The most common nonmalignant gastrointestinal. Editorials, continued

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1 WILLIAM L. HASLER Department of Medicine, Division of Gastroenterology, University of Michigan Health System, Ann Arbor, Michigan References 1. Schey R, Cromwell J, Rao SS. Medical and surgical management of pelvic floor disorders affecting defecation. Am J Gastroenterol 2012;107: Rao SS, Mudipalli RS, Stessman M, et al. Investigation of the utility of colorectal function tests and Rome II criteria in dyssynergic defecation (Anismus). Neurogastroenterol Motil 2004;16: Bharucha AE, Wald A, Enck P, et al. Functional anorectal disorders. Gastroenterology 2006;130: Rao SS. Advances in diagnostic assessment of fecal incontinence and dyssynergic defecation. Clin Gastroenterol Hepatol 2010;8: Raza N, Bielefeldt K. Discriminative value of anorectal manometry in clinical practice. Dig Dis Sci 2009;54: Minguez M, Herreros B, Sanchiz V, et al. Predictive value of the balloon expulsion test for excluding the diagnosis of pelvic floor dyssynergia in constipation. Gastroenterology 2004;126: Reiner CS, Tutuian R, Solopova AE, et al. MR defecography in patients with dyssynergic defecation: spectrum of imaging findings and diagnostic value. Br J Radiol 2011;84: Ratuapli SK, Bharucha AE, Noelting J, et al. Phenotypic identification and classification of functional defecatory disorders using high-resolution anorectal manometry. Gastroenterology 2013;144: Tantiphlachiva K, Rao P, Attaluri A, Rao SS. Digital rectal examination is a useful tool for identifying patients with dyssynergia. Clin Gastroenterol Hepatol 2010;8: Bharucha AE, Klingele CJ, Seide BM, et al. Effects of vaginal hysterectomy on anorectal sensorimotor functions a prospective study. Neurogastroenterol Motil 2012;24: Jones MP, Post J, Crowell MD. High-resolution manometry in the evaluation of anorectal disorders: a simultaneous comparison with water perfused manometry. Am J Gastroenterol 2007;102: Rao SS, Seaton K, Miller M, et al. Randomized controlled trial of biofeedback, sham feedback, and standard therapy for dyssynergic defecation. Clin Gastroenterol Hepatol 2007;5: Chiarioni G, Whitehead WE, Pezza V, et al. Biofeedback is superior to laxatives for normal transit constipation due to pelvic floor dyssynergia. Gastroenterology 2006;130: Rao SS, Valestin J, Brown CK, et al. Long-term efficacy of biofeedback therapy for dyssynergic defecation: randomized controlled trial. Am J Gastroenterol 2010;105: Noelting J, Ratuapli SK, Bharucha AE, et al. Normal values of HRM in healthy women: effects of age and significance of rectoanal gradient. Am J Gastroenterol 2012;107: Reprint requests Address requests for reprints to: Satish S. C. Rao, MD, PhD, FRCP, FACG, AGAF, Georgia Health Sciences University, th Street - BBR2538, Augusta, Georgia srao@ georgiahealth.edu. Conflicts of interest The authors disclose no conflicts by the AGA Institute /$ Preventing Pancreatitis by Protecting the Mitochondrial Permeability Transition Pore See Effects of oxidative alcohol metabolism on mitochondrial permeability transition pore and necrosis in a mouse model of alcoholic pancreatitis, by Shalbueva N, Mareninova OA, Gerloff A, et al, on page 437. The most common nonmalignant gastrointestinal (GI) disorder requiring hospital admission in the United States is acute pancreatitis. 1 Although research into its etiology has made rapid progress over the last 20 years and identified a number of additional environmental 2 and genetic risk factors 3,4 for the disease, the most common cause of pancreatitis immoderate alcohol consumption has remained a mystery on the cellular level. One reason why the question of how ethanol causes pancreatitis has remained so elusive is the simple fact that most animal species, unlike man, can consume excessive amounts of ethanol over extended periods of time without ever developing clinical pancreatitis, 5 although their liver and brain do suffer. In this issue of GASTROENTEROLOGY, Shalbueva et al 6 may have found a possible solution to the enigma of alcohol-induced pancreatitis by joining 2 lines of experimental evidence. The first involves pancreatic acinar cell signal transduction. Thirty-five years ago, it was established that supraphysiologic concentrations of agonists for a subset of G-protein coupled 7-transmembrane protein receptors such as cholecystokinin (CCK) can induce pancreatitis in rodents 7 and man. 8 This observation was used to establish one of the most widely used models of pancreatitis. 5,7 Although initially contributing little to the question of alcohol-induced pancreatitis, supramaximal hormone stimulation, when used in vitro or ex vivo on isolated pancreatic acini, has revealed a number of fundamental cell biological mechanisms involved in cellular injury and pancreatitis. Among them are disturbances in intracellular trafficking 9 and cytoskeletal transport mechanisms, 10,11 premature activation of digestive proteases 12,13 and its dependence on lysosomal hydrolases, 14,15 the role of inflammatory cells, 16 as well as the regulation of most of the above by the cytosolic release of millimolar concen- 265

2 trations of calcium. 17,18 CCK releases calcium from intracellular stores into the cytosol via several second messenger systems that include inositol triphosphate (IP3), nicotinic acid adenine dinucleotide phosphate, and cyclic ADP-ribose. 19 Particularly the apical calcium release near the zymogen granule compartment seems to be critically involved in premature protease activation and the onset of pancreatitis, not only in the hormone-induced variety, 17,18 but also in more clinically relevant models. 20 Of note, 2 protective mechanisms were found to interfere with the steamroller calcium waves (as opposed to regulated oscillations) that run from the apical pole to the basolateral portion of the cell. The first is the divalent cation magnesium, which can counteract calcium s effects. 21 The second is an intracellular belt, formed by a subset of mitochondria, that separates the apical calcium release compartment from the basolateral cytosol, and has been found to act as a very effective calcium sponge that, when damaged, loses its protective barrier function for pathologic cytosolic calcium waves. 22 This brings us to the second line of evidence that Shalbueva et al build on the role of mitochondria. The exocrine pancreas synthesizes and secretes very large amounts of protein and to this end needs to provide large amounts of energy. The organelle for this energy-transducing process is the mitochondrion. Some investigators assume that not all mitochondria are equal because functional differences exist between mitochondria at different subcellular sites 22 and between mitochondria in different organs such as liver and pancreas. 23 Essentially, mitochondria transform chemical energy delivered from substrate oxidation into an electrochemical proton gradient across their inner membrane ( m). The mitochondrial membrane potential can then be used for adenosine triphosphate (ATP) synthesis and for storing Ca (Figure 1). Because ATP is required for virtually every energy-dependent step in pancreatic acinar cell metabolism, mitochondrial dysfunction has been implicated in tissue injury and pancreatitis as early as methods for investigating mitochondria became available. 24 Several pathways in mitochondrial function have been implicated in pancreatitis. Impairment of the outer mitochondrial membrane permits leakage of larger proteins from the space between the inner and outer membranes, including cytochrome C, which in turn leads to activation of caspases in the cytosol. Cytosolic cytochrome C, through a mechanism that involves different cathepsins, 15 induces apoptosis. Apoptosis, a rather neat and regulated form of cell death that does not involve the uncontrolled release of active digestive proteases and cytokines into the interstitial space, is usually followed by tissue regeneration. On the other hand, damage to the outer membrane also leads to a collapse of the inner membrane m, subsequent energy depletion, and certain acinar cell and tissue necrosis. Necrosis not only propagates to neighboring cells but also attracts inflammatory cells, 25 which, in turn, determine the ultimate severity of the disease. Depolarization of the inner mitochondrial membrane, which follows physiologic CCK stimulation, leads to opening of the mitochondrial permeability transition pore (MPTP) complex (of which cyclophillin D [CypD] is a major component), a reduction in reactive oxygen species generation, and the prevention of cytochrome C release. Quite a useful thing in the context of pancreatitis 23,26 unless the opening of the MPTP is not transient but sustained permits an unselective entry of molecules up to 1.5 kda through the inner membrane and into the mitochondrial matrix, causes swelling of the entire organelle, collapse of the m, and invariably cell necrosis. The balance between apoptosis and necrosis has been shown to also determine a shift from mild to severe pancreatitis and can thus depend on the opening status of the MPTP. How does ethanol fit into this mitochondrial program? So far, not at all. The ethanol we consume can essentially be metabolized in 2 ways. 27 In nonoxidative metabolism, the enzyme fatty acid ethyl ester synthase converts ethanol to fatty acid ethyl ester (FAEE). Previous studies have suggested that the metabolite FAEE is the most critical toxic component of alcohol in the pancreas 28 because it can, in itself, stimulate intracellular Ca release via an IP3R-dependent mechanism. 29 This was considered sufficient as an explanation for the ethanol sensitization of the pancreas and its acinar cells towards injury by otherwise physiologic stimuli 30 or to greater severity in experimental pancreatitis models under long-term alcohol exposure. 31 The alternative mechanism was investigated here by Shalbueva et al. In oxidative metabolism (which is largely dependent on mitochondria), ethanol is converted to acetaldehyde via alcohol dehydrogenase and acetaldehyde, in turn, is converted to acetate via aldehyde dehydrogenase The latter is located at the inner mitochondrial membrane and highly dependent on the coenzyme NAD. This mechanism is where the authors found the explanation for the toxic effect of alcohol on pancreatic mitochondria and the injury that leads to necrosis. They investigated the MPTP in isolated pancreatic acini, in an animal model of pancreatitis and in isolated mitochondria, the latter being a rather challenging technique in which Russian scientists have a record of great accomplishment. They also used acini and animals with genetic CypD deletion, a condition in which the MPTP no longer opens properly. What they found is that ethanol causes a collapse of the m that is entirely dependent on the integrity of the MPTP and, in return, independent of FAEEs. Although physiologic concentrations of CCK induced a transient m response without cellular damage, pretreatment with ethanol for 30 or 60 minutes converted the same CCK 266

3 Figure 1. Cholecystokinin (CCK) and ethanol cooperate at the mitochondrial permeability transition pore (MPTP) in sensitizing the pancreatic acinar cell towards injury. CCK, via the CCK receptor (CCKR), releases cyclic ADP-ribose, nicotinic acid adenine dinucleotide phosphate, and inostitol triphosphate (IP3), all of which can induce Ca to be released into the cytosol from stores, either as physiologic Ca oscillations or as cytotoxic Ca waves. When the MPTP is overwhelmed by a Ca wave, this leads to permanent (rather than transient) MPTP opening with the consequence of a collapse of the mitochondrial membrane potential, m, causing permeabilization of the outer mitochondrial membrane, followed by acinar cell necrosis, as well as apoptosis via leakage of cytochrome C into the cytosol. Ethanol is metabolized to either fatty acid ethyl ester (FAEE), which contributes to Ca release via IP3, or undergoes oxidative catalysis by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase 2 (ALDH2). The latter is mitochondrially localized and competes for NAD with the MPTP. In concert, physiologic CCK concentrations together with ethanol can thus convert a transient opening of the MPTP into a permanent one, leading to acinar cell damage. Deletion of cyclophillin D, a major component of the MPTP prevents both, the CCK and the ethanol effect on the MPTP and prevents acinar cell necrosis. Blue arrows indicate effects of 1 component on the other. signal into a sustained decrease in m with subsequent depletion of cellular ATP and acinar cell necrosis. Ethanol-induced MPTP opening was mediated by a decrease in NAD as shown by several lines of evidence including the introduction of NAD in permeabilized cells. The CCK effect on the decrease in m, be it transiently with physiologic concentrations or sustained with supramaximal concentrations is mediated via the release of [Ca ]i. Because both, the effect of ethanol and the effect of CCK was prevented by CypD deletion, depended on a functional intact and readily opening MPTP, albeit via different mechanism. In the case of CCK the process was Ca -dependent and could be suppressed with the Ca chelator BAPTA, whereas for ethanol the effect was Ca -independent and involved NAD depletion through the oxidation of acetaldehyde to acetate at the inner mitochondrial membrane. Oxidative ethanol metabolism thus interferes with the enzymatic machinery needed to maintain the function of the MPTP. What are the consequences of this discovery? First, we finally have a conclusive explanation for why too much alcohol damages the pancreas. Moreover, the clinical observation of pancreatitis onset after a substantial meal in conjunction with too much alcohol suddenly makes sense in that the physiologic CCK released from the small intestine intended to digest the food load is converted into a pathologic stimulus leading to cell necrosis when too much alco- 267

4 hol is consumed simultaneously. It needs to be left to speculation whether or not the ethanol metabolite FAEE also turns the physiologic CCK signaling pattern into a pathologic one, but is seems clear that both CCK and ethanol cooperate at the MPTP via different mechanisms, resulting in sustained MPTP opening, m collapse, energy depletion, and acinar cell necrosis when they join forces. The question remains how this novel information can be used to protect the pancreas from injury. For the first, CCK signal transduction part, there seems to be a working hypothesis. The cytosolic calcium wave that leads to sustained opening of the MPTP can be antagonized by either orally feeding or IV infusion of magnesium. 20 Two multicenter, controlled, clinical trials are currently investigating whether magnesium supplementation can either change the natural history of recurrent pancreatitis (NCT, ISRCTN ) or prevent endoscopic retrograde cholangiopancreatography-induced acute pancreatitis (ISRCTN ). For the ethanol part, intervention seems to be more difficult to achieve. Although NAD restitution is definitely a drug target in many pharmaceutical research laboratories, no clinically effective compound seems to be presently available that could be tested for the treatment or prevention of alcohol-induced pancreatitis. Interfering with MPTP function, for example, by inhibiting CypD, would be an attractive way to pursue but compounds that have been found effective in this context, such as cyclosporine, a calcineurin inhibitor, are not specific for CypD and have been shown to have beneficial effects in some, 32 but negative effects in other models of pancreatitis. 33 Particularly in frequently recurrent pancreatitis after successful alcohol cessation, such a therapeutic option would be most welcome. The jury is still out on whether or not we will be able to translate the finding of Shalbueva et al into a feasible treatment modality. However, thanks to this group of investigators, we now have a welldefined target. MARKUS M. LERCH Department of Medicine A, Greifswald University Medicine, Greifswald, Germany WALTER HALANGK Department of Surgery, Otto-von-Guericke University, Magdeburg, Germany JULIA MAYERLE Department of Medicine A, Greifswald University Medicine, Greifswald, Germany References 1. 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5 inositol trisphosphate-evoked local cytosolic Ca(2 ) signals. EMBO J 1999;18(18): Odinokova IV, Sung KF, Mareninova OA, et al. Mechanisms regulating cytochrome C release in pancreatic mitochondria. Gut 2009;58: Schild L, Matthias R, Stanarius A, et al. Induction of permeability transition in pancreatic mitochondria by cerulein in rats. Mol Cell Biochem 1999;195: Mayerle J, Schnekenburger J, Krüger B, et al. Extracellular cleavage of E-cadherin by leukocyte elastase during acute experimental pancreatitis in rats. Gastroenterology 2005;129: Halangk W, Lerch MM. A unique pancreatic mitochondrial response to calcium and its role in apoptosis. Gut 2009;58: Wilson JS, Apte MV. Role of alcohol metabolism in alcoholic pancreatitis. Pancreas 2003;27: Apte MV, Pirola RC, Wilson JS. Fatty acid ethyl esters alcohol s henchmen in the pancreas? Gastroenterology 2006;130: Criddle DN, Murphy J, Fistetto G, et al. Fatty acid ethyl esters cause pancreatic calcium toxicity via inositol trisphosphate receptors and loss of ATP synthesis. Gastroenterology 2006;130: Orabi AI, Shah AU, Muili K, et al. Ethanol enhances carbacholinduced protease activation and accelerates Ca2 waves in isolated rat pancreatic acini. J Biol Chem 2011;286: Gorelick FS. Alcohol and zymogen activation in the pancreatic acinar cell. Pancreas 2003;27: Muili KA, Ahmad M, Orabi AI, et al. Pharmacological and genetic inhibition of calcineurin protects against carbachol-induced pathological zymogen activation and acinar cell injury. Am J Physiol Gastrointest Liver Physiol 2012;302:G898 G Hackert T, Pfeil D, Hartwig W, et al. Ciclosporin aggravates tissue damage in ischemia reperfusion-induced acute pancreatitis. Pancreas 2006;32: Reprint requests Address requests for reprints to: Markus M. Lerch, MD, FRCP, Department of Medicine A, University Medicine Greifswald, Ferdinand-Sauerbruch-Strasse, Greifswald, Germany. lerch@uni-greifswald.de. Acknowledgments The authors wish to thank Andreas Holtz for secretarial assistance. Conflicts of interest The authors disclose no conflicts. Funding The authors original work is supported by the Alfried-Krupp-von- Bohlen-und-Hahlbach-Foundation (Graduate Schools Tumour Biology and Free Radical Biology), the Deutsche Krebshilfe/Dr Mildred- Scheel-Stiftung (109102), the Deutsche Forschungsgemeinschaft (DFG GRK840-E3/E4, MA 4115/1-2/3, NI 1297/1-1), the Federal Ministry of Education and Research (BMBF GANI-MED A and BMBF ), and the European Union (EU-FP-7: EPC-TM and EU-FP7-REGPOT ) by the AGA Institute /$