Measuring digestive protease activation in the mouse pancreas

Abstract

Intrapancreatic activation of digestive proteases, trypsin and chymotrypsin in particular, is a hallmark of pancreatitis. In experimental rodent models, protease activation is routinely measured from pancreatic homogenates using fluorogenic peptide substrates. Here we investigated the optimal conditions for the determination of intrapancreatic trypsin and chymotrypsin activation elicited by a single intraperitoneal injection of cerulein in C57BL/6N mice. We found that these protease assays were significantly improved by using lower amounts of pancreatic homogenate and exclusion of bovine serum albumin from the assay buffer. Furthermore, pancreatic homogenates had to be freshly prepared and assayed; as freezing and thawing stimulated protease activation. Finally, replacement of the widely used Boc-Gln-Ala-Arg-AMC trypsin substrate with Z-Gly-Pro-Arg-AMC reduced the background activity in saline-treated control mice and thereby increased the extent of cerulein-induced trypsin activation. Using the optimized protocol, we reproducibly measured 20-fold and 200-fold increases in the intrapancreatic trypsin and chymotrypsin activity, respectively, in mice given cerulein.

INTRODUCTION

The development of pancreatitis is intimately linked with the inappropriate intrapancreatic activation of digestive proteases. Human genetic studies indicate that ectopic trypsin activity in the pancreas is responsible for the onset of acute pancreatitis and eventual progression to chronic pancreatitis [1]. Mutations in the high-impact risk genes PRSS1 (encoding human cationic trypsinogen), SPINK1 (encoding serine protease inhibitor Kazal type 1) and CTRC (encoding chymotrypsin C) result in elevated intrapancreatic trypsin levels by various mechanisms. Thus, PRSS1 mutations accelerate autoactivation of trypsinogen to trypsin either directly, by blocking CTRC-mediated trypsinogen degradation or by augmenting CTRC-dependent autoactivation stimulation. Mutations in SPINK1 cause loss of trypsin inhibition and mutations in CTRC decrease protective trypsinogen degradation. Intrapancreatic activation of trypsin and chymotrypsin is also routinely detected in various experimental models of acute pancreatitis in rodents [2–11]. Protease activation is typically measured from pancreas homogenates prepared at various time points after pancreatitis induction. Interestingly, published results vary with respect to the magnitude (fold increase) of trypsin and chymotrypsin activation after induction of pancreatitis, in all likelihood due to differences in the assay protocols used. Experimental conditions for the measurement of intrapancreatic trypsin and chymotrypsin activity are not standardized and arbitrary differences in methodology may lead to suboptimal detection. Therefore, we set out to investigate the optimal conditions for the determination of intrapancreatic protease activation during experimental pancreatitis.

METHODS

Materials.

The fluorescent trypsin substrates Boc-Gln-Ala-Arg-7-amido-4-methylcoumarin (QAR-AMC, catalog number I-1550.0025) and Z-Gly-Pro-Arg-7-amido-4-methylcoumarin (GPR-AMC, catalog number I-1150.0100) and the chymotrypsin substrate Suc-Ala-Ala-Pro-Phe-7-amido-4-methylcoumarin (AAPF-AMC, catalog number I-1465.0050) were obtained from Bachem USA (Torrance, CA). Cerulein (catalog number C9026) was obtained from Sigma-Aldrich (St. Louis, MO).

Animals.

C57BL/6N mice were purchased from Charles River Laboratories (Wilmington, MA) or produced in our breeding facility from the same stock. Both males and females were studied. Mice used for the experiments were 9–11-weeks old. The number of animals shown in the figures represents a pooled value from 3 experiments using 3–7 mice in the cerulein-treated and 1–3 mice in the saline-treated groups, respectively.

Cerulein-induced trypsin and chymotrypsin activation.

Intrapancreatic protease activation was induced by a single intraperitoneal injection of the secretagogue peptide cerulein given in the supramaximal stimulatory dose of 50 μg/kg. Cerulein was dissolved in normal saline at a concentration of 10 μg/mL. Control mice were given normal saline injections. Mice were sacrificed 30 min after the injection and the pancreas was harvested.

Assay of intrapancreatic trypsin and chymotrypsin activity.

Activity of trypsin and chymotrypsin was measured from pancreas homogenates obtained 30 min after a single cerulein (50 μg/kg) or saline injection. Pancreas tissue (40 mg) was homogenized in 1 mL MOPS homogenization buffer (250 mM sucrose, 5 mM MOPS (pH 6.5), 1 mM MgSO₄, 4°C) by a rotorstator homogenizer (Omni Tissue Master 125, Omni International, Kennesaw, GA) in 1.7 mL microcentrifuge tubes. Where indicated, 100 mg tissue was used. Typically, pancreata from 4 mice were homogenized and the homogenates were kept on ice until all 4 were completed. The 4 samples were then centrifuged at 1,000 rcf, at 4°C, for 2 min and the supernatants were saved, kept on ice, and assayed for protease activity. Although we typically measured protease activity immediately after homogenization, we found that the homogenates were stable and yielded the same activity values when kept on ice for several hours. Aliquots (5 μL) of the supernatants were mixed with 45 μL assay buffer containing the nonionic detergent Tween 20 (0.1 M Tris HCl (pH 8.0), 1 mM CaCl₂, 0.05% Tween 20) and 150 μL of 200 μM trypsin or chymotrypsin substrate dissolved in assay buffer was added immediately resulting in 200 μL final assay volume. The increase in fluorescence was followed in a fluorescent plate reader at 380 nm excitation and 460 nm emission wavelengths for 5 min. Rates of substrate cleavage were determined from the linear portion of the curves. Trypsin and chymotrypsin activities were expressed either as relative fluorescent units per sec (RFU/sec) or RFU/sec normalized to the total protein in the assay mix (RFU/sec/mg protein). Where indicated, the amount of the cleared homogenate was varied in the assay, supplemented with assay buffer to a final volume of 50 μL. In some experiments, the assay buffer contained bovine serum albumin and NaCl instead of Tween 20 (50 mM Tris-HCl (pH 8.1), 150 mM NaCl, 1 mM CaCl₂, 0.05 mg/mL bovine serum albumin). In these experiments, the substrates were also dissolved in the same assay buffer.

Statistical analysis.

Results were plotted as individual data points with the mean indicated. Differences of means between two groups were analyzed by two-tailed unpaired t-test. P < 0.05 was considered statistically significant.

Study approval.

Animal experiments were performed at Boston University and the University of California Los Angeles (UCLA) with the approval and oversight of the Institutional Animal Care and Use Committee (IACUC) of Boston University and the Animal Research Committee (ARC) at UCLA, including protocol review and post-approval monitoring. The animal care programs at these institutions is managed in full compliance with the US Animal Welfare Act, the United States Department of Agriculture Animal Welfare Regulations, the US Public Health Service Policy on Humane Care and Use of Laboratory Animals and the National Research Council’s Guide for the Care and Use of Laboratory Animals. Boston University and UCLA have approved Animal Welfare Assurance statements (A3316–01 and A3196–01, respectively) on file with the US Public Health Service, National Institutes of Health, Office of Laboratory Animal Welfare. Both institutions are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

RESULTS AND DISCUSSION

In the present study, we investigated conditions for the measurement of trypsin and chymotrypsin activity in pancreas homogenates from C57BL/6N mice, prepared after a single intraperitoneal injection of cerulein or saline (control). While the results are specific for this type of experiment, the optimized protocol is generally applicable to other experimental conditions in mice or other species. We routinely use a single cerulein injection in mice to elicit intrapancreatic digestive protease activation. Pancreas is harvested 30 min after the injection and immediately homogenized. Other investigators demonstrated that trypsin activity is the highest at this early time point [5, 8, 9]. More importantly, the pancreas is still relatively intact at this time; there is no significant acinar cell death or inflammatory cell infiltration.

Effect of freezing and thawing on protease activity.

We avoid freezing and thawing the pancreas or the pancreatic homogenate and we always determine protease activities from fresh homogenates. We typically process 4 mice at a time; and store pancreas homogenates on ice until all 4 samples have been prepared. We found that freezing the tissue either before or after homogenization resulted in unwanted protease activation, presumably driven by trypsinogen autoactivation. This type of artificial activation was highly variable among samples and was more frequently observed with cerulein-treated pancreata (not shown).

Effect of bovine serum albumin on protease activity.

Laboratories routinely use protease assay buffers, which contain bovine serum albumin to stabilize proteases and to prevent adherence to plastic surfaces. We compared the effect of 0.05 mg/mL albumin on protease activities by replacing it with the detergent Tween 20, which is widely used in analytical protease biochemistry in low concentrations (0.05%) to inhibit non-specific surface absorption. As shown in Figure 1, albumin had a marked inhibitory effect both on trypsin and chymotrypsin activity, in all likelihood due to protease inhibitor contamination of the commercial bovine serum albumin preparations. We found no evidence that bovine serum albumin would directly quench the released fluorophore 7-amino-4-methylcoumarin (AMC). We recommend staying away from albumin and using nonionic detergent in the protease assays.

Figure 1.

The effect of bovine serum albumin (BSA) and the nonionic detergent Tween 20 on trypsin and chymotrypsin activity measurements from pancreas homogenates. Mice were given a single injection of normal saline or cerulein, as described in Methods. Pancreatic homogenates were prepared from 40 mg tissue and protease activity was assayed using 5 μL homogenate in a final volume of 200 μL, in the presence of either 0.05 mg/mL BSA or 0.05% Tween 20, as indicated. A, trypsin activity measured with the GPR-AMC substrate. B, chymotrypsin activity measured with the AAPF-AMC substrate. Individual data points with mean are shown. Differences of means between two groups were analyzed by two-tailed unpaired t-test.

Effect of NaCl on protease assays.

In this protocol, homogenization of pancreas tissue took place in the absence of added NaCl. We found that inclusion of 150 mM NaCl in the protease assay buffers introduced unexplained variability in the activity measurements. We believe this has to do with the sudden change in ionic strength when the homogenate lacking salt is added to the salt-containing assay buffer. Therefore, we recommend to be consistent with the inclusion of NaCl, one should either use it in all buffers starting with homogenization or omit it altogether as we did in the measurements presented here.

Effect of homogenate amount on protease activity.

Next, we set out to optimize how much pancreas tissue should be homogenized and how much homogenate should be assayed to obtain the highest protease activities. We homogenized 40 mg and 100 mg pancreas tissue in 1 mL buffer and then assayed 5 μL homogenate prepared from the 40 mg pancreas versus 20 μL homogenate prepared from the 100 mg pancreas. Surprisingly, markedly higher trypsin and chymotrypsin activity was measured with the 5 μL sample (Figure 2). To characterize this phenomenon in more detail, we took a single homogenate prepared from either 40 or 100 mg pancreas and measured trypsin activity of increasing homogenate amounts, such as 1, 2, 5, 10 and 20 μL. Curiously, protease activities did not increase linearly with increasing homogenate volumes. Instead, linearity was observed up to 5 μL, after which lower than expected activities were measured (Figure 3A). Absolute activities were higher in the sample prepared from 100 mg pancreas (versus that from the 40 mg pancreas), however, the difference was lower than the expected 2.5-fold ratio. When activities were normalized to protein content, the sample prepared from 40 mg pancreas yielded higher values than that from the 100 mg pancreas (Figure 3B). A likely explanation for our observations is that the use of lower homogenate amounts allows for adequate dilution of inhibitory impurities and protease inhibitors present in the homogenate, which interfere with the assay more and more as the amount of homogenate is increased. Taken our findings together, we conclude to recommend homogenizing 40 mg or less pancreas tissue in 1 mL buffer and measuring 5 μL homogenate in 200 μL final assay volume.

Figure 2.

The effect of homogenate concentration on trypsin and chymotrypsin activity measurements from pancreas homogenates. Mice were given a single injection of normal saline or cerulein, as described in Methods. Pancreatic homogenates were prepared either from 100 mg or from 40 mg tissue in 1 mL buffer. Protease activities were then assayed in the presence of Tween 20, using either 20 μL from the 100 mg homogenate or 5 μL from the 40 mg homogenate in a final volume of 200 μL. A, trypsin activity measured with the GPR-AMC substrate. B, chymotrypsin activity measured with the AAPF-AMC substrate. Individual data points with mean are shown. Differences of means between two groups were analyzed by two-tailed unpaired t-test. Data for the 5 μL, 40 mg condition were taken from Figure 1.

Figure 3.

The effect of homogenate concentration on trypsin activity measurements from pancreas homogenates. Mice were given a single injection of normal saline or cerulein, as described in Methods. Pancreatic homogenates were prepared either from 100 mg or from 40 mg tissue in 1 mL buffer. Trypsin activity was assayed in the presence of Tween 20, with the GPR-AMC substrate, using increasing amounts of homogenate (1, 2, 5, 10 and 20 μL) in a final volume of 200 μL. A, measured trypsin activity values; B, trypsin activity values normalized to total protein concentration in the assay mix. Data points represent the average of two measurements from the same sample.

Effect of peptide substrate on trypsin activity.

Many laboratories routinely use the Boc-Gln-Ala-Arg-AMC (QAR-AMC) substrate to assay trypsin activity in pancreas homogenates [12]. When used in low concentrations, this substrate is relatively specific for trypsin and does not react with thrombin. We compared this substrate to the Z-Gly-Pro-Arg-AMC (GPR-AMC) substrate in our assay and found that the background trypsin activity measured in the pancreas from mice given saline injections was significantly lower with the GPR-AMC substrate (Figure 4). As a result of the lower background activity, the fold increase in trypsin activity in mice given cerulein was about 3-fold higher when measured with GPR-AMC versus QAR-AMC. Based on these observations, we recommend the use of the GPR-AMC substrate to determine intrapancreatic trypsin activity. The trypsin activity from cerulein-treated mice measured with the GPR-AMC substrate can be completely blocked by 1–2 μM ecotin or aprotinin added simultaneously with the substrate (not shown). Since at the given concentrations these protease inhibitors do not affect thrombin (ecotin is completely ineffective and K_i for aprotinin is 61 μM), we can safely exclude the possibility that thrombin contamination would interfere with the assay [13–15].

Figure 4.

The effect of the peptide substrate on trypsin activity measurements from pancreas homogenates. Mice were given a single injection of normal saline or cerulein, as described in Methods. Pancreatic homogenates were prepared from 40 mg tissue in 1 mL buffer and trypsin activity was assayed in the presence of Tween 20, using 5 μL homogenate, with QAR-AMC and GPR-AMC substrates, as indicated. Individual data points with mean are shown. Differences of means between two groups were analyzed by two-tailed unpaired t-test. Data for the GPR-AMC substrate were taken from Figure 1.

Conclusions.

Our results indicate that measurements of intrapancreatic trypsin and chymotrypsin activities from pancreas homogenates can be significantly improved by optimizing assay conditions with respect to homogenate preparation and amount assayed, by replacement of bovine serum albumin with Tween 20 in assay buffers and substrate solutions and by the use of the appropriate fluorescent peptide substrate. The optimized protocol prescribes the homogenization of 40 mg fresh pancreas tissue in 1 mL buffer and the assay of 5 μL clarified homogenate in the presence of Tween 20 using the GPR-AMC trypsin substrate and the AAPF-AMC chymotrypsin substrate.