Cellular localization of intrinsic factor in pancreas and stomach of the dog

Summary A cobalamin (vitamin B₁₂)-binding protein has recently been identified in canine pancreatic juice which is biochemically, immunochemically and functionally similar to canine gastric intrinsic factor. However, the cellular sources of both this pancreatic intrinsic factor and gastric intrinsic factor in the dog are not known. Antisera raised against canine gastric intrinsic factor have been used to examine the distribution of intrinsic factors in the canine pancreas and stomach. Immunoreactivity was demonstrated in duct cells but not acinar or endocrine cells in the pancreas, and in fundic peptic and pyloric gastric pit cells in stomach. All immunostaining was abolished by preabsorption of the antisera with purified canine gastric and pancreatic intrinsic factors. A cellular source of pancreatic intrinsic factor has not been previously described, and the demonstration of intrinsic factor-like immunoreactivity in two cell types in the canine stomach contrasts with its localization in a single cell type in the gastric mucosa of other mammalian species. Furthermore, immunoreactivity in pancreatic duct cells was detected at much higher dilutions of antisera than those required for staining of peptic and gastric pit cells. This suggests a higher concentration of antigen, and supports previous evidence that the pancreas is a major source of intrinsic factor in the dog.     

Purification of classical pancreatic lipase from dog pancreas

The purification of canine classical pancreatic lipase from canine pancreatic juice, but not from pancreatic tissue, has been reported previously. Given the logistic difficulties associated with collection of pancreatic juice in dogs and efforts to minimize experiments in live animals the objective of this project was to purify canine classical pancreatic lipase from dog pancreas. Dog pancreata were collected from research dogs that had been sacrificed for unrelated research projects. Pancreatic tissue was delipidated using organic solvents. The delipidated pancreatic extract was further purified by extracting the enzymes in a Tris-buffer containing two different protease inhibitors, benzamindine and phenylmethylsulfonyl fluoride (PMSF), followed by anion exchange chromatography, gel-filtration, and cation exchange chromatography. The purified protein showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of approximately 50.7. Isoelectric focusing showed isoelectric points ranging from 6.0 to 6.2. N-terminal amino acid sequencing of the first 25 amino acid residues showed the sequence Lys-Glu-Val-X-Phe-Pro-Arg-Leu-Gly-X-Phe-Ser-Asp-Asp-Ser-Pro-Trp-Ala-Gly-Ile-Val-Glu-Arg-Pro-Leu. This sequence showed close homology with classical pancreatic lipase in pigs, horses, and human beings. We conclude that canine classical pancreatic lipase can be successfully purified from canine pancreatic tissue.     

Catalytic and kinetic properties of purified high-affinity cyclic AMP phosphodiesterase from dog kidney

High-affinity cyclic AMP phosphodiesterase purified to homogeneity from dog kidney was studied with respect to its stability, its catalytic and kinetic properties, and its sensitivity to pharmacological agents. The enzyme was shown to rapidly lose activity upon dilution to low protein concentrations in aqueous media, but this activity loss was largely prevented by the presence of bovine serum albumin or ethylene glycol. Similarly, maximum activity required bovine serum albumin to be present during incubation for activity analysis. Enzyme activity required a divalent cation; Mg²⁺, Mn²⁺, and Co²⁺ each supported activity, but highest activity was obtained with Mg². The temperature optimum ranged from 30 to 45 °C and depended on substrate concentration; the E_a = 10,600 cal/mol. The pH optimum of the enzyme was broad, with a maximum from pH 8.0 to 9.5. The enzyme exhibits linear Michaelis-Menton kinetics for hydrolysis of cyclic AMP at all substrate concentrations tested and for hydrolysis of cyclic GMP at > 20 μm. The K_m for cyclic AMP hydrolysis was 2 μm, and that for cyclic GMP hydrolysis was 312 μm. The K_i values for the competitive inhibition of hydrolysis of each substrate by the other were similar to their K_m values suggesting a single active site. Cyclic AMP hydrolysis was weakly inhibited by cyclic GMP, cyclic IMP, adenine, and adenosine, but was not inhibited by the mono-, di, or trinucleotides of adenosine, guanosine, or inosine. Activity was competitively inhibited with K_i values in the micromolar range by drugs representative of methylxanthines, isoquinolines, pyrazolopyridines, imidazolidinones, triazolopyrimidines, pyridylethylenediamines, phenothiazines, and calcium antagonists. The results are discussed with reference to the similarities and differences between high- and low-affinity phosphodiesterase forms.     

Sequences of gastrins purified from a single antrum of dog and of goat

Heptadecapeptide gastrins (G17) have been purified and sequenced from a variety of species. However, progastrin (G34) sequences have been determined only for pig and human from purified peptides and for rat from cDNA. Since G34 in most species accounts for only approximately 5% of total antral gastrin, micropurification techniques must be employed to avoid the need for large quantities of antral tissue. Efficient purification methodology yielded 1.5 and 1.3 nmol of G34 from the antrum of a single goat and of a single dog, respectively. The N-terminal pyroglutamyl residues were enzymatically removed and the peptides were sequenced through to the proximity of their COOH-termini. The COOH-terminal sequences of goat and dog G34 were confirmed by sequencing the corresponding deblocked G17 from each animal. The previously published dog G17 sequence was shown to be incorrect. The sequences for dog and goat G34 are: Dog less than ELGLQGPPQLVADLSKKQGPWMEEEEAAYGWMDF# Goat less than ELGLQDPPHMVADLSKKQGPWVEEEEAAYGWMDF# Dog and goat gastrins differ in 3 sites in the 17 amino acid NH2-terminus and only a single site in G17 (the sites of differences are underlined). The ratio for sulfated to non-sulfated antral G17 is 9:1 for the goat and 1:9 for the dog.     

Segregation of histone fractions from purified rat pancreas nuclei by isoelectric focusing.

The present report describes the purification of nuclei from rat pancreas and the use of isoelectric focusing for the segregation of histone fractions, a heterogeneous mixture with respect to charge. This procedure employs gels of 5% polyacrylamide as support matrices, in 6.25 M urea, and spans a pH range of 7.5-10.8. It was, therefore, found adequate for study of the microheterogeneity of histones. The banding pattern of histone fractions in pancreas development using electrofocusing is presented.     

Significance of the peri-insular extracellular matrix for islet isolation from the pancreas of rat,dog, pig,and man

Summary The presence and distribution in the peri-insular region of extracellular matrix, and in particular basement membrane, was investigated in a comparative study comprising pancreata of rat, dog, pig, and man. Basement membrane markers, collagen type-IV and laminin, were determined immunohistochemically. Additional information pertaining to the structural relationships between endocrine and exocrine pancreas, in particular cell-to-cell and cell-to-matrix contacts, was obtained by electron microscopy. In pig, very little periinsular capsule is present, and the structural integration of the porcine islet in the exocrine pancreas almost exclusively depends on cell-to-cell adhesion. In the canine pancreas, the islets are almost completely encapsulated with very little direct exocrine-to-endocrine cell-to-cell contact. In rat and man, the situation is intermediate with a tendency towards predominance of cell-to-matrix adhesion. The intra-insular adhesion mechanisms depend largely on cell-to-cell adhesion in all four species. The ultrastructural results suggest that collagenase preparations employed in islet isolation procedures should be of high purity as to preserve the protease-sensitive intra-islet cell-to-cell adhesion. Under these conditions, however, the endocrine-to-exocrine cell-to-cell contacts will be conserved also, resulting in an exocrine-tissue contamination of the islets of Langerhans. Consequently, additional steps for the effective removal of exocrine tissue and the purification of islets are required.     

Role of leptin in the stomach and the pancreas

Leptin, a 16 kDa protein encoded by the ob gene, is known mainly for its role in the regulation of food intake, body composition and energy expenditure through a central feedback mechanism. Initially leptin was considered as an ob gene product of adipocytes but recently the presence of leptin and its receptors have been revealed in other organs including gastric mucosa and the pancreas and found to be released from these organs by cholecystokinin (CCK), gastrin and ordinary feeding. Furthermore, leptin was found to mimic the action of CCK on gastric and pancreatic integrity, while reducing the food intake and to affect gastric and pancreatic secretion. This report emphasizes the role of leptin originating from the gastrointestinal tract acting synergistically with CCK at the hypothalamus level on the mechanism of food intake and locally on the protection of gastric mucosa and the pancreas against noxious agents and to maintain tissue integrity.     

Long-term effect of somatostatin 14 on mouse stomach, antrum, intestine and exocrine pancreas

Mice were injected 3 times a day for 12 days with 8 micrograms/kg of somatostatin 14 which caused a hypoplasia of parietal and goblet cells, a hypotrophy and hypofunctionality of pancreatic acinar cells with a decrease in lipase and chymotrypsin activities, a decrease in the secretory fuction of the Brunner gland and in the number of dark granules of G cells. Neither villous and microvillous areas nor brush border hydrolase activities were affected. The number of peptic cells and Paneth cells increase as the level of pepsin and lysozyme. Mice were injected 4 times per hour with 2 micrograms/kg of somatostatin. 2 h after the first injection of somatostatin and 90 min after a single injection of tritiated thymidine, fundic, antral, jejunal and ileal labelling indexes strongly decrease (maximal effect in ileum). The inhibitory effect of somatostatin on the digestive epithelial cell proliferation compared to its long-term action only directed on specific cell types evokes probable compensatory mechanisms induced to maintain the equilibrium of the digestive epithelia.     

Histochemical and ultrastructural identification of neurotensin cells in the dog ileum

Summary In the dog ileum, neurotensin cells stained with immunofluorescence or immunoperoxidase proved distinct from argentaffin (EC) cells, glucagon immunoreactive (GLI) cells and pancreatic peptide immunoreactive (PP) cells. Neurotensin cells showed various degrees of reactivity with Grimelius'silver. With electron microscopy, besides EC cells, large granule cells with a thin peripheral rim of Grimelius-reactivity (L cells) and large granule cells with variable Grimelius-reactivity of the core (N cells) were found. On distributive grounds, L cells were identified with GLI cells and N cells were interpreted as neurotensin cells.     

Neuropeptide-hydrolysing activities in synaptosomal fractions from dog ileum myenteric, deep muscular and submucous plexi. Their participation in neurotensin inactivation

The mapping of neuropeptidases in synaptosomal fractions prepared from dog ileum myenteric, deep muscular and submucous plexus was established by means of fluorigenic substrates and specific inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme and aminopeptidases were found in all tissues, the highest amounts being recovered in the submucous preparation. Post-proline dipeptidyl aminopeptidase was obtained in high quantities whatever the tissue source while proline endopeptidase was detected in low amounts and pyroglutamyl-peptide hydrolase was never detectable. The above peptidases were examined for their putative participation in the inactivation of neurotensin by monitoring the effect of specific inhibitors on the formation of the metabolites of labeled neurotensin separated by HPLC. Endopeptidases 24.11, 24.15 and 24.16 were respectively responsible for the formation of neurotensin(1-11), neurotensin(1-8) and neurotensin(1-10) that are devoid of biological activity. The secondary attacks occurring on neurotensin degradation products were the following: cleavage of neurotensin(1-10) into neurotensin(1-8) by angiotensin-converting enzyme; conversion of neurotensin(9-13) into neurotensin(11-13) by post-proline dipeptidyl aminopeptidase; hydrolysis of neurotensin(11-13) into free tyrosine by aminopeptidase(s).     

Calmodulin-binding proteins and calmodulin-regulated enzymes in dog pancreas.

Calmodulin was isolated and purified to homogeneity from dog pancreas. Highly purified subcellular fractions were prepared from dog pancreas by zonal sucrose-density ultracentrifugation and assayed for their ability to bind 125I-calmodulin in vitro. Proteins contained in these fractions were also examined for binding of 125I-calmodulin after their separation by polyacrylamide-gel electrophoresis in SDS. Calmodulin-binding proteins were detected in all subcellular fractions except the zymogen granule and zymogen-granule membrane fractions. One calmodulin-binding protein (Mr 240,000), observed in a washed smooth-microsomal fraction, has properties similar to those of alpha-fodrin. The postribosomal-supernatant fraction contained three prominent calmodulin-binding proteins, with apparent Mr values of 62,000, 50,000 and 40,000. Calmodulin-binding proteins, prepared from a postmicrosomal-supernatant fraction by Ca2+-dependent affinity chromatography on immobilized calmodulin, exhibited calmodulin-dependent phosphodiesterase, protein phosphatase and protein kinase activities. In the presence of Ca2+ and calmodulin, phosphorylation of smooth-muscle myosin light chain and brain synapsin and autophosphorylation of a Mr-50,000 protein were observed. Analysis of the protein composition of the preparation by SDS/polyacrylamide-gel electrophoresis revealed a major protein of Mr 50,000 which bound 125I-calmodulin. This protein shares characteristics with the calmodulin-dependent multifunctional protein kinase (kinase II) recently observed to have a widespread distribution. The possible role of calmodulin-binding proteins and calmodulin-regulated enzymes in the regulation of exocrine pancreatic protein synthesis and secretion is discussed.