Following implementation of Improvement Outcome Guidance (IOG), pancreatic cancer surgery is now ... more Following implementation of Improvement Outcome Guidance (IOG), pancreatic cancer surgery is now mostly performed in major pancreatic referral centers. This may have affected selection for surgery and outcome. We report the effect on workload and mortality rate of centralizing pancreatic surgery for a population of about 3.5 millions to a single Centre. Method: We reviewed activity in 2003 and 2007 (before and after implementation of IOG). Pancreatic surgery was done in 3 hospitals in 2003. In 2007 centralization affected 2.3M population form January, and an additional 1.2M from April 2007. Before centralization the surgical HDU was expanded from 3 to 8 beds. Results: In 2003, there were 67 pancreatic procedures of all types performed by 7 surgeons. In 2007 102 patients diagnosed with a neoplasm were deemed operable, and underwent laparotomy. Only one patient on a cancer pathway failed to meet the 62 day target. Two patients were found to be inoperable, and had palliative bypass. Three surgeons performed 100 resections. A further 10 patients had pancreatic necrosectomy. Of the elective resections 94% were managed in surgical HDU (level 2 critical care). Neo-adjuvant treatment was attempted in 12 borderline pancreatic cancers, 4 were successfully resected, 3 are currently receiving chemotherapy, and in 5 cases there was no improvement and palliative chemoYradio therapy was stopped. Conclusion: Centralization has increased the numbers of patients in this Network offered pancreatic resection for tumor. This has enabled new approaches such as laparoscopic distal pancreatectomy and neoadjuvant treatment for borderline resectable tumors. The inoperability rate is low. Despite wider indications for surgery, the mortality rate has fallen. We conclude that centralization has enabled more patients to benefit from safe surgery for pancreatic neoplasm.
<p>(A) By EM, there was an increase in the number of ZGs. The nucleus (N) and lumen (Lu) of... more <p>(A) By EM, there was an increase in the number of ZGs. The nucleus (N) and lumen (Lu) of one cell from each lower power magnification is labeled (top row). (B) In some cells, ZGs could be seen extending into the basolateral (Bl) region. (C) Quantification of ZG number averaged over 20 fields at 8200X magnification. Each field contained one apical lumen that was positioned in the center of the field of view. Three to five cells converged at each lumen on cross section. (D) The average size of each ZG was unchanged between the two groups. *, P<0.05 relative to WT.</p
<p>(A) Schema for pancreatitis induction with hourly intra-peritoneal injections of (Caer)u... more <p>(A) Schema for pancreatitis induction with hourly intra-peritoneal injections of (Caer)ulein (50 ug/kg). Mice were euthanized 12 hr after the first caerulein injection, and serum amylase. (B) Intra-pancreatic trypsin activity, (C) percent wet weight of pancreas and (D) serum amylase from mice given caerulein were assayed. (E) Representative hematoxylin and eosin sections of the pancreas from caerulein treated mice, along with (F) overall and (G) categorical histological severity scores (n = 3 animals per group). *, P<0.05 relative to saline-treated controls.</p
<p>Isolated acinar cells received (A) caerulein or (B) carbachol over a range of concentrat... more <p>Isolated acinar cells received (A) caerulein or (B) carbachol over a range of concentrations. Secretion was assayed after 30 min. (n = 3).*, P<0.05 relative to WT cells administered the same concentration of agonist.</p
<p>(A) Hematoxylin and eosin staining of pancreas tissue from WT and IP3R2<sup>−/−<... more <p>(A) Hematoxylin and eosin staining of pancreas tissue from WT and IP3R2<sup>−/−</sup> mice. (B) The area of the ZG pool is represented as percent of total cell area (n = 25–40 cells per condition). *, P<0.05 relative to WT.</p
<p>(A) Western blots were performed on pancreatic homogenates from WT and IP3R2<sup>−... more <p>(A) Western blots were performed on pancreatic homogenates from WT and IP3R2<sup>−/−</sup> mice for amylase and actin. Densitometry was expressed as fold increase over WT, normalized to actin (n = 3). (B) Amylase and (C) trypsinogen content were obtained by measuring the activities of each enzyme from lysed acinar cell suspensions of equal volume. Trypsinogen was activated to trypsin by incubating with enterokinase (1 uM) for 15 min prior to activity measurements (n = 3). *, P<0.05 relative to WT.</p
<p>(A) From left to right: Bright field view of an acinus, with a single acinar cell labele... more <p>(A) From left to right: Bright field view of an acinus, with a single acinar cell labeled at the apical (Ap) and basolateral (Bl) regions of interest. Fluorescent images of acini loaded with the Ca<sup>2+</sup> indicator fluo-4 at baseline (1), shortly after stimulation with 1 uM carbachol (2), and subsequent images showing propagation of the Ca<sup>2+</sup> wave from the apical to the basolateral region (3,4). (B) Each paneled image (1–4) also corresponds to a frame along a representative tracing of change in fluorescence over time for each region of interest. (C) Comparisons of the amplitude, latency period, and Ca<sup>2+</sup> wave speed between WT and IP3R2<sup>−/−</sup> cells (n = 25–40 cells in each group). The time from the administration of carbachol to the first Ca<sup>2+</sup> rise in the apical region is the latency period. *, P<0.05 relative to WT.</p
Acute pancreatitis is a painful, life-threatening disorder of the pancreas whose etiology is ofte... more 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 2/2). 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 2/2 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.
Following implementation of Improvement Outcome Guidance (IOG), pancreatic cancer surgery is now ... more Following implementation of Improvement Outcome Guidance (IOG), pancreatic cancer surgery is now mostly performed in major pancreatic referral centers. This may have affected selection for surgery and outcome. We report the effect on workload and mortality rate of centralizing pancreatic surgery for a population of about 3.5 millions to a single Centre. Method: We reviewed activity in 2003 and 2007 (before and after implementation of IOG). Pancreatic surgery was done in 3 hospitals in 2003. In 2007 centralization affected 2.3M population form January, and an additional 1.2M from April 2007. Before centralization the surgical HDU was expanded from 3 to 8 beds. Results: In 2003, there were 67 pancreatic procedures of all types performed by 7 surgeons. In 2007 102 patients diagnosed with a neoplasm were deemed operable, and underwent laparotomy. Only one patient on a cancer pathway failed to meet the 62 day target. Two patients were found to be inoperable, and had palliative bypass. Three surgeons performed 100 resections. A further 10 patients had pancreatic necrosectomy. Of the elective resections 94% were managed in surgical HDU (level 2 critical care). Neo-adjuvant treatment was attempted in 12 borderline pancreatic cancers, 4 were successfully resected, 3 are currently receiving chemotherapy, and in 5 cases there was no improvement and palliative chemoYradio therapy was stopped. Conclusion: Centralization has increased the numbers of patients in this Network offered pancreatic resection for tumor. This has enabled new approaches such as laparoscopic distal pancreatectomy and neoadjuvant treatment for borderline resectable tumors. The inoperability rate is low. Despite wider indications for surgery, the mortality rate has fallen. We conclude that centralization has enabled more patients to benefit from safe surgery for pancreatic neoplasm.
Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute and up to 90% of ch... more Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute and up to 90% of chronic cases, yet the mechanisms leading to alcohol-induced injury remain elusive. An early and critical feature of this disease is the aberrant signaling of Ca2+ within the pancreatic acinar cell. An important conductor of this Ca2+ release is the basolaterally localized, intracellular Ca2+ channel the ryanodine receptor (RYR). In this study, we examined the role of the RYR in mediating Ca2+ signals during alcohol exposure. We hypothesized that alcohol triggers the release of Ca2+ from pathologically leaky, hyper-phosphorylated RYRs. Acinar cells were freshly isolated from rat, loaded with the Ca2+ dye fluo-4, and imaged by time lapse confocal microscopy. Spatial and temporal changes in Ca2+ release were examined after perfusion with the Ca2+-activating agonist carbachol (1 uM) with or without an ethanol concentration clinically achievable after heavy intoxication (100 mM). Ethanol accelerated the speed of the apical to basolateral Ca2+ wave from 9 to 18 um/sec (P<0.0005; n=18-22 cells/group). Ethanol also caused a similar doubling in intra-cellular cAMP levels from 1.5 to 3 fmoles. Acceleration of the acinar cell Ca2+ wave by alcohol was abrogated by the PKA inhibitor PKI (1 uM; P<0.05; n=10-16 cells/group). Using an epitope-specific phospho-antibody that recognizes the PKA site on the RYR, we found that ethanol increased RYR phosphorylation by nearly five-fold (P<0.05). Finally, the alcohol-induced acceleration of Ca2+ wave speed was completely reduced by the RYR inhibitor dantrolene (100 uM; P<0.05; n=10-16 cells/group). In summary, our results have for the first time implicated a pathological role for RYR-Ca2+ release in the predisposition to alcohol-induced pancreatitis via cAMP/PKA hyper-phosphorylation of the RYR.
Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute and up to 90% of ch... more Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute and up to 90% of chronic cases, yet the mechanisms leading to alcohol-induced injury remain elusive. An early and critical feature of this disease is the aberrant signaling of Ca2+ within the pancreatic acinar cell. An important conductor of this Ca2+ release is the basolaterally localized, intracellular Ca2+ channel the ryanodine receptor (RYR). In this study, we examined the role of the RYR in mediating Ca2+ signals during alcohol exposure. We hypothesized that alcohol triggers the release of Ca2+ from pathologically leaky, hyper-phosphorylated RYRs. Acinar cells were freshly isolated from rat, loaded with the Ca2+ dye fluo-4, and imaged by time lapse confocal microscopy. Spatial and temporal changes in Ca2+ release were examined after perfusion with the Ca2+-activating agonist carbachol (1 uM) with or without an ethanol concentration clinically achievable after heavy intoxication (100 mM). Ethanol accelerated the speed of the apical to basolateral Ca2+ wave from 9 to 18 um/sec (P<0.0005; n=18-22 cells/group). Ethanol also caused a similar doubling in intra-cellular cAMP levels from 1.5 to 3 fmoles. Acceleration of the acinar cell Ca2+ wave by alcohol was abrogated by the PKA inhibitor PKI (1 uM; P<0.05; n=10-16 cells/group). Using an epitope-specific phospho-antibody that recognizes the PKA site on the RYR, we found that ethanol increased RYR phosphorylation by nearly five-fold (P<0.05). Finally, the alcohol-induced acceleration of Ca2+ wave speed was completely reduced by the RYR inhibitor dantrolene (100 uM; P<0.05; n=10-16 cells/group). In summary, our results have for the first time implicated a pathological role for RYR-Ca2+ release in the predisposition to alcohol-induced pancreatitis via cAMP/PKA hyper-phosphorylation of the RYR.
American Journal of Physiology-Gastrointestinal and Liver Physiology, 2008
Cytosolic Ca2+(Ca[Formula: see text]) flux within the pancreatic acinar cell is important both ph... more Cytosolic Ca2+(Ca[Formula: see text]) flux within the pancreatic acinar cell is important both physiologically and pathologically. We examined the role of cAMP in shaping the apical-to-basal Ca2+wave generated by the Ca2+-activating agonist carbachol. We hypothesized that cAMP modulates intra-acinar Ca2+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 μM) with or without vasoactive intestinal polypeptide (VIP) or 8-bromo-cAMP (8-Br-cAMP), and then Ca[Formula: see text] was monitored by confocal laser-scanning microscopy. The apical-to-basal carbachol (1 μM)-stimulated Ca2+wave was 8.63 ± 0.68 μm/s; it increased to 19.66 ± 2.22 μm/s (* P < 0.0005) with VIP (100 nM), and similar increases were observed with 8-Br-cAMP (100 μM). The Ca2+rise time after carbachol stimulation was reduced in both regions but to a greater degree in the...
Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute and up to 90% of ch... more Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute and up to 90% of chronic cases, yet the mechanisms leading to alcohol-induced injury remain elusive. An early and critical feature of this disease is the aberrant signaling of Ca2+ within the pancreatic acinar cell. An important conductor of this Ca2+ release is the basolaterally localized, intracellular Ca2+ channel the ryanodine receptor (RYR). In this study, we examined the role of the RYR in mediating Ca2+ signals during alcohol exposure. We hypothesized that alcohol triggers the release of Ca2+ from pathologically leaky, hyper-phosphorylated RYRs. Acinar cells were freshly isolated from rat, loaded with the Ca2+ dye fluo-4, and imaged by time lapse confocal microscopy. Spatial and temporal changes in Ca2+ release were examined after perfusion with the Ca2+-activating agonist carbachol (1 uM) with or without an ethanol concentration clinically achievable after heavy intoxication (100 mM). Ethanol accelerated the speed of the apical to basolateral Ca2+ wave from 9 to 18 um/sec (P<0.0005; n=18-22 cells/group). Ethanol also caused a similar doubling in intra-cellular cAMP levels from 1.5 to 3 fmoles. Acceleration of the acinar cell Ca2+ wave by alcohol was abrogated by the PKA inhibitor PKI (1 uM; P<0.05; n=10-16 cells/group). Using an epitope-specific phospho-antibody that recognizes the PKA site on the RYR, we found that ethanol increased RYR phosphorylation by nearly five-fold (P<0.05). Finally, the alcohol-induced acceleration of Ca2+ wave speed was completely reduced by the RYR inhibitor dantrolene (100 uM; P<0.05; n=10-16 cells/group). In summary, our results have for the first time implicated a pathological role for RYR-Ca2+ release in the predisposition to alcohol-induced pancreatitis via cAMP/PKA hyper-phosphorylation of the RYR.
Following implementation of Improvement Outcome Guidance (IOG), pancreatic cancer surgery is now ... more Following implementation of Improvement Outcome Guidance (IOG), pancreatic cancer surgery is now mostly performed in major pancreatic referral centers. This may have affected selection for surgery and outcome. We report the effect on workload and mortality rate of centralizing pancreatic surgery for a population of about 3.5 millions to a single Centre. Method: We reviewed activity in 2003 and 2007 (before and after implementation of IOG). Pancreatic surgery was done in 3 hospitals in 2003. In 2007 centralization affected 2.3M population form January, and an additional 1.2M from April 2007. Before centralization the surgical HDU was expanded from 3 to 8 beds. Results: In 2003, there were 67 pancreatic procedures of all types performed by 7 surgeons. In 2007 102 patients diagnosed with a neoplasm were deemed operable, and underwent laparotomy. Only one patient on a cancer pathway failed to meet the 62 day target. Two patients were found to be inoperable, and had palliative bypass. Three surgeons performed 100 resections. A further 10 patients had pancreatic necrosectomy. Of the elective resections 94% were managed in surgical HDU (level 2 critical care). Neo-adjuvant treatment was attempted in 12 borderline pancreatic cancers, 4 were successfully resected, 3 are currently receiving chemotherapy, and in 5 cases there was no improvement and palliative chemoYradio therapy was stopped. Conclusion: Centralization has increased the numbers of patients in this Network offered pancreatic resection for tumor. This has enabled new approaches such as laparoscopic distal pancreatectomy and neoadjuvant treatment for borderline resectable tumors. The inoperability rate is low. Despite wider indications for surgery, the mortality rate has fallen. We conclude that centralization has enabled more patients to benefit from safe surgery for pancreatic neoplasm.
<p>(A) By EM, there was an increase in the number of ZGs. The nucleus (N) and lumen (Lu) of... more <p>(A) By EM, there was an increase in the number of ZGs. The nucleus (N) and lumen (Lu) of one cell from each lower power magnification is labeled (top row). (B) In some cells, ZGs could be seen extending into the basolateral (Bl) region. (C) Quantification of ZG number averaged over 20 fields at 8200X magnification. Each field contained one apical lumen that was positioned in the center of the field of view. Three to five cells converged at each lumen on cross section. (D) The average size of each ZG was unchanged between the two groups. *, P<0.05 relative to WT.</p
<p>(A) Schema for pancreatitis induction with hourly intra-peritoneal injections of (Caer)u... more <p>(A) Schema for pancreatitis induction with hourly intra-peritoneal injections of (Caer)ulein (50 ug/kg). Mice were euthanized 12 hr after the first caerulein injection, and serum amylase. (B) Intra-pancreatic trypsin activity, (C) percent wet weight of pancreas and (D) serum amylase from mice given caerulein were assayed. (E) Representative hematoxylin and eosin sections of the pancreas from caerulein treated mice, along with (F) overall and (G) categorical histological severity scores (n = 3 animals per group). *, P<0.05 relative to saline-treated controls.</p
<p>Isolated acinar cells received (A) caerulein or (B) carbachol over a range of concentrat... more <p>Isolated acinar cells received (A) caerulein or (B) carbachol over a range of concentrations. Secretion was assayed after 30 min. (n = 3).*, P<0.05 relative to WT cells administered the same concentration of agonist.</p
<p>(A) Hematoxylin and eosin staining of pancreas tissue from WT and IP3R2<sup>−/−<... more <p>(A) Hematoxylin and eosin staining of pancreas tissue from WT and IP3R2<sup>−/−</sup> mice. (B) The area of the ZG pool is represented as percent of total cell area (n = 25–40 cells per condition). *, P<0.05 relative to WT.</p
<p>(A) Western blots were performed on pancreatic homogenates from WT and IP3R2<sup>−... more <p>(A) Western blots were performed on pancreatic homogenates from WT and IP3R2<sup>−/−</sup> mice for amylase and actin. Densitometry was expressed as fold increase over WT, normalized to actin (n = 3). (B) Amylase and (C) trypsinogen content were obtained by measuring the activities of each enzyme from lysed acinar cell suspensions of equal volume. Trypsinogen was activated to trypsin by incubating with enterokinase (1 uM) for 15 min prior to activity measurements (n = 3). *, P<0.05 relative to WT.</p
<p>(A) From left to right: Bright field view of an acinus, with a single acinar cell labele... more <p>(A) From left to right: Bright field view of an acinus, with a single acinar cell labeled at the apical (Ap) and basolateral (Bl) regions of interest. Fluorescent images of acini loaded with the Ca<sup>2+</sup> indicator fluo-4 at baseline (1), shortly after stimulation with 1 uM carbachol (2), and subsequent images showing propagation of the Ca<sup>2+</sup> wave from the apical to the basolateral region (3,4). (B) Each paneled image (1–4) also corresponds to a frame along a representative tracing of change in fluorescence over time for each region of interest. (C) Comparisons of the amplitude, latency period, and Ca<sup>2+</sup> wave speed between WT and IP3R2<sup>−/−</sup> cells (n = 25–40 cells in each group). The time from the administration of carbachol to the first Ca<sup>2+</sup> rise in the apical region is the latency period. *, P<0.05 relative to WT.</p
Acute pancreatitis is a painful, life-threatening disorder of the pancreas whose etiology is ofte... more 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 2/2). 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 2/2 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.
Following implementation of Improvement Outcome Guidance (IOG), pancreatic cancer surgery is now ... more Following implementation of Improvement Outcome Guidance (IOG), pancreatic cancer surgery is now mostly performed in major pancreatic referral centers. This may have affected selection for surgery and outcome. We report the effect on workload and mortality rate of centralizing pancreatic surgery for a population of about 3.5 millions to a single Centre. Method: We reviewed activity in 2003 and 2007 (before and after implementation of IOG). Pancreatic surgery was done in 3 hospitals in 2003. In 2007 centralization affected 2.3M population form January, and an additional 1.2M from April 2007. Before centralization the surgical HDU was expanded from 3 to 8 beds. Results: In 2003, there were 67 pancreatic procedures of all types performed by 7 surgeons. In 2007 102 patients diagnosed with a neoplasm were deemed operable, and underwent laparotomy. Only one patient on a cancer pathway failed to meet the 62 day target. Two patients were found to be inoperable, and had palliative bypass. Three surgeons performed 100 resections. A further 10 patients had pancreatic necrosectomy. Of the elective resections 94% were managed in surgical HDU (level 2 critical care). Neo-adjuvant treatment was attempted in 12 borderline pancreatic cancers, 4 were successfully resected, 3 are currently receiving chemotherapy, and in 5 cases there was no improvement and palliative chemoYradio therapy was stopped. Conclusion: Centralization has increased the numbers of patients in this Network offered pancreatic resection for tumor. This has enabled new approaches such as laparoscopic distal pancreatectomy and neoadjuvant treatment for borderline resectable tumors. The inoperability rate is low. Despite wider indications for surgery, the mortality rate has fallen. We conclude that centralization has enabled more patients to benefit from safe surgery for pancreatic neoplasm.
Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute and up to 90% of ch... more Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute and up to 90% of chronic cases, yet the mechanisms leading to alcohol-induced injury remain elusive. An early and critical feature of this disease is the aberrant signaling of Ca2+ within the pancreatic acinar cell. An important conductor of this Ca2+ release is the basolaterally localized, intracellular Ca2+ channel the ryanodine receptor (RYR). In this study, we examined the role of the RYR in mediating Ca2+ signals during alcohol exposure. We hypothesized that alcohol triggers the release of Ca2+ from pathologically leaky, hyper-phosphorylated RYRs. Acinar cells were freshly isolated from rat, loaded with the Ca2+ dye fluo-4, and imaged by time lapse confocal microscopy. Spatial and temporal changes in Ca2+ release were examined after perfusion with the Ca2+-activating agonist carbachol (1 uM) with or without an ethanol concentration clinically achievable after heavy intoxication (100 mM). Ethanol accelerated the speed of the apical to basolateral Ca2+ wave from 9 to 18 um/sec (P<0.0005; n=18-22 cells/group). Ethanol also caused a similar doubling in intra-cellular cAMP levels from 1.5 to 3 fmoles. Acceleration of the acinar cell Ca2+ wave by alcohol was abrogated by the PKA inhibitor PKI (1 uM; P<0.05; n=10-16 cells/group). Using an epitope-specific phospho-antibody that recognizes the PKA site on the RYR, we found that ethanol increased RYR phosphorylation by nearly five-fold (P<0.05). Finally, the alcohol-induced acceleration of Ca2+ wave speed was completely reduced by the RYR inhibitor dantrolene (100 uM; P<0.05; n=10-16 cells/group). In summary, our results have for the first time implicated a pathological role for RYR-Ca2+ release in the predisposition to alcohol-induced pancreatitis via cAMP/PKA hyper-phosphorylation of the RYR.
Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute and up to 90% of ch... more Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute and up to 90% of chronic cases, yet the mechanisms leading to alcohol-induced injury remain elusive. An early and critical feature of this disease is the aberrant signaling of Ca2+ within the pancreatic acinar cell. An important conductor of this Ca2+ release is the basolaterally localized, intracellular Ca2+ channel the ryanodine receptor (RYR). In this study, we examined the role of the RYR in mediating Ca2+ signals during alcohol exposure. We hypothesized that alcohol triggers the release of Ca2+ from pathologically leaky, hyper-phosphorylated RYRs. Acinar cells were freshly isolated from rat, loaded with the Ca2+ dye fluo-4, and imaged by time lapse confocal microscopy. Spatial and temporal changes in Ca2+ release were examined after perfusion with the Ca2+-activating agonist carbachol (1 uM) with or without an ethanol concentration clinically achievable after heavy intoxication (100 mM). Ethanol accelerated the speed of the apical to basolateral Ca2+ wave from 9 to 18 um/sec (P<0.0005; n=18-22 cells/group). Ethanol also caused a similar doubling in intra-cellular cAMP levels from 1.5 to 3 fmoles. Acceleration of the acinar cell Ca2+ wave by alcohol was abrogated by the PKA inhibitor PKI (1 uM; P<0.05; n=10-16 cells/group). Using an epitope-specific phospho-antibody that recognizes the PKA site on the RYR, we found that ethanol increased RYR phosphorylation by nearly five-fold (P<0.05). Finally, the alcohol-induced acceleration of Ca2+ wave speed was completely reduced by the RYR inhibitor dantrolene (100 uM; P<0.05; n=10-16 cells/group). In summary, our results have for the first time implicated a pathological role for RYR-Ca2+ release in the predisposition to alcohol-induced pancreatitis via cAMP/PKA hyper-phosphorylation of the RYR.
American Journal of Physiology-Gastrointestinal and Liver Physiology, 2008
Cytosolic Ca2+(Ca[Formula: see text]) flux within the pancreatic acinar cell is important both ph... more Cytosolic Ca2+(Ca[Formula: see text]) flux within the pancreatic acinar cell is important both physiologically and pathologically. We examined the role of cAMP in shaping the apical-to-basal Ca2+wave generated by the Ca2+-activating agonist carbachol. We hypothesized that cAMP modulates intra-acinar Ca2+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 μM) with or without vasoactive intestinal polypeptide (VIP) or 8-bromo-cAMP (8-Br-cAMP), and then Ca[Formula: see text] was monitored by confocal laser-scanning microscopy. The apical-to-basal carbachol (1 μM)-stimulated Ca2+wave was 8.63 ± 0.68 μm/s; it increased to 19.66 ± 2.22 μm/s (* P < 0.0005) with VIP (100 nM), and similar increases were observed with 8-Br-cAMP (100 μM). The Ca2+rise time after carbachol stimulation was reduced in both regions but to a greater degree in the...
Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute and up to 90% of ch... more Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute and up to 90% of chronic cases, yet the mechanisms leading to alcohol-induced injury remain elusive. An early and critical feature of this disease is the aberrant signaling of Ca2+ within the pancreatic acinar cell. An important conductor of this Ca2+ release is the basolaterally localized, intracellular Ca2+ channel the ryanodine receptor (RYR). In this study, we examined the role of the RYR in mediating Ca2+ signals during alcohol exposure. We hypothesized that alcohol triggers the release of Ca2+ from pathologically leaky, hyper-phosphorylated RYRs. Acinar cells were freshly isolated from rat, loaded with the Ca2+ dye fluo-4, and imaged by time lapse confocal microscopy. Spatial and temporal changes in Ca2+ release were examined after perfusion with the Ca2+-activating agonist carbachol (1 uM) with or without an ethanol concentration clinically achievable after heavy intoxication (100 mM). Ethanol accelerated the speed of the apical to basolateral Ca2+ wave from 9 to 18 um/sec (P<0.0005; n=18-22 cells/group). Ethanol also caused a similar doubling in intra-cellular cAMP levels from 1.5 to 3 fmoles. Acceleration of the acinar cell Ca2+ wave by alcohol was abrogated by the PKA inhibitor PKI (1 uM; P<0.05; n=10-16 cells/group). Using an epitope-specific phospho-antibody that recognizes the PKA site on the RYR, we found that ethanol increased RYR phosphorylation by nearly five-fold (P<0.05). Finally, the alcohol-induced acceleration of Ca2+ wave speed was completely reduced by the RYR inhibitor dantrolene (100 uM; P<0.05; n=10-16 cells/group). In summary, our results have for the first time implicated a pathological role for RYR-Ca2+ release in the predisposition to alcohol-induced pancreatitis via cAMP/PKA hyper-phosphorylation of the RYR.
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