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Morphological and functional studies of substance P and somatostatin in the small intestine Accili, Eric Anthony 1992

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MORPHOLOGICAL AND FUNCTIONAL STUDIES OF SUBSTANCE P AND SOMATOSTATIN IN THE SMALL INTESTINE  By  Eric Anthony Accili, B.Sc. (1982), M.Sc. (1987) The University of British Columbia  A thesis submitted in partial fulfillment of the requirements for the degree of  Doctor of Philosophy  in The Faculty of Graduate Studies Department of Physiology  We accept this thesis as conforming to the required standard  Signature(s) removed to protect privacy  The University of British Columbia June 1992  ©  Eric Anthony Accili, 1992  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.  Signature(s) removed to protect privacy  (Signature)  Department of  /4’’ -,y  The University of British Columbia Vancouver, Canada  /97  Date  7  DE-6 (2/88)  ii  ABSTRACT  The regulation of gastrointestinal function is partly dependent on the intrinsic neurones of the gut.  Intrinsic  neurones contain a large number of neurotransmitters, including neuropeptides,  in different combinations.  Neurones have been grouped according to the combination of neurotransmitters they contain and this practice is called chemical coding.  The present studies were carried out to  examine differences in chemical coding and enteric neuronal morphology in the human and canine small intestine, especially the submucosal plexus. for examination were substance P,  The neuropeptides chosen somatostatin and  vasoactive intestinal peptide because of their known involvement in both the physiology and pathophysiology of the small intestine.  Further, primary cultures of  submucosal neurones from human and canine submucosal plexus were utilized to determine whether differences in coding and morphology paralleled differences in somatostatin secretion.  Substance P inununoreactivity (SP—IR), immunoreactivity (SS-IR)  somatostatin  and vasoactive intestinal peptide  immunoreactivity (VIP-1R) have been localized and their distributions have been compared in dog and human small intestine using ilmrLunocytochemistry (ICC). protein gene product 9.5  An antibody to  (PGP) was used to localize all  nerve cell bodies and fibres in the dog and human upper  iii  jejunum.  In addition, the proportions of peptide-containing  neurones were determined by double staining.  Staining with PGP revealed neuronal cell bodies in the submucosal plexus  (SMP)  and the myenteric plexus  (MYP)  as  well as extensive innervation by fibres throughout all regions of the small intestine.  Canine submucosal ganglia  contained 7.7 ± 0.6 neurones per submucosal ganglion ganglia, n ± 0.3  =  (184  6 dogs), while the human ganglia contained 2.9  (185 ganglia counted, n  =  5 donors).  Over 50 % of the  ganglia in the human sections contained 3 or less neurones but over 10 % of the ganglia in canine sections contained 15 or more neurones.  Finally, the canine circular muscle was  shown to possess a distinct deep neural plexus,  in contrast  to that of human circular muscle.  The distribution of SPIR,  SSIR and VIP’IR in canine  and human jejunum was similar, confirming the results of previous studies.  Double staining revealed that SP-IR and  SS-IR were always co-localized in the human, SMP and MYP.  but not canine,  In both species VIPIR was present in a  population of neurones distinct from those containing SP—IR and SS—IR.  In canine ganglia,  in the SMP contained SP-IR, contained VIP-IR.  30% of neurones per ganglion  35% contained SSIR and 30%  In human ganglia,  42% of neurones per  ganglion contained SP-IR and SSIR while 40% contained VIP  iv  These results sugest different functions for SP and SS  IR.  in canine and human enteric ganglia.  The secretion of SSIR, small intestine, First,  from intact preparations of  0 is difficult to interpret for two reasons  SS—IR has been demonstrated in vagal,  submucosal and  myenteric neurones as well as endocrine cells, intestine.  Second,  of the small  enteric neurones have been shown to to  contain the 14 amino acid form of SS-IR (SS-14) while endocrine cells have been shown to contain the 28 amino acid form (SS28).  A dispersed culture of submucosal cells from  human small intestine was developed to examine the localization, release and molecular characteristics of SS IR.  After 72 h, the neurones were shown to be viable and to  sprout neurites containing varicosities suggesting that they retained a morphologic phenotype similar to that observed in situ.  Thirty percent of the submucosal neurones per  ganglion in tissue sections and 35 % of cells per cluster in culture contained SSoIR.  Acetic acid extracts of cultures  contained 1990 ± 809 pg SS-IR/well.  Incubation of cultures  with phorbol 1213-myristate 13-acetate (13-PMA),  an activator  of protein kinase C (PKC), at concentrations up to for 120 mm  M  increased the release of SSIR up to 23 times  the basal level, and up to 27 times the basal level when extracellular K+ was increased from 5 to 10 mM.  Of the  total SSIR released in response to 13-PMA (106 M, K+),  10 mM  59% was present in the medium after 30 iuin and 80%  V  after 60 mm.  Basal release of SS—IR could be reliably  measured only after 120 mm, therefore experiments which examined somatostatin secretion were carried out for this amount of time.  The release of SS-IR by 13-PMà was not due  to non—specific membrane effects since the inactive 4a phorbol at the same concentrations did not alter basal release.  Greater than 90 % of SS—IR present in acid  extracts of cultures and released by I3PMà eluted with the same retention time as synthetic SS’14 on reverse phase high performance liquid chromatography (HPLC)  In summary, the results presented in this thesis have shown that differences exist in the neuropeptide distribution and neuronal.morphology between the canine and human small intestine.  Moreover, SS—IR secretion from human  submucosal neurones in response to SP and the phorbol ester were found to be different from the secretion of SS—IR from canine neurones.  The results suggest that differences  observed in the pattern of secretion in submucosal neurones probably reflect the differences noted in neuropeptide distribution and neuronal morphology.  Furthermore, the  present studies emphasize that the extrapolation of experimental data between species must be made with caution.  vi  TABLE OF CONTENTS  Page ABSTRACT  ii  LIST OF TABLES  xi  LIST OF FIGURES  xii  ACKNOWLEDGEMENTS  xvi  INTRODUCTION  1  I. A.  General Background  1  B.  Background on the Small Intestine  4  C.  Experimental Rationale  16  D.  Hypotheses  18  E.  Specific Objectives  19  II.  IMMUNOCYTOCHEMICAL METHODS Tissue Sections  A.  21 21  1.  Tissue Preparation  21  2.  Protocol  21  a.  Primary antibodies  22  0 b  Secondary Layers  22  i. ii.  Peroxidase  22  Inuuunofluorescence  23  c.  Double Stains  25  d.  Controls  25  B.  Tissue Culture  25  C.  Quantification of Peptide Containing Neurones  26  vii  IMMUNOCYTOCHEMICAL STUDIES  29  III.  CHAPTER ONE.  A.  Introduction  29  B.  Results  33  Human tissue sections  1.  33  a.  Protein gene product 9.5  33  b.  Autofluorescence  40  c.  Single Stains of SS—IR,  d.  Double Stains  SP—IR and VIP—IR.  46  Canine Tissue Sections  2. a.  Protein Gene Product 9.5  b.  9 Single Stains of SS-1R  c.  Double Stains  40  58 58 SPIR and VIP—IR  67 68  C.  Discussion  79  D.  Summary  86  IV.  CHAPTER TWO.  ISOLATION OF CANINE AND HUMAN SUBMUCOSAL NEURONES  87  A.  Introduction  87  B.  Methods  94  1.  Human Donor Experiments.  94  a.  Procurement of Tissue  b.  Isolation of Submucosal Ganglia  94  c.  Elutriation Centrifugation  95  d.  Tissue Culture  96  so  Somatostatin Secretion  99  i. ii. iii.  General Protocol High Potassium Time-course  99 100 100  viii  f.  Somatostatin Extraction  100  g.  Somatostatin Recovery  101  h.  Characterization of Primary Molecular Forms  101  i,  Sample Preparation  101  ii.  Reverse Phase HPLC  101  Dog Experiments  2.  102  a.  Procurement of Tissue  102  b.  Isolation of Submucosal Ganglia  102  c.  Elutriation Centrifugation  103  d.  Tissue Culture  103  e.  SSIR and SP—IR Secretion  104  i.  General Protocol  104  ii.  Acetic acid Extraction  104  iii.  Sep—Pak Extraction and Concentration  104  iv.  SP-IR Recovery  105  Radioimmunoassay  30  a.  Soiuatostatin  i. ii. iii. iv. v. vi. vii. viii. ix. b.  105  Assay Buffer  106  Antibody  106  Standards  106  Preparation of 125 1-Soiuatostatin  106  Separation  107  Assay Protocol  108  Calculation and Presentation of RIA Data  110  Inter- and Intra—assay Variation  110  Testing pH effects  111  Substance P  116  ix  i.  ii. iii. iv.  Assay Buffer  116  Antiserum  116  Assay Protocol  116  Calculations  117  Data Analysis  4.  117  Results  C.  Isolation and Characterization of Submucosal  1.  Cultures a.  2.  Human  119  Dog  121  Somatostatin Secretion from Human Submucosal Neurones  3.  119  144  a.  Effects of Secretagogues  144  b.  Somatostatin Content  165  c,  Somatostatin Recovery  165  d.  Characterization of Molecular Forms  165  Somatostatin and Substance P Secretion from Canine Submucosal Neurones  173  0 D  Discussion  174  E.  Summary  188  GENERAL SUMMARY AND CONCLUSIONS  V.  191  A.  Morphological Data from the Small Intestine  191  B.  Culture Studies  193  VI,  CONCLUSIONS AND FUTURE DIRECTIONS  197  A.  Conclusions and Significance  197  B.  Future Directions  199  1.  Morphological Studies  199  x  2. VII.  Functional Studies REFERENCES  199 203  APPENDIX I  233  APPENDIX II  234  xi  LIST OF TABLES  Number  Title  Page  10  Primary Antibodies  24  2.  Biotinylated Antibodies  27  3.  Fluorophore-conjugated Antibodies  27  4.  Avidin Layers  28  5.  Summary of Neuropeptide Distribution and Co—localization in the Human Small Intestine  6.  Summary of Neuropeptide Distribution and Co—localization in the canine small intestine  7.  8.  Somatostatin Assay Protocol  9.  The Effect of the Calcium lonophore on SS—IR Secretion  78 109  164  Variations in Content of Soluatostatin Immunoreactivity  11.  77  Comparison of Quantification Data Between the Canine and Human SMP  10.  76  168  Similarities and Differences in the Secretion of Somatostatin Immunoreactivity from Canine and Human Submucosal Neurones  181  xii  LIST OF FIGURES  Number  Page  1.  Diagram showing segments of enteric plexuses  7  2.  PGP 9.5 staining of human mucosa  34  3.  PGP 9.5 staining of human muscularis  36  4.  VIP-IR and PGP 9.5 double stain  38  5.  The relative size distribution of human submucosal ganglia of the small intestine  6.  41  A section of human duodenum showing association of autofluorescence and SP’IR  43  7.  Human intestine representative inununostains  47  8.  Human duodenal sections double stained for SS-IR and SP-IR  9.  Human duodenum SMP stained for SS’IR and VIP-IR  10,  52  54  Human duodenum double stained for VIP-IR and SP-IR  56  11.  PGP 9.5 staining of canine duodenum  59  12.  Details of the PGP 9,5 staining in the canine duodenal SN?  13.  Details of the PGP 9.5 staining in the canine duodenum showing the deep muscular plexus  14.  15,  61  63  The relative size distribution of canine submucosal ganglia of the small intestine  65  Representative immunostains of the canine SMP  70  xiii  16.  Double stains of canine submucosal ganglia showing SS-IR and SP-IR  17.  Double stains of canine submucosal ganglia showing SS-IR,  SP—IR  and VIP-IR  18.  Diagram of elutriator and chamber  19.  Somatostatin standard curves  (% bound vs concentration of somatostatin) 20.  122  124  126  Cultures of human duodenal MY? stained SS—IR  25  114  Cultures of human duodenal SM? stained SP-IR and VIP’IR  24.  112  Cultures of human duodenal SMP stained for SS-IR  23.  97  High magnification micrograph of human duodenal neurones stained for SS-IR  22.  74  Somatostatin standard curves (logit % bound vs concentration of somatostatin)  21.  72  128  A time course of the attachment and progression in short term cultures of canine SMP neurones  26.  Phase contrast micrograph of canine submucosal neurones  27.  135  Cultures of canine SM? stained for SS—IR,  28.  131  SP—IR and VIP—IR  137  Cultures of canine SM? neürones stained for VIP-IR  140  xiv  29.  Human and canine SMP cultures double stained for SP-IR and SS-IR  30.  Release of soiuatostatin immunoreactivity (SS—IR)  from submucosal neurones in response to 145  incubation with B-PMA 31.  Release of somatostatin immunoreactivity (SS—IR)  from submucosal neurones in response to  incubation with 13-PMA in 5 mM and 10 mM KC1 32.  147  Release of SS—IR from submucosal neurones in response to 13’-PMA (106 M, after 30,  33.  142  10 mM KC1) 149  60 and 120 mm  Release of SS—IR (% TCC)  from submucosal  neurones in response to incubation with 13—PMA and the inactive 4a-phorbo1 for 120 mm 34.  Release of SS-IR (pg/dish)  152  from submucosal  neurones in response to incubation with 154  substance P for 120 mm 35  Release of SSIR (pg/dish)  from submucosal  neurones in response to incubation with substance P for 120 mm  in the presence or  absence of hexamethonium (hex) 36.  Release of SS-IR (pg/dish)  and atropine (atr)  156  from submucosal  neurones in response to incubation with i3PMA for 120 mm  of substance P  in the presence or absence 158  xv  37.  Release of SS—IR (pg/dish)  from submucosal  neurones in response to incubation with 13—PMA f or 120 38.  mm  in the presence or absence of TTX  Release of SS—IR (pg/dish)  160  from submucosal  neurones in response to incubation with CGRP 162  and methacholine for 120 mm 39.  Release of SS—IR (pg/dish)  from submucosal  neurones in response to incubation 13PMA and carbachol or 120 mm 40.  HPLC profile of SS-IR released from submucosal neurones in response to BPMA  41.  166  169  HPLC profile of SS—IR contained in acetic acid extracts in response to 13PM  171  xvi  ACKNOWLEDGEMENTS  I extend my thanks to Dr. Alison Buchan for her time and cominittment to my work,  and for her perseverance during the  completion of this thesis.  The assistance of Dr.  in providing training and expertise for the HPLC,  Chris McIntosh and imparting  current information on everything known to man, was much appreciated.  Advice on the design of pharmacological studies,  especially the importance of industrial doses of skepticism in science, was graciously offered by Dr. graciously accepted by myself.  Yin Nam Kwok and  As the graduate advisor,  Dr. Ray  Pederson has always put the concerns of students first and I thank him for his enthusiastic interest in both my studies and my hobbies.  I must thank Dr. John Brown because he would feel left  out otherwise and more importantly because he was responsible for cultivating my interest in regulatory peptides and for encouraging me to pursue this interest within the MRC group. Finally,  I am especially grateful to my friends and colleagues  who have helped me with my curricular and extracurricular activities.  1  I.  INTRODUCTION.  A.  General Background.  Neuropeptides have been recognized as potential neurotransmitters since the discovery of the hypothalamic hormones.  These include vasopressin and oxytocin from  magnocellular neurones, 1954),  sequenced by du Vigneaud (1953,  as well as luteinizing hormone releasing hormone,  thyrotropin releasing hormone and somatostatin (SS) parvicellular neurones 1973; Schally et al,  (Guillemin,  from  1978; Brazeau et al,  1973)  Information on their function is difficult to interpret, in part,  because they are members of a larger group of  regulatory peptides that also have hormonal and paracrine actions.  Peptides have been demonstrated to exist in  neurones and endocrine cells, which suggests that they may have endocrine, paracrine, neuroendocrine and neurotransmitter functions (Brown et al, 1953; Chang et al,  1971; Gullemin,  1978)  1971; Feyrter, For example,  SS  has been found in hypothalamic neurones and acts in a neuroendocrine manner to inhibit growth hormone release (Brazeau et al,  1973).  Somatostatin has been localized to  cerebral cortical neurones Krisch,  (Johansson and Ho]cfelt,  1980;  1980) where it has been implicated as a  neurotransmitter (Guillemin,  1978).  Somatostatin has been  2  found in endocrine cells of the stomach, pancreas and small intestine,  al,  indicating a possible role as a hormone  1974; Dubois,  1975; Polak et al,  1975).  paracrine actions have been proposed for SS,  (Luft et  Finally, in addition to  its endocrine actions, based on its presence in endocrine cells particularly those which have long cytoplasmic processes  (Larsson,  1979; Yamada,  1987).  Other examples of  peptides with multicellular origins include substance P, cholecystokinin and neurotensin.  Unlike classical neurotransmitters, the original consideration of neuropeptides as potential neurotransmitters was the result of their localization in neural tissue by immunocytochemistry or radioimmunoassay (Hokfelt,  1991)  most compelling.  and in most cases this evidence remains the The function of the putative neuropeptide  neurotransmitters has been difficult to elucidate.  The  peptides must fulfil several criteria in order to be considered as neurotransmitters including the following (Dockray,  1987):  a.  that the peptide is found in neurones.  b.  that it is concentrated in nerve terminals from which it  can be released by depolarizing stimuli with or without a calcium-dependent mechanism. c.  application of the purified peptide exerts an effect  that is duplicated when the endogenous material is released.  3  d.  a selective antagonist is able to block the actions of  both endogenously released and exogenously applied peptide. e.  there are mechanisms for the breakdown,  reuptake or the  removal of the peptide.  There are difficulties in the identification of neuropeptides as neurotransmitters when compared to classical neurotransmitters such as acetyicholine and noradrenaline.  Unlike classical neurotransmitters which are  synthesized exclusively in nerve terminals, neuropeptides must be synthesized in cell bodies and transported to the nerve terminal where they are stored prior to release. There is presently no evidence for the uptake and reuse of neuropeptides by neurones.  The cell types which contain peptides belong to a family of cells with a common amine handling system present and hence were called APUD (Amine Precursor, Uptake and Decarboxylation)  cells  (Pearse,  1976).  These cells include  neuropeptide containing endocrine cells which share common characteristics with neuropeptide producing neurones.  Peptides have been shown to be members of families coded by the same or similar genes, but become processed in different ways in different cell types. occur at the level of mRNA,  The processing can  for example, by splicing such as  in the case of the neuropeptide calcitonin gene-related  4  peptide (CGRP)  and the hormone calcitonin.  Peptides may  undergo post—translational modifications such as enzymatic cleavage and C—terminal amidation,  as in the case of the  neuropeptide CCK-8 and the hormone CCK-33.  A further complication in the study of neuropeptides is that more than one neurotransmitter can be present in one neurone (Milihorn and Hokfelt,  1988)  substances may be neuropeptides, transmitter  The transmitter  amino acids or a classical  (noradrenalin or acetylcholine) which would  allow the release of three types of neurotransmitters to convey fast, moderate or slow signalling from a single terminal  B.  (Iversen and Goodman,  1986).  Background on the Small Intestine.  The small intestine is innervated by both extrinsic and intrinsic nerves.  The vagus and sacral nerves as well as  the sympathetic ganglia supply the extrinsic parasympathetic and sympathetic innervation, respectively.  However,  the  small intestine has an extensive intrinsic nervous component which, together with the processes of the sympathetic, parasympathetic and sensory neurones, was named the enteric nervous system.  Langley (1921)  considered this to be a  portion of the autonomic nervous system separate from the sympathetic and parasympathetic branches.  5  Enteric neuronal axons are unmyelinated and have been shown to have varicosities along their length,  each of which  represents a nerve ending from which a neurotransmitter can be released (Gabella,  1987).  The enteric nervous system is made up of two ganglionated plexuses called, The submucosal  and myenteric. Meissner  respectively, the submucosal  (1857),  plexus  (SMP), described by  is located in the connective tissue of the  submucosa while the myenteric plexus  (MYP),  lies between the  longitudinal and circular muscle of the muscularis externa (figure 1).  The ganglia of the SMP have been shown to be smaller than those of the MYP,  and it has been suggested that they  contain fewer neurones  (Furness and Costa,  1987; Gabella,  The MYP has been shown to be  1990).  1980; Gabella,  structurally uniform along the length and circumference of the small intestine, and the myenteric ganglia were shown to have their long axis in the direction of the circular muscle (Gabella,  1987).  The submucosal  ganglia were shown to form  smaller meshes, they did not demonstrate polarization with respect to the axes of the small intestine and they showed variation with respect to cell number along the length of the small intestine.  6  The enteric ganglia also show substantial variation between species  (Gabella,  1987;  1990).  The ganglia of the  guinea pig small intestine have- been shown to vary in size, but contain similar types of nerve cells and fibres et al, 1988)  1981; Furness et al,  (Wilson  1984; Bornstein and Furness,  7  Figure 1.  Diagram of a segment of intestine partly  separated into layers showing the arrangement of enteric plexuses. plexus  (1)  (4)  MYP  (2)  longitudinal muscle  circular muscle (7)  plexus  plexus  muscularis mucosa  Costa et al,  1987).  tertiary  deep muscular plexus  submucosal artery  submucosal (9)  (5)  (3)  (10)  mucosa.  (8)  (6)  mucosal  (Adapted from  0  9  In many species including the dog and human,  the ganglia  of the submucosa have been shown to form outer and inner plexuses  (Schabadasch,  1930), the latter which would become  known as Schabadasch’s plexus and the former which would be regarded as conforming to Meissner’s original description of a plexus in the submucosa Scheuerman et al,  1989; Gunn,  1989; Christensen and Rick,  Submucosal  1977).  (Furness et al,  1968;  1987; Stach,  ganglia of the outer plexus were shown  to be more similar to the MYP,  and the shapes, histochemical  staining and patterns of innervation differ from the inner plexus  (Gunn,  1968; Scheuerman et al,  The outer submucosal species.  For example,  1989;  Stach,  1977).  plexus does not exist in all  in the guinea pig,  submucosal nerve  cell bodies do not project to the circular muscle but exclusively to the mucosa where they have been shown to influence water and electrolyte absorption and secretion (Bornstein and Furness,  1988).  The outer SMP is most  evident in the colon of larger species such as the human, pig and sheep (Gunn,  1968; Stach,  1977; Crowe et al,  1992),  whereas this plexus has not been found in the small intestine (Christensen and Rick, of the dog (Furness et al,  1987), with the exception  1989).  Interestingly, the interstitial cells of Cajal have also been found in the outer plexus of the mammalian  10  submucosa  (Christensen and Rick,  first described by Cajal  1987).  These cells were  (1893) who suggested they may be  modified neurones, and have since been found in the muscle layers of the small intestine of several mammals 1987; Rumesson and Thuneberg,  1991)  (Gabella,  including the human.  Their function has been the subject of considerable debate since they share characteristics of both neurones and non neural cells such as fibroblasts (Rogers and Burnstock, 1982).  1966; Thuneberg,  These cells have been implicated in  generating the rhythmic activity of smooth muscle (Thuneberg,  1982; Huizinga,  1991).  This has been supported  by experiments in the dog which demonstrated that interstital cells at the muscle/submucosal interface possess electrical pacemaker activity (Barajas-Lopez et al,  The submucosal  1989).  plexus has been suggested to be involved  primarily in the control of mucosal secretion and absorption of electrolytes 1988).  (Hubel,  1985; Keast et al,  1987; Cooke,  The SMP is tonically active and suppresses ion  transport (Cooke,  1988).  The SMP of the small intestine and  colon has been thought to help regulate motility by conveying sensory information to the MYP 1958; Crema,  1970; Kottegoda,  1970).  (Bulbring et al,  Its innervation of the  inner circular muscle in larger animals such as the dog (Furness et al,  1989) would suggest direct involvement of  the SMP in control of motility.  A portion of circular  muscle innervation of the rat also has been shown to come  11  from the SMP (Ekblad et al,  1987,  1988).  To date,  only the  circular muscle of the guinea pig has been shown not to receive input from the SMP (Smith et al,  1988).  Although  implicated in the control of motility in the colon,  the  function of submucosal innervation of circular muscle has not been examined in detail in the small intestine. However, motoneurones from the submucosal  ganglia have been  shown to innervate and provide inhibitory and excitatory inputs in the circular muscle of the canine colon (Sanders and Smith,  1986)  The submucosal  plexus receives inputs from the  myenteric and extrinsic innervation in the guinea pig (Bornstein et al,  1988)  but most mucosal fibres arise  primarily from the SMP with the exception of some SP containing fibres  (Furness and Costa,  1987).  The majority  of fibres found in the mucosa of canine small intestine also appear to originate in the SMP while the submucosa receives little input from extrinsic sources In the guinea pig,  (Furness et al,  inputs from the MY? may be responsible  for inhibitory synaptic inputs to the SMP 1988),  1989).  (Bornstein et al,  and may utilize SS as the neurotransmitter.  The  function of the MY? in control of ion transport has not been determined.  Substance P  (SP),  somatostatin (SS)  and vasoactive  intestinal peptide (VIP) were the peptides examined in this  12  thesis and are the focus the following discussion.  The  undecapeptide substance P was the first gut neuropeptide to be discovered (von Euler and Gaddum,  1931),  on the basis of  its ability to stimulate atropine resistant contractions of the rabbit ileum.  Substance P was among the first known  neuropeptides to be isolated and sequenced (Chang et al, 1971)  and among the first whose presence in the gut was  demonstrated by immunocytochemistry (Pearse and Polak, 1975)  Somatostatin is a tetradecapeptide originally isolated from bovine hypothalamus as a factor which inhibited growth hormone release (Brazeau et al,  and has since been  1973)  localized in many different regions including the endocrine cells  (Polak et al,  1975)  and neurones  (Keast et al,  1985)  of the gut.  Vasoactive intestinal peptide was isolated from porcine duodenum on the basis of its vasodilatory ability (Said and Mutt,  1970)  and, unlike SP and SS, has been localized  exclusively to neurones including those of the gut (Larsson, 1977)  Somatostatin occurs in two major molecular forms,  SS14  which was demonstrated to predominate in enteric nerves,  and  SS28 which predominates in mucosal endocrine cells in the human small intestine  (Penman et al,  1983; Keast et al,  13  1984; Baldissera et al,  1985).  Exogenous application of  either form has been shown to inhibit both small intestinal motility in the guinea pig (McIntosh et al,  l987a)  as well  as secretion of electrolytes in porcine small intestine (Brown et al,  Inhibition of ion transport was shown  1989).  to be partly mediated by enteric nerves in the guinea pig by SS—14  (Keast et al,  pig (Brown et al,  1987),  1989).  or by both SS—14 and S—28 in the Somatostatin has been shown to  inhibit VIP—stimulated secretion in the dog but not the human, whereas basal secretion was not affected in either the dog or human.  (Keast,  1987; Krejs and and Fordtran,  1980).  Substance P and VIP have been shown to increase secretion and decrease absorption of electrolytes in both canine and human small intestine in vivo (Kres et al, Hubel et al,  1984; McFadden et al,  1986).  1980;  Using human small  intestine in situ, these effects were shown to be direct and not mediated by intrinsic nerves  (Hubel et al,  1984).  Similar studies have not been carried out in the dog,  and  therefore whether or not the secretory effects of VIP and SP were mediated by intrinsic nerves in this animal was not determined.  The effect of SP has been shown to be partly  mediated by intrinsic nerves in several species  al,  1984; Keast et al,  1985; Perdue et al,  1987)  (Hubel et but VIP has  been demonstrated to act directly on the mucosa in all  14  species tested (Cassuto et al, Carey et al,  1983; Binder et al,  1984;  1985).  Substance P and VIP have opposite effects on Vasoactive intestinal peptide  gastrointestinal motility.  has been shown to inhibit motility in human colon (Couture et al,  1981; Furness and Costa,  intestine of the guinea pig  1982)  and the small  (Furness and Costa,  1982).  Substance P was shown to have excitatory effects on the motility of human gastrointestinal muscle (Zappia et al, 1978; Couture et al,  1981)  Intracellular recordings from enteric neurones have revealed fast and slow excitatory postsynaptic potentials (EPSPs)  and fast and slow inhibitory postsynaptic potentials  (IPSPs)  (Wood,  1987).  Since membrane potentials or ionic  conductance can be measured over a period of hours, putative neurotransmitters can be applied and receptors can be characterized using pharmacological methods  1986).  (North,  Substance P has been shown to mimic the non-’cholinergic slow EPSP evoked in enteric neurones by stimulation of axon bundles between enteric ganglia both in situ  al,  1979; Bornstein et al,  culture (Willard,  1990).  1984; Surprenant,  1984)  and in  The signal transduction mechanism  of the SP-mediated EPSP did not involve cAMP 1987).  (Katayama et  (Palmer et al,  Somatostatin has been shown to produce two types of  slow IPSPs in both myenteric and subinucosal neurones  (Mihara  15  et al,  1987).  Interestingly,  SS—14 and SS—28 have been  shown to exert opposite effects in rat neocortical neurones (Wang et al,  1989).  Somatostatin—14 was shown to increase  the delayed rectifier K+ current while SS28 was shown to decrease this current.  Both effects were mediated by GTP-  binding proteins and mediated by distinct receptors. Different effects of SS-l4 and SS—28 have not been demonstrated in enteric neurones.  These neuropeptides have been shown to have effects on neurotransmitter release from enteric neurones.  Substance P  has been shown to stimulate the release of Ach from guinea pig myenteric neurones  (Yau et al,  1986)  release from canine submucosal neurones  and neurotensin (Barber et al,  Substance P has diverse effects on SS—IR release  1989).  depending on the cell type.  It has been shown to inhibit (Buchan et al,  SS—IR release from canine submucosal neurones 1990)  and gastric somatostatin cells  (Kwok et al,  1985)  but  to stimulate SS—IR release from the hypothalamus and pancreas  (Reichlin,  1981).  Vasoactive intestinal peptide  has also been shown to stimulate the release of SS-IR from enteric neurones  (Grider,  1989).  Somatostatin has been  shown to inhibit the release of neurotransmitters, Ach,  from enteric neurones  (Wiley and Owang,  such as  1987).  Receptors for neuropeptides have also been studied using receptor binding assays  (Quirion and Gaudreau,  1985).  The  16  distributions of SP and VIP binding sites in human  al,  1989; Korman et al,  (Mantyh et al, to be similar.  1988;  1989)  (Gates et  and canine small intestine  Zimmerman et al,  1989)  have been shown  In both species, binding of VIP was found  predominantly in the epithelial layer while binding of SP was found mostly in the smooth muscle layers but also in the submucosal arterioles and venules,  and in the epithelium.  Only few studies have detected receptors for any neuropeptides on canine enteric ganglia and these include receptors for opioids and bombesin (Allescher et al, Ahmad et al,  1989; Vigna et al,  1987).  1989;  Substance P binding  sites have been demonstrated in guinea pig enteric ganglia (Bornstein and Burcher,  1987).  Although useful for the  determination of high and low density binding sites,  these  techniques have been shown to be insensitive, probably because of a lack of specific ligands to date.  C.  Experimental Rationale.  The actions and secretion of SS and SP in the submucosal plexus of the small intestine has been difficult to interpret for several reasons.  Peptides such as SS are  present in multiple locations and in multiple molecular forms.  Therefore,  studies which utilize in vivo  experimental models to examine the release or actions of SS and SP become confusing.  Also, the distribution and the  pattern of co—localization of peptides varies between  17  species.  Differences in peptide distribution between  species may result in differences in their function.  The studies presented in this thesis compare the distribution of SP-IR, VIP-IR and SS-IR in the submucosal plexus of the human and canine small intestine. particular,  In  the possibility of co—localization of these  neuropeptides is examined.  Further, the studies describe  the development of dispersed cultures of submucosal ganglia from the human small intestine and utilization of canine and human cultures to study the release of SS-IR and SP-IR. Finally, the studies compare the secretion of SS-IR and the effect of SP on SS-IR secretion between the dog and human, and relate these to differences in their distribution.  18  D.  1  Eypotheses  That interspecies variations in neuropeptide localization  and enteric neuronal morphology exist between the human and canine small intestine.  2.  That differences in neuronal chemical coding and  morphology will be reflected in neuronal function.  3.  That shortterm cultures of human and canine SMP will  provide a model system in which to examine whether differences in neuronal chemical coding and morphology will be reflected in neuronal function.  19  E.  Specific Objectives  1.  To develop dispersed cultures of submucosal neurones  from human small intestine and compare these to cultures of canine small intestine.  2.  To compare the morphology of canine and human small  intestinal innervation using the antibody to protein gene product 9.5.  3.  To compare the distribution of SP,  SS and VIP in canine  and human submucosal neurones using single and double stains,  4.  and to compare the morphology of these neurones.  To compare the total number of neurones and total number  of SPIR,  SS-IR and VIP-IR containing neurones per canine  and human submucosal ganglion using PGP/peptide double stains.  5.  To compare the distribution of SP,  SS and VIP in canine  and human submucosa in culture.  6.  To examine and compare,  the content and secretion of SS—  IR from canine and human submucosal neurones in culture.  7.  To examine the content and secretion of SP—IR from  canine submucosal neurones in culture.  20  8.  To compare the effects of SP on secretion of SS—IR from  human and canine submucosal cultures.  21  II.  IMMUNOCYTOCHEMICAL METHODS  A.  Tissue Sections  1.  Tissue Preparation  Cross—sections of intact intestine (1 cm thick),  and  were fixed in Bouin’s solution  2 stripped submucosa (6 cm  )  for morphological study.  The tissues were fixed (in Bouin’s  solution)  for 2 h, washed and stored in 70% alcohol prior to  processing.  The tissue was dehydrated through graded  alcohols and xylene,  and embedded in wax.  Seven micron  sections were air dried on glass slides at 37°C and the wax removed with xylene followed by clearing through petroleum ether  2.  Protocol  All antibodies were diluted in PBS containing 10% bovine or swine serum while avidin layers were diluted in PBS 0 alone  Primary antibodies were incubated at 4°C,  and all  other procedures were carried out at room temperature.  22  a.  Primary Antibodies  (Table 1).  The tissue sections were incubated for 48—72 h in primary antibody diluted with PBS with 10% horse serum.  The  bound antibodies were localized using peroxidase or immunofluorescence techniques.  b.  Secondary Layers  0 i  Peroxidase  Sections which were utilized for peroxidase staining were previously incubated with a 0.01% solution of hydrogen peroxide to block endogenous peroxidase activity before incubation with the primary antibody.  The endogenous enzyme  becomes blocked because the peroxide oxidizes peroxidase. Incubation with a biotinylated (biotin conjugated)  (Table 2) was then carried out for 1 h (canine  antibody tissue)  secondary  or 2 h (human tissue, canine and human cultures).  A  further incubation was carried out for 1—2 h with a solution containing avidin and biotin-peroxidase  Two methods were used to localize  incubated for 10—30 mm. the resulting complex. of diaiuinobenzidene (0.03 %),  (ABC) previously  The first method utilized a solution  (DAB,  4 mg/ml)  and hydrogen peroxide  in 0.1 M Tris buffer, which was filtered and added  dropwise on slides and cultures to develop the peroxidase reaction.  The second method utilized 100 ml of 0.1 M Tris  23  buffer containing 200 mg dextrose, glucose oxidase (0.3 mg), aimnonium chloride (40 mg)  and DAB  (4 ing)  in which the  sections or cultures were incubated for 1—1.5 h  After  counterstaining with heiuatoxylin, the staining was observed with a Zeiss Axiophot microscope equipped with phase contrast optics.  linmunofluorescence,  ii.  Primary antibodies were visualized by immunofluorescence in two ways.  An indirect method utilized a 12 h incubation  with a fluorophore-conjugated second antibody (Table 3).  A  second method was carried out by incubation with a biotinylated second antibody for 1 h (canine tissue) (human tissue, human and canine cultures) 60 mm  followed by a 45—  incubation with an avidin-fluorophore third layer  (Table 4). (FITC)  or 2 h  The fluorophores used were fluoro—isothiocyanate  and tetramethyl-rhodamine-isothiocyanate (rhodamine).  The FITC and rhodamine staining was observed with a Zeiss Axiophot microscope equipped with epifluorescence, using barrier filters of band width 465-490 nm (green) nm (red), respectively.  and 510-560  24  Table 1.  Primary Antibodies.  Source  Dilution  Species  VIP  CURE  1:1000  mouse  serum  VIP  Peninsula  1:1000  rabbit  serum  Soma  CURE  1:10,000  mouse  ascites  Soma  RPG  1:1000  mouse  ascites  SP  SL  1:2000  rabbit  serum  SP  JP  1:2000  rabbit  serum  SP  RPG  1:1000  guinea pig  serum  Immunonuclear  1:2000  rabbit  serum  Antigen  PGP  CURE Professor J. Walsh, Education  Centre for Ulcer Research and  RPG Regulatory Peptide Group. SL— Professor S.A.  Leeman.  JP— Professor J.M.  Polak.  Type  25  c.  Double Stains.  Double staining was carried out using the techniques described for iimuunofluorescence.  It was necessary to  choose antibodies to peptides raised in species such that cross—reactivity between primary and secondary antibodies did not occur.  Second layers have been affinity purified to  remove potential cross—reacting globulins, developed specifically for double staining.  and have been For example,  primary monoclönal antibodies were localized with secondary antisera raised in goat or donkey rather than the more usual rabbit antimouse IgG to eliminate cross-reactivity with rabbit primary antibodies.  d.  Controls.  Extensive characterization of the antisera/antibodies has been carried out previously to eliminate the possibility of cross-reactivity with other related peptides. addition,  In  incubations using PBS in 10 % serum layers were  carried out to determine the extent of non—specific staining due to the second and third layers.  Be  Tissue Culture.  The cultures were fixed in Bouin’s solution for 15—30 mm, washed with distilled water followed by phosphate  26  buffered saline.  The localization of primary antibodies was  carried out as for tissue sections except for the following changes.  Some cultures were frozen with liquid nitrogen  before washing and/or incubated in first, layers containing Triton X-100  (0.1%),  second and third  in order to lyse the  plasma membrane and allow the optimal penetration of antibodies into the cell.  The durations of first,  second  and third layer incubations were up to 50 % longer than those for tissue sections.  The concentrations of primary  and secondary antibodies, and of avidin third layers were double those used in tissue sections.  C.  Quantification of Peptide—containing Neurones.  In tissue sections, the percentage of the submucosal plexus occupied by ganglia was determined by planimetry in sections stained with PGP 9.5.  Neurones iituuunoreactive for  each peptide were counted in six ganglia per section in 6 sections to a total of 180 qanglia and compared to the total number of neurones in those ganglia.  The total number of  neurones was determined using the PGP 95 antiserum. cultures,  In the  cells immunoreactive for somatostatin were counted  and compared to the total number of neurones in that group or cluster.  27  Table 2.  Biotinylated Antibodies  Source  Antigen  Dilution  Species  Rabbit IgG Rabbit IgG  Vector Co. Jackson Co.  1:300 1:1000  goat donkey  Mouse IgG Mouse IgG  Vector Co. Jackson Co.  1:300 1:1000  horse goat  Table 3.  Antigen  Fluorophore-conjugated Antibodies  Source  Dilution  Species  Rhodamine Rabbit IgG Mouse IgG  Jackson Co. Jackson Co.  1:1000 1:1000  donkey goat  Jackson Co. Jackson Co.  1:1000 1:1000  donkey donky  FITC Rabbit IgG Mouse IgG  28  Table 4.  Layers.  Avidin FITC Avidin rhodaiuine Avidin peroxidase  Avidin Layers  Source  Vector Co. Vector Co. Vector Co.  Dilution  1:1000 1:1000 1:1000  29  CHAPTER ONE.  III.  A.  IMMUNOCYTOCHEMICAL STUDIES  Introduction  Enteric neurones in several mammalian species have been shown to contain a plethora of neuropeptides  al,  1980; Furness and Costa,  1982)  (Schultzberg et  The enteric nervous  system of the guinea pig has been the most thoroughly characterized with respect to the pathways of individual peptide containing neurones  al 1990).  (Costa et al,  1987; Furness et  Differences in the distribution of peptides  within nerve cell bodies of the MYP and external musculature of the dog and guinea pig have been shown Daniel et al, 1989).  1985; Daniel et al,  In particular,  1987;  (Tange,  1983;  Furness et al,  differences in innervation of the  circular muscle of the guinea pig and dog may lie in the submucosa  (Furness et al,  1990).  Canine circular muscle has  been shown to contain SPIR and VIP-IR fibres whose origin was the outer submucosal ganglia,  or Schabadasch’s plexus.  Guinea pig circular muscle receives SP-IR and VIP-IR fibres solely from the MYP.  Similarities exist between the dog and human with respect to enteric submucosal/mucosal neuropeptide distribution.  Single staining of VIP,  SP and SS was  comparable between canine and human submucosa/mucosa. peptide has been found in both submucosal  Each  and myenteric  30  neuronal cell bodies and fibres in canine and human iuucosa/subxnucosa  al,  (Fern  1984; Daniel et al,  et al,  et al;  1985; Keast. et al,  1988; Furness et al,  1983; Keast et  1982; Tange,  1990).  1985;  Wattchow  These studies have shown  that fibres containing VIP iimuunoreactivity  (VIP-IR)  were  more abundant than those containing SP-IR, which in turn were more abundant than those containing SS—IR.  Comprehensive examinations of neurotransmitter combinations in enteric neurones have been carried out in the guinea pig and rat and have revealed distinct differences  (Furness et al,  1989; Pataky et al,  1990).  is often referred to as chemical coding of neurones et al,  1989).  In the SMP of the guinea pig,  groups of neurones were distinguished.  This  (Furness  two major sub  These were  dynorphin/galanin/VIP containing and CCK/CGRP/choline acetyl transferase  (Chat) /galanin/neuropeptide Y  containing neurones 1989).  In the rat,  (Costa et al,  1987;  (NPY) /SS  Furness et al,  the major subgroups were VIP/NPY  containing and SS/SP/CGRP containing neurones 1990).  (Pataky et al,  These studies used multiple staining techniques with  highly specific antisera/antibodies to characterize the neuronal types.  However,  no information was obtained about  whether functional differences could be correlated with differential peptide coding.  31  Earlier studies with canine intestine demonstrated the presence of large ganglia containing a high proportion of neurotensin immunoreactive (NT—IR) Barber,  1987).  neurones  (Buchan and  These NT-IR positive neurones had not been The  demonstrated in either the rat or the guinea pig.  chemical coding of canine SMP neurones probably differs from both the guinea pig and the rat.  The basis for such a  difference may be the diet and size of the animal 1987; Gabella,  (Powell,  1990)  Immunocytochemical studies of peptide co-’localization and chemical coding of human and canine neurones of the small intestine are few (Wattchow et al,  al,  1990).  1988; Furness et  In the human small intestine SP and enkephalin  have been shown to co—exist in fibres which were suggested to be excitatory to the external muscle (Wattchow et al, 1988).  These authors also showed that NPY and VIP co  existed in a separate population of fibres and were suggested to be inhibitory to the external muscle.  In the present study,  immunocytochemical staining was  carried out to examine the hypothesis that there is interspecies variation in the localization of SS-IR,  SP—IR  and VIP—IR between the human and canine small intestine. The availability of highly specific antibodies/antisera raised in different species  (mouse, rabbit and guinea pig)  32  combined with affinity-purified secondary antisera was essential in these experiments.  In order to quantify the number of neurones within a ganglion,  a general neuronal marker was required.  In these  Protein gene product 9.5 is a  studies PGP 9.5 was used.  soluble cytoplasmic protein originally detected in protein extracts of human organs by high resolution two dimensional polyacrylamide electrophoresis (Doran et al,  1983).  The  distribution of PGP is similar to neurone-’specific enolase, since both have been found in neurones and neuroendocrine cells  (Doran et al,  1983).  Polyclonal antibodies to PGP  have demonstrated peripheral nerve cell bodies and nerve fibres with clarity and intensity (Gulbenkian et al, Lauweryns and Van Ranst,  1988; Wilson et al,  1988).  1987;  33  B.  Results  1.  Human Tissue Sections  a.  Protein Gene Product 9.5.  The antibody to PGP strongly stained human neuronal cell bodies and fibres throughout the small intestine.  This  staining revealed the nerve cell bodies of the SMP and nerve fibres throughout the mucosa (figure 2).  Submucosal ganglia  were anatomically separated into those adjacent to the interface with the mucosa and those situated by the circular muscle.  The myenteric ganglia were strongly stained and  there was extensive innervation of the circular and longitudinal muscle  (figure 3).  Quantification of the relative proportion of peptide containing neurones was carried out using double stains of PGP 9.5 in combination with the particular neuropeptide (for an example see figure 4).  The intensity staining was much  greater for PGP than for SP—IR,  SS-IR or VIP-IR and revealed  more abundant nerve fibres found throughout the ganglia. The volume of the myenteric ganglia was larger than that of the submucosal ganglia because of larger amounts of fibres and other neuronal processes  (figure 4).  34  Figure 2. bodies  PGP 9.5 staining of human mucosa.  (large arrows)  Note cell  immediately below muscularis mucosae.  Immunoreactive fibres were present throughout the mucosal layer  (small arrows). x 200.  36  Figure 3.  PGP 9.5 staining of human muscularis  circular muscle,  LM  both muscle layers  =  longitudinal muscle).  (small arrows)  the neurones in the NYP  (CM  =  Note fibres in  and intense staining of  (large arrow). x 100.,  38  Figure 4  a)  VIP-IR neurone  (large arrow)  and fibres in the human  MYP., x 400.  b)  The same ganglion in a double exposure showing VIP-IR  neurone double stained by PGP 9.5 and a single neurone stained by PGP-IR 9.5  (small arrow) x 400.  6E  40  The average number of neurones per ganglion in the SMP was 2.9 ± 0.3  (184 ganglia, n  =  5 donors)  (figure 5)  and made up  only 5% of the submucosa while collagen made up over 85% of this layer.  b.  Autofluorescence.  Certain neurones demonstrated autofluorescence when viewed under ultraviolet light which was often associated with neuropeptide containing neurones  (figure 6).  Autofluorescence was indicative of the high intrinsic amine content of these neurones and fibres. was visible at 3 wavelengths (380,  The autofluorescence  480 and 570nm)  be differentiated from Rhodamine (570 nm)  but could  arid FITC (480 nm)  because these were not observed at the 340 nm wavelength.  c.  Single Staining of SSIR,  SPIR and VIP-IR.  Single staining of human small intestine with antibodies to SS,  SP and VIP revealed a pattern similar to that found  previously in other laboratories. in endocrine cells, (figure 7 a, b,  c).  Somatostatin—IR was found  submucosal ganglia and myenteric ganglia Endocrine cells containing SS—IR were  abundant in the mucosal epithelium,  stained more intensely  than neurones containing SS—IR and were concentrated at the base of the crypts  (see also figure 8 a).  41  Figure 5.  The relative size distribution of human  submucosal ganglia of the small intestine.  Note that over  50 % of ganglia contained 3 or less neurones.  42  70 a C  a  60 50  9-  0  40  0  30  E  D C  20 10 0 0  5  10  15  20  number of neurones per ganglion  25  30  43  Figure 6. a)  a section of human duodenum photographed under  ultraviolet light  (370 rim).  autofluorescence (arrows)  Note the presence of intense  in the SNP ganglion. x 200.  b)  The same area photographed under long wavelength  nm)  to show SP—IR neurones.  the autofluorescent areas  (570  Note the close association of  (arrows) with the positively  stained neurones x 200.  44  44  45  Submucosal and myenteric ganglia also contained SS—IR nerve fibres but these were less abundant than those containing SP-IR or VIP-IR.  Forty two percent of neuronal  cell bodies per human submucosal ganglion contained SS—IR.  Cell bodies containing VIP-IR were again found in myenteric (separate from neurones containing SS—IR) submucosal ganglia (figure 7 d,  e).  and  Occasional nerve cell  bodies were observed in the smooth muscle and submucosa. Fibres containing VIP-IR were found within the ganglia running between other neurones. throughout the MYP,  Fibres were also found  SMP and the smooth muscle layers.  Fibres containing VIP—IR were also found in the mucosa (figure 7 f), and in general were found in similar amounts to SP—IR fibres, and greater than SS—IR fibres.  Forty  percent of neurones per human submucosal ganglion contained VIP-IR.  Endocrine cells containing VIP-IR were not  observed.  Cell bodies containing SP-IR were found in human myenteric  (not shown)  and submucosal ganglia (figure 7 g).  Fibres containing SP-IR were found within the myenteric and submucosal ganglia and seemed to form pericellular baskets around other neurones.  Fibres were also found in the muscle  layers, the SMP, the mucosa and the mucosal villi. Substance P fibres were more plentiful than fibres containing SS—IR and were present in amounts similar to  46  those containing VIP—IR.  Forty two percent of neuronal cell  bodies per human ganglion contained SP-IR while endocrine cells containing SP—IR were not found.  d.  Double Stains.  Substance P—IR and SS-IR were found to completely co exist in the neurones of human submucosal and myenteric ganglia.  (figure 8 a,  b,  c)  Large numbers of fibres were  demonstrated in all layers of the gut which contained only SP—IR while endocrine cells of the mucosa were found to contain only SS-IR.  Neurones containing VIP—IR were demonstrated to be a population completely distinct from those neurones containing SS-IR/SP-IR in both the myenteric and submucosal neurones  (figure 9,  and 10 a, b).  Double stains revealed  neuropeptide containing fibres and varicosities of each neuropeptide distributed around other neuropeptide containing neurones within the ganglia.  Some fibres formed  pericellular baskets around neuropeptide containing neurones.  Certain small ganglia were made up of neurones  which were exclusively of the VIP-IR type or SS-IR/SP-IR type.  47  Figure 7.  Human intestine representative inununostains.  a)  SS—IR endocrine cell  b)  SS—IR SMP neurones  c)  SSIR neurones  (arrow)  x 200. x 200.  (arrow)  (arrows)  and nerve fibres in the MYP x 200.  d)  stained for VIP-IR.  The same ganglion as in  the separation of neurones.  SS-IR (arrow heads), VIP-IR x 200.  (long arrows) e)  Note  VIP-IR neurones  (arrows)  in a ganglion of the SMP x 200.  f)  VIP—IR fibres  (arrows)  in the duodenal mucosa g)  SP-IR neurones  duodenal SMP  (arrows)  in the lamina propria of a villus x 200. and fibres in a ganglion in the x 200.  6t’  50  .1  Ln  52  Figure 8. (FITC) a)  Human duodenal sections double stained for SS—IR  and SP-IR (Rhodamine).  A low magnification overview showing SS-IR epithelial  endocrine cells  (open arrows) which do not contain SP—IR;  SP—IR fibres in the lamina propria and muscularis mucosae (small arrows) which do not contain SS-IR; a ganglion in the SMP  (large black arrow)  containing a neurone double stained  for both SS-IR and SP-IR (large white arrow) x 50.  b)  A higher magnification micrograph of SP-IR in a SMP  neurones co—localized with c)  SS—IR x 500  C,,  54  Figure 9.  Human duodenal SMP stained for SS-IR (Rhodamine,  large arrows)  and VIP-IR (FITC,  small arrows).  Note the  complete separation of the two neurone types and the presence of SS—IR varicosities around the VIP—IR neurone (small arrow heads)  x 500.  Lfl  56  Figure 10.  Human duodenum double stained for VIP-IR (FITC)  and SP-IR (Rhodamine).  a)  A ganglion in the SNP with a single VIPIR neurone x 200.  (small arrow)  b)  A double exposure of the same ganglion showing the VIP  IR neurone  (small arrow)  and two SP—IR neurones  arrows) Note the lack of co—localization.  (larger x 200.  In  58  2.  Canine Tissue Sections  a.  Protein Gene Product 9.5.  The antibody to PGP strongly stained canine neuronal cell bodies and fibres throughout the small intestine.  This  staining revealed the nerve cell bodies of the SMP and nerve fibres throughout the mucosa  (figure 11).  Canine submucosal  ganglia were also present in two distinct groups,  one closer  to the mucosa and one near the interface with the circular muscle (figure 11,  12,  13).  Unlike the human small  intestine, PGP 9.5 staining revealed the presence of a discrete deep muscular plexus,  situated near the outer layer  of the submucosa  Canine myenteric ganglia were  (figure 13).  strongly stained and there was extensive innervation of the circular and longitudinal muscle,  The intensity of staining  was much greater for PGP than for SP”IR,  SS—IR or VIP-IR and  more abundant nerve fibres were apparent throughout the ganglia.  The volume of the myenteric ganglia was larger  than that of the submucosal ganglia again due to large amounts of neuropil i.e. processes, and glia.  fibres and other neuronal  The average number of neurones per  ganglion in the SMP was 7.7 ± 0.6 dog)  and made up  (185 ganglia counted,  n=6  10* of the submucosa while collagen made  up over 80% of this layer (figure 14).  59  PGP 9.5 staining of canine duodenum (MUC  Figure 11. mucosa, CM  =  circular muscle).  =  Note the presence of two  distinct sets of ganglia in the SMP,  one close to the  interface with the mucosa (upper dotted line), the other apposed to the circular muscle (lower dotted line) x 50.  60  61  Figure 12.  Details of the PGP 9.5 staining in the canine  duodenal SMP.  Note the close association of the large  ganglion with the muscularis mucosae mucosae,  CM  =  circular muscle, M  =  (in  =  inuscularis  inucosa). x1OO.  62  63  Figure 13. duodenum. arrows)  Details of the PGP 9.5 staining in the canine Note the band of inununoreactive fibres  (small  at the inner surface of the circular muscle forming  the deep muscular plexus  (dmp) x200.  64  65  Figure 14.  The relative size distribution of canine  submucosal ganglia of the small intestine.  Note that the  majority of ganglia contained between 4 and 7 neurones. Large ganglia (those which contained 10 % of the population 0  >  15 neurones) made up  66  70 0 C, C  50  0 C, ‘4-  0  40  L.  a) -a E  30  C  20  D  10 0 0  5  10  15  20  number of neurones per ganglion  25  30  67  b.  Single Stains of SS—IR,  SP—IR and VIP—IR.  The pattern of single staining of canine small intestine with antibodies to SS,  SP and VIP was again similar to that  found in previous investigations.  Cell bodies containing  SS—IR were found in submucosal ganglia endocrine cells and myenteric ganglia.  (figure 15 a), Submucosal and  myenteric ganglia also contained SS—IR nerve fibres but were less abundant than those containing SP-IR or VIP-IR.  The  SS—IR neurones were often grouped into clusters within the ganglia whose fibres would exit the ganglia in the same direction.  Thirty five percent of neurones per canine  submucosal ganglion contained SS—IR,  Endocrine cells  containing SS-IR were predominant in the region of the crypts of the mucosal epithelium and were stained more intensely than neurones containing SSIR.  Cell bodies containing VIPIR were again found in myenteric and submucosal ganglia. throughout the MYP,  Fibres were also found  SMP and the smooth muscle layers.  Fibres containing VIP-IR were also found in mucosa villi and,  as in the human, were found in numbers similar to SP-IR  fibres and greater than SS-IR fibres.  Thirty one percent of  neurones per canine submucosal ganglion contained VIP-IR. Endocrine cells containing VIP-IR were not observed.  68  Cell bodies containing SP—IR were found in canine submucosal  (figure 15 b)  and myenteric ganglia.  Numerous  SP—IR fibres containing varicosities were found within the myenteric and submucosal ganglia which were distributed amongst other neuronal cell bodies.  As with the other  peptide containing neurones, SP—IR neurones were segregated within the ganglia and their fibres exited the ganglia in the same direction.  Fibres were also found in the mucosa,  the SMP, MYP and muscle layers.  Substance P fibres were  more plentiful than fibres containing SS-IR and were present in amounts similar to those containing VIP-IR.  Thirty  percent of neurones per canine submucosal ganglion contained SP—IR while endocrine cells containing SP-IR were not found.  c.  Double Stains.  The double stains revealed that, unlike the human, and SP—IR were never present in the same neurone a, b).  SS-IR  (figure 16  Neurones containing VIPIR were demonstrated to be a  population completely separate from those neurones containing SS—IR or SP-IR (figure 17 a,  b).  Double stains  also demonstrated neuropeptide containing fibres and varicosities of each neuropeptide distributed around other neuropeptide containing neurones within the ganglia. fibres formed pericellular baskets around neuropeptide containing neurones,  Finally, double stains provided  Some  69  further evidence for the spatial segregation of different neuronal types within the ganglia.  The data obtained from the iimuunocytochemistry of sections of canine and human small intestine are summarized in tables 5,  6 and 7.  70  Figure 15.  Representative iinmunostains of the canine SMP.  Note that the neurons are grouped into small clusters within the larger ganglion  (larger arrows)  and that the fibres of a  cluster appear to exit the ganglion as a unit  (small  arrows). a)  SS—IR  x 200.  b)  SP-IR  x 200.  IL  72  Figure 16, a and b. SS—IR (FITC, arrows).  Double stains of canine SNP ganglia for  small arrows)  and SP—IR (Rhodamine,  longer  Once again note the grouping of the neurones into  clusters of a single type and the merging of exiting fibres (see ‘a’, medium sized arrow) x500,  74  Figure 17. neurones arrows) b)  Double stains of canine SMP ganglia.  (Rhodamine, x 500.  small arrows)  localization.  and SS—IR (FITC,  VIP-IR small  Note the absence of co—localization.  VIP—IR neurones  (FITC,  large arrows)  a)  (Rhodamine, x 500.  large arrows)  and SP—IR  Note the absence of co—  cL  76  Table 5.  Summary of Neuropeptide Distribution and Co  localization in the Human Small Intestine  SS—IR  SP-IR  VIP—IR  Mucosa. +++  endocrine cells nerve fibres  +  ++  Ganglia (SMP and MYP) +++  neurones  nerve fibres  +  +++  +++  ++  +++  yes  no  Co—localization. SS—IR SP-IR  yes  VIP—IR  no  +++  most abundant 0  ++  less abundant.  +  least abundant.  no no  77  Table 6.  Summary of Neuropeptide Distribution and Co  localization in the Canine Small Intestine  SS—IR  SP—IR  VIP-IR  Mucosa. endocrine cells  +++  +  nerve fibres  ++  Ganglia (SMP and MYP) neurones  +++  nerve fibres  +  +++  +++  ++  +++  no  no  Co—localization. SS-IR SP—IR  no  VIP-IR  no  Deep Muscular  no no  ++  —  +++  Plexus  +++  most abundant.  ++  less abundant.  +  least abundant.  0  CD U)  Ii 0  CD  I H  H  1 0 CD U)  CD (0  CD 0  CD  I H  Cl)  Cl) I H  Cl)  0  Q  q  CD ‘1  CD U)  CD i ii 0  (J  0  1+  lD  1+  0  b CD  CD  DI  CD  0  I  CD  CD  CD  rI  CD  w  DI Cl. DI  II  0  I-’  79  C.  Discussion  Staining of tissue sections of canine and human small intestine with PGP 9.5, nerves  a general marker for peripheral  (Gulbenkian et al,  1988; Wilson et al,  1987; Lauweryns and Van Ranst,  1988), was used to localize all  myenteric and submucosal neuronal cell bodies and revealed an extensive network of nerve fibres throughout the small intestine.  The distribution of nerve fibres was similar  between the dog and the human, but canine submucosal ganglia contained,  on average,  a greater number of neurones.  Also,  the density of neurones was greater in the canine SMP than human small intestine based on the smaller amount of collagen in the canine submucosa.  It has been suggested  that the size of the intestine determines the number of neurones per unit of serosal surface and the total number of neurones, with each of these values being parallel to the size (weight)  of the animal  (Gabella,  1987;  1990).  The average size of each submucosal ganglion was larger in canine small intestine than in the human small intestine. These data suggest that the size of submucosal ganglia does not parallel the size of the animal. per submucosal  The number of neurones  ganglion in the human was closer to that of  the rat which has 3-5 per ganglion (Pataky et al, than to that of the dog,  1990),  Sheep probably represent a third  group of animals, based on the low spatial density of  80  neurones in the small intestine and the large size of the enteric ganglia.  The small intestine of these ruminants has  been shown to have a low spatial density of neurones but have neuronal ganglia which contain large numbers of neurones  (Gabella,  1987).  It is generally accepted that mammalian myenteric ganglia contain a greater number of neurones than the submucosal ganglia  (Gabella,  1987; Furness et al,  1987),  although preliminary càunts have indicated that this is not the case in the dog, while in the human the numbers of neurones in submucosal and myenteric ganglia seem to be equal  (data not shown).  A major portion of the volume of.  the iuyenteric ganglia in both the dog and human was made up of neuropil i.e. neuronal fibres and other neuronal processes,  and glia.  It could be argued that 2 dimensional  renditions of 3—D structures do not accurately reflect neuronal number and volume, requiring the use of whole mount preparations.  However, analysis of sections cut  perpendicular and parallel to the direction of the circular muscle provided a reasonable construction of both plexi.  The myenteric ganglia were in an orderly array with their long axis travelling in the same direction as the circular muscle as noted in previous studies 1987).  (Gabella,  Whether the submucosal ganglia possessed a similar  orientation could not be discerned from tissue sections but  81  most likely these ganglia were less structured, demonstrated in previous studies  as has been  1990).  (Gabella,  The presence of SS—IR in submucosal and myenteric neurones,  and endocrine cells of the mucosa,  and the small  numbers of SS—IR containing nerve fibres in canine and human small intestine tissue sections was in agreement with the results of previous studies  al, 1986).  1985; Keast et  (Daniel et al,  Nerve fibres throughout all layers of the small  intestine and neurones of the myenteric and submucosal plexus contained SP-IR and VIP-IR, as demonstrated previously (Tange,  1983; Daniel et al,  1985).  Mucosal  endocrine cells did not contain SP-IR, although this has been reported in both the human and canine small intestine (Daniel et al,  1985; Keast et al,  out by these authors,  l985)  In studies carried  endocrine cells containing SP—IR were  few in number and stained less intensely than those endocrine cells containing SSIR and SPIR.  Only the small  intestine of the marmoset has been shown to contain large numbers of endocrine cells which stained brightly for SP-IR (Keast et al,  1985).  The double staining experiments revealed the co existence of SP—IR and SS—IR in neurones of human, canine,  but not  small intestine which has not been previously  demonstrated.  Neurones containing VIPIR were distinct from  those containing SP-IR and SS-IR in both species, which is  82  in agreement with previous studies which have demonstrated no co-existence between SP-IR and VIP-IR fibres in the muscularis externa throughout the canine and human gastrointestinal tract (Wattchow et al,  1988; Furness et al,  1989)  Two separate populations of motoneurones have been proposed by these authors to innervate the external muscle of the human gastrointestinal tract, one containing VIP-IR and neuropeptide Y-IR and the other containing SP-IR and enkephalin—IR (Wattchow et al,  Fibres containing  1988).  VIP—IR and NPY—IR were called inhibitory by Wattchow et al, (1988),  since both peptides have been shown to inhibit  motility of human gastrointestinal muscle 1981; Furness et al,  1982; Allen et al,  (Couture et al,  1987).  The presence  in the human small intestine of nerves which are excusively inhibitory is consistent with the results obtained in this study.  Fibres containing SP—IR and enkIR were called excitatory by Wattchow et al,  (1988),  since these peptides  have been shown to be excitatory on human gastrointestinal smooth muscle (Zappia et al,  1978; Couture et al,  1981).  The presence of nerves which are exclusively excitatory in the human small intestine is not supported by the present study since neural SS-IR, which has been shown to inhibit motility in the small intestine (McIntosh et al,  1987a), was  83  found to co-exist in all SP-IR containing neurones in both the myenteric and submucosal ganglia.  It may be argued that  SS—IR may only act as an interneuronal neurotransmitter and thus not affect motility by acting directly on the muscle, in a physiological setting.  An indirect action of SS on  muscle, by inhibiting enteric neurones, has not been proven and furthermore each peptide may potentially affect adjacent neurones containing excitatory and inhibitory neurotransmitters making the terms “excitatory” and “inhibitory” neurone inappropriate (Cooke,  1989).  The interspecies alteration in peptide profiles demonstrated in this study parallels that seen between the guinea pig and the rat (Costa et al, 1990).  1987; Pataky et al,  All neurones containing SS—IR were found to co—exist  with SP—IR in the rat small intestine which was similar to the human small intestine (Pataky et al,  1990).  The  proportion of neurones containing SP-IR and VIP-IR in the rat (46 and 42 %, respectively) and human also similar.  (42 and 39 %) was  There were two major differences between the  rat and the human in the distribution of peptides examined in this study.  Not all rat SP-IR neurones contained SS-IR,  and SS—IR neurones made up only 18 % of submucosal ganglia rather than 40 % as in the human.  The similarities in  peptide distribution between the rat and human small intestine were in addition to the similarity in total number of neurones per ganglion.  84  SP-IR and SS-IR have been shown to  In the guinea pig,  exist in separate neurones similar to canine submucosal ganglia (Costa et al,  1987).  The proportion of neurones  which contain SS-IR was similar between these species  (29 %  in the guinea pig and 32 % in the dog), whereas the proportion containing SP-IR and VIPIR was different.  The  proportions of canine submucosal neurones containing SP—IR and VIP—IR were 32 % and 30 % respectively, but were 11 % and 45 % respectively in the guinea pig.  Thus,  even in  those species in which there were similarities in chemical coding of the neurones,  significant differences in  ganglionic composition (i.e. the proportions of peptide— containing neurones)  occurred.  There is general agreement  that interspecies variation in peptide localization of the enteric plexi will be present in almost all cases et al,  (Furness  1989).  In human tissues, VIP—IR fibres innervated SP-IR/SS-IR containing neurones and SP—IR fibres innervated VIP—IR neurones.  In addition,  SP—IR containing fibres were  associated with SP—IR containing neurones, providing anatomical evidence for interganglionic regulation.  In canine tissues,  SP-IR fibres were distributed  throughout the larger ganglia surrounding neurones containing both VIP—IR and SS-IR.  The results provided  85  anatomical evidence for the action of SP on SS—IR secretion. It should be cautioned that anatomical evidence cannot be taken as conclusive,  since data presented in this study do  not support an action of SP on SS—IR secretion in the human, even though SP-IR fibres were found apposed to SS-IR containing neurones.  Fibres located in all regions were shown to contain only SP—IR in both canine and human tissue.  In human tissue, the  co-existence of SP—IR and SS-IR in neuronal cell bodies, but not fibres,  could have been due to low levels of SS—IR.  Another possibility for the lack of co-existence in fibres was that those containing SP—IR were extrinsic fibres, as sensory afferents of the vagus.  such  Substance P has been  shown to commonly be present in vagal afferents in the small intestine and several other organs of many species where it does not co-exist with SS—IR (Costa et al,  1987).  The source of SP-IR and VIP-IR fibres in the outer layer of circular muscle in the canine small intestine has been shown to be in the MYP, whereas fibres in the inner layer and the innervation of the muscularis mucosae and mucosal layer originated in the submucosal  plexus  (Furness et al,  1989)  In both species, there was a spatial difference in the localization of neuronal types.  In the canine sections, the  86  SS-IR, VIP-IR and SP—IR were clearly segregated within the ganglion.  In the human sections, segregation was more  extreme, with small ganglia (2-4 cells)  being made up  exclusively of VIP-IR or SS-IR/SP-IR neurones.  D.  Summary  The data collected have supported the existence of major interspecies variations in chemical coding of enteric neurones between human and canine small intestine.  They  have confirmed the existence of two distinct plexuses in the submucosa of the canine small intestine.  The presence of  neuropeptide containing nerve fibres and varicosities within enteric ganglia has provided a morphological basis for neuropeptide actions on other neurones.  Finally,  it was  demonstrated that different neuronal types are segregated within enteric ganglia, and send processes in the same direction.  The factors which determine differences in  gastrointestinal gross anatomy and function have been suggested to be related to diet (Powell, the animal  (Gabella,  1990).  1987)  and size of  Thus, the gastrointestinal  tract of omnivores such as rats and humans are more similar than the gastrointestinal tract of carnivores such as the dog, or ruminants such as sheep.  In other words, the  interspecies differences observed in this study may be due, in part, to differences in diet.  87  ISOLATION OF CANINE AND HUMAN SUBMUCOSAL  CHAPTER TWO.  IV.  NEURONES  A.  Introduction  The release of SS—IR and SP-IR from the mammalian small intestine using in situ and in vivo experimental models has been demonstrated (Andersson et al, 1984; Manaka et al,  1989)  1982; Donnerer et al,  but the results have been  difficult to interpret for two reasons.  First,  SS-IR and  SP-IR have been found in three different groups of cells, namely neurones of the submucosal  and myenteric plexi,  and  endocrine cells of the canine and human small intestine (Keast et al, Thus,  1986; Keast et al,  1985; Daniel et al,  1985).  a peptide released from endocrine and/or neural cells  could play a role in the physiological homeostasis of the small intestine.  Second,  SSIR is present in the small  intestine in two primary molecular forms, namely SS—14 and SS-28.  Substance P is processed from the tachykinin gene  which can express three different peptides, and it is not known which are expressed in different cell types 1987).  Therefore,  (Dockray,  it has become necessary to establish  models that circumvent difficulties associated with in vivo experiments in order to understand differential release of peptides from neurones or endocrine cells.  88  Organotypic cultures from small intestine have been developed using segments of the gut wall which contain the enteric plexuses in association with muscle layers and connective tissue (Gershon et al, the myenteric and submucosal  1980).  The separation of  plexuses from the gut wall  using a combination of enzymatic treatment and microdissection,  and the maintenance of explants in tissue  culture was later carried out (Jessen et al,  1978;  1983).  Dispersed cultures of rat small intestine have also been developed and have been extensively characterized with respect to their morphological, pharmacological and electrophysiological properties Willard and Nishi,  1985 a,b).  (Nishi and Willard,  1985;  These authors found that  enteric neurones maintained in tissue culture possessed morphological, pharmacological and electrophysiological properties similar to those possessed by neurones in situ.  More recently, methods to examine the release of peptides from isolated cultured cells obtained from mucosal (Barber et al, 1989)  1986)  and submucosal layers  (Barber et al,  of the small intestine have been developed and these  offer certain advantages over in vivo studies.  Acutely  dissociated ganglia from myenteric neurones of guinea pigs (Grider,  1989)  submucosal  al,  1989)  and a newly developed short-term culture of  neurones from canine small intestine  (Buchan et  have been used specifically to study the release  of SS—IR from enteric nerves,  The use of acutely  89  dissociated neurones for secretion studies is limited because nerve fibres are not present after isolation.  Thus,  the release of neurotransmitter would have to be from the cell soma for which there is presently no evidence in vivo.  The control of peptide release in vivo is mediated via receptor regulation and/or a membrane voltage dependent mechanism.  In culture studies, the role of receptors can be  mimicked by the addition of specific pharmacological agents. These drugs activate second messenger pathways normally associated with receptor activation (Berridge,  1985).  Three  such drugs are commonly utilized to activate different intracellular pathways.  The phorbol esters activate protein  kinase C, the calcium ionophore (A23187)  increases  intracellular calcium and forskolin increases intracellular cAMP.  The effects of these drugs have been examined in cultures of canine SMP neurones.  Of particular interest was  the action of phorbol 12-myristate 13-acetate (i3—PMA) which, in the presence of the calcium ionophore A23187,  stimulated  the release of SS—IR from canine submucosal neurones in culture (Buchan et al,  1990).  Phorbol esters,  such as B—  PMA, have been shown to activate protein kinase C (PKC) (Blumberg,  1981).  Protein kinase C is normally activated by  diacyiglycerol formed from phospholipase C-mediated cleavage of membrane phospholipids, which also produces  90  inositol triphosphate (Nishizuka,  1986).  The activation of  PKC has been shown to stimulate the release of hormones and neurotransmitters  (Kaczmarek,  1987).  The release of SS—IR  by phorbol esters has also been demonstrated using dispersed cultures of fetal rat hypothalamus and cortex (Peterfreund and Vale,  1983).  The addition of the calcium ionophore or the phorbol ester alone was shown not to increase the secretion of SS—IR from canine submucosal neurones (Buchan et al,  1990).  This  indicated that increases in calcium per se or activation of PKC were not sufficient to stimulate SSIR secretion.  Interestingly, the addition of forskolin did not stimulate the secretion of SS—IR from canine cultures, although it was able to stimulate the release of neurotensin from similar cultures 1990).  (Barber et al,  1989; Buchan et al,  Previous experiments carried out using gliafree  cultures of rat cerebral neurones have shown that adenylate cyclase activation by forskolin was not sufficient to stimulate SSIR secretion (Tapia-Arancibia et al,  1988).  Substance P has been shown to inhibit SS—IR release from canine submucosal neurones somatostatin cells  (Buchan et al,  (Kwok et al,  1988)  1990)  and gastric  and to stimulate SS—IR  release from the hypothalamus and pancreas  (Reichlin,  1981).  The effect of SP on SS—IR secretion from canine neurones was  91  unexpected,  since it has been shown to stimulate the release  of NT from similar cultures  (Barber et al,  1989).  The  inhibitory actions of SP on SS—IR release in canine cultures were likely indirect since this peptide has also been shown to release Ach from myenteric neurones 1987),  (Wiley and Owang,  increase intracellular calcium in dorsal horn  neurones (Womack et al,  1988)  and produce EPSPs in  submucosal neurones (Surprenant,  1984).  Its action on SSIR  secretion from human submucosal neurones was examined in the present study and compared to its effects on canine submucosal neurones.  The experiments outlined in the following chapter describe the development of a system to isolate human neurones from the upper small intestine and to maintain these neurones in tissue culture.  These experiments test  the hypothesis that the difference in chemical coding between canine and human neurones is reflected in their function.  Specifically, the effects of the phorbol ester,  the calcium ionophore and SP on the secretion of SS-IR from human submucosal neurones were examined.  Preliminary  experiments which examine the release of SPIR from canine neurones are also described.  In addition to substance P, a variety of agents were tested for their ability to modify SS-IR secretion. Receptor independent secretagogues, the calcium ionophore  92  A23187 and the phorbol ester i3-PMA, were utilized to + and PKC activity, respectively. 2 increase intracellular Ca It was expected that A23187 would require the conconunitant addition of 13—PMA to stimulate secretion  ,  since increasing  intracellular calcium was insufficient in canine neurones. The phorbol, 4a-phorbol,  is similar in structure to 13-PMA  but does not stimulate PKC activity in vitro (Blumberg, 1981).  Therefore this was used as a control for non  specific membrane effects,  since phorbols are highly  lipophilic and may cause non-specific destabilization of lipid membranes.  Experiments were carried out using 10 mM KC1, concommitantly with the phorbol ester.  alone and  This level of  potassium would produce a depolarization of 20 my, as determined by the Goldman equation using “normal” values for intracellular and extracellular ions. reasons.  This was done for two  First, this determined whether a small  depolarization would stimulate the secretion of SS—IR from human submucosal neurones,  Second, to determine whether a  small depolarization would render the neurones more sensitive to stimulation by the phorbol ester.  It has been shown that cholinergic neurones are present in the submucosal plexus and may be involved in the release of other neurotransmitters (Barber et al,  1989).  In order  to determine the role of cholinergic neurones in the  93  secretion of SS—IR two agonists and two antagonists were used.  Carbachol and methacholine are non—specific  cholinergic agonists.  Methacholine is more potent, has less  nicotinic activity and has been shown to be more effective in stimulating the release of antral SS—IR (Buchan et al, 1991).  Hexamethonium and atropine are nicotinic and  muscarinic antagonists and were used to block both exogenous and endogenous cholinergic substances.  Cholinergic agonists  have been shown to produce both excitatory and inhibitory effects in enteric neurones  (North et al,  1985)  therefore  the overall effects of the antagonists on SS—IR secretion were not predictable.  These antagonists were also used to  determine whether the effects of SP were direct or indirect via endogenous cholinergic action.  Calcitonin gene-related peptide (CGRP) has been shown to co—exist with SP in primary sensory neurones,  and has been  shown to stimulate the release of SS—IR in other systems (Dockray,  1987).  Therefore,  its effects on SS—IR secretion  from human submucosal neurones were examined.  94  B.  Methods  1.  Human Donor Experiments  a.  Procurement of Tissue  Twelve to fifteen inches of upper small intestine was obtained from multiple organ donors in association with the Pacific Organ Retrieval for Transplantation (PORT)  program.  Previous to surgical removal, the small intestine was perfused with Eurocollins all red blood cells.  (see appendix 2)  at 4°C to remove  The donors were pre—screened for  transplantation and therefore had no known pathophysiological conditions.  b.  Isolation of Submucosal Ganglia  The duodenal bulb was not taken nor was the initial portion of duodenum which contained Brunner’s glands since the submucosa could not be properly separated in these areas.  The mucosa and muscle layers were removed by blunt  dissection, the remaining submucosa was washed in Hanks’ Balanced Salt Solution (HBSS) albumin (BSA)  containing 0.1% bovine serum  and 20 mM N-2-hydroxyethyl piperazine-N-2-  ethane sulphonic acid (HEPES),  and chopped finely.  The  Hank’s buffer was used throughout the isolation for washing tissue between periods of incubation.  The medium used for  95  incubation consisted of Basal Medium Eagle (BME) 0.1% BSA,  containing  20 mM HEPES and collagenase, and was gassed with  5% CO 2 in 02.  The pH of the incubation and washing media  was strictly maintained between 7.0 and 7.4,  low enough to  inhibit collagen reassembly yet remaining within physiological limits.  Each 10 g of tissue was incubated  with 50 ml of the incubation medium in 200 ml flasks, continually shaken in a water bath at 200 Hz and maintained at 37°C throughout each incubation period. was carried out in three stages. incubated for 30 mm  The isolation  First, the tissue was  in incubation medium containing 600  U/ml collagenase (type XI)  and 4 inN Cad . 2  The partly  digested tissue was washed and centrifuged for 5-10 mm 200 x g to remove collagen,  fat and debris.  further digested for two periods of 60 mm, washing and centrifugation.  at  The tissue was each followed by  The suspension was then  filtered through a 240 u Nytex mesh and resuspended in HBSS.  c.  Elutriation Centrifugation (figure 18)  Elutriation centrifugation utilizes centrifugal force and flow, which act in opposing directions, to separate cells on the basis of their volume.  Different fractions of  cells can be removed by altering the flow rate of fluid passing through the elutriation chamber by altering the pump rate or by changing the speed of the centrifuge. Appropriate flow rates and centrifugation speeds were  96  The cell suspension was loaded into  determined empirically.  an elutriator rotor (Beckman) 25 mi/mm.  at  2500 rpm at a flow rate of  Flow was supplied by a pump  (Cole Parmer,  Masterfiex Model 7520-20) with a quick-loading head (Cole Parmer, model 7021—20)  fitted with silicon tubing (Cole  Parmer, type 6411-16),  equipped with a pressure gauge.  The  pump flow rates were calibrated before each experiment at 2500 rpm.  A fraction was collected at 2200 rpm at a flow  rate of 35 mi/mm  which contained fibroblasts, red blood  cells and cell fragments.  A second fraction was collected  at 800 rpm and a flow rate of 100 mi/mm  which contained  single ganglia and clusters of two or three cells as well as undigested collagen fibres and fragments of blood vessels. Approximately 90% of the neurones were viable,  as  demonstrated by trypan blue exclusion.  d.  Tissue Culture  The final fraction was again centrifuged at 200 x g for 5—10 mm  to concentrate the cells and resuspended in growth  6 cells/mi and medium at a density of approximately 1x10 plated on 12 well plates coated with rat tail collagen at imi/well.  Single cells were counted and numbers were  estimated for clusters of cells in order to obtain total counts.  The growth medium consisted of Dulbecco’s Modified  Eagl&s Medium (DMEM)  containing 5.5 mM glucose and  supplemented with 20 mM HEPES,  2 mM glutamine,  200 mM  97  Figure 18.  Diagram of elutriator rotor and chamber.  Variations in flow rates and rpm allow cells to be separated on the basis of their volume.  98  Vewh’g Port Sample lnjCCtiOn Pressure Gaugc  Bubble Trap  Pump  Buffer Reservoir  Standard Elutriacion Chamber  ‘eb  elutriation boundary  I eb andr mm  -  Centrlugal Force  I Axis of Ret3tion Srobe tamp  II  99  cytosine i3—D-arabinofuranoside, nerve growth factor CS-7S,  8 tLg/ml insulin,  100 Jhg/ml gentamicin,  20 ng/ml 1 jg/m1  4 ug/ml fungizone and 5% fetal calf serum.  hydrocortisone,  The cells were maintained in culture for 72 h at 37°C after which immunocytochemistry (ICC), release experiments, extraction and HPLC were carried out.  e.  Somatostatin Secretion  i.  General Protocol.  The cells were washed with 1 ml of release medium which consisted of DMEM containing 5,5 inN glucose, and 0.1% BSA.  1.0% aprotinin  A 585 jl aliquot of release medium and 15 ,tl  of drug or peptide solution was added to each well volume  600 J.Ll).  =  (total  The drug or peptide solutions were  prepared at 40 times the desired final concentration in dimethyl sulfoxide (DMSO)  and release medium.  The highest  ratio of DMSO to release medium was 1:400 and at this concentration the release of SS—IR was not affected. cells were incubated for 120 mm,  The  after which the release  medium was removed, centrifuged to remove any particulate matter,  and stored at —70°C for radioimmunoassay (RIA).  This procedure was used for 13—PMA, peptides.  4a—phorbol and all  Comparisons were carried out using the same  preparation i.e.  in paired experiments.  100  ii.  High potassium.  Experiments were carried out using release medium augmented with potassium chloride (KC1) potassium concentration of 10 mM.  This resulted in a  depolarization of approximately 20 my, Goldman equation.  in order to obtain a  as determined by the  The osmolality of the release medium was  346 mOsm/kg and 337 mOsm/kg, with and without the additional KC1, respectively, and therefore no adjustment to the composition of the medium was made.  Time—course  iii.  Experiments were carried out to determine the time course of SS—IR secretion in response to i3—PMA (10—6 M, mM KCL).  10  13-’PMA was incubated in separate sets of wells for  60 and 120  mm,  15,  30,  f.  Somatostatin Extraction.  Each well was extracted to determine the content of SS— IR per well and the variability of SS—IR between wells (n=8).  The isolated ganglia were dislodged from the bottom  of each well using a rubber spatula after the addition of 600 j.l of 2N acetic acid and boiled for 15 mm.  The  extracts were centrifuged and the supernatant was frozen at -70°C for RIA and HPLC analysis.  101  Somatostatin Recovery  g.  The recovery of SS—IR was determined in two ways. Firstly,  SS  (500 pg) was added to release medium and  incubated with submucosal cultures for 2 h.  Secondly,  SS  (500 pg) was dissolved in 2 N acetic acid, boiled and lyophilized.  h.  SS-IR was then measured by RIA,  Characterization of Primary Molecular Forms.  i  Sample Preparation  Extracts and release samples were added to a Waters 18 column that had been primed by the addition of 5 Sep-pak C mls of 100% acetonitrile (ACN) trifluoroacetic acid (TFA)  containing 0.1%  followed by 5 ml of 100% dH O 2  with 0.1% TFA and dried with 5 ml of air.  The sample and  extracts were added to the column, washed with 1.5 ml of O and eluted with 1.5 ml of 70% ACN containing 0.1% TFA, 2 dH The samples were gassed with 100% nitrogen to remove the ACN,  frozen at -70°C and lyophilized.  ii,  Reverse Phase HPLC.  The samples were reconstituted in distilled water for subsequent reverse phase HPLC.  Reverse phase HPLC was  carried out on Waters equipment consisting of a 3.9 x 30 mm  102  -Bondapak C 18 column,  a model 512 WISP, two model 510 pumps  and a model 441 absorbance detector. 35%)  A gradient of ACN (28-  in 0.1% TFA run over a 10 minute period was used at a  flow rate of 1 ml/min and 0.5 ml fractions were collected. The fractions were lyophilized, reconstituted in assay buffer and SS-IR was measured by RIA.  Synthetic SS-14 and  SS—28 were used for calibration of the column.  2.  Dog Experiments  a.  Procurement of Tissue  Mature mongrel dogs were sedated with phentanol triperidol  (0.1 mi/kg)  given with atropine (0.05 mg/kg),  anaesthetized with sodium pentobarbital prepared for abdominal surgery.  ( 30 mg/kg) Lv, and  The animal was bled from  the abdominal vena cava, the upper small intestine was removed and immediately placed in a container of ice—cold Hank’s buffer.  b.  Isolation of Submucosal Ganglia  The initial preparation of the tissue was similar to that which was carried out with the human small intestine. The isolation was carried out in two 1 h stages. tissue was incubated for 60 mm  First, the  in incubation medium  containing 300 U/mi collagenase (type XI,  Sigma).  The  103  partly digested tissue was washed and centrifuged for 5-10 mm  at 200 x g to remove collagen,  fat and debris.  tissue was further digested for 60 mm and centrifugation.  The  followed by washing  The suspension was then filtered  through a 240 p. Nytex mesh and resuspended in HBSS.  c.  Elutriation Centrifugation  The cell suspension was loaded into an elutriator rotor (Beckman)  at  2500 rpm at a flow rate of 25 ml/min.  A  fraction was collected at 2200 rpm at a flow rate of 35 mi/mm  which contained fibroblasts, red blood cells and cell  fragments.  A second fraction was collected at 1600 rpm and  a flow rate of 100 ml/min which contained single ganglia and clusters of two or three cells as well as undigested collagen fibres and fragments of blood vessels. Approximately 90% of the neurones were viable as demonstrated by trypan blue exclusion.  In several  experiments, a third fraction which contained large groups of cells,  fragments of blood vessels and collagen fibres was  collected at 800 rpm and a flow rate of  0 d  >  100 mi/mm.  Tissue Culture  The procedure for tissue culture of canine submucosal neurones was identical to that for human neurones.  104  e.  SS—IR and SP-IR Secretion  i.  General Protocol  The cells were washed with 1 ml of release medium which consisted of DMEM containing 5.5 itiN glucose, and 0.1% BSA.  1.0% aprotinin  A 1 ml aliquot of release medium and 10 /hl of  drug or peptide were added to each well.  Drugs or peptides  were prepared at 100 times the desired final concentration in distilled water.  The cells were incubated for 45 mm  after which the release medium was removed, pipetted into Eppendorf tubes containing 110 jl concentrated HC1, centrifuged to remove any particulate matter and stored at 70°C for RIA.  Comparisons were carried out using the same  preparation i.e.  ii.  in paired experiments.  Acetic Acid Extraction  Canine submucosal cells were extracted to determine the total cell content of SS-IR and SP-IR in the same manner as SSIR from human submucosa.  iii.  Sep—Pak Extraction and Concentration  Samples were extracted as previously described McIntosh,  1990).  (Kwok and  Release and acetic acid extract samples  were applied to Sep Pak C 18 cartridges which had been primed  105  with 10 ml of acetonitrile containing 0.1% trifluoroacetic acid (TFA),  The  O and 10 ml 1% BSA. 2 followed by 10 ml dH  O and 1 ml 20% 2 cartridge was then washed with 10 ml dH acetonitrile containing 0.1% TFA.  Two ml of 50%  acetonitrile containing 0.1% TFA were added to the column to elute the peptides.  The samples were lyophilized using a  speed vac, and stored at —70°C for RIA. cartridge was used no more than twice,  Each Sep Pak a procedure which has  been shown not to alter the recovery of substance P and somatostatin.  iv.  SP-IR Recovery  Known amounts of SP-IR (500 or 1000 pg) were added to release medium and incubated with canine submucosal cells for 1 h in wells which either did or did not contain the enzyme inhibitors captopril (20 jM)  3.  and bacitracin (20 ,.LN).  Radioinuuunoassay  Radioimmunoassay (RIA) was used to measure SS—IR and SP— IR in release medium and cell extracts of submucosal cultures, and the techniques for each have been previously described (Kwok and McIntosh,  1990; McIntosh et al,  1987b).  106  a.  Somatostatin  i.  Assay buffer  Sodium barbital  (4.90 g),  sodium acetate (0.32 g)  and  ethylmercurithiosalicyclic acid sodium salt (inerthiolate; The pH was adjusted  O. 2 0.10 g) were dissolved in 700 ml dH to 7.4 with HC1, Aprotinin  and this stock buffer was stored at 4°C.  (trasylol;  100 K.I.U.)  and BSA (Pentex;  5.0 g/l)  were added to the stock buffer for preparation of assay buffer.  ii.  Antibody  The SS-IR was assayed in duplicate using a monoclonal antibody (SOMA 3)  to somatostatin (Buchan et al,  detects both SS-14 and SS28  (McIntosh,  1987).  1985)  which  The antibody  was prepared from crude mouse ascites as previously described and kept at 4°C until use.  This stock solution  was diluted with assay buffer to obtain a final titre of 6 for the assay. 4x10  The antibody has been shown to not  cross—react with GIP, motilin, gastrin or substance P.  0 ui  Standards  Synthetic cyclic somatostatin was dissolved in 0.1 M acetic acid, diluted to 100 J.Lg/ml using dH O containing 2  107  0.05% BSA (Pentex)  and aliquots of 50 jl  lyophilized and stored at -20°C.  (5 JLg) were  An aliquot was dissolved  O, and serially diluted in assay buffer on 2 in 100 ji of dH the day of the assay to obtain standards ranging from 3.9 to 500 pg/mi.  iv.  Preparation of 125 1-Soiuatostatin.  -somatostatin was iodinated using the 1 Synthetic Tyr chloramine-T method, purified initially by adsorption to 6 cpm. silica and lyophilized in aliquots of 1x10  Aliquots  were further purified on the day of the assay using a CM-52 Sephadex column previously equilibrated with 0.002 M ammonium acetate.  An aliquot of 1—somatostatin 25 was ‘  dissolved in 0.002 M ammonium acetate, applied to the column and eluted using 0.2 N ammonium acetate at a flow rate of 1 ml/min.  One or two peak fractions were counted, neutralized  with 2 N NaOH and diluted to 30003500 cpm/100 Jhl in assay buffer.  v.  Separation  Activated charcoal  (1.25%)  and dextran  (0.25%) were  dissolved in 005 N phosphate buffer and this mixture was stirred for at least one h after the addition of 0.1% charcoal—extracted plasma  (CEP).  108  vi.  Assay Protocol  (Table 8)  Assays were carried out on a refrigerated Table, maintained at approximately 4°C, using 12 x 75 borosilicate glass tubes.  Total count, non—specific binding (NSB),  zero  binding and standard tubes were assayed in triplicate, while samples were assayed in dup1icate  samples  If necessary,  were diluted with assay buffer so that concentrations fell within the most sensitive portion of the standard curve. For release experiments, to NSB,  100 jl of release medium was added  zero and standard tubes in place of assay buff er  After a 72 h period of equilibration at 4°C,  separation  of bound and free peptide was carried out by adding 1 ml of charcoal slurry to all except “total count” tubes. were vortexed and centrifuged at 3000 rpm for 30 mm  Tubes at 4°C.  Tubes were decanted, dried over absorbant paper and counted on a gamma counter.  The remaining pellet contained free  iodinated and cold peptide.  109  Table 8.  TUBES  NSB  Somatostatin Assay Protocol  zero  sample or standard  total count  F____________________________ buffer  standard or sample  antibody  300 j.Ll  200 l  100 Ll  —  ———  100 j.Ll  100 j.Ll  100 jl  label  100 J.Ll  100 jl  100 jl  100 1  total volume  400 J.Ll  400 l  400 JA1  100 Jhl  110  V  Calculation and Presentation of RIA Data  vii.  % bound  =  100 X (C NSB  -  C sample)  C total where C  =  % NSB  100 X (C total  =  cpm C NSB)  C total  A standard curve was plotted of % bound versus Esomatostatin] on semilogarithmic paper and/or was transformed into a logit—log plot using a RIA software program (RIA Analysis v 1.0).  Sample concentrations were  determined from the original standard curve or by the RIA program using the mean of duplicate counts.  viii.  Inter— and Intra-Assay Variation  Samples containing known amounts of somatostatin could not be kept for long periods of time because of a loss in immunoreactivity and therefore were not used as controls. Inter—assay variation was determined by comparing multiple standard curves while intra—assay variation was determined by comparing 55—IR standards randomly placed within the assay.  Both inter—assay and intra—assay were less than 10%,  111  ix.  Testing pH Effects.  Acetic acid (2N) was normally used to extract SS-IR from tissue and cultures and samples were diluted 1:20 and 1:40 for measurement in RIA. acetic acid alone (2N,  Therefore, the effects of adding diluted 1:20 and 1:40)  standard curve were examined.  to the SS-IR  The plots of % bound versus  [somatostatin] showed that the “zero binding” was reduced from 50% to 36%, reduced  and the linear portion of the curve was  when the 1:40 dilution was used in the standard  curve (figure 19).  In addition, the nonspecific binding of  the label was increased when the 1:40 dilution was used. The standard curve could be corrected when the pH of the 1:20 and 1:40 dilutions of 2N acetic acid was adjusted to 7.4 with sodium hydroxide.  It should be noted that the  standard curve produced using the 1:40 dilution without the pH adjustment could be utilized to determine sample SS-IR concentrations.  Interestingly,  linear logit—log plots of  the 1:40 undiluted, and the ‘normal’  standard curves were  more similar than the plots of % bound vs. concentration (figure 20).  This illustrates the inability of linear  transformations of assay data to assess the usefulness of a standard curve.  112  Figure 19.  Somatostatin standard curves  (% bound vs  concentration of somatostatin), without acetic acid, with acetic acid (1:40 dilution)  and with acetic acid (1:40 and  1:20 dilution) where pH was adjusted with NaOH.  113  o  50  o  40 —S  Std. Curve 1/40 dilution 1/20 dilution pH adjusted with NaOH 1/40 dilution pH adjusted with NoCH  0 0  x  30  0  20 10 0— 3.9  500  [somatostatin] (pg/mi)  114  Figure 20.  Somatostatin standard curves  (logit % bound vs  concentration of somatostatin), without acetic acid, with acetic acid (1:40 dilution) 1:20 dilution)  and with acetic acid (1:40 and  0 pH adjusted with NaOH  11  7  Std. Curve • 1/40 dilution 1/20 dilution pH adjusted with NaCH 1/40 dilution pH adjusted with NaOH  o  6 5, 0  4,  a 6  2 ‘  1 0 —1•  0  —3,  I  39  I  I  I  500  [somatostatin] (pg/mi)  116  b.  Substance P  The substance P RIA has been described previously and McIntosh,  1990)  (Kwok  and was identical to that for  somatostatin with the following differences.  i.  Assay buffer  0 Gelatin was used in place of BSA in the assay buffer  ii.  Antiserum  The antiserum KGPO5 was raised in guinea pig using haemocyanin-conjugated SP and was used at a final dilution of 1:180,000. 100% with SP, kassinin, 11), 7),  <  The antiserum has been shown to cross—react SP-(3--l1)  and physalaemin,  0.07% with SP methyl ester,  SP—(7—11)  and eledoisin,  <  < <  0.3% with 0.04% with SP—(5—  0.009% with SP-(1—4),  SP—free acid, neurokinin A, neurokinin B,  SP—(l-  bombesin and  somatostatin.  iii.  Assay protocol  1-Substance P was added to each RIA tube 2-3 h after 125 the addition of the antiserum. (plastic)  Also, polypropylene  tubes were used rather than borosilicate tubes  117  since the latter were shown to result in a loss of measurable SP-IR due to the adsorption of peptides to glass.  iv.  Calculations  The RIA data were analyzed in the same way as for somatostatin.  4.  Data Analysis  The total amount of SSIR or SPIR per well was determined by adding the amounts extracted and released. Peptide release was calculated as a percent of total cell content (%TCC)  in the following way:  % TCC  =  [r/(e + r])  where  x  100  r  =  amount of SS-IR released  e  =  amount of SS—IR extracted  (e+r)  =  total SS—IR per well  All values are given as means ± SE and n always refers to the number of donors/dogs.  The Mann Whitney U analysis  was used for statistical comparisons of secretion data and differences were considered significant at the p<0.05 level.  118  To test human extract data, utilized.  a one way ANOVA was  119  C.  Results  1.  Isolation and Characterization of Submucosal Cultures  a.  Human  The isolation protocol described produced the highest yield of viable cells.  Using less collagenase (300 U/ml)  and increasing the time of digestion always resulted in more undigested tissue and a smaller yield of viable cells. Increasing the collagenase (1000 U/nil)  and decreasing the  time also resulted in a lower yield of viable cells. Maintaining the pH below 7.4 reduced the reaggregation and gelation of collagen.  Likewise,  several washing and  centrifugation steps were required to remove digested collagen and reduce gelation.  The post-collagenase digest  consisted of single cells, clusters of cells, blood vessels, red blood cells  (RBCs)  fragments of  and satellite cells.  Following filtration and elutriation, the suspension contained no RBC5,  fewer fragments,  fewer single cells and  less debris but individual ganglia could be seen clearly under the microscope  Cells adhered to the collagen substrate overnight and their viability in culture remained  >  90% for up to 5 days  as shown by trypan blue exclusion conducted throughout this period.  After 5 days, the viability decreased and the cells  120  became detached from the collagen.  Cells aggregated around  individual ganglia and there was abundant neurite outgrowth after 72 h in culture.  The individual clusters of cells  were linked by neurite extensions and resembled the submucosal Therefore,  plexus in situ  (see canine isolation).  ICC, release experiments and HPLC extractions  were carried out at this time point.  An initial plating  6 cells/mi/plate was chosen and was found density of 1-2x10 to be optimal for the survival of the neurones for 72 h.  At  densities of 5—8 x io cells/mi, the majority of cells would not adhere while at densities of 3—5 x io cells/mi the cells would detach from the collagen substrate after 1-3 days.  The mitotic inhibitor cytosine arabinoside was included in the growth medium and effectively prevented the overgrowth of fibroblasts.  A sheet of fibroblasts to  support the attachment of neurones was not required with the use of plates coated with rat tail collagen.  Hematoxylin staining of sections of stripped submucosa confirmed that all mucosal and muscle tissue had been removed (not shown).  Human neurones were phase bright,  sprouted neurites which contained varicosities along their length and made anatomical connections to other cells after 72h.  121  Human cultures contained neurones which stained for SS IR (figure 21 a, b).  In human cultures,  35 % of all cells  per cluster contained SS—IR in culture, which was similar to the amount found in tissue sections  Positively stained  fibres were abundant and varicosities present along the length of the fibres also contained SS—IR (figure 22 a, b, c).  Submucosal neurones and fibres also contained SPIR and  VIP—IR (figure 23 a, b).  Myenteric cultures also contained neurones which stained for SS—IR (figure 24), as well as SPIR and VIPIR neurones 0 These neurones sprouted extensive neurites with varicosities which contained SS-IR,  b  SP-IR and VIPIR..  Dog  The isolation and culture of canine SMP differed from that of human tissue in several ways.  The amount of  collagenase and overall incubation time required to disperse the submucosa were less than for the human submucosa. was due to a lesser amount of collagen contained in the canine submucosa.  This  122  Figure 21 a and b  High magnification micrograph of  cultures of human duodenal neurones, grown on coverslips, stained for SS—IR. x 5OO  124  Figure 22. IR.  Cultures of human duodenal SMP stained for SS—  Note the presence of IR neurones  varicosities a)  x 100  b)  x 200  c)  x 200.  (small arrows).  (large arrows)  and  It-I -I  * fr 4  e  fr ,.fr  A-  4 .a  126  Figure 23. a)  limaunostained cultures of human duodenal SMP.  SP—IR neurone  (arrow). x 200  b)  VIP’IR neurones  (arrow)  and varicosities  (small arrows) x200,  -  •i  ‘4  *  .  128  Figure 24. IR.  Cultures of human duodenal MYP stained for SS  Note the presence of IR—neurones  varicosities a)  x 100  b)  x 200,  (small arrows).  (large arrows)  and  —‘  4  4  CD  I  V  A  .0  130  Three different protocols were tested for elutriation of Fraction 1 contained primarily single cells,  canine tissue.  including neurones and red blood cells.  Fraction 2  contained mainly ganglia and groups of 2—5 cells.  Fraction  3 was eluted according to the method used for human submucosa, cells.  and contained large fragments and large groups of  The viability of all fractions was  >  95% immediately  after elutriation, but fractions 2 and 3 contained the cells of interest.  The elutriation procedure for fraction 2 was First, only single ganglia were  chosen for two reasons. present in fraction 2, cultures.  Second,  similar to the situation in the human  the cells in fraction 3 would flatten and  sprout neurites but became detached from the collagen substrate after 2-3 days.  An examination of the time—course and progression of short—term cultures is shown in figure 25.  Clusters were  phase bright, and attached to the plates after 24 h in culture but no sprouting was observed (figure 25 a).  After  72 h in culture, ganglionic structures formed and made interconnections  (figure 25 b).  After 120 h, many of the  cells in the ganglionic structures were dead (figure 25 c). Therefore,  72 hwas selected as the time when ICC and  secretion experiments were carried out  131  Figure 25.  A time—course of the attachment and progression  in short—term cultures of canine SMP neurones, a)  Day 1,  (arrow) b)  24 h after plating neurones clusters attached  but no sprouting of neurones was observed x 200.  Day 3,  72 h after plating, neuronal clusters formed  ganglionic type structures 2OO.  c)  Day 5,  (arrow) with interconnections x  120 h after plating, the majority of the  cells in the ganglion—like structures were dead x 200.  132  ‘33  £  I.  134  V  Phase contrast microscopy revealed that the canine neurones were phase bright,  sprouted neurites which  contained varicosities along their length and made anatomical connections to other cells after 72 h  (figure  Canine cultures contained neurones which stained for  26).  SS—IR (figure 27 a, b), (figure 27 d,  SP—IR (figure 27 c)  figure 28).  and VIPIR  Positively stained fibres were  observed and varicosities present along the length of the fibres also contained SSIR and SPIR.  Double stains of human cultures demonstrated co localization of SS—IR and SP-IR (figure 29 a,b).  Double  stains of the canine cultures revealed separate populations of SS-IR and SP-IR neurones  (figure 29 c,d)  Thus,  the  neurones in culture displayed the same phenotype as neurones  in situ with respect to SPIR and SSIR.  Interestingly, the  canine neurones also demonstrated the segregation of neuronal types normally observed in neuronal ganglia in  situ.  135  Figure 26. neurones. bodies  Phase contrast micrograph of canine submucosal Note the presence of phase bright neuronal cell  (large arrows)  and extensive sprouting of neurites  containing varicosities along their length  (small arrows) x 200.  Cl  137  Figure 27.  Immunostained cultures of canine SMP.  a)  SS—IR neurones x 200  b)  SS-IR neurones x 200  C)  SP—IR neurones x 200  d)  VIP-IR neurones x 200. Note the extensive sprouting of neurites.  138  w  ‘a  b  139  ‘C  140  Figure 28.  Cultures of canine SMP neurones stained for VIP  IR (arrows), which are located within ganglion-like clusters of cells x 100  142  Figure 29.  Double stains of human and canine cultures of  SMP for SP—IR (Rhodamine)  a)  Human SP—IR neurones and b)  arrows)  c)  and SS—IR (FITC).  x 2OO,  (small arrows)  localization.  (large  Note the co—localization of SS—IR and SP-IR  Canine SP—IR neurones  neurones  SS—IR neurones  Also,  (large arrows)  x 100.  and d)  SS—IR  Note the lack of co—  note the grouping of each type of  neurone reminiscent of what is observed in situ.  144  2.  Somatostatin Secretion from Human Submucosal Neurones.  a.  Effects of Secretagogues  The addition of B—PMa at concentrations of io8,  io  M caused significant increases in the release of  and  SS—IR (figure 30).  Increasing the potassium concentration  from 5 to 10 mM resulted in a further increase in the mean value of SS—IR released in response to i3—PMA, not statistically significant (figure 31).  but this was  The basal level  of SS-IR over the two hour time period was 16 ± 6 24 ± 4  (n=1l)  added KC1,  (n=6)  and  pg/600l using release medium with and without  respectively.  Basal release of SS-IR could be  measured only after 120 minutes.  The variability of SS—IR  release in response to 13—PMA and 10 mM KC1 was determined in 3 wells and was found to be  5%  <  release levels was found to be  <  (n=3).  Variation in basal  3% between wells  (n=6).  Of the total SS-IR released in response to 13’PMA (106 M,  10 mM KC1)  after 30 mm, (n=3)  after 120 mm,  59% was present in the medium  and 80% was present after 60 mm  (figure 32)  145  Figure 30.  Release of somatostatin immunoreactivity  as a percent of total cell content  (SSIR)  (% TCC) from submucosal  neurones in response to incubation with i3-PMA for 120 mm =  6 donors).  (n  “C” is the basal level of SSIR after 120 mm.  Values are means ± S.E.  146  30 *  C) C)  25 *  20  j)  *  15 10 5 0 C  —8  —7  log [fiPMA]  147  V  Figure 31.  Release of SS—IR  (% TCC) from submucosal  neurones in response to incubation with 8—PHA for 120 mm 5 mM and 10 mM KC1  (n=6 donors).  Values are means ± S.E.  *Significantly different from basal release  (p  O.05)  in  148  —  5mM K+ 10mM K+  C) C) * * *  ) LI)  *  *  *  15  10 5 0  C  —8  —7 log [PMA]  —6  149  Figure 32.  Release of SS—IR  (% TCC) from submucosal  6 M, neurones in response to 8—PMA (io’ 60 and 120  mm  (n  =  3 donors).  10 mM KC1)  after 30,  Control values represent the  amounts of SS—IR released in the absence of B—PMA over 120 mm.  Values are ± means S.E.  150  •=. flPMA (106 M, 10 mM KCL) 0—0  control  50 40 0  C),  (I)  1 0 0  30  60  90  Time (mm)  120  150  151  The addition of the inactive 4a-phorbol at concentrations of io , 8  lO and io 6 M did not cause  significant increases in the release of SS—IR  (figure 33)  (n=3)  The addition of substance P did not cause any increase in SS—IR release in comparison to basal  (figure 34).  The  addition of SP in the presence of hexamethonium (106 M) atropine  (106 M)  secretion, levels  did not cause any change in the SS-IR  but did result in more variation of the basal  (figure 35).  The addition of SP  affect 13-PMA stimulated SS—IR release  The effects of tetrodotoxin (TTX, M)  and  (1O N)  did not  (figure 36).  l0’6 M)  on 13-PMA  (106  —stimulated SS—IR release were examined in one experiment  (figure 3  (figure 37).  The secretion of SS—IR in response  was partly attenuated by TTX.  The effects of CGRP 6 io N and methacholine 1o 6 N on SS—IR release were examined in one experiment The secretion of SS-IR was stimulated by CGRP, affected by methacholine.  (figure 38). but not  Methacholine attenuated CGRP  induced secretion of SS-IR.  The calcium ionophore  (10—6 N,  5 x 10-6 N)  was tested in  two donors and it was found to not have any effect on SS—IR secretion  (Table 9).  152  Figure 33.  Release of SS-IR  (% TCC) from submucosal  neurones in response to incubation with 13—PMA and the inactive 4a—phorbol for 120 mm means ± S.E.  (n  =  3 donors).  Values are  153  50  — 4phorboI f3PMA  40 0 0  30 20  3  (I)  10  C  —8  —7  —6  log [drug]  154  Figure 34  Release of SSIR (pg/dish)  from submucosal  neurones in response to incubation with substance P for 120 mm  (n  =  4 donors)  Values are means ± S,E.  155  50 U,  40 30  ri)U, 1 0 C  —9  —8  —7  —6  Log [Substance P]  156  Figure 35.  Release of SS—IR (pg/dish)  from submucosal  neurones in response to incubation with substance P for 120 mm  in the presence or absence of hexamethonium (hex)  atropine  (atr)  (n  =  4 donors)  and  Values are means ± S.E.  17  50  UI  C  40 30  L  C’,  20 10  C  hex 6 + 6 otr  —9  —8  —7  —6  Log [Substance P]  ÷ 6 + atr hex 6  158  Figure 36.  Release of SS-IR (pg/dish)  from submucosal  neurones in response to incubation with i3-PMA for 120 mm the presence or absence of substance P Values are means ± S.E,  (n  =  3 donors).  in  159  — PMA PMA  +  SP (1Q— M) 7  400 350 •  300 250 200 150  U)  (1)  100 50 0  C  —9  —8  Log [PMA]  —7  160  Figure 37.  Release of SS—IR (pg/dish)  from submucosal  neurones in response to incubation with 13—PMA for 120 mm the presence or absence of TTX (n  =  1 donor).  in  16].  900 800 700 600 500 00 300 200 100 0• c  6 -nx--  —6  -6 +TrX— 6  log [fiPMA]  162  Figure 38.  Release of SS-IR (pg/dish)  from submucosal  neurones in response to incubation with CGRP and methacholirie for 120 mm  (n  =  1 donor).  163  1  1 100 80 cn  60 40 20  C  6 CGRP  6 Methcholine  CGRP 6 + 6 Methchoflne  P  .D  w  o.  IJ ‘J  I  00  P  x  LYI  %  P o  M I..)  I  P  I-I  0  rt I-C  () 0.  CD  CD  0  II CD  CD  I-a.  O-  -  .Q  ‘t  —.  0 CD  cn 0  0  I-a.  0  I-a  w  I-C CD  0  0  ‘a  0  C) I I-J C) I-’  CD  c1  H w CD C) II CD 1.  I-h O  0  a  I-h CD  CD  CD  S  CD  b.  1  165  The effects of carbachol one donor  (figure 39)  (1o  10 M)  were tested in  and it appeared to increase the  secretion of SS-IR.  b.  Somatostatin Content (Table 9)  The amount of SS—IR per well was found to be 1990 ± 809 per well; however the variability of SS-IR content between wells within each donor was found to be (Table 10).  Also,  <  3%  (n  =  6 donors),  incubation with different concentrations  of 13-PMA, with or without increased KCL,  did not  significantly alter SS-IR content (n=6)  c.  Somatostatin Recovery.  The recovery of SS—IR from release medium after incubation with the cultures was greater than 95% and after extraction with 2 N acetic acid was greater than 90%.  d.  Characterization of molecular forms.  The majority (> 90%)  of SS—IR in acid extracts of the  neurones in culture and released in response to 13—PMA eluted with the same retention time as synthetic SS-l4 on HPLC (figure 40,  41).  166  Figure 39.  Release of SSIR (pg/dish)  from submucosal  neurones in response to incubation with 13—PHA and carbachol for 120 mm  (n  =  1 donor)  167  400  -PMA Carbcchc  .  300  -Q  200 ci)  10:  og [dose]  168  Table 10.  Variations in content of somatostatin immunoreactivity  Somatostatin content of extracts (pg/dish) control  3359 ± 1441  10 mM KC1  3131 ± 1253  13—PMA io6 M  3483 ± 1429  M  3221 ± 1259  io’8 M  3276 ± 1332  6 M, 1o  10 inN KC1  3475 ± 1367  H  3286 ± 1327  io8 H  3307 ± 1444  169  Figure 4O  HPLC profile of SS—IR released from subinucosal  neurones in response to B—PMA (106 M). pooled medium from 6 wells  Sample represents  SS—28 and SS-l4 markers  indicate the elution position of synthetic peptide  170  SS—14 1500 SS—28  ‘1’  35  1000  C/)  ci) =  500  28  C 0 -‘C)  0  -  0  10  15  Elution Volume (ml)  I 20  25  171  Figure 41.  HPLC profile of SS—IR contained in acetic acid  extracts in response to B—PMA (106 M). pooled extracts from 2 control wells.  Sample represents SS—28 and SS-l4  markers indicate the elution position of synthetic peptide.  172  SS—14 1500 SS—28  ‘1’  35  ‘if 1000  (I) (J)  500  28  ___•II_•i.r_I.•_II..I  Li  0  5  10  1 .Ii_IIi. 15  Elution Volume (ml)  20  25  S  173  3.  Somatostatin and Substance P Secretion from Canine  Submucosal Neurones.  SS-IR and SP-IR were extracted according to the protocol used for SP-IR previously  (Kwok and McIntosh,  1980)  The total cell content of SSIR was 1200  measurable by RIA.  ± 210 pg/well and for SP—IR was 810 ± 107 pg/well The basal release of SP—IR was 4.5 ± 0.4 for SS—IR was 8.1 ± 1.5 IR was found to be of SPIR was  >  and were  >  (% TCC)  (n=3).  (n=3).  ( % TCC) and that The recovery of SS  90% using this procedure.  The recovery  95 % with or without the presence of  bacitracin and captopril.  Depolarization by 55 mM K did  not increase SP—IR secretion (n=3).  174  V  D.  Discussion  In the present study,  submucosal neurones from human and  canine small intestine were isolated and maintained in tissue culture and the localization and release of SS—IR were examined.  A vigorous digestion procedure was required  to obtain a high yield of neuronal ganglia from human tissue.  Gelation and reaggregation of collagen occurred due  to the large amount of collagen present in the human submucosa.  Dimeric collagen has been shown to undergo a  reversible gelation as temperature and pH increase  (with  significant changes in turbidity at pH=7.4 and T=28°C), the process also being dependent on the concentration of free collagen (Yurchenko and Furthmayr,  1984).  Therefore,  the pH  of the washing and incubation medium was kept below 7.4,  and  the cells were washed and centrifuged to reduce the concentration of collagen.  If these procedures were not  followed, the yield of viable neurones was insufficient to perform secretion studies.  The method of short—term culture  described was adapted from previous work carried out using canine small intestine (Barber et al, 1989).  1986; Buchan et al,  In order to obtain neuronal ganglia, human tissue  required a more vigourous digestion than canine tissue. thicker submucosa in the human small intestine was the primary reason for this difference.  A  175  The average yield of neurones was two 12 well plates at a concentration of 1-2 x 106 cells/well.  This meant that  only 12 conditions could be tested in duplicate for each experiment.  The human and canine submucosal neurones attached overnight, were phase bright as demonstrated by phase contrast microscopy,  sprouted processes which contained  varicosities along their length and made anatomical connections with other cells.  This indicated that after  isolation and culture the neurones were viable and did not alter their normal morphologic phenotype.  Time—course  studies of canine cultures demonstrated that the cells in culture formed aggregates after 72 h in culture.  This type  of aggregate formation has been observed previously in explants of enteric neurones maintained in tissue culture (Jessen et al,  1983).  Electron microscopic examination of  the aggregates examined by these authors showed that the cellular contacts inside of them were similar to those seen in enteric ganglia in situ.  The staining pattern of SS-IR in human cultured submucosal neurones was similar to that observed in tissue sections, suggesting that peptide localization was unchanged after isolation and culture.  Nerve fibres and varicosities  positively staining for SS-IR and SP-IR were commonly observed in the cultures.  The more intense staining of SS—  176  IR fibres in culture probably relates to the lack of obscuring collagen which surrounds the neurones in situ.  Somatostatin and SP—IR were co—localized in human, but not canine,  submucosal neurones which parallels the location  0 of these neuropeptides in tissue sections  Thus,  the  isolation of the ganglia and maintenance of submucosal neurones in tissue culture did not alter the expression of SP—IR and SS—IR.  The results presented have shown for the first time that activation of PKC by 13-PMA markedly stimulates the release Release of SS—IR by  0 of SS—IR from human enteric neurones  activation of PKC using phorbol esters has been reported with dispersed cultures of fetal rat hypothalamus and cortex (Peterfreund and Vale, (Buchan et al,  1983)  and canine jejunal submucosa  1989)  The predominant molecular form of SS—IR present in acid extracts of the cultures was SS-14.  This supports the work  of other groups that SS—14 is the the major molecular form in acid extracts of human enteric nerves 1985; Keast et al,  1986; Penman et al,  (Baldissera et al,  1983)  Furthermore,  the predominant form of SS-IR released in response to 13—PMA was also found to elute with the same retention time as SS— 14 using HPLC.  Central neurones have been shown to contain  and release SS-14  (Bonanno et al,  1988).  Somatostatin  177  endocrine cells of the human gut have been shown to contain SS-28  predominantly  (Baskin and Ensinck,  1984).  The lack  of measurable SS—28 in the human submucosal cultures further indicated the absence of endocrine cell contamination.  The release of SSIR by B-PMA was not due to non specific membrane effects since the inactive 4a-phorbol did not significantly alter basal release.  The secretion of SS—  IR was probably due to activation of protein kinase C (Nishizuka,  although effects other than P1CC activation  1986)  1982).  cannot be ruled out (Castagna et al,  The release of SS-IR in response to i3PMA (106 M with 10 mM KC1)  reached a plateau over the incubation period so  that 59% of the SS-IR released after 120 minutes was present after 30 mm  and 80% of the total SS—IR released was present  after one hours  Therefore, the rate of release of SS—IR  decreased after the first 30 minutes of the incubation period.  The decrease in SS—IR release may have been due to  depletion of SS-IR from the cells, down-regulation of protein kinase C or autocrine regulation by SS.  Autocrine  regulation of SS has been demonstrated in.the pancreas and stomach  (McIntosh,  1985).  Phorbol esters have been shown to  down-regulate PKC activity involved in norepinephrine release from rat brain synaptosomes  (Oda et al,  release period was extended to 120 mm  1991).  The  to allow measurement  178  of basal SS—IR levels which were not detectable at earlier periods.  To examine whether neuronal depolarization would augment basal or phorbol ester-stimulated SS—IR secretion, the extracellular concentration of potassium was doubled to 10mM, which should have caused a small but sustained depolarization of neurones, increased,  Basal release of SSIR was not  implying that attenuation of membrane polarity is However, there was an  not sufficient to generate release.  indication that the neurones were more sensitive to stimulation by 13-PMA after potassium depolarization although this effect was not statistically significant.  Furthermore,  preliminary experiments have demonstrated that TTX only partly attenuated BPMA-stimulated release of SS-IR, which suggests a mechanism only partly dependent on membrane depolarization.  Previous studies demonstrated that even  high levels of potassium (> 50 mM)  did not evoke the release  of vasoactive intestinal peptide or calcitonin gene—related peptide from enteric nerves Burnstock,  (Belai et al,  1988; Besson et al,  1983).  1987; Belai and  The results presented  in this thesis imply that exocytosis of peptide-containing vesicles requires the activation of second messenger cascades  (e.g. protein kinase C activation)  membrane depolarization.  in addition to  179  The release of SS—IR by B—PMA from human neurones was not affected by the presence of the calcium ionophore A23187, unlike canine neurones in which stimulation of SSIR secretion by 13—PMA occurred only in the presence of the ionophore (Buchan et al,  1989)  (see Table 11).  Interestingly, the basal secretion of SS-IR in the dog was 23 fold higher than that in the human cultures suggesting that there was a tonic basal stimulation of SS-IR secretion. It is probable that due to this background stimulation, the canine neurones required both activation of PKC and influx of calcium (by A23187)  (by iS-PHA)  to further stimulate SSIR  release.  The calcium ionophore on its own had no effect on SS--IR secretion in human cultures as was the case in canine neurones  (Buchan et al,  1990).  The ability of the calcium  ionophore to stimulate the release of NT from canine cultures has suggested that different mechanisms must be involved in the release of different peptides,  It is possible that I3-PMA affected the release of other neurotransmitters present in the cultures which in turn may have altered the release of SS-IR.  For example,  SP has been  shown to release Ni from canine submucosal neurones et al,  1989).  (Barber  The release of SP by 13—PMA presumably would  not have affected the release of SS—IR, since the present results have shown that exogenous SP had no effect.  The  180  release of other neuropeptides from similar cultures in response to 13—PMA remains to be determined.  While SP—IR neurones were present in human submucosal cultures, the addition of exogenous SP did not inhibit SS-IR release.  Substance P has been found to inhibit SS-IR  release from neurones of canine submucosal cultures and endocrine cells of the perfused rat stomach (Buchan et al, 1990; Kwok et al,  1988)  (see Table 11).  Conversely,  substance P has been shown to stimulate the release of SS—IR from the hypothalamus and pancreas  (Reichlin,  1981).  The  variation in SP effect and co—localization of SP—IR and SS IR observed in the human but not dog may reflect an underlying difference in the regulation of SS-IR secretion.  Different effects of SP, on canine versus human submucosal neurones, were probably due to a combination of direct and indirect effects on SS—IR containing neurones. Support for direct SP effects on submucosal neurones comes from experiments using isolated mucosa/submucosa preparations which have shown that SP—mediated increases in the secretion of C1 1985; Perdue et al,  ions were TTX—sensitive 1987).  In addition,  (Keast et al,  substance P has  been shown to release neurotransmitters such as acetylcholine from guinea pig myenteric neurones Owang et al,  1987)  and  (Wiley and  181  Table 11.  Similarities and differences in the secretion of  somatostatin immunoreactivity from canine and human submucosal neurones  *  increase  no effect  13-PMA  13-PMA  Human  Dog  +  increase  A23187  decrease  Sp  Buchan et al,  #  Data not shown  increase  no effect no effect  SP + PMA  *  #  1990  182  neurotensin from canine submucosal neurones  (Barber et al,  1989)  The signal transduction mechanism for the SPmediated slow EPSP in myenteric neurones was shown not to involve cAMP (Palmer et al,  1987)  but substance P has been shown to  increase levels of intracellular calcium in dorsal horn neurones and pancreatic acinar cells Gallacher et al,  1990).  (Womack et al,  In both cell types,  1988;  SP was  suggested to increase cytoplasmic calcium by activating protein kinase C.  The activation of PKC has been shown to  stimulate SS—IR secretion from a variety of neurones including submucosal neurones in this study.  Substance P has been shown to produce a slow EPSP in myenteric nerves submucosal nerves  (Katayama et al, (Surprenant,  1979; Willard,  1989)  1984; Mihara et al,  and  1985).  The electrophysiological effects of SP in combination with its stimulatory effects on PKC activation and neurotransmitter secretion suggest that a direct action of SP on SS-IR containing neurones would have caused stimulation of SS-IR secretion,  The data obtained in the  present human study and the results previously shown in the dog do not support a direct action of SP on SSIR containing neurones.  183  An indirect action of SP caused by the concoimnitant release of another neurotransmitter, for example Ach,  is a  more probable explanation for the results observed in both canine and human neuronal cultures.  The release of Ach from  enteric neurones by SP has been demonstrated (Keast et al, 1985)  and also provides a possible explanation for  differences in the effects of SP on SS—IR secretion between canine and human cultures.  The release of various  transmitters suggests that SP may have both excitatory and inhibitory effects  (see section below on effects of Ach)  which could be why no SPef feet was observed in the human cultures.  Acetylcholine has been shown to excite enteric neurones (Wood,  1970; North and Nishi,  1976).  These neurones  exhibited a postsynaptic fast nicotinic EPSP North,  1973; Hirst et al,  1974; Surprenant,  (Nishi and 1984)  and a slow  1 receptor (North et al, EPSP by activation of a N  1985).  In  addition, Ach was shown to produce a presynaptic IPSP by activation of a N 2 receptor (North and Tokimasa,  1982)  which  also inhibits the release of Ach and non—cholinergic neurotransmitters (Morita et al,  1982).  The effects of Ach  release on SS—IR secretion would then depend on the number and distribution of different cholinoceptor types.  Further,  cholinergic effects would depend on the anatomical relationship between SS—IR and Ach containing neurones. results of this study do not provide evidence for a  The  184  difference in cholinergic receptor number and distribution, or in Ach distribution between the canine and human neurones.  However, they have clearly demonstrated a  difference in the anatomical relationship of SS—IR neurones between these species.  There was large variability in the basal SS-IR secretion of human submucosal neurones in the presence of hexamethonium and atropine.  Preliminary experiments have  demonstrated the release of SS—IR in response to the muscarinic agonist carbachol.  However, the muscarinic  agonist methacholine had no effect on its own and attenuated i3-PMA stimulated release of SS-IR.  These experiments serve  to illustrate that the effect of endogenous Ach and exogenous muscarinic agonists was variable.  Further  experiments using cholinergic agonists and antagonists are required to determine the precise role of cholinergic neurones in the secretion of SS—IR from submucosal neurones.  Immunohistochemical identification of neurones containing choline acetyl transferase (Chat) has not been carried out in human or canine enteric neurones but studies in the guinea pig indicated that at least 50 % of enteric neurones were cholinergic (Furness et al,  al,  1991).  The prevalence of fast postsynaptic EPSPs  (mediated by Ach) (Wood,  1987)  1984; Steele et  during.stimulation of presynaptic fibres  also argues in favor of a large population of  185  cholinergic enteric neurones.  The lack of a suitable Chat  antibody or antiserum has made it impractical to carry out studies to examine the localization of this enzyme in submucosal cultures.  The levels of added SP or SS,  or secreted SP—IR or SS-IR  in the present study may have been altered as a consequence of degradation by proteolytic enzymes,  e.g. the lack of SP  action on SS—IR secretion from human submucosal neurones could be attributed to the degradation of either added SP and/or secreted SS—IR.  The recovery of  >  95 % of exogenous  SS—IR from release medium after incubation with human or canine submucosal cells suggested that significant imiuunoreactive SS was not lost during the 2 h incubation period.  Substance P recovery was also found to be complete  with or without the addition of bacitracin and captopril. These results indicate that minimal levels of proteolytic degradation occurred in the submucosal cultures.  The  peptidases responsible for degradation of SP and SS have been found in or on glia 1983) et al,  (Bunnett,  and endothelial cells 1982).  1987; Lentzen et al,  (Defendini et al,  1983; Takada  Endothelial cells were not present in the  cultures while the amount of qua was small due to the presence of the mitotic inhibitor cytosine arabinoside.  The  basolateral membrane of enterocytes has been suggested to be a primary region for the degradation of both SS-28 and SS-14 (Weber et al,  1986)  and it is probable that particular  186  capillary beds and circulatory enzymes contribute to the degradation of neuropeptides since their half-life in blood is short  (Bunnett,  1987).  These results have suggested that  the use of dispersed neuronal cultures significantly reduced the levels of proteolytic enzymes  Previous studies have  reported a lack of degradation of SS-IR in cultured rat brain neurones  (Lucius and Mentlein,  1991)  and neurotensin  in cultured canine submucosal neurones (Barber et al,  1989).  Isolation methods utilizing collagenase have been shown to specifically damage muscarinic receptors on neurones and various cell separation methods using hypertonic solutions have been implicated in general receptor damage (Guarnieri et al,  1975).  However, the lack of SP effect on SS-IR  release from human cultures was not likely due to receptor damage for several reasons.  Receptor damage due to  hyperosmolality was avoided since cell separation in these experiments was carried out using elutriation which permits the use of isotonic solutions et al,  1975).  (Meinstrich,  1983; Guarnieri  Once the cells have been isolated, the  receptors would have regenerated in culture, as has been shown for the nicotinic acetylcholine receptor (Hartzell et  al,  1973).  Further support comes from experiments using  canine submucosal neurones which have been isolated and maintained in tissue culture in a similar fashion and were able to respond to receptor dependent secretagogues such as SP  (Barber et al,  1989; Buchan et al,  1989).  Finally,  187  preliminary experiments using CGRP and carbachol have shown increases in SS—IR secretion suggesting,  but not confirming,  the presence of functional receptors on the neurones.  As mentioned, the neurones in tissue culture sprouted processes which were associated with varicosities along their length.  In addition, the proportion of human neurones  containing SS—IR was similar to that in tissue sections. These results have suggested that the culture and isolation procedures did not alter their normal morphologic and immunocytochemical phenotype.  Neurones dissociated from rat  MYP have also been shown to retain morphological and immunocytochemical properties after having grown in culture for 4—8 weeks  (Nishi and Willard,  1985).  These authors have  also demonstrated that dissociated myenteric neurones in vitro retain normal pharmacological and electrophysiological properties  ‘-p  (Willard and Nishi,  1985a;  1985b).  188  E.  Summary  These data have demonstrated that neurones of the SMP of the human small intestine can be isolated and maintained in tissue culture for 72 h.  The neurones were viable and  sprouted neurites which contained varicosities along their length and which made anatomical connections with other This indicated that the neurones retained a  cells.  morphologic phenotype in culture. SS—IR,  ‘normal’  The cultures contained  SP—IR and VIP—IR which were present in cell bodies,  neurites and varicosities.  The proportion of SS—IR neurones  was similar in culture and in situ and these neurones contained SP-IR which reflected the co—localization observed  in situ.  This indicated that the isolation and culture  conditions did not alter the expression of these neuropeptides.  A major advantage of these cultures was that  there were no SS—IR endocrine cells present and therefore experiments were carried out which examined SS—IR secretion from neurones exclusively.  The presence of varicosities  suggested that the release of SS-IR was regulated,  since  regulated peptide secretion from unmyelinated nerve fibres has been shown to occur at this level.  The secretion of SS—  IR from these neurones was stimulated by 13—PMA activation of PKC in a time-dependent fashion.  Somatostatin—14 was the  predominant form of SS—IR present in the neurones and released by 13-PMA.  Potassium depolarization had no effect  on SS—IR secretion but seemed to make the neurones more  189  sensitive to stimulation by 13-PMA, statistically significant.  although this was not  Preliminary experiments  suggested that TTX could attenuate the 8-PMA-stimulated secretion of SS-IR.  This would further support the  contention that membrane depolarization was only partly responsible for the secretion of SS—IR from these neurones in response to activation of PKC.  Further experiments are  required to determine the role of membrane depolarization in Unlike the situation in the dog,  the secretion of SS-IR.  the calcium ionophore A23187 was not required to elicit the secretion of SS—IR in the human but,  like the dog,  ionophore on its own did not release SSIR.  the  The inhibitory  effect of SP on basal SS—IR secretion observed in canine cultures cultures.  (Buchan et al, In addition,  1990)  did not occur in the human  SP did not have an effect on 13-PHA--  stimulated SS—IR release.  The difference in SP effect  between the canine and human cultures is correlated to the difference in the co-localization of SP-IR and SS-IR between canine and human submucosal neurones  .  Preliminary  experiments suggested that cholinergic agonists have mixed actions on the secretion of SS—IR from these neurones.  The  stimulation of SS-IR secretion by CGRP and carbachol indicated that the neurones were responsive to receptor dependent stimulation in addition to receptor independent stimulation by i3-PMA.  190  Human myenteric cultures exhibited characteristics similar to those observed in submucosal cultures. neurites and varicosities contained SS-IR,  Neurones,  0 SP-IR and VIP’IR  The canine cultures exhibited similar characteristics to human cultures.  These cultures contained SP-IR and SS—IR as  demonstrated by RIA, and were present in distinct neurones as demonstrated by ICC, which reflected their localization  in situ.  The release of SPIR was measurable but was not  0 stimulated by potassium depolarization  The recovery of SP  IR incubated with canine cultures was close to 100 % and was not affected by the enzyme inhibitors bacitracin or captopril.  191  V.  GENERAL SUMMARY AND CONCLUSIONS  Hypotheses 1..  That interspecies variations in neuropeptide  localization and enteric neuronal morphology exist between the human and canine small intestine.  A.  Morphological Data from the Small Intestine  1.  The human and canine small intestine contain two  different sub—groups of submucosal ganglia based on morphological examination.  One group was nearer the mucosa  and the other was nearer the circular muscle.  There were no  differences noted in their morphology or chemical coding.  2.  Canine and human submucosal ganglia contained neuronal  types which were segregated into clusters of a predominant type.  In canine submucosa,  large ganglia were composed of  several such clusters of different neuronal types.  In human  submucosa, the situation was more dramatic in that some small ganglia contained one neurone type exclusively.  These  results have both developmental and functional implications. From a developmental standpoint, the existence of such clusters suggests that neurones migrating into the enteric plexi could be pre—prograimued to differentiate,  for example,  into SS—IR neurones rather than receive signals to differentiate after arrival.  The embryonic neurone after  arrival at a specific location would resume division to  192  produce clusters of a single type within the plexus. Alternatively, the existence of the clusters could result from the parallel tracking of axons growing out of the embryonic ganglia.  In this case, the phenotype would be  dependent on the target innervated but all neurones with axons in the same bundle would differentiate into the same type.  3.  The canine, but not the human,  small intestine has a  characteristic deep muscular plexus that was separate from other sets of fibres in the circular muscle layer 0  This  implies that neural control of circular muscle differs between the dog and human.  4.  Immunocytochemical studies have demonstrated,  for the  first time, that SP-IR and SS-IR are co-localized in the human, but not canine, intestine.  enteric neurones of the small  Furthermore, canine. ganglia were shown to be  substantially larger than their human counterparts.  The  difference in chemical coding of neurones and the difference in ganglion size may be indicative of differences in the function of submucosal neurones.  Conclusions.  These data support the hypothesis that interspecies variations in neuropeptide localization and enteric neuronal  193  morphology exist between the human and canine small intestine.  In addition, these studies have shown that there  are differences between canine and human submucosal neurones with respect to ganglion size, chemical coding of submucosal neurones and the innervation of circular muscle.  It has  been suggested that interspecies differences in gut morphology are related to diet (Powell,1987; Gross, 1990).  and size (Gabella,  1986)  The interspecies differences  observed in the present study support the contention that animals which have similar diets have similar enteric ganglia with respect to neuropeptide distribution and neuronal number.  Moreover, the results have emphasized that  comparisons of the results of physiological experiments carried out in different species should be made with caution.  Rypothesis 2.  That differences in neuronal chemical coding  0 and morphology will be reflected in neuronal function  Hypothesis 3.  That short-term cultures of human and canine  SMP will provide a model system in which to examine such 0 differences  B.  Culture Studies  1.  These experiments have demonstrated,  for the first time,  that adult human submucosal neurones can be isolated and  194  maintained in tissue culture.  Cell cultures eliminate the  problems associated with peptides present in both submucosal and myenteric neurones, and enteroendocrine cells.  2.  The neurones sprouted neurites which contained  varicosities,  suggesting that the secretion of neuropeptides  is from varicosities and can be regulated.  This provides an  advantage over secretion studies carried out with acutely dissociated neurones which do not have neuronal fibres. Secretion from neurones which do not possess nerve fibres suggests that secretion of neuropeptides occurs from the cell body for which there is presently no evidence using in vivo experimental techniques.  Previous studies and the  present evidence have suggested that SSIR secretion was independent of the cell body.  Thus,  the regulation of SSIR  secretion is suggested to be at the level of the varicosity. These data have also suggested that a small depolarization renders the varicosities more sensitive to a secretory stimulus.  3.  The difference in neuronal localization of SSIR and  SP—IR between the canine and human small intestine also suggested a difference in function.  Substance P did not  inhibit the secretion of SSIR from human neurones as was the case in canine neurones  (Buchan et al,  1990).  This  supports the hypothesis that the differences observed in SP-  195  IR and SS—IR localization are related to differences in SS IR secretion and SP actions.  4.  The phorbol ester, 13—PMA, was able to stimulate the  secretion of SS—IR from human neurones in the absence of the A23l87, unlike canine neurones which required the presence of the calcium ionophore (Buchan et al,  1990).  This further  supports the hypothesis that canine and human submucosal neurones differ with respect to the regulation of SS-IR secretion.  5.  The effects of CGRP and carbachol suggested that  receptor dependent secretagogues were able to stimulate SS IR secretion.  This suggested that functional receptors were  present on the neurones after isolation and tissue culture but further experiments are required to confirm this possibilty.  Conclusions.  These results support the hypothesis that dispersed cultures of submucosal neurones are useful models to examine neuronal function.  The cultures provided advantages over in  victh experiments for studying the secretion of neural SS-IR. The regulation of SS-IR was shown to be different in canine and human submucosal neurones and this difference was  196  related to the difference in the localization of SSIR and SP-IR i.e.  a difference in neuropeptide phenotype.  197  CONCLUSIONS AND FUTURE DIRECTIONS  VI.  A.  Conclusions and Significance  The data presented and previous studies suggest that the morphology of the small intestine reflects diet i.e. structure and function are closely linked.  The corollary to  this statement is that animals having similar diets have similar gastrointestinal tracts.  The similarity in  structure is illustrated by the correspondence in the small intestine of omnivores groups  (rat and human)  (e.g. ruminants).  compared to other  Genetic similarity does not seem  to be a prerequisite except where it confers a preference in diet.  An example of this is seen in two new world monkeys  similar in size, the howler and spider monkeys 1986).  (Milton,  Both species are plant eaters, but howler monkeys  pass food through their digestive system at half the rate of This reflects the larger colon of the  spider monkeys. howler monkey.  Although the diets of both species are plant  based, the spider monkey eats mostly fruit and meets its nutritional requirements by ingesting large volumes of food. The howler monkey eats less and is able to ferment quantities of plants present in the colon.  There are many  examples which show that species can rapidly respond to changes in dietary quality by altering the features of the gut (Gross et al,  1986).  Alterations in the gut no doubt  198  include changes in the morphology of enteric neurones and the neurotransmitters they contain.  The experiments presented in this thesis have demonstrated that differences exist between canine and human small intestine with regard to the morphology of the SMP, the localization of SP, SS and VIP in enteric nerves and the actions of SP and P1CC—activation on enteric nerves. Differences in the secretion and absorption of electrolytes and in the control of motility between the canine and human small intestine (outlined in the general introduction)  are  probably related to the differences observed in the present experiments.  For example,  SP increases the secretion of  electrolytes in the small intestine by a mechanism which is TTX sensitive in some mammals but not the human (Hubel et  al,  1984; Keast et al,  1987).  It is possible that SP exerts  its TTX sensitive effects by the indirect inhibition of neural SS—IR release similar to that which is observed in the canine small intestine.  The necessity and usefulness of combining morphological and physiological experimental techniques to study the function of the small intestine was exemplified by the results obtained in the present studies.  Moreover, they  have suggested possibilities for further examination of the morphology of the small intestine, characterization of the neuronal cultures themselves, differences in SP action and  199  SS—IR release from the cultures and regulation of other neurotransmitters present in the cultures.  Finally,  functional studies of the small intestine can be carried out which examine whether the differences in morphology correlate with neurotransmitter regulation  B.  Future Directions  1.  Morphological Studies  The differences in neuropeptide distribution and plexus morphology between the human and canine enteric nervous system will probably be more extensive than reported in this thesis.  Further characterization of the distribution of  other known enteric neuropeptides will allow the neurone types to be identified in a manner similar to that which has been carried out previously in the rat and guinea pig (Furness et al,  2.  1989; Pataky et al,  1990).  Functional Studies  With the use of dispersed neuronal cultures of the SMP, it has been possible to study the secretion of a peptide which is produced by several cell types.  It is possible  that the regulation of SS-IR secretion from neurones is 0 different from that in endocrine cells  Furthermore,  differences in the modulation of secretion could reflect  200  different physiological roles for endocrine and neuronal SSIR.  Parallel studies of SS—IR secretion from endocrine cell  and neuronal cultures would provide an excellent model system in which to define different release patterns.  Although the data collected demonstrated both stimulation and inhibition, a role for cholinergic transmission in neural SS—IR release from human SMP neurones was indicated.  Further experiments are required to  elucidate the precise actions of cholinergic agonists and antagonists on SSIR secretion.  Testing the actions of the  cholinergic agonists and antagonists on canine enteric neurones would be useful for comparison to human neurones. Access to an antibody for Chat would permit the determination of the proportion of cholinergic neurones present enteric neurones in culture and in situ.  The overall actions of SP on SSIR secretion were suggested to be indirect, based on canine and human experiments. way.  This hypothesis can be tested in the following  First, the effects of SP on cellular events,  such as  ionic currents and calcium transients, can be examined in neurones known to contain SS-IR.  Second,  the effects of SP  on the release of other neurotransmitters, such as Ach, be determined.  Neurotransmitters can then be tested for  their ability to affect the cellular events of SS-IR neurones.  can  201  The present study and previous studies  (Buchan et al,  1990) have suggested that somatostatin secretion from canine and human submucosal neurones is modulated differently by The secretion of SS-IR can be further  the phorbol ester.  characterized by examining the response to depolarization by levels of potassium higher than those used in the present experiments.  It should be noted that depolarization of  enteric neurones by potassium may not result in the The lack of neurotransmitter release in  secretion of SS-IR.  response to high potassium has been noted in other preparations where it has been suggested that calcium entry is mediated by receptor-operated calcium channels Belai et al,  (e.g.  1987).  The cultures utilized in the present experiments were shown to contain neurones which possessed a morphologic phenotype similar to that observed in situ,  It would be  useful to compare ionic currents and pharmacological responses of neurones to known neurotransmitters in order to confirm the similarity of neurones maintained in tissue culture and in situ.  The calcium dependence of SS-’IR secretion from submucosal neurones is necessary to support its role as a neurotransmitter,  The examination of calcium transients in  SSIR neurones by the use of dyes such as Fura2 would be  202  necessary to confirm the calcium dependence of SS—IR secretion.  The actions of PKC stimulation on SS-IR secretion suggests that neuropeptides which activate this pathway also stimulate SS-IR secretion.  Gastrin-releasing peptide is an  example of a neuropeptide,  found in enteric neurones, which  activates this pathway in a variety of cell types.  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Chemical  CHEMICAL SOURCES  Source  BDH Acetic acid BDH Acetone Baker Ammonium acetate Fisher Anuuonium chloride Gibco Aanphotericin B Miles Aprotinin Gibco Basal medium eagle (powder) Sigma Bovine serum albumin (fraction V) Sigma, Miles Bovine serum albumin (RIA grade) Fisher Calcium chloride Sigma Calcium ionophore (A23187) Fisher Carbon decolourizing neutral (Activated charcoal) Sigma Chioramine T Sigma Collagenase (type I, XI) Gibco Cytosine B-D-arabinoside Pharmacia Dextran T—70 BDH Diaminobenz idine Sigma Dimethylsuf oxide Gibco Dulbecco’s modified Eagle Medium Commercial Alcohol Ethanol Gibco Fetal calf serum Formaldehyde (histology grade) Fisher Sigma Forskolin Sigma Gelatin Gentamycin sulphate Sigma Glucose (50 % commercial solution) Abbott Glucose oxidase Sigma Glutamine Sigma Hank’s balanced salt solution (powder) Gibco Hematoxylin Fisher HEPES Fisher Hydrocortisone Sigma Hydrogen peroxide Fisher Imidazole Sigma Insulin Sigma Lithium carbonate Fisher Magnesium sulphate Fisher Collaborative Research Nerve growth factor Normal swine serum Gibco Parraff in (paraplast) Monoject Permount Fisher Petroleum ether Fisher Phenol red Sigma Phorbol esters (I3PMA, 4aphorbol) Sigma  234  Source  Chemical  Picric acid Potassium chloride Potassium phosphate (monobasic) Sephadex CM—52 Sodium acetate Sodium barbital Sodium bicarbonate Sodium chloride Sodium hroxide Sodium iodide Sodium merthiolate Sodium metabisulphite Sodium pentobarbital Sodium phosphate Sodium pyruvate Somatostatin Substance P Tris-HCL Triton X-100 Xylene  APPENDIX II Na 10  ,  K 115,  Osmolarity pH  =  7.0  =  BDH Fisher Fisher Pharmacia Fisher Baker Fisher Fisher Fisher Amersham Eastman Kodak Fisher Gibco Fisher Gibco Peninsula Peninsula Sigma Fisher Fisher  Eurocollins buffer (mmol/L)  Cl 15, HCO 3 10, P0 4 577, glucose 195  330 mosm/kg  

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