Pharmacological and genetic inhibition of calcineurin protects against carbachol-induced pathological zymogen activation and acinar cell injury.
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.
Department of Pediatric Gastroenterology, Hepatology, and Nutrition, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, Pennsylvania, USA.
Kamaldeen A
KA
Muili
Mahwish
M
Ahmad
Abrahim I
AI
Orabi
Syeda M
SM
Mahmood
Ahsan U
AU
Shah
Jeffery D
JD
Molkentin
Sohail Z
SZ
Husain
D061354:Acinar Cells / Q000187:drug effects* / Q000201:enzymology; D000681:Amylases / Q000378:metabolism; D000818:Animals; D019703:Calcineurin / Q000235:genetics* / Q000502:physiology; D065095:Calcineurin Inhibitors; D002217:Carbachol / Q000037:antagonists & inhibitors* / Q000633:toxicity*; D002766:Cholecystokinin / Q000494:pharmacology; D002918:Chymotrypsin / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004792:Enzyme Precursors / Q000378:metabolism*; D005260:Female; D005838:Genotype; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D018722:Nicotinic Agonists / Q000633:toxicity*; D010179:Pancreas / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D014357:Trypsin / Q000378:metabolism
D065095:Calcineurin Inhibitors; D004792:Enzyme Precursors; D018722:Nicotinic Agonists; D002217:Carbachol; D004247:DNA; D002766:Cholecystokinin; D007770:L-Lactate Dehydrogenase; C120817:calcineurin phosphatase; D019703:Calcineurin; C557393:protein phosphatase 3, catalytic subunit, beta isoform, mouse; D010744:Phosphoric Monoester Hydrolases; D000681:Amylases; D002918:Chymotrypsin; D014357:Trypsin