction was monitored at 405 nm on a Micro ELISA reader (MR 580, Dynatech; Alexandria, Virginia). To determine the regional specificity of the motilin antisera an inhibition ELISA was performed. In this instance motilin antisera (first antibody layer) were incubated at 4^  C for 24 h with either uncleaved motilin, the C-terminal fragment, the N-terminal motilin fragment, or no peptide. A l l other aspects of the ELISA were as described above. IMMUNOCYTOCHEMISTRY  i ) TISSUE FIXATION Antisera were also tested immunocytochemically on sections of rat duodenum, ileum, jejunum, cerebellum, and porcine duodenum. Three methods of fixation were used for rat tissues. A). Boulns Fresh samples of rat duodenum, ileum, and jejunum were rinsed with PBS and immediately placed in Bouins fixative (75% picric acid, 25% v/v of a 37% solution of formaldehyde in distilled water, and 3% glacial acetic acid) for up to 18 h. After fixing In Bouins the tissue was dehydrated and embedded In paraffin using a tissue processor - 2 5 -(Flsher Histomatic; model 166). Bouins fixed tissues were sectioned at 5pm and mounted on uncoated glass s l i d e s by heating to 37^ C overnight. B ). Benzoquinone f i x a t i o n Rats anaesthetized with urethane were perfused through the descending aorta with PBS u n t i l r e l a t i v e l y free of red blood c e l l s (approximately 100ml). Immediately following perfusion with PBS, animals were fixed by perfusing with 250ml of a f r e s h l y prepared solution of 0.4% benzoquinone i n PBS, pH 7.2-7.4. The benzoquinone solution was shielded from l i g h t , and perfusion pressure was approximately 150 cm 1^0. A f t e r perfusion tissues were removed and post-fixed i n the benzoquinone s o l u t i o n for 2 h. Excess benzoquinone was removed by soaking i n several changes of PBS + 5% sucrose. Tissues were stored i n PBS + 5% sucrose u n t i l sectioned. C). Paraformaldehyde Whole rats were fixed (as described for benzoquinone) with a 4% solu t i o n of paraformaldehyde i n 0.1M phosphate, ImM Mg . Tissue was postfixed overnight i n paraformaldehyde, and stored i n 5% sucrose as above. Benzoquinone and paraformaldehyde fixed tissues were cut at lO^jm on a cryostat and mounted on formol-gelatine coated s l i d e s ( i n t e s t i n e ) , or suspended In PBS, 0.2% azide (cerebellum). i i ) STAINING Tissue sections either mounted on s l i d e s (small i n t e s t i n e ) or free f l o a t i n g (cerebellum) were incubated f o r 24 h at 4° C with m o t i l i n antisera i n d i l u t i o n s ranging from 1:100 to 1:5000. Antisera used i n stainin g Bouins fixed material were d i l u t e d i n PBS with 1% BSA. Antisera used i n staini n g benzoquinone or paraformaldehyde fixed tissues were dilut e d i n 50mM T r i s with 0.3 % T r i t o n x-100. Hybridoma supernatants were immunocytochemically screened neat and at d i l u t i o n s of 1:10 and 1:50 i n PBS with 1% BSA. Following the i n i t i a l incubation, s l i d e s were washed 3 times for 5 min periods i n PBS, and then incubated for 1 h with flu o r e s c e i n l a b e l l e d goat antiserum to rabbit IgG (Calblochem; La J o l l a , C a l i f o r n i a ) or to flu o r e s c e i n l a b e l l e d rabbit anti-guinea pig IgG (Miles-Yeda; N a p e r v i l l e , I l l i n o i s ) d i l u t e d 1:200 i n PBS (the host species of the primary antisera are indicated i n table 1). Sections were then washed 3 times as above and subsequently mounted i n glycerine PBS (3:1). Sections were examined with a fluorescence microscope under u l t r a v i o l e t l i g h t ( 220). A l t e r n a t i v e l y a peroxidase immunostain was used i n conjunction with guinea pig antiserum, or a peroxidase-antiperoxidase s t a i n was used with rabbit antiserum. Where the f i r s t layer consisted of a guinea pig derived antiserum, a peroxidase conjugated rabbit a n t i -guinea pig serum (Dako; Santa Barbara, C a l i f o r n i a ) at 1:200 was applied as the second layer and incubated for 30 min. The s t a i n was then developed by immersing the s l i d e s i n PBS buffer pH 7.3; containing 0.01% hydrogen peroxide and 0.05% 3,3'diaminobenzidine tetrahydrochloride (DAB) (BDH chemicals; Toronto, Ontario). Where the f i r s t layer consisted of a rabbit derived antiserum ( i e : 13-3) a goat a n t i - r a b b i t serum (Calblochem; La J o l l a , C a l i f o r n i a ) was applied at 1:100 and incubated for 30 min. The second layer was then thoroughly washed off and a rabbit peroxidase anti-peroxidase complex (Dako; -27-Santa Barbara, C a l i f o r n i a ) applied at 1:200. Slides were then developed as above and counterstained with haematoxylin. TISSUE EXTRACTION Wistar rats were anaesthetized with pentobarbitol; lOOul/lOOg, and the proximal 40-50 cm of small i n t e s t i n e removed and immediately frozen i n l i q u i d nitrogen. These tissues were then either immediately used or stored at -70^ C for l a t e r extraction. Frozen tissue was f i r s t boiled f or 10 min i n an equal volume (w/w) of d i s t i l l e d water, allowed to cool then homogenized for 2 min (Tekmar Tissumizer; C i n c i n n a t i , Ohio). A f t e r homogenization an equal volume of 4% t r i f l u o r o a c e t i c acid was added, and the suspension l e f t s t i r r i n g at 4^ C for 12 h. The acid extract was then centrifuged at 15,000 x g for 90min, the supernatant pr e c i p i t a t e d with NaCl (30g/100ml), and the tissue p e l l e t re-extracted as above. The s a l t p e l l e t was c o l l e c t e d by centrifugation and extracted 2x i n methanol pH 6.0 (25ml/g). Methanol soluble material was p r e c i p i t a t e d with 2 volumes cold ether and c o l l e c t e d by c e n t r i f u g a t i o n . The ether p r e c i p i t a t e was dried under a stream of ^ and then dissolved i n 0.1% TFA to a concentration of 15mg/ml. The dissolved ether p r e c i p i t a t e was then loaded i n 50 ml aliquots onto Sep-Pak C^g cartridges (Waters Associates; Mississauga, Ontario) using a syringe driven by a Harvard pump at a flow rate of approximately l.Oml/min. Aft e r loading, the cartridges were washed with 5ml 0.1% TFA followed by 5 ml of 5% a c e t o n i t r i l e , 0.1% TFA. Cartridges were then eluted with 2ml 50% a c e t o n i t r i l e i n 0.1% TFA. A c e t o n i t r i l e was eliminated from the 50% eluate by evaporation under a stream of N_. -28-CHROMATOGRAPHIC CHARACTERIZATION OF RAT INTESTINAL IR-M A) ION EXCHANGE CHROMATOGRAPHY The Sep-Pak concentrated extract was dialyzed overnight i n 0.02M ammonium acetate adjusted to pH 5.5 with d i l u t e a c e t i c a c i d . The dialyzed concentrate was then loaded onto a SP-Sephadex-C25 column (1.5x30 cm) and eluted with 150ml of 0.02M ammonium acetate pH 5.5. The column was then eluted with a further 110ml of 0.3M ammonium acetate, adjusted to pH 8.0 with 0.02M ammonia. Flow rate was approximately 1.5ml/mln. Fractions (3ml) were monitored for absorbance at 280 nm and assayed for IR-M by RIA. B) GEL FILTRATION CHROMATOGRAPHY The SP-Sephadex-C25 f r a c t i o n s containing the IR-M were pooled and applied to a Biogel P-10 (200-400 mesh) gel f i l t r a t i o n column (2.5 x 20 cm). The column was eluted with 0.2M a c e t i c acid at a flow rate of 0.6 ml/min. Fractions (3ml) were c o l l e c t e d and monitored f o r absorbance at 280 nm and assayed f o r IR-M by RIA. C) HPLC The IR-M peak obtained from the gel f i l t r a t i o n step was pooled and l y o p h i l i z e d . The l y o p h i l i z e d material was reconstituted with 0.1% TFA and applied to a 3.9mm x 30cm ^i-Bondapak column (Waters; Mlssissauga, Ontario). The column was eluted at a flow rate of 1.5 ml/min and developed with a l i n e a r gradient of w a t e r / a c e t o n i t r i l e ; 0.1% TFA 15-45% over 30 min. Fractions were c o l l e c t e d every 0.5 min and assayed for IR-M by RIA. -29-Porcine motilin submitted to an extraction protocol similar to that of rat intestinal tissue was chromatographed as an external and internal standard for comparison to rat intestinal IR-M. SMOOTH MUSCLE CONTRACTILE ACTIVITY New Zealand White rabbits of either sex were anaesthetized with a solution of 30% urethane 1% alpha-chloralose i . v . , and the duodenum and the proximal 5cm of jejunum removed rapidly and placed in Krebs' ringer bicarbonate at 22^C. The solution (in mM) was composed of KCl 4.4, CaCl2 2.5, MgSO^  1.2, KH2P04 1.5, NaHC03 25, NaCl 120, dextrose 9.0. Choline chloride was added to a final concentration of 10 uM and the solution maintained at 37^  C and gassed with 95% 0 2 , 5% C02« Longitudinal segments of duodenum in lengths of 1.5-2.0cm were suspended vertically in a 5 ml organ chamber containing Krebs ringer bicarbonate. The upper end of the strips was connected to a force-voltage transducer (Grass model TF03; Quincy, Massachusetts) and the signal amplified (Grass model 7DA) and recorded on a chart recorder (Grass model 79WU). Tissues were suspended and adjusted to a tension of approximately lg, then allowed to equilibrate for 15 min. Following the in i t ia l equilibration, porcine motilin at 10 M^ was applied and rinsed out as soon as a maximal response was seen. The preparation was then allowed to equilibrate for a further 20-30 min. After final equilibration the preparation was tested for desensitization to porcine motilin by administering a subsequent dose of motilin within 4-5 min of a previous dose. The response to the second dose was then compared to that of the f irst . In each experiment of this type a dose of acetylcholine was given both before and after motilin -30-admlnlstration as a means of gauging the s e n s i t i v i t y of the muscle s t r i p over the experimental period. In a s i m i l a r experiment rat i n t e s t i n a l extract was administered to the muscle bath within 4-5 min of a previous dose of porcine m o t i l i n , and the c o n t r a c t i l e response compared to that induced by the extract without p r i o r administration of m o t i l i n . -31-RESULTS MIGRATING MYOELECTRIC COMPLEX Regular patterns of myoelectric a c t i v i t y i n the i n t e s t i n e of the fasted rat have been recorded from c h r o n i c a l l y implanted bipolar electrodes. Polyphasic e l e c t r i c a l complexes with a frequency of 4-5/sec ( f i g IC) occurred superimposed on a background of regular and continuous slow waves with a frequency ranging from 40-43/min ( f i g . I B ) . The polyphasic e l e c t r i c a l complexes occurred i n c y c l i c association with the slow waves and were represented by 3 recognizable phases. 1) A 4-5 min period of almost complete absence of spike bursts followed by, 2) a 1-1.5 min period of i r r e g u l a r spike burst occurring at a frequency ranging from 6-24/min. 3) The period of I r r e g u l a r spiking a c t i v i t y was followed by a 2-3 min period of intense spiking a c t i v i t y during which spike bursts occurred with every slow wave (f i g . I D ) . To improve the recognition of myoelectric patterns recorded the number of spike bursts per 20 sec i n t e r v a l was summed and plotted as "integrated a c t i v i t y " . The duodenal myoelectrical pattern exhibited by fed rats ( f i g 2A) consisted of i r r e g u l a r spike bursts occurring with an average frequency of 15-18/mln. The integrated myoelectrical pattern i n the fed rat was noticeably d i f f e r e n t from that seen i n the fasted rat ( f i g 2B) Intravenous ( i . v . ) glucose administered at a dose of 10-mg.kg 1 min v i a a cannula i n t o the jugular vein had no e f f e c t on the period or i n t e n s i t y of myoelectric complexes recorded from the duodenum of fasted rats ( f i g 3A). A single o r a l glucose dose of lg«kg , however, ra p i d l y resulted i n the d i s r u p t i o n of the fasted pattern of myoelectric a c t i v i t y and the appearance of continuous spike bursts at -32-submaximal frequency (fig. 3B), a pattern characteristic of the fed state. Intravenous administration of 6 mg cysteamine in 0.25 ml saline had no effect on the fasted pattern of periodic activity. Administration of 11 mg cysteamine ln 0.25 ml saline, however, prolonged the third phase of periodic activity (spike bursts at maximal intensity) so that the period of quiescence between complexes was abolished (fig. 2B). Intravenous administration of 5 ug motilin In 0.25 ml saline and 35 pg motilin in 0.25 ml saline did not affect the fasted pattern of periodic activity recorded from the duodenum of the fasted rat. Similarly, administration of 500 ul motilin antiserum M03 diluted 1:1 in saline had no affect on the fasted pattern of myoelectric activity (fig 4). HYBRIDOMA PRODUCTION In general Balb/c mice showed a moderate to strong immune response to subcutaneous injection of motilin-KLH conjugate in complete Freunds adjuvant. This is depicted by the high anti-motilin titre seen in mouse serum 28 days after primary Injection of motilin-KLH conjugate (50 ug motilin equivalent) (fig 5). Fusions were also successful, resulting in greater than 50% of the primary fusion colonies with a positive reaction to porcine motilin upon screening with ELISA. However, subsequent ICC screening of approximately 101 "ELISA-positive" clones on sections of rat duodenum and ileum, produced only negative results. - 3 3 -ImV Ssec 1mV 0.5sec 2mV <M> wii»n SMIUKI^HIH* • in. mi mn Hun )iimini)i |mn a mni i 2mV 20sec IH-li-t Of- 1 1 1 1 I Mr-20sec - 3 3 b -Figure 1. A) Diagramatic illustration of electrode insertion into the intestinal smooth muscle. A 27 gauge needle was used as a trocar to insert stainless steel wire (75 um in diameter) through the muscle layer. The wire was tied off close to the intestinal wall, and proliferation of serosal tissue achieved fixation and insulation within 5 days. B) Electromyogram (expanded scale) showing slow waves with a frequency of 42/min. C) Expanded scale showing polyphasic spike burst with a frequency of approximately 4-5/sec. D) Electromyogram (reduced scale) showing 6 min continuous recording from duodenal bipolar electrode. Two phases of activity are depicted, a 2.4 min period of spike burst with every slow wave, followed by approximately 3 minutes of quiescence. 6mg c y s l « « m l n « » 3 0 4 0 TIMC (MINI Figure 2. A) Histogram of integrated a c t i v i t y (spike bursts/20 sec i n t e r v a l ) recorded from the duodenum of a fed r a t . The frequency of spike bursts are submaximal and display no d i s t i n c t pattern. Representative of 3 t r i a l s . B) Histogram of integrated a c t i v i t y recorded from the duodenum of a fasted r a t . Spike bursts occur at maximal frequency f o r periods of 2-3 min, followed by 6-7 min of quiescence and a return to maximal a c t i v i t y . 6 mg cysteamine ( i . v . i n 200ul saline) had no e f f e c t on t h i s pattern, but 11 mg cysteamine disrupted the pattern by eliminating the period of quiescence. Representative of 2 t r i a l s . -35 -o to JO >o <o so •> ro TIWC I MIN) Fi gure 3 . A) Histogram depicting integrated activity (spike bursts/20sec interval) recorded from the duodenum of a fasted rat. The administration of 10 mg-kg min ^ glucose i . v . had no effect on the myoelectric pattern. Representative of 2 tr ia ls . B) Histogram of integrated activity from the duodenum of a fasted rat. The -1 administration of oral glucose (lg.kg) was followed by a disruption of the fasted pattern of activity and the introduction of a fed pattern of myoelectric activity. Representative of 2 tr ials . - 3 6 -ifig Motilin I TIME (MINI 70 80 SO 100 110 120 130 TIME (MINI Figure 4. Histogram depicting 135 min continuous recording of integrated a c t i v i t y (spike bursts/20sec i n t e r v a l ) i n a fasted r a t . The intravenous administration of porcine m o t i l i n or antiserum directed against porcine m o t i l i n had no ef f e c t on the myoelectric pattern. DILUTION Figure 5. Enzyme linked immunosorbent assay for anti-porcine-motilin activity in serum from 3 mice. Mouse A and B were injected with motilin-KLH congugate (equivalent to 50pg motilin) 21 days prior to assay. Normal mouse was from the same l itter but received no motilin. After subsequent injections of motilin-KLH conjugate, spleen cells from mouse A were used for fusion. -38-CNBr CLEAVAGE OF NATURAL PORCINE MOTILIN The lyophilized CNBr reaction mixture was redissolved in 500 ul 0.1% TFA and centrifuged. In a typical experiment 100 ul of the resuspended cleavage material was injected into the HPLC system and eluted with a linear gradient of 17% to 57% acetonitrile over 15 min. Six major peaks were identified and labelled A through F (fig. 6A). Application of a CNBr blank (a reaction mixture identical to that used for motilin cleavage but containing no motilin) to the HPLC system and elution in the same manner identified no spurious peaks attributable to impurities in the CNBr (fig.6C). Similarly, addition of 20ug motilin to the CNBr cleavage material showed uncleaved motilin eluting in the approximate position of peak C (fig.6B). Five separate runs of the cleavage material were made through the HPLC system. Peaks A through F were collected separately for each run. Corresponding peaks for each run were pooled and lypophilized. Each of the pooled peaks were then reconstituted in 200 ul of HPLC water (0.1% TFA) and re-chromatographed on the HPLC system. The re-chromatographed material was isocratically eluted (no change in solvent concentration) at 26% acetonitrile. A representative chromatogram of the three major peaks is shown in figure 2. Isocratic elution of peak E demonstrated a minor contaminant with an elution time of approximately 6 min (fig 7A). This contaminant probably resulted from an overlap in peak collection during the in i t ia l chromatograph. The improved resolution with the re-chromatographed material, however, allowed for a more precise peak collection and consequently improved purification of fractions. Isocratic elution of peak B indicated that this fraction was collected relatively free of contamination from adjoining peaks (fig 7B). Peak F was re-chromatographed both with and without the addition of a 6 pg standard of uncleaved porcine motilin. Under these conditions (26% acetonitrile) motilin eluted at approximately 5.5 min. (fig 7C). Peak F appears to have been contaminated with material from peak E during the in i t i a l collection, as isocratic elution reveals two peaks, one with an elution time identical to peak E (fig 7C). Al l major peaks from isocratically eluted material were collected and lyophilized. Lyophilized material was then reconstituted in 50 mM acetic acid. IDENTIFICATION OF MOTILIN FRAGMENTS A. ) U.V. Spectra Ultra violet absorption spectra of peaks A through F revealed that the major tyrosine component was contained in fraction E (fig 8). Thus this fraction was designated as the N-terminal fragment. The only other fraction to show a minor peak at 280 nm was fraction D (not shown). The other two major peaks, B and F, did not exhibit any adsorption peaks at 280 nm. Either peak B or peak F was therefore designated as the C-terminal fragment. B. ) N-Terminal Residue Determination Cleavage at the methionyl residue with CNBr yields a 13 amino acid N-terminal fragment (CN 1), and a 9 amino acid C-terminal fragment (CN 2) . CN1 H2N-Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Leu-Gln-Arg-Met-COOH CN2 H9N-Gln*-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln-C00H * Motilin heterogeneity is possibly demonstrated by the presence of glutamic acid at position 14 (88b). -40-The amino groups available for dansylation in the C-termlnal fragment include the alpha-amino of the N-terminal glutamine as well as the ep8llon-amino groups of the lysine residues at position 16 and 20. The only amino group available for dansylation in the N-terminal motilin fragment is the alpha-amino of phenylalanine at the N-terminus. The OH group on the aromatic side chain of tyrosine at position 7 also provides a reactive site for dansylation within the N-terminal region 1-13. DNS-tyrosine and DNS-phenylalanine were the only DNS-amino acids detected for peak E from the HPLC fraction, thus confirming this fraction as the N-terminal fragment (fig 9A). Peak B was the only HPLC fraction yielding epsilon-DNS-lysine. DNS-tyrosine and DNS-phenylalanine were not detected in fraction B, thus confirming this fraction as the C-terminal motilin fragment. Of the minor peaks A, C, D, only peak D showed a slight absorbance peak at 280 nm which, in conjunction with co-elution of motilin at similar times (fig 6B), suggests that peak D represents uncleaved motilin. Dansylation of fraction A, C, and D, however proved inconclusive. Fraction F is suspected as the homoserine-lactone variant of the N-terminal motilin fragment as this fraction demonstrated the presence of low levels of DNS-phenylalanine and DNS-tyrosine. The lack of an absorbance peak at 280 nm for fraction F may simply reflect a low fragment concentration. - 4 1 -F l g u r e 6. Chromatograms o f CNBr c l e a v e d p o r c i n e m o t i l i n , o b t a i n e d by r e v e r s e phase HPLC w i t h a l i n e a r g r a d i e n t c o n s i s t i n g o f w a t e r / a c e t o n i t r i l e . A ) chromatogram o f 200ul CNBr c l e a v e d m o t i l i n . B) chromatogram o f l O O u l CNBr c l e a v e d m a t e r i a l w i t h a d d i t i o n o f 20ug i n t e r n a l m o t i l i n s t a n d a r d . C) chromatogram of l O O u l of CNBr b l a n k . - 4 2 -ISOCRATIC ELUTION OF CNBr C L E A V A G E F R A G M E N T S 0 ) u." => < o CM CM CO m < 0.2 0.05 0 0.05 • 0 -0.05 Bug Motilin • i i • i i i i i i 0 6 12 18 Elution Time (min) FlRure 7. Chromatograms of major pooled peaks from the in i t i a l HPLC separation of CNBr cleaved porcine motilin. Chromatograms were obtained by reverse phase HPLC Isocratically eluted with 26% acetonitrile. A) chromatogram of peak "E". B) chromatogram of peak "B". C) chromatogram of peak "F" with addition of an internal standard of 6ug porcine motilin. -43-U.V. SPECTRA OF HPLC FRACTIONS B,E,F 0,10 • • • • » • • i t 280 240 280 320 280 Wavelength (nm) Figure 8. Ultra violet spectra of major peaks from in i t ia l HPLC separation of CNBr cleaved porcine motilin. Left) UV spectra of peak "B" . Centre) UV spectra of peak "E", with absorbance peak at 280 nm indicating presence of tyrosine residue. Right) UV spectra of peak o LYS DNS-OH TYR ° L U O DNS-NH , 0 o 3 PHE O 1 t B D DNS-OH A v" + I TYR o PHE o Figure 9. Thin layer chromatography of dansyl amino acids generated from CNBr cleavage fragments of porcine motilin. Chromatograms were obtained by spotting hydrolysed material onto 5x5 cm polyamide plates and running as described in the methods section. A) standard containing DNS-tyrosine, DNS-lysine, DNS-phenylalanine, DNS-glutamic acid and DNS-amide. B) chromatogram of DNS-amino acids from peak "E". C) chromatogram of DNS-amino acids from peak "B". D) chromatogram from peak "F". Solvents and direction of solvent migration is as indicated by arrows. -45-CHARACTERIZATION OF MOTILIN ANTISERA Antisera 72x and 74 were produced i n guinea pigs by immunization with porcine m o t i l i n conjugated to bovine serum albumin (BSA) using the carbodiimlde method. Antisera 13-3, Mx, and M03 were produced i n rabbits by immunization with porcine m o t i l i n conjugated to BSA using the carbodiimlde method. Pre-incubation of antisera 72x and 13-3 with the C-terminal fragment of m o t i l i n p r i o r to appl i c a t i o n to the ELISA ( f i g 10), produced i n h i b i t i o n curves i d e n t i c a l to those produced by pre-incubation with uncleaved m o t i l i n , i n d i c a t i n g that these antisera are highly s p e c i f i c f o r the C-terminal region of m o t i l i n . These antisera also demonstrate a minor component with N-terminal s p e c i f i c i t y , as pre-incubation with the N-terminal fragment produced a maximal i n h i b i t i o n of between 10-20%. Antisera 74 and M03 also demonstrate a primary s p e c i f i c i t y directed toward the C-terminal region of m o t i l i n , however, a r e l a t i v e l y high proportion of the antibodies i n these antisera are also capable of detecting the N-terminal region of m o t i l i n ( f i g 11). RADIOIMMUNOASSAY Three d i f f e r e n t antisera were used for RIA's: 13-3, 72X, and M03. The other antisera or monoclonal antibodies l i s t e d i n table I were not appropriate f o r RIA. Of the three antisera used, 13-3 and 72X were able to detect IR-M peptides i n the Sep-Pak concentrated extract of rat i n t e s t i n a l tissue (Fig 12) Both antisera 13-3 and 72X demonstrated s i m i l a r binding c h a r a t e r i s t i e s f o r IR-M i n rat i n t e s t i n a l extracts, as s e r i a l d i l u t i o n of th i s extract produced curves p a r a l l e l to the standard curve. Antisera 13-3 demonstrated considerably higher t i t r e than antisera 72x, 13-3 was therefore employed i n the RIA used to monitor the extraction procedures. -46-AS 13—3 dilution: 1:16000 [FRAGMENT / MOTILIN) n g / m l 90 1 1 80 : 70-C D 60-C D 50-40-C O 30-HN: 20-10-0-A S 7 2 - X dilution: 1:2000 101 102 [FRAGMENT/MOTILIN) n g / m l Figure 10. Regional specificity of antisera 72X, and 13-3 as demonstrated with the Enzyme Linked Immunosorbent Assay. Each antiserum was pre-incubated for 24 h at 4° C with either natural porcine motilin, N-terminal fragment 1-13, C-terminal fragment 14-22, or no peptide. The antisera were then applied to motilin coated micro-titre plates and the assay developed as described in the methods. Inhibition of immunoreactivity was calculated as a percentage of phosphatase activity in wells containing motilin antisera that had not been pre-incubated with motilin or motilin fragments. -47-A S M 0 3 U i •— i •— i • — — " i 10° 101 IO2 IO3 [FRAGMENT/MOTILIN] ng/ml Figure 11. Regional specificity of antisera M03, and 74 as demonstrated with the Enzyme Linked Immunosorbent Assay. Each antiserum was pre-incubated for 24 h at 4^  C with either natural porcine motilin, N-terminal fragment 1-13, C-terminal fragment 14-22, or no peptide. The antisera were then applied to motilin coated micro-ti tre plates and the assay developed as described in the methods. Inhibition of immunoreactivity was calculated as a percentage of phosphatase activity in wells containing motilin antiserum that had not been pre-incubated with motilin or motilin fragments. -48-CD CD CD CD a 3 2 -1 -0 --2 AS 13-3 dilution: 1:500,000 10° - MOTILIN • GUT EXTRACT 101 10 2 (MOTILIN) pg/ml 103 B AS 72-X dilution: 1:40.000 2.0 1.0-1 CD CD CO 5 0.0-1 CD ~ J -1.0 -2.0 1 0 ° • GUT EXTRACT PORCINE M 0 T L N 101 10 2 [MOTILIN] pg/ml 10 3 Figure 12. RIA standard curves and serial dilutions of Sep-Pak concentrated rat intestinal extracts.(A) antisera 13-3, (B) antisera 72X - 4 9 -ICC RESULTS AS// code type host pig i n t . / r a t i n t . / r a t cer. RIA d i l u t i o n s p e c i f i c i t y 1 72x poly Gp _____ , , _____ + 1:40k C-term 2 13-3 poly Rb + + 1:500k C-term 3 23B mono My + n/a > ? 4 M03 poly Rb ———- i i _____ „ , 1:10k C/N-term 5 74 poly Gp _____ _____ n/a C/N-term-6 Mx poly Rb n/a ? TABLE I. The antiserum employed ; the host species i n which they were produced (Gp,gui nea pig. Rb, r a b b i t . My, mouse myeloma NS1.); t h e i r a b i l i t y to react with c e l l s In tissue from pig i n t e s t i n e (pig i n t . ) , rat i n t e s t i n e (rat i n t . ) , or rat cerebellum (rat cer.); the a b i l i t y of antisera to detect IR-M i n rat i n t e s t i n a l extracts by RIA; and the antigenic s p e c i f i c i t y of the antisera as determined by binding to C-terminal or N-terminal fragments of porcine m o t i l i n . (n/a= antiserum not 125 applicable to RIA; no displacement of I-motilin) - 5 0 -TISSUE EXTRACTION The purification steps in the crude extraction of IR-M from 560g of rat intestinal tissue are shown in table II. For both the methanol extraction and Sep-Pak chromatography, steps, yields of IR-M were approximately 70% of the previous step. In both of these steps IR-M in side fractions accounted for the remaining 30%. Yield after SP-Sephadex C-25 chromatography, however, fe l l to 32% of IR-M in the previous step, and the early eluting fraction from this column accounted for less than one quarter of the remaining 68%. Overall purification after SP-Sephadex C25 chromatography was approximately 19 fold. CHROMATOGRAPHIC CHARACTERIZATION OF RAT INTESTINAL IR-M  GEL FILTRATION CHROMATOGRAPHY Fractions //53-70 from SP-Sephadex C25 chromatography (fig 13A) were pooled and lyophilized. The lyophilized fraction was redissolved in 2ml 0.2M acetic acid and 80ul sample of this material further diluted to 0.5ml and applied to the Biogel P-10 column. The motilin like immunoreactive peak co-eluted with porcine motilin indicating a similar molecular size (fig 13B). Recovery of Immunoreactive material in fractions #10.-12 was approximately 90% of that applied to the column. HPLC The pooled IR-M peak (fractions #10-12) from gel filtration chromatography were chromatographed on the HPLC system. Elution with a linear gradient of water/acetonitrile; 0.1% TFA 15-50% over 30 min, yielded an early peak that did not correspond to the e l u t i o n time for porcine m o t i l i n ( f i g 14). CONTRACTILE ACTIVITX Fractions number 53 to 70 from SP-Sephadex C25 chromatography were pooled and l y o p h i l i z e d . The l y o p h i l i z e d material was reconstituted with 2ml dl^O. Half of t h i s sample was retained for further chromatographic steps and the other h a l f u t i l i z e d i n c o n t r a c t i l i t y studies. The molar concentration of t h i s material, i f s i m i l a r i n molecular weight to porcine m o t i l i n , was approximately 7 x 10~^M. F i f t y m l c r o l i t r e aliquots were introduced to a 5 ml organ bath. Consequently the f i n a l concentration was equivalent to 7 x 10 ^M. The c o n t r a c t i l e a c t i v i t y induced by t h i s material was not equivalent to that observed with a 10 dose of porcine m o t i l i n ( f i g 15). The concentrated tissue extract produced a b r i e f c o n t r a c t i l e response, and the preparation quickly returned to base l i n e a c t i v i t y a f t e r a single washing. The response to 10 m o t i l i n , however, was more prolonged. Overall muscle tension did not return to baseline even a f t e r repeated washings, and i n t r i n s i c c o n t r a c t i l e a c t i v i t y changed from a rhythmic to a more phasic type of response ( f i g 15). Segments of rabbit duodenum demonstrated desensitization to porcine m o t i l i n . Response to a 10 dose of m o t i l i n was s i g n i f i c a n t l y reduced when repeated within 4-5 min of a previous dose ( f i g 16). The response of the preparation to 10 acetylcholine was used as an index of tissue s e n s i t i v i t y . Acetylcholine induced contraction was s i m i l a r before and a f t e r m o t i l i n treatment, demonstrating that desensitization was not accompanied by a loss of o v e r a l l tissue s e n s i t i v i t y . Pretreatment of the duodenal segment with a 10 dose of -52-porcine motilin did not diminish the contractile activity of a subsequent dose of extracted IR-M (fig 17). IMMUNOCYTOCHEMISTRY Rigorous attempts to immunostain rat intestinal tissue for IR-M failed to produce positive results with any of the antisera employed. Two of the antisera, however, were capable of staining IR-M cells in porcine jejunum (table 1). An example of this staining and the ability to inhibit the reaction by pre-ihcubation* of the antiserum with porcine motilin is given in figure 18. DISTRIBUTION OF IR-M IN THE RAT SMALL INTESTINE Tissue from five different regions of the rat small intestine was removed and Immediately frozen in liquid nitrogen. The tissue was pulverized while frozen using a mortar and pestel, then extracted for 6 h in 2% TFA. After centrifugation aliquots were lyophilized then reconstituted in assay buffer and assayed for IR-M. The two antisera capable of recognizing rat intestinal IR-M (table I), gave strikingly different distributions. Most notably antiserum 13-3 detected large amounts of IR-M in the duodenum, whereas antiserum 72X did not detect any IR-M in the duodenum (table III). -53-FRACTION ' (3ml) I MOTILIN I t II II 21 f RUCTION ' (2 ml) Figure 13. (A) SP-Sephadex-C25 chromatography of Sep-Pak concentrated rat Intestinal extract. (B) Gel filtration (Bio-gel P-10 200-400 mesh) of pooled fractions 53-70 from SP-Sephadex-C25. -54-/ porcine motilin IR-M (ng/ml) 0 0.2 0.1 I 20 40 60 FRACTION # (0.75 ml) 80 Figure 14. HPLC profile of rat intestinal extract after fi ltration. Figure 15. C o n t r a c t i l e response of i s o l a t e d rabbit duodenal muscle s t r i p to rat i n t e s t i n a l t i s s u e extract and 10~7M porcine m o t i l i n . (W"wash) - 5 6 -MOTILIN A c h Figure 16. M o t i l i n induced de s e n s i t i z a t i o n of rabbit duodenal mucscle. Histogram represents the mean + S.E.M. (N-4) c o n t r a c t i l e response as a percentage of the o r i g i n a l response to a 10 dose of m o t i l i n . The second response was s i g n i f i c a n t l y reduced compared to the response observed when the same dose was given 4 min e a r l i e r . * s i g n i f i c a n t to p-0.01 (ANOVA). W - wash -57-EXTRACT A c h Figure 16. Motilin induced desensitization of rabbit duodenal mucscle. Histogram represents the mean + S.E.M. (N-A) contractile response as a percentage of the original response to a 10 M^ dose of motilin. The second response was significantly reduced compared to the response observed when the same dose was given A min earlier. * significant to p=0.01 (ANOVA). W = wash - 5 8 -• * • IP I -58b-Flgure 18. S e r i a l sections of porcine duodenum (10 um) . A) Arrow indicates peroxidase anti-peroxidase stained c e l l s using m o t i l i n antiserum 13-3. B) S e r i a l section stained i d e n t i c a l to plate "A", but antiserum 13-3 was incubated overnight at i n lug/ml porcine m o t i l i n . - 5 9 -A 280 IR-M Step STEP (total) (total) IR-M/A280 Purification Purification Yield acid extract 14242 2800 ng 0.2 100Z methanol 2325 2085 0.9 4.3 4.3 74 Sep-Pak 790 1416 1.78 8.9 2.1 68 SP-Sephadex- 120 450 • 3.75 18.8 2.1 32 C25 Table II. Partial purification of rat intestinal IR-M from 560g tissue. Total absorbance: A280 dilution volume. Yield at each step was calculated as a percentage of the total IR-M detected i n the previous step. - 6 0 -Antiserum TISSUE 13-3 72X corpus 1.45 +0.21 ND (ng/g wet weight) duodenum 4.96+0.55 ND jejunum 1 3.65+0.28 1.01+0.22 jejunum 2 3.62+0.22 1.35+0.53 ileum 3.77+0.33 1.64+0.21 control 0.52+0.15 ND Table III. Distribution of IR-M In the rat corpus and proximal small bowel. (Values are means + standard error, N=4-6. jejunum 1 = proximal 30 cm, jejunum 2 = distal 30 cm. Control = abdominal muscle.) -61-t Discussion 1) INTERDIGESTIVE INTESTINAL MYOELECTRIC ACTIVITY IN THE RAT The Interdigestive migrating myoelectric complex (MMC) has been observed i n several mammalian species i n c l u d i n g the rat (17,46,48,81,96). Exogenous porcine m o t i l i n has been shown to increase the frequency of MMC i n both man (97) and dog (48,101). Both species demonstrate peaks i n endogenous m o t i l i n correlated with phase III of the MMC i n the upper duodenum and stomach (49,50,96). Species differences i n the ph y s i o l o g i c a l control of the MMC appear to be associated with differences i n eating habits and food sources (98). Periodic myoelectric a c t i v i t y seen i n fasted herbivores i s not disrupted by normal feeding. In omnivores such as the pi g , periodic a c t i v i t y i s not disrupted by normal feeding, but can be altered by high-calorie meals. Rat dog and man are a l l c l a s s i f i e d as carnivores, and i n these species normal feeding abolishes the periodic myoelectrical a c t i v i t y associated with the fasted state. There i s an interspecies r e l a t i o n s h i p between eating habits and food source, and changes i n the m o t i l i t y pattern seen with the fed and fasted state. This r e l a t i o n s h i p , however, does not nec e s s a r i l y Imply s i m i l a r i t i e s i n the mechanisms f o r c o n t r o l l i n g these m o t i l i t y patterns. There are considerable s i m i l a r i t i e s i n the presentation of MMC's i n both man and dog, i n c l u d i n g four distinguishable phases and a period of approximately 100 min (100). Exogenous m o t i l i n e l i c i t s premature a c t i v i t y fronts i n both species (97,48). In dog, disappearance of MMC's with immunoneutralization of m o t i l i n (55,74), and the persistence of MMC's with fundic denervation (94) c l e a r l y indicates that m o t i l i n i s acting as a humoral modulator of i n t e r d i g e s t i v e g a s t r i c c o n t r a c t i l e a c t i v i t y . Other aspects of m o t i l i n physiology, such as i t s e f f e c t s on g a s t r i c emptying (23,27,82) and stimuli for release (23,61) are not s i m i l a r i n man and dog. As i s seen i n both man and dog, i n t r a g a s t r i c glucose and other nutrients (81) disrupt periodic i n t e r d i g e s t i v e a c t i v i t y i n the r a t . There i s , however, no data a v a i l a b l e f o r comparison of the rat to man or dog with respect to the e f f e c t s of exogenous m o t i l i n , endogenous m o t i l i n , or other peptides and neural elements on i n t e r d i g e s t i v e periodic a c t i v i t y . Recordings of rat i n t e r d i g e s t i v e myoelectric a c t i v i t y i n t h i s study are comparable to previous observations although MMC periods observed, 7-10 min, were somewhat shorter than has been previously recorded; 8-12 min (72), 14-18 min (81), 11-18 min (99). Disruption of motor complexes by the administration of cysteamine i s also comparable to previous reports (72), although dosage and route of administration are not s i m i l a r for the two studies. S i m i l a r l y , o r a l glucose was observed to cause disruption of motor complexes. Not previously reported, was the observation that i . v . glucose had no e f f e c t on motor complexes i n the r a t . This r e s u l t i n d i c a t e s that luminal presentation of nutrients i s required f o r changes i n m o t i l i t y patterns. Similar r e s u l t s are seen i n the dog where i n t e r r u p t i o n of the myoelectric complex i s dependent on the p h y s i c a l and chemical nature of the food being presented to the duodenum. When the i n t e s t i n e i s by-passed and n u t r i t i o n i s given parenterally there i s no d i s r u p t i o n of periodic a c t i v i t y (98). The administration of m o t i l i n and m o t i l i n antiserum had no e f f e c t on the i n t e r d i g e s t i v e myoelectric a c t i v i t y i n the r a t . Similar r e s u l t s have been observed i n the pig (18,17). The observation that porcine m o t i l i n has no e f f e c t on the MMC i n rats may be due to species differences i n the structure of m o t i l i n . Conversely, the lack of an e f f e c t of porcine m o t i l i n i n the rat may represent a fundamental difference i n the p h y s i o l o g i c a l control mechanisms governing i n t e r d i g e s t i v e a c t i v i t y i n the rat as compared to dog and man. Such a difference i s exemplified i n the pig where MMC's occur during the digestive period (18), and periodic f l u c t u a t i o n s of plasma m o t i l i n are absent unless the animal i s fasted for prolonged periods (16). Before the structure of canine m o t i l i n was published i t was suggested that a l l 22 amino acids of m o t i l i n were important to i t s b i o l o g i c a l a c t i v i t y . This hypothesis was based on g a s t r i c c o n t r a c t i l e a c t i v i t y of synthetic porcine m o t i l i n fragments i n fasted conscious dogs (51). M o t i l i n fragments 1-6 and 12-22 had no c o n t r a c t i l e a c t i v i t y , while fragment 7-22 demonstrated minimal a c t i v i t y , approximately 1/300 that of i n t a c t m o t i l i n . Replacement of the N-terminal residue of m o t i l i n also resulted i n a loss of a c t i v i t y to l e s s than 1/300 of the natural peptide. S u r p r i s i n g l y , the recent c h a r a c t e r i z a t i o n of canine m o t i l i n i n d i c a t e s that i t d i f f e r s from porcine m o t i l i n at 5 of i t s 22 amino acids (75,80). The amino acid differences occur at positions 7, 8, 12, 13 and 14; hi s t i d i n e : t y r o s i n e , s e r i n e : g l y c i n e , l y s i n e : a r g i n i n e , isoleucine:methionine, arginine:glutamine, r e s p e c t i v e l y (canine:porcine). Amino acid switches at positions 8, 12 and 13 represent only minor changes i n s t r u c t u r e . The replacement of an aromatic with a basic residue at p o s i t i o n 7 and switching of a basic for an a c i d i c residue at p o s i t i o n 14, however, represent s i g n i f i c a n t s t r u c t u r a l a l t e r a t i o n that might be expected to change the t e r t i a r y structure of canine m o t i l i n . Based on e a r l i e r structure a c t i v i t y observations (51) these differences i n canine m o t i l i n would also be expected to s i g n i f i c a n t l y reduce the potency of this peptide. As the b i o l o g i c a l potencies of canine and porcine m o t i l i n are i d e n t i c a l (75), i t must be concluded that the pharmacophore of porcine m o t i l i n does not include the e n t i r e 22 amino acid sequence, and that t h i s b i o l o g i c a l l y active sequence i s not affected by the amino acid changes seen i n canine m o t i l i n . Three generalizations can be drawn from the above discussion: 1) Man and dog, both c l a s s i f i e d as carnivores, display s i m i l a r c h a r a c t e r i s t i c s with respect to the r o l e of m o t i l i n i n the control of i n t e r d i g e s t i v e g a s t r i c c o n t r a c t i l e a c t i v i t y . 2) The r a t , also c l a s s i f i e d as a carnivore, displays c h a r a c t e r i s t i c s i n d i c a t i n g that control of i n t e r d i g e s t i v e c o n t r a c t i l e a c t i v i t y may be s i m i l a r to man and dog. 3) Amino acid sequence differences between canine and porcine m o t i l i n do not a f f e c t the b i o l o g i c a l a c t i v i t y of m o t i l i n i n the dog. The rat i s ostensibly within the same c l a s s i f i c a t i o n as man and dog i n that i t i s a carnivore and i n t e r d i g e s t i v e MMC's are disrupted by the luminal presentation of c e r t a i n n u t r i e n t s . Therefore, a natural extension of the above generalizations would be to suggest that the e f f e c t s of porcine m o t i l i n i n the rat should be s i m i l a r to m o t i l i n induced effects i n man and dog. As t h i s does not appear to be the case i t must be concluded that the rat displays fundamental differences from other carnivores in the mechanisms controlling Interdigestive activity. i i ) PORCINE-MOTILIN MONOCLONALS DO NOT DETECT IR-M IN THE RAT Several reports have indicated that antisera directed against porcine-motilin were capable of specifically staining rat cerebellar tissue (19,68). Certain motilin antisera were also capable of detecting IR-M in extracts of rat intestine (68,54,66). Based on these observations i t appeared reasonable to assume that a monoclonal antibody directed against rat intestinal IR-M could be produced using porcine motilin as the original antigen. One fusion, utilizing murine B-cells primed with porcine motilin, produced over 100 motilin positive clones none of which reacted with rat intestinal tissue. None of the serum, samples collected from motilin immunized mice prior to fusion were capable of immunochemically staining rat intestinal tissue. Although ICC results were negative for mouse serum prior to fusion i t was speculated that the polyclonal antiserum could possibly contain a very small fraction with the ability to bind rat IR-M, and that this small fraction although not evident in the polyclonal state, could become amplified through fusion and subsequent cloning. The failure to produce a monoclonal antibody capable of reacting with rat intestinal tissue, while inconclusive in itself , does suggest that the ability of polyclonal antisera to detect IR-M in rat intestinal extracts is artifactual in nature. -66-111) CHARACTERIZATION OF RAT INTESTINAL IR-M Motilin was originally isolated from a side fraction produced during the extraction of secretin from hog intestinal mucosa (62,63). The technique originally used for the extraction of secretin involved boiling of the tissue for 8-10 minutes. The rationale for boiling the tissue includes denaturing proteolytic enzymes as well as precipitation of the bulk of the nitrogenous mass. Following the boiling step the tissue was homogenized then acid extracted. Basic peptides were removed from the acid extract by adsorption on alginic acid. Peptides were eluted from the alginic acid and precipitated with NaCl. The ethanol soluble fraction of this concentrate was subjected to gel filtration chromatography, and the smaller molecular weight fractions extracted into methanol. The methanol soluble fractions were retained for further purification of secretin. After methanol extraction the crude secretin was subjected to ion exchange chromatography on carboxymethylcellulose (CMC). The early eluting fractions from the CMC step were the side fractions that provided the starting material for motilin extraction (13,62). Gastrointestinal hormones are distributed diffusely throughout the gastrointestinal tract. The diffuse nature of the gastro-enteropancreatic neuroendocrine system has been the major obstacle to isolation of gastrointestinal regulatory peptides. As the pig is utilized for human consumption this species provided large quantities of starting material for the original isolation of motilin. Until recently, limited amounts of available tissue coupled with low recoveries have prevented isolation of gut peptides from species that - 6 7 -are not used for human consumption. Advances in purification and microsequence analysis, however, have contributed to the recent isolation and characterization of canine intestinal motilin (80). The technique utilized for the extraction of canine motilin s t i l l relies on boiling the tissue to denature proteolytic enzymes, and extraction of basic peptides with acid. Major differences in technique are seen in the methods of concentrating the various fractions. In the original isolation of porcine motilin, NaCl precipitation and lyophllization were employed, whereas Sep-Pak cartridges (Waters), and evaporation under reduced pressure were used as means of concentration in the isolation of canine motilin. Significant losses occur with salt precipitation and lyophllization as motilin is Invariably trapped within precipitates in both cases. These losses are greatly reduced by keeping the motilin containing fractions in an aqueous phase, as is seen with the extraction of canine motilin (80). Another major technical advance that greatly facilitated the microisolation of canine motilin was the use of high pressure liquid chromatography (HPLC). The sensitivity and resolution of HPLC greatly exceeds that possible with classical methods of column chromatography. In this study 560g of rat intestinal tissue was extracted. In accordance with established techniques for the extraction of gastrointestinal peptides, the tissue was first boiled for 10 min in order to denature proteolytic enzymes (62,63). After boiling, followed by extraction into 2.0% TFA, the yield was approximately 3ng/g tissue wet weight. From table III i t can be seen that the average IR-M content detected in the rat small bowel by antiserum 13-3 is -68-approxlmately 4ng/g tissue wet weight. The approximately 25% reduction in IR-M detected in the bulk tissue extract as compared to the smaller samples used in the distribution study, probably reflects the trapping of immunoreactive material within the cellular debris during centrifugation. In the distribution study, tissues were extracted in proportionally larger volumes of 2.0% TFA. Although extraction into a larger volume of dilute acid appears to provide a better in i t ia l yield of immunoreactive material, the larger volumes proved to be too unwieldy in the larger extractions to justify the extra 25% recovery. The observation that certain gastrointestinal peptides, including motilin, are soluble in low molecular weight alcohols was applied to the second stage of motilin extraction, although Mutt (62,63) applied methanol extraction to a much later step in purification of secretin (62). Extraction into methanol and subsequent precipitation with ether provided a significant concentration step as well as a four fold purification (table II). Application of methanol soluble material to Sep-Pak cartridges provided another two fold purification and further concentration. Loss of IR-M in both the methanol and Sep-Pak steps was approximately 30%. These losses could be largely accounted for in the immunoreactive content of methanol insoluble material and side fractions from Sep-Pak chromatography. With SP-Sephadex-C25 chromatography yield dropped to a prohibitive 32% of the previous step. Only 15-25% of the missing IR-M could be accounted for in the early eluting peak from SP-Sephadex-C25. The remaining immunoreactive material remained either strongly bound to the sulfonyl-propyl residues or lost its immunoreactive properties on passage through the column. The substantial drop in yield with SP-Sephadex-C25 chromatography prevented further quantitative separation as a relatively large quantity of immunoreactive material was required for assessment of contractile activity. A small aliquot of the later eluting peak from SP-Sephadex-C25 material, however, was retained for qualitative comparisons to the elution profiles of porcine motilin on gel filtration and HPLC. Gel filtration chromatographs of rat intestinal extracts yielded an IR-M peak that co-eluted with porcine motilin. This observation is in accordance with previously published observations (68), and indicates that rat intestinal IR-M has a molecular size similar to porcine motilin. When pooled IR-M from gel filtration was chromatographed by reverse phase HPLC, rat intestinal IR-M did not co-elute with porcine motilin. This observation is in disagreement with previously published results (54,68). There are two possible reasons for this discrepancy. First , the extraction protocol employed in this study may have altered rat intestinal IR-M resulting In a different elution profile on reverse phase HPLC. This possibility was examined by subjecting porcine motilin to a similar extraction procedure, then chromatographing "extracted" and "unextracted" porcine motilin. The elution profile of extracted porcine motilin was identical to that of porcine motilin before extraction. A more likely reason for the discrepancy between the chromatographic properties of rat intestinal IR-M observed in this study and those of previous reports is that a -70-more rigorous extraction protocol is employed in this work which presumably results in a more highly purified form of IR-M being applied to the HPLC column. Chromatographic profiles of crude extracts on HPLC can be highly variable and resolution is often poor with concentrated crude samples as non-covalent bonding between peptides can form complexes that are difficult to dissociate. A number of immunocytochemical studies have shown that motilin-containlng cells of the mammalian gastrointestinal tract were located predominantly in the duodenum and jejunum (44,71). Subsequent reports have indicated a more widespread distribution and have identified motilin immunoreactivity in the mucosa of the oesophagus, stomach, ileum, and colon, as well as peripheral locations such as pancreas, gall bladder, adrenals and kidney in some mammals (33,104). The nature of motilin containing cells has been investigated*in a number of studies. Motilin immunoreactivity has been described as primarily located to a sub-population of enterochromaffin (EC) cells (77), primarily in non-EC cells (44), or located in variable proportions to both EC cells and non-EC cells (33,24). The reasons for discrepancies in motilin distribution and cell type have been variously described as 1) differences in immunoreactivity of antisera (53), 2) motilin heterogeneity (24), or 3) variation in EC cell content dependent on stages of maturation of EC cells (26). The identification of IR-M in rat intestinal tissue is similarly plagued with discrepancies. Originally, Insignificant amounts of IR-M were detected by RIA in rat intestinal extracts (88,105). Subsequently, highly variable levels of IR-M in rat intestinal tissue have been reported (54,68). Consistent with e a r l i e r reports we have also detected r e l a t i v e l y low l e v e l s of IR-M i n rat i n t e s t i n a l t i s s u e . The apparent d i s t r i b u t i o n and r e l a t i v e amounts, however, vary s i g n i f i c a n t l y dependent on the antiserum used. As with discrepancies i n m o t i l i n l o c a l i z a t i o n and d i s t r i b u t i o n i n other species t h i s i s presumably due to v a r i a t i o n i n antisera s p e c i f i c i t y and\or heterogeneity of IR-M. The s p e c i f i c i t y of the antisera used i n th i s study was only roughly gauged according to the a b i l i t y of eit h e r N-terminal (1-13) or C-terminal (14-22) amino acid fragments of m o t i l i n to cross-react with the a n t i s e r a . The c r o s s - r e a c t i v i t y of these fragments was assessed by f i r s t incubating the antisera with the fragment and subsequently determining the r e s u l t i n g i n h i b i t i o n of binding to uncleaved m o t i l i n . Of the antisera thst were assessed i n t h i s manner (antiserum MX and monoclonal 23B yielded non-specific r e s u l t s ) there appeared to be two general groups; p r i m a r i l y C-terminal directed, or C-terminal directed with a large proportion also recognizing the N-termlnal. Antisera 13-3 and 72X are primarily C-terminal d i r e c t e d . With both of these antisera binding to uncleaved porcine m o t i l i n was less than 10% i n h i b i t e d when preincubated with the N-terminal fragment at a concentration of approximately 500 ng/ml f o r 72X and 10 ng/ml f o r antiserum 13-3. Maximal i n h i b i t i o n of binding to i n t a c t porcine m o t i l i n occurred when these antisera were pre-incubated with the C-terminal fragment at the same concentrations. Antisera M03 and 74, however, demonstrated approximately 50% and 40% i n h i b i t i o n of binding, respectively, when -72-pre-Incubated with the N-terminal fragments at the same concentrations that induced maximal i n h i b i t i o n using the C-terminal fragment. The observation that rat i n t e s t i n a l IR-M i s detected only by antisera which are predominately C-terminal s p e c i f i c i s consistent with e a r l i e r observations (88) where the highest concentrations of IR-M i n rat i n t e s t i n e were detected using a C-terminal s p e c i f i c antiserum. This i s suggestive of homology between the C-terminal region of porcine m o t i l i n and an unspecified region of rat i n t e s t i n a l IR-M. However, the rough approximation of antisera s p e c i f i c i t y to eith e r the C-terminal or N-terminal i n t h i s study and that of Shin e t . a l . (88) allows for only the most ten t a t i v e speculation with respect to possible homologies. For instance, both t h i s study and that of Shin et. a l . (88) demonstrated that two apparently C-terminal directed a n t i s e r a could detect widely varying amounts of IR-M. Shin e t . a l . further characterized his C-terminal antisera i n t o e i t h e r s e n s i t i v e or i n s e n s i t i v e to amino acid s u b s t i t u t i o n s at p o s i t i o n 15. Similar ch a r a c t e r i z a t i o n was not attempted i n t h i s study, but presumably s i m i l a r differences i n antisera 72 and 13-3 may e x i s t and thus could explain the d i f f e r e n t d i s t r i b u t i o n s observed. Rat i n t e s t i n a l extracts were compared to natural porcine m o t i l i n 125 for t h e i r a b i l i t y to displace I - m o t i l i n i n the m o t i l i n RIA. Representative figures are shown i n f i g u r e 12A and 12B. The I n h i b i t i o n caused by the addition of s e r i a l d i l u t i o n of rat i n t e s t i n a l extract i s p a r a l l e l to the porcine m o t i l i n standard curve. This suggests that both antisera 13-3 and 72X have s i m i l a r binding c h a r a c t e r i s t i c s f o r porcine m o t i l i n and rat i n t e s t i n a l IR-M. - 7 3 -125 The s p e c i f i c displacement of I - m o t i l i n by rat i n t e s t i n a l IR-M was further examined by adding a standard amount of porcine m o t i l i n to rat i n t e s t i n a l extracts. The increased displacement of 1 2 " ' l - m o t i l i n r e s u l t i n g from the addition of porcine m o t i l i n to samples of rat I n t e s t i n a l extract was additive and l i n e a r . These r e s u l t s are a further i n d i c a t i o n that rat i n t e s t i n a l extracts did not i n t e r f e r e with the binding of porcine m o t i l i n to the antisera used. Attempts to immunostain for m o t i l i n i n rat i n t e s t i n a l tissue have produced only negative r e s u l t s . There are several possible explanations f o r the lack of m o t i l i n - l i k e staining i n the rat i n t e s t i n e . I t i s possible that the major epitopes of rat m o t i l i n are unavailable to the antisera i n the i n s i t u preparation. This explanation seems u n l i k e l y i n that antisera which are appropriate for RIA of Gl peptides are i n general capable of detecting those peptides by immunocytochemical techniques. A l t e r n a t i v e l y , i t i s possible that tissue f i x a t i o n i s d i s r u p t i v e to rat i n t e s t i n a l m o t i l i n . Again, t h i s seems u n l i k e l y as a v a r i e t y of f i x a t i v e s were employed, and m o t i l i n -l i k e immunoreactivity was observed i n tissues from other species (pig) fixed i n an i d e n t i c a l manner. The t h i r d and most p l a u s i b l e explanation i s that the amino acid sequence of rat i n t e s t i n a l m o t i l i n i s d i f f e r e n t from that of porcine m o t i l i n and as a r e s u l t does not cross react i n s i t u with antiserum raised against porcine m o t i l i n . This hypothesis i s supported by the observation that p a r t i a l l y p u r i f i e d and concentrated rat i n t e s t i n a l IR-M produces c o n t r a c t i l e a c t i v i t y i n rabbit duodenal muscle s t r i p s that i s d i f f e r e n t from that induced by porcine m o t i l i n . Furthermore, there was no i n d i c a t i o n of c r o s s - d e s e n s i t i z a t i o n to rat -74-intestinal extract by pretreatment of the muscle strips with porcine motilin. This suggests that contractile activity induced by porcine motilin is not mediated by pathways similar to the contractile activity induced by rat . intestinal extracts of IR-M. These observations coupled with the lack of immunocytochemical staining for IR-M in rat intestinal tissue indicate that assayable levels of IR-M in rat tissue extracts may be due to nonspecific interaction of peptides or peptide fragments with the assay. Of interest in this regard are several recent reports disputing the existence of motilin in rat brain. Motilin-like immunoreactivity in rat brain was either not detected (RIA) (34), not inhibited (ICC) by addition of porcine motilin (54,67), or did not chromatograph (HPLC) similar to porcine motilin (54). These results suggest that localization of motilin in rat brain tissue (68,4) may also result from non-specific binding of an uncharacterized fraction of the antisera. In support of this view i t has been observed that several monoclonal antibodies raised against porcine motilin do not stain CNS tissue in a variety of species including the rat (S. Vincent , personal communication). In summary, the results presented here suggest that intestinal motilin, l f i t exists in the rat, is present in a form that is very different from porcine motilin. The possibility that the motilin-like immunoreactive material detected in the rat intestine is artifactual has been suggested by the Inability to generate monoclonal antibodies demonstrating anti-motilin activity in rat intestinal tissue. It is possible that fractions of the polyclonal antisera 13-3 and 72X react with peptide fragments generated during the extraction of intestinal -75-tissue. A computer search of known peptide sequences (4) has revealed that a portion of rabbit skeletal muscle tropomyosin (TM), 142-150, bears a striking similarity to the middle portion of porcine motilin (PM), 9-17. PM 9-17. Glu-Leu-Gln-Arg-Met-Gln-Glu-Lys-Glu TM 142-150 . Glu-Leu-Gln-Glu-Met-Gln-Leu-Lys-Glu Although intact or heat denatured TM did not cross-react with motilin antisera, specific fragments of digested TM were not rigorously tested (4). Furthermore, only known peptide sequences have been screened for possible homology with motilin. The majority of cellular proteins have not been sequenced, and the possibility remains of other peptide fragments with homologies to one or more regions of porcine motilin. Of interest in this regard is- the observation that pharmacologically active opioid peptide sequences can be derived from enzymatically treated bovine blood, and that these novel peptides originate from both cytochrome b and haemoglobin (7,8). It also appears unlikely that a motilin-like peptide is involved in the control of interdigestive periodic activity in the rat, as exogenous porcine motilin had no effect on the MMC in the rat. In species such as man and dog where motilin does appear to act as a modulator of MMCs, the biologically active site of motilin is conserved. According to current phylogenetlc theory the order Artiodactyl (pigs) and Carnivora (dogs) show divergent evolution from an early common ancestor. As motilin has been isolated and characterized in both the pig and dog, i t is apparent that the -76-ancestral m o t i l i n - l i k e peptide must have existed p r i o r to divergence. The same common ancestor that gave r i s e to the pig and the dog also gave r i s e to the primates of which the order Rodentia ( r a t s ) are an early divergent lineage. Consequently, a m o t i l i n - l i k e peptide might be expected to exist i n both rats and man. As previously discussed there are reasonably good data suggesting the existence of m o t i l i n i n man (plasma IR-M l e v e l s correspond to MMC cycles, and exogenous m o t i l i n induces premature MMCs). From the data presented i n t h i s study, the converse i s true f o r the r a t . The presence of m o t i l i n In widely divergent species such as pig and dog, and the high p r o b a b i l i t y that m o t i l i n e x i s t s i n a c l o s e l y related species (man) in d i c a t e s that the probable lack of m o t i l i n i n the rat i s due to evolutionary factors that have selected f o r divergence of the ancestral m o t i l i n - l i k e gene i n t h i s species. It i s apparent that m o t i l i n i s r e l a t i v e l y l a b i l e with respect to changes i n i t s amino acid sequence; 23% difference between canine and porcine m o t i l i n . The fact that m o t i l i n i s poorly conserved between species further supports the hypothesis that t h i s peptide may have been r e l a t i v e l y quickly selected out of c e r t a i n species. As previously mentioned, i n t e r r u p t i o n of periodic a c t i v i t y by the luminal presentation of nutrients i s c h a r a c t e r i s t i c of carnivores, but not of herbivores, and omnivores such as the pig w i l l d i splay c h a r a c t e r i s t i c s of eit h e r group depending on i t s d i e t . In the r a t , periodic a c t i v i t y i s also disrupted by food, placing t h i s species i n t o the catagory i n c l u d i n g carnivores. C l a s s i f y i n g the rat as a carnivore, however, i s dubious i f not misleading. The vast majority of rodents, due primarily to s i z e r e s t r i c t i o n s , are herbivores and although the rat may be a f a c u l t a t i v e carnivore i t i s l i k e l y to have evolved from a s t r i c t l y herbivorous lineage. The extended periods of almost continuous food intake c h a r a c t e r i s t i c of herbivores, may represent a sel e c t i v e pressure against the expression of m o t i l i n , and/or allow mutations leading to the expression of an e n t i r e l y d i f f e r e n t peptide, both f u n c t i o n a l l y and immunologically. In conclusion, two of the a n t i s e r a empolyed i n t h i s study recognize an IR-M component of rat i n t e s t i n a l extracts. This component, however, i s l i k e l y to be an a r t i f a c t possibly r e s u l t i n g from a protein fragment that has some homology with the C-terminal portion of m o t i l i n . Thus a large scale extraction of rat i n t e s t i n a l tissue f o r m o t i l i n u t i l i z i n g a n t i s e r a 13-3 or 72x i n the detection method, i s not warranted. 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