Fig. 1
The acinar-ductal functional unit. The acinar cells secrete an isotonic NaCl-rich fluid. Cl− enters the acinar cells through basolateral Na+-K+-2Cl− cotransporters, which can operate due to the Ca2+-activated K+ channels and the Na+/K+ pump. Thus, the net transport across the basolateral membrane is uptake of Cl−. Cl− leaves the acinar cells via the Ca2+-activated Cl− channels in the apical (luminal) membrane. Whereas Cl− is transported through the acinar cells, Na+ moves from the interstitial fluid to the acinar lumen via the leaky tight junctions and water follows through both the tight (leaky) junctional pathway and through the cells [water movements not shown in order not to overcrowd the diagram]. (The secretion processes are activated by a rise in the cytosolic Ca2+ concentration evoked by stimulation of acetylcholine and/or cholecystokinin receptors on the basolateral acinar cell membrane, which results in the production of intracellular Ca2+-releasing messengers [not shown in this figure]). The zymogen granules of the acinar cells contain hundreds of proteins and bioactive molecules including more than 20 different digestive (pro)enzymes. These molecules are stored in an acidic fluid composed by ion transport mechanisms shown in the figure. The ductal cells secrete an isotonic HCO3 −-rich fluid and this process is mediated by the basolateral and apical ion transport mechanisms indicated. (This process is stimulated by the hormone secretin, which evokes intracellular production of cyclic AMP [not shown in this figure]). Luminal Cl− and Ca2+, guanylin, angiotensin II, and ATP stimulate HCO3 − efflux from ductal cells, whereas Na+ and HCO3 − in the lumen, and most probably some unknown bioactive molecules secreted by acinar cells, inhibit HCO3 − influx. This coordinated finely tuned process will provide around 2 l/day of an isotonic solution rich in bicarbonate and digestive enzymes. CFTR cystic fibrosis transmembrane conductance regulator, Gua guanylin, ATII angiotensin II, ATP adenosine-5′-triphosphate, ZG zymogen granules, TJ tight (leaky) junctions
Importantly, if the coordinated finely tuned secretion is damaged by either of the cell types, severe pancreatic damage may occur which will of course not be confined to the impaired cell type (Hegyi et al. 2011b). For example, impaired ductal fluid secretion and lower intraluminal pH which occurs in cystic fibrosis (CF) lead to acinar cell dysfunction too (Freedman et al. 2001), whereas mutations of cationic trypsinogen (PRSS1) (Whitcomb et al. 1996), trypsin inhibitors (SPINK1) (Witt et al. 2000), or chymotrypsin C (CTRC) (Rosendahl et al. 2008) can elevate the risk of (or lead to) chronic pancreatitis in which case the ductal bicarbonate secretion is also impaired.
In this chapter, we uniquely bring the acinar and ductal cells together and summarize our current knowledge of their communication paths in physiology and pathophysiology.
2 Physiological Aspects of the Acinar-Ductal Interactions
The pancreas has a “tree-and branch-like” structure with secretory endpieces in the form of acini connected to branches of small ducts merging into larger ducts finally connected to the main pancreatic duct. Acinar cells within individual acinar units are electrically coupled via gap-junctional channels (Petersen and Findlay 1987). Ultrastructural studies show that the density of gap junctions between adjacent acinar cells is extremely high with the major part of the lateral plasma membranes dominated by these structures (Meda et al. 1983), which accounts for the complete electrical coupling (coupling ratio between adjacent cells = 1) between acinar cells within individual acinar units (Iwatsuki and Petersen 1978). On the other hand, no electrical coupling has been found between acinar cells in different neighboring acinar units, indicating the absence of gap-junctional channels between acinar and duct cells (Iwatsuki and Petersen 1978). There is also direct chemical coupling between acinar cells within individual acinar units as demonstrated by direct visualization of the movements of the fluorescent dyes, fluorescein or Lucifer Yellow (mol. wt. 457) injected into one acinar cell to adjacent cells, eventually spreading to the whole acinar unit (Iwatsuki and Petersen 1979; Findlay and Petersen 1983). Lucifer Yellow injected into acinar cells was never detected in duct cells, confirming the absence of gap-junctional coupling between adjacent acinar and duct cells (Findlay and Petersen 1983). Sustained intra-acinar injection of Lucifer Yellow revealed a finite limit to the extent of dye spread and serial sections of pancreatic tissue fixed after complete dye spread allowed reconstruction of the whole acinar network, which turned out to consist of ~100 to 250 cells (Findlay and Petersen 1983) in reasonable agreement with the electrophysiological analysis indicating that there might be up to 500 well-coupled cells in individual acinar units (Iwatsuki and Petersen 1978). Acinar cells within individual acinar units can be acutely and reversibly uncoupled, both electrically and chemically, by supramaximal stimulation with acetylcholine (ACh) or cholecystokinin (CCK) (Iwatsuki and Petersen 1978; Petersen and Findlay 1987). The functional importance of the gap-junctional communication between acinar cells is not understood, but sharing of the relatively sparse functional innervations and/or sharing of the relatively low density of high-conductance Ca2+-activated K+ channels may be important (Petersen and Findlay 1987). The uncoupling observed in response to supramaximal stimulation is most likely a protective mechanism designed to isolate a cell that experiences a dangerously high level of cytosolic-free Ca2+ or a low intracellular pH (Petersen and Findlay 1987). The absence of direct coupling between acinar and duct cells may be functionally important as it allows separate fine regulation of these two cell types without messengers, for example, inositol triphosphate (IP3) and cyclic adenosine monophosphate (cAMP) generated in one cell type necessarily invading the other type.
3 Composition of Pancreatic Juice Secreted by Acinar Cells to the Ductal Lumen
The neurotransmitter acetylcholine and the neuropeptide cholecystokinine stimulate the acinar cells to secrete an isotonic NaCl and protein-rich juice into the ductal system (Petersen 2008).
3.1 Pancreatic Digestive Enzymes and Other Proteins
The acinar cells secrete over 20 different enzymes including more than 10 different proteases (which is the major enzyme product of acinar cells), lipases, ribonucleases, amylases, and hydrolases (Keller and Allan 1967; Rinderknecht 1993; Whitcomb and Lowe 2007). They are generally synthesized and secreted in an inactive form (proenzymes) in order to avoid autodigestion of the organ. The physiological pancreatic enzyme cascade starts only in the duodenum with the activation of trypsinogen by enteropeptidases; subsequently, the active trypsin will activate the other proenzymes. To avoid inappropriate intrapancreatic activation of proteases, pancreatic acinar cells secrete trypsin inhibitors (pancreatic secretory trypsin inhibitor [PSTI]) (Kazal et al. 1948) which are capable of trapping active trypsin. Adenosine-5′-triphosphate (ATP) was found to be co-released with enzymes in response to cholinergic and hormonal stimuli (Haanes and Novak 2010). ATP is transported via vesicular nucleotide transporters which can be stimulated by elevation of exogenous pH or Cl− concentration (Haanes and Novak 2010). Recently, proteomic analyses of rat zymogen granules (ZGs) found 371 different proteins including enzymes, membrane proteins, juice proteins, transporters, and channels (Rindler et al. 2007). Although additional proteomics analysis confirmed the large quantity of bioactive molecules (Chen and Andrews 2008, 2009), their physiological roles need to be understood.
3.2 Ions
The acinar cells secrete little in the absence of nervous or hormonal stimulation, but are activated by ACh released from parasympathetic nerve endings surrounding the acini or by the hormone CCK. Both these physiological stimuli activate mechanisms that result in repetitive cytosolic Ca2+ spikes driving both acinar fluid and enzyme secretion. The intracellular Ca2+ signaling mechanisms have been extensively reviewed (Petersen 1992, 2005; Petersen and Tepikin 2008) and will therefore not be dealt with in any detail here.
The isotonic NaCl-rich fluid produced by the acinar cells in response to stimulation is the result of ion transports occurring across both the basolateral and apical plasma membranes. At the basolateral membrane, the crucial transport proteins are the Ca2+– and voltage-activated K+ channels (surprisingly not expressed in mouse or rat pancreatic acinar cells), but clearly present and important in, for example, pig and human pancreatic acinar cells (Petersen and Maruyama 1984; Petersen et al. 1985), the Na+-K+ pump and the Na+-2Cl−-K+ cotransporter, functioning together as a Cl− uptake mechanism, whereas at the apical (luminal) membrane the most important transporter is the Ca2+-activated Cl− channel (Petersen and Findlay 1987; Park et al. 2001). Cl− thus passes through the acinar cells, whereas Na+ moves from the interstitial fluid to the acinar lumen via the intercellular pathway through the leaky tight junctions (Petersen and Findlay 1987). The crucially important exclusive localization of the Ca2+-activated Cl− channels (CLCs) at the apical membrane has been demonstrated directly in studies combining high-resolution Ca2+ imaging and patch clamp current recording, in which it was shown that the Cl− channels could only be activated by specifically uncaging caged Ca2+ near the apical, but not the basolateral membrane (Park et al. 2001).
The membrane of pancreatic acinar ZGs also contains several ion channels, which may or may not play a role in the formation of the fluid content secreted by acinar cells into the ductal lumen. Protons are taken up into the ZG via a H+-ATPase pump (Thevenod et al. 1989; Behrendorff et al. 2010) which is electrically balanced by influx of Cl− (Pazoles and Pollard 1978; Gasser et al. 1988) via diisothiocyanostilbene disulfonate (DIDS)-sensitive CLC-2 or CLC-3 Cl− channels (Thevenod 2002; Kelly et al. 2005). Inwardly rectifying K+-8 (IRK-8) (Kelly et al. 2005), potassium voltage-gated channel, subfamily Q, member 1 (KCNQ1) (Braun and Thevenod 2000; Lee et al. 2008) and two-pore domain weakly inward rectifying (TWIK-2) (Rindler et al. 2007) K+ channels were shown to be the major proteins driving K+ inside the granule. The DIDS-sensitive Ca2+-activated CLCA channel will drive HCO3 − inside the granules which may promote exocytosis (Quinton 2001; Thevenod et al. 2003). Proteomic analyses suggested additional type of transporters such as Cl−/K+ cotransporter and Na+/K+ ATPase; however, their functional confirmation still needs to be done (Rindler et al. 2007).
Although it might be tempting to suggest that acinar fluid secretion is a consequence of the exocytotic insertion of ZG membrane into the apical plasma membrane, evidence from the (in many ways rather similar) parotid gland suggests that this is unlikely to be true. In the parotid gland, exocytotic enzyme secretion is principally stimulated by activation of beta-adrenergic receptors, whereas fluid secretion is mainly stimulated by activation of muscarinic and alpha-adrenergic receptors. It has been shown that beta-adrenergic stimulation hardly produces any conductance changes in the acinar membranes, whereas cholinergic and alpha-adrenergic stimulation massively increases the membrane conductance (Iwatsuki and Petersen 1981). When, during continuous beta-adrenergic stimulation – evoking a steady and high level of amylase secretion – cholinergic stimulation is applied, this results in a dramatic increase in acinar membrane conductance with only a very minor increase in amylase secretion (Iwatsuki and Petersen 1981). This is incompatible with the view that the conductance changes necessary for the acinar fluid secretion are due to insertion of granule membrane into the apical plasma membrane, but suggests strongly that fluid secretion is due to activation of ion channels in the plasma membrane entirely unconnected with the ZGs.
4 Sensing Mechanisms of the Ductal Cells
There are many molecules/ions secreted by the acinar cells that regulate the physiological process of pancreatic ductal bicarbonate secretion. In this chapter, we summarize the current knowledge of the effects of acinar fluid contents on ductal secretion and highlight future lines of research to understand the regulation of pancreatic fluid secretion.
4.1 Ions in the Pancreatic Juice Effecting Pancreatic Ductal Secretion
As we discussed above, pancreatic acinar cells secrete an isotonic juice including Na+, Cl−, H+, Ca2+ into the pancreatic ducts (Petersen 2008). The ductal secretory processes are very much dependent on the ionic composition of the luminal fluid. Elevation of the luminal Cl− and Ca2+ concentration stimulates HCO3 − efflux via the Cl−/HCO3 − exchangers elevating the luminal HCO3 − concentration, whereas elevation of the Na+ concentration stimulates HCO3 − influx thus decreasing the luminal HCO3 − concentration in the pancreatic ductal tree. HCO3 − in an autocrine manner dose dependently inhibits both the (cystic fibrosis transmembrane conductance regulator) CFTR Cl− channel and the Cl−/HCO3 − exchangers.
4.2 Chloride and Bicarbonate
Elevation of the Cl− concentration in the ductal lumen will stimulate the HCO3 − secretion providing the anionic substrate of the Cl−/HCO3 − exchangers from the luminal side (Park et al. 2010). In addition, luminal Cl− is also essential for the activity of CFTR Cl− channel (Wright et al. 2004), which highlights the crucial importance of acinar Cl− secretion. Generally, in physiological circumstances, the acinar fluid contains 25 mM HCO3 − and 135 mM Cl−, which will reach the apical membrane of the proximal duct where the Cl−/HCO3 − exchange starts (Park et al. 2010). By the time the fluid leaves the pancreas, the ratio has been turned around to 140 mM HCO3 − and 20 mM Cl−, due to the ductal activity of the CFTR Cl− channel and the Cl−/HCO3 − exchangers (Park et al. 2010). It is important to highlight that the high HCO3 − and the low Cl− concentration will inhibit both the CFTR Cl− channel and the Cl−/HCO3 − exchangers, thereby preventing bicarbonate reabsorption via the apical membrane of the duct cells (Wright et al. 2004; Park et al. 2010).
4.3 Sodium
Much less is known about the physiological importance of intraluminal Na+. It has been shown that there are Na+/H+ exchangers (NHE2 and 3) (Marteau et al. 1995; Lee et al. 2000; Rakonczay et al. 2006) and Na+/HCO3 − cotransporters (Luo et al. 2001; Park et al. 2002) on the apical membrane of the ductal cells. Generally the activity of both transporter types will decrease the luminal HCO3 − concentration, thus retrieving luminal bicarbonate. There is evidence suggesting that the epithelial electrolyte and water secretion at the cellular level is not only mediated by “stimulatory pathways” but also by “inhibitory pathways” (Hegyi et al. 2003; Hegyi and Rakonczay 2007; Kemeny et al. 2011). Such inhibitory pathways may be important in terms of reducing the hydrostatic pressure within the ductal lumen (preventing leakage of enzymes into the parenchyma of the pancreas), and in terms of switching off pancreatic secretion after the digestion has finished (Aponte et al. 1989). Substance P (Hegyi et al. 2003, 2005b; Kemeny et al. 2011), basolaterally applied ATP (Ishiguro et al. 1999), and 5-hydroxytryptamine (Suzuki et al. 2001) have all been shown to inhibit secretion from isolated pancreatic ducts, but their effects on the apical sodium transporters are unknown.
4.4 Hydrogen
One of the more recent findings in the physiology of acinar fluid secretion is that acinar cells co-release H+ during exocytosis causing a significant acidosis in the proximal tubes of the pancreatic ducts (Behrendorff et al. 2010). Physiological concentrations of the secretagogue CCK (10–20 pM) decrease the luminal pH up to 1 pH unit and cause short-lasting extracellular acidifications during exocytosis (Behrendorff et al. 2010). Then, the protons are quickly neutralized by the alkaline fluid secreted by the proximal ductal epithelia (Hegyi et al. 2011a). Importantly, a pathophysiologically relevant concentration of the CCK-analogue cerulein (100 nM) decreases the luminal pH up to 2 pH unit. This long-lasting extracellular acidification is followed by disruption of tight junctions (Behrendorff et al. 2010). Furthermore, a decrease of luminal pH speeds up the autoactivation of trypsinogen inside the lumen and decreases the HCO3 − secretion of the ductal cells, which all together will promote inflammatory disease of the pancreas (Pallagi et al. 2011). In order to ensure the physiological homeostasis of the pancreas and to prevent such a proton-induced damage, the duct cells should have a physiological proton-sensing mechanism to restore luminal pH (Hegyi et al. 2011a).
Four main classes of acid-sensing ion channels have been identified in the gastrointestinal tract (Holzer 2007), namely, the acid-sensing ion channels (ASICs) (Page et al. 2005), the two-pore domain potassium channels (KCNKs) (Holzer 2003), the transient receptor potential ion channels of the vanilloid subtype (TRPVs) (Liddle 2007), and the ionotropic purinoceptor (P2X) (Henriksen and Novak 2003). Although the two latter ones have been identified in the pancreas, there is still no convincing evidence available to prove their involvement in the acid-mediated regulation of HCO3 − secretion.
4.5 Calcium
A close parallelism can be observed between the calcium and protein concentration of the pancreatic juice during secretin and CCK stimulation (Goebell et al. 1973; Gullo et al. 1984). Generally, the calcium concentration can reach 1–3 mM Ca2+ (Goebell et al. 1973; Gullo et al. 1984) and can act as a paracrine signaling molecule (Bruce et al. 1999; Racz et al. 2002). Ca2+-sensing receptors have been identified on the apical membrane of the pancreatic ducts in the human pancreas and adenocarcinoma cell lines (Racz et al. 2002). Activation of this G-protein-coupled receptor increases HCO3 − and fluid secretion (Bruce et al. 1999). Since 70 % of the proteins in the acinar fluid are proteases (Petersen 2008), this mechanism is crucially important in order to elevate the intraluminal pH which inhibits trypsinogen activation (Pallagi et al. 2011).
4.6 Molecules in the Pancreatic Juice Affecting Pancreatic Ductal Secretion
4.6.1 Guanylin/Uroguanylin
One of the best examples of an autocrine regulatory mechanism in pancreatic ducts is the guanylin-regulated electrolyte secretion. The 15-amino acid peptide guanylin is highly expressed in centroacinar and proximal ductal cells (Kulaksiz et al. 2001), whereas the 16-amino acid peptide uroguanylin, besides the above-mentioned places, is also expressed in the interlobular ducts (Kulaksiz and Cetin 2001). Both peptides are secreted into the ductal lumen and stimulate the guanylate cyclase-C receptor which will stimulate the CFTR Cl− channel via elevation of the intracellular cyclic guanosine monophosphate (cGMP) level and activation of protein kinase II (Kulaksiz and Cetin 2001; Kulaksiz et al. 2001).
4.6.2 Angiotensin II
Angiotensin II can be detected in the pancreatic juice, and angiotensin I and II receptors can be found on the apical side of pancreatic ductal cells (Leung et al. 1997), suggesting a local regulatory effect of the oligopeptide hormone in a paracrine fashion. Since angiotensin was not detectable either in the ductal or acinar cells (Regoli et al. 2003), but in the glucagon-secreting islet cells (Regoli et al. 2003), it is more than likely that besides the acinar-ductal interaction there is an islet-ductal regulatory process. Activation of angiotensin I or II receptors elevates intracellular Ca2+ level [Ca2+]i and cAMP level and therefore activates CFTR current stimulating HCO3 − secretion (Leung et al. 1999; Tahmasebi et al. 1999; Lam and Leung 2002; Leung 2007). However, the physiological role of the islet-ductal regulatory process remains unclear.
4.6.3 Adenosine-5′-triphosphate
Adenosine-5′-triphosphate can be detected in nano-molar concentration range in human pancreatic juice (Kordas et al. 2004). CCK-8, but not secretin, was found to stimulate both ATP-consuming and ATP-generating enzyme secretions (Yegutkin et al. 2006). Luminal ATP was also shown to activate the G-protein-coupled P2Y and the ligand-gated ion channel P2X expressed on the apical membrane of duct cell (Luo et al. 1999; Henriksen and Novak 2003; Novak 2008). Alpha-d-glucose-1-phosphate uridylyltransferase (UTP) were found to stimulate pancreatic ductal HCO3 − secretion via intracellular Ca2+ elevation (Ishiguro et al. 1999; Szucs et al. 2006).
It is clear that in parallel with the enzymes, the acinar cells secrete many substances that stimulate pancreatic ductal fluid and bicarbonate secretion. The main benefit of the ductal fluid secretion is to decrease the transit time of the pancreatic proenzymes in the ductal tree, whereas, the very important physiological role of HCO3 − is to preserve trypsinogen in its inactive form in the ductal lumen. This acinar-ductal coordinated secretion is absolutely essential in order to prevent the pancreas from being digested by its own enzymes. Therefore, it is not surprising that derangement of this process can lead to pancreatic inflammatory diseases.
5 Pathophysiological Aspects of Acinar-Ductal Interactions
The most important diseases of the exocrine pancreas are pancreatitis and CF. Acute pancreatitis (AP) is initiated by a pathological increase in protease activity inside the acinar cells, which causes autodigestion and ultimately is capable of destroying the acinar cells, the whole pancreas, and the surrounding tissue (Petersen and Sutton 2006). Repeated attacks of AP leads to chronic pancreatitis, where the normal-functioning exocrine cells are replaced by a fibrous matrix, which is cancer promoting (Petersen et al. 2011a). In CF, pancreatic HCO3 − secretion in the ducts is impaired resulting in blockage of the ducts, which in turn destroys the acinar cells and results in failure to secrete adequate amounts of digestive enzymes. In both cases, the physiologically important normal interaction between acinar and duct cells is disrupted. In the human pancreas, it has been shown that primary ductal blockade leads to intra-acinar trypsinogen activation (Murphy et al. 2008) demonstrating directly the importance of duct function for preservation of normal acinar cell function (Fig. 2).
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Fig. 2
Pathophysiological aspects of acinar-ductal interactions. Pancreatitis-inducing factors evoke Goblet cell (mucin-secreting cell) metaplasia as a replacement of the ductular and sometimes the acinar epithelium. This is often seen in chronic pancreatitis. Parallel to the metaplasia, the toxic agents also decrease HCO3 − as well as fluid secretion by ductal cells, by which mechanism the formation of aggregated protein plugs is promoted. The protein plug will obstruct the outflow of pancreatic fluid, which will lead to the elevation of luminal pressure which further decreases HCO3 − secretion and inhibits the outflow of enzymes from the ductal tree. Ductal blockade causes trypsinogen activation inside acinar cells. The decrease of luminal pH will strongly enhance autoactivation of luminal trypsinogen, which will inhibit CFTR channels and anion exchanger via luminal PAR2. This will further elevate the viscosity of pancreatic juice. Moreover, luminal acidosis will cause ZG dilatation and damage acinar endocytosis, which further elevates the protein concentration of the luminal content. These vicious cycles are crucial driving forces of the irreversible progressive chronic inflammation of the pancreas. CFTR cystic fibrosis transmembrane conductance regulator, ATP adenosine-5′-triphosphate, ZG zymogen granules, TJ tight junction, T trypsin, PAR2 protease-activated receptor 2
6 Pathophysiological Changes in the Composition of Ions and Other Molecules in the Pancreatic Juice
6.1 Development of Protein Gel in the Ductal Lumen
One of the leading histological features of chronic pancreatitis is the formation of a protein gel in the fine pancreatic ducts (Sarles et al. 1965). Importantly this morphological observation is visible without damage of acinar cells or malformation of the duct of Wirsung (Sarles et al. 1965), suggesting that this mechanism may be responsible not only for the progression but also for the initiation of chronic pancreatitis. Ductal mucinous hyperplasia can be seen in 62 % of the patients suffering from chronic pancreatitis (Allen-Mersh 1985). In addition, Goblet-cell metaplasia is described as a replacement of the ductular and sometimes the acinar epithelium (Walters 1965). Despite the increase in the number of mucin-secreting cells, diminished HCO3 − secretion is also a well known and very early defect in chronic pancreatitis (Braganza and Rao 1978; Freedman 1998). Therefore, the viscosity of pancreatic juice is elevated due to the elevation of the mucin concentration in the ductal lumen and to the decreased fluid and bicarbonate secretion by ductal cells (Ko et al. 2012). Since HCO3 − is essential for mucin swelling and hydration, by reducing Ca2+ cross-linking in mucins, the low concentration of HCO3 − and high concentration of mucin will promote the formation of aggregated protein plugs (Chen et al. 2010). The protein plug will obstruct the outflow of pancreatic fluid, which will lead to higher pressure in the proximal direction from the plug. The elevated pressure will further decrease HCO3 − secretion and inhibit the outflow of enzymes from the ductal tree (Suzuki et al. 2001). The decrease of luminal pH will strongly enhance the autoactivation of trypsinogen, which will inhibit CFTR Cl− channels and anion exchanger via luminal protease-activated receptor 2 (PAR2) (Pallagi et al. 2011). This will further elevate the viscosity of pancreatic juice. This vicious cycle is one of the main driving forces of the irreversible progressive chronic inflammation of the pancreas. Importantly, the R122H mutation in the cationic trypsin causes the same inhibition of bicarbonate secretion as the normal trypsin via the PAR2 receptor (Ko et al. 2012). Since the autoactivation of trypsinogen is more enhanced in the “gain of function” mutations of PRSS1 (Sahin-Toth and Toth 2000; Simon et al. 2002), this process can be even more important in hereditary pancreatitis.
6.2 Changes in Luminal pH (pHL)
Physiologically, as discussed in our earlier chapter, the luminal pH of pancreatic ducts depends on both acinar and ductal cell functions. Acinar cells can decrease the luminal pH down to 6.8 by secreting protons (Bhoomagoud et al. 2009), whereas pancreatic ductal cells can elevate the pH up to around 8.0 (Argent 2006). However, in pathophysiological states, pHL can be decreased either due to enhanced proton secretion from the proximal part (Bhoomagoud et al. 2009) or bile reflux (Opie 1901) or injection of a contrast solution from the distal part of the duct (Noble et al. 2008). pHL can also be decreased by decreased pancreatic ductal secretion (Wang et al. 2006; Hegyi et al. 2011b) and/or inflammation (Toyama et al. 1997; Patel et al. 1999). Noble et al. (2008) showed very elegantly that pH dropping below 7.0 causes pancreatic inflammation, whereas elevation – that is, correction – of the acidic luminal pH is beneficial (Freedman et al. 2001; Ko et al. 2010). Overall, irrespective of the manner by which luminal acidosis occurs, pH decrease is harmful, whereas pH elevation is beneficial for the pancreas.
6.3 The Effects of the Luminal Fluid Changes on Acinar Cells
The drop in luminal pH may not only induce or exacerbate pancreatic inflammation but also impair the physiological secretory function of the acinar cells. Glycoprotein 2 (GP2) release is around four times higher at pH 8.3 than 6.0 suggesting that bicarbonate secretion (alkalization of pHL) regulates enzymatic cleavage of GP2, which is one of the major ZG membrane proteins in pancreatic acinar cells (Freedman et al. 1994, 1998a). Since amylase secretion (representing exocytosis) is only 20 % higher, whereas horseradish peroxidase uptake (representing endocytosis) is around eight times higher at pH 8.3 than at pH 6.0 (Freedman et al. 1998a), this suggests that endocytosis is much more strongly affected by pHL than exocytosis. When ductal elements were isolated together with acinar cells, the anion exchanger DIDS inhibited GP2 as well as enzyme release from the acinar cells, clearly suggesting that bicarbonate secretion is essential for regulated enzyme secretion (Freedman et al. 1994). Besides the damage of endocytosis, acinar luminal dilatation with a marked reduction of ZGs can also be observed at pH 6.0 (Freedman et al. 1998a, b, 2001), which can be reversed by elevating the pH up to 8.3 (Scheele et al. 1996).
7 The Effects of the Main Pancreatitis-Inducing Factors on Acinar and Ductal Cells
Besides some genetic alterations, almost all cases of pancreatitis are due to a stress/toxic factor, which initiates pancreatic damage. In AP, 80–90 % of the stress is induced either by excessive ethanol consumption or by bile stones obstructing the outflow of pancreatic fluid, which besides the elevation of intraductal pressure may result in bile reflux into the ductal tree (Topazian and Pandol 2009). The remaining 10–20 % of the stress is shared between iatrogen, metabolic, infectious, neoplastic, and traumatic factors. Concerning chronic pancreatitis, 70 % of the cases are caused by alcohol, whereas, the rest is developed mostly due to other metabolic (e.g., hyperlipidemia, hypercalcemia), obstructive or autoimmune stress (Owyang and MJ 2009). Most recently, smoking was also shown to be an independent and dose-dependent factor for both acute and chronic pancreatitis (Alexandre et al. 2011). Generally, the pancreatitis-inducing factors in low concentration cause oscillatory calcium elevation and stimulate ductal bicarbonate and fluid secretion representing a ductal-defense mechanism. The stimulated secretion elevates the HCO3 − concentration, that is, inhibits autoactivation of trypsinogen and washes out the toxic factors/enzymes from the pancreas. However if the toxic factors reach the cells in high concentrations, they will induce toxic sustained calcium signals (Voronina et al. 2002; Venglovecz et al. 2008), mitochondrial damage (Mukherjee et al. 2008; Maleth et al. 2011) with a consequent ATP depletion both in acinar and ductal cells. This results in inhibited bicarbonate and fluid secretion in ductal cells (Hegyi et al. 2011b), intra-acinar and intraductal trypsinogen activation (Sherwood et al. 2007; Pallagi et al. 2011), ER stress (Kubisch and Logsdon 2008), and secretory block (Thrower et al. 2010).
8 The Effects of Pancreatitis-Inducing Factors on Ductal Cells
8.1 The Effects of Bile Acids on Ductal Cells
The effects of bile acids on the pancreatic ductal tree depend on the bile concentration and may be different in different parts of the duct system (Venglovecz et al. 2012). Concerning the main pancreatic ducts, bile acids in a concentration above 15 mM cause cell death with a consequent disruption of ductal integrity (Armstrong et al. 1985). Bile acids in concentrations between 2 and 15 mM are also toxic. They make the ducts permeable to molecules above 20,000 Da (Farmer et al. 1984; Armstrong et al. 1985), whereas normally the ducts are impermeable to molecules above 3,000 Da. Therefore, bile acids elevate the permeability of the epithelial barrier making the pancreas more vulnerable. Moreover, bile acids in these concentrations also elevate the permeability of the main duct to both Cl− and HCO3 − (Reber et al. 1979; Reber and Mosley 1980) causing loss of HCO3 − concentration in the main duct. Bile acids in a concentration less than 2 mM have stimulatory effects both on Cl− and K+ conductances via Ca2+-dependent mechanism.
Concerning the intra-/interlobular ducts, the same stimulatory and inhibitory pattern can be observed; however, these ducts are much more sensitive to bile than the main duct. In addition, there is a big difference between the effects of nonconjugated and conjugated bile acids. The conjugated bile acids have no effects on the secretory function of ductal cells at or below 1 mM concentration; however, the nonconjugated ones cause dual effects. Nonconjugated bile acids at a concentration of 100 μM stimulate bicarbonate secretion in a Ca2+-dependent manner (Venglovecz et al. 2008; Venglovecz et al. 2011a), whereas at 1 mM concentration they damage the mitochondria (Maleth et al. 2013), deplete intracellular ATP, and block both the basolateral and apical ion transport mechanisms (Venglovecz et al. 2008; Ignath et al. 2009; Maleth et al. 2011).
8.2 The Effects of Ethanol and Its Metabolites on Ductal Cells
There is much less data available concerning the effects of ethanol and their metabolites on pancreatic ductal cells. Yamamoto et al. (2003) showed that ethanol in low concentration augmented the stimulatory effect of secretin, whereas in high concentration they inhibited the secretory rate (Yamamoto et al. 2003). We have recently investigated the effects of ethanol, fatty acids (FAs), fatty acid esters, and acetaldehyde on pancreatic ductal secretion (Venglovecz et al. 2011b; Hegyi et al. 2012). Our preliminary experiments showed that ethanol at a low concentration (10 mM) stimulates, whereas at a high concentration it inhibits ductal HCO3 − concentration. Acetaldehyde has no effects, but both fatty acids (FAs) and fatty acid ethyl esters (FAEEs) strongly inhibit the CFTR Cl− channel and the Cl−/HCO3 − exchanger (Venglovecz et al. 2011b; Hegyi et al. 2012). However, these intracellular signaling pathways need further investigation.
8.3 The Effects of Virus Infection on Ductal Cells
Viruses have been shown to induce AP. We have shown that intact pancreatic ducts can be infected with pseudorabies virus which is an alpha-herpesvirus (Hegyi et al. 2005a). The virulent strain of the virus was able to stimulate pancreatic bicarbonate secretion around four- to fivefold, suggesting a washout defense mechanism of the ductal epithelia.
9 The Effects of Pancreatitis-Inducing Factors on Acinar Cells
The key challenge in understanding the initiation of pancreatitis is to unravel the chain of intracellular events following exposure of the acinar cells to agents known to be instrumental in provoking an attack of AP, namely, bile acids, alcohol, FAs, and products of alcohol and FAs (Petersen et al. 2011a). The end point is the inappropriate intracellular protease activation causing necrosis, but there is still much uncertainty about the intermediary steps.
9.1 The Effects of Bile Acids on Acinar Cells
Bile acids can reach acinar cells either from the basolateral side or, if the ductal secretion is damaged, from the luminal side. The first important step in understanding the primary action of bile acids on the acinar cells was the finding that these substances evoke increases in the cytosolic Ca2+ concentration ([Ca2+]i), due to primary release from intracellular stores and subsequent activation of Ca2+ entry (Voronina et al. 2002). Further studies revealed that Ca2+ was released from both the endoplasmic reticulum (ER) and acid stores (Gerasimenko et al. 2006). The Ca2+ release occurs through both IP3 and ryanodine receptors (Voronina et al. 2002; Gerasimenko et al. 2006; Petersen et al. 2011b). Bile acid concentrations that are easily within a pathophysiologically relevant range can evoke sustained global [Ca2+]i elevations (Voronina et al. 2002; Gerasimenko et al. 2006) and cause Ca2+-dependent cell death (Kim et al. 2002). Bile acids evoke mitochondrial depolarization (Voronina et al. 2004) and a reduction in the intracellular ATP level (Voronina et al. 2010), which causes inhibition of both sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and plasma membrane Ca2+ ATPase pump (PMCA) creating a vicious circle driving the cell to further damage (Barrow et al. 2008). The intracellular ATP level determines whether cell death occurs by apoptosis or necrosis. This has been demonstrated in patch clamp whole cell recording experiments, in which direct supply of ATP to the cell interior converts bile acid–induced necrosis to apoptosis (Booth et al. 2011). Patch clamp experiments revealed that bile acids also induce a Ca2+-independent cationic current, even at a stimulus intensity not evoking any major increase in the intracellular Ca2+ level (Voronina et al. 2004), but the significance of this phenomenon is currently unclear. Some of the bile acid effects may be mediated via G-protein-coupled cell surface bile acid receptors as they were found to play a role in the initiation of intra-acinar Ca2+ elevation and zymogen activation (Perides et al. 2010). Pancreatic acinar calcineurin may also play a role in the development of the Ca2+ elevation suggesting that calcineurin inhibition may be a therapeutical possibility against bile-induced acinar cell damage (Muili et al. 2013). Notably, bile acids also increase intracellular and mitochondrial reactive oxygen species (ROS) concentrations promoting acinar cell apoptosis (Booth et al. 2011) suggesting a possible defense mechanism against the more severe cell necrosis. However, it should be noted that oxidant stress imposed on top of a steady small elevation of [Ca2+]i can induce a significant amount of necrosis (Ferdek et al. 2012). Proteome analyses of AR42J cells showed that taurolithocholic acid (TLC) induced upregulation of 23 and downregulation of 16 proteins suggesting further mechanisms involved in the toxicity of bile acids on acinar cells (Li et al. 2012). Besides the effects of bile acids on ductal and acinar cells, experiments on Toll-like receptor 4-deficient mice suggest that leukocytes are also involved in the bile-induced pancreatic damage. The bile-induced pancreatic damage and pancreatic and lung myeloperoxidase activities were decreased in the receptor-deficient animals (Awla et al. 2011). Overall, the effects of bile acids on pancreatic acinar cells are complex and at this stage it is still difficult to judge the relative importance of several of the processes that have been indicated by often very different experimental protocols. It is, however, abundantly clear that excessive intracellular Ca2+ release, and therefore intracellular Ca2+ toxicity, is the central feature of the pathological bile actions, in both the ducts and the acini (Fig. 3).
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