Membrane interactions between secretion granules and plasmalemma in three exocrine glands.

Three types of membrane interactions were studied in three exocrine systems (the acinar cells of the rat parotid, rat lacrimal gland, and guinea pig pancrease) by freeze- fracture and thin-section electron microscopy: exocytosis, induced in vivo by specific pharmacological stimulations; the mutual apposition of secretory granule membranes in the intact cell; membrane appositions induced in vitro by centrifugation of the isolated granules. In all three glandular cells, the distribution of intramembrane particles (IMP) on the fracture faces of the luminal plasmagranule membrane particles (IMP) on the fracture faces of the lumenal plasmalemma appeared random before stimulation. However, after injection of secretagogues, IMP were rapidly clearly from the areas of granule- plasmalemma apposition in the parotid cells and, especially, in lacrimocytes. In the latter, the cleared areas appeared as large bulges toward the lumen, whereas in the parotid they were less pronounced. Exocytotic openings were usually large and the fracture faces of their rims were covered with IMP. In contrast, in stimulated pancreatic acinar cells, the IMP distribution remained apparently random after stimulation. Exocytoses were established through the formation of narrown necks, and no images which might correspond to early stages of membrane fusion were revealed. Within the cytoplasm of parotid and lacrimal cells (but not in the pancreas), both at rest and after stimulation, secretion granules were often closely apposed by means of flat, circular areas, also devoid of IMP. In thin sections, the images corresponding to IMP-free areas were close granule-granule and granule-plasmalemma appositions, sometimes with focal merging of the membrane outer layers to yield pentalaminar structures. Isolated secretion granules were forced together in vitro by centrifugation. Under these conditions, increasing the centrifugal force from 1,600 to 50,000 g for 10 min resulted in a progressive, statistically significant increase of the frequency of IMP-free flat appositions between parotid granules. In contrast, no such areas were seen between freeze-fractured pancreatic granules, although some focal pentalaminar appositions appeared in section after centrifugation at 50 and 100,000 g for 10 min. On the basis of the observation that, in secretory cells, IMP clearing always develops in deformed membrane areas (bulges, depressions, flat areas), it is suggested that it might result from the forced mechanical apposition of the interacting membranes. This might be a preliminary process not sufficient to initiate fusion. In the pancreas, IMP clearing could occur over surface areas too small to be detected. In stimulated parotid and lacrimal glands they were exceptional. These structures were either attached at the sites of continuity between granule and plasma membranes, or free in the acinar lumen, with a preferential location within exocytotic pockets or in their proximity. Experiments designed to investigate the nature of these blisters and vesicles revealed that they probably arise artifactually during glutaraldehyde fixation. In fact, (a) they were large and numerous in poorly fixed samples but were never observed in thin sections of specimens fixed in one step with glutaraldehyde and OsO(4); and (b) no increase in concentration of phospholipids was observed in the parotid saliva and pancreatic juice after stimulation of protein discharge, as was to be expected if release of membrane material were occurring after exocytosis.

Isolation, characterization, and distribution of an unusual pancreatic human secretory protein.

An unusual protein was isolated from acid extracts of normal human pancreas and pancreatic secretion in the form of uniform 7-10-nm long single threads without visible axial periodicity or other structure, as seen in the electron microscope. It accounts for as much as 300 micrograms/ml in some pancreatic secretions as measured by specific radioimmunoassay. The protein undergoes a freely reversible, pH dependent, globule-fibril transformation, being stable in the fibril form between pH 5.4 and 9.2. The monomer at acid pH has an apparent molecular weight of approximately 14,000 and consists of a single polypeptide chain, the amino acid composition of which is rich in aromatic amino acids and lacks carbohydrate, fatty acid, and phosphate. The amino acid sequence of 45 residues from the amino terminus shows no homology with any other reported protein sequences other than that of the A chain of the bovine pancreas thread protein (reported elsewhere). A sensitive radioimmunoassay employing monoclonal antibodies against human pancreatic thread protein failed to detect the antigen in a wide range of human tissues other than pancreas, nor was the antigen measurable in normal human sera. Immunohistochemistry utilizing these antibodies revealed the antigen as a component of the cytoplasm of some but not all the pancreatic acinar cells. A physiologic function has not yet been determined for this protein.

Secretory pancreatic stone protein messenger RNA. Nucleotide sequence and expression in chronic calcifying pancreatitis.

The pancreatic stone protein and its secretory form (PSP-S) are inhibitors of CaCO3 crystal growth, possibly involved in the stabilization of pancreatic juice. We have established the structure of PSP-S mRNA and monitored its expression in chronic calcifying pancreatitis (CCP). A cDNA encoding pre-PSP-S has been cloned from a human pancreatic cDNA library. Its nucleotide sequence revealed that it comprised all but the 5' end of PSP-S mRNA, which was obtained by sequencing the first exon of the PSP-S gene. The complete mRNA sequence is 775 nucleotides long, including 5'- and 3'- noncoding regions of 80 and 197 nucleotides, respectively, attached to a poly(A) tail of approximately 125 nucleotides. It encodes a preprotein of 166 amino acids, including a prepeptide of 22 amino acids. No overall sequence homology was found between PSP-S and other pancreatic proteins. Some homology with several serine proteases was observed in the COOH-terminal region, however. The mRNA levels of PSP-S, trypsinogen, chymotrypsinogen, and colipase in CCP and control pancreas were compared. PSP-S mRNA was three times lower in CCP than in control, whereas the others were not altered. It was concluded that PSP-S gene expression is specifically reduced in CCP patients.

Demonstration and immunochemical characterization of carcinoembryonic antigen in human pancreatic juice.

Pancreatic juice collected from 10 patients without evidence of malignant disease of the pancreas or other organs was pooled, extracted, and fractionated by Sepharose 6-B and Sephadex G-200 gel filtration. The carcinoembryonic antigen (CEA) activity in the material was demonstrated and studied by: a) radioimmunoassay, b) competitive binding to antibodies against CEA, c) precipitin inhibition, and d) Ouchterlony analysis. The immunochemical identity of the active material to CEA purified from liver metastases of colon cancer was demonstrated.

Isolation and characterization of colipase from porcine and human pancreatic juice by immunoaffinity chromatography.

Pure colipase was prepared by immunoaffinity chromatography from porcine and human pancreatic juice. A single form of the porcine colipase was obtained, having the structural and biological properties of previously characterized porcine procolipase A. Two forms of activated colipase (N-terminal Gly) were isolated from human pancreatic juice by the same procedure. The existence of two forms of activated colipase might arise from rapid activation of a precursor form of human colipase during collection of the pancreatic juice.

Concanavalin A variants of the fetoacinar pancreatic protein in the developing human pancreas and other biological sources.

Two new sources for the fetoacinar pancreatic protein (FAP protein) are described in this study: amniotic fluids taken at 18 weeks gestation, and pancreatic juices from patients with pancreatic pathology. The FAP protein from different biological sources showed two kinds of molecular heterogeneity: (a) molecular weight, and (b) lectin-binding affinity. By Western blot the protein was shown to exist either as a doublet (the higher-Mr component at 110 kDa and the second in the range 80-100 kDa) or as a single band (110 kDa) depending on the source. By chromatography on Con A-Sepharose the protein could be separated into two variants, reactive and nonreactive. Most of the protein was present as the Con A-nonreactive variant. The Western-blot patterns of both variants in a given sample were identical. The FAP protein expression had an oncodevelopmental character; maximal concentration was seen in middle-gestation fetal pancreas extracts. Expression of the FAP protein Con A variants followed the same developmental pattern as that of total FAP protein, and their relative amounts remained almost constant during fetal growth. Evidence is given for the presence of lectin and molecular-weight heterogeneities of the protein as well as for the lack of a developmental pattern for the expression of these variants.

Isolation and characterization of sulfated glycoprotein from human pancreatic juice.

Sulfated glycoprotein was isolated by precipitation from dialyzed human pancreatic juice and purified by ion-exchange chromatography followed by repeated gel chromatography. The sulfated glycoprotein was obtained as a sulfated glycoprotein-lipid complex by Sepharose CL-2B chromatography. Lipids aggregating with the sulfated glycoprotein were glycolipids such as ceramide trihexoside, and simple lipids such as cholesterol and cholesterol ester. This glycoprotein was resistant to digestion with mucopolysaccharidases or alpha-amylase, and consisted of 60% (w/w) protein and 40% sugars. The polypeptide core was characterized by a high content of serine, threonine, aspartic acid and glycine, but lacked cysteine. Its sugar components were N-acetylglucosamine, N-acetylgalactosamine, galactose, fucose and sialic acid. Absorption at 1240 cm-1 and 820 cm-1 by infrared spectroscopy indicated the presence of a sulfate ester group. All the carbohydrate chains of this sulfated glycoprotein, which are polydisperse and heterogeneous, were O-glycosidically linked through N-acetylgalactosamine to a protein core.

Binding between immobilized anti-colipase purified antibodies and colipase. Radioimmunoassay of colipase from pig plasma and pancreatic juice.

Procedures for purification of porcine colipase II (Gly6-Gly89) and for obtaining purified anti-colipase antibodies are described. The interactions between antibodies immobilized on an Ultrogel AcA 22 column and colipase were investigated and colipase radioimmunoassay carried out. The immobilized antibody-colipase binding was preserved in the presence of mixed micelles, lipase, or both when added to the elution mixture. Bound colipase maintained its capability of interacting with mixed micelles, but not with lipase in either the presence or the absence of mixed micelles. It could be inferred that the antigenic site(s) is independent of the interfacial recognition site and close to the site of lipase recognition. Results are reported suggesting that one or both colipase histidyl residue-containing sequences are involved as antigenic determinant(s). Immunoreactive colipase, bound to a macromolecular protein complex, was found in the plasma of pig. This finding could be explained by an endocrine 'leakage' of colipase from the exocrine pancreatic cell rather than by passage through the intestinal mucosa.

Isolation and partial characterization of the pancreatic secretory trypsin inhibitor in the rat.

Isolation in a 55% yield of the low molecular weight pancreatic secretory trypsin inhibitor was achieved by gel filtration of an acid extract of whole inactive rat pancreas juice on Sephadex G-50 at pH 2.5 followed by desalting and ion-exchange chromatography on SP Sephadex C-50 at pH 4.5. Two distinct chromatographic fractions were obtained, labeled fraction 1 and 2. Fractions 1 and 2 showed three, respectively two, distinct closely migrating cationic bands on gel electrophoresis in barbital buffer, pH 8.6. Each fraction demonstrated one band on polyacrylamide disc electrophoresis at pH 4.6. The inhibitor is homogenous on gel filtration and on the basis of its stoichiometry with active site titrated rat anionic trypsin. Its molecular weight is approx. 6024. The amino acid composition is included. Rat pancreatic secretory trypsin inhibitor is trypsin-specific and interacts on a 1:1 molar basis with rat trypsin. It is a good inhibitor of bovine trypsin but a poor inhibitor of human cationic trypsin and its binding to trypsin is reversible by acidification. Like other inhibitors of this sort, it is present in about 0.1-0.2% of the total protein content of the juice, and normally exists in its free form. A simple procedure for the production of antiserum to the inhibitor which is a poor antigen is also described.

N-terminal sequence extension in the glycosylated forms of human pancreatic stone protein. The 5-oxoproline N-terminal chain is O-glycosylated on the 5th amino acid residue.

The pancreatic stone protein isolated from human calculi (PSP) derives from the immunoreactive protein forms detected in human pancreatic juice (PSP S2-5) through the tryptic cleavage of the Arg-11-Ile-12 bond. Among the eleven amino acids of the PSP S2-5 N-terminal extension Z-E-A-Q-T-E-L-P-Q-A-R, the first residue is an oxoproline and the fifth, a threonine, bears the single carbohydrate chain of the protein molecules. Variations in the glycan chain composition account for the differences in the Mr of PSP S2-5. The PSP S2-5 forms are very soluble in aqueous solutions between the pH values 5.0-9.0, whereas the proteolysated form is scarcely soluble.

Rejection of kidney and pancreas after pancreas-kidney transplantation.

On the basis of 26 combined pancreas-kidney transplants we questioned whether both organs undergo rejection simultaneously. Reliable diagnosis of pancreas-graft rejection was made possible by monitoring exocrine graft function, including quantitative measurements of pancreatic juice, its amylase content, and pancreatic juice cytology. In addition, diagnosis of pancreas rejection was based on regular flow studies, daily urinary neopterin excretion, and a retrospective analysis of the clinical course. Clinical symptoms, blood chemistry, and, primarily, histology were used to assess rejection of the kidney allograft. In 18 cases the kidney and pancreas were rejected together; in 8 cases the kidney or the pancreas was rejected. Although both organs were rejected at the same time in most cases, either organ can be rejected alone. Thus, the kidney cannot be used to monitor the pancreas allograft in every case.

Fate of the major zymogen granule membrane-associated glycoproteins from rat pancreas. A biochemical and immunocytochemical study.

A glycoprotein resembling the major zymogen granules membrane-associated glycoprotein (GP-2) from the exocrine rat pancreas was detected in a 200 000 g sedimentable subfraction from pancreatic secretion. This glycoprotein had a slightly smaller molecular weight than GP-2 (70 000 D versus 75 000 D), gave a line of identity with GP-2 in a double immunodiffusion test against anti GP-2, and tryptic peptide charts of both glycoproteins were largely similar. Immunofluorescence cytochemistry showed that GP-2 antigens were exclusively located in the exocrine pancreas, where they were preferentially found in the perimeter of secretory granules and in the acinar lumina. These results suggest the possibility of loss of GP-2 from exocrine cells during secretion and raise doubts as to the status of GP-2 as a true membrane glycoprotein.

Immunoreactive somatostatin in human pancreatic secretion.

Somatostatin in human pancreatic juice collected during endoscopic retrograde cholangiopancreatography following secretin injection was determined by RIA. The mean immunoreactive somatostatin (IRS) levels in the pancreatic juice of 13 non-diabetics was 78 +/- 11 (SE) pg/ml. The IRS levels in 4 non-insulin-dependent diabetics ranged from 843 to 2286 pg/ml with a mean of 1559 +/- 392 (SE) pg/ml. This was significantly greater than non-diabetic values. The IRS in the pancreatic juice was immunologically indistinguishable from somatostatin14 but consisted of 2 major components, one corresponding to somatostatin14 and the other being of a molecular size of approximately 3000 daltons.

Immunoreactive human epidermal growth factor in human pancreatic juice.

Human epidermal growth factor (hEGF), which stimulates the growth of a variety of cells in culture, has recently been isolated from human urine. In the present study, we identified significant amounts of immunoreactive (IR) hEGF (mean +/- SE, 2.3 +/- 0.09 ng/ml; n = 3) in human pancreatic juice. The IR-hEGF materials were immunologically indistinguishable from standard hEGF, although they were only 5% as active in receptor binding to human placental membrane as in RIA. Gel exclusion chromatography of the pancreatic juice under neutral and acidic conditions revealed three distinct IR-hEGF components with different molecular sizes. Incubation of 125I-labeled hEGF with either the pancreatic juice or the high molecular weight component(s) yielded no aggregation or degradation products. These data suggest that fully immunoreactive but much less bioactive hEGF-like substances which are heterogeneous in size are secreted into human pancreatic juice. Their tissue(s) of origin and physiological functions, if any, remain to be elucidated.

The disulfide bridges of the immunoreactive forms of human pancreatic stone protein isolated from pancreatic juice.

Following the complete sequence elucidation of human pancreatic stone protein (immunoreactive form PSP S1 isolated from pancreatic juice) [(1987) Eur. J. Biochem. 168, 201-207], the location of the three S-S bridges of the protein was investigated. The cystine-containing peptides, detected after the separation of the peptic or chymotryptic digests on SP-Sephadex or Sephadex G-50, were submitted to Edman degradation and/or to oxidation. The cysteic peptides after separation on SP-Sephadex or Sephadex G-50 were characterized by their amino acid compositions. The pairing of the half-cystines: Cys 3-Cys 14, Cys 31-Cys 129 and Cys 104-Cys 121 was determined. The same experiments carried out with PSP S2-5 (other immunoreactive forms) gave an identical characterization.

Purification and complete amino acid sequence of canine pancreatic secretory trypsin inhibitor.

Pancreatic secretory trypsin inhibitor (PSTI) was purified from canine pancreatic juice by HPLC. Canine PSTI inhibited bovine trypsin activity stoichiometrically and strongly with a dissociation constant of below 10(-9) M. The amino acid sequence of canine PSTI was determined by conventional methods. It had one more amino acid residue at the amino-terminus than other mammalian PSTIs, i.e. human, porcine, bovine and ovine.

Effect of dietary fiber supplementation on the secretory function of the exocrine pancreas in the dog.

The effects on pancreatic secretion of a diet supplemented with wheat bran for one month were studied in five dogs provided with chronic pancreatic and gastric fistulae. Dogs were subjected before and after bran administration to a continuous intravenous infusion of secretin (GIH, 0.5 U/kg/h) alone and a combination of secretin with cerulein (25 or 100 ng/kg/h). Exocrine pancreatic secretions under basal conditions were collected during the intravenous infusions of normal saline. Wheat bran supplementation for 4 wk to the standard diet in dogs increased pancreatic juice flow rate along with bicarbonate and amylase output whether the secretion was unstimulated or stimulated with caerulein alone or combined with secretin. No significant changes in the concentration of pancreatic bicarbonate, amylase, chymotrypsin, and proteins were noted under stimulated conditions. Furthermore, lipase concentration and output were dramatically reduced. These data indicated that wheat bran supplemented to the standard diet in dogs affects the exocrine pancreatic secretion whether it was unstimulated or stimulated by secretin alone or combined with cerulein.

Picolinic acid in milk, pancreatic juice, and intestine: inadequate for role in zinc absorption.

Picolinic acid (PA) was measured by high pressure liquid chromatography in human milk and other fluids and tissues. Skimmed human milk, intestinal homogenates from human infants and rats, and human and rat pancreatic juice were ultrafiltered and analyzed by high pressure liquid chromatography using an anion-exchange column. Identity of sample components was verified by comparing retention times with those of pure nicotinic acid and PA. The detection limit for PA was 2.5 microM. Human milk contained less than 3.7 microM PA. PA was undetectable in human infant or rat intestine or in human or rat pancreatic juice. The extremely low concentration of PA in milk and its apparent absence in pancreatic juice and intestine provide additional evidence that PA is not the low molecular weight zinc binding ligand of human milk and that it does not have an important physiological role in intestinal zinc absorption.

Monoclonal antibodies to pancreatic stone protein. Radioimmunoassay and immunological comparison with trypsin 1.

Monoclonal antibodies were prepared against pancreatic stone protein, a protein which inhibits calcium carbonate precipitation. Two monoclonal antibodies designated D4 and 2E7 were characterized. Immunoadsorbant columns, obtained by linkage of these monoclonal antibodies to Affigel 10, have been used to isolate immunoreactive forms of pancreatic stone protein from nonactivated human pancreatic juice. These monoclonal antibodies permitted us to test the possible immunological relationship between pancreatic stone protein and human trypsin 1. No immunological similarity was found, in agreement with our previous results, and it was established that pancreatic stone protein is a novel protein and not a degradation product of human trypsin(ogen) 1.

Evidence for the existence of procolipase in chicken pancreas and pancreatic juice.

Purified antibodies raised against chicken colipase were coupled to Sepharose 4B and colipase was isolated in a single step by immunoaffinity chromatography from an extract of chicken pancreas prepared under conditions where trypsin activation is avoided. The purified protein has a single amino terminal residue of alanine and its biochemical properties are similar to those of the precursor form of colipase (procolipase) previously isolated from porcine and equine pancreas or pancreatic juice. Further evidence for the existence of procolipase was obtained from kinetic studies of the hydrolysis of the Intralipid emulsion by untreated and trypsin-treated chicken pancreatic juice.