Ultrastructural changes in the rat exocrine pancreas after jejunoileal bypass

The influence of a 90% jejunoileal bypass on the rat exocrine pancreas was studied by morphometrical procedures. In sham-operated animals exocrine acinar cells accounted for 80.3% of the pancreas volume. These cells are composed of 9.9% nuclei, 8.4% mitochondria, 12.2% zymogen granules, 0.3% lipid droplets and 69.2% of a compartment ("ERGLS") composed of endoplasmic reticulum, ribosomes, Golgi areas, lysosomes and the cytoplasmic ground substance. Intestinal bypass did not change the volume density of exocrine cells nor that of nuclei in the cells during the three postoperative months. The means nuclear diameter was approximately the same in both groups. However, the volume density of secretory granules diminished by 50%. This was accompanied by a decrease in mean granular diameter, but not in their numerical density. The volume density of lipid droplets increased 10 fold, that of mitochondria increased slightly from the 15th postoperative day but significantly from the 45th day. The remaining cellular compartment composed of "ERGLS" was not modified by intestinal bypass. These findings suggest that a 90% jejunoileal bypass induces major changes in the composition of pancreatic acinar cells but not in their size.     

Immunohistochemical localization of exocrine enzymes in normal rat pancreas.

On the basis of morphological and biochemical differences, the exocrine pancreatic tissue has been divided in peri- and teleinsular regions. In the present study the enzymatic profile of these regions has been investigated by the immunofluorescent technique using antibodies against nine pancreatic enzymes (alpha-amylase, lipase, chymotrypsinogen A, trypsinogen, elastase, carboxypeptidase A and B, DNase and RNase A). These antibodies were specific to their antigens without cross reaction. By immunofluorescence, most acinar cells of the normal rat pancreas were positive to the nine enzymes tested. However, an inhomogeneity in the staining pattern was found; specifically, the cells located in the periinsular region of many islets showed a brighter fluorescence than acinar cells in the teleinsular tissue. These data add a new parameter to describe the inhomogeneity of the exocrine pancreas.     

Amino acid transport in the rat exocrine pancreas

Summary Amino acid transport and incorporation have been studied in vitro in rat pancreatic lobules after maximal and supramaximal hormonal stimulation with caerulein. Incorporation into proteins was increased already after 30 and 120 min of maximal stimulation, but was decreased after the infusion of a supramaximal dose. Uptake of neutral amino acids was monitored using labeled leucine and -aminoisobutyric acid (AIB). In the case of leucine the free pool was consistently reduced after maximal stimulation, while supramaximal doses led to an increase which could be potentiated by the addition of 2mM tetracaine. Using AIB, a significant increase in the intracellular pool was observed after maximal stimulation, conversely a decrease after supramaximal stimulation. Release of labeled leucine and AIB from preloaded lobules during incubation in the cold was significantly reduced after maximal secretory stimulation, but was found enhanced by 200 to 300 percent after supramaximal stimulation. No fine structural alterations at junctional complexes or at both the lateral and luminal plasma membranes were observed after maximal stimulation except an increased number of exocytotic figures at the luminal face. However, supramaximal stimulation led to progressive rarefaction of the tight junctional network and disintegration of the gap junctions. Concomitantly, an equal distribution of membrane particles on both faces of the plasma membrane together with a random occurrence of exocytotic figures were observed.Supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (SFB 122, project C 5). Dedicated to Professor Dr. Gerhard Petry, Marburg, on the occasion of his 65th birthday     

Amino acid transport in the rat exocrine pancreas

Summary The two calcium antagonistic agents lanthanum and tetracaine cause severe disturbances in the secretory process of the exocrine pancreas, including inhibition of the rate of protein synthesis and exocytosis. The former effect resulted mainly from the inhibition of amino acid transport. Lanthanum in a concentration up to 1 mM inhibited transport of different species of amino acids in an unspecific way whereas tetracaine interfered specifically with the Na⁺-dependent transport system for neutral amino acids (¹⁴C--amino-isobutyric acid). Na⁺-independent transport of neutral amino acids (³H-leucine) was not affected. Transport inhibition was correlated to the activity of the Na⁺, K⁺-ATPase system which was measured in isolated plasma membrane fractions. At higher concentrations (5–10 mM) some uptake of lanthanum into the cells by limited endocytosis was observed. At lower concentrations lanthanum seemed to bind exclusively to certain components of the plasma membrane, mainly at the lateral and basal cell surface. Even at a concentration of 5–10 mM, no binding to the apical surface occurred. Similarly, no binding of lanthanum was observed to the limiting membrane of isolated zymogen granules, while mitochondria, contained in the same fraction, showed considerable binding affinity. The action of lanthanum and tetracaine on membrane carrier systems did not affect the interior organization of the plasma membrane. Particle density and distribution in freeze-fracture replicas as well as the submembrane microfilamentous-microtubular system and the junctional elements remained unaffected.Supported by a grant from the Deutsche Forschungsgemeinschaft (Ke 113/10). The expert technical assistance of Miss Helga Hollerbach and Miss Hiltraud Hosser and the editorial help of Mrs. Gisela Lesch is gratefully acknowledged     

Beta adrenergic receptor localization in rat brain by light microscopic autoradiography

The characteristics of ³H-dihydroalprenolol (³H-DHA) binding to mounted tissue sections of rat brain were studied. The binding had all the characteristics of a beta-receptor. It was reversible, saturable (K_D 2.3 nM, B_max 23 fmol/mg tissue, wet wt) and was inhibited only by beta-adrenergic drugs. The forebrain binding had the properties of a beta-1 receptor, while cerebellar binding had beta-2 characteristics. Beta-adrenergic receptors were widely distributed in the rat brain. High concentrations were localized in the superficial layers of the neocortex, in the caudate-putamen, nucleus accumbens, olfactory tubercles, substantia nigra, nucleus interpeduncularis, subiculum and pia mater. Areas containing intermediate concentrations of receptors included the cerebellum, hippocampus and thalamus. Areas containing low concentrations of receptors included the hypothalamus, amygdala, brainstem and medulla. Co-incubation with low concentrations of zinterol to preferentially block beta-2 receptor resulted in an inhibition of most of the binding to the cerebellum and pia matter and produced only a small and generalized decrease in the rest of the brain. Beta receptors were found in many areas known to contain noradrenergic nerve terminals. Paradoxically some areas with high densities of dopaminergic nerve terminals, had high densities of receptors. Results of electrophysiologica and lesion studies are also discussed.     

Studies on secretory glycoproteins in the rat exocrine pancreas

Summary Using a double-label technique on isolated rat pancreatic lobules, the rate of synthesis and discharge of regular and fucosylated secretory proteins was studied under control conditions and after in vivo prestimulation with caerulein. Both labeled leucine and fucose were incorporated into pancreatic proteins at a linear rate, which was potentiated by in vivo stimulation. In pulse-chase experiments both regular and fucosylated secretory proteins were discharged into the medium in parallel. The in vivo pretreatment with caerulein caused an earlier discharge and increased the total amount released. Kinetic analysis of unstimulated (baseline) discharge of both classes of secretory proteins indicated a striking in vitro sensitivity by the previous in vivo treatment with caerulein.The biochemical data were compared to the fine structure of the Golgi complex under both control and prestimulated conditions. The Golgi stacks were composed of four to six individual cisternae which in some cases were connected by intercisternal pores. Transporting vesicles were observed fusing along the total length of the outermost cisterna on both the cis- and transside and with the lateral ends of the intermediate cisternae. Under control conditions only the last trans-cisterna contained some electron opaque material; in vivo prestimulation led to distension and filling of all cisternae in an individual Golgi-unit. Numerous stages of transformation of the last transcisterna into condensing vacuoles were observed, lending support to the hypothesis that during packaging of secretory products the membranes of the Golgi complex undergo a continuous turnover.Supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn-Bad-Godesberg (Ke 113/10). The competent technical assistance of Miss Hiltraud Hosser and Miss Helga Hollerbach and the editorial help of Miss Annemarie Erben is gratefully acknowledged     

Ultrastructural analysis of the effect of vagotomy on the state of the exocrine portion of the rat pancreas

Dynamics of reactions in the main components of the exocrine part of the rat pancreas (capillary, nerve, pancreocyte) was subjected to ultrastructural analysis at bilateral subdiaphragmatic vagotomy within 24 hours--30 days. It was stated that certain ultrastructural shifts developed in all the components mentioned above; at first they are within the limits of physiological parameters (the first three days), and then destructive processes occur (up to destruction of a part of cells) reaching their maximum by the end of the first--beginning of the second week and having a focal character. Then compensatory-recovery rearrangements begin to predominate, and by the 30th day they result in a considerable but not yet complete normalization of the ultrastructural organization of the gland. Three stages (phases) can be mentioned in the development of the neurodistrophic process: the first--stress reaction of the organ and the whole organism to the operation; the second--predominance of destructive changes; the third--development of compensatory-adaptive processes. Possible mechanisms of the rearrangements occurring are discussed.     

Studies on secretory glycoproteins in the rat exocrine pancreas

Summary A fetuin, fucosyl transferase has been identified in the smooth microsomal fraction from the rat exocrine pancreas. This enzyme is involved in the glycosylation of secretory proteins and is bound to membranes, predominantly of the Golgi complex. Optimal in vitro conditions for the assay of the enzyme activity were established: a pH of 5.5–6.0, a temperature of 21° C and concentrations of Mg^{+ +} at 5.0 mM and ATP at 2.0 mM.Supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (Ke 113/10). Dedicated to Professor Helmut Ferner, Vienna, on the occasion of his 65^th birthday.     

Immunohistochemical localization of glandular kallikrein in the endocrine and exocrine human pancreas

Fab fragments from two new monospecific anti-human tissue kallikrein sera were examined for their capacity to inhibit the functional activities of purified human urinary kallikrein and purified human pancreatic kallikrein. Fragments from a new anti-urinary kallikrein serum and from an anti-pancreatic kallikrein serum yielded mixed inhibition of kinin-generating activity and minimal inhibition of esterolytic activity. In contrast to the previously described "active site directed" anti-urinary kallikrein, these new antisera demonstrated little specificity for epitopes near the enzymatic site of urinary or pancreatic kallikrein. When used to localize kallikrein antigen in human pancreas obtained at surgery, IgG fractions of the new anti-kallikrein sera yielded moderate acinar and ductal staining in the absence of pretreatment of the tissue with trypsin or pronase. Short incubation with 0.125 mg/ml of either enzyme permitted the discrete localization of islet beta cell kallikrein antigen, while increased pronase concentrations decreased kallikrein antigen in both islets and exocrine tissue and led to islet destruction. Both antibody specificity and tissue preparation influence kallikrein localization in human pancreas.     

Ultrastructural, morphometrical and immunocytochemical analyses of the exocrine pancreas in a hibernating dormouse

Pancreatic acinar cells of euthermic, hibernating and arousing individuals of the hazel dormouse Muscardinus avellanarius (Gliridae) have been observed at the electron-microscopic level and analysed by means of ultrastructural morphometry and immunocytochemistry in order to investigate possible fine structural changes of cellular components during periods of strikingly different degrees of metabolic activity. During hibernation, the cisternae of the rough endoplasmic reticulum (RER) flatten assuming a parallel pattern, the Golgi apparatus is extremely reduced and the mitochondria contain many electron-dense particles. The cell nuclei appear irregularly shaped, with deep indentations containing small zymogen granules. They also contain abundant coiled bodies and unusual constituents, such as amorphous bodies and dense granular bodies. Large numbers of zymogen granules occur in all animals. However, the acinar lumina are open and filled with zymogen only in euthermic animals, whereas, in hibernating and arousing individuals, they appear to be closed. Morphometrical analyses indicate that, in pancreatic acinar cells, nuclei and zymogen granules significantly decrease in size from euthermia to hibernation, probably reflecting a drastic decrease of metabolic activities, mainly protein synthesis and processing. In all the studied animals, immunocytochemistry with specific antibodies has revealed an increasing gradient in α-amylase content along the RER-Golgi-zymogen granule pathway, reflecting the protein concentration along the secretory pathway. Moreover, during deep hibernation, significantly larger amounts of α-amylase accumulate in RER and zymogen granules in comparison to the other seasonal phases analysed. Upon arousal, all cytoplasmic and nuclear constituents restore their euthermic aspect and all morphometrical and immunocytochemical parameters exhibit the euthermic values, thereby indicating a rapid resumption of metabolic activities.     

Circadian rhythmicity in the rat exocrine pancreas: chronomorphological patterns

Circadian rhythmicity of the structural morphometric model of rat endocrine pancreas has been studied in 24 Wistar female rats, four months old, kept in LD 12:12. The following parameters were evaluated: the volume fractions of nucleus and cytoplasm of exocrine cells, the size distribution and number in unit tissue volume of acinar cell nuclei, the mean nuclear diameter, the shape coefficient of glandular acini (that is the ratio acinar area/perimeter2 which indicates the shifting of structures from circularity). A statistically significant circadian rhythm was demonstrated for the shape coefficient of glandular acini. Results obtained in the present experiment are compared with data recorded in a previous study.     

Ultrastructural localization of osteocalcin in rat tooth germs by immunogold staining

Summary Osteocalcin was localized by indirect immunogold staining of thin frozen sections of rat tooth germs which had been fixed by different methods. Acrolein fixation proved to be satisfactory considering the preservation of fine structure and antigenicity. In odontoblasts, osteocalcin was found to be localized in the cisternae of the rough endoplasmic reticulum and Golgi apparatus. Few positive transport vesicles were found. Staining for osteocalcin in odontoblastic processes was only observed after strong fixation and was intense in odontoblasts engaged in early dentine formation. Predentine was slightly positive in the neighbourhood of positive processes. Matrix vesicles were negative and strong osteocalcin labeling of dentine seemed to appear after the onset of mineralization.     

Ultrastructural localization of osteocalcin in rat tooth germs by immunogold staining

Osteocalcin was localized by indirect immunogold staining of thin frozen sections of rat tooth germs which had been fixed by different methods. Acrolein fixation proved to be satisfactory considering the preservation of fine structure and antigenicity. In odontoblasts, osteocalcin was found to be localized in the cisternae of the rough endoplasmic reticulum and Golgi apparatus. Few positive transport vesicles were found. Staining for osteocalcin in odontoblastic processes was only observed after strong fixation and was intense in odontoblasts engaged in early dentine formation. Predentine was slightly positive in the neighbourhood of positive processes. Matrix vesicles were negative and strong osteocalcin labeling of dentine seemed to appear after the onset of mineralization.     

Ultrastructural localization of laminin in rat sensory ganglia

We adapted immunocytochemical methods for localization of laminin to examine its disposition in neural tissue at the ultrastructural level. In dorsal root ganglia, laminin was found in basal laminae of the satellite and Schwann cells ensheathing neuronal perikarya and nerve fibers, respectively, and around blood vessels. Within the basal lamina, the immunostain was found in the lamina lucida and lamina densa. Occasional immunostained coated pits were identified in satellite and Schwann cells, but virtually no intracellular label was seen even in freeze-thawed/detergent-permeabilized specimens. In the perineurium, only the basal lamina of the inward-facing surface of the inner-most cell layer was usually stained.