Studies on the guinea pig pancreas. Parallel discharge of exocrine enzyme activities.

131

24-h intravenous caerulein infusion studies in the rat were combined with in vitro amino acid incorporation studies followed by high- resolution separation of proteins by two-dimensional isoelectric focusing and SDS gel electrophoresis to study the extent to which persistent changes in the biosynthesis of exocrine pancreatic proteins are regulated by cholecystokinin-like peptides. Beginning in the third hour of optimal hormone infusion at 0.25 microgram kg-1 h-1, changes were observed in the synthetic rates of 12 proteins, which progressed over the course of the 24-h study. Based on coordinate response patterns, exocrine proteins could be classified into four distinct groups. Group I (trypsinogen forms 1 and 2) showed progressive increases in synthetic rates reaching a combined 4.3-fold increase over control levels. Group II (amylase forms 1 and 2) showed progressive decreases in synthesis to levels 7.1- and 14.3-fold lower than control levels, respectively. Group III proteins (ribonuclease, chymotrypsinogen forms 1 and 2, procarboxypeptidase forms A and B, and proelastase 1) showed moderate increases in synthesis, 1.4-2.8-fold, and group IV proteins (trypsinogen 3, lipase, proelastase 2, and unidentified proteins 1-4) did not show changes in synthesis with hormone stimulation. Regulation of protein synthesis in response to caerulein infusion was specific for individual isoenzymic forms in the case of both trypsinogen and proelastase. The ratio of biosynthetic rates of trypsinogen forms 1 + 2 to amylase forms 1 + 2 increased from a control value of 0.56 to 24.4 after 24 h of hormonal stimulation (43.5-fold increase). Biosynthetic rates for an unidentified protein (P23) with an Mr = 23,000 and isoelectric point of 6.2 increased 14.2- fold, and the ratio of synthesis of P23 to amylase 2 increased 200-fold during caerulein infusion. During hormone stimulation the anticoordinate response in the synthesis of pancreatic glycosidases (decreased synthesis) and serine protease zymogens (increased synthesis) explain previous observations that showed little change in rates of total protein synthesis under similar conditions.

Section: Measurement Of Protein Synthesismentioning

confidence: 99%

Hormonal stimulation in the exocrine pancreas results in coordinate and anticoordinate regulation of protein synthesis.

Schick¹,

Kern²,

Scheele³

1984

131

“…Mechanically dissociated tumor cells or pancreatic lobules (9) prepared from normal Fischer 344 rats were pulse-labeled for 10 rain with [3HIleucine (5 #Ci/ ml) in KRH at 37 ° C, washed twice with KRH containing an excess of unlabeled leucine (4 mM final concentration), and resuspended in the same medium. After addition of secretagogues, cells or lobules were incubated for 2 h at 37°C with constant agitation under 100% 02.…”

Section: Assay For Secretory Protein Dischargementioning

confidence: 99%

Biochemical analysis of secretory proteins synthesized by normal rat pancreas and by pancreatic acinar tumor cells.

Iwanij

Jamieson

1982

We have examined the secretogogue responsiveness and the pattern of secretory proteins produced by a transplantable rat pancreatic acinar cell tumor. Dispersed tumor cells were found to discharge secretory proteins in vitro when incubated with hormones that act on four different classes of receptors: carbamylcholine, caerulein, secretin-vasoactive intestinal peptide, and bombesin. With all hormones tested, maximal discharge from tumor cells was only about one-half that of control pancreatic Iobules, but occurred at the same dose optima except for secretin, whose dose optimum was 10-fold higher. Biochemical analysis of secretory proteins discharged by the tumor cells was carried out by crossed immunoelectrophoresis and by two-dimensional isoelectric focusing-SDS polyacrylamide gel electrophoresis. To establish a baseline for comparison, secretory proteins from normal rat pancreas were identified according to enzymatic activity and correlated with migration position on two-dimensional gels.Our results indicate that a group of basic polypeptides including proelastase, basic trypsinogen, basic chymotrypsinogen, and ribonuclease, two out of three forms of procarboxypeptidase B, and the major lipase species were greatly reduced or absent in tumor cell secretion. In contrast, the amount of acidic chymotrypsinogen was notably increased compared with normal acinar cells. Although the acinar tumor cells are highly differentiated cytologically and express functional receptors for several classes of pancreatic secretagogues, they show quantitative and qualitative differences when compared with normal pancreas with regard to their production of secretory proteins.The acinar cell of the mammalian pancreas is highly differentiated and adapted for the synthesis, packaging, and discharge of secretory proteins. The anatomic organization of organdies within the cell expedites the secretory process in that the protein synthetic apparatus is located in the basal pole of the cell while secretory granules accumulate in the apical pole. Under physiologic conditions, hormones interact with receptors located on the basal plasmalemma, which in turn leads to granule release at the apical membrane. Specialized regions of the plasma membrane may play a role in directing vectorial release of secretory proteins. Evidence for such specialization includes differences in intramembrane particle distribution between basolateral and apical membranes as shown by freezefracture electron microscopy (1) as well as by marked differences in distribution of lectin binding sites (2). As we have shown in previous papers (3, 4), rat pancreatic acinar tumor cells lack polarized organization of the plasmalemma as deter-734 mined by either freeze-fracture techniques or lectin binding. Though the acinar tumor ceils contain the cellular machinery for the production and packaging of secretory proteins, it is not known whether the lack of polar organization of the plasmalemma compromises their response to peptide secretagogues.In this investigation we sough...

“…Antibodies to normal rat pancreatic secretory proteins were raised as follows. Pancreatic lobules were prepared from the glands of normal Fischer 344 rats and stimulated in vitro with 10 -~ M carbamylcholine according to Scheele (10) except that BSA and SBTI were omitted from the medium that consisted of fortified KRH (11). Secretion released into the medium was subjected to high-speed centrifugation (100,000 g,vg for 30 min) in order to remove particulate matter.…”

Section: Immunocytochemistrymentioning

confidence: 99%

Structural characterization of a rat acinar cell tumor.

Iwanij

Hull

Jamieson

1982

A transplantable acinar cell tumor of the rat pancreas has been examined by light and electron microscopy. The tumor cells, though highly cytodifferentiated and characterized by the presence of abundant rough-surfaced endoplasmic reticulum, elements of the Golgi complex, and zymogen granules, undergo mitosis in a manner similar to that seen in the developing pancreas. Cells in the parenchyma of the tumor grow as disarrayed cords and sheets, are randomly oriented with respect to each other, and do not form acinar structures. However, when in contact with the adventitial surface of blood vessels, the tumor cells palisade and form a polarized layer of cells with their zymogen granule-rich poles oriented away from the vessel lumen. Only in this area of the tumor is a basal lamina present that underlies the basal plasmalemma of the reoriented epithelial cells. Freeze-fracture electron microscopy of tumor cells in the parenchyma shows extensive disruption of tight junctions whose sealing strands are randomly distributed over the entire plasmalemma. Gap junctions are infrequent and when present are often enclosed by tight-junctional strands. Intramembrane particles are randomly distributed over the cell surface. Both the absence of basal lamina and derangement of the junctional complexes may account in part for the altered morphogenesis of this tumor.The pancreatic acinar cell has for many years been used in the study of the biosynthesis, packaging, and discharge of secretory proteins. Though many of the steps leading to the discharge of digestive enzymes and proenzymes have been identified (1, 2), the details of controls involved in the release mechanism are still obscure.The role of tissue organization and its influence upon cell function has been under consideration in our laboratory (3-5). One approach to this problem has been to examine the developing rat pancreas in an attempt to correlate the concomitant events of histogenesis and cytodifferentiation with secretagogue response (6). In this paper we characterize morphologically a pancreatic acinar cell tumor, first described by Reddy and Rao (7, 8), that shows a high level of cytodifferentiation without organization into acinar structures. This system, therefore, provides an opportunity to study the relationship between epithelial organization and the stimulated release of secretory proteins. We report here on the morphologic features of the tumor obtained from passages 14 through 20 using light and electron microscopy and freeze-fracture techniques. With this morphotogic baseline, we describe in the subsequent papers of this series the functional and biochemical properties of the tumor cells. Portions of this study have been published in abstract form (9). MATERIALS AND METHODS Tumor TransplantationFischer 344 Sprague-Dawley rats bearing tumors in the tenth passage were the generous gift of Drs. J. K. Reddy and M. S. Rao of Northwestern University. The tumors were passaged in weanling Sprague-Dawley Fischer 344 rats (HarlanSprague Dawley, Madison, Wl) a...