Evaluation of Pancreatic Amylase mRNA upon Cholinergic Stimulation of Secretion
Département de Pathologie et Biologie Cellulaire, Université de Montréal, Montréal, Québec, Canada
Correspondence to: Dr. Moïse Bendayan, Department of Pathology and Cell Biology, Université de Montréal, CP 6128 Succursale Centre-ville, Montréal, Québec, Canada H3T 1J4. E-mail: moise.bendayan{at}umontreal.ca
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Summary |
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Key Words: amylase cholinergic stimulation pancreas mRNA in situ hybridization RNasegold Northern blotting
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Introduction |
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Extensive work has been carried out on the effects of secretagogues on protein synthesis and secretion using either in vivo or in vitro systems. Some studies reported increases in pancreatic enzyme secretion without apparent effects on the rate of protein synthesis (Hokin and Hokin 1954; Campagne and Gruber 1962
; Bauduin et al. 1969
), whereas others reported increases in both synthesis and secretion (Morisset and Webster 1971
; Webster et al. 1974
). Increases in secretion with decreases in synthesis were also reported (Mongeau et al. 1976
; Lahaie 1986
; Perkins et al. 1991
; Yuan et al. 1999
). Decreases in rates of synthesis are due to either a decrease in amino acid incorporation, a depletion of specific mRNA molecules, or a deficiency in protein translation at the polysomal level. Discrepancies among all these studies are partially due to differences in experimental protocols and to the fact that most studies were aimed at the understanding of acute pancreatitis induced by supramaximal infusion or multiple injections of secretagogues.
In this study we evaluated changes in RNA and specific mRNA molecules upon stimulation and inhibition of secretion. Amylase mRNA in rat pancreas was evaluated after a single injection of carbachol, a cholinergic agonist of ACh, and after fasting conditions known to lead to inhibition of secretion. Two different techniques of molecular morphology were applied to evaluate and quantitate changes in RNA labeling in the rat pancreatic acinar cells. We performed the RNase Agold cytochemical technique known to reveal RNA molecules at the ultrastructural level (Bendayan 1981). Quantitation of the labeling revealed that, after a short period of stimulation and under fasting conditions, RNA labeling at the rough endoplasmic reticulum (RER) level decreased. On the other hand, non-radioactive in situ hybridization (ISH), using digoxigenin (DIG)-labeled oligonucleotide (Gingras and Bendayan 1995
), revealed drastic decreases in amylase mRNA also at the RER level. The advantages of these approaches reside in their high resolution and their ability to quantitate RNA labeling at the subcellular level. The ISH carries a further advantage in its higher specificity, revealing solely amylase mRNA. Finally, non-radioactive Northern blotting, a more sensitive technique, confirmed the drastic decreases in amylase mRNA after stimulation of secretion under fasting conditions. The combination of these techniques demonstrates that, after a very short period of stimulation of secretion, the levels of amylase mRNA as well as total RNA drastically decreased, which could reflect a drop in amylase synthesis at the translational level.
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Materials and Methods |
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Stimulation of secretion was performed by a single IP injection of carbamyl ß-methylcholine chloride (carbachol) (Sigma-Aldrich; Oakville, Ontario, Canada) at a final concentration of 12 mg/kg body weight. The animals were then anesthetized with urethane and pancreatic tissue was excised 5, 15, or 30 min after induction of secretion.
Fasted animals were deprived of food for either 12 or 48 hr but had free access to drinking water and were kept in individual cages (Bendayan et al. 1985). The animals were housed and handled according to the guidelines from the Canadian Council on Animal Care (CCAC).
Tissue Preparation
Light Microscopy
Portions of pancreatic tissue were sampled, fixed in Bouin's fluid for 24 hr and embedded in paraffin. Five µm-thick sections were mounted in diethyl pyrocarbonate (DEPC)-treated water on Superfrost Plus slides (Fisher Scientifique; Montréal, Québec, Canada) and kept at room temperature (RT) until used for in situ hybridization (ISH).
Electron Microscopy
Fragments of the pancreatic tissues were fixed by immersion with 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for 2 hr at 4C. After fixation the tissue samples were processed through dehydration in methanol and embedding in Lowicryl K4M at 30C (Canemco; St-Laurent, Québec, Canada), as previously described (Bendayan 1995). Sectioning was performed using DEPC-treated water to avoid RNA degradation and thin tissue sections were mounted on Parlodioncarbon-coated grids. The sections were then processed for electron microscopic cytochemistry.
In Situ Hybridization
Probe Preparation
The amylase single-stranded DNA oligonucleotide probe is a 30-mer sequence (3' ACA ACC GTC ACT ACC GTT TCG AGT GAA GAG 5') complementary to nucleotides 14271457 of pancreatic amylase mRNA specific to rat (MacDonald et al. 1980). It was chemically synthesized on an Automated DNA Synthesizer and purified on a Sep-Pak cartridge (Millipore Canada; Mississauga, Ontario, Canada). After purification the oligonucleotide was tagged by tailing at the 3' end with digoxigenin (DIG). This procedure was carried out using the 3' end-labeling kit (Roche Diagnostics; Laval, Québec, Canada) and by following the different steps previously described (Schmitz et al. 1991
). We combined 4 µl 5 x tailing buffer, 4 µl CoCl2 (5 mM final), 1µl DIGUTP, 1µl dCTP (0.5 mM final) (Pharmacia Biotech; Baie d'Urfé, Québec, Canada), 1 µl 30-mer oligonucleotide (100 pmol), 8 µl Millipore water, and 1 µl terminal transferase (50 U). Incubation was carried out at 37C for 15 min, and after transfer to ice the reaction was stopped by adding 2 µl of the stop solution (200 µl 0.2 M EDTA, pH 8.0, and 1 µl 2% glycogen solution). The probe was then precipated by adding 2.5 µl 4 M LiCl and 75 µl prechilled absolute ethanol and kept at 20C. A dot-blotting experiment was carried out to verify the labeling of the amylase probe. Briefly, 5 µl of 50, 25, 10, and 5 ng of the labeled oligonucleotide was fixed by UV crosslinking onto nitrocellulose membrane and incubated with the anti-DIGalkaline phosphatase 1:5000 for 30 min at RT. The color reaction was revealed by the NBT and X-phosphate provided in the nucleic acid detection kit (Boehringer Mannheim; Laval, Québec, Canada). Positive reaction confirmed the tagging of the probe. As expected, the intensity of the reaction decreased proportionally to the concentration of the probe.
Light Microscopy
This protocol was previously described in detail (Gingras and Bendayan 1995). Paraffin sections were rehydrated, incubated in proteinase K solution (15 µg/ml in 0.05 M Tris buffer, pH 7.6), fixed in 0.4% paraformaldehyde in PBS at 4C for 20 min, rinsed in DEPC-treated water, and incubated for 1 hr at 37C in the prehybridization buffer [0.6 M NaCl, 30% deionized formamide (v/v), and 150 µg/ml salmon sperm DNA]. Hybridization was performed using the 30-mer synthetic oligonucleotide at a concentration of 200 ng/ml in the prehybridization buffer, overnight at 37C in a humid chamber. The tissue slides were then washed at 37C twice for 10 min in the following solutions: 2 x SSC/30% formamide and 0.2 x SSC/30% formamide. Before the detection steps the slides were incubated in 0.05 M Tris buffer, pH 7.6, 0.15 M NaCl, 2 mM MgCl2 (TBS) containing 0.1% bovine serum albumin (BSA) and 0.1% Triton X-100 for 15 min. Detection of the DIG-labeled hybrids was carried out with an anti-DIGalkaline phosphatase-conjugated antibody 1:600 (Roche Diagnostics) in TBS/0.1% BSA for 30 min at RT. The slides were then washed twice for 5 min in TBS/0.1% BSA followed by an incubation with the substrate, which consisted of 45 µl NBT and 35 µl X-phosphate in 10 ml of alkaline buffer (0.1 M Tris-HCl, 0.15 M NaCl, pH 9.5). The tissue slides were mounted with 50% glycerol in PBS and examined with a Leitz Orthoplan microscope (Leica; St-Laurent, Québec, Canada). Controls of specificity were performed by either omitting the probe or by treating the sections with RNase (100 µg/ml in 2 x SSC containing 10 nM MgCl2), 1 hr at 37C, before the hybridization step. All labeling experiments were carried out in parallel. Semi-serial sections of the different tissues were incubated at the same time under the same conditions of length of time and temperature.
Electron Microscopy
Ultrathin Lowicryl tissue sections of pancreas from the animals submitted to the different experimental conditions were processed for ISH following the protocol described in detail previously (Gingras and Bendayan 1995). The tissue sections were hybridized overnight at 37C in the prehybridization buffer containing 400 ng/ml of the DIG-labeled oligonucleotide complementary to amylase. Section were washed in PBS, then incubated with a solution of 1% ovalbumin in PBS for 5 min and transferred to a drop of the anti-DIG polyclonal antibody 1:5 (Roche Diagnostics) in PBS for 2 hr at RT. The section were then washed in PBS, incubated 5 min with 1% ovalbumin in PBS, and transferred to a drop of 10 nm protein AGgold complex (Ghitescu et al. 1991
) for 1 hr at RT. The sections were then rinsed with PBS and water, stained with uranyl acetate, and observed with a Philips 410 LS electron microscope (FEI Systems Canada; St-Laurent, Québec, Canada). Controls of specificity were performed by omitting the probe or by treating the sections with RNase before the hybridization step.
RNaseGold
The RNasegold cytochemical labeling was previously shown to be a useful probe to reveal specifically RNA-containing structures on tissue sections. The RNase A was chosen in the present study for its strong labeling of the RER, the site of protein synthesis (Bendayan 1981; Cheniclet and Bendayan 1990
). The RNase Agold complex was prepared according to conditions previously determined (Bendayan 1984
; Cheniclet and Bendayan 1990
), using RNase A (Sigma-Aldrich) and 10 nm gold particles. Ultrathin Lowicryl sections of pancreatic tissues from the different experimental conditions were incubated on drops of the RNase Agold complex for 30 min at RT, washed, and stained with uranyl acetate. Controls of specificity were performed by either heating the RNase Agold complex to 100C before labeling or by treating the sections with RNase A before incubation with the RNase Agold complex (Bendayan 1984
).
Quantitative Evaluations
Densities of the various labelings obtained by electron microscopy over the different cellular compartments of the pancreatic acinar cells were evaluated using the Videoplan 2 image processing system (Carl Zeiss; Oberkochen, Germany). At least 25 electron micrographs recorded at x30,000 final magnification were analyzed for each animal tissue in each protocol. Labeling densities are expressed either per unit of area occupied by the compartment (number of gold particles per µm2) or per length of membrane (number of gold particles per µm). To proceed with comparative evaluations of the results, labeling protocols of the different tissue sections were carried out in parallel under identical conditions with the same reagent solutions. For comparative purposes, the labeling of the RER was evaluated in terms of length and surface. Being a linear structure, it is more logical to refer to length units. However, to compare its labeling to that of nuclei and granules, a labeling by surface units had to be evaluated.
RNA Extraction and Northern Blotting
The entire pancreas was removed. Tissues from at least five animals submitted to each experimental condition were pooled and frozen immediately in liquid nitrogen. Rat pancreatic tissue contains large amounts of endogenous RNases and tissue RNA is known to undergo spontaneous autolysis upon sampling. This required particular attention during the procedure to ensure recovery of intact RNA suitable for Northern blot hybridization (Gill et al. 1996). We therefore modified the protocol as follows: 100 mg of frozen tissue was transferred directly into 10 ml of ice-cold TRIzol reagent (Invitrogen; Burlington, Ontario, Canada) and homogenized with a Polytron homogenizer (Brinkmann Instruments; Mississauga, Ontario, Canada). Total cellular RNA isolation with TRIzol reagent was performed according to the protocol described by Invitrogen. At the end of the protocol, the RNA pellet was suspended in DEPC-treated water containing 0.05% RNAsin (Promega; Madison, WI), measured spectrophotometrically at 260 nm, and used the same day. Twenty µg of total RNA samples was mixed in 1 x MOPS buffer (0.02 M 3-(N-morpholino) propanesulfonic acid, pH 7.0, 0.005 M sodium acetate, and 0.001 M EDTA, pH 8.0) containing 8% formaldehyde (v/v) and 50% deionized formamide (v/v), denatured by heating at 60C for 15 min, and loaded onto a 1% agaroseformaldehyde gel (1% agarose in MOPS buffer and 6% formaldehyde). Electrophoresis was performed at 60 V for 5 hr with a constant recirculation of the buffer. The gels were then soaked for 20 min in 20 x SSC (3 M NaCl, 0,3 M sodium citrate, pH 7.0) and RNA was transferred to positively charged nylon membranes (Roche Diagnostics) by classic capillary blotting in 20 x SSC (Thomas 1980
). The membranes were rinsed in DEPC-treated water, baked for 30 min at 120C, and prehybridized for 1 hr at 37C in DIG Easy Hyb solution (Roche Diagnostics). Hybridization was performed overnight at 37C with 400 ng/ml of the DIG-labeled amylase probe in prehybridization solution. Membranes were washed twice for 10 min in 2 x SSC containing 0.01% SDS at RT and twice for 15 min in 0.1 x SSC, 0.01% SDS at 68C. The membranes were rinsed for 2 min at RT in maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, pH 7.5) and incubated for 30 min in the blocking solution [maleic acid buffer, 10% blocking reagent (Roche Diagnostics)]. The hybrids were detected by chemiluminescence. The membranes were incubated for 30 min at RT with an anti-DIGalkaline phosphatase antibody 1:5000 in blocking solution and then washed twice for 15 min in washing buffer [0.1 M maleic acid, 150 mM NaCl, pH 7.5, 0.3% (v/v) Tween-20]. Membranes were then equilibrated for 5 min at RT in detection buffer (0.1 M Tris-HCl, 0.15 M NaCl, pH 9.5) and incubated for 5 min at RT with the chemiluminescent substrate CSPD (Roche Diagnostics) followed by a second incubation for 15 min at 37C. Finally the membranes were exposed to Kodak X-Omat film (Perkin-Elmer Life Sciences; Woodbridge, Ontario, Canada). RNA size was determined by comparing with the migration of an RNA ladder DIG-labeled (0.37.4 kb) (Roche Diagnostics).
For internal controls we chose to reveal the 28S RNA. Indeed, some of the conventional housekeeping genes generally used as controls, such as actin and glyceraldehyde-3-phosphate dehydrogenase (GAPD), are not appropriate for our study. Recent reports (Yuan et al. 1999) have revealed major changes of these proteins in pancreatic tissue submitted to stimulation of secretion and have underlined caution in using ß-actin and GAPD as internal RNA controls. rRNAs should be preferred as RNA loading controls for Northern blot analysis (Bhatia et al. 1994
; Gong et al. 1996
; Yuan et al. 1999
).
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Results |
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The strongest positive signals were generated by tissues of control fed rats (Tables 1 and 2). Animals fasted for 12 hr demonstrated a decrease in RER labeling, which became more drastic after 48 hr of fasting (Table 1; Figure 5a). Interestingly, the labeling over nuclei increased significantly in pancreatic tissue of 48 hr-fasted animals (Table 2; Figure 5a). After 5 min of cholinergic stimulation we found very low labeling density over the RER (Table 1; Figure 5b). Similarly, the labeling over nuclei was also the lowest compared with the other experimental conditions. At 15 and 30 min after stimulation of secretion, recovery of the RER labeling took place (Table 1). Very low levels of background labeling were found over the secretory granules (Table 2). Control experiments confirmed the specificity of these results. Pretreatment of the pancreatic tissue sections with RNase resulted in an almost complete elimination of the RER and nuclear labeling (Figure 3c).
Northern Blotting
The presence of amylase mRNA in rat pancreas under fed, 5-min cholinergic stimulation, and starvation conditions was evaluated by Northern blotting and is reported in Figure 6. Only one band was recovered at 1.5 kb, corresponding to the size of the amylase mRNA (MacDonald et al. 1980
). The levels of pancreatic amylase mRNA decreased drastically after 5 min of cholinergic stimulation. Those after starvation were also found to be lower than those in control tissues.
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Discussion |
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Two different techniques were applied to reveal and quantitate RNA and mRNA molecules in pancreatic acinar cells under experimental conditions: RNasegold, which reveals a wide range of RNA molecules, and ISH for more specific detection of a particular mRNA. Indeed, the ISH technique has allowed the detection of specific nucleic acid chains, in our case the amylase mRNA. Once performed at the electron microscopic level, it revealed the precise ultrastructural localization and allowed quantitation of amylase mRNA molecules in cellular compartments. The high-resolution signal was assigned to the RER and nuclei of the acinar cells, which substantiates results obtained by light microscopy. Under experimental conditions, the pattern of labeling remained the same although the intensity of labeling varied from one condition to the other. After stimulation of secretion, the labeling for mRNA was drastically reduced, particularly at the level of the RER. Similarly, prolonged fasting also led to reduced levels of labeling. Control protocols, particularly those using RNase treatment, consolidate our results demonstrating good specificity. Furthermore, the use of an antisense DIG riboprobe, synthesized from a plasmid containing a portion of the rat pancreatic amylase cDNA and amplified by PCR (generously supplied by Dr Richard Blouin from the Département de Biologie, Université de Sherbrooke), yielded the same pattern of labeling, confirming the validity of the results obtained with our DIG-tagged probe.
On the other hand, we used the RNase Agold complex for the ultrastructural detection of RNA molecules in a postembedding approach, which allows the preferential detection of pyrimidine bases in different RNA molecules such as mRNA, tRNA, and rRNA (Cheniclet and Bendayan 1990). The fact that RNase Agold displays affinity for a wide array of RNA molecules renders its application less specific than that of ISH. However, the results reflect more widespread changes occurring at translational and transcriptional sites. The results obtained with RNase Agold paralleled closely those generated by ISH, with major reductions of cytoplasmic labeling under stimulation of secretion and fasting conditions. The increase in RNA labeling found at the level of the nuclei under starvation conditions differs from the ISH results and must reflect increases in transcriptional activities for proteins other than amylase, triggered by such a stress condition.
It is well established that the Northern blotting technique is more sensitive than ISH for the detection of specific mRNA molecules. The results obtained by Northern blotting confirmed those generated by ISH, with significant decreases in amylase mRNA levels after carbachol stimulation of secretion and under fasting conditions. However, the evident drawback of such an approach is the lack of ability to assign particular cell types and cell compartments to the molecules revealed.
The results obtained after performing ISH agree in demonstrating very low levels of cytoplasmic amylase mRNA after cholinergic stimulation. Nuclei, on the other hand, still retained some labeling. Lowest cytoplasmic levels were detected 5 min after stimulation and recovery took place gradually over time. This was well demonstrated by the quantitative evaluations and is in line with some previous studies demonstrating that stimulation of pancreatic secretion by secretagogues generates a decrease in protein synthesis (Mongeau et al. 1976; Lahaie 1986
; Perkins et al. 1991
; Yuan et al. 1999
). This decline in pancreatic enzyme synthesis appears to be caused by inhibition of translational and posttranscriptional activities (Wicker et al. 1985
; Perkins et al. 1991
,1997
; Perkins and Pandol 1992
; Sans and Williams 2002
,2004
; Sans et al. 2003
). However, some of these studies reported no major changes in mRNA levels (Wicker et al. 1985
; Perkins et al. 1991
; Perkins and Pandol 1992
), which might be explained by the fact that measurements of enzyme mRNA were carried out more than 30 min after the initial stimulation of secretion, at a time point where we already observed a recovery of amylase mRNA. Moreover, most of the studies dealing with secretagogues to evaluate pancreatic enzyme synthesis and secretion have used either supramaximal infusion or several consecutive injections of secretagogues, leading to complex modifications in intracellular signaling pathways. In contrast, we used a single injection of carbachol at physiological doses that led to the simultaneous increase in protein secretion and decrease in synthesis. Because stimulation of intracellular protein kinase signaling pathways requires multiple injections or infusion of secretagogues (Duan and Williams 1994
; Duan et al. 1995
; Dabrowski et al. 1996
; Turner et al. 2001
; Williams 2001
; Williams et al. 2002
), we can assume that after our single injection of carbachol the MAPK pathways were not stimulated and that the PI3K-PKB-mTOR pathway might have been inhibited due to the decrease in cytoplasmic RNA (Sans and Williams 2002
). It was demonstrated that inhibition of protein synthesis after stimulation of secretion is due not to a lack of amino acids but rather to a decrease in translational activity (Mongeau et al. 1976
), which is now explained by the demonstration of a drastic decrease in mRNA at the RER level.
Upon fasting of the animals a weak decrease in amylase mRNA was registered at the RER and nuclear levels. These results confirmed previous studies demonstrating that food deprivation or change in diet composition leads to a decrease in protein synthesis (Black and Webster 1973; Giorgi et al. 1984
; Iovanna et al. 1991
). According to RNasegold results, RER RNA decreases, whereas that in nuclei increases, reflecting stimulation in transcriptional activities of some specific proteins due to the condition of acute stress.
In conclusion, our study has demonstrated the strengths and advantages of performing ISH at the electron microscopic level with the possibility of quantitatively analyzing levels of labeling in different specific cell compartments and of comparative evaluations. By combining different morphological techniques and Northern blotting, we have shown that stimulation of secretion, as well as fasting conditions, induces drastic decreases in RER RNA and in amylase mRNA.
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Footnotes |
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Literature Cited |
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