Histamine H1-receptor in heart: unique electrophoretic mobility and autoradiographic localization.

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.

Cell differentiation in intestinal adenomatosis of pigs studied by histochemistry of laminin and enzymes of epithelial and subepithelial tissue.

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.

Expression of J1/tenascin in the crypt-villus unit of adult mouse small intestine: implications for its role in epithelial cell shedding.

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, neurotensin6-13 and neuromedin N: primary structures and receptor activity.

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.

Identification and characterization of N-glycosylated and phosphorylated variants of proenkephalin A-derived peptides in bovine adrenal medulla, spinal cord and ileum.

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.

Glutamate receptors of the N-methyl-D-aspartate subtype in the myenteric plexus of the guinea pig ileum.

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)

Isolation of glycoprotein D from herpes simplex virus type 1 by gel filtration high performance liquid chromatography.

Rabbit kidney (RK-13) and human jejunum and ileum (I-407) cells infected with herpes simplex virus type 1, strain F, were radiolabelled with [14C]glucosamine or [35S]methionine for 24 h. The cells were extracted with 1% Triton X-100 and the extracts were separated by gel filtration high performance liquid chromatography. Monoclonal antibody immunoprecipitation of the fractions collected from the column revealed a monomeric glycoprotein D (gD) of 52 - 56,000 molecular weight from RK-13 cells and two monomeric forms of gD, 54,000 and 58,000 molecular weight, from I-407 cells. Densitometry scanning of the autoradiograms from SDS-PAGE showed gD from the RK-13 host cells to be 98.7% pure with the [35S]methionine label and 97.0% pure with the [14C]glucosamine. On the other hand, gD from the I-407 host cells was only 78.6% with the [35S]methionine label and 96% pure with the [14C]glucosamine. This method could provide a means for the isolation of native gD for structural and immunological studies.

Tyrosine hydroxylase activity in sympathetic nervous system of rats with streptozocin-induced diabetes.

The activity of tyrosine hydroxylase (TOH), the rate-limiting enzyme in norepinephrine biosynthesis, was measured in selected sympathetic ganglia to develop a quantitative measure of sympathetic autonomic neuropathy in streptozocin-induced diabetic rats. Surprisingly, TOH activity was elevated twofold in diabetic prevertebral ganglia innervating the alimentary tract (i.e., superior mesenteric, celiac, and inferior mesenteric), which has terminal processes that develop neuroaxonal dystrophy in this model system. TOH activity of paravertebral ganglia (i.e., superior cervical and stellate) with nonalimentary targets was not increased in the same animals. Increased TOH activity in the prevertebral ganglia 1) developed within the 1st wk of diabetes and persisted for 10 mo, 2) did not represent a change in TOH affinity for d-1,6-methyl-5,6,7,8- tetrahydropterine cofactor, 3) was prevented by both nicotinamide pretreatment and early institution of insulin therapy, and 4) did not develop as a result of classical transsynaptic induction. Pair-feeding experiments confirmed that the most likely cause of increased TOH activity in this system was the marked hypertrophy and hyperplasia of the diabetic bowel resulting from compensatory hyperphagia. We conclude that TOH activity does not represent a suitable marker for sympathetic autonomic neuropathy in this experimental system. Rather, the increase appears to be an example of a selective increase in the synthesis of neurotransmitter enzymes, possibly in response to increased trophic support provided by the expanded target, i.e., the hypertrophic gut. The additional synthetic stress imposed on prevertebral neurons by the expansion of the innervation of the alimentary target coupled with the complex diabetic metabolic milieu may contribute to the development and selective distribution of dystrophic axonopathy to the innervation of the alimentary tract.

The histochemical specificity of high iron diamine-alcian blue.

The specificity of the High Iron Diamine-Alcian Blue pH 2.5 (HID-AB 2.5) procedure was examined in tissue sites containing sialogycoproteins alone or differing proportions of sialo- and sulphosialoglycoproteins. Studies with HID in differing final concentrations of hydrochloric acid or sodium chloride confirmed that staining is dependent upon both the pH and the ionic strength of the dye bath and demonstrated a marked heterogeneity in the pKa of the anionic groups of sialosulphoglycoproteins. Use of the sequence High Iron Diamine-Alcian Blue pH 1.0 demonstrated that complete or almost complete staining of O-sulphate esters occurred when HID was prepared in water (final pH 1.3). However, under these conditions HID-AB 2.5 was shown to be non-specific because only black HID staining was observed in sites containing large quantities of sialic acids. This non-specificity was due either to the masking of Alcian Blue staining by HID and/or the black HID staining of anionic groups other than sulphate. These results may account for some of the conflicting data obtained by different groups of investigators who have studied 'transitional mucosa' in human colonic diseases. Caution should be used in drawing conclusions from the use of HID-AB 2.5 without confirmatory evidence from other more specific procedures.

EGF- and TGF-alpha-like peptides in human fetal gut.

The presence of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) immunoreactivities in fetal human tissues was studied immunohistochemically at different gestational ages. EGF and TGF alpha immunoreactivities were detected from the 20th gestational wk. EGF immunoreactivity was limited to the small intestine, but TGF alpha immunoreactive cells were present in the colon also. According to radioreceptor assay, the intestine of a 19-wk-old human fetus contained 10 times more EGF receptor-binding substance than EGF, as measured by immunofluorometric assay. Chromatographic analysis suggests that TGF alpha-like peptides account for at least part of this activity, as so argues in favor of the presence of TGF alpha- and EGF-like peptides in the human fetal gut. Whether they are synthesized in the fetus is yet unknown.

Meal-induced peptide tyrosine tyrosine inhibition of pancreatic secretion in the rat.

In the present studies we examined the distribution, release, and biological actions of peptide tyrosine tyrosine (PYY) in the rat. The concentration and distribution of PYY was highest in the ileum and colon as determined by both radioimmunoassay of rat tissue extracts and immunocytochemistry. An ultrastructural comparison of rat and dog colonic PYY cells revealed a bipolar distribution of peptide-containing secretory granules in both species. Serum PYY and pancreatic exocrine secretory responses were monitored after presentation of a meal to meal-trained rats (n = 12). A significant increase in PYY concentrations was not observed until 120 min after meal presentation, a delayed response similar to that previously observed in the dog. PYY responses were also observed in rats after perfusion of the intestine at the level of the duodenum and ileum with an 80 mOsm micellar solution of sodium oleate. Duodenal instillations of the fatty acids resulted in a maximum PYY response after 120 min, whereas rats subject to ileal perfusion of fat exhibited maximum PYY release within the first hour. In other experiments, infusion of exogenous PYY at 100 pmol.kg-1.h-1, which reproduced plasma PYY levels observed after a meal and perfusion of the gut with fat, significantly inhibited CCK-stimulated bile pancreatic volume (P less than 0.02), protein (P less than 0.01), and amylase (P less than 0.01) output. These studies demonstrate a bipolar distribution of PYY-containing secretory granules in cells of the jejunal, ileal, and colonic mucosa, and show that PYY is released in response to a meal in amounts sufficient to inhibit cholecystokinin-stimulated pancreatic secretion. Evidence is presented that PYY may mediate the delayed inhibition of pancreatic secretion that is observed in the rat after ingestion of a meal.

Pigment resembling atmospheric dust in Peyer's patches.

Terminal ileal pigmentation was observed during colonoscopy, in surgically resected specimens, and autopsy cases. Microscopically, black pigment was seen within macrophages in the lamina propria and submucosa, closely related to the Peyer's patches. Three ilia from autopsies with no macroscopic pigmentation showed deposits following digestion and X-ray microanalysis. X-ray microanalysis of tissue sections and digestates revealed a heterogenous population of particles. Approximately one third of the particles contained calcium and phosphorus and were considered endogenous. The rest of the particles were predominantly aluminum and magnesium-rich silicates, which were considered exogenous. Analysis of particulate extracted from lungs and ilea of four autopsy cases demonstrated remarkable similarities in composition. These findings suggest that the ileal deposits are derived from atmospheric dust. This pigment is believed to migrate into the Peyer's patches through the M cells of the follicle associated epithelium, although other mechanisms for pigment deposition cannot be ruled out.

Measurement and chromatographic characterization of prodynorphin-derived peptides in the guinea-pig ileum.

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.

Evidence for regulation of peptide-YY release by the proximal gut.

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.

The effect of feeding different protein-free diets on the recovery and amino acid composition of endogenous protein collected from the distal ileum and feces in pigs.

Nine barrows with an average initial weight of 60 kg were fitted with simple T-cannulas at the distal ileum. The animals were fed four different protein-free diets according to an incomplete latin square design. Diet 1 (control diet) consisted of 79.7% cornstarch, 10% sucrose, 3% Alphafloc (a source of cellulose), 3% canola oil and a vitamin-mineral premix. Diets 2, 3 and 4 contained, respectively, 4% pectin, an additional 7% cellulose and an additional 10% canola oil, each included at the expense of cornstarch. Feces were collected during 3 d following a 7-d adaptation period. Thereafter, digesta were collected during two 24-h periods with a 24-interval between periods. The pigs were fed 800 g of feed twice at 0800 and 2000. Added pectin increased (P less than .05) the recovery of endogenous protein in ileal digesta from 19.8 diet (diet 1) to 24.0 g per kg dry matter intake. This increment was largely due to increases (P less than .05) in glycine and proline from 1.9 to 2.4 and from 6.2 to 8.4 g per kg dry matter intake, respectively. In feces, only added cellulose increased (P less than .05) excretion of endogenous protein (8.4 vs 11.1 g/kg DM intake) and of most amino acids. Including additional fat did not affect the quantity of endogenous protein and amino acids recovered in ileal digesta or feces. Small but significant differences (P less than .05) were observed in the amino acid composition of endogenous protein recovered in ileal digesta when the different protein-free diets were fed.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of protein status of the pig on the recovery and amino acid composition of endogenous protein in digesta collected from the distal ileum.

Barrows with an average initial weight of 55 kg were fitted with simple T-cannulas at the distal ileum. The animals were fed a protein-free diet that consisted of 79.7% cornstarch, 10% sucrose, 3% Alphafloc (a source of cellulose), 3% canola oil and a vitamin-mineral premix. The pigs were fed 700 g of diet twice each day, at 0800 and 2000. A balanced amino acid mixture or a saline solution was administered intravenously while the protein-free diet was fed. Ileal digesta were collected for 24 h following a 7-d adaptation period. The administration of amino acids reduced (P less than .05) the recovery of endogenous protein from 18.5 to 12.7 g per kg dry matter intake. For the amino acids, the reduction was only significant (P less than .05) for proline, from 3.6 to .6 g per kg dry matter intake. If the total endogenous protein losses are assumed to be constant and the differences in the amino acid composition of non-reabsorbed endogenous protein, as observed in this study, are used to calculate true ileal digestibilities, differences in the digestibilities of the indispensable amino acids are large (up to 7.4 percentage units for threonine). The amino acid composition of endogenous protein determined in pigs fed a protein-free diet and parenterally administered with amino acids should provide a better estimate for the calculation of true amino acid digestibilities when based on the determination of true protein digestibility by the 15N-isotope dilution technique.

Distribution and molecular forms of glucagon-like peptide in the dog.

Using glucagon-like peptide-1 N-terminus and C-terminus directed antisera, we investigated concentration and molecular forms of GLP-1 immunoreactivity (IR) in extracts of various tissues of the dog. GLP-1 IR measured with C-terminus-directed antiserum R2337 (GLP-1 IR-CT) was high in the ileum, appendix, jejunum, colon, and gastric fundus and body. GLP-1 IR measured with N-terminus-directed antiserum R1043 (GLP-1 IR-NT) was high only in the pancreas, and gastric fundus and body. Only GLP-1 IR-CT was found in the hypothalamus, thalamus and medulla oblongata. No immunoreactive materials were detected in the liver, spleen and kidney. Gel-filtration with Sephadex G-50 showed two peaks of both GLP-1 IR-CT and GLP-1 IR-NT, at 10kd and at the position of GLP-1 (1-36 amide) in the pancreatic extract, and one peak at 10kd in the stomach extract. Ileal extracts showed 3 peaks of GLP-1 IR-CT at 10kd, at the position of GLP-1(1-36 amide) and GLP-1(7-36 amide), respectively, but GLP-1 IR-NT was coeluted with GLP-1(1-36 amide). Hypothalamic extracts showed a single peak at the position of GLP-1(7-36 amide). These results suggest that processing of preproglucagon differs in different organs, and that the main GLP-1-related products are a large molecular form and GLP-1(1-36 amide) or GLP-1(1-37) in the pancreas, and GLP-1(7-36 amide) or GLP-1 (7-37) in the ileum and hypothalamus.