Ethanol Enhances Carbachol-induced Protease Activation and Accelerates Ca²⁺ Waves in Isolated Rat Pancreatic Acini

Abstract

Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute cases and 70–90% of chronic cases, yet the mechanisms leading to alcohol-associated pancreatic injury are unclear. An early and critical feature of pancreatitis is the aberrant signaling of Ca²⁺ within the pancreatic acinar cell. An important conductor of this Ca²⁺ is the basolaterally localized, intracellular Ca²⁺ channel ryanodine receptor (RYR). In this study, we examined the effect of ethanol on mediating both pathologic intra-acinar protease activation, a precursor to pancreatitis, as well as RYR Ca²⁺ signals. We hypothesized that ethanol sensitizes the acinar cell to protease activation by modulating RYR Ca²⁺. Acinar cells were freshly isolated from rat, pretreated with ethanol, and stimulated with the muscarinic agonist carbachol (1 μm). Ethanol caused a doubling in the carbachol-induced activation of the proteases trypsin and chymotrypsin (p < 0.02). The RYR inhibitor dantrolene abrogated the enhancement of trypsin and chymotrypsin activity by ethanol (p < 0.005 for both proteases). Further, ethanol accelerated the speed of the apical to basolateral Ca²⁺ wave from 9 to 18 μm/s (p < 0.0005; n = 18–22 cells/group); an increase in Ca²⁺ wave speed was also observed with a change from physiologic concentrations of carbachol (1 μm) to a supraphysiologic concentration (1 mm) that leads to protease activation. Dantrolene abrogated the ethanol-induced acceleration of wave speed (p < 0.05; n = 10–16 cells/group). Our results suggest that the enhancement of pathologic protease activation by ethanol is dependent on the RYR and that a novel mechanism for this enhancement may involve RYR-mediated acceleration of Ca²⁺ waves.

Introduction

Pancreatitis is a life-threatening inflammatory disorder of the pancreas that leads to more than 30,000 deaths per year (1). Alcohol-associated pancreatitis accounts for 30% of acute cases and 70–90% of chronic cases. Further, alcoholic pancreatitis carries the highest mortality rate among all etiologies (2). However, the mechanisms by which ethanol mediates pathology are largely unknown.

Alcohol can exert diverse effects on the pancreas. Ethanol exposure has been linked to abnormal blood flow, leading to ischemic changes, and increased sphincter of Oddi dysfunction, resulting in pancreatic duct hypertension (2).

In addition, ethanol appears to directly predispose the acinar cell to pathological changes including oxidant stress (3), membrane fragility (4), mitochondrial uncoupling (5, 6), and basolateral exocytosis (7). Several lines of evidence also link ethanol to aberrant Ca^{2+ 3} signaling (6). High amplitude, aberrant, intracellular Ca²⁺ waves that propagate from the apical to basolateral region of the acinar cell predispose to early features of pancreatitis, particularly intra-acinar protease activation (8, 9). We know that intracellular Ca²⁺ release is responsible for the onset of this aberrant Ca²⁺ signal (10). In addition, we have previously shown that the ryanodine receptor (RYR),⁴ a major intracellular Ca²⁺ channel, is localized to the basolateral region of the acinar cell and, more importantly, is linked to the onset of pathologic protease activation (8).

Although ethanol has been reported to cause small increases in resting acinar cell Ca²⁺ and low amplitude Ca²⁺ transients, more prominent changes were associated with its metabolites, notably fatty acid ethyl esters (FAEEs) (6, 11). Recent studies demonstrate that administration of FAEEs evoked large Ca²⁺ transients that were IP3R-sensitive (5, 6). However, the effect of ethanol on RYR Ca²⁺ has not been examined.

Cellular Ca²⁺ homeostasis is governed by the interaction of a number of factors, including Ca²⁺ pools, release channels, pumps, exchangers, and buffers (12). Following secretagogue-induced stimulation of acinar cells, the initial rise in Ca²⁺ is predominantly controlled by two types of endoplasmic reticulum Ca²⁺ channels: 1) the inositol 1,4,5-trisphosphate receptor (IP3R) channel, which is activated by inositol 1,4,5-trisphosphate (IP3); and 2) the RYR, which is structurally similar to the IP3R but responds to different ligands (13). We and others have shown that the RYR is excluded from the apical region but localized to the basal region, importantly, where pancreatic zymogens, particularly proteases, are first activated during acute pancreatitis (8, 14). Cytosolic Ca²⁺ is the most potent activator, thus making the RYR the prototypic Ca²⁺-induced Ca²⁺ release channel.

We have linked RYR Ca²⁺ release in the basal region of the acinar cell to pathologic, premature intra-acinar protease activation, a critical and primary event in the pathogenesis of pancreatitis (15). Protease activation is dependent on cytosolic Ca²⁺ signals. This is supported by evidence demonstrating that the cytosolic Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) completely blocks activation (9, 16, 17). In the current study, we examined the role of ethanol in modulating the RYR Ca²⁺ signal and its effect on mediating protease activation. Our data demonstrate that ethanol 1) enhances intracellular protease activation and 2) accelerates carbachol-induced Ca²⁺ waves. Both effects of ethanol appear to be working through the RYR.

EXPERIMENTAL PROCEDURES

Preparation of Pancreatic Acini for Enzyme Kinetics/Secretion Assays

Groups of pancreatic acinar cells were isolated as described previously (8) with modifications. Briefly, Sprague-Dawley rats were euthanized using a protocol approved by the Animal Care and Use Committee. The pancreas was removed and minced for 5 min in Dulbecco's modified Eagle's medium (DMEM)/F12 1× buffer without phenol (Invitrogen) containing 15 mm HEPES (pH 7.4), 120.61 mm NaCl, 4.16 mm KCl, 0.301 mm MgCl₂, 1.05 mm CaCl₂, 17.51 mm dextrose, 0.05 m HCl, 2 mm sodium pyruvate, and 2.5 mm glutamine, plus 0.1% BSA and 2 mg/ml type-4 collagenase (Worthington). The tissue was briefly oxygenated and incubated for 5 min at 37 °C with shaking (90 rpm); buffer was removed and replaced with fresh collagenase buffer and then briefly oxygenated and incubated for 35 min. The tissue digest was triturated and filtered through a 300-μm mesh (Sefar American, Depew, NY). Acinar cells were washed three times with collagenase-free buffer. Acinar cells were allowed to equilibrate for 5 min at 37 °C before treatment.

Preparation of Pancreatic Acini for Ca²⁺ Imaging

Groups of pancreatic acinar cells were isolated as described previously (8) with minor modifications. Briefly, Sprague-Dawley rats weighing 50–100 g (Charles River Laboratories, Wilmington, MA) were euthanized using a protocol approved by the Animal Care and Use Committee. The pancreas was removed and minced for 5 min in buffer containing 20 mm HEPES (pH 7.4), 95 mm NaCl, 4.7 mm KCl, 0.6 mm MgCl₂, 1.3 mm CaCl₂, 10 mm glucose, and 2 mm glutamine, plus 1% BSA, 1× minimum Eagle's medium non-essential amino acids (Invitrogen), 200 units/ml type-4 collagenase (Worthington), and 1 mg/ml soybean trypsin inhibitor. The tissue was incubated for 60 min at 37 °C with shaking (90 rpm). The digest was transferred to a 15-ml conical tube and washed three times with collagenase-free and BSA-free buffer. The cells were vigorously shaken and then filtered through a 300-μm mesh (Sefar American) to separate the cells into smaller clusters.

Stimulation of Acini and Enzyme Activation Assays

Acini were pretreated with ethanol (1, 10, 50, 100 mm) or dantrolene (10, 50, 100 μm) at 37 °C for 15 and 30 min, respectively. Acini were then stimulated with the acetylcholine analog carbachol for 60 min at 37 °C. Protease activity assays were performed at room temperature using fluorogenic substrates as described previously (8). Briefly, 50 μl of 400 μm enzyme substrate were added to each homogenized sample, and accumulation of fluorescence was measured over 10 min using a fluorescent plate reader (Infinite M200, Tecan) at 380-nm excitation and 440-nm emission wavelengths. The trypsin substrate was supplied by Peptides International (Louisville, KY) and had the amino acid sequence Boc-Gln-Ala-Arg-MCA. Chymotrypsin substrate was supplied by Calbiochem and had the amino acid sequence Suc-Ala-Ala-Pro-Phe-AMC. Protease activity was collected as relative fluorescent units/second, normalized to total amylase (relative fluorescent units/second/microgram of amylase), and expressed as a -fold increase relative to either the control or a maximum condition. Amylase secretion was measured at room temperature from the media and cell homogenates using a Phadebas kit (Magle Life Sciences, Lund, Sweden). Total amylase values were used for normalization of chymotrypsin and trypsin results, and the percentage of amylase secretion into the media was calculated as a measure of enzyme secretion.

Detection of Cellular Ca²⁺ Signals

Acinar cells were loaded at room temperature with the high affinity Ca²⁺-sensing dye fluo-4/AM (K_Ca = 345 nm; Molecular Probes). Cells were then pretreated with or without one or more of the following for 30 or 60 min at room temperature: ethanol (100 mm) and the RYR inhibitor dantrolene (100 μm). Acinar cells were plated on acid-washed glass coverslips and then mounted on a perifusion chamber. Thereupon, they were stimulated at room temperature with the muscarinic agonist carbachol at the concentrations indicated. A Zeiss LSM510 laser scanning confocal microscope was used with a 63×, 1.4 numerical aperture objective. The dye was excited at 488-nm wavelength, and emission signals of >515 nm were collected at frame speeds of 200–300 ms/frame. Fluorescence from individual acinar cells as well as apical and basal subcellular regions was recorded. For experiments performed at 37 °C, cells were loaded at room temperature and mounted on a Zeiss 710 heating stage. Images were collected as described above.

Determination of Ca²⁺ Wave Speed

Apical and basal regions of interest in the acinar cell recordings were chosen using the ImageJ software (National Institutes of Health), and mean fluorescence over time in each region was graphed. Ca²⁺ wave speed was calculated by dividing the distance along the long axis of the acinar cell by the time it took for the Ca²⁺ wave to travel from the apical to the basal region.

Cell Injury Assays

Acinar cell injury was quantified as lactate dehydrogenase release using the non-radioactive cytotoxicity assay (Promega, Madison, WI). Absorbance was measured at 490 nm within 15 min of stopping the enzyme reaction. Results are expressed as the percentage of lactate dehydrogenase released into the media.

Statistics

Data represent mean ± S.E. of at least three individual experiments with multiple cells from each experiment. Statistical significance was determined by Student's t test analysis.

RESULTS

Ethanol Enhances Carbachol-induced Protease Activation

Supraphysiologic concentrations of the secretagogues cholecystokinin (CCK) and acetylcholine (ACh), or their analogs, induce pathologic intra-acinar protease activation. It is known that ethanol enhances CCK-induced protease activation within acinar cells (18). However, there are conflicting reports as to whether the cholinergic pathways are similarly affected by ethanol (19, 20). It would be of particular interest to know this because ACh stimulation of acinar cells may be a more physiologically relevant situation than CCK stimulation because 1) ACh is the primary stimulant for pancreatic secretion in human acinar cells and 2) even in rodents, neurogenic pathways that locally secrete ACh likely contribute to a major component of CCK-induced secretion as well as pancreatitis (20, 21). In addition, ethanol was recently shown to disrupt ACh-induced acinar cell events such as secretion by redirecting exocytosis from the apical to the basolateral membrane (22). For these reasons, we examined whether ethanol can modulate intracellular events initiated by stimulation with the ACh analog carbachol. Assays for trypsin and chymotrypsin activity were measured because the generation of these proteases closely follows first order kinetics over a 30–60-min period of incubation (18).

Treatment of acinar cells with ethanol alone (100 mm) for 15 min had no effect on protease activity, a result consistent with previous work (18). However, administration of ethanol (100 mm) for 15 min before physiologic carbachol (1 μm) stimulation caused a 2-fold increase in both trypsin and chymotrypsin activities above unstimulated controls (p < 0.02; Fig. 1). This effect was also observed during supraphysiologic stimulation with carbachol (1 mm) (p < 0.05; supplemental Fig. 1A) as well as with 1 h of ethanol pretreatment (p < 0.05; supplemental Fig. 2). To confirm that ethanol does not interfere with the enzymatic assays, ethanol was added to cells after homogenizing. As expected, direct application of ethanol to homogenates did not affect protease activation (supplemental Fig. 3).

FIGURE 1.

Ethanol enhances carbachol-induced pancreatic protease activation. Acinar cells were pretreated with ethanol (100 mm) for 15 min prior to carbachol (Carb, 1 μm) stimulation. Activities of the proteases relative to carbachol alone were measured. A and B, trypsin (A) and chymotrypsin (B) were normalized to total amylase content (n = 4). *, p < 0.05 with respect to carbachol alone.

To know whether the enhancement of protease activation by ethanol is seen over a broad range of concentrations, acinar cells were incubated with biologically relevant ethanol concentrations (1–100 mm) prior to stimulation with carbachol (1 μm). These ethanol concentrations are achievable in serum during acute ingestion, corresponding to blood alcohol concentrations of 0.005–0.5%. We observed a concentration-dependent increase in chymotrypsin activity with ethanol (p < 0.01; Fig. 2). For all subsequent experiments, we used 100 mm concentration to provoke the maximal ethanol effect. These results indicate that biologically relevant ethanol concentrations sensitize the pancreatic acinar cell to carbachol-induced protease activation.

FIGURE 2.

Ethanol causes a concentration-dependent enhancement of protease activation induced by carbachol. Acinar cells were treated with ethanol for 15 min prior to carbachol (Carb, 1 μm) stimulation. Activity of chymotrypsin was normalized to total amylase content and represented relative to carbachol alone (n = 3). *, p < 0.01 with respect to carbachol alone.

Dantrolene Reduces Ethanol-enhanced Protease Activation but Does Not Affect Amylase Secretion

In previous work, we have shown that aberrant Ca²⁺ signaling is mediated by the intracellular Ca²⁺ channel, the RYR (8). In the current study, we tested the hypothesis that ethanol triggers protease activation through RYR-mediated aberrant Ca²⁺ signals. Pancreatic acinar cells were pretreated with the RYR inhibitor dantrolene for 30 min prior to ethanol incubation. We observed that the enhancement of protease activity by ethanol was abrogated by dantrolene during physiologic carbachol stimulation (p < 0.01; Fig. 3). The reduction with dantrolene was also seen during supraphysiologic carbachol stimulation (p < 0.05; supplemental Fig. 1) and 1 h of ethanol pretreatment (p < 0.05; supplemental Fig. 2). The predominance of RYR1 in rat acinar cells substantiated the use of dantrolene (8), which selectively inhibits RYR1 over other isoforms (23). Further, unlike other RYR inhibitors, dantrolene does not disrupt mitochondrial pathways at concentrations above 100 μm. Nevertheless, in limited studies with lower concentrations, dantrolene (10–100 μm) caused a concentration-dependent reduction in the enhancement of protease activation by ethanol (p < 0.01; Fig. 4). The results indicate that ethanol sensitizes the pancreatic acinar cell to enhanced protease activation via the RYR.

FIGURE 3.

Ethanol-enhanced protease activation is dependent on the RYR. Acinar cells were pretreated with the RYR inhibitor dantrolene (Dant, 100 μm) for 30 min prior to ethanol (100 mm) and carbachol (Carb, 1 μm) administration. A and B, activities of the proteases trypsin (A) and chymotrypsin (B) were normalized to total amylase content and represented relative to carbachol alone (n = 3). *, p < 0.005; **, p < 0.01, relative to carbachol alone and carbachol with ethanol, respectively.

FIGURE 4.

Dantrolene reduces ethanol-enhanced protease activation over a range of inhibitory concentrations. Acinar cells were pretreated with dantrolene (Dant, 10–100 μm) for 30 min prior to ethanol (100 mm) and carbachol (Carb, 1 μm) administration (n = 3). Activity of chymotrypsin was normalized to total amylase content and represented relative to the maximum condition. *, p < 0.005; #, p < 0.01, relative to carbachol alone and carbachol with ethanol respectively.

Early pancreatic acinar cell injury is thought to result from a combination of intracellular protease activation and concurrent secretory inhibition of those enzymes from the acinar cell. Cosen-Binker and Gaisano (24) demonstrated that pretreatment of isolated acinar cells with ethanol for 1 h caused an inhibition of amylase secretion. However, we found that brief pretreatment did not affect either constitutive or regulated (i.e. carbachol-stimulated) enzyme secretion (Fig. 5). These data are consistent with our hypothesis that RYR-mediated Ca²⁺ signaling from the basolateral region enhances protease activation, but does not affect apical secretory events.

FIGURE 5.

Amylase secretion is unaffected by short term ethanol incubation or RYR inhibition. Acinar cells were pretreated with dantrolene (Dant, 100 μm) for 30 min prior to ethanol (100 mm) and carbachol (Carb, 1 μm) administration (n = 3). The percentage of amylase release was assayed from cell lysate and media samples.

We also demonstrated that cells pretreated with ethanol prior to supraphysiologic carbachol stimulation undergo cell injury, evidenced by an increase in lactate dehydrogenase release. However, dantrolene treatment completely abrogated the enhancement by ethanol (p < 0.05; supplemental Fig. 1B).

Supraphysiologic Carbachol Stimulation Leads to Increased Speed of the Apical to Basolateral Ca²⁺ Wave as Compared with Physiologic Stimulation

Acinar cells perfused with carbachol evoke a Ca²⁺ wave that originates in the apical region and propagates in a coordinated fashion to the basolateral region. Although there also exists a coordinated release of Ca²⁺ between cells (25–27), each individual cell propagates an apical to basolateral Ca²⁺ wave. We examined the speed of the acinar cell Ca²⁺ wave induced by carbachol with or without ethanol because, among other factors in the Ca²⁺ toolbox (28), the speed is dependent on the serial activation of the basolaterally localized intracellular Ca²⁺ channel the RYR (29). Tracings from apical and basolateral regions of interest from multiple cells in each field were used to calculate the speed of this Ca²⁺ transient, as described under ”Experimental Procedures.“ Physiologic carbachol (1 μm) evoked a Ca²⁺ wave speed of 9 μm/s, whereas supraphysiologic (1 mm) stimulation, a concentration that causes pathologic protease activation, markedly increased the speed to 43 μm/s (p < 0.05; Fig. 6). Although no causal relationship has been established, the results correlate an increase in Ca²⁺ wave speed with a change from increasing carbachol concentrations that cause protease activation.

FIGURE 6.

Supraphysiologic carbachol stimulation leads to increased speed of the apical to basolateral Ca²⁺ wave as compared with physiologic stimulation. A, acinar cells were stimulated with carbachol (1 μm). From left to right, bright field view of an acinus labeled at the apical (A) and basolateral (B) regions of interest from an acinar cell. Cells were loaded with the Ca²⁺ indicator fluo-4 (5 μm). Upon stimulation with physiologic carbachol (1 μm), subsequent images show the initiation of the Ca²⁺ signal in the apical region followed by propagation to the basal region. B, each paneled image (1–4) corresponds to a frame along a representative tracing of change in fluorescence over time for each region of interest. Left and right arrows show time of first Ca²⁺ rise in the apical and basal regions, respectively. Est. [Ca²⁺]_i, estimated [Ca²⁺]_i; min, minimum; max, maximum. C and D, cells were stimulated with supraphysiologic carbachol (1 mm). E, quantitation of difference in Ca²⁺ wave speed between the two carbachol conditions (n = 10 cells in each). *, p < 0.05.

Ethanol Accelerates the Speed of the Carbachol-induced Ca²⁺ Wave via the RYR

Next, we examined whether ethanol affected Ca²⁺ waves generated by physiologic carbachol (1 μm). Cells were pretreated with ethanol (100 mm) for 30 min prior to carbachol (1 μm) stimulation. We observed that cells pretreated with ethanol still evoked a Ca²⁺ transient that began in the apical region and propagated to the basolateral region; however, Ca²⁺ wave speed was significantly enhanced above control levels (p < 0.05; Fig. 7). This effect was also observed after 1 h of ethanol pretreatment or at 37 °C (p < 0.05; supplemental Figs. 4 and 5). Ethanol had no effect on the apical or basolateral Ca²⁺ peak amplitude (supplemental Fig. 7). In addition, ethanol caused a 2-fold increase in Ca²⁺ wave speed after supraphysiologic carbachol (1 mm) stimulation (p < 0.05; supplemental Fig. 8). To know whether the RYR mediates the ethanol-induced acceleration of Ca²⁺ waves, acinar cells were pretreated with dantrolene (100 μm). In this condition, the acceleration of Ca²⁺ wave speed by ethanol was abrogated (p < 0.05; Fig. 8), indicating a dependence on the RYR.

FIGURE 7.

Ethanol accelerates the physiologic carbachol-stimulated Ca²⁺ wave. A, acinar cells were treated with or without ethanol (100 mm) for 30 min prior to carbachol (1 μm) stimulation. From left to right, bright field view of an acinus labeled at the apical (A) and basolateral (B) regions of interest from an acinar cell. Cells were loaded with the Ca²⁺ indicator fluo-4 (5 μm). Upon stimulation with physiologic carbachol (1 μm), subsequent images show the initiation of the Ca²⁺ signal in the apical region followed by propagation to the basal region. B, each paneled image (1–4), corresponds to a frame along a representative tracing of change in fluorescence over time for each region of interest. Left and right arrows show time of first Ca²⁺ rise in the apical and basal regions, respectively. Est. [Ca²⁺]ⁱ, estimated [Ca²⁺]ⁱ; min, minimum; max, maximum. C and D, cells were pretreated with ethanol (100 mm) for 30 min. E, quantitation of difference in Ca²⁺ wave speed between the two carbachol conditions (n = 13 cells in each). *, p < 0.005.

FIGURE 8.

The acceleration of Ca²⁺ wave speed by ethanol is dependent on the RYR. In addition to pretreatment with ethanol (100 mm), a third group of acinar cells was incubated with dantrolene (Dant, 100 μm) for 30 min prior to carbachol (Carb, 1 μm) stimulation (n = 13–14 cells in each group). *, p < 0.005; **, p < 0.0005, with respect to carbachol alone and carbachol with ethanol, respectively.

DISCUSSION

Our study advances four important findings that relate ethanol to early events in pancreatitis. 1) Ethanol accelerates the speed of the apical to basolateral Ca²⁺ wave generated by physiologic carbachol (1 μm), a previously undescribed effect of ethanol. 2) An increase in Ca²⁺ wave speed correlates with conditions that cause protease activation (e.g. supraphysiologic carbachol). 3) Ethanol enhances carbachol-induced protease activation. 4) Both the acceleration of Ca²⁺ wave speed and the enhancement of protease activation are dependent on RYR activation.

Effect of Ethanol on the Acinar Cell

Ethanol has multiple effects on the acinar cell (Fig. 9). Cosen-Binker et al. (30) have shown that ethanol inhibits enzyme secretion stimulated by submaximal carbachol concentrations. The reduction is associated with a partial redirection of zymogen granule exocytosis to the basolateral membrane. Ethanol phosphorylates PKC α, leading to phosphorylation of a SNARE protein, Munc18C. This results in displacement of Munc18C and allows a SNARE complex to form between zymogen granules and the basolateral membrane, thereby permitting basolateral exocytosis (30). Satoh et al. (31) showed that another PKC isoform, PKC ϵ, could be phosphorylated by ethanol and that this caused NF-κB activation. In addition, ethanol causes lysosomal membrane fragility, resulting in the leakage of lysosomal enzymes capable of activating trypsinogen within the cytosol (32).

FIGURE 9.

Proposed mechanism by which ethanol evokes pathological effects on the pancreatic acinar cell. In the presence of carbachol, ethanol targets the RYR to induce aberrant Ca²⁺ waves that propagate from the apical to basolateral region of the acinar cell. This change in the shape of the Ca²⁺ signal predisposes to premature intra-acinar protease activation and results in pancreatic injury, leading to pancreatitis. ER, endoplasmic reticulum.

Effect of Ethanol on Protease Activity

Our results demonstrate that ethanol pretreatment enhances intra-acinar protease activation by carbachol. The findings confirm recent work by Lugea et al. (20). Previous studies from Gorelick and colleagues (18, 33) demonstrated that ethanol sensitizes the acinar cell to CCK-induced protease activation, but they were not able to detect sensitization of ethanol to carbachol using a carboxypeptidase A1 zymogen conversion assay (33). However, both our study and that of Lugea et al. (20) used selective fluorescent protease substrates, which are more sensitive in measuring changes in zymogen activity. We used a physiologic secretagogue concentration (1 μm), which evokes maximal pancreatic enzyme secretion, only a small amount of protease activation, and no acinar cell damage. The pathologic effects of ethanol on this condition were observed over a clinically relevant range of ethanol concentrations (10–100 mm) that are achievable in serum during intoxication (34).

Effect of Ethanol on Ca²⁺ Signaling

Ethanol also affects acinar cell Ca²⁺. By itself, a concentration of 850 mm was required to evoke a small Ca²⁺ transient (6). There is also evidence that ethanol either directly or through generation of reactive oxygen species causes Ca²⁺ influx (35, 36). However, several reports implicate a role for ethanol metabolites as the actual mediators of aberrant Ca²⁺ signaling in acinar cells (34, 37). Ethanol is readily metabolized by tissues into oxidative metabolites, notably acetaldehyde, and the non-oxidative metabolites, phosphatidylethanol and FAEEs (38).

Role of FAEEs in Pathology

Notably, FAEEs are found at highest levels in the pancreas, as produced by pancreatic acinar cells (37), and can induce pancreatic damage (39, 40) and intra-acinar protease activation (6, 41). In more recent studies, administration of FAEEs evoked large Ca²⁺ transients that were both IP3R-sensitive and RYR-sensitive (42). Further, sustained cytosolic Ca²⁺ levels by FAEEs were associated with necrosis (5, 6).

We confirmed that ethanol alone (100 mm) did not evoke a Ca²⁺ transient. However, brief pretreatment nearly doubled the speed of the apical to basolateral Ca²⁺ wave induced by physiologic carbachol. To our knowledge, this is the first demonstration that ethanol accelerates the speed of the apical to basolateral acinar cell Ca²⁺ wave. Further, an increased Ca²⁺ wave speed was associated with conditions leading to protease activation. It is intriguing to postulate how this acceleration of Ca²⁺ waves might decode signals leading to pancreatitis. Specifically, what pathologic targets are modulated by the change in the shape of the Ca²⁺ signal? A prototypic demonstration is that the frequency of Ca²⁺ oscillations can cause differential changes in gene expression (43). Although ethanol did not affect the frequency of acinar cell Ca²⁺ oscillations (supplemental Fig. 6D), the concept is proof of principal that apparently subtle biophysical changes in the Ca²⁺ signal can translate to biologically important outcomes. Putative targets of acinar cell Ca²⁺ pathology could include the Ca²⁺-activated phosphatase calcineurin, which has recently been shown to mediate protease activation and pancreatitis (17, 44), the calpains (45–47), or the classical protein kinase C (48). Future work using targeted deletion of these proteins as well as simultaneous imaging of both Ca²⁺ and protease activation will help examine pathways linking an acceleration of Ca²⁺ waves to pancreatitis.

Origins and Determinants of the Ca²⁺ Wave

Two theories explain the origins of the Ca²⁺ wave. Kasai et al. (49, 50) postulated that high sensitivity IP3Rs at the apical ”trigger zone“ generate the Ca²⁺ transient, which then propagates throughout the cell as a result of Ca²⁺-induced Ca²⁺ release from low sensitivity IP3R in the granular and basal regions. A second theory postulates that RYRs distributed in the basolateral pole are responsible for the Ca²⁺ wave propagation once a Ca²⁺ transient is triggered by apical IP3Rs (25, 26, 29). Our results, demonstrating that RYR inhibition abrogates the acceleration of Ca²⁺ waves by ethanol, support the latter possibility. These data also suggest that ethanol, either directly or indirectly, causes the RYR to shift to a higher subconductance state for Ca²⁺ opening.

RYR as a Mediator of Pathology

The current work is consistent with our previous findings demonstrating that 1) high amplitude Ca²⁺ transients in the basolateral region induced by supraphysiologic carbachol are dependent on the RYR (8), 2) RYR inhibition reduces protease activation in those conditions (8), and 3) in vivo administration of the RYR inhibitor dantrolene alleviates disease outcomes of pancreatitis (51). Factors that might modulate RYR opening in the acinar cell include phosphorylation, nitrosylation, and oxidation. The former two post-translational modifications are associated with disorders of Ca²⁺ leak in myocytes, leading to cardiac arrhythmias and muscular dystrophy, respectively (52).

Although the RYR can be phosphorylated at different sites, it will be interesting to examine whether a specific PKA site extensively described in cardiomyocytes is modulated by ethanol. This is because cAMP has been shown to accelerate the acinar cell Ca²⁺ wave in an RYR-dependent manner (53).

Although the RYR may play a central role in accelerating acinar cell Ca²⁺ waves, there are several other potential modifiers. Activation of PKC, without regard to isoform specificity, or lowering cytosolic pH can reduce Ca²⁺ wave speed (54, 55). Conversely, inhibition of mitochondria (25) or sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA) pumps (27) increases wave speed. It will be of interest to know whether ethanol affects one or more of these pathways to modulate Ca²⁺ waves or whether its metabolites are involved.

In summary, we report that ethanol accelerates the speed of acinar cell Ca²⁺ waves induced by carbachol and that it also enhances carbachol-induced protease activation. Both effects of ethanol are dependent on the RYR. We speculate that RYR inhibition with dantrolene might, therefore, function as treatment for alcohol-induced pancreatitis, a disease for which only supportive care is currently available.