- Email: [email protected]
Can Too Much Acid Sour Your Pancreas?
Editorials continued
Can Too Much Acid Sour Your Pancreas?
See “Reducing extracellular pH sensitizes the acinar cell to secretagogue-induced pancreatitis responses in rats” by Bhoomagoud M, Jung T, Atladottir J, et al, on page 1083.
A
cute pancreatitis afflicts approximately 50,000 Americans annually, is associated with significant morbidity and mortality, and has limited treatment options. Treatment is supportive, and in patients with potentially severe acute pancreatitis includes aggressive fluid resuscitation, hemodynamic monitoring, enteral feeding, and tempered use of endoscopic retrograde cholangiopancreatography.1,2 To develop specific prevention and treatment options for acute pancreatitis, there has been considerable interest in developing and characterizing animal models. Premature activation of pancreatic proteases, especially trypsin, is an important initiating component of the disease. Current interest on how and where the activation of trypsin occurs focuses on missorting of digestive and lysosomal enzymes and altered autophagy. Evidence is robust that acidic organelles from lysosomes/endosomes participate in intracellular trypsin activation and the initiation of cellular models of pancreatitis because dissipating local acidic compartments in acinar cells prevents premature trypsin activation. Owing to the potential importance of intracellular acidic compartments and clinical data “linking” acidosis to pancreatitis, Bhoomagoud et al,3 in this issue of Gastroenterology, evaluated how both an acidic environment in vitro and systemic acidosis in vivo affect secretagogue induced zymogen activation and acinar cell injury. Reducing the pH of an acinar cell suspension had no effect on its own, but potentiated secretagogue-induced trypsin and chymotrypsin activation and cell damage. Moreover, in vivo acid infusion had similar effects. The authors conclude that this effect was because of changes in intracellular pH. However, to evaluate their experiments and pose further questions, we first review acinar cell pH regulation and then examine how the acidic milieu investigated by Bhoomagoud et al3 might affect trypsin activation with resultant cell damage, thereby contributing to acute pancreatitis in human diseases associated with acidosis. Homeostatic mechanisms are designed to maintain the constancy of the “milieu intérieur.” Cells must maintain their cytoplasmic pH in response to changes in plasma pH, a continued acid load induced by cell metabolism, passive influx of H⫹, and for epithelia, the presence of transepithelial transport (for a comprehensive review of cellular pH homeostasis, the reader is referred to Boron4). Within the cell, the relatively constant cytoplasmic pH allows the maintenance of organelles
with differing pH’s such as the lysosome where a vacuolar H⫹-ATPase pumps H⫹ into the vacuolar lumen. Mean intracellular pH (pHi) for most cells, including acinar cells, is around 7.0. Rodent acinar pHi values vary from 6.77 to 7.28, as measured by the distribution of weak base,5 NMR,6 and fluorescent probes.7–11 Acinar cells, similar to other cells, contain proteins and small molecules that provide intrinsic buffer capacity to cells. The CO2-HCO3⫺ system also conveys buffering amounting to slightly over half of cellular buffering. Hence, when cells are studied in a TRIS or HEPES buffer without CO2 or HCO3⫺, not all buffering is evaluated. In addition to the intrinsic buffer capacity, cells defend their pH by specific transport mechanisms. The main acid extruding mechanism is Na⫹–H⫹ exchange that is activated by a falling pHi and Ca2⫹ mobilizing secretagogues. This and other key cellular mechanisms (Figure 1) are responsible for maintaining a relatively constant acinar cell pHi, even when extracellular pH (pHe) varies from 7.2 to 7.5. Within cells, the relatively constant cytoplasmic pH allows maintaining organelles of different pHs. The lysosome pumps H⫹ into the vacuolar lumen with the vacuolar H⫹-ATPase and the pH falls until active influx is balanced by passive efflux through the membrane. Lysosomes/endosomes have a pH around 5.0. In chromaffin or islet  cells secretory granules are acidic, which allows them to take up and sequester amines. In exocrine cells, trans-Golgi cisternae and condensing vacuoles (immature granules) are acidic, but mature granules are not.12 These organelle acidic compartments can be dissipated with H⫹-ATPase inhibitors such as bafilomycin or H⫹ ionophores such as monensin. Where does the change in pHe studied by Bhoomagould et al3 affect trypsin activation and cell damage? The location could be extracellular, cytoplasmic, or in the lysosome. Changes in pHe can alter receptor ligand interaction or signal transduction. Cholecystokinin (CCK) receptor binding to pancreatic membranes has a pH optima of 5.5 and lowering pHe should increase CCK binding.13 CCK is a Ca2⫹ mobilizing secretagogue, but in intact acinar cells decreasing pHe reduced Ca2⫹ influx, the height of the sustained Ca2⫹ plateau and the amount of intracellular Ca2⫹ stores.8,14 Thus, it seems unlikely that the effect of lowering pHe on trypsin activation and cell damage is mediated extracellularly. However, in contrast with the work of Bhoomagoud et al,3 other acute in vitro studies reported less amylase secretion (rather than no change) when pHe decreased below 7.0,5,8,14 perhaps owing to a decrease in Ca2⫹ signaling. Changes in cytoplasmic pH could affect trypsin activation. Although Bhoomagould et al3 did not measure cytosolic pH it 779
Editorials continued
Figure 1. Simplified model of acinar cell pH regulation and trypsinogen activation. The basolateral membrane adjacent to capillaries is on the left and the luminal or secretory membrane is on the right. The basolateral Na⫹-K⫹ ATPase is responsible for maintaining a low intracellular Na⫹, high K⫹ and a negative intracellular potential. Thus both Na⫹ and H⫹ are diffusing into the cell down their electrochemical gradient and the energy stored in the Na⫹ gradient is used to pump H⫹ out by the Na⫹-H⫹ exchanger or HCO3⫺ in by the Na⫹-HCO3⫺ cotransporter. Cl- is close to electrochemical equilibrium and at rest the Cl⫺-HCO3⫺ exchanger serves to move HCO3⫺ out of the cell. Note when cells are incubated in HEPES Ringer medium not all of these mechanisms are activated. Lysosomes maintain an acid pH by virtue of a vacuolar(v)-type H⫹-ATPase. The low pH environment is necessary for activation of trypsinogen by cathepsins.
is reasonable to assume it was acidic. However, it would be useful to measure pHi and to investigate the effects of HEPES and HCO3⫺ buffers. They concluded that the effect of lowering pHe was cytoplasmic because adding Na-propionate, a weak acid, acidified the cytoplasm measured with BCECF, even though the pHe was constant. However, the effect of 50 mmol/L propionate on pHi was much greater (a pHi of 6) than expected for reducing pHe from 7.4 to 7.0 which might decrease pHi by only 0.1–0.2 units. Propionate (25 mmol/L), which should decrease pHi to a lesser extent, had only a small effect on trypsin activation. Thus, further studies comparing pHe, pHi, and trypsin activation are warranted. Because the cytoplasm provides the external milieu for the organelles, it is possible that lowering cytoplasmic pH will lower lysosomal/ endosomal vacuolar pH and thereby increase activity of lysosomal cathepsin enzymes that activate trypsin. The findings that concanamycin A and bafilomycin blocked augmentation of trypsin activation by low pHe suggests a lysosomal site for trypsin activation might be important. It would be useful to confirm that concanamycin did not affect cytoplasmic pH. Overall, the studies of Bhoomagould et al3 indicate that further investigation focused on pancreatic acinar cytoplasmic and organelle pH regulation are worthwhile. However, 780
in vivo studies are necessary to determine the clinical relevance of pH regulation to pancreatitis. Bhoomagould et al,3 in their preliminary studies, found that acidosis enhanced cerulein-induced pancreatitis. To interpret the clinical significance of these findings, we review the incidence and possible pathogenesis of acute pancreatitis associated with metabolic acidoses, specifically the clinical conditions referred to by Bhoomagoud et al3: organic acidemias, lactic acidosis induced by hypovolemia and reverse transcriptase inhibitor (NRTI) therapy for HIV, and diabetic ketoacidosis (DKA). Branched-chain organic acidemias, including the classic form propionic acidemia, are a group of inborn errors of metabolisms characterized by impaired catabolism of amino acids, generation of toxic intermediates and development of ketoacidosis, coma, death, or neurologic sequelae. Acute pancreatitis occurs in up to 6.5% of affected children15 and is a known side effect of the branched-chain organic compound valproate, an antiepileptic medication.16 Kahler et al15 speculated that branched-chain organic compounds may predispose to acute pancreatitis by impairing mitochondrial function. Although Bhoomagoud et al3 show that sodium propionate sensitizes rats to pancreatic zymogen activation and injury, the relationship between propionic aci-
Editorials continued
demia and clinical acute pancreatitis remains unclear because we identified only 5 individual cases in the literature, and only 2 cases had imaging or histologic evidence of pancreatitis.17,18 Lactic acidosis develops from cell hypoxia and disorders of cell metabolism. Lactic acidosis resulting
from hypoperfusion and hypoxic injury of organs is a useful prognostic indicator and a tool to monitor the adequacy of fluid resuscitation in critically ill patients.19 In severe acute pancreatitis, volume depletion is a leading contributor to necrotizing pancreatitis and requires early, aggressive fluid resuscitation.1,2,20 Niederau et al21 investigated the importance of treating hypovolemia, hypoxia, and acidosis and reported that survival from a murine model of severe acute pancreatitis improved dramatically by fluid resuscitation but not by correcting hypoxia and acidosis.21 A systematic review provides additional insight that treating lactic acidosis with bicarbonate has no clear impact on clinical outcomes.22 Hence, lactic acidosis in acute pancreatitis is usually a secondary effect of hypoperfusion and hypoxic injury, and should be treated with fluid resuscitation. NRTI treatment for HIV is known to cause lactic acidosis and pancreatitis. Didanosine, combined with
ribavarin (another NRTI that activates didanosine), has a high frequency of hyperlactatemia (23%), acute pancreatitis (28%), and hyperamylasemia (51%) in patients with HIV and hepatitis C.23 NRTIs may predispose to acute pancreatitis by inducing moderate hypertriglyceridemia and/or by inhibiting mitochondrial oxidative phosphorylation in a variety of cell types,24 including the exocrine pancreas,25 which could predispose to cellular necrosis. Hence, it seems more plausible that NRTI associated lactic acidosis is a sequela of a mitochondrial disorder that predisposes to pancreatitis rather than the possibility that lactic acidosis predisposes to or causes acute pancreatitis. DKA has an unclear relationship with acute pancreatitis. A prospective study clarified this relationship
by investigating 100 consecutive episodes of DKA.26 The diagnosis of acute pancreatitis was based on conclusive computed tomographic imaging findings in patients with either abdominal pain and/or ⬎3-fold elevation of serum lipase or amylase levels.26 Eleven percent of patients had acute pancreatitis, but patients had a higher frequency of nonspecific hyperamylasemia (21%) and/or hyperlipasemia (29%). Hypertriglyceridemia was present in 30% and was the most common cause of acute pancreatitis. Other etiologies of acute pancreatitis were alcohol, drug induced, and idiopathic. Acute pancreatitis was associated with more severe metabolic acidosis and a higher mean blood glucose, findings most likely attributable to the composite effects of DKA and acute pancreatitis, because both conditions cause hyperglycemia, hypovolemia, and metabolic acidosis. Because 64% of
cases had an identifiable cause of acute pancreatitis, it seems unlikely that acidosis or DKA triggered acute pancreatitis. Conversely, it is conceivable in some cases that acute pancreatitis triggered DKA or that an independent insult triggered DKA and acute pancreatitis simultaneously. Currently, immediate treatment of suspected severe acute pancreatitis focuses on aggressive fluid resuscitation to restore perfusion of organs and correct hypoxia and acidosis. There are insufficient clinical data to appraise and substantiate the postulated paradigm that metabolic acidemias predispose to or cause acute pancreatitis. To clarify the role of metabolic acidemia in acute pancreatitis, further research is necessary using in vitro and in vivo animal models and careful human studies.
JOHN A. WILLIAMS Department of Molecular and Integrative Physiology University of Michigan Ann Arbor, Michigan MATTHEW J. DIMAGNO Department of Internal Medicine Division of Gastroenterology and Hepatology University of Michigan Ann Arbor, Michigan References 1. Pandol SJ, Saluja AK, Imrie CW, et al. Acute pancreatitis: bench to the bedside. Gastroenterology 2007;132:1127–1151. 2. DiMagno MJ, Wamsteker EJ, Debenedet AT. Advances in managing acute pancreatitis. F1000 Medicine Reports 2009. 3. Bhoomagoud M, Jung T, Atladottir J, et al. Reducing extracellular pH sensitizes the acinar cell to secretagogue-induced pancreatitis responses in rats. Gastroenterology 2009;137:1083–1092. 4. Boron WF. Regulation of intracellular pH. Adv Physiol Educ 2004; 28:160 –179. 5. Preissler M, Williams JA. Pancreatic acinar cell function: measurement of intracellular ions and pH and their relation to secretion. J Physiol 1981;321:437– 448. 6. Dufresne M, Bastie MJ, Vaysse N, et al. The amiloride sensitive Na⫹/H⫹ antiport in guinea pig pancreatic acini. Characterization and stimulation by caerulein. FEBS Lett 1985;187:126 –130. 7. Hellmessen W, Christian AL, Fasold H, et al. Coupled Na⫹-H⫹ exchange in isolated acinar cells from rat exocrine pancreas. Am J Physiol 1985;249:G125–136. 8. Muallem S, Pandol SJ, Beeker TG. Modulation of agonist-activated calcium influx by extracellular pH in rat pancreatic acini. Am J Physiol 1989;257:G917–924. 9. Carter KJ, Rutledge PL, Steer ML, et al. Secretagogue-induced changes in intracellular pH and amylase release in mouse pancreatic acini. Am J Physiol 1987;253:G690 – 696. 10. Bastie MJ, Williams JA. Gastrointestinal peptides activate Na(⫹)-H⫹ exchanger in AR42J cells by increasing its affinity for intracellular H⫹. Am J Physiol 1990;258:G958 –966. 11. Muallem S, Loessberg PA. Intracellular pH-regulatory mechanisms in pancreatic acinar cells. II. Regulation of H⫹ and HCO3- transporters by Ca2(⫹)-mobilizing agonists. J Biol Chem 1990;265:12813– 12819. 781
Editorials continued
12. Orci L, Ravazzola M, Anderson RG. The condensing vacuole of exocrine cells is more acidic than the mature secretory vesicle. Nature 1987;326:77–79. 13. Steigerwalt RW, Williams JA. Characterization of cholecystokinin receptors on rat pancreatic membranes. Endocrinology 1981;109: 1746 –1753. 14. Tsunoda Y, Stuenkel EL, Williams JA. Characterization of sustained [Ca2⫹]i increase in pancreatic acinar cells and its relation to amylase secretion. Am J Physiol 1990;259:G792– 801. 15. Kahler SG, Sherwood WG, Woolf D, et al. Pancreatitis in patients with organic acidemias. J Pediatr 1994;124:239 –243. 16. Badalov N, Baradarian R, Iswara K, et al. Drug-induced acute pancreatitis: an evidence-based review. Clin Gastroenterol Hepatol 2007;5:648 – 661. 17. Bultron G, Seashore MR, Pashankar DS, et al. Recurrent acute pancreatitis associated with propionic acidemia. J Pediatr Gastroenterol Nutr 2008;47:370 –371. 18. Burlina AB, Dionisi-Vici C, Piovan S, et al. Acute pancreatitis in propionic acidaemia. J Inherit Metab Dis 1995;18:169 –172. 19. Schuster HP. Prognostic value of blood lactate in critically ill patients. Resuscitation 1984;11:141–146. 20. Gardner TB, Vege SS, Pearson RK, et al. Fluid resuscitation in acute pancreatitis. Clin Gastroenterol Hepatol 2008;6:1070 –1076. 21. Niederau C, Crass RA, Silver G, et al. Therapeutic regimens in acute experimental hemorrhagic pancreatitis. Effects of hydration, oxygenation, peritoneal lavage, and a potent protease inhibitor. Gastroenterology 1988;95:1648 –1657. 22. Forsythe SM, Schmidt GA. Sodium bicarbonate for the treatment of lactic acidosis. Chest 2000;117:260 –267.
23. Moreno A, Quereda C, Moreno L, et al. High rate of didanosinerelated mitochondrial toxicity in HIV/HCV-coinfected patients receiving ribavirin. Antivir Ther 2004;9:133–138. 24. Brinkman K, ter Hofstede HJ, Burger DM, et al. Adverse effects of reverse transcriptase inhibitors: mitochondrial toxicity as common pathway. AIDS 1998;12:1735–1744. 25. Yeo TK, Kintner J, Armand R, et al. Sublethal concentrations of gemcitabine (2=,2=-difluorodeoxycytidine) alter mitochondrial ultrastructure and function without reducing mitochondrial DNA content in BxPC-3 human pancreatic carcinoma cells. Hum Exp Toxicol 2007;26:911–921. 26. Nair S, Yadav D, Pitchumoni CS. Association of diabetic ketoacidosis and acute pancreatitis: observations in 100 consecutive episodes of DKA. Am J Gastroenterol 2000;95:2795–2800.
Reprint requests Address requests for reprints to: John A. Williams, MD, PhD, Department of Molecular & Integrative Physiology, University of Michigan, 7708 MS II, Ann Arbor, Michigan 48109-5622. email: [email protected]. Or to: Matthew J. DiMagno, MD, Department of Internal Medicine, Division of Gastroenterology and Hepatology, 6510 MSRB 1, Ann Arbor, Michigan, 48109. email: [email protected]. Conflicts of interest The authors disclose no conflicts. © 2009 by the AGA Institute 0016-5085/09/$36.00 doi:10.1053/j.gastro.2009.07.036
Finding and Killing the CRABs of Pancreatic Cancer
See “Combined targeted treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer” by Mueller M–T, Hermann PC, Witthauer J, et al, on page 1102.
P
ancreatic ductal adenocarcinoma (PDA) is a rapidly lethal disease, largely because it is resistant to therapeutic intervention. Potential explanations for medical failure in PDA include classical pharmacological resistance,1 insufficient drug delivery,2 and innate cellular features such as activated survival pathways.3 Recently, several groups reported that a subpopulation of PDA cells possess enhanced resilience to various stresses, and such cells are termed cancer stem cells (CSC), because their tenaciousness could lead logically to the continual regeneration of the neoplasm.4,5 The CSC premise posits that tumors contain a hierarchical arrangement of cells to ensure tumor propagation, with the core or “stem” population directing a pattern of asymmetric proliferation and differentiation akin to the developmental models proposed for stem cells in normal tis782
sues.6,7 The small fraction of CSCs are projected to have a slower proliferation rate than the majority of the bulk tumor, thus minimizing the effects of cytotoxic drugs owing to longer times available for DNA damage repair. In addition, CSCs have been reported to express drug efflux pumps,8,9 thereby contributing to the evasion of cytotoxic and targeted drugs. Such characteristics of CSCs offer explanations for both therapeutic resistance and tumor dormancy, central problems in the treatment of most incurable neoplasms. Two prior studies had reported the isolation of CSCs from PDA, characterized by an increased ability of that population to survive orthotopic transplantation in an immunocompromised mouse. CSCs expressed either CD24/ 44/ESA4 or CD1335 and, consistent with the concept that CSCs harbor therapeutic resistance properties, gemcitabine treatment increased the proportion of the tumor cells that express these markers.5 Given the resistance of these CSC to therapeutic intervention, Mueller et al10 set out to design rational therapeutic strategies based upon the known or suspected biology of CSCs, as reported in this issue of GASTROENTEROLOGY. Because CSCs were previously reported
Can Too Much Acid Sour Your Pancreas?
See “Reducing extracellular pH sensitizes the acinar cell to secretagogue-induced pancreatitis responses in rats” by Bhoomagoud M, Jung T, Atladottir J, et al, on page 1083.
A
cute pancreatitis afflicts approximately 50,000 Americans annually, is associated with significant morbidity and mortality, and has limited treatment options. Treatment is supportive, and in patients with potentially severe acute pancreatitis includes aggressive fluid resuscitation, hemodynamic monitoring, enteral feeding, and tempered use of endoscopic retrograde cholangiopancreatography.1,2 To develop specific prevention and treatment options for acute pancreatitis, there has been considerable interest in developing and characterizing animal models. Premature activation of pancreatic proteases, especially trypsin, is an important initiating component of the disease. Current interest on how and where the activation of trypsin occurs focuses on missorting of digestive and lysosomal enzymes and altered autophagy. Evidence is robust that acidic organelles from lysosomes/endosomes participate in intracellular trypsin activation and the initiation of cellular models of pancreatitis because dissipating local acidic compartments in acinar cells prevents premature trypsin activation. Owing to the potential importance of intracellular acidic compartments and clinical data “linking” acidosis to pancreatitis, Bhoomagoud et al,3 in this issue of Gastroenterology, evaluated how both an acidic environment in vitro and systemic acidosis in vivo affect secretagogue induced zymogen activation and acinar cell injury. Reducing the pH of an acinar cell suspension had no effect on its own, but potentiated secretagogue-induced trypsin and chymotrypsin activation and cell damage. Moreover, in vivo acid infusion had similar effects. The authors conclude that this effect was because of changes in intracellular pH. However, to evaluate their experiments and pose further questions, we first review acinar cell pH regulation and then examine how the acidic milieu investigated by Bhoomagoud et al3 might affect trypsin activation with resultant cell damage, thereby contributing to acute pancreatitis in human diseases associated with acidosis. Homeostatic mechanisms are designed to maintain the constancy of the “milieu intérieur.” Cells must maintain their cytoplasmic pH in response to changes in plasma pH, a continued acid load induced by cell metabolism, passive influx of H⫹, and for epithelia, the presence of transepithelial transport (for a comprehensive review of cellular pH homeostasis, the reader is referred to Boron4). Within the cell, the relatively constant cytoplasmic pH allows the maintenance of organelles
with differing pH’s such as the lysosome where a vacuolar H⫹-ATPase pumps H⫹ into the vacuolar lumen. Mean intracellular pH (pHi) for most cells, including acinar cells, is around 7.0. Rodent acinar pHi values vary from 6.77 to 7.28, as measured by the distribution of weak base,5 NMR,6 and fluorescent probes.7–11 Acinar cells, similar to other cells, contain proteins and small molecules that provide intrinsic buffer capacity to cells. The CO2-HCO3⫺ system also conveys buffering amounting to slightly over half of cellular buffering. Hence, when cells are studied in a TRIS or HEPES buffer without CO2 or HCO3⫺, not all buffering is evaluated. In addition to the intrinsic buffer capacity, cells defend their pH by specific transport mechanisms. The main acid extruding mechanism is Na⫹–H⫹ exchange that is activated by a falling pHi and Ca2⫹ mobilizing secretagogues. This and other key cellular mechanisms (Figure 1) are responsible for maintaining a relatively constant acinar cell pHi, even when extracellular pH (pHe) varies from 7.2 to 7.5. Within cells, the relatively constant cytoplasmic pH allows maintaining organelles of different pHs. The lysosome pumps H⫹ into the vacuolar lumen with the vacuolar H⫹-ATPase and the pH falls until active influx is balanced by passive efflux through the membrane. Lysosomes/endosomes have a pH around 5.0. In chromaffin or islet  cells secretory granules are acidic, which allows them to take up and sequester amines. In exocrine cells, trans-Golgi cisternae and condensing vacuoles (immature granules) are acidic, but mature granules are not.12 These organelle acidic compartments can be dissipated with H⫹-ATPase inhibitors such as bafilomycin or H⫹ ionophores such as monensin. Where does the change in pHe studied by Bhoomagould et al3 affect trypsin activation and cell damage? The location could be extracellular, cytoplasmic, or in the lysosome. Changes in pHe can alter receptor ligand interaction or signal transduction. Cholecystokinin (CCK) receptor binding to pancreatic membranes has a pH optima of 5.5 and lowering pHe should increase CCK binding.13 CCK is a Ca2⫹ mobilizing secretagogue, but in intact acinar cells decreasing pHe reduced Ca2⫹ influx, the height of the sustained Ca2⫹ plateau and the amount of intracellular Ca2⫹ stores.8,14 Thus, it seems unlikely that the effect of lowering pHe on trypsin activation and cell damage is mediated extracellularly. However, in contrast with the work of Bhoomagoud et al,3 other acute in vitro studies reported less amylase secretion (rather than no change) when pHe decreased below 7.0,5,8,14 perhaps owing to a decrease in Ca2⫹ signaling. Changes in cytoplasmic pH could affect trypsin activation. Although Bhoomagould et al3 did not measure cytosolic pH it 779
Editorials continued
Figure 1. Simplified model of acinar cell pH regulation and trypsinogen activation. The basolateral membrane adjacent to capillaries is on the left and the luminal or secretory membrane is on the right. The basolateral Na⫹-K⫹ ATPase is responsible for maintaining a low intracellular Na⫹, high K⫹ and a negative intracellular potential. Thus both Na⫹ and H⫹ are diffusing into the cell down their electrochemical gradient and the energy stored in the Na⫹ gradient is used to pump H⫹ out by the Na⫹-H⫹ exchanger or HCO3⫺ in by the Na⫹-HCO3⫺ cotransporter. Cl- is close to electrochemical equilibrium and at rest the Cl⫺-HCO3⫺ exchanger serves to move HCO3⫺ out of the cell. Note when cells are incubated in HEPES Ringer medium not all of these mechanisms are activated. Lysosomes maintain an acid pH by virtue of a vacuolar(v)-type H⫹-ATPase. The low pH environment is necessary for activation of trypsinogen by cathepsins.
is reasonable to assume it was acidic. However, it would be useful to measure pHi and to investigate the effects of HEPES and HCO3⫺ buffers. They concluded that the effect of lowering pHe was cytoplasmic because adding Na-propionate, a weak acid, acidified the cytoplasm measured with BCECF, even though the pHe was constant. However, the effect of 50 mmol/L propionate on pHi was much greater (a pHi of 6) than expected for reducing pHe from 7.4 to 7.0 which might decrease pHi by only 0.1–0.2 units. Propionate (25 mmol/L), which should decrease pHi to a lesser extent, had only a small effect on trypsin activation. Thus, further studies comparing pHe, pHi, and trypsin activation are warranted. Because the cytoplasm provides the external milieu for the organelles, it is possible that lowering cytoplasmic pH will lower lysosomal/ endosomal vacuolar pH and thereby increase activity of lysosomal cathepsin enzymes that activate trypsin. The findings that concanamycin A and bafilomycin blocked augmentation of trypsin activation by low pHe suggests a lysosomal site for trypsin activation might be important. It would be useful to confirm that concanamycin did not affect cytoplasmic pH. Overall, the studies of Bhoomagould et al3 indicate that further investigation focused on pancreatic acinar cytoplasmic and organelle pH regulation are worthwhile. However, 780
in vivo studies are necessary to determine the clinical relevance of pH regulation to pancreatitis. Bhoomagould et al,3 in their preliminary studies, found that acidosis enhanced cerulein-induced pancreatitis. To interpret the clinical significance of these findings, we review the incidence and possible pathogenesis of acute pancreatitis associated with metabolic acidoses, specifically the clinical conditions referred to by Bhoomagoud et al3: organic acidemias, lactic acidosis induced by hypovolemia and reverse transcriptase inhibitor (NRTI) therapy for HIV, and diabetic ketoacidosis (DKA). Branched-chain organic acidemias, including the classic form propionic acidemia, are a group of inborn errors of metabolisms characterized by impaired catabolism of amino acids, generation of toxic intermediates and development of ketoacidosis, coma, death, or neurologic sequelae. Acute pancreatitis occurs in up to 6.5% of affected children15 and is a known side effect of the branched-chain organic compound valproate, an antiepileptic medication.16 Kahler et al15 speculated that branched-chain organic compounds may predispose to acute pancreatitis by impairing mitochondrial function. Although Bhoomagoud et al3 show that sodium propionate sensitizes rats to pancreatic zymogen activation and injury, the relationship between propionic aci-
Editorials continued
demia and clinical acute pancreatitis remains unclear because we identified only 5 individual cases in the literature, and only 2 cases had imaging or histologic evidence of pancreatitis.17,18 Lactic acidosis develops from cell hypoxia and disorders of cell metabolism. Lactic acidosis resulting
from hypoperfusion and hypoxic injury of organs is a useful prognostic indicator and a tool to monitor the adequacy of fluid resuscitation in critically ill patients.19 In severe acute pancreatitis, volume depletion is a leading contributor to necrotizing pancreatitis and requires early, aggressive fluid resuscitation.1,2,20 Niederau et al21 investigated the importance of treating hypovolemia, hypoxia, and acidosis and reported that survival from a murine model of severe acute pancreatitis improved dramatically by fluid resuscitation but not by correcting hypoxia and acidosis.21 A systematic review provides additional insight that treating lactic acidosis with bicarbonate has no clear impact on clinical outcomes.22 Hence, lactic acidosis in acute pancreatitis is usually a secondary effect of hypoperfusion and hypoxic injury, and should be treated with fluid resuscitation. NRTI treatment for HIV is known to cause lactic acidosis and pancreatitis. Didanosine, combined with
ribavarin (another NRTI that activates didanosine), has a high frequency of hyperlactatemia (23%), acute pancreatitis (28%), and hyperamylasemia (51%) in patients with HIV and hepatitis C.23 NRTIs may predispose to acute pancreatitis by inducing moderate hypertriglyceridemia and/or by inhibiting mitochondrial oxidative phosphorylation in a variety of cell types,24 including the exocrine pancreas,25 which could predispose to cellular necrosis. Hence, it seems more plausible that NRTI associated lactic acidosis is a sequela of a mitochondrial disorder that predisposes to pancreatitis rather than the possibility that lactic acidosis predisposes to or causes acute pancreatitis. DKA has an unclear relationship with acute pancreatitis. A prospective study clarified this relationship
by investigating 100 consecutive episodes of DKA.26 The diagnosis of acute pancreatitis was based on conclusive computed tomographic imaging findings in patients with either abdominal pain and/or ⬎3-fold elevation of serum lipase or amylase levels.26 Eleven percent of patients had acute pancreatitis, but patients had a higher frequency of nonspecific hyperamylasemia (21%) and/or hyperlipasemia (29%). Hypertriglyceridemia was present in 30% and was the most common cause of acute pancreatitis. Other etiologies of acute pancreatitis were alcohol, drug induced, and idiopathic. Acute pancreatitis was associated with more severe metabolic acidosis and a higher mean blood glucose, findings most likely attributable to the composite effects of DKA and acute pancreatitis, because both conditions cause hyperglycemia, hypovolemia, and metabolic acidosis. Because 64% of
cases had an identifiable cause of acute pancreatitis, it seems unlikely that acidosis or DKA triggered acute pancreatitis. Conversely, it is conceivable in some cases that acute pancreatitis triggered DKA or that an independent insult triggered DKA and acute pancreatitis simultaneously. Currently, immediate treatment of suspected severe acute pancreatitis focuses on aggressive fluid resuscitation to restore perfusion of organs and correct hypoxia and acidosis. There are insufficient clinical data to appraise and substantiate the postulated paradigm that metabolic acidemias predispose to or cause acute pancreatitis. To clarify the role of metabolic acidemia in acute pancreatitis, further research is necessary using in vitro and in vivo animal models and careful human studies.
JOHN A. WILLIAMS Department of Molecular and Integrative Physiology University of Michigan Ann Arbor, Michigan MATTHEW J. DIMAGNO Department of Internal Medicine Division of Gastroenterology and Hepatology University of Michigan Ann Arbor, Michigan References 1. Pandol SJ, Saluja AK, Imrie CW, et al. Acute pancreatitis: bench to the bedside. Gastroenterology 2007;132:1127–1151. 2. DiMagno MJ, Wamsteker EJ, Debenedet AT. Advances in managing acute pancreatitis. F1000 Medicine Reports 2009. 3. Bhoomagoud M, Jung T, Atladottir J, et al. Reducing extracellular pH sensitizes the acinar cell to secretagogue-induced pancreatitis responses in rats. Gastroenterology 2009;137:1083–1092. 4. Boron WF. Regulation of intracellular pH. Adv Physiol Educ 2004; 28:160 –179. 5. Preissler M, Williams JA. Pancreatic acinar cell function: measurement of intracellular ions and pH and their relation to secretion. J Physiol 1981;321:437– 448. 6. Dufresne M, Bastie MJ, Vaysse N, et al. The amiloride sensitive Na⫹/H⫹ antiport in guinea pig pancreatic acini. Characterization and stimulation by caerulein. FEBS Lett 1985;187:126 –130. 7. Hellmessen W, Christian AL, Fasold H, et al. Coupled Na⫹-H⫹ exchange in isolated acinar cells from rat exocrine pancreas. Am J Physiol 1985;249:G125–136. 8. Muallem S, Pandol SJ, Beeker TG. Modulation of agonist-activated calcium influx by extracellular pH in rat pancreatic acini. Am J Physiol 1989;257:G917–924. 9. Carter KJ, Rutledge PL, Steer ML, et al. Secretagogue-induced changes in intracellular pH and amylase release in mouse pancreatic acini. Am J Physiol 1987;253:G690 – 696. 10. Bastie MJ, Williams JA. Gastrointestinal peptides activate Na(⫹)-H⫹ exchanger in AR42J cells by increasing its affinity for intracellular H⫹. Am J Physiol 1990;258:G958 –966. 11. Muallem S, Loessberg PA. Intracellular pH-regulatory mechanisms in pancreatic acinar cells. II. Regulation of H⫹ and HCO3- transporters by Ca2(⫹)-mobilizing agonists. J Biol Chem 1990;265:12813– 12819. 781
Editorials continued
12. Orci L, Ravazzola M, Anderson RG. The condensing vacuole of exocrine cells is more acidic than the mature secretory vesicle. Nature 1987;326:77–79. 13. Steigerwalt RW, Williams JA. Characterization of cholecystokinin receptors on rat pancreatic membranes. Endocrinology 1981;109: 1746 –1753. 14. Tsunoda Y, Stuenkel EL, Williams JA. Characterization of sustained [Ca2⫹]i increase in pancreatic acinar cells and its relation to amylase secretion. Am J Physiol 1990;259:G792– 801. 15. Kahler SG, Sherwood WG, Woolf D, et al. Pancreatitis in patients with organic acidemias. J Pediatr 1994;124:239 –243. 16. Badalov N, Baradarian R, Iswara K, et al. Drug-induced acute pancreatitis: an evidence-based review. Clin Gastroenterol Hepatol 2007;5:648 – 661. 17. Bultron G, Seashore MR, Pashankar DS, et al. Recurrent acute pancreatitis associated with propionic acidemia. J Pediatr Gastroenterol Nutr 2008;47:370 –371. 18. Burlina AB, Dionisi-Vici C, Piovan S, et al. Acute pancreatitis in propionic acidaemia. J Inherit Metab Dis 1995;18:169 –172. 19. Schuster HP. Prognostic value of blood lactate in critically ill patients. Resuscitation 1984;11:141–146. 20. Gardner TB, Vege SS, Pearson RK, et al. Fluid resuscitation in acute pancreatitis. Clin Gastroenterol Hepatol 2008;6:1070 –1076. 21. Niederau C, Crass RA, Silver G, et al. Therapeutic regimens in acute experimental hemorrhagic pancreatitis. Effects of hydration, oxygenation, peritoneal lavage, and a potent protease inhibitor. Gastroenterology 1988;95:1648 –1657. 22. Forsythe SM, Schmidt GA. Sodium bicarbonate for the treatment of lactic acidosis. Chest 2000;117:260 –267.
23. Moreno A, Quereda C, Moreno L, et al. High rate of didanosinerelated mitochondrial toxicity in HIV/HCV-coinfected patients receiving ribavirin. Antivir Ther 2004;9:133–138. 24. Brinkman K, ter Hofstede HJ, Burger DM, et al. Adverse effects of reverse transcriptase inhibitors: mitochondrial toxicity as common pathway. AIDS 1998;12:1735–1744. 25. Yeo TK, Kintner J, Armand R, et al. Sublethal concentrations of gemcitabine (2=,2=-difluorodeoxycytidine) alter mitochondrial ultrastructure and function without reducing mitochondrial DNA content in BxPC-3 human pancreatic carcinoma cells. Hum Exp Toxicol 2007;26:911–921. 26. Nair S, Yadav D, Pitchumoni CS. Association of diabetic ketoacidosis and acute pancreatitis: observations in 100 consecutive episodes of DKA. Am J Gastroenterol 2000;95:2795–2800.
Reprint requests Address requests for reprints to: John A. Williams, MD, PhD, Department of Molecular & Integrative Physiology, University of Michigan, 7708 MS II, Ann Arbor, Michigan 48109-5622. email: [email protected]. Or to: Matthew J. DiMagno, MD, Department of Internal Medicine, Division of Gastroenterology and Hepatology, 6510 MSRB 1, Ann Arbor, Michigan, 48109. email: [email protected]. Conflicts of interest The authors disclose no conflicts. © 2009 by the AGA Institute 0016-5085/09/$36.00 doi:10.1053/j.gastro.2009.07.036
Finding and Killing the CRABs of Pancreatic Cancer
See “Combined targeted treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer” by Mueller M–T, Hermann PC, Witthauer J, et al, on page 1102.
P
ancreatic ductal adenocarcinoma (PDA) is a rapidly lethal disease, largely because it is resistant to therapeutic intervention. Potential explanations for medical failure in PDA include classical pharmacological resistance,1 insufficient drug delivery,2 and innate cellular features such as activated survival pathways.3 Recently, several groups reported that a subpopulation of PDA cells possess enhanced resilience to various stresses, and such cells are termed cancer stem cells (CSC), because their tenaciousness could lead logically to the continual regeneration of the neoplasm.4,5 The CSC premise posits that tumors contain a hierarchical arrangement of cells to ensure tumor propagation, with the core or “stem” population directing a pattern of asymmetric proliferation and differentiation akin to the developmental models proposed for stem cells in normal tis782
sues.6,7 The small fraction of CSCs are projected to have a slower proliferation rate than the majority of the bulk tumor, thus minimizing the effects of cytotoxic drugs owing to longer times available for DNA damage repair. In addition, CSCs have been reported to express drug efflux pumps,8,9 thereby contributing to the evasion of cytotoxic and targeted drugs. Such characteristics of CSCs offer explanations for both therapeutic resistance and tumor dormancy, central problems in the treatment of most incurable neoplasms. Two prior studies had reported the isolation of CSCs from PDA, characterized by an increased ability of that population to survive orthotopic transplantation in an immunocompromised mouse. CSCs expressed either CD24/ 44/ESA4 or CD1335 and, consistent with the concept that CSCs harbor therapeutic resistance properties, gemcitabine treatment increased the proportion of the tumor cells that express these markers.5 Given the resistance of these CSC to therapeutic intervention, Mueller et al10 set out to design rational therapeutic strategies based upon the known or suspected biology of CSCs, as reported in this issue of GASTROENTEROLOGY. Because CSCs were previously reported