Experiments have been carried out to determine whether intracellular transport of pancreatic secretory proteins is obligatorily coupled to protein synthesis or whether it is a separable process which can be independently regulated. To this intent, guinea pig pancreatic slices were pulse labeled with leucine-3H for 3 rain and incubated post-pulse for 37 rain in chase medium containing cycloheximide up to concentrations sufficient to inhibit protein synthesis by 98 %. In controls, newly synthesized secretory proteins are transported over this interval to condensing vacuoles of the Golgi complex. Since the latter are recovered in the zymogen granule fraction upon cell fractionatlon, intracellular transport was assayed by measuring the amount of protein radioactivity found in the zymogen granule fraction after a (3 + 37) min incubation. The results indicated that at maximum inhibition of protein synthesis (5 X 10 -4 M cycloheximide), transport proceeded with an efficiency ~80% of control. Parallel radioautographic studies on intact slices confirmed these data and further indicated that all the steps of intracellular transport, including discharge to the acinar lumen, were independent of protein synthesis. We conclude that: (1) transport and protein synthesis are separable processes; (2) intracellular transport is not the result of a continuous delivery of secretory proteins from attached polysomes to the cisternae of the rough endoplasmic reticulum; and (3) transport is not dependent on the synthesis of "specific" nonsecretory proteins within the time limits tested.

Our previous observations on the synthesis and transport of secretory proteins in the pancreatic exocrine cell were made on pancreatic slices from starved guinea pigs and accordingly apply to the resting, unstimulated cell. Normally, however, the gland functions in cycles during which zymogen granules accumulate in the cell and are subsequently discharged from it in response to secretogogues. The present experiments were undertaken to determine if secretory stimuli applied in vitro result in adjustments in the rates of protein synthesis and/or of intracellular transport. To this intent pancreatic slices from starved animals were stimulated in vitro for 3 hr with 0.01 mM carbamylcholine. During the first hour of treatment the acinar lumen profile is markedly enlarged due to insertion of zymogen granule membranes into the apical plasmalemma accompanying exocytosis of the granule content. Between 2 and 3 hr of stimulation the luminal profile reverts to unstimulated dimensions while depletion of the granule population nears completion. The acinar cells in 3-hr stimulated slices are characterized by the virtual complete absence of typical condensing vacuoles and zymogen granules, contain a markedly enlarged Golgi complex consisting of numerous stacked cisternae and electron-opaque vesicles, and possess many small pleomorphic storage granules. Slices in this condition were pulse labeled with leucine-3H and the route and timetable of intracellular transport assessed during chase incubation by cell fractionation, electron microscope radioautography, and a discharge assay covering the entire secretory pathway. The results showed that the rate of protein synthesis, the rate of drainage of the rough-surfaced endoplasmic reticulum (RER) compartment, and the over-all transit time of secretory proteins through the cells was not accelerated by the secretogogue. Secretory stimulation did not lead to a rerouting of secretory proteins through the cell sap. In the resting cell, the secretory product is concentrated in condensing vacuoles and stored as a relatively homogeneous population of spherical zymogen granules. By contrast, in the stimulated cell, secretory proteins are initially concentrated in the flattened saccules of the enlarged Golgi complex and subsequently stored in numerous small storage granules before release. The results suggest that secretory stimuli applied in vitro primarily affect the discharge of secretory proteins and do not, directly or indirectly, influence their rates of synthesis and intracellular transport.

Large populations (up to 600/cell) of spherical, electron-opaque granules ~ 0.3 to 0.4/~ in diameter are characteristically found in muscle fibers of mammalian atria. They are absent in muscle fibers of the ventricles. The granules arc concentrated in the sarcoplasmic core and occur in lesser numbers in the sarcoplasmic layers between myofibrils and under the plasma membrane. Their intimate association with a central voluminous Golgi complex and the frequent occurrence of material reminiscent of the granular content within the cisternae of the Golgi complex suggest that the latter is involved in the formation of the atrial granules. Atrial granules are larger and more numerous in smaller species (rat, mouse), and generally smaller and less numerous in larger mammals (dog, cat, human); they are absent from the atrial fibers of very young fetuses (rat) but are present in those of newborn animals. A small population of bodies containing glycogen particles and remnants of the endoplasmic reticulum and mitochondria occurs in the sarcoplasmic cores of atrial as well as ventricular muscle fibers in the rat; they contain acid phosphatase and thus appear to be residual bodies of autolytic loci. Their frequency increases with the age of the animal. Typical lipofuscin pigment granules, which are known to contain acid phosphatase and are found in the sarcoplasmic cores in old animals (cat, dog and human), are presumed to arise by progressive aggregation and fusion of small residual bodies.

It has been established by electron microscopic radioautography of guinea pig pancreatic exocrine cells (Caro and Palade, 1964) that secretory proteins are transported from the elements of the rough-surfaced endoplasmic reticulum (ER) to condensing vacuoles of the Golgi complex possibly via small vesicles located in the periphery of the complex. To define more clearly the role of these vesicles in the intracellular transport of secretory proteins, we have investigated the secretory cycle of the guinea pig pancreas by cell fractionation procedures applied to pancreatic slices incubated in vitro. Such slices remain viable for 3 hr and incur minimal structural damage in this time. Their secretory proteins can be labeled with radioactive amino acids in short, well defined pulses which, followed by cell fractionation, makes possible a kinetic analysis of transport. To determine the kinetics of transport, we pulse-labeled sets of slices for 3 min with leucine-' 4 C and incubated them for further +7, +17, and +57 min in chase medium. At each time, smooth microsomes (=peripheral elements of the Golgi complex) and rough microsomes (= elements of the rough ER) were isolated from the slices by density gradient centrifugation of the total microsomal fraction. Labeled proteins appeared initially (end of pulse) in the rough microsomes and were subsequently transferred during incubation in chase medium to the smooth microsomes, reaching a maximal concentration in this fraction after +7 min chase incubation. Later, labeled proteins left the smooth microsomes to appear in the zymogen granule fraction. These data provide direct evidence that secretory proteins are transported from the cisternae of the rough ER to condensing vacuoles via the small vesicles of the Golgi complex.

We have studied a transplantable carcinoma of the rat pancreas [Reddy, J. K. & Rao, M. S. (1977) Science 198,[78][79][80] Classically, the BL has been viewed as a host barrier through which a malignant tumor must gain the ability to invade. In fact, BL is normally a specialized product of the overlying epithelial cells (3-5), which also plays a central role as a stabilizer of epithelial form and orientation in embryogenesis (3) and is maintained throughout adult life. Thus, it is possible that neoplastic disorganization of epithelial architecture as well as malignant invasion may result either from loss of maintenance of this epithelial scaffolding or through the acquisition ofsome new transformed cell product that compromises its structural integrity.In order to investigate the role of BL in the maintenance of organized tissue structures as well as neoplastic disorganization, we have studied a transplantable carcinoma of the rat exocrine pancreas as a model system. This tumor is composed of cytologically differentiated acinar cells that have lost their normal epithelial organization. The tumor was discovered, with associated metastatic foci, in nafenopin-treated rats in the laboratory of Reddy and Rao (6) and was kindly provided to us for study. As the BL is a complex of different collagenous and noncollagenous macromolecules, the distribution of two ubiquitous BL constituents, the glycoprotein laminin (Lm) (7) and type IV collagen (type IV) (8), was studied in both the tumor and normal pancreas, using indirect immunofluorescence. A preliminary account of these studies has appeared in abstract form (9). MATERIALS AND METHODSExperimental System. Weanling male F344 Sprague-Dawley rats (Harlan-Sprague-Dawley, Madison, WI) were inoculated subcutaneously or intraperitoneally with a mechanically prepared suspension of the pancreatic acinar carcinoma in isotonic saline. All tumors used in this study were between the 18th and 23rd passage, 1-4 cm in diameter, and displayed consistent growth characteristics and morphology. In our laboratory, the tumor grows as a nonmetastatic carcinoma. Normal pancreas was obtained from either tumor-bearing animals or non-tumor-bearing animals; the distribution of Lm and type IV was identical in both.Light

The present study describes a novel phenomenon in pancreatic acinar cells undergoing regulated exocytosis. When acinar cell preparations were challenged with the secretagogue carbamylcholine, a subpopulation of zymogen granules became coated with filamentous actin. These zymogen granules were always in proximity of the acinar cell apical membrane (the site of exocytosis) but did not appear to have fused yet. They were distinct from regular zymogen granules not only because of their association with filamentous actin, but also because the majority of them lacked the zymogen granule marker rab3D, a small GTPase implicated in regulated exocytosis. The apparent loss of rab3D, presumed to result from the release of rab3D from the granule membranes, could be prevented by agents that modulate the actomyosin system as well as by GTP [␥S]. These data suggest that zymogen granules engaging in exocytosis become coated with actin before fusion and that this actin coating is tightly coupled to the release of rab3D. We propose that rab3D is involved in the regulation of actin polymerization around secretory granules and that actin coating might facilitate the movement of granules across the subapical actin network and toward their fusion site.

No abstract

Rab3D, a member of the Rab3 subfamily of the Rab/ypt GTPases, is expressed on zymogen granules in the pancreas as well as on secretory vesicles in mast cells and in the parotid gland. To shed light on the function of Rab3D, we have generated Rab3D-deficient mice. These mice are viable and have no obvious phenotypic changes. Secretion of mast cells is normal as revealed by capacitance patch clamping. Furthermore, enzyme content and overall morphology are unchanged in pancreatic and parotid acinar cells of knockout mice. Both the exocrine pancreas and the parotid gland show normal release kinetics in response to secretagogue stimulation, suggesting that Rab3D is not involved in exocytosis. However, the size of secretory granules in both the exocrine pancreas and the parotid gland is significantly increased, with the volume being doubled. We conclude that Rab3D exerts its function during granule maturation, possibly by preventing homotypic fusion of secretory granules.