Ca2+ Influx Channel Inhibitor SARAF Protects Mice From Acute Pancreatitis

Aran Son, Malini Ahuja, Daniella M. Schwartz, Arpad Varga, William Swaim, Namju Kang, Jozsef Maleth, Dong Min Shin, Shmuel Muallem

Research output: Contribution to journalArticle

Abstract

Background & Aims: Pancreatitis is characterized by increased influx of Ca2+ into acinar cells, by unknown mechanisms. Inhibitors of Ca2+ influx channels could be effective in treating acute pancreatitis, but these have deleterious side effects that can result in death. We investigated the expression patterns and functions of acinar cell Ca2+ channels and factors that regulate them during development of acute pancreatitis, along with changes in the channel inactivator store-operated calcium entry–associated regulatory factor (SARAF). We investigated whether SARAF is a target for treatment of acute pancreatitis and its status in human with pancreatitis. Methods: We generated mice that expressed SARAF tagged with hemagglutinin, using CRISPR/Cas9 gene editing, and isolated acinar cells. We also performed studies with Saraf–/– mice, Saraf zf/zf mice, mice without disruption of Saraf (control mice), and mice that overexpress fluorescently labeled SARAF in acinar cells. We analyzed interactions between stromal interaction molecule 1 (STIM1) and SARAF in HEK cells stimulated with carbachol using fluorescence resonance energy transfer microscopy and immunoprecipitation. Mice were given injections of caerulein or L-arginine to induce pancreatitis. Pancreatic tissues and blood samples were collected and levels of serum amylase, trypsin, tissue damage, inflammatory mediators, and inflammatory cells were measured. We performed quantitative polymerase chain reaction analyses of pancreatic tissues from 6 organ donors without pancreatic disease (controls) and 8 patients with alcohol-associated pancreatitis. Results: Pancreatic levels of Ca2+ influx channels or STIM1 did not differ significantly between acinar cells from mice with vs. without pancreatitis. By contrast, pancreatic levels of Saraf messenger RNA and SARAF protein initially markedly increased but then decreased during cell stimulation or injection of mice with caerulein, resulting in excessive Ca2+ influx. STIM1 interacted stably with SARAF following stimulation of HEK or mouse acinar cells with physiologic levels of carbachol, but only transiently following stimulation with pathologic levels of carbachol, leading to excessive Ca2+ influx. We observed reduced levels of SARAF messenger RNA in pancreatic tissues from patients with pancreatitis, compared with controls. SARAF knockout mice developed more severe pancreatitis than control mice after administration of caerulein or L-arginine, and pancreatic acinar cells from these mice had significant increases in Ca2+ influx. Conversely, overexpression of SARAF in acini reduced Ca2+ influx, eliminated inflammation, and reduced severity of acute pancreatitis. Conclusions: In mice with pancreatitis, SARAF initially increases but is then degraded, resulting in excessive, pathological Ca2+ influx by acinar cells. SARAF knockout mice develop more severe pancreatitis than control mice, whereas mice that express SARAF from a transgene in acinar cells develop less-severe pancreatitis. SARAF therefore appears to prevent pancreatic damage during development of acute pancreatitis. Strategies to stabilize or restore SARAF to acinar cells might be developed for treatment of pancreatitis.

Original languageEnglish
Pages (from-to)1660-1672.e2
JournalGastroenterology
Volume157
Issue number6
DOIs
Publication statusPublished - 2019 Dec

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Pancreatitis
Acinar Cells
Calcium
Ceruletide
Carbachol
Knockout Mice
Arginine
Clustered Regularly Interspaced Short Palindromic Repeats
Pancreatic Diseases
Messenger RNA
Fluorescence Resonance Energy Transfer
Injections
Hemagglutinins
Amylases
Transgenes
Immunoprecipitation
Trypsin
Microscopy

All Science Journal Classification (ASJC) codes

  • Hepatology
  • Gastroenterology

Cite this

Son, A., Ahuja, M., Schwartz, D. M., Varga, A., Swaim, W., Kang, N., ... Muallem, S. (2019). Ca2+ Influx Channel Inhibitor SARAF Protects Mice From Acute Pancreatitis. Gastroenterology, 157(6), 1660-1672.e2. https://doi.org/10.1053/j.gastro.2019.08.042
Son, Aran ; Ahuja, Malini ; Schwartz, Daniella M. ; Varga, Arpad ; Swaim, William ; Kang, Namju ; Maleth, Jozsef ; Shin, Dong Min ; Muallem, Shmuel. / Ca2+ Influx Channel Inhibitor SARAF Protects Mice From Acute Pancreatitis. In: Gastroenterology. 2019 ; Vol. 157, No. 6. pp. 1660-1672.e2.
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abstract = "Background & Aims: Pancreatitis is characterized by increased influx of Ca2+ into acinar cells, by unknown mechanisms. Inhibitors of Ca2+ influx channels could be effective in treating acute pancreatitis, but these have deleterious side effects that can result in death. We investigated the expression patterns and functions of acinar cell Ca2+ channels and factors that regulate them during development of acute pancreatitis, along with changes in the channel inactivator store-operated calcium entry–associated regulatory factor (SARAF). We investigated whether SARAF is a target for treatment of acute pancreatitis and its status in human with pancreatitis. Methods: We generated mice that expressed SARAF tagged with hemagglutinin, using CRISPR/Cas9 gene editing, and isolated acinar cells. We also performed studies with Saraf–/– mice, Saraf zf/zf mice, mice without disruption of Saraf (control mice), and mice that overexpress fluorescently labeled SARAF in acinar cells. We analyzed interactions between stromal interaction molecule 1 (STIM1) and SARAF in HEK cells stimulated with carbachol using fluorescence resonance energy transfer microscopy and immunoprecipitation. Mice were given injections of caerulein or L-arginine to induce pancreatitis. Pancreatic tissues and blood samples were collected and levels of serum amylase, trypsin, tissue damage, inflammatory mediators, and inflammatory cells were measured. We performed quantitative polymerase chain reaction analyses of pancreatic tissues from 6 organ donors without pancreatic disease (controls) and 8 patients with alcohol-associated pancreatitis. Results: Pancreatic levels of Ca2+ influx channels or STIM1 did not differ significantly between acinar cells from mice with vs. without pancreatitis. By contrast, pancreatic levels of Saraf messenger RNA and SARAF protein initially markedly increased but then decreased during cell stimulation or injection of mice with caerulein, resulting in excessive Ca2+ influx. STIM1 interacted stably with SARAF following stimulation of HEK or mouse acinar cells with physiologic levels of carbachol, but only transiently following stimulation with pathologic levels of carbachol, leading to excessive Ca2+ influx. We observed reduced levels of SARAF messenger RNA in pancreatic tissues from patients with pancreatitis, compared with controls. SARAF knockout mice developed more severe pancreatitis than control mice after administration of caerulein or L-arginine, and pancreatic acinar cells from these mice had significant increases in Ca2+ influx. Conversely, overexpression of SARAF in acini reduced Ca2+ influx, eliminated inflammation, and reduced severity of acute pancreatitis. Conclusions: In mice with pancreatitis, SARAF initially increases but is then degraded, resulting in excessive, pathological Ca2+ influx by acinar cells. SARAF knockout mice develop more severe pancreatitis than control mice, whereas mice that express SARAF from a transgene in acinar cells develop less-severe pancreatitis. SARAF therefore appears to prevent pancreatic damage during development of acute pancreatitis. Strategies to stabilize or restore SARAF to acinar cells might be developed for treatment of pancreatitis.",
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Son, A, Ahuja, M, Schwartz, DM, Varga, A, Swaim, W, Kang, N, Maleth, J, Shin, DM & Muallem, S 2019, 'Ca2+ Influx Channel Inhibitor SARAF Protects Mice From Acute Pancreatitis', Gastroenterology, vol. 157, no. 6, pp. 1660-1672.e2. https://doi.org/10.1053/j.gastro.2019.08.042

Ca2+ Influx Channel Inhibitor SARAF Protects Mice From Acute Pancreatitis. / Son, Aran; Ahuja, Malini; Schwartz, Daniella M.; Varga, Arpad; Swaim, William; Kang, Namju; Maleth, Jozsef; Shin, Dong Min; Muallem, Shmuel.

In: Gastroenterology, Vol. 157, No. 6, 12.2019, p. 1660-1672.e2.

Research output: Contribution to journalArticle

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T1 - Ca2+ Influx Channel Inhibitor SARAF Protects Mice From Acute Pancreatitis

AU - Son, Aran

AU - Ahuja, Malini

AU - Schwartz, Daniella M.

AU - Varga, Arpad

AU - Swaim, William

AU - Kang, Namju

AU - Maleth, Jozsef

AU - Shin, Dong Min

AU - Muallem, Shmuel

PY - 2019/12

Y1 - 2019/12

N2 - Background & Aims: Pancreatitis is characterized by increased influx of Ca2+ into acinar cells, by unknown mechanisms. Inhibitors of Ca2+ influx channels could be effective in treating acute pancreatitis, but these have deleterious side effects that can result in death. We investigated the expression patterns and functions of acinar cell Ca2+ channels and factors that regulate them during development of acute pancreatitis, along with changes in the channel inactivator store-operated calcium entry–associated regulatory factor (SARAF). We investigated whether SARAF is a target for treatment of acute pancreatitis and its status in human with pancreatitis. Methods: We generated mice that expressed SARAF tagged with hemagglutinin, using CRISPR/Cas9 gene editing, and isolated acinar cells. We also performed studies with Saraf–/– mice, Saraf zf/zf mice, mice without disruption of Saraf (control mice), and mice that overexpress fluorescently labeled SARAF in acinar cells. We analyzed interactions between stromal interaction molecule 1 (STIM1) and SARAF in HEK cells stimulated with carbachol using fluorescence resonance energy transfer microscopy and immunoprecipitation. Mice were given injections of caerulein or L-arginine to induce pancreatitis. Pancreatic tissues and blood samples were collected and levels of serum amylase, trypsin, tissue damage, inflammatory mediators, and inflammatory cells were measured. We performed quantitative polymerase chain reaction analyses of pancreatic tissues from 6 organ donors without pancreatic disease (controls) and 8 patients with alcohol-associated pancreatitis. Results: Pancreatic levels of Ca2+ influx channels or STIM1 did not differ significantly between acinar cells from mice with vs. without pancreatitis. By contrast, pancreatic levels of Saraf messenger RNA and SARAF protein initially markedly increased but then decreased during cell stimulation or injection of mice with caerulein, resulting in excessive Ca2+ influx. STIM1 interacted stably with SARAF following stimulation of HEK or mouse acinar cells with physiologic levels of carbachol, but only transiently following stimulation with pathologic levels of carbachol, leading to excessive Ca2+ influx. We observed reduced levels of SARAF messenger RNA in pancreatic tissues from patients with pancreatitis, compared with controls. SARAF knockout mice developed more severe pancreatitis than control mice after administration of caerulein or L-arginine, and pancreatic acinar cells from these mice had significant increases in Ca2+ influx. Conversely, overexpression of SARAF in acini reduced Ca2+ influx, eliminated inflammation, and reduced severity of acute pancreatitis. Conclusions: In mice with pancreatitis, SARAF initially increases but is then degraded, resulting in excessive, pathological Ca2+ influx by acinar cells. SARAF knockout mice develop more severe pancreatitis than control mice, whereas mice that express SARAF from a transgene in acinar cells develop less-severe pancreatitis. SARAF therefore appears to prevent pancreatic damage during development of acute pancreatitis. Strategies to stabilize or restore SARAF to acinar cells might be developed for treatment of pancreatitis.

AB - Background & Aims: Pancreatitis is characterized by increased influx of Ca2+ into acinar cells, by unknown mechanisms. Inhibitors of Ca2+ influx channels could be effective in treating acute pancreatitis, but these have deleterious side effects that can result in death. We investigated the expression patterns and functions of acinar cell Ca2+ channels and factors that regulate them during development of acute pancreatitis, along with changes in the channel inactivator store-operated calcium entry–associated regulatory factor (SARAF). We investigated whether SARAF is a target for treatment of acute pancreatitis and its status in human with pancreatitis. Methods: We generated mice that expressed SARAF tagged with hemagglutinin, using CRISPR/Cas9 gene editing, and isolated acinar cells. We also performed studies with Saraf–/– mice, Saraf zf/zf mice, mice without disruption of Saraf (control mice), and mice that overexpress fluorescently labeled SARAF in acinar cells. We analyzed interactions between stromal interaction molecule 1 (STIM1) and SARAF in HEK cells stimulated with carbachol using fluorescence resonance energy transfer microscopy and immunoprecipitation. Mice were given injections of caerulein or L-arginine to induce pancreatitis. Pancreatic tissues and blood samples were collected and levels of serum amylase, trypsin, tissue damage, inflammatory mediators, and inflammatory cells were measured. We performed quantitative polymerase chain reaction analyses of pancreatic tissues from 6 organ donors without pancreatic disease (controls) and 8 patients with alcohol-associated pancreatitis. Results: Pancreatic levels of Ca2+ influx channels or STIM1 did not differ significantly between acinar cells from mice with vs. without pancreatitis. By contrast, pancreatic levels of Saraf messenger RNA and SARAF protein initially markedly increased but then decreased during cell stimulation or injection of mice with caerulein, resulting in excessive Ca2+ influx. STIM1 interacted stably with SARAF following stimulation of HEK or mouse acinar cells with physiologic levels of carbachol, but only transiently following stimulation with pathologic levels of carbachol, leading to excessive Ca2+ influx. We observed reduced levels of SARAF messenger RNA in pancreatic tissues from patients with pancreatitis, compared with controls. SARAF knockout mice developed more severe pancreatitis than control mice after administration of caerulein or L-arginine, and pancreatic acinar cells from these mice had significant increases in Ca2+ influx. Conversely, overexpression of SARAF in acini reduced Ca2+ influx, eliminated inflammation, and reduced severity of acute pancreatitis. Conclusions: In mice with pancreatitis, SARAF initially increases but is then degraded, resulting in excessive, pathological Ca2+ influx by acinar cells. SARAF knockout mice develop more severe pancreatitis than control mice, whereas mice that express SARAF from a transgene in acinar cells develop less-severe pancreatitis. SARAF therefore appears to prevent pancreatic damage during development of acute pancreatitis. Strategies to stabilize or restore SARAF to acinar cells might be developed for treatment of pancreatitis.

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Son A, Ahuja M, Schwartz DM, Varga A, Swaim W, Kang N et al. Ca2+ Influx Channel Inhibitor SARAF Protects Mice From Acute Pancreatitis. Gastroenterology. 2019 Dec;157(6):1660-1672.e2. https://doi.org/10.1053/j.gastro.2019.08.042