We undertook studies to determine whether secretagogue action on the exocrine pancreas and parotid is accompanied by phosphorylation of proteins in intact cells. For this purpose, rat pancreatic, and parotid Iobules were preincubated with 32p~ for 45 min at 37°C, washed, and then incubated at 37°C in the presence or absence of secretagogues that effect discharge through different second messengers. Among a variety of polypeptides exhibiting enhanced phosphorylation in pancreatic Iobules upon a 30-s incubation in the presence of the secretagogues carbamylcholine, cholecystokinin octapeptide, or secretin, one species with an Mr of 29,000 was especially notable for three reasons: (a) its enhanced level of phosphorylation was dependent on the dose of secretagogue used and was still apparent after incubation for 30 min at 37°C; (b) an analogous phosphorylated polypeptide was observed in isoproterenolstimulated parotid Iobules; and (c) in both tissues its selective dephosphorylation was observed upon termination of stimulation by administration of atropine to carbamylcholine-stimulated pancreatic Iobules and propranolol to isoproterenol-stimulated parotid Iobules. These results suggest that the phosphorylation of one protein with an Mr of 29,000 is closely correlated both temporally and in a dose-dependent fashion with secretagogue action in both the exocrine pancreas and parotid.The mechanism whereby secretagogues are able to elicit a characteristic biological response in their respective target ceils is unclear at the moment. There is now increasing evidence that protein phosphorylation is involved in mediating the effects of a variety of the actions of hormones in many diverse enzymological and physiological processes (1). To gain further insight into the mechanism of secretagogue action in exocrine glands, we examined the relationship between protein phosphorylation and hormone action in the intact cell to determine whether this covalent modification mediates or modulates any of the biological actions of secretagogues.The acinar cells of the exocrine pancreas and parotid offer good systems for studying secretagogue effects on endogenous protein phosphorylation since homogeneous cell preparations can be prepared, several different secretagogues exist which elicit discharge through different intracellular messengers, and the effects of these hormones on calcium and cyclic nucleotide levels during secretion are well characterized (2, 3, 4). Since cAMP, cGMP, and Ca 2+ have been implicated in the secretion of exportable proteins from the exocrine pancreas, protein kinases are plausible targets for these putative secretagogue mediators and, in fact, both cAMP and cGMP-dependent protein kinases have been partially purified from homogenates of rat pancreas (5, 6).We examined the relationship between endogenous protein phosphorylation and secretagogue action in situ in gland lobules under physiological conditions using the rat exocrine pancreas and parotid as model systems. A preliminary note on this research has...

In the preceding paper, we demonstrated that the endogenous phosphorylation of a protein with a molecular weight of 29,000 was enhanced by various secretagogues in rat pancreatic and parotid Iobules, the phosphorylation of this protein correlating both temporally and in a dose-dependent fashion with secretory protein discharge. In the present study, we established a specific methodology to characterize this phosphoprotein. Once established, this 29,000-dalton phosphoprotein was then followed selectively and quantitatively throughout subcellular fractionation procedures. Analysis of two-dimensional polyacrylamide gels demonstrated that proteins with similar mobilities (Mr 29,000; pl > 8.4) were affected by cholecystokinin octapeptide and isoproterenol in rat pancreatic and parotid Iobules, respectively, suggesting that the same 29,000-dalton phosphoprotein was covalently modified in both tissues. Cellular fractionation studies using differential velocity and sucrose density gradient centrifugation revealed that the 29,000-dalton phosphoprotein copurified with the rough microsomal fraction of pancreas and was highly enriched in ribosomal fractions of both pancreas and parotid. Electrophoresis in two dimensions confirmed that the 29,000-dalton polypeptide that was resolved directly from stimulated cells and from ribosomal fractions exhibited a common mobility, and apparent identity of the species was strongly suggested when the 29,000-dalton polypeptides from both sources were compared by peptide mapping following limited digestion with Staphylococcus aureus V8 protease. This phosphoprotein was tentatively identified as ribosomal protein S6 after analysis by pH 8.6/4.2 two-dimensional PAGE.In the previous paper (l), we reported that the endogenous phosphorylation of a protein with an Mr of 29,000 was markedly stimulated in response to secretagogue application in intact cells from both the rat exocrine pancreas and parotid and was dephosphorylated upon termination of secretagogue effects. To gain additional insight into the role that this phosphoprotein may. play in secretagogue action, we here describe the subcellular localization as well as the characterization of this protein by two-dimensional PAGE and limited proteolytic digestion using Staphylococcus aureus V8 protease. The results indicate that the 29,000-dalton protein whose phosphorylation is stimulated by hormone application in pancreatic and parotid lobules is localized to a highly enriched ribosomal fraction and is likely the small ribosomal subunit protein $6. A preliminary note on this research has been published (2). MATERIALS AND METHODS MaterialsAll chemicals used were of reagent grade and were obtained from the following sources: isoproterenol hydrochloride, deoxycholic acid, and Nonidet P-40 from Sigma Chemical Co. (St. Louis, MO); ampholytes from Bio-Rad Laboratories (Richmond, CA). All other reagents were obtained as described previously (l). MethodsSAMPLE PREPARATION: 32el-labeled rat pancreatic and parotid lobules were prepared and in...

SUMMARY Background Primary sclerosing cholangitis (PSC) is characterised by progressive inflammatory and fibrotic destruction of the biliary ducts. There are no effective medical therapies and presently high dose ursodeoxycholic acid is no longer recommended due to significant adverse events in a recent clinical trial. Cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction is associated with PSC in both children and adults. Since CFTR dysfunction leads to altered fatty acid metabolism, specifically reduced docosahexaenoic acid (DHA), we hypothesised that DHA supplementation might be an effective therapy for patients with PSC. Aim To determine the safety and efficacy of oral DHA supplementation for the treatment of PSC. Methods We conducted a 12 month open-label pilot study to evaluate safety of oral DHA and its effects on serum alkaline phosphatase as a primary outcome measure in 23 patients with PSC. DHA was administered orally at 800 mg twice per day. Secondary outcomes included changes in other liver function tests and fibrosis biomarkers. Results A 1.7-fold increase in serum DHA levels was observed with supplementation. The mean alkaline phosphatase level (±S.E.) at baseline was 357.8 ± 37.1 IU compared to 297.1 ± 23.7 IU (P < 0.05) after 12 months of treatment. There were no changes in other liver function tests and fibrosis biomarkers. No adverse events were reported. Conclusions Oral DHA supplementation is associated with an increase in serum DHA levels and a significant decline in alkaline phosphatase levels in patients with PSC. These data support the need for a rigorous trial of DHA therapy in PSC.

Previous work from our laboratory has demonstrated that neurohumoral stimulation of the exocrine pancreas is associated with the phosphorylation of the Mr 29,000 ribosomal protein $6. In a cell-free system using pancreatic postmicrosomal supernatant as the kinase donor, we found that the following co-factors stimulate the phosphorylation of the Mr 29,000 ribosomal protein: calcium with calmodulin, calcium with phosphatidyl serine, and cAMP. These findings suggest that the pancreas contains a calcium-calmodulin-dependent protein kinase (CaM-PK) that can phosphorylate the Mr 29,000 ribosomal protein. A CaM-PK activity was partially purified sequentially by ion exchange, gel filtration, and calmodulin-affinity chromatography. Phosphorylation of the Mr 29,000 ribosomal protein by the partially purified CaM-PK was dependent on the presence of both calcium and calmodulin and not on the other co-factors. The CaM-PK fraction contained a phosphoprotein of Mr 51,000 whose phosphorylation was also dependent on calcium and calmodulin. When *251-calmodulin-binding proteins from the CaM-PK fraction were identified using electrophoretic transfers of SDS-polyacrylamide gels, a single Mr 51,000 protein was labeled. The preparation enriched in CaM-PK activity contained an Mr 51,000 protein that underwent phosphorylation in a calciumcalmodulin-dependent manner and an Mr 51,000 calmodulin-binding protein. It is therefore possible that the CaM-PK may comprise a calmodulin-binding phosphoprotein component of Mr 51,000.Neurohumoral stimulation of the exocrine pancreas by agents such as carbachol and cholecystokinin results in alterations in protein phosphorylation (1-4) and intracellular calcium levels (5, 6). Recent studies from this laboratory have demonstrated that an Mr 29,000 ribosomal protein which has been putatively identified as $6 in pancreas undergoes phosphorylation in close association with secretagogue action. This phosphorylation is stimulated in situ in 32Pi-labeled pancreatic lobules by carbachol and cholecystokinin and in pancreatic homogenates by calcium and cAMP using [-r-32p]ATP as the phosphate donor (2-4). In this report we describe a calciumcalmodulin-dependent protein kinase (CaM-PK ~) in rat pancreatic postmicrosomal supernatant that phosphorylates the Mr 29,000 ribosomal protein. Partial purification of this CaM-PK activity yielded both an Mr 51,000 calcium-binding protein (CBP) and an Mr 51,000 phosphoprotein which underwent phosphorylation in a calcium-calmodulin-dependent manner. This CaM-PK activity resembled calmodulin-regulated kinases that have recently been characterized in liver Abbreviations used in this paper: CaM-PK, calcium-calmodulindependent protein kinase; CBP, calmodulin-binding protein.using glycogen synthase as substrate (7,8) and in brain using synapsin I (9) and tubulin (10) as substrates. MATERIALS AND METHODSTissue Preparation: Pancreata were obtained from fed, male, Sprague-Dawley rats ( 150-200 g) following decapitation. The pancreata were homogenized in 0.3 M sucrose contai...

In the preceding papers, we demonstrated that the endogenous phosphorylation of a 29,000-dalton protein is stimulated in response to secretagogue application to intact cells from the rat exoorine pancreas and parotid and dephosphorylated upon termination of secretagogue action. One-and two-dimensional gel analysis of a2P~-Iabeled pancreatic and parotid Iobules as well as their respective subcellular fractions revealed that the same protein was covalently modified in both tissues and was localized to the ribosomal fraction.To identify the intracellular second messengers which may mediate or modulate the phosphorylation of the 29,000-dalton protein in intact cells, the effects of Ca 2÷, cAMP, and cGMP on the endogenous phosphorylation of this protein were assessed in subcellular fractions from the rat pancreas and parotid. Our results demonstrate that the phosphorylation of the 29,000-dalton polypeptide may be regulated by both Ca 2+ and cAMP in the pancreas and in the parotid. No cGMP-dependent protein phosphorylation was found in either tissue. As in the in situ phosphorylation studies, the Ca 2÷-and cAMP-dependent phosphorylation of this same protein was localized to the ribosomal fraction. The cAMP-dependent protein kinase activity was found primarily in the postmicrosomal supernatant in contrast to the Ca2+-dependent protein kinase that appeared to be tightly associated with the substrate in addition to being present in the postmicrosomal supernatant. The data suggest that, in cells from the exocrine pancreas and parotid, secretagogues may regulate the phosphorylation of the 29,000-dalton protein through Ca 2+ and/or cAMP.We have demonstrated that the endogenous phosphorylation of a ribosomal protein (Mr 29,000) is reversibly altered in a hormone-dependent fashion in intact pancreatic and parotid acinar cells (1, 2). Since the binding of secretagogues with their receptors in these cells results in the release of intracellular messengers such as Ca 2+ and/or cyclic nucleotides, the effects of these putative second messengers on protein phosphorylation were assessed in homogenates from the parotid and pancreas and in pancreatic subcellular fractions to gain a better understanding of how hormones regulate the phosphorylation of the Mr 29,000 protein. The present experiments demonstrate that the 29,000-dalton protein, which has been identified as ribosomal protein $6, can act as a major substrate for both Ca ~+-and cAMP-dependent protein kinases. A preliminary note on this research has been published (3). MATERIALS AND METHODS MaterialsAll chemicals used were of reagent grade and were obtained from the following sources: adenosine 3':5'-cyclic monophosphate, guanosine 3':5'-cyclic monophosphate, ATP, Tris, and maleic acid were obtained from Sigma Chemical Co. (St. Louis, MO); [),-a2P]ATP (5-10 Ci/mmol, l Ci = 3.7 x l0 ~° Bq) was obtained from New England Nuclear (Boston, MA); all other reagents were obtained as described previously (1). MethodsSAMPLE PREPARATION: Female Sprague-Dawley rats weighing 100-1...