Pharmacological and genetic inhibition of calcineurin protects against carbachol-induced pathological zymogen activation and acinar cell injury

Abstract

Acute pancreatitis is a major health burden for which there are currently no targeted therapies. Premature activation of digestive proenzymes, or zymogens, within the pancreatic acinar cell is an early and critical event in this disease. A high-amplitude, sustained rise in acinar cell Ca(2+) is required for zymogen activation. We previously showed in a cholecystokinin-induced pancreatitis model that a potential target of this aberrant Ca(2+) signaling is the Ca(2+)-activated phosphatase calcineurin (Cn). However, in this study, we examined the role of Cn on both zymogen activation and injury, in the clinically relevant condition of neurogenic stimulation (by giving the acetylcholine analog carbachol) using three different Cn inhibitors or Cn-deficient acinar cells. In freshly isolated mouse acinar cells, pretreatment with FK506, calcineurin inhibitory peptide (CiP), or cyclosporine (CsA) blocked intra-acinar zymogen activation (n = 3; P < 0.05). The Cn inhibitors also reduced leakage of lactate dehydrogenase (LDH) by 79%, 62%, and 63%, respectively (n = 3; P < 0.05). Of the various Cn isoforms, the β-isoform of the catalytic A subunit (CnAβ) was strongly expressed in mouse acinar cells. For this reason, we obtained acinar cells from CnAβ-deficient mice (CnAβ-/-) and observed an 84% and 50% reduction in trypsin and chymotrypsin activation, respectively, compared with wild-type controls (n = 3; P < 0.05). LDH release in the CnAβ-deficient cells was reduced by 50% (n = 2; P < 0.05). The CnAβ-deficient cells were also protected against zymogen activation and cell injury induced by the cholecystokinin analog caerulein. Importantly, amylase secretion was generally not affected by either the Cn inhibitors or Cn deficiency. These data provide both pharmacological and genetic evidence that implicates Cn in intra-acinar zymogen activation and cell injury during pancreatitis.

Figures

Fig. 1.

Calcineurin (Cn) inhibitors at varying concentrations reduce intra-acinar zymogen activation. Acinar cells were treated with FK506 (A), Cn inhibitory peptide (CiP) (B), or cyclosporine A (CsA) (C) at the indicated concentrations for 30 min before carbachol (1 mM) stimulation. Activities of trypsin (left) and chymotrypsin (right) were measured 30 min after carbachol stimulation and normalized to total amylase content (n = 3). *P < 0.05 with respect to control or carbachol alone, respectively. Data are means ± SE. RFU, relative fluorescence units.

Fig. 2.

Cn inhibitors reduce intra-acinar zymogen activation over a range of carbachol concentrations, but they do not affect acinar cell enzyme secretion. Acinar cells were pretreated with FK506 (10 μM), CiP (10 μM), or CsA (1 μM) for 30 min before carbachol (0.1 μM-1.0 mM) stimulation. Activities of trypsin (A) and chymotrypsin (B) were measured 30 min after carbachol stimulation and normalized to total amylase content (n= 3). C: biphasic amylase secretion curve with or without the Cn inhibitors (n = 3). *P < 0.05 with respect to control or carbachol alone, respectively. Data are means ± SE.

Fig. 3.

Cn inhibitors reduce cell injury induced by carbachol. Acinar cells were pretreated with FK506 (10 μM), CiP (10 μM), or CsA (1 μM) for 30 min before a 2-h carbachol (1 μM or 1 mM) incubation. Cell injury was measured as percent lactate dehydrogenase (LDH) released from acinar cells (n = 3). #P < 0.001 with respect to 1 mM carbachol. Data are means ± SE.

Fig. 4.

The β-isoform of the catalytic A subunit (CnAβ) isoform is expressed in pancreatic acinar cells, and CnAβ-deficient mice have reduced pancreatic and acinar cell Cn phosphatase activity.A: DNA gel of PCR from acinar cell cDNA using Cn isoform-specific primers. B: Cn phosphatase activity from whole pancreas, acinar cells, and splenic lymphocytes. #P< 0.01 with respect to wild-type (WT). Data are means ± SE.

Fig. 5.

CnAβ-deficient mice have reduced intra-acinar zymogen activation and cell injury induced by carbachol. Acinar cells from WT or CnAβ−/− mice were treated with carbachol (0.1 μM-1.0 mM) for 30 min, and trypsin (A) and chymotrypsin (B) activities were normalized to total amylase content (n = 3). #*P< 0.05 with respect to the unstimulated condition or WT acinar cells, respectively. C: acinar cells were treated with 1 mM carbachol for 2 h, and cell injury was measured as percent LDH released (n = 2). #P < 0.001, with respect to WT acinar cells receiving 1 mM carbachol. D: biphasic amylase secretion curve (n = 3). Data are means ± SE.

Fig. 6.

CnAβ-deficient mice have reduced intra-acinar zymogen activation and cell injury induced by caerulein. Acinar cells from WT or CnAβ−/− mice were treated with caerulein (0.01–100 nM) for 30 min, and trypsin (A) and chymotrypsin (B) activities were normalized to total amylase content (n = 3). #*P< 0.05 with respect to the unstimulated condition or WT acinar cells, respectively. C: acinar cells were treated with 100 nM caerulein for 2 h, and cell injury was measured as percent LDH released (n = 2). #P < 0.001, with respect to WT acinar cells receiving 100 nM caerulein. D: biphasic amylase secretion curve (n = 3). Data are means ± SE.