Structure-Function Relationships in the Pancreatic Acinar Cell

39.3.1

Extracellular Matrix

Typical of all epithelia, acinar units and ducts of the pancreas are embedded in an ECM. All cells found in the ECM synthesize ECM molecules; ECM precursor or stem cells are derived from embryonic mesenchyme. Thus, the resident cells of the ECM, with the exception of transients such as phagocytic and immune cells, are capable of forming all of the cellular and molecular elements of the ECM.

The ECM has a number of functions including to organize and support the epithelium. Collagen and elastin fibrils provide tensile strength and resiliency, respectively, for the gland proper while reticular fibrils of Type III collagen form a meshwork that supports the parenchyma as it does in all glandular structures. The proteoglycans in which the structural elements of the gland's ECM are embedded serve as a reservoir for water and electrolytes. The ECM binds cytokines and growth factors and so makes them available when needed. Tissue viscosity/stiffness is largely a function of the ECM and can signal to cells in the matrix as well as epithelial cells to modulate gene expression, tissue development, and differentiation. Mechanisms linking ECM signaling to the nucleus for regulating transcriptional activity have been described. Although the importance of tissue stiffness has been recognized in other tissues, such as the liver, it has not been studied in the pancreas, but should have important roles in the development, maintenance of the differentiated state, regeneration, and responses to fibrosis.

The ECM also acts as a physical barrier for infectious agents and is the site where phagocytic and immune cells play their roles during infections and inflammatory processes. A number of nonepithelial cell types including macrophages, stellate (Ito) cells, mast cells, and cells of the T and B lymphocytic line are embedded in the ECM. The formation of the majority of the ECM appears to be under the control of the stellate cell. In the resting state, prominent Vitamin A-filled vesicles in their cytoplasm can identify these cells. After activation, the Vitamin-A laden vesicles disappear and stellate cells proliferate and produce collagen. Such stimulation can occur during development, tissue repair, or regeneration. Some pathologic conditions, such as chronic pancreatitis and pancreatic cancers, are associated with persistent stellate cell activation and excess pancreatic fibrosis and positive feedback for proliferation between pancreatic cancer and stellate cells is described.

Integrating the ECM with the epithelial cells of the pancreas is the BM ( Fig. 39.5 ). The BM directly underlies the basal plasma membrane of the epithelium and is produced by the epithelial cells. It is composed of a cage of Type IV collagen in which is embedded a number of BM matrix components including negatively charged heparin sulfate proteoglycans and linker molecules such as laminin, nidogen, and perlecan. The linker molecule laminin has an important role in integrating the structure and function of the overlying epithelium with the ECM. Laminin is a multivalent molecule with binding sites for integrins on the basal plasma membrane of epithelial cells, proteoglycans, ECM collagen and the surfaces of ECM cells such as fibroblasts, adipocytes, and capillary endothelial cells. Because of these multivalent functions and the molecules to which it binds, the epithelium and the ECM are effectively a structural continuum.

The extensive functional interactions between epithelial cells and the ECM is often mediated by proteins of the integrin family. These transmembrane proteins are located in the basal plasma membranes of virtually all epithelial cells. In this location, they serve as receptors for various extracellular ligands, such as laminin, fibronectin, and collagens. Although they traditionally have been viewed as mechanical links between the overlying epithelium and the BM at focal contacts, and hence to the ECM, their function is much broader. By virtue of the fact that the cytoplasmic portion of most integrins are linked indirectly to the actin cytoskeleton and to organized signal arrays within the cell, integrins are able to transmit information from the extracellular environment into the cell. This information takes a variety of forms that are now being understood, including activation of signaling pathways for growth and differentiation and activation of transcription. Thus, signals from outside the cell arising in the external environment/ECM can affect cell function in the short term such as potentially changing the patterns of transcription of secretory proteins, and they may have long-term effects such as progression to neoplasia.

39.3.2

Intercellular Junctions

All cells in an epithelium, including the duct and acinar cells of the pancreas, are attached to each other by junctional complexes. These are functionally classified into three groups: (1) sealing or tight junctions; (2) adhering junctions, and (3) communicating or gap junctions. These structures are distinguished by their distribution, morphologic appearance, protein composition, and function.

39.3.2.1

Tight Junctions

Among the junctional complexes, sealing or tight junctions are always found closest to the lumen of glands and ducts and are classically described as providing an impermeable barrier to the intermixing of ions and other molecules between luminal contents and the interstitial space. As shown in Fig. 39.6 , transmission electron microscopy demonstrates that tight junctions are discrete structures within closely opposed regions of plasma membrane. Filamentous bands on the cytoplasmic side of tight junctions represent regions of attachment of cytoplasmic actin filaments. Freeze-fracture preparations that reveal the distribution of proteins within membranes show that tight junctions are composed of interdigitating bands that form a contiguous collar around the apical region of the cell ( Fig. 39.6 B).

Over a dozen proteins contribute to the tight junction including occludins, ZO1 and ZO2, and the claudins. The latter is a family of tetra-spanning membrane proteins, the claudins (from the Latin claudere “to close”). This family of proteins are the major determinant tight-junction permeability. Homophilic and heterophilic claudin complexes form strong adhesive structures between epithelial cells. Claudins are expressed in an epithelial-specific fashion; for example, claudin 16 is highly expressed in epithelial cells of the proximal tubules of the kidney, but little is found in other tissues. In the pancreas, claudin 2 is found only in junctions of the duct epithelia, claudin 5 is limited to junctions between acinar cells, whereas claudin 3 and claudin 4 are in both locations. Notably, tissue claudin expression shows plasticity that can change paracellular permeability in adult tissues.

Claudins are central to the structural functional basis of tight junctions and central to forming a highly selective barrier that both restricts the paracellular movement of large and small molecules and facilitates passage of certain ions between the lumen and the interstitial space. Ion selectivity of the paracellular pathway is determined by the charges of amino acids on the extracellular domain of tight-junction claudins. Claudin mutations are the cause of several human diseases including familial hypomagnesemia caused by mutations in claudin 16 (also known as paracellin-1) in distal tubules of the kidney. More recently, variants in claudin 2 have been found to be a risk factor for developing alcoholic pancreatitis as well as nonalcoholic pancreatitis, though the mechanism remains unclear.

ZO1 and ZO2 and possibly other proteins such as cingulin indirectly link claudins and possibly occludins to the actin cytoskeleton. Because of this association, it has been suggested that actin microfilaments, containing myosin II, may also be involved in the modulation of the paracellular pathway in response to signals. A variety of signaling molecules including cytokines can upregulate or downregulate paracellular permeability. These functions are effected by changing the levels of junctional proteins or by effecting their phosphorylation. For example, TGF-α increases permeability while EGF decreases permeability. The tight junction and other components of junctional complexes that forms an impermeant barrier ( Fig. 39.6 C) become disordered early in the course of experimental pancreatitis.

In addition to its central role in restricting the bidirectional movement of soluble molecules, tight junctions also help to establish and maintain acinar cell polarity by preventing the intermixing of membrane proteins and lipids between the apical and basolateral domains of epithelial cells by diffusion.

39.3.2.3

Gap Junctions

Epithelial cells are functionally connected to each other through gap junctions that allow direct communication between the cytoplasm of adjacent acinar cells ( Fig. 39.7 ). A distinct protein family known as connexins assembles in hexamers to form small transmembrane pores between cells known as connexons. Large rounded groupings of connexons forming gap junctions are found in the lateral membranes of adjacent cells. The connexon pores demonstrate size and ionic selectivity and do not allow molecules with molecular weights > 1000 to pass. Their main physiological functions are to coordinate signaling between cells by allowing the movement of signaling molecules such as calcium, inositol trisphosphate (IP3), and cAMP through the junction. Thus, hormonal signals impinging on one or a few cells in an acinus are coupled to synchronize the exocytosis of zymogen granules. The channels also close rapidly in response to intracellular acidification or high levels of cytosolic calcium, preventing spread of damage between cells in a wounded epithelium.

The importance of acinar cell gap junctions has been examined using chemical inhibitors and mutant mice that are deficient in specific connexins and studied in the pancreas and other tissues. Several generalizations can be drawn from these studies. First, pancreatic acinar cells are coupled by gap junctions containing connexins 26 and 32 that allow molecular messengers, including calcium and other signaling molecules, to spread from one cell to the next. In mice lacking connexin 32 the number of gaps junctions is greatly reduced and intercellular communication assessed using a transferable dye is inhibited. Interestingly, genetic absence of connexin 32 caused increased pancreatic enzyme secretion, but electrical coupling was maintained. These findings suggest that gap junctions might suppress pancreatic secretion, but electrical coupling can be mediated by gap junctions formed only by connexin 26. Notably, the connexin 32 ^−/− mice exhibited a much more severe form of caerulein [a cholecystokinin (CCK) analogue] hyperstimulation pancreatitis than wild-type controls. These studies suggest that the overall effect of the gap junctions formed by connexin 32 in pancreatic acinar cells is to dampen both basal secretion and the pancreatitis response to hyperstimulation. Although intracellular pH and calcium levels can affect gap-junction function, the extracellular environment can also affect the biogenesis of this organelle. Thus, reducing extracellular pH affects the turnover rate of acinar cell connexon-32 and gap-junction coupling. Such a response could worsen pancreatitis responses if blood flow is reduced, causing tissue acidosis, during acute pancreatitis. Finally, an unexplored contribution to pancreatic injury and inflammation may come from hemi-channels formed by connexons or a related family of protein known as pannexins. The hemi-channels are plasma membrane pores that are not connected to opposing cells. Although normally closed, they could open under pathologic conditions releasing cellular ATP into the interstitium and stimulate innate immunity.

39.4.1

Protein Synthesis

The synthesis of new secretory proteins occurs on and in the ER. Two ER forms can be distinguished morphologically and have distinct functions: the rough ER (RER) and smooth ER (SER). The RER can be recognized by the ribosomes on its cytoplasmic surface, while the SER lacks ribosomes ( Figs. 39.8 and 39.9 ). Specialized regions that are contiguous with the RER and SER have been termed transitional elements. The ER exit sites, locations in the transitional ER, a specialized domain in which both the membrane and soluble proteins are transported from the ER in COPII coated vesicles (see Section 39.4.4 ). The RER, transitional elements, and SER enclose ( Fig. 39.9 ) a contiguous lumen known as the intracisternal space. Within this space, nascent export proteins, such as those destined for zymogen granules and the lysosome, begin their folding and covalent modifications as described below (see Section 39.4.3 ). The acinar cell has an extensive RER to meet its requirements for high levels of secretory protein synthesis. Although the RER is most evident at the base of the acinar cell, it extends up to the apical region of the cell ( Figs. 39.1 and 39.8 ). The width of the RER cisterna ranges from 20 to 80 nm and enlarges during active protein synthesis; all cisternae appear to be contiguous. Sites of active protein synthesis are marked by regularly placed rosettes of electron dense ~ 30 nm ribosomes that are attached to the cytosolic face of the ER membrane ( Fig. 39.8 ). These represent areas active in translation and are ribosomes associated with messenger RNA (mRNA).

Morphologic and functional studies provide evidence that there are mechanisms for regional specialization within the ER. For example, in some regions of the ER ribosomes are attached to the cytoplasmic face at regular intervals (RER) while other regions of the ER are devoid of ribosomes. Functional specialization of the ER is also demonstrated by calcium signaling. Thus, the initial increase in cytosolic calcium in the acinar cell occurs at the apex of the cell and represents release from specific regions of the ER. This particular phenomenon results from the localization of the IP3 calcium channel to the SER at the apical-most region of the acinar cell (see below). Distinct structural links between the ER and other membrane compartments have been observed by electron microscopy, but insight into how these contacts are formed and their physiologic functions has only recently been described. Contacts between the ER and mitochondria and ER and plasma membrane are among those that have been described. For example, pancreatic acinar cell calcium homeostasis involving the apical region of the cell revolves around distinct apically distributed IP3 receptors (ER) that release calcium into the cytosol and closely approximated regulators of calcium entry into the ER (Orai1, STIM, and others) that are activated when depletion of ER calcium is sensed. Specific proteins, such as extended synaptotagmin I, are required to establish these specialized membrane domains that bring the ER and plasma membrane into close stable proximity. Membrane contacts, similar to those that tether the ER and mitochondria and that facilitate the exchange of glycerol lipids between opposed membranes, are likely to participate in content exchange between other tethered cellular organelles.

The ribosome is formed by large and small subunits of the ribosome macromolecular complexes of proteins and specific ribosomal RNA; the large and small subunit can be distinguished by transmission electron microscopy. New protein synthesis takes place in a groove that lies between the two subunits and is directed by mRNA. Specific groups of soluble proteins regulate the initiation, elongation, and termination phases of protein synthesis. The first synthesized component of a secretory protein is a specific amino-terminal extension known as the signal sequence. The signal sequence is 15–50 amino-acid peptides that contains a hydrophobic core. Also known as a leader sequence , the signal sequence allows nascent proteins to enter the ER through a macromolecular multiprotein channel known as the translocon. This signal sequence binds to a signal recognition particle (SRP) that directs the connection of the early nascent protein and mRNA to the ER by attachment to its receptor, SRP receptor. After attachment, the SRP is released and the signal peptide enters the ER protein-conducting channel. As a nascent secretory protein enters the ER, the signal sequence is removed by a resident protease, trapping the protein in the ER lumen. The translocon also mediates the insertion of transmembrane proteins into the ER membrane and has a role in protein degradation (discussed elsewhere). In addition to its critical role in the initial steps of protein synthesis, the ER has other specialized functions that include cell signaling, protein folding, and specific protein modifications.

39.4.2

Vectorial Movement of Proteins

The sequential movement of nascent proteins between secretory compartments was first demonstrated using the pancreatic acinar cell and pulse-chase protocols. In this type of study, isolated cells or tissues were transiently incubated with tracer amounts of a radioactive amino acid and then this was washed away and replaced with unlabeled amino acids. Nascent proteins incorporate the pulse of radioactive amino acids and can be followed as they traverse the cell. The labeled proteins can be detected using autoradiography or by assaying isolated subcellular fractions for radioactive amino acids incorporated into proteins ( Fig. 39.12 ). This approach demonstrated that nascent proteins moved through the acinar cell in a vectorial manner over time from their site of synthesis in the ER to be stored in zymogen granules. The classic studies demonstrating the stepwise movement of new secretory proteins used a pulse of tracer amounts of a radioactive amino acid followed by a large excess of the nonradioactive form of the amino acid to label nascent protein. The itinerary of the newly synthesized radio-labeled protein was tracked over time using electron microscopy with autoradiography ( Fig. 39.12 ) as well as cell-fractionation studies. These studies showed that nascent proteins quickly left the ER to reside in the Golgi complex, then moved from the Golgi complex to condensing vacuoles (CV) [immature secretory granules (ISGs)], and finally to zymogen granules. Note that it takes almost 60 min for the first nascent protein to reach zymogen granules. Subsequent studies have examined the conditions for nascent proteins entering the ER, the regulation of vesicular movement between compartments, and the distinct protein modifications that occur in specific organelles and are summarized below.

Nascent secretory proteins are subject to a variety of modifications; many of these occur within specific cellular organelles. Protein modifications often require the activity of accessory resident proteins that are concentrated in specific organelles. The mechanisms that restrict resident proteins to specific organelles are not fully understood. However, many soluble resident ER proteins contain the amino acid sequence KDEL or a similar sequence that binds the ER resident KDEL receptor, a seven-transmembrane domain protein. While the KDEL receptor and KDEL proteins can escape from the ER and travel to the Golgi, they are efficiently retrieved using retrograde vesicular transport back to the ER. Similarly, a KKxx cytoplasmic sequence that binds to COPI proteins enables ER membrane proteins to return to the ER from the Golgi complex. The KDEL receptor has a structure similar to that of a heterotrimeric G-protein and may function to regulate both antegrade and retrograde movement of KDEL containing proteins by interacting with distinct signaling pathways.

39.4.3