Enzyme cytochemistry of rat organs after uremia with special reference to proteases

Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed an increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and beta-N-acetyl-D-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and gamma-glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.     

Ultrastructural studies of the ontogeny of fetal human and porcine endocrine pancreas, with special reference to colocalization of the four major islet hormones.

Most, if not all, endocrine cells seem capable of synthesizing and storing more than one hormone. Such cellular colocalization of hormones can be due either to the presence of two or more specific granules within the cells or to colocalization of the hormones within a single granule. The present study was performed to clarify the subcellular localization of insulin, glucagon, somatostatin, and pancreatic polypeptide within the endocrine cells of the human and porcine pancreas during fetal development, with special reference to possible colocalization of the hormones. The tissue specimens were processed for ultrastructural cytochemistry using Lowicryl as embedding medium. An immunogold labeling technique was used with two parallel, but not interacting, antibody chains. Sections from each specimen were double labeled in different combinations giving a complete covering of the four major islet hormones. During fetal life (50-90 days prenatally in porcine pancreas, 14 weeks gestation in the human pancreas) several hormones were demonstrated, not only in the same endocrine cells, but also in the same secretory granules (polyhormonal granules). Costorage of insulin, glucagon, somatostatin, and pancreatic polypeptide was demonstrated in granules in pancreatic endocrine fetal cells. At an early fetal stage, the endocrine cells contained either dense, round granules or pale, heteromorphous granules. With increasing age and maturation of the endocrine cells, structural differentiation of the secretory granules was found to be associated with a gradual disappearance of the polyhormonal granules. The first genuine monohormonal cell to appear in the porcine fetus was the pancreatic polypeptide cell (at 70 days gestation); it was followed by the somatostatin-producing endocrine cell. Mature insulin- and glucagon-producing cells were only demonstrated after birth. Thus, in the adult pancreatic endocrine cells, each specific endocrine cell type produced only one of the four classical hormones. The present investigation demonstrated that the endocrine cells of the fetal, but not the adult, pancreas are able to synthesize all the major islet hormones, and that these peptides are costored in the same granule. The data obtained support the concept of a common precursor stem cell for pancreatic hormone-producing cells.     

Enzyme cytochemistry of rat organs after uremia with special reference to proteases

Summary Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed and increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and -N-acetyl-d-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and -glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the German Research Foundation (Sfb 174)     

Reaction of dipeptidylpeptidase IV with substrate-analogous azapeptides.

The reaction of dipeptidyl peptidase IV (EC 3.4.14.5.) with azapeptide substrates containing azaalanine or azaproline in the P1-position was investigated. Accumulation of a fairly stable acyl-enzyme could be shown for ester substrates. Ala-AzaPro-pNA is a very poor substrate of DP IV and does not accumulate an acyl-enzyme. DP IV does not react with active-site titrants for trypsin-like serine proteases.     

Reaction rate measurements of proteases and glycosidases with chromogenic methods

Simultaneous azo-coupling and indigogenic methods were evaluated for the quantitative histochemical assay of the plasma membrane proteases gamma-glutamyl transpeptidase (EC 2.3.2.2) and dipeptidyl peptidase IV (EC 3.4.14.5) and the glycosidases maltase-glucoamylase and glucoamylase (EC 3.2.1.20) in decidual cells, jejunal enterocytes and renal proximal tubulocytes. Using kinetic (continuous) microdensitometry, a linear increase in the final reaction product was found from 3 up to 10 min, depending on the substrate concentration and the plasma membrane glycosidase or protease under investigation. Combined continuous and end point (static) microdensitometry revealed a linear relationship between the section thickness (enzyme concentration) and final reaction product up to 12 microns for the proteases and up to 16 microns for the glycosidases. Apparent Km and Vmax values were calculated with a computerized version of the direct linear plot and compared with the results obtained with the linear transformations according to Lineweaver-Burk, Eadie-Hofstee and Hanes. Apparent Km and Vmax values for the proteases were calculated separately for each animal and were 1.82 mM and 1.02 mM and 2.43 arbitrary units (a.u.) and 1.67 a.u. (gamma-glutamyl transpeptidase, decidua) and 0.42 mM and 0.38 mM and 0.29 and 0.26 a.u. (dipeptidyl peptidase IV, decidua). For the alpha-D-glucosidases, the corresponding values were 0.23 mM and 0.15 a.u. (kidney) and 0.55 mM and 0.20 a.u. (jejunum). The results show the suitability of the indigogenic methods for quantitative histochemical measurements of plasma membrane alpha-D-glucosidases, whereas the simultaneous azo-coupling procedures seemed to be less suitable for the quantification of surface membrane proteases, due to, for example, interactions of diazonium salts with amino acid or peptide substrates, reaction products and peptide activators.     

Lateral diffusion of luminal membrane components during secretion in parotid acinar cells of the rat

Summary The movements of the molecular components of the luminal plasma membrane during exocytotic secretion in parotid acinar cells were examined. For immunocytochemical study, we used an antiserum of dipeptidyl peptidase IV as a marker for the components of the luminal plasma membrane of acinar cells. In unstimulated acinar cells, dipeptidyl peptidase IV immunoreactivity is restricted to the luminal plasma membrane. However, after secretion was stimulated with a -adrenergic agonist, isoproterenol, immunostaining became detectable on the membrane of discharged granules. Freeze-fracture images showed that the density of intramembrane particles on the P-fracture leaflets of discharged granule membranes is much higher than that of undischarged granule membranes during secretion. These results suggest that in parotid acinar cells of the rat, the components of the luminal plasma membrane move laterally, during secretion, to the membranes of discharged granules.     

Coexistence of pancreatic polypeptide (PP)-and glucagon-immunoreactivity in pancreatic endocrine cells of mouse

Immunocytochemical double staining techniques were used to study PP- and glucagon-like-immunoreactivity in pancreatic endocrine cells of mouse. An antiserum against FMRFamide appeared to react with all PP-immunoreactive endocrine cells. With fluorescence microscopy most PP/FMRFamide-immunoreactive cells also showed glucagon-immunoreactivity, but cells containing only PP- or glucagon-like substances were found as well. The proportion of cells containing PP-, glucagon, and both immunoreactivities varied strongly from islet to islet in all parts of the pancreas. Using an electron microscopical immunogold double staining procedure on Lowicryl-embedded pancreas, PP/FMRFamide- and glucagon-immunoreactivity appeared to be present in the majority of endocrine A cells; both immunoreactivities were randomly distributed within the granules of these cells. Cells containing only PP/FMRFamide- or glucagon-immunoreactivity were also found. Glucagon- and a faint FMRFamide-immunoreactivity was also observed in osmicated epon-embedded tissue. Independent of their immunoreactivity all positive cells showed the same round electron dense secretory granules.     

Coexistence of pancreatic polypeptide (PP)-and glucagon-immunoreactivity in pancreatic endocrine cells of mouse

Summary Immunocytochemical double staining techniques were used to study PP- and glucagon-like-immunoreactivity in pancreatic endocrine cells of mouse. An antiserum against FMRFamide appeared to react with all PP-immunoreactive endocrine cells. With fluorescence microscopy most PP/FMRFamide-immunoreactive cells also showed glucagon-immunoreactivity, but cells containing only PP-or glucagon-like substances were found as well. The proportion of cells containing PP-, glucagon, and both immunoreactivities varied strongly from islet to islet in all parts of the pancreas.Using an electron microscopical immunogold double staining procedure on Lowicryl-embedded pancreas, PP/FMRFamide-and glucagon-immunoreactivity appeared to be present in the majority of endocrine A cells; both immunoreactivities were randomly distributed within the granules of these cells. Cells containing only PP/FMRFamide-or glucagon-immunoreactivity were also found. Glucagon-and a faint FMRFamide-immunoreactivity was also observed in osmicated epon-embedded tissue. Independent of their immunoreactivity all positive cells showed the same round electron dense secretory granules.     

Reaction of Dipeptidylpeptidase IV with Substrate-Analogous Azapeptides

Abstract

The reaction of dipeptidyl peptidase IV (EC 3.4.14.5.) with azapeptide substrates containing azaalanine or azaproline in the P₁-position was investigaled. Accumulation of a fairly stable acyl-enzyme could be shown for ester substrates. Ala-AzaPro-pNA is a very poor substrate of DP IV and does not accumulate an acyl-enzyme. DP IV does not react with active-site titrants for trypsin-like serine proteases.     

Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV

The biogenesis of three intestinal microvillar enzymes, maltase-glucoamylase (EC 3.2.1.20), aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. The earliest detectable forms of the enzymes were polypeptides of Mr 225000, 140000 and 115000 respectively. These were found to represent the enzymes in a 'high-mannose' state of glycosylation, as judged by their susceptibility to treatment with endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96). After about 40-60 min of chase, maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV were further modified to yield the mature polypeptides of Mr 245000, 170000 and 137000 respectively, which were expressed at the microvillar membrane after 60-90 min of chase. The fact that the enzymes before reaching the microvillar membrane were found in a Ca2+-precipitated membrane fraction (intracellular and basolateral membranes), but not in soluble form, indicates that during biogenesis maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV are transported and assembled in a membrane-bound state.     

Proteases in the human full-term placenta

Summary Aminopeptidase A, not yet defined aminopeptidases and endopeptidases, dipeptidyl peptidase I, II and IV, -glutamyl transferase and oxytocinase were investigated in the normal human full-term placenta using qualitative (catalytic) cytochemistry, isoelectric focusing, immunocytochemistry and kinetic fluorometry. Aminopeptidase A could be visualized cytochemically in the smooth muscle cells of the chorionic plate, stem villi and basal plate blood vessels. Aminopeptidases were found in connective tissue fibres of the chorionic plate, villous stroma, basal plate and paraplacenta. Dipeptidyl peptidase IV was detected at the same sites as the aminopeptidases and, in addition, in amniotic epithelial cells, fibroblasts of the villous stroma, endothelium of chorinic plate and villous blood vessels as well as in the basophilic cytotrophoblast cells (x-cells) of the basal plate and paraplacenta, and it possibly also occurred in some domains of the plasma membrane of the syncytiotrophoblast and cytotrophoblast cells. The x-cells surrounded the fetus in the form of a dipeptidyl peptidase IV-positive shell at the border to the mother. The enzyme represented the first specific marker for x-cells. Dipeptidyl peptidase I and II were primarily found in Hofbauer cells (macrophages) of the villous stroma, but also in the syncytiotrophoblast, other villous stromal cells and cells of the chorionic and basal plate. -Glutamyl transferase was present in some connective tissue elements of the chorionic plate. Oxytocinase and endopeptidases were not detected. Isoclectric focusing of proteases revealed different molecular forms of dipeptidyl peptidase IV in the paraplacenta and villous tree, while the aminopeptidases shared the same pattern in both regions. Immunocytochemical staining of dipeptidyl peptidase IV in the villous tree resembled the pattern obtained by catalytic cytochemistry except for the blood vessel endothelium and the x-cells of the basal plate. Fluorometrically, all proteases were more active in the villous tree than in the paraplacenta. The kinetic measurements revealed the highest hydrolysis rates for dipeptidyl peptidase IV followed by the aminopeptidases. In contrast to eatalytic cytochemistry all proteases were detectable when using fluorometry.Supported by the German Research Foundation (Sfb 174)A preliminary account of this work was presented at a Symposion on Progress in General, Applied and Diagnostic Histochemistry (Smolenice, Czechoslovakia on March 24–28, 1986).     

Proline residues in the maturation and degradation of peptide hormones and neuropeptides

The proteases involved in the maturation of regulatory peptides like those of broader specificity normally fail to cleave peptide bonds linked to the cyclic amino acid proline. This generates several mature peptides with N-terminal X-Pro-sequences. However, in certain non-mammalian tissues repetitive pre-sequences of this type are removed by specialized dipeptidyl (amino)peptidases during maturation. In mammals, proline-specific proteases are not involved in the biosynthesis of regulatory peptides, but due to their unique specificity they could play an important role in the degradation of them. Evidence exists that dipeptidyl (amino)peptidase IV at the cell surface of endothelial cells sequesters circulating peptide hormones which are then susceptible to broader aminopeptidase attack. The cleavage of several neuropeptides by prolyl endopeptidase has been demonstrated in vitro, but its role in the brain is questionable since the precise localization of the protease is not clarified.     

Intestinal digestive resistance of immunodominant gliadin peptides

Two recently identified immunodominant epitopes from alpha-gliadin account for most of the stimulatory activity of dietary gluten on intestinal and peripheral T lymphocytes in patients with celiac sprue. The proteolytic kinetics of peptides containing these epitopes were analyzed in vitro using soluble proteases from bovine and porcine pancreas and brush-border membrane vesicles from adult rat intestine. We showed that these proline-glutamine-rich epitopes are exceptionally resistant to enzymatic processing. Moreover, as estimated from the residual peptide structure and confirmed by exogenous peptidase supplementation, dipeptidyl peptidase IV and dipeptidyl carboxypeptidase I were identified as the rate-limiting enzymes in the digestive breakdown of these peptides. A similar conclusion also emerged from analogous studies with brush-border membrane from a human intestinal biopsy. Supplementation of rat brush-border membrane with trace quantities of a bacterial prolyl endopeptidase led to the rapid destruction of the immunodominant epitopes in these peptides. These results suggest a possible enzyme therapy strategy for celiac sprue, for which the only current therapeutic option is strict exclusion of gluten-containing food.     

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax): an ultrastructural study. I. The primordial cord and the primitive,single and primordial islets

The primordial cord and the primitive, single and primordial islets present in the 3 earliest stages of the developing endocrine pancreas of sea bass were studied ultrastructurally. The primordial cord consisted of type I and II cells and was included in the gut. Besides these cell types, X cells were seen in the primitive islet. The single islet was made up of type I, II, III and IV cells. A correlation between these endocrine cell-types and cells previously identified immunocytochemically, was established. Type I, II, III and IV cells, correlated respectively with SST-25-, insulin-, SST-14- and glucagon-immunoreactive cells, and could be related to the D₁, B, D₂ and A cells, respectively, of older larvae and adult sea bass. Each cell type shows characteristic secretory granules from its first appearance. A progressive development of the organelles and an increase in the number and size of the secretory granules, whose ultrastructure also varied, was observed in the endocrine cells of the primordial cord and the succeeding islets. In 25-day-old larvae at the beginning of the fourth developmental stage, the primordial islet, the first ventral islet found, was close to a pancreatic duct and blood vessel, and consisted of type I and II cells whose ultrastructure was similar to that of the type I and II cells in the primordial cord. These data suggest a ductular origin for the pancreatic endocrine cells in the ventral pancreas. It is suggested that although endocrine cells undergo mitosis, their increase in number during the earliest development stages is principally due to the differentiation of surrounding cells.     

Proline-specific dipeptidyl peptidase from the blue blowfly Calliphora vicina hydrolyzes in vitro the ecdysiostatic peptide trypsin-modulating oostatic factor (Neb-TMOF)

To elucidate the mechanisms of inactivation of the ecdysiostatic peptide trypsin-modulating oostatic factor (Neb-TMOF) in the blue blowfly Calliphora vicina, we investigated its proteolytic degradation. In homogenates and membrane and soluble fractions, this hexapeptide (sequence: NPTNLH) was hydrolyzed into two fragments, NP and TNLH, suggesting the involvement of a proline-specific dipeptidyl peptidase. The dipeptidyl peptidase activity was highest in the late larval stage. It was purified 240-fold from soluble fractions of pupae of mixed age and classified on the basis of several catalytic properties as an invertebrate homologue of mammalian dipeptidyl peptidase IV (EC 3.4.14.5). Fly dipeptidyl peptidase IV has a molecular mass of 200 kDa, showed a pH optimum of 7.5–8.0 with the chromogenic substrate Gly-Pro-4-nitroanilide, and cleaved other chromogenic substrates with penultimate Pro or, with lower activity, Ala. It liberated Xaa-Pro dipeptides from the N-terminus of several bioactive peptides including substance P, neuropeptide Y, and peptide YY but not from bradykinin, indicating that the peptide bond between the two proline residues was resistant to cleavage. Fly dipeptidyl peptidase belongs to the serine class of proteases as the mammalian enzyme does; the fly enzyme, however, is not inhibited by several selective or nonselective inhibitors of its mammalian counterpart. It is suggested that dipeptidyl peptidases exert a regulatory role for the clearance not only of TMOF in flies but for other bioactive peptides in various invertebrates. Arch. Insect Biochem. Physiol. 37:146–157, 1998. © 1998 Wiley-Liss, Inc.     

Histochemistry of some proteases in the normal rabbit, pig and ox corneas

The distribution of activities of membrane aminopeptidases (aminopeptidase M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV), gamma-glutamyltransferase (GGT) and lysosomal exopeptidases (dipeptidyl peptidase I (DPP I), dipeptidyl peptidase II (DPP II)) was investigated in rabbit, ox and pig corneas. Cryostat sections of snap-frozen corneas treated with chloroform-acetone (4 degrees C) were used for the demonstration of membrane-bound enzymes and sections of corneas fixed in 4% paraformaldehyde (4 degrees C) for the demonstration of lysosomal enzymes. In activities of proteases species differences were found. The rabbit cornea was most active, followed by ox and pig corneas. Individual corneal layers reacted differently. Of membrane proteases a high APM activity was found in keratocytes, whereas epithelium and endothelium were negative. On the other hand, APA and GGT were active in the epithelium and endothelium. Their activities in keratocytes were less pronounced. DPP IV activity was demonstrated in some keratocytes beneath the epithelium only. Lysosomal enzymes DPP I and DPP II were active in all corneal layers. The epithelium displayed the highest activity. Differences in activities in the centro-peripheral and epithelio-endothelial directions were found. DPP I, DPP II, and APM were most active in the limbal region in all corneal layers.     

Involvement of plasma membrane dipeptidyl peptidase IV in fibronectin-mediated adhesion of cells on collagen

Dipeptidyl peptidase IV is an exopeptidase found in the serum and in plasma membranes of most animal tissues. The role of this enzyme in cell-matrix interaction of BHK cells and hepatocytes grown on collagen-coated surfaces was investigated by three different approaches. 1) Glass surfaces were derivatized with bovine serum albumin which resulted in a cell-repulsing substratum. When it was further modified with Gly-Pro-Ala tripeptide, which is a substrate for dipeptidyl peptidase IV, BHK fibroblasts spread on it rapidly. The spreading could be inhibited by addition of free Gly-Pro-Ala or other substrates of the enzyme as well as by an inhibitor peptide Val-Pro-Leu. It was not influenced by tripeptides which were neither substrates nor inhibitors of dipeptidyl peptidase IV. 2) The addition of Gly-Pro-Ala to seeded cells slowed down the initial process of cell spreading on denatured collagen in the presence of fibronectin. The presence of both collagen and fibronectin was a necessary precondition for the spreading of cells in a manner sensitive to Gly-Pro-Ala. 3) Antiserum raised against mouse liver dipeptidyl peptidase IV added to the medium delayed the spreading of rat hepatocytes on denatured collagen in the presence of fibronectin in a manner similar to when Gly-Pro-Ala was added to the medium. These observations lead to the conclusion that plasma membrane dipeptidyl peptidase IV may be involved in the initial phase of fibronectin-mediated cell spreading on collagen.     

5′-AZA induces Ngn3 expression and endocrine differentiation in the PANC-1 human ductal cell line

Neurogenin 3 is necessary for endocrine cell development in the embryonic pancreas and has been shown to induce transdifferentiation duct cells from adult pancreas toward a neuro-endocrine phenotype. Here we discovered that the demethylating agent 5′-Azadeoxycytidine (AZA) induced Ngn3 expression and endocrine differentiation from the PANC-1 human ductal cell line. The expression of markers specific to mature islet cells, i.e., glucagon and somatostatin, was also observed. In addition, we demonstrated that growth factors (betacellulin and soluble factors released during pancreas embryogenesis) increased the level of maturation. Our studies revealed that the PANC-1 model system may provide a basis for elucidating the ductal/endocrine differentiation.     

Metabolism of glucagon by dipeptidyl peptidase IV (CD26)

Glucagon is a 29-amino acid polypeptide released from pancreatic islet alpha-cells that acts to maintain euglycemia by stimulating hepatic glycogenolysis and gluconeogenesis. Despite its importance, there remains controversy about the mechanisms responsible for glucagon clearance in the body. In the current study, enzymatic metabolism of glucagon was assessed using sensitive mass spectrometric techniques to identify the molecular products. Incubation of glucagon with purified porcine dipeptidyl peptidase IV (DP IV) yielded sequential production of glucagon(3-29) and glucagon(5-29). In human serum, degradation to glucagon(3-29) was rapidly followed by N-terminal cyclization of glucagon, preventing further DP IV-mediated hydrolysis. Bioassay of glucagon, following incubation with purified DP IV or normal rat serum demonstrated a significant loss of hyperglycemic activity, while a similar incubation in DP IV-deficient rat serum did not show any loss of glucagon bioactivity. Degradation, monitored by mass spectrometry and bioassay, was blocked by the specific DP IV inhibitor, isoleucyl thiazolidine. These results identify DP IV as a primary enzyme involved in the degradation and inactivation of glucagon. These findings have important implications for the determination of glucagon levels in human plasma.