Departments of 1Internal Medicine and 2Molecular and Integrative Physiology, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0362
Submitted 26 August 2003 ; accepted in final form 9 October 2003
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ABSTRACT |
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nitric oxide; cholecystokinin; carbachol
Effects of NO are strongly influenced by the site of NO production, the amount of NO produced, and the specific NOS isoform involved in NO synthesis (35). Each of the three NOS isoforms was named for its initial localization in vascular endothelium (eNOS), neurons (nNOS), and macrophages [inducible NOS (iNOS)], and each primarily regulates vascular tone, neurotransmission, and immune defense, respectively. In the pancreas, the constitutive NOS isoforms (eNOS and nNOS) have been localized to endothelial cells (eNOS), neurons (nNOS), and islets (nNOS), but it is controversial whether either is present in exocrine cells. Many NOS inhibitors are available, but none is completely selective for one enzyme. Conceivably, one or more NOS isoforms could influence pancreatic secretion; nNOS in either the brain or pancreas could regulate neurotransmission, and eNOS could affect pancreatic blood flow.
To clarify the effect of NO on pancreatic secretion, we first developed an in vivo murine model to establish normal values of pancreatic secretion. Using maximal stimulatory doses of CCK-8 (160 pmol·kg-1·h-1) and carbachol (100 nmol·kg-1·h-1), we examined the effect of nonspecific NOS inhibition or genetic deletion of individual NOS isoforms on in vivo murine pancreatic secretion. Because the localization of NOS isoforms in the pancreas is controversial, we functionally assessed whether acinar events alone were sufficient to explain the effect of NOS on pancreatic secretion by examining secretagogue-stimulated amylase release from isolated pancreatic acini (52).
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MATERIALS AND METHODS |
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Animal care and selection. All experiments were approved by the University of Michigan Committee on Use and Care of Animals. All mice were obtained from Jackson Laboratory. Animals were 6-8 wk old, weighed 18-24 g, and were maintained in a climate-controlled room kept at 22°C, exposed to a 12:12-h light-dark cycle, fed standard laboratory chow, and given water ad libitum. The effect of selective NOS deletion on experimental in vivo and in vitro pancreatic secretion was studied in mice with targeted gene deletion of nNOS (15), eNOS (47), and iNOS (28). The genetic background for eNOS and iNOS knockout mice was C57BL/6J and for nNOS knockout mice was B6129SF2/J. Control mice were age- and sex-matched and of identical genetic background.
In vivo pancreatic secretion. Mice were anesthetized with a combination of ketamine (80 mg/kg ip) plus xylazine (Rompun; 5 mg/kg ip) to produce 30-45 min of anesthesia, redosing with 1/3 the original calculated dose of ketamine but without xylazine. A surgical, x4 magnification, Leica MZ6 stereomicroscope was used for microsurgery procedures. A polyethylene (PE)-10 cannula was inserted into the right internal jugular vein for secretagogue infusion. A superficial, 1.5- to 2.0-cm midline abdominal incision was made, and a PE-10 catheter was inserted into the extraduodenal, intrapancreatic common bile-pancreatic duct. The abdominal wound was covered with saline gauze, and the mouse was placed on a protective pad overlying a heating pad to maintain body temperature at 37°C.
Experiments began after a 30-min stabilization period. Combined bile-pancreatic (mixed) juice was collected every 15 min. In separate experiments, selective bile duct cannulation was performed to assess the relative contribution of bile to mixed-juice protein output. Juice volume was measured, and protein concentration was determined spectrophotometrically by using protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Basal protein output was obtained from the second of two 15-min baseline, mixed-juice samples. The secretagogue intravenous infusion rate was set at 100 µl/h using a Harvard PHD 2000 Push/Pull syringe pump, and dose response studies were performed with CCK-8 (40-400 pmol·kg-1·h-1) and carbachol (50-500 nmol·kg-1·h-1) by using 0.9% sodium chloride as a control vehicle. Protein output (µg/min) was expressed as %basal, and statistical analysis was performed on the pooled peak CCK-8 or carbachol response between 45 and 75 min. Secretagogue doses found to produce maximal protein output were used to determine the effect of nonselective NOS blockade and selective NOS deletion on pancreatic secretion. Blood pressure was measured by using a Capto SP844 physiological pressure transducer and PowerLab data acquisition instrument (www.ADInstruments.com).
In vitro pancreatic amylase secretion. Pancreatic acini were prepared by collagenase digestion (6, 52) of mouse pancreata, suspended in HEPES-Ringer buffer with Eagle's minimal essential amino acids, 1 mg/ml BSA, and 0.1 mg/ml soybean trypsin inhibitor and were equilibrated with 100% O2. Viability of acini was >95% on the basis of trypan blue exclusion. Amylase secretion was determined by incubating dispersed pancreatic acini at 37°C for 30 min with graded concentrations of CCK-8 or carbachol. After centrifugation, amylase activity in the supernatant and total acinar pellet was determined by using Phadebas reagent (Pharmacia Diagnostics, Atlanta, GA), and amylase secretion was expressed as %total content.
Nonselective NOS inhibition. In vivo, C57BL/6J mice were pre-treated with a single injection of N-nitro-L-arginine (L-NNA) (10 mg/kg ip) in 0.9% sodium chloride (pH 7.4) 30 min before beginning CCK-8 or carbachol stimulation. L-NNA is the stable, active hydrolysis product of N
-nitro-L-arginine methyl ester (L-NAME) and is known to inhibit all NOS isoforms and allow for relatively constant NOS inhibition in vivo (1-20 mg/kg iv or ip) and in vitro (0.1-1.0 mM) (12). This L-NNA dose is similar to doses used previously to nonselectively inhibit rat pancreatic NOS (1, 31). The control group received an equal volume of 0.9% sodium chloride. In vitro, dispersed pancreatic acini from C57BL/6J mice were preincubated 30 min with L-NNA (10-4 M), a dose similar to that used in other cell types (5), before incubation with secretagogues.
Statistical analysis. The data reported represent means ± SE from multiple determinations obtained from three or more experiments. Statistical comparisons were performed with Student's t-test when comparing only two groups. When comparing three or more groups ANOVA was used followed by post hoc testing with Fisher's protected least significant differences test by using StatView software (SAS Institute, Cary, NC). Statistical significance was assumed for P < 0.05.
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RESULTS |
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In bile duct cannulation experiments, the average basal bile protein output was 1.75 ± 0.43 µg/min or 40% of basal mixed juice protein pooled from CCK-8 and carbachol experiments. Neither carbachol nor CCK stimulation affected bile protein output (Fig. 2, A and B).
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NOS blockade reduces in vivo stimulated pancreatic secretion. The effect of NOS blockade on in vivo pancreatic secretion was studied in C57BL/6J mice. The dose of L-NNA (10 mg/kg ip) employed is similar to doses used to inhibit rat pancreatic NOS (1, 31) and exerts an observable hemodynamic effect; L-NNA increased the systolic blood pressure in an anesthetized mouse from 85 to 105 mmHg over 30 min, similar to the effect of NOS inhibition in rats (2), cats (41), and humans (24). In this study, L-NNA caused a 45% reduction in the maximal CCK-8-stimulated in vivo bile-pancreatic protein output from 45-75 min (Fig. 3A). A more profound effect of L-NNA on secretion was observed during the first 15-min stimulation period when secretion above basal was nearly abolished. From 30 to 90 min, L-NNA reduced CCK-8-stimulated secretion by 52%. L-NNA similarly inhibited maximal carbachol-stimulated in vivo bile-pancreatic output, which was reduced by 40% between 45 and 75 min (Fig. 3B). In addition, the reduction in carbachol-stimulated secretion invoked by L-NNA at both 30- to 45- and 30- to 90-min intervals paralleled that observed with CCK-8 stimulation. Basal bile-pancreatic protein output and basal bile protein output were unaffected by L-NNA. Basal bile-pancreatic protein output averaged 5.12 ± 0.74 µg/min pooled from CCK-8 and carbachol data (n = 13), and basal bile protein output averaged 1.27 ± 0.18 µg/min, which was 25% of basal bile-pancreatic protein output.
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Constitutive NOS isoforms differentially affect CCK-8-stimulated in vivo pancreatic secretion. To determine the NOS isoform responsible for the inhibitory effect of L-NNA on CCK-8-stimulated in vivo pancreatic secretion, similar experiments were performed in mice with individual gene deletions for eNOS, nNOS, and iNOS along with age- and sex-matched controls. Similar to the inhibitory effects of NOS blockade, eNOS deletion caused a 44% reduction in CCK-8-stimulated in vivo bile-pancreatic protein output with a larger effect on the initial 15-min period (Fig. 4A). By contrast iNOS deletion had no effect (Fig. 4B). Somewhat unexpectedly, nNOS deletion increased protein secretion by 91% throughout the time course of stimulation (Fig. 4C). Basal bile-pancreatic protein output was unaltered by eNOS and iNOS gene deletion and averaged 4.66 ± 0.69 µg/min (n = 11) pooled from the CCK-8- and carbachol-stimulated eNOS-deleted group and averaged 4.75 ± 1.05 µg/min (n = 4) pooled from the CCK-8-stimulated iNOS-deleted group. Because the nNOS-deficient mice and the respective control mice were on a B6129SF2/J background, CCK-8 dose response experiments were also performed with the B6129SF2/J strain. These mice had a similar dose-dependent pattern of secretion to the C57BL/6J mice, achieving maximal secretion at 160 pmol·kg-1·h-1, although the maximal CCK-8-stimulated protein output was somewhat less than that observed for C57BL/6J mice (data not shown). Basal bile-pancreatic protein output was unaltered by nNOS gene deletion and averaged 3.65 ± 0.59 µg/min (n = 12) pooled from the CCK-8 and carbachol data, compared with an average of 4.07 ± 0.76 µg/min (n = 16) for the B6129SF/2 control mice data pooled from the CCK-8 and carbachol experiments.
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Carbachol-stimulated in vivo pancreatic secretion is reduced by eNOS deletion. To determine whether the effects of NOS isoform deletion extended to other secretagogues, we carried out carbachol-stimulated in vivo pancreatic secretion studies in mice with individual gene deletions for the constitutive NOS isoforms, eNOS, or nNOS, along with age- and sex-matched controls. Similar to the inhibitory effects of NOS blockade, eNOS deletion reduced carbachol-stimulated in vivo bile-pancreatic protein output by 45% (Fig. 5A). This inhibition was similar to results with CCK-8. By contrast, genetic deletion of nNOS had no statistically significant effect on carbachol-stimulated in vivo bile-pancreatic protein output (Fig. 5B), which differed from the augmentation in secretion due to nNOS deletion in response to CCK-8 stimulation (Fig. 4C). As previously noted, basal bile-pancreatic protein output was unaltered by eNOS and nNOS gene deletion.
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NOS blockade and eNOS deletion do not affect in vitro stimulated pancreatic secretion. Because the intrapancreatic localization of the constitutive NOS species (eNOS and nNOS) is controversial, we studied in vitro stimulated pancreatic secretion by dissociated pancreatic acini to determine whether acinar events alone were sufficient to explain the inhibitory effects of NOS blockade and eNOS deletion on in vivo pancreatic secretion. In contrast to in vivo experiments, NOS blockade (Fig. 6A) and eNOS deletion (Fig. 6B) had no affect on CCK-8-stimulated amylase release from dispersed pancreatic acini. Similarly, NOS blockade had no affect on carbachol-stimulated amylase release from dispersed pancreatic acini (data not shown).
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DISCUSSION |
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In vivo secretion experiments performed with C57BL/6J mice showed that both CCK-8 and carbachol dose-dependently increased bile-pancreatic protein output, which was maximal at 160 pmol·kg-1·h-1 CCK-8 and 100 nmol·kg-1·h-1 carbachol. Both secretagogues showed a similar 315% increase in stimulated in vivo pancreatic secretion. Unfortunately, a meaningful comparison to other murine studies of pancreatic secretion is not possible, because few have been published and they utilize different methodologies, including the method of juice collection and the drug type, dose, and route of delivery (36, 39, 48). Our results of pancreatic secretion, however, are similar to experiments in rats (30, 32). In rats, which lack a gallbladder, there is a similar in vivo dose-dependent increase in bile-pancreatic protein secretion in response to intravenous CCK-8 (30) and no significant increase in bile protein output (32). Despite methodological differences, it is interesting that our results of bile output are similar to results of two mouse studies that showed no increase in bile-acid output after either carbachol (500 nmol/kg sc) (36) or CCK-8 (1 nmol/kg sc) (48); however, members of the same research group showed that a lower dose of CCK-8 (100 pmol/kg sc) significantly increased bile-acid output (36). Thus the results we obtained of stimulated in vivo bile-pancreatic and bile protein outputs are generally consistent with the results of others.
Across species (13, 24, 37, 41, 51), including humans (23), NOS inhibition reduces pancreatic protein secretion arising from vagal electrical stimulation (13) and from the peptides CCK (41); caerulein (37, 51); secretin (41); VIP (13); and combinations of CCK, caerulein, and secretin (23, 24). In the present study, L-NNA reduced CCK-8- and carbachol-stimulated in vivo pancreatic protein secretion but not in vivo basal pancreatic protein secretion. The latter finding appears to be species dependent, because NOS inhibition reduces basal protein secretion in rats (37, 51) but not in dogs (24) and cats (41). In vitro, L-NNA did not affect CCK-8- or carbachol-stimulated pancreatic acinar amylase release, in agreement with most studies performed in rats (20, 24, 37, 57) and dogs (24). We interpret these data as suggesting that nonacinar cell types mediate the functional effects of NOS blockade observed in vivo.
Previous pancreatic NOS localization studies have failed to fully pinpoint the candidate cell types involved in NO-dependent pancreatic protein secretion. Attempts to localize NOS can be affected by tissue fixation techniques (e.g., paraformaldehyde vs. acetone) (22, 53), the NOS detection method, and the animal species studied. NOS has frequently been visualized by using NADPH-diaphorase (NADPH-d) activity as a histochemical marker and by using immunocytochemistry with NOS-specific antibodies; however, NADPH-d activity may be catalyzed by non-NOS enzymes (53).
Pancreatic endothelial cells from diverse species show eNOS staining associated with NADPH-d activity (22, 53), although nonpancreatic endothelium may express nNOS as well (14, 42). Pancreatic neuronal tissue appears to exclusively express nNOS and is associated with NADPH-d activity (22, 53), but in brain, some neuronal cell populations were reported to express eNOS (8). Multiple studies (7, 9, 10, 22, 53) show NADPH-d activity associated with nNOS or nonselective NOS in intrapancreatic ganglia cell bodies and thin varicose nerve fibers with processes running among acini, ducts, blood vessels, islets, and stroma. Islet cell NOS activity occurs in isolated mouse pancreatic islets (43), but data on the constitutive expression of islet cell NOS is conflicting (10, 44, 50, 53). Pancreatic acinar cell NOS expression is less clear. Most studies show absence of both NADPH-d activity and NOS (eNOS or nNOS) staining in pancreatic acini of rats (50, 53), dogs (50), humans (53), and pigs (10). Recently, both NADPH-d activity and eNOS staining were observed in rat acinar cells by treating unfixed tissue with acetone before histo- and immunocytochemistry and by using a rabbit polyclonal antibody directed against a different epitope (22) than the mouse monoclonal antibody used by Worl et al. (53). Two additional studies showed nNOS immunoreactivity in acinar cells of bovine (38) and rat (55) pancreas but without studying NADPH-d activity. Our preliminary Western blotting and immunohistochemical studies employed monoclonal and polyclonal antibodies similar to those used by Konig et al. (22) and utilized the eNOS and nNOS gene-deleted mouse pancreas as negative controls (M. J. DiMagno, Y. Hao, S. A. Ernst, J. A. Williams, and C. Owyang, unpublished observations). Western blotting showed that nNOS is absent from acinar cells, and eNOS is either absent or present as only a small fraction of total pancreatic eNOS. Our immunohistochemical studies failed to clarify pancreatic NOS expression, because eNOS and nNOS staining in the control pancreas was not significantly altered by selective eNOS or nNOS gene deletion, although the expected absence of eNOS and nNOS protein expression in the eNOS and nNOS gene-deleted mice was found by Western blotting. Although these studies fail to clarify the localization of NOS in the exocrine pancreas, the in vivo and in vitro pancreatic secretion studies provide evidence that regardless of the presence or absence of NOS in acinar cells, the functional impact of NOS on pancreatic secretion involves nonacinar cell types.
NOS may influence carbachol- and CCK-8-stimulated in vivo pancreatic protein secretion through affects on the vasculature, the nervous system, the endocrine system, or other nonacinar cell types. Tankel et al. (49) hypothesized that reductions in pancreatic microvascular blood flow (PMBF) can reduce pancreatic secretion. NOS inhibition has been shown to reduce in vivo stimulated pancreatic protein secretion and PMBF (24, 41), although the association between secretory and circulatory events was secretagogue dependent (41). In the present study, NOS inhibition and specifically eNOS gene deletion reduced in vivo stimulated pancreatic protein secretion, but in contrast to prior studies using NOS inhibitors (13, 23-25, 37, 41, 51), the present study was performed in mice and examined the effects of single NOS isoforms. Because eNOS plays an important role in endothelial function in part by modulating vasodilation and blood perfusion of organs (35), it is conceivable that reduced eNOS enzyme activity (by NOS inhibition or eNOS gene deletion) could impair murine PMBF and interfere with tissue oxygen delivery or extraction for cell metabolism and thereby reduce in vivo stimulated pancreatic protein secretion. How eNOS is regulated in pancreatic secretion is unknown, but it is conceivable that metabolic products of acinar cells affect eNOS activity, reinforcing the notion that acinar and nonacinar interactions occur and are important during pancreatic secretion.
Nonvascular mechanisms may also explain the effect of eNOS on pancreatic protein secretion, because parallel changes in pancreatic secretion and PMBF do not always occur (18, 40, 41). First, secretin (3) and isosorbide dinitrate (56) stimulated in vivo pancreatic protein secretion at much lower doses than required to augment PMBF. A clearer dissociation of pancreatic protein secretion from PMBF has also been observed in multiple unrelated studies. For example, no change in PMBF was reported despite observing that NOS inhibitors reduce secretin-stimulated in vivo pancreatic secretion in cats (41) and that the NO donor sodium nitroprusside increases in vivo pancreatic protein secretion (17, 41). To explain this dissociation between stimulated pancreatic protein secretion and PMBF, and the relationship of NO to both, Patel et al. (41) proposed that NO may exert its principal effect through a nonvasodilatory mechanism, possibly by modulating vagal stimulation of secretory cells or directly stimulating acinar cells. Although an effect on neurotransmission has not been specifically ascribed to the eNOS isoform, this potential role is conceivable on the basis of evidence that eNOS has been detected in central nervous system (CNS) neural cells (8), that eNOS-derived NO may play a role as a retrograde neurotransmitter in the CNS (19), and that NO arising from nonneural eNOS-containing cells may diffuse freely between cells to act directly on intracellular targets (27). Without specifically implicating eNOS (or nNOS), the role of NOS in neurotransmission rather than vasodilation was examined by Holst et al. (13), who showed that NOS inhibition reduces vagal-stimulated pancreatic protein secretion in pigs without affecting vasodilatation, which was attributed to the effects of VIP, a hormone with potent effects on PMBF and weak activity as a secretagogue (26). In addition, Klein et al. (21) showed that truncal vagotomy in dogs reduced secretin-stimulated in vivo pancreatic secretion without altering PMBF.
Independent of vascular and neural-mediated effects, NOS inhibition, and specifically eNOS gene deletion, may influence exocrine pancreatic secretion through effects on insulin resistance and/or insulin release, both of which may be regulated at peripheral and/or CNS locations. In rats, NOS inhibitors administered by an intravenous route induce acute insulin resistance (2) and intracerebroventricular administration induces both peripheral insulin resistance and defects in insulin secretion (45). The effect of individual NOS isoforms on insulin resistance (but not insulin secretion) was examined by using hyperinsulinemic-euglycemic clamp studies, which showed that insulin resistance was exhibited by nNOS and to a greater extent by eNOS gene-deleted mice (46). Because insulin strongly potentiates CCK (4, 29)- and secretin (29)-stimulated in vivo pancreatic secretion in rats, the presence of insulin resistance may partially explain why eNOS gene deletion reduces stimulated in vivo murine pancreatic secretion. It is less likely that impaired insulin release would explain the effects of eNOS gene deletion on pancreatic secretion because 1) eNOS gene-deleted mice have normal baseline glucose and insulin levels (46); 2) constitutive expression of nNOS rather than eNOS has been identified in islet cells (10, 44, 50); and 3) islet cells appear to withhold insulin on exogenous (i.e., eNOS-generated) NO stimulation (43) but to release insulin on endogenous (nNOS-generated) NO stimulation (43, 44).
In contrast to eNOS, the genetic deletion of the nNOS isoform increased secretagogue-stimulated in vivo pancreatic secretion. This effect is more likely mediated by altered peripheral or central neurotransmission. nNOS-derived NO plays a role in CNS neurotransmission and may play an independent or integrated role in the peripheral nervous system neurotransmission, specifically in the pancreas, in which nNOS is expressed in many postganglionic neurons (7, 9, 10, 22, 53), which are nitrergic and cholinergic. The effect of nNOS gene deletion on pancreatic secretion may allow enhanced nerve terminal acetylcholine release during CCK-8- but not carbachol-stimulated in vivo pancreatic protein secretion. Support for this hypothesis arises from two studies (11, 34). In a nonpancreatic murine study, NOS inhibition, and specifically nNOS gene deletion, enhanced the electrically evoked release of acetylcholine from ex vivo terminal ileum myenteric plexuslongitudinal muscle preparations (34), leading to enhanced cholinergic muscular contractions. Investigation of such an effect in pancreas segments showed that the NO donor sodium nitroprusside as well as 8-bromoadenosine 3',5'-cGMP inhibited electrically evoked release of amylase in a calcium-dependent manner and failed to inhibit ACh-induced amylase secretion (11).
In summary, these studies provide evidence that nonselective NOS inhibition and eNOS gene deletion reduce CCK-8- and carbachol-stimulated in vivo pancreatic secretion by modulating nonacinar cell events. In contrast, nNOS gene deletion augments CCK-8- but not carbachol-stimulated in vivo pancreatic secretion, suggesting that eNOS plays a dominant stimulatory role and that nNOS plays a minor role, possibly acting as a negative feedback mechanism. Because of undisputed immunohistochemical localization of eNOS to the vasculature and nNOS to pancreatic nerves, it seems most likely that eNOS acts on pancreatic microvasculature to increase blood flow and that nNOS tonically inhibits acetylcholine release from pancreatic neurons. Further studies are necessary to focus more directly on these loci.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-48419 (to C. Owyang), DK-41122 (J. A. Williams), DK-60416 (M. J. DiMagno), and P30-DK-34933 to the Michigan Gastrointestinal Peptide Center and by the Michigan Center for Integrative Genomics.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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