![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Department of Nutritional Sciences, University of Wisconsin, Madison, WI 53706
3To whom correspondence should be addressed. E-mail: groby{at}nutrisci.wisc.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
|---|
KEY WORDS: • exocrine pancreas • phosphorylation • CRHSP-28 • secretion • Ca2+-signaling
The exocrine pancreas is responsible for the synthesis, storage and regulated secretion of digestive enzymes that are essential for macronutrient digestion in the small intestine. As with all digestive functions, pancreatic secretion is most highly stimulated after ingestion of a meal. Indeed, the presence of macronutrients within the small intestine evokes the release of gastrointestinal hormones from enteroendocrine cells and stimulates parasympathetic neural reflexes that directly activate acinar and duct cells within the pancreas [reviewed in (1)]. Digestive enzymes are secreted into the pancreatic ductal system where they are delivered to the duodenum.
To date, a majority of our understanding of acinar cell biology has come from studies utilizing acutely isolated cultures of acini, which have a robust secretory response to neurotransmitter and hormone stimulation. Using the acinar cell model, we previously isolated and characterized a Ca2+-responsive heat stable protein (CRHSP)-283 (2), which is a member of the TPD52 protein family (3). Introduction of recombinant CRHSP-28 into permeabilized acinar cells reconstituted Ca2+-stimulated digestive enzyme secretion, establishing an integral role for the protein in the exocytotic pathway (4). Supporting this, CRHSP-28 and related TPD52 molecules were shown to interact with annexin VI (5) and MAL2 (6) and various SNARE proteins (7), all of which are important for membrane trafficking and fusion events in cells.
In acini, CRHSP-28 undergoes a rapid increase in serine phosphorylation after stimulation with high, supraphysiologic concentrations of cholecystokinin-octapeptide (CCK-8) (2). The present study expands these findings by demonstrating that CRHSP-28 phosphorylation occurs in vivo after infusion of a secretory dose of caerulein, a CCK analog, and also immediately after ingestion of meal. This diet-induced post-translational modification of CRHSP-28 may be used as a molecular indicator of acinar cell Ca2+ signaling in vivo; moreover, it underscores the physiologic relevance of CRHSP-28 in modulating pancreatic function.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
|---|
CCK-8, JMV-180 and caerulein were purchased from Research Plus (Bayonne, NJ); 12-o-tetradecanoylphorbol-13-acetate (TPA), 8-(4-chlorophenylthio)-3',5'-cyclic adenosine monophosphate (CPT-cAMP), 8-bromo-cyclic guanosine monophosphate (cGMP) and all other chemicals were obtained from Sigma Chemical (St. Louis, MO). The AIN-93M diet was purchased from Harlan Teklad (Madison, WI). Camostat was kindly provided by ONO Pharmaceutical (Osaka, Japan). The anti-CRHSP-28 polyclonal antibodies were characterized previously (8).
Preparation and treatment of acinar cells.
Pancreatic acinar cells were isolated from adult, male Sprague-Dawley rats by collagenase digestion as previously described (9). Acini were suspended in a buffer consisting of (mmol/L) 10 HEPES, 137 NaCl, 4.7 KCl, 0.56 MgCl2, 1.28 CaCl2, 0.6 Na2HPO4, 5.5 D-glucose, 2 L-glutamine and an essential amino acid solution. The buffer was supplemented with 0.1 g/L soybean trypsin inhibitor, 1 g/L bovine serum albumin, gassed with 100% O2 and adjusted to pH 7.4. Acini were incubated at 37°C for 1 h before initiating experiments. After the indicated treatments, cells were sonicated in isoelectric focusing (IEF) buffer containing 9 mol/L urea, 0.4 mL/L Nonidet P40, 0.1mL/L 2-mercaptoethanol, and 0.2mL/L ampholytes [isoelectric point (pI) range 3–10] and protein concentrations were determined using BioRad reagent (Hercules, CA).
Isoelectric focusing and immunoblotting.
IEF was conducted in slab gels composed of 65 g/L acrylamide, 2 g/L N,N'methylene-bis-acrylamide, 1 mL/L glycerol, 8 mol/L urea and ampholytes (pI 2.5–5 and 3–10). Samples were resolved using a Pharmacia Biotech Multiphor II apparatus and proteins were transferred to nitrocellulose. Immunoblotting was conducted with anti-human CRHSP-28 antibodies (1 µg/mL), and detected by enhanced chemiluminescence using a horseradish peroxidase-conjugated donkey anti-rabbit IgG secondary antibody (1:5000). The intensity of the CRHSP-28 phospho-isoforms on the immunoblots was quantified by densitometric analysis using a PDI model DNA35 scanner interfaced with the Protein and DNA Imageware System (Huntington Station, NY).
Stimulation of exocrine pancreas in vivo.
The University of Wisconsin Committee on Use and Care of Animals approved all studies involving animals. Rats were acclimated for 3 d and consumed the AIN-93M diet ad libitum before the initiation of the feeding studies. For hormone stimulation, 250- to 300-g male Sprague-Dawley rats were deprived of food over night and anesthetized with 2% isoflurane in 100% oxygen. A catheter was placed in the jugular vein for infusion of either 9 g/L NaCl as a control, a bolus secretory dose of caerulein equivalent to 0.1 µg/(kg body · h) or a supramaximal dose of caerulein equivalent to 10 µg/(kg body · h) (10). At 15 min postinfusion, pancreatic tissue was quickly removed and homogenized in ice-cold IEF buffer. For feeding studies, rats were maintained on a 12-h light/dark cycle. After a 24-h food deprivation, rats were fed at the initiation of the dark cycle (1800 h) 15 g of a test diet composed of AIN-93 (11), a ratio of 40 g casein:60 g AIN-93, or AIN-93 supplemented with 0.1% camostat, a synthetic trypsin inhibitor peptide. All meals contained 0.1% vanilla extract. At 15 min postingestion, rats were anesthetized with CO2, killed by decapitation and the pancreas was quickly removed and processed for IEF. Each hormone infusion or feeding experiment was performed 3 separate times and included rats from each treatment group. The statistics are based on a total of 9 rats (n = 3 rats/treatment group) in both the infusion and feeding studies. There was 100% survival of all rats during treatment. All phosphorylation determinations were conducted in duplicate.
Statistics.
The quantified intensity of the CRHSP-28 phospho-isoforms was expressed as a percentage of total CRHSP-28 present in each sample. Data were arcsin-transformed to account for unequal variance and significant difference from control values was established at P < 0.05 as determined using an unpaired Student’s t test. Data are presented as means ± SEM.
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
|---|
CRHSP-28 phosphorylation in 32P-labeled acinar cells was previously shown to occur in response to nanomolar concentrations of CCK-8 (2,8), which are considered to represent supraphysiologic levels of the hormone in vivo. To determine whether CRHSP-28 phosphorylation occurs in response to physiologic levels of CCK-8, isoelectric focusing and immunoblotting were used to analyze its phosphorylation after treatment of isolated acini with various concentrations of the hormone (Fig. 1). This technique is based on our previous finding (2,8) that CRHSP-28 exists as three phospho-isoforms after isoelectric focusing in 32P-labeled cells, consisting of a basic nonphosphorylated isoform (
-isoform) and 2 more acidic isoforms (ß- and 
-isoforms), each of which is radiolabeled on serine residues (2). Thus, the CCK-8-induced acidic migration of CRHSP-28 seen in isoelectric focusing gels may be used as a direct measure of its phosphorylation in intact cells (2,8).
|
-isoform, and to lesser extent, in the phosphorylated ß-isoform (Fig 1). Treatment of cells with increasing concentrations of CCK-8 for 2 min evoked a marked acidic shift in CRHSP-28 to the most highly phosphorylated -isoform, indicative of an increase in its phosphorylation. Indeed, CCK-8 treatment decreased the intensity of the -isoform with a reciprocal increase in the intensity of the most highly phosphorylated -isoform. Minimal phosphorylation was detected at 10 pmol/L and a maximal effect at 300 pmol/L CCK-8, with a 50% effective concentration (EC50) of 
30 pmol/L. Consistent with a previous study examining the time course of CRHSP-28 phosphorylation in 32P-labeled acini (2), CCK-8–induced CRHSP-28 phosphorylation was maximal 2 min after treatment with CCK and then returned to basal levels over 30 min (data not shown). Rat pancreatic acinar cells express the A-form of the CCK receptor (CCK-A), which possesses at least two affinity states (12). The high affinity state of the CCK-A receptor is thought to mediate the physiologic effects of CCK and responds to picomolar levels of the hormone, whereas the low affinity state responds to nanomolar levels of CCK (12). That the EC50 for CCK-8-dependent CRHSP-28 phosphorylation was in the low picomolar range suggested that the hormone was acting through the high affinity binding site of the receptor. To confirm these findings, the synthetic peptide JMV-180, which is a high affinity agonist and low affinity antagonist of the CCK-A receptor (12,13), was utilized (Fig. 2). Treatment of acini with high concentrations of JMV-180 to activate the high affinity state of the receptor strongly stimulated CRHSP-28 phosphorylation. Conversely, pretreatment of cells with JMV-180 to block the low affinity sites before treatment with 10 nmol/L CCK-8 did not inhibit the stimulatory effects of CCK-8. Collectively, these data indicate that CRHSP-28 phosphorylation in acinar cells occurs in response to physiologic stimulation of the high affinity state of the CCK-A receptor.
|
To identify the intracellular signaling mechanism that regulates CRHSP-28 phosphorylation in acini, various pharmacologic agents that by-pass receptor occupation and induce cellular signaling pathways were utilized (Fig. 3). Treatment of acini with CPT-cAMP to activate cAMP-mediated regulatory proteins, 8-bromo-cGMP to activate protein kinase G or the phorbol ester TPA to activate protein kinase C had no effect on CRHSP-28 phosphorylation. Conversely, treatment of acini with the Ca2+ ionophore ionomycin markedly stimulated CRHSP-28 phosphorylation to levels consistent with those seen after CCK-8 treatment. Additionally, CCK-8-stimulated CRHSP-28 phosphorylation was completely inhibited in cells that were pretreated with the intracellular Ca2+-buffer BAPTA-AM (data not shown), further confirming the Ca2+-dependnecy of CRHSP-28 regulation. Thus, in agreement with the Ca2+-dependent regulatory effects of CRHSP-28 on acinar cell digestive enzyme secretion (4), these data demonstrate that the acute phosphorylation of the protein is also dependent on a rise in free cellular Ca2+.
|
Utilization of isoelectric focusing and immunoblotting abolished the need for radioisotopes to study CRHSP-28 phosphorylation, therefore allowing us to investigate its regulation in vivo (Fig. 4). Isoflurane-anesthetized rats were given intravenous infusions of caerulein, a CCK analog with an extended half-life. Phosphorylation of CRHSP-28 in the basal state was similar to that seen under basal conditions in isolated acini. Infusion of rats with secretory or supraphysiologic concentrations of caerulein increased CRHSP-28 phosphorylation by >100% over controls within 15 min. These data both confirmed and expanded our results in isolated acini, demonstrating that hormone activation of the pancreas leads to the acute regulation of CRHSP-28 phosphorylation.
|
6–8 g of semipurified AIN-93M diet induced CRHSP-28 phosphorylation by >150% over basal levels. Phosphorylation was maximal at 15 min postfeeding, with modest effects seen at 30 min (data not shown). Investigation of intestinal contents revealed the diet was clearly present within the proximal small bowel at 15 min, indicating the intestinal phase of digestion had begun.
|
In conclusion, CRHSP-28, a member of the TPD52 protein family (3), was shown to interact with a number of important regulatory molecules governing membrane trafficking and fusion events in epithelia and endocrine cells (5–7). Consistent with a regulatory role in apical membrane trafficking, CRHSP-28 is highly expressed in the apical cytoplasm of epithelial cells throughout the digestive system (8). Because membrane trafficking events dictate the protein composition of the brush boarder in epithelia, this process is responsible not only for the regulated secretion of proteins in acinar cells but also the insertion of nutrient transporters, digestive enzymes and ion channels orientated toward the intestinal lumen in enterocytes. The acute effect of CRHSP-28 on digestive enzyme secretion from acini was shown previously to be dependent on increased intracellular Ca2+ (4). Supporting this, the present data establish that CRHSP-28 phosphorylation is also modulated by elevated intracellular Ca2+. Moreover, these data show that phosphorylation is directly coupled to hormonal stimulation and the consumption of a meal. These findings underscore the physiologic importance of CRHSP-28 function in digestive epithelia and provide a method by which the acute effects of nutrient ingestion may be analyzed at the cellular level.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 G.E.G. was supported by a National Science Foundation Award (MCB-0094154) and a USDA Cooperative State Research Education and Extension Service (CSREES) Program award (WISO4221 and WIS04444)
4 Abbreviations used: CCK, cholecystokinin; CCK-8, cholecystokinin-octapeptide; cGMP, 8-bromo-cyclic guanosine monophosphate; CPT-cAMP, 8-(4-chlorophenylthio)-3',5'-cAMP; CRHSP, calcium-regulated heat stable protein; EC50, 50% effective concentration; IEF, isoelectric focusing; pI, isoelectric point; TPA, 12-o-tetradecanoylphorbol-13-acetate.
Manuscript received 2 June 2003. Initial review completed 17 July 2003. Revision accepted 29 July 2003.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
|---|
1. Owyang, C. (1994) Neurohormonal control of the exocrine pancreas. Curr. Opin. Gastroenterol. 10:491-495.
2. Groblewski, G. E., Wishart, M. J., Yoshida, M. & Williams, J. A. (1996) Purification and identification of a novel calcium-regulated heat-stable protein. J. Biol. Chem. 271:31502-31507.
3. Byrne, J. A., Mattei, M. G. & Basset, P. (1996) Definition of the tumor protein D52 (TPD52) gene family through cloning of D52 homologues in human (hD53) and mouse (mD52). Genomics 35:523-532.[Medline]
4. Thomas, D. H., Taft, W. B., Kaspar, K. M. & Groblewski, G. E. (2001) CRHSP-28 regulates Ca2+-stimulated secretion in permeabilized acinar cells. J. Biol. Chem. 276:28866-28872.
5. Thomas, D. H., Kaspar, K. M., Taft, W. B., Weng, N. & Groblewski, G. E. (2002) Identification of annexin VI as a CRHSP-28 binding protein in pancreatic acinar cells. J. Biol. Chem. 277:39456-39502.
6. Wilson, S. H., Bailey, A. M., Nourse, C. R., Mattei, M. G. & Byrne, J. A. (2001) Identification of MAL2, a novel member of the mal proteolipid family, through interactions with TPD52-like proteins in the yeast two-hybrid system. Genomics 76:81-88.[Medline]
7. Proux-Gillardeaux, V., Galli, T., Callebaut, I., Mikhailik, A., Calothy, G. & Marx, M. (2003) D53 is a novel endosomal SNARE-binding protein that enhances interaction of Syntaxin 1 with the Synaptobrevin 2 complex in vitro. Biochem. J. 370:213-221.[Medline]
8. Groblewski, G. E., Yoshida, M., Hongren, Y., Williams, J. A. & Ernst, S. A. (1999) Immunolocalization of CRHSP28 in exocrine digestive glands and gastrointestinal tissues of the rat. Am. J. Physiol. 276:G219-G226.
9. Wishart, M. J., Groblewski, G. E., Goke, B. J., Wagner, A.C.C. & Williams, J. A. (1994) Secretagogue regulation of pancreatic acinar cell protein phosphorylation shown by two-dimensional electrophoresis. Am. J. Physiol. 267:G676-G686.
10. Groblewski, G. E., Grady, T., Mehta, N., Lambert, H., Logsdon, C. D., Landry, J. & Williams, J. A. (1997) Cholecystokinin stimulates heat shock protein-27 phosphorylation in rat pancreas both in vivo and in vitro. Gastroenterology 112:1354-1361.[Medline]
11. American Institute of Nutrition (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123:1939-1951.
12. Williams, J. A. & Blevins, G. T. (1993) Cholecystokinin and regulation of pancreatic acinar cell function. Physiol. Rev. 73:701-723.
13. Groblewski, G. E., Wang, Y., Ernst, S. A., Kent, C. & Williams, J. A. (1995) Cholecystokinin stimulates the down-regulation of CTP-phosphocholine cytidylytransferase in pancreatic acinar cells. J. Biol. Chem. 270:1437-1442.
14. Guan, D. & Green, G. M. (1996) Significance of peptic digestion in rat pancreatic secretory response to dietary protein. Am. J. Physiol. 271:G42-G47.[Medline]
15. Reeve, J. R., Jr., Green, G. M., Chew, P., Eysselein, V. E. & Keire, D. A. (2003) CCK-58 is the only detectable endocrine form of cholecystokinin in rat. Am. J. Physiol. 285:G255-G265.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
