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Histamine H1-receptors, visualized in the guinea pig heart by autoradiography using [125I]iodobolpyramine as a specific probe, are abundant in the nodal tissue and cardiac vessels but also occur heterogeneously in the myocardium. Following photoaffinity labeling with [125I]iodoazidophenpyramine and electrophoresis, the ligand binding domain of the heart H1-receptor was shown to be present on a major 68-kDa and a less abundant 54- to 58-kDa protein. The 68-kDa protein displayed a molecular size higher in heart than in all other tissues (56 kDa). This indicates the existence of at least two isoforms of the H1-receptor; the cardiac isoform, however, was pharmacologically indistinguishable from the common isoform studied in cerebellar membranes using available ligands. Its distinct electrophoretic properties suggest that the cardiac isoform may have a unique function.

The distribution of enzymes and laminin was examined in ileal tissue from pigs suffering from intestinal adenomatosis to reveal the nature of the lesion. A disruption of the normal and specific pattern of distribution was found. Thus, the normal ileal epithelium was characterised by brush border enzymes: alkaline phosphatase, magnesium-dependent adenosine triphosphatase (Mg-ATPase), fluoride resistant acid phosphatase and 5'-nucleotidase; enzymes of the basolateral border: Mg-ATPase; and cytoplasmic enzymes: beta-glucuronidase, non-specific esterase and acid phosphatase. Subepithelial fibroblasts seemed to be characterised by 5'-nucleotidase. Laminin was present as a continuous band under the surface and crypt epithelium, somewhat thicker in the former. In contrast, the branching proliferating crypts of intestinal adenomatosis largely lacked enzymes characteristic of both villus and crypt cells. Reactions for the subepithelial components, laminin and fibroblasts were also reduced. The deficient differentiation of the epithelial as well as subepithelial components in porcine intestinal adenomatosis distinguish the condition from crypt hyperplasia and indicate an adenoma-like character.

The localization of the extracellular matrix recognition molecule J1/tenascin was investigated in the crypt-villus unit of the adult mouse ileum by immunoelectron microscopic techniques. In the villus region, J1/tenascin was detected strongly in the extracellular matrix (ECM) between fibroblasts of the lamina propria. It was generally absent in the ECM at the interface between subepithelial fibroblasts and intestinal epithelium, except for some restricted areas along the epithelial basal lamina of villi, but not of crypts. These restricted areas corresponded approximately to the basal part of one epithelial cell. In J1/tenascin-positive areas, epithelial cells contacted the basal lamina with numerous microvillus-like processes, whereas in J1/tenascin-negative areas the basal surface membranes of epithelial cells contacted their basal lamina in a smooth and continuous apposition. In order to characterize the functional role of J1/tenascin in the interaction between epithelial cells and ECM, the intestinal epithelial cell line HT-29 was tested for its ability to adhere to different ECM components. Cells adhered to substratum-immobilized fibronectin, laminin and collagen types I to IV, but not to J1/tenascin. When laminin or collagen types I to IV were mixed with J1/tenascin, cell adhesion was as effective as without J1/tenascin. However, adhesion was completely abolished when cells were offered a mixture of fibronectin and J1/tenascin as substratum. The ability of J1/tenascin to reduce the adhesion of intestinal epithelial cells to their fibronectin-containing basal lamina suggests that J1/tenascin may be involved in the process of physiological cell shedding from the villus.

Canine neurotensin (NT) and neuromedin N (NMN) were isolated from extracts of ileal mucosa using radioimmunoassay for detection. The structures determined were consistent with those predicted by earlier cDNA work. The molar ratio of NT to NMN was ca. 7, suggesting that the NT/NMN precursor, which contains one copy of each peptide, undergoes complex posttranslational processing or that other NT-precursors lacking NMN exist. In addition to NT, small quantities of NT6-13 and NT2-13 were obtained. Native and synthetic preparations of these peptides were indistinguishable in a radioreceptor assay employing rat brain membranes and 125I-labeled NT; NT6-13 was ca. 8-times more potent than NT and NMN was about one-sixth as potent as NT. NT6-13 was also ca. 10 times more potent than NT in inhibiting spontaneous contractile activity in longitudinally-oriented smooth muscle strips of porcine jejunum. Preparations of intestinal N-cells as well as N-cell vesicles also appeared to contain NT2-13 and NT6-13; however, it is not yet clear whether these peptides are utilized physiologically or simply represent metabolites of NT. These results suggest that further work on the processing of NT precursor and on biologic abilities of partial sequences of NT could be fruitful.

The healing of ileal and colonic anastomoses is compared in rabbits. The intestinal segment that contains the anastomosis shows a temporary loss of strength, which is reflected in a decreased bursting pressure. This loss of strength is accompanied by a massive loss of collagen, measured as hydroxyproline, both in ileum and in colon. In ileum, hydroxyproline concentrations, expressed on a dry weight basis, are lowered by 30 per cent, one day after operation. Thereafter, they rise again, after seven days reaching a level that is 40 per cent enhanced as compared with unwounded tissue. Maximal decrease in colon, measured two days after operation, is 40 per cent. After seven days hydroxyproline levels are back at preoperative values. In colon, significant loss of hydroxyproline is also apparent in the intestinal segment proximal to the anastomosis. This phenomenon does not occur in ileum. These results clearly demonstrate that after ileal anastomosis a loss of collagen occurs similar to that in colonic anastomoses. The fact that the loss of collagen is less extensive and more rapidly restored may be important in explaining the lesser incidence of leakage encountered after surgery of the small intestine.

Mcrovilli membranes have been isolated from dog jejunal and ileal enterocytes. Proteins were analysed by polyacrylamide gel electrophoresis after solubilization with sodium dodecylsulphate. The recovery of the membrane fraction with this purification method was found to be 22% and the specific activity of sucrase increases 19 folds in the membrane fraction. Microvilli membrane proteins after polyacrylamide gel electrophoresis were seprated in 21 bands, most of them with a molecular weight higher than 70 000. Seven bands with molecular weight from 150 000 to more than 340 000, were found to be glycoproteins.

Guinea-pig ileum was dissected and the mucosa, submucosa and external musculature extracted with aqueous acetic acid for measurement of four prodynorphin-derived peptides, namely dynorphin A 1-8, dynorphin A 1-17, dynorphin B, and alpha-neoendorphin. The peptide-like immunoreactive material extracted from the external musculature was characterized by multi-dimensional chromatographic analysis and compared to synthetic porcine standards. The chromatographic methods utilized were: reversed-phase high performance liquid chromatography (RP-HPLC), using two different eluants; cation exchange high performance liquid chromatography (CE-HPLC) and gel filtration chromatography. The dynorphin A 1-8-like immunoreactive material was homogeneous and coeluted with the standard in all chromatographic modes. The dynorphin A 1-17-like and dynorphin B-like immunoreactive material was heterogeneous but showed a peak that coeluted with synthetic standard in all chromatographic modes. The alpha-neoendorphin-like immunoreactive material also appeared to be heterogeneous with the major component on CE-HPLC coeluting with the synthetic peptide standard while the major component on RP-HPLC eluted differently. It was concluded that the guinea-pig ileum contains immunoreactivity for peptides derived from all coding regions of the prodynorphin gene and that these peptides may be present in multiple immunoreactive forms.

Eight patients with inflammation of the skin about the cutaneous stoma responded to local application of a 20% cholestyramine ointment in a short period of time. Two children treated with the hydrophilic base (Aquaphor) alone did not respond to treatment. The ileal bile acid patterns of two children with ileostomies were found to be different from that of the control group. Ileostomy patients had a higher concentration for total bile acids and a higher percentage of free bile acids.

A novel blood group H-active triglycosylceramide has been isolated from rat small intestine. It was present exclusively in the epithelial cells. The structure was established by mass spectrometry, NMR spectroscopy and degradative methods to the Fucp alpha 1 leads to 2Galp beta 1 leads to 4Glcp beta 1 leads to 1Cer. The lipophilic part was made up of mainly trihydroxy base (phytosphingosine) and 16 : 0--24 : 0 fatty acids.

The localization of 3H-opiates in the myenteric plexus of the guinea pig ileum is subject to systematic artifact when stretch preparations of the myenteric plexus are dipped into liquid Kodak NTB-3 emulsion for autoradiography. The cause of the artifact was determined to be a discontinuous distribution, or retraction, of emulsion over plexuses. The apposition of frozen freeze-dried ileal sections to dried photographic emulsion avoids this source of error.

Neurotensin levels in pancreas, but not other tissues, were increased in diabetic animals. The concentration of pancreatic immunoreactiveneurotensin was 138% higher in streptozotocin-diabetic rats, and 68% higher in genetically diabetic (db/db) mice as compared to their respective control animals. Daily administration of insulin (10-15 IU/kg) to diabetic rats completely reversed this effect, and pancreatic neurotensin levels in these animals returned to control values. These findings suggest that elevated levels of pancreatic neurotensin may contribute to some of the metabolic and hormonal disturbances occurring in diabetes.

The activity of neuraminidase prepared from the mucinase complex of Vibrio cholerae was measured by a new, semi-quantitative goblet-cell assay. The counts of normal, alcianophilic, sialomucin-containing goblet cells (purple-stained) and neutral mucosubstance-containing goblet cells (magenta-stained) in serial sections of ileum were compared before and after neuraminidase treatment. The procedure provides a more natural assessment of the action of V. cholerae neuraminidase on the viscous intestinal mucus.

The C- and N-terminal fragments of substance P were compared to the parent molecule with respect to their ability to: (a) contract the isolated guinea pig ileum, (b) induce salivation in the rat, (c) excite single cat dorsal horn neurones, and (d) induce scratching by intracranial injections in mice. C-terminal fragments as small as the heptapeptide were potent SP agonists on all assay systems. C-terminal fragments containing five amino acids or less were, at most, only weakly active. The C-terminal hexapeptide was a potent SP receptor stimulant on the isolated guinea pig ileum and, when directly applied by microiontophoresis, on cat dorsal horn neurons. However, the same compound was only 2-5% as potent as substance P in eliciting salivation and scratching in vivo, an indication that this fragment may be especially labile to enzymatic degradation. N-terminal fragments were totally inactive on the isolated guinea pig ileum. On the rat salivation and central nervous system assays, however, N-terminal fragments were capable of weak SP-like activity. It is concluded that SP receptors exist in multiple forms which we have labelled SP1 and SP2 receptors for those insensitive or sensitive to N-terminal fragments, respectively.

To examine the distribution of cholecystokinins (CCKs) along the small intestine we examined the nature of CCKs in samples of jejunum, mid-intestine and ileum from human and porcine intestine. CCKs in intestinal mucosa were extracted by boiling in both neutral and acid conditions, and subjected to high pressure liquid chromatography (HPLC) to separate the forms of CCK followed by radioimmunoassay of separate fractions. In neutral extracts of human intestine CCK immunoreactivity totalled 119.4, 22.9 and less than 1 ng/g in jejunum, mid-intestine and ileum, whilst in acid extracts the corresponding values were 65.3, 47.4 and less than 1 ng/g. Amounts of CCK extracted from porcine mucosa were of similar magnitude. In neutral extracts material co-chromatographing on HPLC with synthetic porcine CCK 8 predominated, whilst in acid extracts material co-chromatographing with CCKs 33/39 was the major form. These forms of human and porcine CCKs extracted from the mucosa behaved similarly to CCK 8 and CCK 33/39 standards on HPLC, in the radioimmunoassay and on molecular exclusion chromatography - suggesting marked similarity of the CCKs in the two species. In both species there was a marked change in the ratios of CCK 8: CCK 33/39 down the intestine from 1: 0.8 in human jejunum to 1: 5.6 in mid-intestine and from 1: 1.5 in porcine jejunum to 1: 5.8 in mid-intestine. These observations may explain the changing patterns of CCKs in circulation with time after ingestion of a fat meal and the greater impairment of CCK 8 than CCK 33/39 release observed in coeliac disease.

Arylazido aminopropionyl ATP (ANAPP3), a photoaffinity analogue of adenosine 5'-triphosphate, photoactivated with visible light (+hv), specifically and irreversibly antagonized ATP contractions of the guinea pig vas deferens. ANAPP3 (30 microM) antagonized responses to exogenously added ATP in untreated, and in tissues pretreated with indomethacin (2.9 microM) and 6-(2-hydroxy-5-nitrobenzyl)-thio guanosine (10 microM). It was of interest to see if this pharmacological antagonist of ATP could be used to assess the validity of the purinergic nerve hypothesis by allowing a differentiation between, or proof of the identity of, responses to ATP and the non-adrenergic inhibitory transmitter in guinea pig stomach fundus. After photoactivation (+hv) in the organ bath and subsequent washout, ANAPP3 (30 and 100 microM) failed to antagonize relaxant responses to ATP (1.0 - 1000 microM) in fundic strips. In addition ANAPP3 failed to antagonize ATP-induced inhibition of the twitch response in electrically stimulated guinea pig ileum longitudinal muscle strips. We conclude that ANAPP3 does not antagonize all actions of ATP, which may limit its usefulness in assessing the above hypothesis. Results with this compound suggest that ATP excitatory receptors may differ from those mediating relaxation and other ATP actions.

A procedure is reported which is well applicable for the quantitative determination of glycogen, especially in cases of a small glycogen content in a small amount of tissue. The method consists of extraction of glycogen by boiling potassium hydroxide, precipitation with ethanol, hydrolysis with sulphuric acid, and determination of the glycogen in form of glucose obtained with orthotoluidine.

1. Inhibition constant (Ki) were determined for a range of opioid standards using two binding assays; [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin ([3H]-GLYOL) binding to guinea-pig brain membranes in HEPES buffer and [3H]-naloxone binding to rat whole brain membranes in Krebs/HEPES buffer. 2. These values were compared with affinity measurements determined by antagonism of GLYOL on the rat isolated vas deferens preparation and by the receptor occlusion technique of Furchgott on the guinea-pig ileum longitudinal muscle, myenteric plexus preparation. 3. Agonists demonstrated markedly reduced binding affinity in the [3H]-naloxone binding assay where binding was conducted in the presence of sodium. 4. A strong correlation was obtained between Ki values from the [3H]-naloxone binding assay and affinity values determined in both isolated tissue preparations. Ki values obtained from [3H]-GLYOL binding did not correlate well with affinity data determined by isolated tissue techniques. 5. These findings suggest that functionally relevant receptors exhibit low agonist affinity.

Treatment with formaldehyde gas and HCl vapor, simultaneously or in sequence, induces fluorescence with indoles, including tryptophan residues of peptides, as is evident from studies on protein droplet models. Among cells that display intense formaldehyde-HCl-induced fluorescence are pancreatic exocrine cells, gastric chief cells, Paneth cells and enterochromaffin cells. Peptide hormone-producing cells that can be visualized by the formaldehyde-HCl treatment include gastrin cells and glucagon cells. The simultaneous procedure has proved superior to the sequential procedure. Simultaneous formaldehyde-HCl treatment appears to be a useful method for the demonstration of tryptophan residues of peptides and proteins. It seems more sensitive than previously described indole methods.

The changes occurring in the ileal wall and in enteroglucagon cells were studied in a rat model of intestinal adaptation, obtained by the transposition of a segment of distal ileum into the mid-duodenum (6 rats, compared with 6 transected controls). After 40 days, the transposed ileal segment, compared to the equivalent segment in the controls, showed striking increase in weight, especially of the epithelium (1,585 +/- 127 vs. 305 +/- 42 mg, mean +/- SEM, p less than 0.0005). The calculated weight of enteroglucagon cells in the segment showed a smaller, but significant increase (1.7 +/- 0.3 vs. 0.8 +/- 0.2 micrograms, p less than 0.05). Plasma enteroglucagon was markedly raised (239 +/- 28 vs. 61 +/- 7.1 pmol/l, p less than 0.0005) and showed a greatly increased meal-stimulated response (1,521 +/- 284 vs. 83 +/- 43 pmol, p less than 0.0005), thus suggesting hyperactivity of enteroglucagon cells.

The stoichiometry of the two heavy chains of myosin in smooth muscle was determined by electrophoresing extracts of native myosin and of dissociated myosin on sodium dodecyl sulfate (SDS) 4%-polyacrylamide gels. The slower migrating heavy chain was 3.6 times more abundant in toad stomach, 2.3 in rabbit myometrium, 2.0 in rat femoral artery, 1.3 in guinea pig ileum, 0.93 in pig trachea and 0.69 in human bronchus, than the more rapidly migrating chain. Both heavy chains were identified as smooth muscle myosin by immunoblotting using antibodies to smooth muscle and non-muscle myosin. The unequal proportion of heavy chains suggested the possibility of native isoforms of myosin comprised of heavy-chain homodimers. To test this, native myosin extracts wer electrophoresed on non-dissociating (pyrophosphate) gels. When each band was individually analysed on SDS-polyacrylamide gel the slowest was found to be filamin and the other bands were myosin in which the relative proportion of the heavy chains was unchanged from that found in the original tissue extracts. Since this is incompatible with either a heterodimeric or a homodimeric arrangement it suggests that pyrophosphate gel electrophoresis is incapable of separating putative isoforms of native myosin.

The interaction of VIP (binding to specific receptors and stimulation of cyclic AMP accumulation) with ileal epithelial cells and the levels of the neuropeptide in the ileal segment were determined after colectomy (removal of cecum and colon followed by ileorectal anastomosis) in the rat. The number of VIP receptors (but not the affinity) and the efficiency (but not the potency) of the neuropeptide upon stimulation of cyclic AMP accumulation in ileal epithelial cells increased 21 (but not 7) days after colectomy, whereas VIP ileal levels followed an inverse pattern. These changes could be interpreted in terms of a consequence or a cause of some of the phenomena that appear after colectomy, i.e., chronic watery diarrhea.

Enzyme-linked immunosorbent assays (ELISAs) with antibodies specific to either cholera enterotoxin (CT) of Vibrio cholerae or heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli were developed to detect LT and CT, respectively. With these ELISA systems, LT and CT could be detected only with the respective specific antibody. Both antibody ELISA and ganglioside ELISA were used for differential detection of LT and CT, but the former method seemed to be more specific. By this ELISA, as little as 0.1 ng of purified LT or CT could be detected per ml. The type of toxins in fluids in intestinal loops of experimental animals challenged with living cells of either V. cholerae or LT-producing E. coli was identified correctly by this ELISA. These results suggest that the ELISA systems reported here could be used to detect and differentiate CT and LT in unknown samples; it could also be used for assaying toxins in stool specimens for the diagnosis of diarrhea due to V. cholerae or LT-producing E. coli directly, without or before bacterial isolation.

The indirect immunoperoxidase method was used to describe time of appearance, morphology and topography of 5-hydroxy- tryptamine-like immunoreactive (5-HT-LI) cells in the gut of chick embryos, newborn and adult chickens. The earliest cells were seen in the ileum at 11 days, in the caeca at 14 and in the colon at 9 days. At first appearance they were ovoid or pyramidal but later became more irregular because of the numerous apical and basal processes. The peak of cell concentration at hatching, was in the ileal samples, whereas in the colon these cells were also abundant in adults both throughout the villi and the glands. In sections of adult ileum, on the contrary, they could be found mainly in the glands.

The excitatory amino acids L-glutamate and N-methyl-D-aspartate (NMDA) produced contractions of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum over the concentration range of 3 X 10(-6) to 10(-3) M. The contractile response to L-glutamate and NMDA, but not carbamyl choline, was blocked noncompetitively by 0.6 mM Mg++. In the absence of Mg++, concentration-dependent increases in contractile force also were produced by, in order of potency, L-aspartate, L-homocysteate and D-glutamate, but not by quisqualate, kainate or quinolinate. L-Glutamate was competitively antagonized by the selective NMDA receptor antagonists D-2-amino-5-phosphonovalerate and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (3 X 10(-6)-3 X 10(-5) M), as well as by the nonselective excitatory amino acid antagonist gamma-D-glutamylglycine (3 X 10(-4) M). Glutamic acid diethyl ester (3 X 10(-4) M) noncompetitively antagonized L-glutamate. L-Glutamate was not blocked by gamma-D-glutamylaminomethyl sulphonate (3 X 10(-4) M), an antagonist which preferentially antagonizes kainate and quisqualate. In addition, the phencyclidine-like drugs etoxadrol (10(-7)-10(-5) M), dextromethorphan (10(-6)-10(-5) M) and 5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine (10(-9)-10(-7) M) noncompetitively antagonized L-glutamate. The (+) isomer of 5-methyl-10, 11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine was approximately 10-fold more potent than the (-) isomer in antagonizing L-glutamate. The present results demonstrate that receptors for the excitatory amino acid L-glutamate are present in the guinea pig myenteric plexus and are of the NMDA subtype.(ABSTRACT TRUNCATED AT 250 WORDS)

The bioactive fraction of "enteroglucagon" has been isolated from the porcine jejuno-ileum on the basis of its glucagon-like action in liver. High performance liquid chromatography analysis followed by Dansylation of peptide fragments obtained after enzymatic digestion of both this peptide and glucagon suggests that the bioactive "enteroglucagon" contains the glucagon molecule plus a C-terminal extension under the from of the octapeptide. Lys-Arg-Asn-Lys-Asn-Asn-Ile-Ala-COOH. The presence in the native molecular of a structural difference of modification as compared to glucagon in the 1-11 fragment is possible.

The synthesis of beta h-endorphin -(1--15), -(1--21), -(1--26), -(1--28) and -(1--29) have been accomplished by the solid-phase method. The opiate activities of the peptides were measured in the guinea pig ileum assay. Their relative potencies were beta h-endorphin (beta h-EP), 1.00; Met-enkephalin, 0.25: beta h-EP-(1--15), 0.17; beta H-EP-(1--21), 0.46; beta H-EP-(1-26), 0.48; beta h-EP-(1--28), 0.89; beta h-EP-(1--29), 1.04.

Collagen content of small intestinal anastomosis in the rat was investigated in relation to previously reported changes in suture-holding capacity. The decrease in suture-holding capacity during the first 3 days previously observed, the inflammatory phase, did not correlate with collagen content during this period. However, the increase in suture-holding capacity from the third to the seventh days, the phase of fibroplasia, correlated well with an increased collagen content in the intestinal wall in both the ileum and jejunum. Anastomotic strength and collagen content correlated well after the third day. Deposition of collagen, not only in the wound but also in the intestinal wall and within the loop of the suture during the phase of fibroplasia, is the likely explanation for the rapid increase of suture-holding capacity during this period.

A bioactive form of enteroglucagon has been isolated from porcine jejuno-ileum according to its glucagon-like effect in liver. Enzymatic digestion followed by HPLC, dansylation and partial sequence analysis strongly suggests that this peptide contains the glucagon molecule (1--29) elongated at the C-terminal end by the octapeptide Lys-Arg-Asn-Lys-Asn-Ile-Ala-COOH and possibly modified in the N-terminal region. A specific action of bioactive enteroglucagon, increase in cAMP, has been found in the acid-secreting fundic part of the rat stomach. The term "oxyntomodulin" is therefore proposed to describe this peptide.

Bile acids are supposed to promote colonic cancer. In Crohn's disease, colonic carcinomas are relatively rare. We, therefore, compared ileal and right colonic mucosal bile acids analysed by gas-liquid chromatography in 8 patients with ileal Crohn's disease (14-48 yrs.) and 7 patients with right colonic carcinoma (28-77 yrs.) who underwent surgery. In both ileal and colonic mucosa, nonsulphated bile acid concentrations were somewhat higher in Crohn's disease (20.98 micrograms/g +/- 4.77 SEM; 12.09 micrograms/g +/- 2.55) than in colonic carcinoma (16.06 micrograms/g +/- 3.46; 7.75 micrograms/g +/- 4.28). In ileal mucosa, percentages of lithocholic and deoxycholic acids were slightly higher in colonic carcinoma (3.9%; 23.2%) than in Crohn's disease (1.1%; 14.9%). In colonic mucosa, carcinoma patients had more lithocholic (7.6%) and less deoxycholic acid (11.9%) than patients with Crohn's disease (1.7%; 20.3%). Bile acid sulphate esters were similar in both diseases (ca. 3.0 micrograms/g in ileal, 1.4 micrograms/g in colonic mucosa). Our results show that ileal and right colonic mucosal nonsulphated bile acids tend to be even lower in right colonic carcinoma than in Crohn's disease. This agrees well with our earlier findings of low mucosal bile acid concentrations in patients with left colonic carcinoma (Tokai J Exp Clin Med 8: 59-69, 1983) and does not support the assumption that bile acids are envolved in right colonic carcinogenesis.

Adrenergic, cholinergic, and serotoninergic nerves were studied in the myenteric plexus of ileum and colon from streptozotocin-treated rats, an animal model of juvenile-onset diabetes. In view of clinical reports implicating diabetic autonomic neuropathy as the cause of gastrointestinal dysfunction in diabetes mellitus, neurochemical and histochemical techniques were used to study changes in the innervation of the gut. In the myenteric plexus of the ileum from diabetic animals, adrenergic nerves displayed signs of degeneration and the brightness of fluorescence in serotoninlike immunoreactive nerves was lower. Cholinergic nerves, however, did not display any signs of reduction in the ileum, and both choline acetyltransferase and acetylcholinesterase activities per centimeter were increased. In contrast, in the proximal colon 8 wk after induction of diabetes, neurochemical assays revealed significant increases in noradrenaline and serotonin levels as well as choline acetyltransferase activity, although no obvious changes in the pattern of innervation could be detected histochemically. The results indicate that changes do occur in the innervation of the gut of the streptozotocin-diabetic model shortly after the induction of diabetes, although they differ significantly in the ileum and colon; these may be of relevance to the types of gastrointestinal dysfunction displayed in human diabetes.

1 Muscarinic receptors from the brain of rat, guinea-pig and frog have been labelled with tritiated propylbenzilylcholine mustard ([3H]-PrBCM). 2 After solubilisation and sodium dodecyl sulphate (SDS)-polycarylamide gel electrophoresis a single radiolabelled peak was seen which was completely suppressed when labelling was carried out in the presence of 10(-6) M atropine. The results suggest the presence of a single polypeptide chain with a molecular weight of similar to or approximately 80,000. 3 In the case of intestinal smooth muscle multiple labelled peaks were found. Precautions against proteolysis led to the identification of a major peak with a molecular weight in the region of 80,000.

Based on the assumption that somatostatin may inhibit peptide release through junctional complexes or through local circulation, an immunfluorescent technique for somatostatin and GLI in the gut was applied in order to investigate whether suppression of GLI release by i.v. administration of somatostatin was a physiological effect of somatostatin or not. Somatostatin-immunoreactive cells (GIF-cells) in the human and canine intestine had no direct cellular contacts with GLI-immunoreactive cells (GLI-cells). This finding suggests that somatostatin in the intestine does not inhibit GLI release through junctional complexes between GIF- and GLI-cells. As to the local circulation, most of GIF-cells in the canine intestine were distributed in the deeper portion of the intestinal gland which corresponds to the upstream sides of the local blood supply of the intestinal gland, as reported by REYNOLD et al. The ratio of GIF-cells to total cells (GIF-cells + GLI-cells) was 68% in the duodenum and 25% in the ileum. In contrast a limited number of GIF-cells was found in the human duodenum where a few GLI-cells were distributed and a few GIF-cells were seen in the human ileum where a large number of GLI-cells were located. Findings in the dog suggest the possibility that somatostatin inhibits GLI release from GLI-cells through the local circulation system of intestinal glands. However, findings in humans suggest that the same possibility does not apply to the human gut. Differences of population density of intestinal GIF-cells between humans and dogs indicate that the functional meaning of GIF-cells may vary from one species to another.

1. In intestinal mucosal tissue a transferrin-like immunoreactivity (TLIR) was determined after loading with 59Fe-(FeCl3) in vivo. The scraped-off mucosal tissue was homogenized and centrifuged at 100 000 X g. The supernatant was fractionated chromatographically. The TLIR was determined by the Mancini-test. 2. Extrapolated to infinite dilutions of the homogenate the following contents of the TLIR were calculated per cm of the intestinal segment: 28, 20, and 13, microgram/cm in the duodenum, jejunum, and ileum of iron-deficient animals and 15, 14, and 12 microgram/cm in the corresponding intestinal segments of normal rats, respectively. 3. The content of TLIR was compared with the iron uptake of the mucosal tissue from the intestinal lumen during absorption. An "iron turnover number" of 1.6 +/- 0.2 iron atoms per min during the absorption in duodenal and jejunal segments and of 0.5 +/- 0.1 in ileal segments of either iron-deficient and normal animals was calculated for the mucosal transferrin represented by the TLIR.

Peptide-YY (PYY) is a novel enteric peptide that is structurally related to pancreatic polypeptide and neuropeptide-Y. The objectives of the present experiments were to characterize the following aspects of PYY metabolism: the distribution of PYY in the canine gastrointestinal tract, the release of PYY in response to oral ingestion of a mixed meal or intraduodenal (ID) administration of oleic acid, the effect of ileocolectomy on the release of PYY in response to ID administration of oleic acid when transit of chyme to the distal ileum and colon is prevented, the effect of interruption of intramural neural pathways of the small bowel on the release of PYY, and the effect of iv cholecystokinin on the release of PYY. The results of these experiments demonstrate that PYY immunoreactivity is distributed primarily in the terminal ileum, colon, and rectum. Circulating levels of PYY increase significantly (P less than 0.05) within 10-30 min after ingestion of a meal or to ID administration of a fatty acid. Complete interruption of the flow of chyme to the site of PYY-containing cells (i.e. ileum-colon) did not block the release of PYY; however, ileocolectomy abolished the release of PYY in response to ID administration of oleic acid. Severance of intramural neural pathways along the small bowel did not alter the release of PYY in response to an oral meal. Intravenous administration of graded doses of cholecystokinin stimulated the release of PYY in a dose-related manner. The results of these experiments indicate that the release of PYY from the distal ileum and colon is controlled, at least in part, by an extramural neural, endocrine, or a combination of both types of mechanisms which originate in the foregut.

Like many other neuropeptides, vasopressin is not confined to the hypothalamic neurohypophysial system. Furthermore, vasopressin was found to be a potent vasoconstrictor in the rat jejunum, reducing myenteric artery flow. These associations were the basis of this investigation on the presence of vasopressin in the gastrointestinal (GI) tract by both RIA and immunohistochemistry. Portions of the gastrointestinal tract and pancreatic islets of the rat were extracted with 0.1 N HCl for RIA measurements of AVP content. Similar portions from the male cat GI tract were used for immunohistochemistry studies. Acid extracts of the GI tract were found to contain immunoreactive AVP with the highest concentration (pg/mg protein) in the fundus portion of the stomach (15.0 +/- 1.6) and slightly lower values down along the antrum-pylorus portion (6.7 +/- 0.6), proximal jejunum (8.6 +/- 0.2), distal ileum (9.7 +/- 0.3) and colon (11.9 +/- 0.5). In the pancreatic islets the concentration was much higher (72.0 pg/mg protein). The extract inhibition curves showed parallelism with the appropriate standard preparation of AVP in the specific RIA. Immunohistochemical localization showed IR-AVP in the nerve fibers around the myenteric plexus of the second portion of the duodenum. It was also found in fibers starting from where the myenteric plexus goes through the layer of muscle fibers, penetrating the submucosa and duodenal mucosa, ending near the capillaries situated along the basal side of the villous epithelium cells. Similar IR-AVP activity was found in cells located in the mucosal epithelium of the duodenum, jejunum, ileum, colon and rectum.(ABSTRACT TRUNCATED AT 250 WORDS)

The alpha 2-adrenergic receptors in rabbit ileal mucosal membranes can be identified by using [3H]clonidine. [3H]Clonidine bound to a homogeneous population of sites (30-120 fmoles/mg protein) with a KD of 2.2 nM at 25 degrees. Alpha-adrenergic agonists and antagonists competed with [3H] clonidine for the binding sites with an order of potency typical for alpha 2-receptors. Mg2+, Ca2+, or Mn2+ (2.4 mM) markedly increased the binding of [3H]clonidine. At the maximally effective concentration, Mg2+ increased both the binding affinity of [3H]clonidine and the number of receptor sites. Both NaCl and GppNHp, the guanyl nucleotide, inhibited [3H]clonidine binding. NaCl decreased the binding affinity of [3H]clonidine, with no appreciable effect on the number of receptor sites. These findings indicate that ileal mucosal alpha 2-receptors can exist in multiple affinity states, which can be regulated by divalent cations, NaCl, and guanyl nucleotides. It appears that NaCl and GppNHp regulate alpha 2-receptors in ileal mucosa by different mechanisms.

Ligatin, a receptor that recognizes phosphorylated sugars, was isolated from plasma membranes of mouse macrophages, rat ileum, and rat brain. Several acidic hydrolases including N-acetyl beta-D-glucosaminidase (beta-NAG) were solubilized with this receptor. The solubilized beta-NAG bound to ligatin in vitro as demonstrated by affinity chromatography using the immobilized receptor. beta-N-Acetyl D-glucosaminidase-ligatin complexes were dissociated by low concentrations of mannose 6-phosphate (Man6P) and/or glucose 1-phosphate (Glc 1P). The effectiveness of these two phosphomonosaccharides varied depending on the source of the enzyme: ileal beta-NAG-ligatin complexes showed a four-fold preferential dissociation with Man6P; macrophage complexes showed a 160-fold preferential dissociation with Glc 1P. Brain complexes dissociated with nearly equal preference for Man6P and Glc 1P. Heterologous complexes displayed the specificity characteristic of the source of the enzyme regardless of the source of the ligatin. Treatment of the solubilized hydrolases with endoglucosaminidase H released phosphorous-32 label from these enzymes and prevented binding of beta-NAG to ligatin. However, treatment of the solubilized hydrolases with alkaline phosphatase reduced the binding of beta-NAG to ligatin by no more than 30%. This apparent resistance of beta-NAG to dephosphorylation was consistent with the chromatographic behavior of QAE of 3H-labeled acidic oligosaccharides isolated from the solubilized hydrolases. The oligosaccharides that contain phosphorylated hexose were less acidic than phosphomonoesters and were insensitive to alkaline phosphatase until subjected to acid hydrolysis. These results suggested the presence of a phosphodiester on beta-NAG analogous to the NAC glucosamine 1 P6 mannose present on beta-glucuronidase isolated from mouse lymphoma cells (Tabas I, Kornfield, S: J Biol Chem 255: 6633, 1980).

The effects of chloroquine diphosphate, a drug with "'membrane-stabilizing" properties, were studied on basal ileal absorption and on ileal secretion induced by increased intracellular cAMP levels and calcium (serotonin). The studies were performed on rat (in vivo) and rabbit ileum (in vitro). Intraluminal chloroquine (10(-4) M) reversed cholera toxin- and theophylline-induced secretion in rat ileum but did not alter the cholera toxin- and theophylline-induced increases in cAMP content. Addition of chloroquine (10(-4) M) to the mucosal surface of rabbit ileum did not alter basal active electrolyte transport or the serotonin-induced decreased Na and Cl absorption but inhibited the theophylline-induced C1 secretion. Addition of chloroquine (10(-4)) M) to the serosal surface stimulated net Na and Cl absorption. This effect may involve intracellular calcium. Chloroquine increased the rabbit ileal calcium content and decreased 45Ca2+ influx from the serosal surface. Both the mucosal and serosal effects of chloroquine described led to a net increase in absorptive function of the intestine and should prove useful in developing treatment of diarrheal diseases.

Using immune electron microscopy, an attempt has been made to visualise viral particles in Crohn's disease tissue and faeces. No particles resembling viruses were observed, but in the absence of a specific antiserum a negative result does not exclude the presence of a virus, particularly if it is sparsely distributed. Compared with controls, an abundance of a 12 nm particle was found in all Crohn's disease tissue and one small intestinal lymphoma. This particle has been identified as ferritin and is unlikely to be of aetiological significance.

Controversy exists regarding the mechanism and degree of mucosal alterations occurring in defunctionalized segments of intestine. This study compares the results of a quantitative analysis of mucosal components, including Paneth cells and immunocytes, between in-continuity and defunctionalized (Thiry-Vella) segments of rat ileum. The micrometer component quantitator was used for the light microscopic morphometric analysis. Intracellular lysozyme and IgA were identified employing the unlabeled antibody enzyme immunohistochemical staining technique. The vol% of the ileal mucosal components of animals from the control group and from the in-continuity segments of the experimental group were comparable. Analysis of the Thiry-Vella fistulae, however, revealed a statistically significant decrease in the vol% of columnar epithelial cells and increase in the vol% of lysozyme-containing Paneth cells and interstitium of the lamina propria. Since Thiry-Vella fistulae are neurovascularly intact, mucosal alterations imply a causal relationship to interaction with chyme. The data suggest that chyme has both a stimulatory (on the columnar epithelium) and suppressive (on the the Paneth cell population) effect. The vol% of IgA-containing Paneth cells and the percentage of the lamina propria represented by IgA-containing immunocytes were also substantially decreased. Normally secretory IgA is the immunoglobulin of highest concentration intraluminally and among immunocytes within the lamina propria, presumably in response to local antigenic stimulation. The presence of immunoglobulin within Paneth cells may reflect the phagocytosis of immunoglobulin complexed antigens. The data suggest that the degree of local antigenic stimulation is decreased in Thiry-Vella fistulae.

Recent studies have shown that during its biosynthesis in bovine adrenal medulla, the opioid precursor proenkephalin A, may be both N-glycosylated and phosphorylated. To investigate whether these chemical modifications were common to proenkephalin A processing in other tissues, we have sought to characterize enkephalin-containing peptides from bovine adrenal medulla, spinal cord and ileum. The peptides were identified using antiserum L189, specific for the C-terminus of Met-enkephalin Arg6Gly7Leu8 (MERGL), and L152, specific for the C-terminus of Met-enkephalin Arg6Phe7 (MERF). Glycosylated MERGL-immunoreactive peptides of 23, 20, 16 and 13 kDa were identified in adrenal medulla using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and concanavalin A-Sepharose affinity chromatography. Sephadex G50 gel filtration fractionated the glycosylated peptides into two immunoreactive peaks. Similar peaks of concanavalin A-binding MERGL immunoreactivity were detected in extracts of spinal cord and ileum, although there were differences in relative proportions of the two peaks. Antiserum L152 identified phosphorylated N-terminally extended variants of MERF when boiling water extracts of adrenal medulla, spinal cord and ileum were separated by anion exchange chromatography. In adrenal medulla these peptides were more than 99% phosphorylated, whereas in both ileum and spinal cord there was a relatively higher proportion of the unphosphorylated peptide. The results indicate that N-glycosylation and phosphorylation of proenkephalin A occurs in adrenal medulla, spinal cord and ileum, although there are tissue-specific differences in the relative proportions of the modified and unmodified peptides.

To compare Staphylococcus aureus delta-toxin with cholera toxin, which is known to increase cellular cyclic adenosine 3',5'-monophosphate (cAMP), studies were undertaken to determine the effect of delta-toxin on the cAMP content of guinea pig ileum maintained in vitro. Concentrations of delta-toxin as low as 0.40 mug/ml increased cAMP levels in guinea pig ileum after 2 h of incubation. Histological damage was seen in ileum exposed for 2 h to delta-toxin concentrations of 100 mug/ml. As little as 3 mug of delta-toxin increased vascular permeability in guinea pig skin. Permeability changes became evident within 5 min and were maximal within 6 h, whereas those produced by cholera toxin required 24 h to become maximal. Benadryl did not interfere with the ability of these toxins to alter vascular permeability. Purified egg lecithin reduced the effectiveness of delta-toxin in the skin but did not inhibit cholera toxin. Delta-toxin in concentrations as low as 0.1 mug/ml caused dislodgement of HeLa cells in tissue cultures. Therefore, delta-toxin appears unique in being the only bacterial toxin, currently known to alter water absorption in the ileum, that is capable of both increasing cAMP levels and being cytotoxic. These findings suggest a possible role for delta-toxin in the pathogenesis of staphylococcal enteritis.

Delta sleep-inducing peptide-like immunoreactivity (DSIP-LI) has previously been demonstrated in brain neurons and in endocrine cells of the pituitary and the adrenal medulla. By means of three different antisera against synthetic DSIP we now describe the occurrence and distribution of DSIP-LI in several gut endocrine cells. The human gut was the richest source, where DSIP-LI was located in gastrin/CCK, secretin and PYY/glicentin cells. The rat and pig gut harbour a moderate number of immunoreactive cells in the antral mucosa but in the intestines DSIP-LI-containing cells were very few. By radioimmunoassay, the concentration of DSIP-LI was determined in extracts of various gut regions from man, pig and rat. The highest concentrations were found in all human specimens compared with corresponding samples in the pig and rat. In all three species, high-performance liquid chromatography revealed a single peak of DSIP-like material with approximately the same retention time as DSIP 3-9. Taken together, the present results provide evidence for the presence of DSIP-LI in gut endocrine cells in man, pig and rat; the human gut seems to be the richest source of DSIP-like peptides.

Immunohistochemical studies using an antiserum to a pure porcine vasoactive intestinal peptide, possessing no cross reactivity against the related hormones glucagon, secretin, and gastrin-inhibitory peptide, revealed a wide distribution of vasoactive intestinal peptide cells throughout the entire length of the mammalian and avian gut. The highest numbers of cells were present in the small intestine and more particularly in the large intestine in all species investigated. Three types of endocrine cell in the mammalian gut are sufficiently widely distributed to be considered as the sites for production of vasoactive intestinal peptide. In the avian gut there are only two identifiable cell types. Sequential immunofluorescence and silver staining showed, in the bird, that the enterochromaffin (EC) cell was not responsible. This procedure could not be used in our mammalian gut samples but here serial section immunofluorescence for enteroglucagon and vasoactive intestinal peptide indicated that the two cells were not identical and that each was differently localized in the mucosa. These results leave the D cell of the Wiesbaden classification as the most likely site for the production of vasoactive intestinal peptide. The final identification must come from successful immune electron cytochemistry but this has not yet been achieved.

Neurotensin is a 13-amino acid peptide that is widely distributed in central and peripheral tissues of various mammalian species. In peripheral tissues, the highest concentration of neurotensin-like immunoreactivity is found in the ileum, where it is present in endocrine-like cells and nerve fibres. The longitudinal smooth muscle layer of the guinea pig ileum, where neurotensin has both a direct relaxant and an indirect contractile action, has been used extensively as a biological assay system for neurotensin. We report here that the majority of specific 3H-neurotensin binding sites is present in the guinea pig ileum circular smooth muscle layer, which is known to be innervated by a large proportion of the ileal non-adrenergic inhibitory nerves. Neurotensin produces a dose-dependent, tetrodotoxin-resistant relaxation, whereas the relaxation produced by field stimulation of the inhibitory nerves is frequency-dependent and tetrodotoxin-sensitive. The calcium-dependent potassium channel blocker apamin inhibits both the neurotensin- and nerve stimulation-induced muscle relaxation. Incubation of the circular smooth muscle preparation with a neurotensin antiserum substantially inhibited the nerve stimulation-induced relaxation, indicating a direct relationship between the effects of neurotensin and of nerve stimulation.