The ryanodine receptor is expressed in human pancreatic acinar cells and contributes to acinar cell injury.

Physiological calcium (Ca(2+)) signals within the pancreatic acinar cell regulate enzyme secretion, whereas aberrant Ca(2+) signals are associated with acinar cell injury. We have previously identified the ryanodine receptor (RyR), a Ca(2+) release channel on the endoplasmic reticulum, as a modulator of these pathological signals. In the present study, we establish that the RyR is expressed in human acinar cells and mediates acinar cell injury. We obtained pancreatic tissue from cadaveric donors and identified isoforms of RyR1 and RyR2 by qPCR. Immunofluorescence staining of the pancreas showed that the RyR is localized to the basal region of the acinar cell. Furthermore, the presence of RyR was confirmed from isolated human acinar cells by tritiated ryanodine binding. To determine whether the RyR is functionally active, mouse or human acinar cells were loaded with the high-affinity Ca(2+) dye (Fluo-4 AM) and stimulated with taurolithocholic acid 3-sulfate (TLCS) (500 µM) or carbachol (1 mM). Ryanodine (100 µM) pretreatment reduced the magnitude of the Ca(2+) signal and the area under the curve. To determine the effect of RyR blockade on injury, human acinar cells were stimulated with pathological stimuli, the bile acid TLCS (500 µM) or the muscarinic agonist carbachol (1 mM) in the presence or absence of the RyR inhibitor ryanodine. Ryanodine (100 µM) caused an 81% and 47% reduction in acinar cell injury, respectively, as measured by lactate dehydrogenase leakage (P < 0.05). Taken together, these data establish that the RyR is expressed in human acinar cells and that it modulates acinar Ca(2+) signals and cell injury.

IP3 receptor type 2 deficiency is associated with a secretory defect in the pancreatic acinar cell and an accumulation of zymogen granules.

Acute pancreatitis is a painful, life-threatening disorder of the pancreas whose etiology is often multi-factorial. It is of great importance to understand the interplay between factors that predispose patients to develop the disease. One such factor is an excessive elevation in pancreatic acinar cell Ca(2+). These aberrant Ca(2+) elevations are triggered by release of Ca(2+) from apical Ca(2+) pools that are gated by the inositol 1,4,5-trisphosphate receptor (IP3R) types 2 and 3. In this study, we examined the role of IP3R type 2 (IP3R2) using mice deficient in this Ca(2+) release channel (IP3R2(-/-)). Using live acinar cell Ca(2+) imaging we found that loss of IP3R2 reduced the amplitude of the apical Ca(2+) signal and caused a delay in its initiation. This was associated with a reduction in carbachol-stimulated amylase release and an accumulation of zymogen granules (ZGs). Specifically, there was a 2-fold increase in the number of ZGs (P<0.05) and an expansion of the ZG pool area within the cell. There was also a 1.6- and 2.6-fold increase in cellular amylase and trypsinogen, respectively. However, the mice did not have evidence of pancreatic injury at baseline, other than an elevated serum amylase level. Further, pancreatitis outcomes using a mild caerulein hyperstimulation model were similar between IP3R2(-/-) and wild type mice. In summary, IP3R2 modulates apical acinar cell Ca(2+) signals and pancreatic enzyme secretion. IP3R-deficient acinar cells accumulate ZGs, but the mice do not succumb to pancreatic damage or worse pancreatitis outcomes.

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 ß-isoform of the catalytic A subunit (CnAß) was strongly expressed in mouse acinar cells. For this reason, we obtained acinar cells from CnAß-deficient mice (CnAß-/-) 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 CnAß-deficient cells was reduced by 50% (n = 2; P < 0.05). The CnAß-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.

Ethanol enhances carbachol-induced protease activation and accelerates Ca2+ waves in isolated rat pancreatic acini.

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.

Dantrolene mitigates caerulein-induced pancreatitis in vivo in mice.

Acute pancreatitis is a painful, inflammatory disorder for which adequate treatments are lacking. An early, critical step in its development is the aberrant signaling of Ca(2+) within the pancreatic acinar cell. This Ca(2+) release is modulated by the intracellular Ca(2+) channel the ryanodine receptor (RYR). We have previously shown that RYR inhibition reduces pathological intra-acinar protease activation, an early marker of pancreatitis. In this study, we examined whether pretreatment with the RYR inhibitor dantrolene attenuates the severity of caerulein-induced pancreatitis in mice. Immunofluorescent labeling for RYR from mouse pancreatic sections showed localization to the basolateral region of the acinar cell. After 1 h of caerulein hyperstimulation in vivo, dantrolene 1) reduced pancreatic trypsin activity by 59% (P < 0.05) and 2) mitigated early ultrastructural derangements within the acinar cell. Eight hours after pancreatitis induction, dantrolene reduced pancreatic trypsin activity and serum amylase by 61 and 32%, respectively (P < 0.05). At this later time point, overall histological severity of pancreatitis was reduced by 63% with dantrolene pretreatment (P < 0.05). TUNEL-positive cells were reduced by 58% (P < 0.05). These data suggest that the RYR plays an important role in mediating early acinar cell events during in vivo pancreatitis and contributes to disease severity. Blockade of Ca(2+) signals and particularly RYR-Ca(2+) may be useful as prophylactic treatment for this disease in high-risk settings for pancreatitis.

Changing referral trends of acute pancreatitis in children: A 12-year single-center analysis.

BACKGROUND: Acute pancreatitis is a painful inflammatory disorder known to occur in children. Recent reports, primarily on the basis of adult data, have suggested an increasing incidence. However, pediatric studies are limited. OBJECTIVE: The study was performed to examine the frequency of acute pancreatitis in a pediatric population from 1994 to 2007 and to characterize etiologies by age subsets. PATIENTS AND METHODS: In this retrospective study, cases of pancreatitis were identified by ICD-9 codes and subjected to inclusion criteria. RESULTS: Two hundred and seventy-one cases of pancreatitis met inclusion criteria. Mean age of the subjects was 13.1 +/- 5.6 years. The recurrence rate was 15.3%. Biliary disease was the most common etiology (32.6%). Acute pancreatitis cases evaluated at a single tertiary care center increased 53% between 1995 to 2000 and 2001 to 2006 (P < 0.02). However, when cases were normalized by all annual pediatric emergency department visits for all medical reasons, the increase was reduced to 22% and lost statistical significance (P = 0.16). The rise was not associated with a change in etiologies or body mass index (BMI). CONCLUSIONS: This is the first report demonstrating that an increase in pediatric pancreatitis may in part be due to growing referrals to tertiary care centers. The data on etiologies, particularly with regard to differing ages, may be helpful in managing children who present with acute pancreatitis.

Protease activation during in vivo pancreatitis is dependent on calcineurin activation.

The premature activation of digestive proenzymes, specifically proteases, within the pancreatic acinar cell is an early and critical event during acute pancreatitis. Our previous studies demonstrate that this activation requires a distinct pathological rise in cytosolic Ca(2+). Furthermore, we have shown that a target of aberrant Ca(2+) in acinar cells is the Ca(2+)/calmodulin-dependent phosphatase calcineurin (PP2B). In this study, we hypothesized that PP2B mediates in vivo protease activation and pancreatitis severity. To test this, pancreatitis was induced in mice over 8 h by administering hourly intraperitoneal injections of the cholecystokinin analog caerulein (50 microg/kg). Treatment with the PP2B inhibitor FK506 at 1 and 8 h after pancreatitis induction reduced trypsin activities by greater than 50% (P < 0.005). Serum amylase and IL-6 was reduced by 86 and 84% relative to baseline (P < 0.0005) at 8 h, respectively. Histological severity of pancreatitis, graded on the basis of pancreatic edema, acinar cell vacuolization, inflammation, and apoptosis, was reduced early in the course of pancreatitis. Myeloperoxidase activity from both pancreas and lung was reduced by 93 and 83% relative to baseline, respectively (P < 0.05). These data suggest that PP2B is an important target of the aberrant acinar cell Ca(2+) rise associated with pathological protease activation and pancreatitis.

Cyclic AMP accelerates calcium waves in pancreatic acinar cells.

Cytosolic Ca(2+) (Ca(i)(2+)) flux within the pancreatic acinar cell is important both physiologically and pathologically. We examined the role of cAMP in shaping the apical-to-basal Ca(2+) wave generated by the Ca(2+)-activating agonist carbachol. We hypothesized that cAMP modulates intra-acinar Ca(2+) channel opening by affecting either cAMP-dependent protein kinase (PKA) or exchange protein directly activated by cAMP (Epac). Isolated pancreatic acinar cells from rats were stimulated with carbachol (1 muM) with or without vasoactive intestinal polypeptide (VIP) or 8-bromo-cAMP (8-Br-cAMP), and then Ca(i)(2+) was monitored by confocal laser-scanning microscopy. The apical-to-basal carbachol (1 muM)-stimulated Ca(2+) wave was 8.63 +/- 0.68 microm/s; it increased to 19.66 +/- 2.22 microm/s (*P < 0.0005) with VIP (100 nM), and similar increases were observed with 8-Br-cAMP (100 microM). The Ca(2+) rise time after carbachol stimulation was reduced in both regions but to a greater degree in the basal. Lag time and maximal Ca(2+) elevation were not significantly affected by cAMP. The effect of cAMP on Ca(2+) waves also did not appear to depend on extracellular Ca(2+). However, the ryanodine receptor (RyR) inhibitor dantrolene (100 microM) reduced the cAMP-enhancement of wave speed. It was also reduced by the PKA inhibitor PKI (1 microM). 8-(4-chloro-phenylthio)-2'-O-Me-cAMP, a specific agonist of Epac, caused a similar increase as 8-Br-cAMP or VIP. These data suggest that cAMP accelerates the speed of the Ca(2+) wave in pancreatic acinar cells. A likely target of this modulation is the RyR, and these effects are mediated independently by PKA and Epac pathways.

Caerulein-induced intracellular pancreatic zymogen activation is dependent on calcineurin.

Aberrant cytosolic Ca(2+) flux in pancreatic acinar cells is critical to the pathological pancreatic zymogen activation observed in acute pancreatitis, but the downstream effectors are not known. In this study, we examined the role of Ca(2+)-activated protein phosphatase 2B (or calcineurin) in zymogen activation. Isolated pancreatic acinar cells were stimulated with supraphysiological caerulein (100 nM) with or without the calcineurin inhibitors FK506 or cell-permeable calcineurin inhibitory peptide (CiP). Chymotrypsin activity was measured as a marker of zymogen activation, and the percent amylase secretion was used as a measure of enzyme secretion. Cytosolic Ca(2+) changes were recorded in acinar cells loaded with the intermediate Ca(2+)-affinity dye fluo-5F using a scanning confocal microscope. A 50% reduction in chymotrypsin activity was observed after pretreatment with 1 microM FK506 or 10 microM CiP. These pretreatments did not affect amylase secretion or the rise in cytosolic Ca(2+) after caerulein stimulation. These findings suggest that calcineurin mediates caerulein-induced intra-acinar zymogen activation but not enzyme secretion or the initial caerulein-induced cytosolic Ca(2+) signal.