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Morphological and functional studies of substance P and somatostatin in the small intestine

University of British Columbia

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. The neuropeptides chosen for examination were substance P, 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 immunoreactivity (SP—IR), somatostatin immunoreactivity (SS-IR) and vasoactive intestinal peptide immunoreactivity (VIP-1R) have been localized and their distributions have been compared in dog and human small intestine using immunocytochemistry (ICC). An antibody to protein gene product 9.5 (PGP) was used to localize all nerve cell bodies and fibres in the dog and human upper 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 (184 ganglia, n = 6 dogs), while the human ganglia contained 2.9 ± 0.3 (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, but not canine, SMP and MYP. In both species VIPIR was present in a population of neurones distinct from those containing SP—IR and SS—IR. In canine ganglia, 30% of neurones per ganglion in the SMP contained SP-IR, 35% contained SSIR and 30% contained VIP-IR. In human ganglia, 42% of neurones per ganglion contained SP-IR and SSIR while 40% contained VIP IR. These results sugest different functions for SP and SS in canine and human enteric ganglia. The secretion of SSIR, from intact preparations of small intestine, is difficult to interpret for two reasons First, SS—IR has been demonstrated in vagal, submucosal and myenteric neurones as well as endocrine cells, of the small intestine. Second, 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 M for 120 mm 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, 10 mM K+), 59% was present in the medium after 30 iuin and 80% 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.

Thesis/Dissertation

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2008-12-12

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http://hdl.handle.net/2429/2877

Physiology

University of British Columbia

UBCV

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