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-Methylated Polyamine Analogues Prevent Trypsinogen Activation and Pancreatitis-Associated Mortality
From the Department of Biotechnology and Molecular Medicine,* A.I. Virtanen Institute for Molecular Sciences, and the Department of Chemistry, University of Kuopio, Kuopio, Finland; the Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland; the Division of Medical Biology, Institute of Pathology, University of Rostock, Rostock, Germany; and Engelhardt Institute of Molecular Biology,¶ Russian Academy of Sciences, Moscow, Russia
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Abstract |
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Genetically modified mice, mainly gene-disrupted mice, have been used to study the mediators of acute pancreatitis.2 The studies have proven the existence of numerous mediators in the immune system but failed to demonstrate that inactivation of any of these alone would prevent the local pancreatic or remote organ damage. It thus seems that an efficient therapy to treat acute pancreatitis would consist of a combination of drugs affecting different mediator pathways or metabolic routes.
The polyamines spermidine and spermine and their diamine precursor putrescine are necessary for normal cellular growth and differentiation, but their exact physiological functions remain to be proven.3
The first indication of the association of polyamines in pancreatic integrity was obtained recently with transgenic rats that overexpressed heavy metal-inducible metallothionein promoter (MT)-driven gene encoding spermidine/spermine N1-acetyltransferase (SSAT), the key enzyme in polyamine catabolism. We showed that activation of polyamine catabolism in these rats caused extensive depletion of spermidine and spermine and resulted in the development of severe acute pancreatitis within 24 hours.4
Using this rat model, we were also able to show that a polyamine analogue, -methylspermidine (MeSpd), which is supposed to fulfill the putative cellular functions of spermidine but is resistant to SSAT-dependent catabolism, prevented acute pancreatitis when administered before the induction of the transgene.5
Polyamine catabolism-induced retardation of liver regeneration could likewise be restored by a prior administration of the polyamine analogue.5,6
We recently showed that another polyamine analogue, namely bis--methylspermidine (Me2Spm), was effective in supporting growth both in vivo and in vitro.6
The aim of the present study was to investigate whether the changes in polyamine pools occur in other rat models of experimental pancreatitis and in human pancreatitis, to investigate the possible effects of pharmacological intervention with -methylated polyamine analogues on experimental pancreatitis including our transgenic rat model, and to elucidate the mechanism of pancreatitis caused by activation of polyamine catabolism using acinar cell culture.
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Materials and Methods |
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MeSpd7 and Me2Spm8 were synthesized as described earlier. The polyamine analogues were dissolved in saline, neutralized, and administered intraperitoneally in a volume of 0.1 ml/kg body weight. Zinc sulfate heptahydrate was purchased form Merck (Darmstadt, Germany) and administered intraperitoneally in distilled water. L-Arginine was a product of Sigma (St. Louis, MO) and was dissolved in saline immediately before use. Cerulein (Takus) was obtained from Pharmacia & Upjohn, Erlangen, Germany.
Patients
Human pancreatic specimens were obtained at the Tampere University Hospital from patients undergoing pancreaticoduodenal resection for a mass in the head of the pancreas (14 patients) or necrosectomy for acute necrotizing pancreatitis (two patients). In the patients with surgical resection, a 2 x 2 mm2 specimen of pancreas was cut from the resection line. In the patients with necrosectomy, a 2 x 2 mm2 specimen was cut immediately from the best preserved area of the removed part of pancreas. The sample was immediately immersed in liquid nitrogen and stored in –70°C before assays.
The human specimens were grouped as follows: group 1 (n = 4): normal pancreas, from patients with bile duct or duodenal adenocarcinoma; group 2 (n = 2): acute pancreatitis, from patients with acute necrotizing pancreatitis and extensive necrosis (alcoholic etiology); group 3 (n = 4): chronic pancreatitis (alcoholic etiology); group 4 (n = 6): chronic secondary pancreatitis, from patients with pancreatic carcinoma or ampullary adenocarcinoma. The study was approved by the Institutional Review Board of Tampere University Hospital, Finland.
Animals
The production of transgenic Wistar rats harboring metallothionein (MT) I promoter-driven SSAT gene construct has been described.4 Severe necrotizing pancreatitis was induced by activating the transgene expression via the MT I promoter with the administration of a nontoxic dose (10 mg zinc/kg i.p.) of zinc in a volume of 0.2 ml/kg body weight.4 The therapeutic effects of MeSpd and Me2Spm were studied by injecting the analogues at 4 and 8 hours after induction of pancreatitis, ie, at times when first signs of pancreatitis were evident. Nontransgenic Wistar rats were used in the studies of L-arginine- and cerulein-induced pancreatitis. L-Arginine was administered intraperitoneally as a single dose of 2.5 g/kg, which produced necrotizing pancreatitis within 24 hours as described by Tani and colleagues.9 Cerulein was given as seven hourly intraperitoneal injections of 50 µg/kg.10 The preventive effect of MeSpd was studied by injecting the analogue at 20 and 4 hours before induction of pancreatitis to allow accumulation of the MeSpd. Both male and female rats were used in the experiments. Blood for hematocrit measurement was collected from femoral vein into heparinized glass microcapillaries (Miles Inc., Elkhart, IN). At the end of the experiments, the rats were sacrificed by decapitation and the blood was drained onto heparinized dishes. Alternatively, the rats were anesthetized with CO2, thoracotomized, and blood was collected by puncturing the heart before decapitation. The pancreas was removed, the tail of the organ was fixed for histology, and the rest frozen in liquid nitrogen and stored at –70°C until analyzed. Where indicated, other tissues were taken after the removal of the pancreas and handled similarly. The Institutional Animal Care and Use Committee of the University of Kuopio and the Provincial Government approved the animal experiments.
Isolation and Analyses of Pancreatic Acini
Dispersed acini (intact secretory units of 20 to 50 acinar cells) were freshly prepared from the pancreas of male syngeneic and transgenic rats by collagenase (Serva, Heidelberg, Germany) digestion.11 Acinar biovolume was determined by a CASY system (Schärfe, Reutlingen, Germany) and adjusted to a concentration of 2 mm3/ml. Acini were incubated in a culture medium containing 24.5 mmol/L HEPES, 96 mmol/L NaCl, 6 mmol/L KCl, 1 mmol/L MgCl2, 2.5 mmol/L Na2HPO4, 0.5 mmol/L CaCl2, 11.5 mmol/L glucose, 5 mmol/L sodium pyruvate, 5 mmol/L sodium glutamate, and 5 mmol/L sodium fumarate, with minimum essential medium (1% v/v) and bovine serum albumin, fraction V (1% w/v), at pH 7.4.12 At a temperature of 37°C, acini were gently mixed (50 rpm) and periodically gassed with oxygen.
In the first experiment, we examined the effect of Zn2+ on intracellular trypsinogen activation. Pancreatic acini of untreated syngeneic and transgenic rats were isolated as described above. Freshly prepared acini were incubated for 2 hours by addition of ZnSO4 to the culture medium at different concentrations (0 to 250 µmol/L). At the end of incubation acini were washed and intracellular trypsinogen activation was analyzed.
In the second experiment, we examined the influence of the polyamine analogue Me2Spm on zinc-induced trypsinogen activation. Therefore, syngeneic and transgenic rats were treated by administration of Me2Spm (25 mg/kg i.p.) as two doses 24 and 4 hours before sacrifice. Moreover, Me2Spm (1 mmol/L) was added to all buffers of acinar isolation and incubation in vitro. As described in the first experiment, isolated acini were incubated with ZnSO4 (50 µmol/L or 100 µmol/L) for 2 hours after analysis of trypsinogen activation. In parallel to all in vitro experiments, separate aliquots of acini were incubated without ZnSO4 but stimulated with the cholecystokinin analogue cerulein (10 nmol/L, 60 minutes). Under these conditions intracellular trypsinogen activation reached a maximum.
To measure intracellular trypsinogen activation, acini were washed, resuspended in fresh medium (without zinc or cerulein), transferred to 96-well microtiter plates, and the cell-permeable and trypsin-specific substrate (CBZ-Ile-Pro-Arg)2-rhodamine-110 (Molecular Probes, Eugene, OR) was added (10 µmol/L). Intracellular tryptic substrate cleavage and the released fluorochrome rhodamine-110 were quantified by cytofluorometry continuously for 60 minutes (excitation, 485 nm; emission, 530 nm; CytoFluor 2350; Millipore, Bedford, MA).13
Trypsinogen activation corresponds to the linear increase of rhodamine fluorescence (
F) per time interval (t) and was expressed as slope of linear regression (F/t).14
Zinc-induced trypsinogen activation was calculated as a percentage of the maximum activatable trypsinogen detected by the corresponding cerulein experiments.
Analytical Methods
SSAT activity was assayed according to Bernacki and colleagues.15
Polyamines, their acetylated derivatives and the methylated polyamine analogues were determined by high-performance liquid chromatography as described by Hyvönen and colleagues.16
Plasma -amylase and alanine aminotransferase (ALAT) activities were determined using the analyzer system Microlab 200 from Merck (Darmstadt, Germany). Hematocrit values were measured in blood samples taken from femoral vein using an Ames microspin centrifuge (Bayer Diagnostic GmbH, Speer, Germany) according to the manufacturer’s instructions.
Histological Analyses
Formalin-fixed tissue specimens were embedded in paraffin, cut into 5-µm-thick slices and stained with hematoxylin and eosin. The sections were evaluated according to Niederau and colleagues.17
For necrosis and vacuolization, the scores refer to an approximate percentage of cells involved. Definitions for histological evaluation in Table 1
in terms of necrosis and/or vacuolization are normal (0 to 5% of necrosis), mild (5 to 30% of necrosis), moderate (30 to 50% of necrosis), and severe (>50% of necrosis).
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The data are expressed as means ± SD. One-way analysis of variance with Dunnett’s post hoc test for multiple comparisons was used for statistical analyses with the aid of the software package GraphPad Prism 4.0 (GraphPad Software, Inc., San Diego, CA). Survival data were analyzed using Fisher’s exact test and the Kaplan-Meier method with log-rank test.
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Results |
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To study whether activation of polyamine catabolism could be observed in other pancreatitis models besides our transgenic model, which is specifically directed to induction of SSAT, we induced necrotizing pancreatitis in normal rats with an excessive dose of L-arginine. As seen in Table 1
, the treatment caused threefold increase in SSAT activity, and consequently, 
50% depletion of spermidine and spermine with a 40-fold accumulation of putrescine. These changes are distinct characteristics of activated polyamine catabolism. Pancreatitis was assessed on the basis of elevated plasma amylase activity and histological scoring representing mild to severe pancreatitis manifested as edema, vacuolization, presence of inflammatory cells, and appearance of extensive necrosis (in average 35 to 60% cells involved). Despite the replenishment of total polyamine pools, pretreatment of the rats with MeSpd before induction of pancreatitis did not prevent the activation of polyamine catabolism, as evidenced by increased SSAT activity and depleted pools of the natural polyamines, although to a lesser extent than with L-arginine alone (Table 1)
. However, plasma amylase activity was nearly normalized in rats treated with the polyamine analogue and the histological status of pancreas was clearly improved. The range of pancreatic necrosis in these animals was 0 to 30%. Essentially similar results were obtained with another polyamine analogue, Me2Spm at 25 mg/kg in an identical experimental setting (results not shown).
Polyamine Catabolism in Cerulein-Induced Pancreatitis
Mild edematous pancreatitis with variable level of edema and vacuolization but virtually without necrosis was caused by cerulein. Activation of polyamine catabolism was also evident in this model of pancreatitis (Table 1)
. In addition to depleted spermidine and spermine pools, accumulation of putrescine was seen. Traces of N1-acetylspermidine, which is normally undetectable in nontransgenic animals, were also detected in cerulein-treated animals (not shown). Plasma amylase activity was highly elevated in cerulein-treated rats. The histological hallmarks observed were edema, inflammation, and abundant intracellular vacuolization with minimal necrosis. Pretreatment of the rats with MeSpd did not change the polyamine pattern, the histological status or amylase activity from those seen in rats treated with cerulein alone (Table 1)
. MeSpd was not detectable at the time of sacrifice (Table 1)
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Treatment of Polyamine Catabolism-Induced Pancreatitis in Transgenic Rats
Our earlier studies showed that pretreatment of transgenic rats with MeSpd before induction of pancreatitis totally prevented the disease. We now investigated the possibility to interfere with the pathogenesis by treating the rats with a polyamine analogue as late as several hours after induction of pancreatitis. The choice of the polyamine analogue used here was based on preliminary experiments in which spermine analogue bismethylspermine proved to be as efficient as MeSpd in supporting cellular function.6
Table 2
summarizes the results of 24- and 48-hour treatment of rats with zinc alone or followed by administration of Me2Spm given at the time when the induction of polyamine catabolism has clearly commenced (50% depletion of spermidine at 4 hours) and the first signs of developing pancreatitis (edema, inflammation, and occasional necrosis) are evident. Immense induction of SSAT was followed by near total depletion of spermidine and spermine by 24 hours. Indicative of greatly enhanced acetylation of spermidine by SSAT, there was an increase in the N1-acetylspermidine pool, which is normally undetectable (Table 2)
. Treatment with Me2Spm had little effect on the depleted concentrations of the natural polyamines but led to compensatory accumulation of Me2Spm and its oxidation product MeSpd. Analysis of the rats at 48 hours after zinc gave similar results (Table 2)
.
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50% of cells in zinc-treated animals when compared to untreated animals (Figure 1, a and b)
. Necrosis appeared to be less extensive (10 to 50% of the cells involved) after Me2Spm (Figure 1c)
. In correlation with the therapeutic effect, there was less ascites fluid on necropsy in the intraperitoneal cavity of animals that responded to the analogue treatment. After a 48-hour treatment with zinc, the pancreas exhibited massive necrosis and inflammation (Figure 1d)
that was alleviated by the supplementation of Me2Spm (Figure 1e)
. A representative pancreatic section of an animal treated similarly with zinc and the spermidine analogue, MeSpd, likewise showed only partial necrosis (Figure 1f)
. Whether or not treated with the polyamine analogues, there were no overt macroscopical or histological abnormalities in other tissues examined (liver, heart, kidney, and lung) apart from occasional foci of inflammatory cells seen in liver and lung (not shown). It should also be noted that tissue polyamine levels in heart, kidney, and lung remained virtually normal in zinc-treated animals (not shown), excluding the possibility that local polyamine depletion caused tissue failure in these organs.
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. Both values were significantly elevated in zinc-treated rats with pancreatitis. ALAT activity decreased to some extent whereas the hematocrit value was totally normalized by Me2Spm treatment (Table 3)
. Without any intervention the overall mortality of zinc-treated rats was 100% within 72 hours (Figure 2)
. Treatment of rats with either MeSpd or Me2Spm after zinc dramatically protected them from early death as well as from later mortality for up to 2 weeks of follow-up (Figure 2)
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. The protective effect of MeSpd was essentially similar to that caused by Me2Spm (Figure 1h)
. Due to expression of MT in liver, hepatic polyamine catabolism is activated in MT-SSAT transgenic animals by zinc, which may contribute to any potential pancreatitis-related hepatic damage. However, the histological changes after zinc treatment were only mild and transient, and liver totally recovered after 2 weeks in rats receiving polyamine analogues. Kidney, heart, and lung were practically normal in the surviving animals, even in those exhibiting sectional fibrosis of pancreas. Polyamine Analysis of Human Pancreatic Specimens
A small number of human pancreatic biopsies were analyzed for polyamine contents. Normal specimens (group 1) revealed polyamine levels comparable to those reported earlier18 for healthy human pancreas, ie, 4 ± 1, 140 ± 63, and 136 ± 50 nmol/mg DNA for putrescine, spermidine, and spermine, respectively. Concentrations of spermidine and spermine constituted only 2 and 24% of normal levels, respectively, in group 2 (acute pancreatitis) whereas they were normal in group 3 (chronic alcoholic pancreatitis). Interestingly, in group 4 (chronic secondary pancreatitis), an accumulation of putrescine (3.5-fold compared with normal) together with decreased levels of spermidine (39% of normal) and spermine (78% of normal) were found. Acetylated spermidine was not detectable in any of the samples.
Protease Activation in Isolated Acini
Intracellular activation of digestive zymogens is the initiating factor in the course of acute pancreatitis. To investigate the hypothesis that a zinc-induced activation of polyamine catabolism and decrease of spermidine and spermine pools leads to an intracellular activation of trypsinogen in isolated acinar cells, we used the cell-permeable and trypsin-specific substrate (CBZ-IlePro-Arg)2-rhodamine-110 as described previously.13
Trypsinogen activation by a supramaximal concentration (10 nmol/L) of cerulein revealed an identical activation pattern in acini from syngenic and transgenic animals with or without pretreatment with Me2Spm (not shown). Treatment of the acini with different concentrations of ZnSO4 for 120 minutes to induce transgene expression resulted in a dose-dependent and significantly higher trypsinogen activation in the acini from transgenic animals in comparison with syngenic acini (Figure 3a)
. Furthermore, pretreatment of the animals with Me2Spm completely blocked the intracellular trypsinogen acti-vation, indicating that the depletion of intracellular polyamine pools is responsible for trypsinogen activation (Figure 3b)
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Discussion |
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. Also, other mediators besides polyamine depletion may have a critical role in the mechanism of edematous pancreatitis caused by this secretagogue. This conclusion is supported by our finding that Me2Spm prevented zinc-induced but not cerulein-induced trypsinogen activation in isolated acini (Figure 3)
. In both pancreatitis models in which substantial necrosis develops (transgenic SSAT induction model and L-arginine model), polyamine supplementation reduced necrosis, suggesting that polyamine changes are associated with the development of necrosis. Spermine and spermidine levels appeared to be decreased in human necrotizing pancreatitis, although the small number of clinical samples available does not allow us to draw conclusions of the involvement of polyamine catabolism in human pancreatitis.
-Methylated analogues of spermidine and spermine have been considered metabolically more stable than the natural polyamines. They are not substrates for SSAT and MeSpd is a poor substrate for spermine synthase.22
In contrast with the stability of MeSpd, Me2Spm is, however, converted to MeSpd to some extent in vivo.6
These analogues appear to fulfill cellular functions of polyamines in promoting the conversion of B-DNA to Z-DNA and restoring growth of polyamine-depleted cells.22,23
Our recent work with MeSpd was the first report of a methylated polyamine analogue being used in vivo.5
Only recently, with a sufficient supply of Me2Spm, have we been able to study the potential of this analogue in supporting pancreatic integrity. The present results distinctly show that Me2Spm is at least as effective as MeSpd in its protective action. Most importantly, the analogues given 4 to 8 hours after the induction of acute pancreatitis alleviated pancreatitis, especially acinar cell necrosis, prevented hemoconcentration and dramatically improved the survival of the animals. The finding that Me2Spm was partially metabolized to MeSpd also in pancreas, by the action of polyamine oxidase and/or spermine oxidase, offers the advantage that depletion of both spermidine and spermine can be overcome by the administration of Me2Spm. It should be noted that similar therapy with the natural polyamines is not feasible, as they would undergo rapid degradation due to activated acetylation.
According to our accumulated experience with the SSAT-overexpressing transgenic rat model, development of severe pancreatitis is inevitable when the cellular concentration of spermidine falls clearly below the value of 1000 pmol/mg tissue weight. Polyamine catabolism-induced severe pancreatitis was associated with ultimate death of the transgenic animals unless depleted pools of polyamines were substituted by administration of polyamine analogues. The specific cause of death of zinc-treated rats with pancreatitis is not clear at the moment, as other tissues studied up to 48 hours after administration of zinc showed little histopathological changes. The minor histological changes and metabolic effects seen in liver (Table 3)
in this transgenic animal model were most likely not related to severe pancreatitis but were associated with hepatic SSAT induction because the metallothionein promoter-driven SSAT transgene is also expressed in liver. However, our earlier studies showed that MeSpd and Me2Spm restored early liver regeneration5,6
suggesting that polyamine analogues may also protect liver from potential damage during the course of pancreatitis. This was now verified in the long-term survivors whose livers showed normal appearance. It is possible that the methylated polyamine analogues exert a protective effect in tissues other than pancreas and liver. Hemoconcentration resulting from enhanced vascular permeability and manifested as abnormally high hematocrit values 24 hours after induction of pancreatitis (Table 3)
is likely to cause hypovolemic shock, hypercoagulability, and microthrombi, leading to deterioration of tissue microcirculation and functional activity. The finding that hematocrit values were normalized by the treatment with Me2Spm strongly suggests that the surviving animals are protected from systemic complications because of less severe pancreatitis. In fact, although methylated polyamine analogues alleviate pancreatic damage their life-supporting effect may primarily be targeted to systemic phenomena. This hypothesis is supported by recent reports showing that acylhomospermines and lysine-spermine conjugates may be useful in the treatment of endotoxic shock24,25
and that oral spermine administration may have therapeutic effect in systemic inflammation and intestinal damage in a mouse endotoxin model.26
Severe distortion of polyamine homeostasis is likely to exert a deleterious effect in tissue integrity and/or function. Exocrine pancreatic tissue has the highest spermidine concentration in the mammalian body27 and the molar ratio of spermidine and spermine is also exceptionally high in pancreas,4 which is typical of a tissue with high rate of protein synthesis or active proliferation. Profound depletion of polyamines destabilizes DNA, interferes with transcription, and results in cell death via activation of caspase-3 and disruption of the mitochondrial membrane potential.28 Polyamines participate in the maintenance of tissue structure via nonspecific ionic or more specific molecular interactions with cellular components and play a role in immune response and contribute to acute phase inflammation.29
Acute pancreatitis is caused by premature activation of the inactive zymogen enzymes with its subsequent consequences of radical formation and cytokine release. The initial factors triggering this activation have not been clarified.30 It is possible that the higher polyamines directly inhibit protease activity.31 Their depletion would therefore result in a direct activation of the digestive enzymes. Our present results show that induction of polyamine catabolism in acinar cells is clearly associated with intracellular trypsinogen activation.
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Acknowledgements |
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Footnotes |
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Supported by the Academy of Finland (project number 50317).
Accepted for publication September 21, 2005.
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References |
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-methylspermidine. Bioorgan Khim (Moscow) 2004, 30:441-445-methyl- and ,'-dimethylspermine. Bioorgan Khim (Moscow) 2005, 31:183-188This article has been cited by other articles:
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  M. T. Hyvonen, T. A. Keinanen, M. Cerrada-Gimenez, R. Sinervirta, N. Grigorenko, A. R. Khomutov, J. Vepsalainen, L. Alhonen, and J. Janne Role of Hypusinated Eukaryotic Translation Initiation Factor 5A in Polyamine Depletion-induced Cytostasis J. Biol. Chem., November 30, 2007; 282(48): 34700 - 34706. [Abstract] [Full Text] [PDF]
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