The acinar cell secretes digestive proteins in response to stimulation
To study secretion at the cellular level, investigators use enzymatically separated single acini (15 to 100 cells) or mechanically dissected single lobules (250 to 1000 cells). The measure of secretion is the release of digestive proteins into the incubation medium. The amount released over a fixed time interval is expressed as a percentage of the total content at the outset of the experiment. Because amylase is released in a fully active form, it is common to use the appearance of amylase activity as a marker for secretion by acinar cells.
When the acinar cells are in an unstimulated state, they secrete low levels of digestive proteins via a constitutive secretory pathway. Acinar cells stimulated by neurohumoral agents secrete proteins via a regulated pathway. Regulated secretion by isolated acinar cells in vitro is detected within 5 minutes of stimulation and is energy dependent. During a 30- to 60-minute stimulation period, acinar cells typically secrete 5 to 10 times more amylase than with constitutive release. However, during this period of regulated secretion, the cells typically secrete only 10% to 20% of the digestive proteins stored in their granules. Moreover, acinar cells are able to increase their rate of protein synthesis to replenish their stores.
The acinar cell exhibits two distinct patterns of regulated secretion: monophasic and biphasic (Fig. 43-3A). Increasing levels of an agonist that generates a monophasic dose-response relationship (e.g., gastrin-releasing peptide [GRP]) causes secretion to reach a maximal level that does not fall with higher concentrations of the agent. In contrast, increasing levels of a secretagogue that elicits a biphasic dose-response relationship (e.g., cholecystokinin and carbachol) causes secretion to reach a maximal level that subsequently diminishes. As discussed below, this biphasic response may reflect the presence of functionally separate high-affinity and low-affinity receptors, and is related to the pathogenesis of acute pancreatitis. N43-1
FIGURE 43-3 Effects of neurohumoral agents on amylase release from isolated pancreatic acini. (A, Data from Jensen RT: Receptors on pancreatic acinar cells. In Johnson LR [ed]: Physiology of the Gastrointestinal Tract. New York, Raven Press, 1994, pp 1377–1446; B, data from Burnham DB, McChesney DJ, Thurston KC, Williams JA: Interaction of cholecystokinin and vasoactive polypeptide on function of mouse pancreatic acini in vitro. J Physiol 349:475–482, 1984.)
Contributed by Fred Gorelick
Acute pancreatitis is an inflammatory condition that may cause extensive local damage to the pancreas as well as compromise the function of other organs such as the lungs. The most common factors that initiate human acute pancreatitis are alcohol ingestion and gallstones. However, other insults may also precipitate acute pancreatitis. Hypertriglyceridemia, an inherited disorder of lipid metabolism, is one such culprit. Less commonly, toxins that increase ACh levels, such as cholinesterase inhibitors (some insecticides) or the sting of scorpions found in the Caribbean and South and Central America, may lead to pancreatitis. Supraphysiological levels of ACh probably cause pancreatitis by overstimulating the pancreatic acinar cell.
Experimental models of pancreatitis suggest a primary defect in protein processing and acinar cell secretory function. More than 100 years ago, it was found that treating animals with doses of ACh that are 10 to 100 times greater than those that elicit maximal enzyme secretion causes “hyperstimulation” pancreatitis. The same type of injury can be generated by CCK. The injury in this model appears to be linked to two events within the acinar cell: (1) Zymogens, in particular proteases, are pathologically processed within the acinar cell into active forms. In this model, the protective mechanisms outlined in Table 43-3 are overwhelmed, and active enzymes are generated within the acinar cell. (2) Acinar cell secretion is inhibited, and the active enzymes are retained within the cell. Although premature activation of zymogens is probably an important step in initiating pancreatitis, other events are important for perpetuating injury, including inflammation, induction of apoptosis, vascular injury, and occlusion that results in decreased blood flow and reduced tissue oxygenation (ischemia).
Knowledge of the mechanisms of acute pancreatitis may lead to effective therapies. In experimental models, administration of serine protease inhibitors that block the activation of pancreatic zymogens improves the course of the acute pancreatitis. In some clinical forms of pancreatitis, prophylactic administration of the protease inhibitor gabexate appears to reduce the severity of the disease.
Acetylcholine and cholecystokinin mediate the regulated secretion of proteins by pancreatic acinar cells
Although at least a dozen different receptors are present on the plasma membrane of the pancreatic acinar cell, the most important in regulating protein secretion is the M3 muscarinic acetylcholine (ACh) receptor (see p. 341), located on the basolateral membrane and also found in many glandular tissues.
Two closely related receptors for cholecystokinin (CCK) are distinguished by their structure, affinity for ligands, and tissue distribution (see p. 867). Although both CCK receptors may be activated by CCK or gastrin, the CCK1 receptor (previously known as CCKA, encoded by the CCK1R gene) has a much higher affinity for CCK than for gastrin, whereas the CCK2 receptor (previously known as CCKB, encoded by CCK2R gene) has approximately equal affinities for CCK and gastrin. In some species, both forms of the CCK receptor are present on the acinar cell. Although CCK1 receptors are present in human acinar cells, their function is unknown. N43-2
Affinity States of the Cholecystokinin Receptors
Contributed by Fred Suchy
An important feature of both CCK receptors is their ability to exist in both a high-affinity and a low-affinity state. Low (picomolar) concentrations of CCK activate the high-affinity forms of the CCK receptors and stimulate secretion. Conversely, supraphysiological (10- to 100-fold higher) concentrations of CCK activate the low-affinity forms of the receptors and inhibit secretion. These two affinity states (i.e., activated by different concentrations of CCK) of each of the two CCK receptors generate distinct second-messenger signaling patterns. It is likely that under physiological conditions, only the high-affinity states of the CCK or muscarinic receptor are activated. Stimulation of the lower-affinity states by supraphysiological concentrations of either CCK or ACh not only inhibits enzyme secretion but also may injure the acinar cell. N43-1
M3 and CCK receptors have many similarities: both are basolateral, both are linked to the Gαq heterotrimeric G protein, both use the phospholipase C (PLC)/Ca2+ signal-transduction pathway (see pp. 58–60), and both lead to increased enzyme secretion from the acinar cell.
Numerous other receptors—including those for gastrin-releasing peptide (GRP; see p. 868), calcitonin gene–related peptide (CGRP; see p. 1067), insulin (see pp. 1035–1050), secretin (see pp. 886–887), somatostatin (see pp. 993–994), and vasoactive intestinal peptide (VIP; see Table 41-1)—are also found on the pancreatic acinar cell. Although these other receptors may also play a role in regulating secretion, protein synthesis, growth, and transformation, their precise physiological functions remain to be clearly defined.
Activation of receptors that stimulate different signal-transduction pathways may lead to an enhanced secretory response. For example, as shown in Figure 43-3B, simultaneous stimulation of the high-affinity CCK receptor (which acts via [Ca2+]i) and the VIP receptor (which acts via cAMP) generates an additive effect on secretion. Alternatively, acinar cells that have previously been stimulated may become temporarily refractory to subsequent stimulation. This phenomenon is known as desensitization.
Ca2+ is the major second messenger for the secretion of proteins by pancreatic acinar cells
Much of the pioneering work on the role of intracellular Ca2+ in cell signaling has been performed on the pancreatic acinar cell (Fig. 43-4A). Generation of a cytosolic Ca2+ signal is a complex summation of cellular events (see p. 60). Even when the acinar cell is in the resting state, the cytosolic free Ca2+ level ([Ca2+]i) oscillates slowly. Maximal stimulatory (i.e., physiological) concentrations of CCK or ACh increase the frequency of the oscillations (see Fig. 43-4B) but have less effect on the amplitude. This increase in the frequency of [Ca2+]i oscillations is required for protein secretion by acinar cells. In contrast, a supramaximal (i.e., hyperstimulatory) concentration of CCK or ACh generates a sudden [Ca2+]i spike that is 2 to 10 times greater than that seen with physiological stimulation and eliminates further [Ca2+]i oscillations. This [Ca2+]i spike and the subsequent absence of oscillations are associated with an inhibition of secretion that appears to be mediated by disruption of the cytoskeletal components that are required for secretion.
FIGURE 43-4 Stimulation of protein secretion from the pancreatic acinar cell. A, The pancreatic acinar cell has at least two pathways for stimulating the insertion of zymogen granules and thus releasing digestive enzymes. B, Applying a physiological dose of CCK (i.e., 10 pM) triggers a series of [Ca2+]i oscillations. However, applying a supraphysiological concentration of CCK (1 nM)—a dose that could initiate pancreatitis—elicits a single large [Ca2+]i spike, halts the oscillations, and decreases amylase secretion. Recall from Figure 43-3 that high levels of CCK also are less effective in causing amylase secretion. AC, adenylyl cyclase; CaM, calmodulin; PIP2, phosphatidylinositol 4,5-bisphosphate; PK, protein kinases other than PKA and PKC; PP, phosphoprotein phosphatases. (B, Data from Tsunoda Y, Stuenkel EL, Williams JA: Characterization of sustained [Ca2+]i increase in pancreatic acinar cells and its relation to amylase secretion. Am J Physiol 259:G792–G801, 1990.)
Secretin, VIP, and CCK increase cAMP production and thus activate protein kinase A (PKA) in pancreatic acinar cells (see Fig. 43-4A). Low concentrations of CCK cause a transient stimulation of PKA, whereas supraphysiological concentrations of CCK cause a much more prominent and prolonged increase in [cAMP]i and PKA activity. One of the effects of cAMP is to enhance secretion that has been stimulated by activation of Ca2+-dependent pathways (see Fig. 43-3B). N43-3 ACh has little, if any, effect on the cAMP signaling pathway.
Effect of cAMP on the EPAC/RAP1 Pathway
Contributed by Fred Gorelick, Emile Boulpaep, Walter Boron
cAMP can also activate the EPAC/RAP1 pathways, but the physiologic consequences of such activation have not been defined. RAP1 is a small Ras-like GTPase. EPAC is the guanine nucleotide exchange factor (GEF) for RAP1 (see p. 56).
As illustrated in Figure 43-4A, the most important effectors of intracellular second messengers are the protein kinases. Stimulation of CCK and muscarinic receptors on the acinar cell leads to the generation of similar Ca2+ signals and activation of calmodulin-dependent protein kinases (see p. 60) and members of the protein kinase C (PKC) family (see pp. 60–61). Activation of secretin or VIP receptors increases [cAMP]iand thus activates PKA. These second messengers probably also activate phosphoprotein phosphatases, as well as other protein kinases not depicted in Figure 43-4A. Some protein targets of activated kinases and phosphatases in the pancreatic acinar cell are involved in regulating secretion, whereas others mediate protein synthesis, growth, transformation, and cell death.
In addition to proteins, the pancreatic acinar cell secretes a plasma-like fluid
Besides secreting proteins, acinar cells in the pancreas secrete an isotonic, plasma-like fluid (Fig. 43-5). This NaCl-rich fluid hydrates the dense, protein-rich material that the acinar cells secrete. The fundamental transport event is the secretion of Cl− across the apical membrane. N43-4 For transcellular (plasma-to-lumen) movement of Cl− to occur, Cl− must move into the cell across the basolateral membrane. As in many other Cl−-secreting epithelial cells (see p. 139), in the acinar cell basolateral Cl− uptake occurs via an Na/K/Cl cotransporter. The Na-K pump generates the Na+ gradient that energizes the Na/K/Cl cotransporter. The K+ entering through the Na-K pump and via the Na/K/Cl cotransporter exits through K+ channels that are also located on the basolateral membrane. Thus, a pump, a cotransporter, and a channel are necessary to sustain the basolateral uptake of Cl− into the acinar cell.
FIGURE 43-5 Stimulation of isotonic NaCl secretion by the pancreatic acinar cell. Both ACh and CCK stimulate NaCl secretion, probably through phosphorylation of basolateral and apical ion channels.
Cl− Secretion into Pancreatic Secretory Vesicles
Contributed by Fred Gorelick
As we have seen in the text (see p. 882), the secretory vesicles in the pancreatic acinar cell fuse with the apical membrane in the process of exocytosis. In the process, these cells release their protein into the lumen of the acinus.
Before the exocytotic event, these secretory vesicles, vacuolar-type H pumps in the vesicle membrane, use the energy of ATP hydrolysis to transport H+ from the cytosol to the lumen of the vesicle. This transport process sets up both a chemical gradient for H+ (inside acid) and an electrical gradient (inside positive) across the vesicle membrane. Cl− channels in the vesicle membrane can then allow Cl− to flow into the vesicle lumen, in parallel with the H+, so that the overall process is HCl movement from cytosol to vesicle lumen. Water follows by osmosis.
The above “secretion” of minute amounts of Cl− into the secretory vesicle may contribute to the hydration of proteins within the granule before vesicle fusion.
The rise in [Cl−]i produced by basolateral Cl− uptake drives the secretion of Cl− down its electrochemical gradient through channels in the apical membrane. As the transepithelial voltage becomes more lumen negative, Na+ moves through the cation-selective paracellular pathway (i.e., tight junctions) to join the Cl− secreted into the lumen. Water also moves through this paracellular pathway, as well as via aquaporin water channels on the apical and basolateral membranes. Therefore, the net effect of these acinar cell transport processes is the production of an isotonic, NaCl-rich fluid that accounts for ~25% of total pancreatic fluid secretion.
Like the secretion of protein by acinar cells, secretion of fluid and electrolytes is stimulated by secretagogues that raise [Ca2+]i. In the pancreas, activation of muscarinic receptors by cholinergic neural pathways and activation of CCK receptors by humoral pathways increase the membrane conductance of the acinar cell. A similar effect is seen with GRP. Apical membrane Cl− channels and basolateral membrane K+channels appear to be the effector targets of the activated Ca2+ signaling pathway. Phosphorylation of these channels by Ca2+-dependent kinases is one likely mechanism that underlies the increase in open channel probability that accompanies stimulation.