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T1934 Ca2+ Signaling via G Protein Coupled P2-Receptors in Activated Pancreatic Stellate Cells
T1933
oscillation frequency. Conclusion: Acidic extracellular pH enhances certain features of calcium signals in the pancreatic acinar cell, which may be responsible for the increased sensitivity to pancreatitis.
AGA Abstracts
Identification of Epi64b as a GTPase-Activating Protein for Rab27b in Mouse Pancreatic Acini Yanan Hou, Xuequn Chen, John A. Williams
T1936
Background: Previous studies have shown that the small G-proteins Rab27B and 3D are present on the pancreatic zymogen granules and support their participation in digestive enzyme secretion. However, regulation of their activity is poorly understood. Recently, the Tre-2/Bub2/Cdc16 (TBC) domain has been recognized as the hallmark of Rab GTPaseactivating proteins (GAPs). Two TBC proteins, EPI64 and 64B were identified as having specific GAP activity for Rab27A. We therefore investigated the role of three related TBC domain-containing proteins as potential GAPs for Rab27B in pancreatic acinar cells. Methods: Isolated pancreatic acini were prepared from mouse pancreas and amylase secretion was measured by amylase assay. Expression of TBC domain proteins EPI64, 64B and 64C in pancreatic acini was examined by RT-PCR. Effects of EPI64B over-expression on GTP-bound state of Rab27B and Rab3D were assessed by pulldown assay using GST-SHD for Rab27B and GST-Rim for Rab3D, respectively. Results: Both EPI64 and 64B were found to be expressed in mouse pancreatic acini by RT-PCR. Mouse EPI64 and 64B sequences were cloned into an adenoviral vector with epitope tags. Over-expression of EPI64B in isolated mouse pancreatic acini by adenoviral delivery decreased the GTP binding of endogenous Rab27B by 69%±17% (mean±SD), while the GTP-bound state of Rab3D was not affected. We found that membrane-associated fraction of Rab27B was also reduced to a similar extent in the pancreatic acini over-expressed with EPI64B. EPI64B over-expression enhanced the basal level of amylase release by 37%±10%. The entire dose response curve of CCK-stimulated amylase release was elevated by EPI64B over-expression with a maximal increase of 59%±20% at a CCK concentration of 300 pM. EPI64 had no effects on the GTP binding state of Rab27B or Rab3D proteins or on amylase release. Conclusions: EPI64B is a potential GTPase-activating protein (GAP) for Rab27B and may play an important role in regulating the activity of Rab27B in mouse pancreatic acinar cells. Moreover, the data showed that reducing the amount of active Rab27B enhanced amylase secretion.
Ethanol Impaired the Assembly and Disassembly of Actin Cytoskeleton and Cell-Cell Adhesion via RHOA and Catenin P120 in CCK-Stimulated Pancreatic Acini Tomoyuki Iwata, Fumihiko Nozu, Michio Imawari Background: Although ethanol is the common risk factor for pancreatitis, precise interactions of actin cytoskeleton, cell-cell adhesion proteins and ethanol have not been clarified in pancreatic acini. Aim: We attempted to evaluate the interaction of RhoA, catenin p120 (p120), F-actin and ethanol in CCK-stimulated pancreatic acini. Methods: Isolated acini were obtained from male Sprague-Dawley rats. Intact acini were preincubated with ethanol or Rho inhibitor, pravastatin and incubated with CCK. The protein expressions and the association of RhoA and p120 were analyzed by Western immunoblotting and immunoprecipitation. The translocation of RhoA was examined by cell fractionation assay. Amylase secretion was also measured. The distribution of RhoA, p120 and F-actin were analyzed by conforcal laser microscopy. Results: CCK (10 pM - 10 nM) enhanced the expressions and association of RhoA and p120. Although pretreatment of 5 mM ethanol, 5 and 10 μM pravastatin did not alter the expressions and association of RhoA and p120 in CCK-treated pancreatic acini, the pretreatment of 20 mM ethanol and 30 μM pravastatin inhibited the expressions and association. Furthermore, cell fractionation assay was performed. In resting state, RhoA was mainly located in the cytosol fraction. RhoA translocated from the cytosol fraction to the membrane fraction after CCK (10 pM, 10 nM) stimulation. On the other hand, pretreatment of 20 mM ethanol and 30 μM pravastatin inhibited the translocation of RhoA. 5 and 10 μM pravastatin did not inhibit dose response of amylase secretion in CCK (10 pM - 10 nM) stimulation without altering basal secretion, the pretreatment of 20 mM ethanol and 30 μM pravastatin inhibited amylase secretion. Conforcal microscopy images showed that RhoA was diffusely distributed throughout of acinar cell and p120 was localized to the lateral plasma membrane in the resting state. F-actin located in apical site and lateral site in the resting state. After 10 pM CCK stimulation, RhoA, p120 and F-actin were mainly located to apical site. After 10 nM CCK stimulation, the distributions of these proteins to apical site were diminished. Pretreatment of 5 mM ethanol and 5 and 10 μM pravastatin did not alter distributions of the proteins in the resting state and after CCK (10 pM, 10 nM) stimulation. The pretreatment of 20 mM ethanol and 30 μM pravastatin inhibited the distributions of these proteins to apical site and lateral site in the resting state and after CCK (10 pM, 10 nM) stimulation. Conclusion: We conclude that ethanol impaired the assembly and disassembly of actin cytoskeleton and cell-cell adhesion via RhoA and p120 in CCK-stimulated pancreatic acini.
T1934 Ca2+ Signaling via G Protein Coupled P2-Receptors in Activated Pancreatic Stellate Cells Jan K. Hennigs, Oliver Seiz, Julia M. Spiro, Marc J. Berna, Hans Klose, Andrea Pace BACKGROUND: There is growing evidence that extracellular nucleotide induced signaling confers to fibrogenesis in liver and pancreas. P2 purine and pyrimidine receptors are pivotal mediators of inflammatory and pro-fibrogenic signals in liver and most likely pancreas as well. However, a specific analysis of the underlying signaling components in activated pancreatic stellate cells, the most important cell type in pancreatic fibrosis, has not been performed so far. METHODS: Rat pancreatic stellate cells (PSC) were freshly isolated and cultured until auto-activation. FURA-2-laden PSC were stimulated by various nucleotides including AMP, ADP, ATP, UDP and UTP and real-time intracellular Ca2+ responses were monitored under various buffer conditions using a cuvette fluorimeter. Additionally, expression of classical components of intracellular Ca2+ signaling such as G proteins and Phospholipase C (PLC) subunits was evaluated by reverse transcription PCR. Rats were cared according to the guidelines and under supervision of the local animal welfare board. RESULTS: A broad variety of Ca2+ signaling components are expressed in activated PSC: All four known PLC-stimulating G alpha subunits Gq, G11, G14 and G15 are present on mRNA basis in activated PSC as were all three IP3 receptor subunits. Additionally, transcripts of PLC-beta subunits 1,3 and 4 were detectable by RT-PCR. In increasing magnitude, purines AMP, ADP and ATP elicited detectable rises in intracellular Ca2+ concentrations. Stimulation of PSC by ATP lead to intracellular Ca2+ increases mediated through both P2X and P2Y receptors. Ca2+ was mostly released from internal stores as shown by preceding depletion of PSCs from extra- and intracellular Ca2+ stores or by preincubation with PLC inhibitor U73122. Pyrimidines triggered more sustained Ca2+ signals than purines. Whereas UTP mediated Ca2+ signals were generated by activation of P2Y receptors only, surprisingly, UDP stimulated P2X receptors as well. DISCUSSION: Not surprisingly, activated pancreatic stellate cells feature a plethora of elements from the Ca2+ signaling toolkit and functionally express a broad variety of P2 nucleotide receptors. Purines and pyrimidines elicit robust intracellular Ca2+ signals likely contributing to the fibrogenetic potential of this cells.
T1937 Extracellular Signal-Regulated Kinase 1/2 Signaling Pathway in Rostral Nucleus Tract Solitarius (NTS) Mediates CCK-Stimulated Pancreatic Enzyme Secretion Yuanxu Lu, Phattrawan Pisuchpen, Shi-Yi Zhou, Angela Y. Chen, Xinyi Wang, Chung Owyang The high- and low-affinity CCK-A receptors are present on distinct vagal afferent fibers which mediate pancreatic enzyme secretion and satiety respectively. We hypothesize that these two groups of nodose ganglia neurons project to different regions of the NTS which in turn relay signals to other parts of the brainstem and hypothalamus to mediate different functions. The objective of this study is to define the intracellular signaling pathways in the NTS that relay and integrate signals from high-affinity vagal CCKA receptors. Recently we showed that most of the NTS neurons activated by gastrointestinal stimuli contain both NMDA and AMPA glutamate receptors which are coupled to the extracellular signal-regulated kinase 1/2 signaling (ERK1/2) pathways. We showed by Western blot that ip administration of JMV-180 which acts as an agonist on high-affinity CCKA receptor and as an antagonist on low affinity CCK receptors caused a dose-dependent increase in pERK1/2 in the NTS. A maximum of 2.6-fold increase was observed at 30 min after JMV-180 (1000 μg/kg ip). Majority of the increase (2/3) came from the rostral NTS whereas the remaining (1/3) were from the caudal NTS. Administration (ip) of CCK8 (10 μg/kg) which acts on both high- as well as low-affinity CCKA receptors caused a similar increase in pERK1/2 but with the 2/ 3 distribution in the caudal and 1/3 in the rostral NTS. Combined administration of JMV180 and CCK8 shifted to a mostly rostral distribution. The expression of RAS, the upstream activator of ERK1/2 has a similar regional distribution pattern as pERK1/2 in response to JMV-180. A similar increase was observed in the A-type voltage dependent K+ channel Kv4.2. The channel is specifically phosphorylated at Thr602 by ERK, resulting in decreased opening of the channel and a relative depolarization of the neuron. The preferential activation of rostral vs caudal NTS neurons by JMV-180 was confirmed by co-staining of pERK and cFos in NTS. Using a rat model with a pancreatic-biliary cannula, we showed that JMV180 (ip) caused a dose-dependent increase in pancreatic protein secretion, reaching a peak at 30″. Microinjection of the MEK inhibitor U0126 (2 μg in 5 μl) into the fourth ventricle reduced JMV-180 evoked pancreatic protein secretion by 45% and this was accomplished by 50% reduction of the phosphorylated Kv4.2 protein. In conclusion, we showed that stimulation of high- and low-affinity vagal CCKA receptors caused differential activation of the rostral vs caudal NTS. The ERK 1/2 signaling pathway in the rostral NTS through phosphorylation of Kv4.2 mediates CCK-stimulated pancreatic enzyme secretion.
T1935 Low pH Enhances Calcium Signaling in the Pancreatic Acinar Cell Anamika Chaudhuri, Ahsan U. Shah, Sohail Z. Husain, Fred S. Gorelick, Michael H. Nathanson Background: Recent data from our lab demonstrates that low pH sensitizes the pancreatic acinar cell to secretagogue-induced zymogen activation and injury. While pathologic calcium signaling underlies aberrant intracellular zymogen activation and cell injury in the acinar cell, the relationship between the injurious effects of acidic conditions and calcium signaling has not been investigated. Aim: To examine the effects of acidic pH on physiologic calcium signaling in the acinar cell. Methods: Isolated rat acinar cells were loaded with the calcium dye, fluo-4, perfused with either pH 7.4 or pH 7.0 HEPES buffer, and treated with physiologic concentrations (10 pM) of the cholecystokinin analogue, cerulein. Changes in cytosolic calcium were measured with time lapse confocal microscopy and normalized to baseline fluorescence. Results: The frequency of calcium oscillations significantly decreased under low pH conditions, from 1.03 +/- 0.07 oscillations per minute at pH 7.4 to 0.63 +/- 0.05 oscillations per minute at pH 7.0, p < 0.05. Cells treated with low pH buffer demonstrated higher amplitude oscillations, with an increase from 455% +/- 59% baseline at pH 7.4 to 655% +/- 76% baseline at pH 7.0, p < 0.05. The effects of low extracellular pH on oscillation frequency and amplitude were not affected by perfusing with calcium free medium containing 10 mM EGTA. Pretreating acini with the ryanodine receptor inhibitors, dantrolene (75 μM) and ryanodine (100 μM) reduced the effects of low pH on amplitude, but did not affect
AGA Abstracts
S-610
oscillation frequency. Conclusion: Acidic extracellular pH enhances certain features of calcium signals in the pancreatic acinar cell, which may be responsible for the increased sensitivity to pancreatitis.
AGA Abstracts
Identification of Epi64b as a GTPase-Activating Protein for Rab27b in Mouse Pancreatic Acini Yanan Hou, Xuequn Chen, John A. Williams
T1936
Background: Previous studies have shown that the small G-proteins Rab27B and 3D are present on the pancreatic zymogen granules and support their participation in digestive enzyme secretion. However, regulation of their activity is poorly understood. Recently, the Tre-2/Bub2/Cdc16 (TBC) domain has been recognized as the hallmark of Rab GTPaseactivating proteins (GAPs). Two TBC proteins, EPI64 and 64B were identified as having specific GAP activity for Rab27A. We therefore investigated the role of three related TBC domain-containing proteins as potential GAPs for Rab27B in pancreatic acinar cells. Methods: Isolated pancreatic acini were prepared from mouse pancreas and amylase secretion was measured by amylase assay. Expression of TBC domain proteins EPI64, 64B and 64C in pancreatic acini was examined by RT-PCR. Effects of EPI64B over-expression on GTP-bound state of Rab27B and Rab3D were assessed by pulldown assay using GST-SHD for Rab27B and GST-Rim for Rab3D, respectively. Results: Both EPI64 and 64B were found to be expressed in mouse pancreatic acini by RT-PCR. Mouse EPI64 and 64B sequences were cloned into an adenoviral vector with epitope tags. Over-expression of EPI64B in isolated mouse pancreatic acini by adenoviral delivery decreased the GTP binding of endogenous Rab27B by 69%±17% (mean±SD), while the GTP-bound state of Rab3D was not affected. We found that membrane-associated fraction of Rab27B was also reduced to a similar extent in the pancreatic acini over-expressed with EPI64B. EPI64B over-expression enhanced the basal level of amylase release by 37%±10%. The entire dose response curve of CCK-stimulated amylase release was elevated by EPI64B over-expression with a maximal increase of 59%±20% at a CCK concentration of 300 pM. EPI64 had no effects on the GTP binding state of Rab27B or Rab3D proteins or on amylase release. Conclusions: EPI64B is a potential GTPase-activating protein (GAP) for Rab27B and may play an important role in regulating the activity of Rab27B in mouse pancreatic acinar cells. Moreover, the data showed that reducing the amount of active Rab27B enhanced amylase secretion.
Ethanol Impaired the Assembly and Disassembly of Actin Cytoskeleton and Cell-Cell Adhesion via RHOA and Catenin P120 in CCK-Stimulated Pancreatic Acini Tomoyuki Iwata, Fumihiko Nozu, Michio Imawari Background: Although ethanol is the common risk factor for pancreatitis, precise interactions of actin cytoskeleton, cell-cell adhesion proteins and ethanol have not been clarified in pancreatic acini. Aim: We attempted to evaluate the interaction of RhoA, catenin p120 (p120), F-actin and ethanol in CCK-stimulated pancreatic acini. Methods: Isolated acini were obtained from male Sprague-Dawley rats. Intact acini were preincubated with ethanol or Rho inhibitor, pravastatin and incubated with CCK. The protein expressions and the association of RhoA and p120 were analyzed by Western immunoblotting and immunoprecipitation. The translocation of RhoA was examined by cell fractionation assay. Amylase secretion was also measured. The distribution of RhoA, p120 and F-actin were analyzed by conforcal laser microscopy. Results: CCK (10 pM - 10 nM) enhanced the expressions and association of RhoA and p120. Although pretreatment of 5 mM ethanol, 5 and 10 μM pravastatin did not alter the expressions and association of RhoA and p120 in CCK-treated pancreatic acini, the pretreatment of 20 mM ethanol and 30 μM pravastatin inhibited the expressions and association. Furthermore, cell fractionation assay was performed. In resting state, RhoA was mainly located in the cytosol fraction. RhoA translocated from the cytosol fraction to the membrane fraction after CCK (10 pM, 10 nM) stimulation. On the other hand, pretreatment of 20 mM ethanol and 30 μM pravastatin inhibited the translocation of RhoA. 5 and 10 μM pravastatin did not inhibit dose response of amylase secretion in CCK (10 pM - 10 nM) stimulation without altering basal secretion, the pretreatment of 20 mM ethanol and 30 μM pravastatin inhibited amylase secretion. Conforcal microscopy images showed that RhoA was diffusely distributed throughout of acinar cell and p120 was localized to the lateral plasma membrane in the resting state. F-actin located in apical site and lateral site in the resting state. After 10 pM CCK stimulation, RhoA, p120 and F-actin were mainly located to apical site. After 10 nM CCK stimulation, the distributions of these proteins to apical site were diminished. Pretreatment of 5 mM ethanol and 5 and 10 μM pravastatin did not alter distributions of the proteins in the resting state and after CCK (10 pM, 10 nM) stimulation. The pretreatment of 20 mM ethanol and 30 μM pravastatin inhibited the distributions of these proteins to apical site and lateral site in the resting state and after CCK (10 pM, 10 nM) stimulation. Conclusion: We conclude that ethanol impaired the assembly and disassembly of actin cytoskeleton and cell-cell adhesion via RhoA and p120 in CCK-stimulated pancreatic acini.
T1934 Ca2+ Signaling via G Protein Coupled P2-Receptors in Activated Pancreatic Stellate Cells Jan K. Hennigs, Oliver Seiz, Julia M. Spiro, Marc J. Berna, Hans Klose, Andrea Pace BACKGROUND: There is growing evidence that extracellular nucleotide induced signaling confers to fibrogenesis in liver and pancreas. P2 purine and pyrimidine receptors are pivotal mediators of inflammatory and pro-fibrogenic signals in liver and most likely pancreas as well. However, a specific analysis of the underlying signaling components in activated pancreatic stellate cells, the most important cell type in pancreatic fibrosis, has not been performed so far. METHODS: Rat pancreatic stellate cells (PSC) were freshly isolated and cultured until auto-activation. FURA-2-laden PSC were stimulated by various nucleotides including AMP, ADP, ATP, UDP and UTP and real-time intracellular Ca2+ responses were monitored under various buffer conditions using a cuvette fluorimeter. Additionally, expression of classical components of intracellular Ca2+ signaling such as G proteins and Phospholipase C (PLC) subunits was evaluated by reverse transcription PCR. Rats were cared according to the guidelines and under supervision of the local animal welfare board. RESULTS: A broad variety of Ca2+ signaling components are expressed in activated PSC: All four known PLC-stimulating G alpha subunits Gq, G11, G14 and G15 are present on mRNA basis in activated PSC as were all three IP3 receptor subunits. Additionally, transcripts of PLC-beta subunits 1,3 and 4 were detectable by RT-PCR. In increasing magnitude, purines AMP, ADP and ATP elicited detectable rises in intracellular Ca2+ concentrations. Stimulation of PSC by ATP lead to intracellular Ca2+ increases mediated through both P2X and P2Y receptors. Ca2+ was mostly released from internal stores as shown by preceding depletion of PSCs from extra- and intracellular Ca2+ stores or by preincubation with PLC inhibitor U73122. Pyrimidines triggered more sustained Ca2+ signals than purines. Whereas UTP mediated Ca2+ signals were generated by activation of P2Y receptors only, surprisingly, UDP stimulated P2X receptors as well. DISCUSSION: Not surprisingly, activated pancreatic stellate cells feature a plethora of elements from the Ca2+ signaling toolkit and functionally express a broad variety of P2 nucleotide receptors. Purines and pyrimidines elicit robust intracellular Ca2+ signals likely contributing to the fibrogenetic potential of this cells.
T1937 Extracellular Signal-Regulated Kinase 1/2 Signaling Pathway in Rostral Nucleus Tract Solitarius (NTS) Mediates CCK-Stimulated Pancreatic Enzyme Secretion Yuanxu Lu, Phattrawan Pisuchpen, Shi-Yi Zhou, Angela Y. Chen, Xinyi Wang, Chung Owyang The high- and low-affinity CCK-A receptors are present on distinct vagal afferent fibers which mediate pancreatic enzyme secretion and satiety respectively. We hypothesize that these two groups of nodose ganglia neurons project to different regions of the NTS which in turn relay signals to other parts of the brainstem and hypothalamus to mediate different functions. The objective of this study is to define the intracellular signaling pathways in the NTS that relay and integrate signals from high-affinity vagal CCKA receptors. Recently we showed that most of the NTS neurons activated by gastrointestinal stimuli contain both NMDA and AMPA glutamate receptors which are coupled to the extracellular signal-regulated kinase 1/2 signaling (ERK1/2) pathways. We showed by Western blot that ip administration of JMV-180 which acts as an agonist on high-affinity CCKA receptor and as an antagonist on low affinity CCK receptors caused a dose-dependent increase in pERK1/2 in the NTS. A maximum of 2.6-fold increase was observed at 30 min after JMV-180 (1000 μg/kg ip). Majority of the increase (2/3) came from the rostral NTS whereas the remaining (1/3) were from the caudal NTS. Administration (ip) of CCK8 (10 μg/kg) which acts on both high- as well as low-affinity CCKA receptors caused a similar increase in pERK1/2 but with the 2/ 3 distribution in the caudal and 1/3 in the rostral NTS. Combined administration of JMV180 and CCK8 shifted to a mostly rostral distribution. The expression of RAS, the upstream activator of ERK1/2 has a similar regional distribution pattern as pERK1/2 in response to JMV-180. A similar increase was observed in the A-type voltage dependent K+ channel Kv4.2. The channel is specifically phosphorylated at Thr602 by ERK, resulting in decreased opening of the channel and a relative depolarization of the neuron. The preferential activation of rostral vs caudal NTS neurons by JMV-180 was confirmed by co-staining of pERK and cFos in NTS. Using a rat model with a pancreatic-biliary cannula, we showed that JMV180 (ip) caused a dose-dependent increase in pancreatic protein secretion, reaching a peak at 30″. Microinjection of the MEK inhibitor U0126 (2 μg in 5 μl) into the fourth ventricle reduced JMV-180 evoked pancreatic protein secretion by 45% and this was accomplished by 50% reduction of the phosphorylated Kv4.2 protein. In conclusion, we showed that stimulation of high- and low-affinity vagal CCKA receptors caused differential activation of the rostral vs caudal NTS. The ERK 1/2 signaling pathway in the rostral NTS through phosphorylation of Kv4.2 mediates CCK-stimulated pancreatic enzyme secretion.
T1935 Low pH Enhances Calcium Signaling in the Pancreatic Acinar Cell Anamika Chaudhuri, Ahsan U. Shah, Sohail Z. Husain, Fred S. Gorelick, Michael H. Nathanson Background: Recent data from our lab demonstrates that low pH sensitizes the pancreatic acinar cell to secretagogue-induced zymogen activation and injury. While pathologic calcium signaling underlies aberrant intracellular zymogen activation and cell injury in the acinar cell, the relationship between the injurious effects of acidic conditions and calcium signaling has not been investigated. Aim: To examine the effects of acidic pH on physiologic calcium signaling in the acinar cell. Methods: Isolated rat acinar cells were loaded with the calcium dye, fluo-4, perfused with either pH 7.4 or pH 7.0 HEPES buffer, and treated with physiologic concentrations (10 pM) of the cholecystokinin analogue, cerulein. Changes in cytosolic calcium were measured with time lapse confocal microscopy and normalized to baseline fluorescence. Results: The frequency of calcium oscillations significantly decreased under low pH conditions, from 1.03 +/- 0.07 oscillations per minute at pH 7.4 to 0.63 +/- 0.05 oscillations per minute at pH 7.0, p < 0.05. Cells treated with low pH buffer demonstrated higher amplitude oscillations, with an increase from 455% +/- 59% baseline at pH 7.4 to 655% +/- 76% baseline at pH 7.0, p < 0.05. The effects of low extracellular pH on oscillation frequency and amplitude were not affected by perfusing with calcium free medium containing 10 mM EGTA. Pretreating acini with the ryanodine receptor inhibitors, dantrolene (75 μM) and ryanodine (100 μM) reduced the effects of low pH on amplitude, but did not affect
AGA Abstracts
S-610