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 Ca(2+) within the pancreatic acinar cell. An important conductor of this Ca(2+) is the basolaterally localized, intracellular Ca(2+) 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 Ca(2+) signals. We hypothesized that ethanol sensitizes the acinar cell to protease activation by modulating RYR Ca(2+). 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 Ca(2+) wave from 9 to 18 μm/s (p < 0.0005; n = 18-22 cells/group); an increase in Ca(2+) 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 Ca(2+) waves.

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.

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.

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.

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.

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.

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.

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.