Evidence for a precursor for TRH in the neonatal rat pancreas

Immunoreactive TRH-OH is present at low concentrations in acid extracts from 2- days old rat pancreas. The sequential treatment of these extracts with trypsin and carboxypeptidase A is followed by a three- and ten-fold increase in TRH-OH IR respectively. The molecular weight of the protein that gives rise to TRH-OH after enzymatic treatment ranges between 30000 and 40000 daltons. The appearance of TRH-OH in the tryptic digest suggests that TRH-OH is the COOH-terminal sequence of this protein. These results are the first evidence that TRH biosynthesis occurs through a large molecule precursor. However, this is an indirect demonstration since TRH cannot be generated under these conditions due to the lack of enzymatic amidation activity.     

Ontogenesis of TRH mRNA in the rat pancreas

The rapid changes in TRH levels in the rat pancreas during the neonatal period make this organ an interesting model for the study of the regulation of TRH biosynthesis. Pancreatic RNAs were isolated by the guanidinium thiocyanate method and layered onto CsCl cushion. Northern blot preparations were hybridized with 32P labeled TRH cDNA probe. Pancreatic TRH mRNA was first detected in 19-day old fetuses and reached the highest level on day 0, then decreased, being barely detectable 14 days after birth. The neonatal injection of streptozotocin induced a dramatic drop of TRH mRNA levels 24 hours later. This result suggests that the peculiar evolution of TRH level in pancreas is partly due to the evolution of the expression of the TRH gene.     

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.     

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     

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     

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.     

Effect of pectin on the function of the rat exocrine pancreas

The author studied the effect of three weeks' administration of a 5% pectin concentration in the standard (Larsen) diet on basal and cholecystokinin octapeptide-(CCK8-) stimulated pancreatic exosecretion in rats. Protein, amylase and trypsinogen output determined in pancreatic juice obtained under basal conditions and after CCK8 stimulation of the exocrine pancreas showed no statistically significant differences between rats fed on the standard and the pectin-enriched Larsen diet. Our observations and studies by other authors show that the direct effect of dietary fibre on the exocrine function of the pancreas is an open question as far as further research is concerned.     

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     

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.     

Cytological effects of ionophore-induced stimulation on the exocrine pancreas of the rat

Summary Rat-pancreas lobules were incubated with the ionophore A-23187 in the presence of Ca²⁺. After 90 min, some of the acini were partially or almost completely depleted of their zymogen granules while others had the appearance of resting acini. With few exceptions, the cells of a given acinus were degranulated to a comparable level. Slight dispersion of the zymogen granules was noticed in cells incubated in a Ca²⁺-free medium containing EGTA with or without A-23187. In the presence of Ca²⁺ the secretory response obtained with the ionophore was comparable to that observed with 10^-5M urecholine. The results obtained provide cytological evidence that the secretory response is only partially determined at the membrane-receptor level and that other mechanisms intervene between cytosol Ca²⁺ increase and exocytosis.     

Regulation of TRH release by the cultured neonate rat pancreas

The TRH secretory responsiveness of the pancreatic islet cell clusters from newborn rat in organ culture was studied. Basal TRH secretion was stable over a 9-day period. The response to various secretagogues was tested on day 4. TRH secretion was stimulated by high potassium-induced depolarization and also through both cAMP and protein kinase-C dependent pathways. Like insulin, TRH release was stimulated by glucose and arginine and inhibited by somatostatin. These data suggest the existence of a common mechanism for TRH and insulin secretion by the pancreatic β-cells.     

Effects of chronic administration of somatostatin on rat exocrine pancreas

We studied the effects of somatostatin on synthesis of pancreatic DNA, RNA and protein and on pancreatic weight and contents of DNA, protein, amylase and chymotrypsinogen in rats. In short term synthesis studies, rats were injected with 100 micrograms . kg-1 somatostatin or 0.15 M NaCl (control) at times 0, 8 and 16 h. Eight rats from each treatment group were killed 2, 4, 8, 12, 16, 20 and 24 h after beginning treatment. Incorporation rates in vivo of [3H]thymidine into DNA, [3H]uridine into RNA and [14C]phenylalanine into total protein were significantly depressed by somatostatin. In long term studies, four groups of 12 rats were injected every 8 h for 5 days with 0.15 M NaCl or 11, 33 or 100 micrograms . kg-1 somatostatin. Body weight was unaffected but pancreatic contents of DNA, protein and enzymes were significantly decreased by somatostatin. Administration of somatostatin inhibits DNA, RNA and protein synthesis in exocrine pancreas with resulting decreases in DNA and enzyme contents.     

Effect of glucagon on the secretory process in the rat exocrine pancreas

Summary Glucagon was infused into conscious rats in doses of 10 to 80 g/h for periods up to 24 h. The effect on the secretory process of the exocrine pancreas was studied in vitro using isolated pancreatic lobules. A pronounced inhibition of the rate of protein synthesis and discharge of stored and newly synthesized proteins combined with increased enzyme content in the pancreas were observed after 30 min infusion. This effect was absent after longer infusion periods of up to six hours. After 12 to 24 h infusions a marked degranulation and decrease in enzyme content was observed. While the rate of protein synthesis was not significantly enhanced, both the basal and stimulated discharge of enzymes from the pancreas were increased. The results suggest a biphasic response of the pancreas to prolonged glucagon infusion.Dedicated to Professor Helmut Ferner, Vienna, Austria, on the occasion of his 65th birthday     

Studies on intracellular transport of secretory proteins in the rat exocrine pancreas

Summary Prolonged secretory stimulation of the exocrine pancreas in the rat by in vivo infusion of caerulein leads to a rapid degranulation of the organ associated with a progressive reduction in the size of the zymogen granules. During the first six to twelve hours of stimulation Golgi complexes are enlarged and several structural forms of multivesicular bodies are found indicating a lysosomal degradation of membrane material in the Golgi area. Maximum secretory activity is obtained after a 24 hour infusion, Golgi complexes appear fragmented, the secretory granules measure only 1/3 to 1/4 their normal size. Thereafter, in spite of a continuous stimulation, the exocrine cells regranulate progressively up to 72 hours of infusion. This regranulation is associated with massive enlargement of the Golgi complexes.The phasic adaptation of the exocrine pancreas to prolonged stimulation, concluded from the structural studies, was confirmed by biochemical analysis of protein synthesis, intracellular transport and enzyme discharge. Pancreatic protein synthesis as measured by the incorporation of tritiated leucine remained unchanged during the first six hours of stimulation, then increased reaching a maximum of 230% of the control levels after 24 hours of infusion. After 48 and 72 hours the rate of protein synthesis decreased again to normal values. Most pronounced changes were observed in the kinetics of intracellular transport of newly synthesized proteins. Using pulse-chase incubation of prestimulated pancreatic lobules, the rate of transition of secretory proteins through the cell increased consistently with prolonged infusion periods reaching maximal acceleration after 24 hours. Newly synthesized proteins were transported and segregated up to ten times faster than in controls. After a maximum at 24 hours transport returned to normal rates after 72 hours of infusion. Enzyme secretion, measured for amylase, followed a similar pattern of stimulation.The results suggest a phasic adaptation of the exocrine pancreatic cell to prolonged stimulation. They demonstrate for the first time the possibility of an acceleration of intracellular transport by means of secretagogues.Dedicated to Professor W. Bargmann on the occasion of his 70th birthday.Supported by a grant from Deutsche Forschungsgemeinschaft (Ke 113/8). A preliminary communication was presented at the 9th annual meeting of the European Society for Clinical Investigation, Rotterdam (April 24–26, 1975). The expert technical assistance of Miss Helga Hollerbach and Miss Hiltraud Hosser is gratefully acknowledged.