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.

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.

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.

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).

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.

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.

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).

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.

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, pH_L 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). pH_L 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.

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 pH_L) 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 pH_L 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).

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).

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.

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.

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).