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S D Freedman

Publications and source records attributed to S D Freedman.

At least 19 recordsLinked to original sources
PubMed2001

Pancreatic acinar cell dysfunction in CFTR(-/-) mice is associated with impairments in luminal pH and endocytosis.

BACKGROUND & AIMS: We have previously shown that endocytosis at the apical plasma membrane in pancreatic acinar cells is coupled to ductal bicarbonate secretion into the lumen. We hypothesized that decreased bicarbonate secretion in cystic fibrosis (CF) inhibits apical endocytosis. The aim of this study was to determine in cftr(-/-) mice (1) if the pH of the pancreatic juice is acidic compared with wild-type (WT) controls, (2) if there is a selective block in endocytosis, and (3) if alkalinization of the luminal fluid reverses this defect. METHODS: Fluid secretion and pH of pancreatic juice were measured. Exocytosis, endocytosis, and morphology were compared in pancreatic lobules from cftr(-/-) and WT mice. RESULTS: Pancreatic juice pH was 8.12 +/- 0.06 in WT mice compared with 6.60 +/- 0.04 in cftr(-/-) mice. Although cholecystokinin-stimulated amylase secretion was not significantly different, endocytosis was markedly inhibited in cftr(-/-) compared with WT mice. Cleavage of GP2, a GPI-anchored protein tightly associated with activation of endocytosis, was also decreased. Incubation of lobules from cftr(-/-) mice at pH 8.3 reversed the luminal dilatation. CONCLUSIONS: These data indicate that apical endocytosis is selectively impaired in cftr(-/-) mice, which explains, in part, the luminal dilatation observed at the apical plasma membrane. In vitro alkalinization of luminal fluid led to reversal of defects in membrane dynamics, restored coupled exocytosis and endocytosis, and abolished the luminal dilatation in this animal model of CF. Acidic pH changes in luminal secretions may play a role in the pancreatic membrane dysfunction observed in CF.

Amylases↗
PubMed2000

Advances in nutritional management of chronic pancreatitis.

Nutrition has an important role in the management of chronic pancreatitis (CP), with two main goals for treatment of patients. The first goal is to provide optimal nutrition support and the second is to decrease pain by minimizing stimulation of the exocrine pancreas. Because cholecystokinin (CCK) stimulates secretion from the exocrine pancreas, one approach is to decrease CCK levels through modulation of diet. If postprandial pain is a limiting factor, alternative enteral therapies that minimally stimulate the pancreas may be beneficial. Nutritional counseling, antioxidants, and pancreatic enzymes may play a role in effective management of CP as well. In addition, because idiopathic CP is associated with cystic fibrosis gene mutations, therapies directed toward cystic fibrosis may also benefit these patients.

Chronic Disease↗
PubMed2000

Mechanisms to explain pancreatic dysfunction in cystic fibrosis.

This article focuses on three potential mechanisms by which pancreatic dysfunction occurs in cystic fibrosis. These include (1) obstruction of pancreatic ducts by inspissated plugs, (2) inhibition of endocytosis in acinar cells, and (3) imbalance in membrane lipids in cystic fibrosis regulated cells. Any of these abnormalities alone or in combination may explain the development of pancreatic exocrine insufficiency.

Cystic Fibrosis↗
PubMed2000

GP2, a GPI-anchored protein in the apical plasma membrane of the pancreatic acinar cell, co-immunoprecipitates with src kinases and caveolin.

We previously showed that endocytosis at the apical plasma membrane (APM) of the pancreatic acinar cell is activated by the cleavage of GP2, a GPI-linked protein, from the apical cell surface. This endocytic process, as measured by horseradish peroxidase uptake into pancreatic acinar cells, is blocked by the tyrosine kinase inhibitors genistein and tyrphostin B42 as well as by disruption of actin filaments with cytochalasin. This suggests that the cleavage of GP2 from the cell membrane may activate endocytosis through a tyrosine kinase-regulated pathway. However, the mechanism by which GP2 and tyrosine kinases act together to activate endocytosis at the APM remains unknown. In this study, we demonstrate that pp60, p62yes, caveolin, and annexin, which have previously been implicated in endocytosis in other cell lines, were present in high abundance in GPI-enriched membranes by Western blot analysis. pp60, p62yes, and caveolin all co-immunoprecipitated with GP2 except annexin. An 85-kDa protein whose tyrosine-dependent phosphorylation is correlated with the activation of endocytosis in intact acinar cells also was present in these immunoprecipitates. This suggests that in pancreatic acini, GP2 may exist in a complex with src kinases, caveolin, and an 85-kDa phosphorylated substrate to regulate endocytosis at the APM.

Animals↗
PubMed2000

Fatty acids in cystic fibrosis.

Cystic fibrosis (CF) is associated with deficiencies in certain essential fatty acids. These deficiencies have been studied in plasma, red blood cells, and mucus and were previously thought to be a result of malnutrition or malabsorption. More recent studies have indicated that these deficiencies are independent of nutritional status. However, these studies examined fatty acids in plasma but not in CF-regulated tissues. In the pancreas, lungs, and ileum of CF knock-out mice, membrane-bound arachidonic acid levels have been shown to be increased while docosahexaenoic acid levels are decreased. This lipid abnormality is reversed following oral administration of docosahexaenoic acid (DHA). In addition, DHA therapy reverses the increased neutrophil infiltration in the lungs of CF knock-out mice. Further studies are required to determine the mechanism by which CF gene mutations lead to this lipid abnormality.

Animals↗
PubMed1999

A membrane lipid imbalance plays a role in the phenotypic expression of cystic fibrosis in cftr(-/-) mice.

A deficiency in essential fatty acid metabolism has been reported in plasma from patients with cystic fibrosis (CF). However, its etiology and role in the expression of disease is unknown. The objective of this study was to determine whether alterations in fatty acid metabolism are specific to CF-regulated organs and whether they play a role in the expression of disease. A membrane lipid imbalance was found in ileum, pancreas, and lung from cftr(-/-) mice characterized by an increase in phospholipid-bound arachidonic acid and a decrease in phospholipid-bound docosahexaenoic acid (DHA). This lipid imbalance was observed in organs pathologically affected by CF including lung, pancreas, and ileum and was not secondary to impaired intestinal absorption or hepatic biosynthesis of DHA. As proof of concept, oral administration of DHA to cftr(-/-) mice corrected this lipid imbalance and reversed the observed pathological manifestations. These results strongly suggest that certain phenotypic manifestations of CF may result from remediable alterations in phospholipid-bound arachidonic acid and DHA levels.

Administration, Oral↗
PubMed1999

Endocytosis at the apical plasma membrane of pancreatic acinar cells is regulated by tyrosine kinases.

We have shown that endocytosis at the apical plasma membrane of pancreatic acinar cells is regulated by the pH of the acinar lumen and is associated with cleavage of GP2, a glycosyl phosphatidylinositol-anchored protein. The aim of this study was to determine the transduction pathway by which endocytosis is activated. Apical endocytosis was studied in rat pancreatic acini by prestimulation with cholecystokinin followed by measurement of horseradish peroxidase (HRP) uptake. Lanthanum, staurosporine, and forskolin had no effect on HRP uptake. Cytochalasin D significantly inhibited endocytosis, indicating a dependence on actin filament integrity. Genistein and the specific tyrphostin inhibitor B42 also inhibited HRP uptake, implicating tyrosine kinases in the regulation of HRP uptake. With the use of an Src kinase-specific substrate, Src kinase activity was temporally related to activation of endocytosis. The tyrosine-dependent phosphorylation of an 85-kDa substrate in both rat and mouse pancreatic acini correlated with Src kinase activation and pH-dependent regulation of HRP uptake. These results indicate that apical endocytosis in acinar cells is associated with tyrosine kinase activation and is dependent on the actin cytoskeleton.

Animals↗
PubMed1998

Acinar lumen pH regulates endocytosis, but not exocytosis, at the apical plasma membrane of pancreatic acinar cells.

A two-step exocytosis/endocytosis protocol was used in rat pancreatic acini to study membrane trafficking events at the apical plasma membrane (APM) as a function of extracellular pH. Exocytosis, as measured by cholecystokinin (CCK)-8-induced release of amylase into the incubation medium, was relatively insensitive to changes in extracellular pH from 5.5 to 9.0. In contrast, endocytosis, as measured by temperature-dependent uptake of horseradish peroxidase (HRP), was robust at pH values between 6.5 and 8.3 but abolished at acidic pH values of 5.5 to 6.0. Energy metabolism and cell viability were maintained during pH 6-induced cessation of HRP uptake, and the vesicular block could be reversed upon raising the luminal pH to 7.4. Histochemical and morphometric studies of HRP uptake examined by electron microscopy indicated that extracellular pH regulates endocytosis at the apical plasma membrane. At pH 6.0 in prestimulated cells, HRP uptake at the APM was abolished, and acinar lumen membranes remained markedly dilated with decreased density of microvilli and "arrested" exocytic images. At pH 7.4, HRP was taken up into endolysosomal structures within the Golgi complex, and acinar lumen membranes were contracted. Cleavage of GP2, a glycosyl phosphatidylinositol-anchored protein, was associated with the pH-dependent activation of HRP uptake. These studies demonstrate that acinar lumen pH regulates endocytic but not exocytic activity at the APM and suggest that alkalinization of the acinar lumen by duct cells is required for retrieval of exocytic membranes into the acinar cell via vesicular uptake mechanisms. The role of acid-base interactions within the acinar lumen provides a novel basis for understanding the cellular and luminal defects observed within the exocrine pancreas in cystic fibrosis.

Animals↗
PubMed1998

Cleavage of GPI-anchored proteins from the plasma membrane activates apical endocytosis in pancreatic acinar cells.

Using rat pancreatic acini, we have recently shown that apical endocytosis is inhibited at pH 6.0 and progressively activated as the pH is increased to 8.3. Endocytotic activity correlated with the release of GP2, a GPI-linked protein, from the apical plasma membrane. To determine whether the cleavage of GPI-anchored proteins from the membrane of rat acinar cells was responsible for activation of endocytosis, cells at pH 6.0 were incubated with PI-specific phospholipase C (PI-PLC). PI-PLC treatment reversed the inhibition of endocytosis observed at pH 6.0. Reactivation of endocytosis correlated with PI-PLC-induced release of GP2 but not cleavage of phospholipids in cellular membranes. Furthermore, administration of diacylglycerol or phorbol esters had no effect on reactivation of endocytosis. PI-PLC did not alter intracellular pH or calcium levels. Two proteins were identified as GPI-linked proteins on the cell surface. One was GP2, whose release from the apical plasma membrane correlated with apical endocytosis of horseradish peroxidase (HRP). The other protein, identified by Western blotting using an antibody directed against a cryptic determinant exposed on GPI-linked proteins after cleavage with PI-PLC, has a molecular weight of 98000 in nonreducing SDS gels and 54000 in reducing SDS gels. By nondenaturing gel electrophoresis and staining with naphthylphosphate, this protein was found to be alkaline phosphatase. In contrast to GP2, alkaline phosphatase was not endogenously released at pH values of 7.4 or 8.3, conditions that activate endocytosis of HRP under physiological conditions. By electron microscopic evaluation, incubation of cells at pH 6.0 with PI-PLC led to induction of HRP uptake into vesicles at the apical pole of the cell, a reduction in apical plasma membranes, and a concomitant contraction of the acinar lumen space. Internalized HRP accumulated in the Golgi region of the cell. These results suggest that the cleavage of GPI-anchored proteins from the apical plasma membrane activates apical endocytosis.

Alkaline Phosphatase↗
PubMed1998

Chronic ethanol administration selectively impairs endocytosis in the rat exocrine pancreas.

Release of GP2, a glycosyl phosphatidylinositol-linked protein on the apical plasma membrane of the pancreatic acinar cell, is associated with activation of endocytosis. Released GP2 is also an integral component of intraductal plugs in patients with alcohol-induced chronic pancreatitis. Our purpose was to determine the effect of ethanol on exocytosis and endocytosis and its association with release of membrane-bound GP2. Rats were fed Lieber-DeCarli diets with and without ethanol for 2 weeks. Endocytosis was then assessed in acini by measuring horseradish peroxidase (HRP) uptake, GP2 release by Western blotting, and exocytosis by measuring amylase release. In ethanol-fed rats, HRP uptake was inhibited by 90% compared to that in control rats. In contrast, no significant difference in cholecystokinin-stimulated amylase secretion was found. In vitro, ethanol inhibited HRP uptake in a dose-dependent manner, with 50% inhibition at 50 mM ethanol. Despite the inhibition of endocytosis, GP2 release increased linearly over 60 min and was significantly higher from acini incubated with ethanol compared to controls. These data indicate that ethanol selectively inhibits endocytosis in pancreatic acinar cells. The release of GP2 into the pancreatic duct was no longer coupled to endocytosis in animals fed ethanol.

Amylases↗
PubMed1996

Pancreatic dysfunction in cystic fibrosis occurs as a result of impairments in luminal pH, apical trafficking of zymogen granule membranes, and solubilization of secretory enzymes.

Recent progress in understanding the luminal biochemistry of regulated pancreatic exocrine secretion, including acid-base interactions between acinar and duct cells and pH-dependent processes that regulate membrane trafficking (endocytosis) at the apical plasma membrane, have led to the development of in vitro models of cystic fibrosis in the rat exocrine pancreas. Based on investigations in these model systems, a unifying hypothesis is presented that proposes that pancreatic dysfunction in cystic fibrosis occurs as a result of progressive acidification of the acinar and duct lumen, which leads to secondary defects in (i) apical trafficking of zymogen granule membranes and (ii) solubilization of secretory (pro)enzymes. By directly acidifying the pH of the acinar lumen in cholescystokinin-stimulated acini, the early cytological findings observed in cystic fibrosis, including (i) massive dilatation of the acinar lumen, (ii) decreased appearance of zymogen granules, (iii) loss of the apical pole of the acinar cell, and (iv) persistent aggregation of secretory (pro)enzymes released into the luminal space, have been reproduced in primary cultures of pancreatic tissue.

Animals↗
PubMed1994

Acid-base interactions during exocrine pancreatic secretion. Primary role for ductal bicarbonate in acinar lumen function.

The role of acid-base interactions during coordinated acinar and duct cell secretion in the exocrine pancreas is described. The sequence of acid-base events may be summarized as follows: (1) Sorting of secretory proteins and membrane components into the regulated secretory pathway of pancreatic acinar cells is triggered by acid- and calcium-induced aggregation and association mechanisms located in the trans-Golgi network. (2) Cholecystokinin-stimulated exocytosis in acinar cells releases the acidic contents of secretory granules into the acinar lumen. (3) Secretin-stimulated bicarbonate secretion from duct and duct-like cells neutralizes the acidic pH of exocytic contents, which leads to dissociation of protein aggregates and solubilization of (pro)enzymes within the acinar lumen. (4) Stimulated fluid secretion transports solubilized enzymes through the ductal system. (5) Further alkalinization of acinar lumen pH accelerates the enzymatic cleavage of the glycosyl phosphatidyl-inositol anchor associated with GP2 and thus releases the GP2/proteoglycan matrix from lumenal membranes, a process that appears to be required for vesicular retrieval of granule membranes from the apical plasma membrane and their reuse in the secretory process. We conclude that the central function of bicarbonate secretion by centroacinar and duct cells in the pancreas is to neutralize and then alkalinize the pH of the acinar lumen, sequential process that are required for (a) solubilization of secreted proteins and (b) cellular retrieval of granule membranes, respectively.

Acid-Base Equilibrium↗
PubMed1994

Role of the GP2/THP family of GPI-anchored proteins in membrane trafficking during regulated exocrine secretion.

Identification and characterization of the GP2/THP family of GPI-anchored membrane proteins associated with apical secretory membranes suggest that this new class of GPI-linked proteins plays a critical role in regulated protein secretion and ion transport in polarized epithelial cells in pancreas, liver, lung, kidney, and gastrointestinal tract. Based on recent information obtained from the world literature and from our own investigations we present the following two hypotheses capable of unifying previously diverse observations. Hypothesis 1 is that formation of GP2 tetramers in the acidic milieu of the trans-Golgi network (TGN) organizes a GP2/proteoglycan (PG) matrix tightly associated with the luminal surface of zymogen granule (ZG) membranes, and proposes that this matrix functions in (a) membrane sorting during granule assembly in the TGN, (b) inactivation of ZG membranes during the storage phase of secretion, and (c) regulated trafficking of ZG membranes from the apical plasma membrane (APM) after exocytosis. Hypothesis 2 is that the acinar lumen constitutes a distinct physiologic compartment for coupled biochemical reactions between acinar and duct cells. Because the acidic pH of the TGN plays a critical role in condensation of secretory proteins, alkalinization of the acinar lumen is required for (a) neutralization of the acidic pH of exocytic contents and (b) solubilization of aggregated (pro)enzymes. Further alkalinization appears to be required for pH-dependent release of the GP2/PG matrix from the APM, a process that may regulate internalization of ZG membranes for reuse during secretion. Taken together, the two hypotheses suggest that luminal factors including acid-base interactions and matrix assembly and disassembly processes perform critical functions during regulated storage and release of pancreatic (pro)enzymes. The requirement that coupling reactions be coordinated through the actions of separate hormones [cholecystokinin (CCK) and secretin] on divergent epithelial cells (acinar and duct cells, respectively) provides a new appreciation for the importance of combined CCK and secretin stimulation during pancreatic secretion in response to food intake.

Animals↗
PubMed1994

Nonparallel secretion of GP-2 from exocrine pancreas implies luminal coupling between acinar and duct cells.

The in vivo and in vitro secretion of glycoprotein-2 (GP-2), a glycosyl phosphatidylinositol (GPI)-anchored protein from the rat exocrine pancreas, was characterized. GP-2 was secreted in a nonparallel manner compared with amylase, a marker of secretory enzymes. Attenuated GP-2 secretion correlated with hormones that stimulated exocytosis in acinar cells. Augmented GP-2 secretion correlated with hormones that stimulated fluid and bicarbonate secretion from ductal elements. Immunofluorescence studies identified an enriched pool of GP-2 tightly bound to the apical membranes of acinar cells in addition to zymogen granules. This non-zymogen granule pool appears to represent the source of GP-2 released from acinar cells in a nonparallel manner. With the use of dispersed pancreatic acini largely devoid of ductal elements, GP-2 release was found to be augmented by alkaline pH. Thus GP-2 secretion appears to be modulated by two discrete cellular processes: 1) delivery of prereleased GP-2 within zymogen granules to the ductal lumen by exocytic mechanisms and 2) enzymatic release of GPI-anchored GP-2 from the luminal membranes, a kinetic process that appears to be regulated by secretin- or carbachol-induced secretion of bicarbonate.

Amylases↗
PubMed1994

Apical membrane trafficking during regulated pancreatic exocrine secretion--role of alkaline pH in the acinar lumen and enzymatic cleavage of GP2, a GPI-linked protein.

The GP2/THP family of glycosyl phosphatidylinositol (GPI)-anchored proteins is targeted to apical secretory compartments in polarized epithelial cells. We demonstrate in the rat exocrine pancreas that enzyme-mediated release of GP2 from acinar cell membranes represents a pH-dependent process regulated by bicarbonate secreted from ductular cells. Release of GP2 from secretin-stimulated pancreatic lobules, which retain intralobular ducts, was inhibited by (i) bicarbonate substitution, (ii) chloride substitution, and (iii) DIDS, a potent inhibitor of chloride-bicarbonate exchange. These inhibitory effects were not observed in preparations of pancreatic acini devoid of ductal elements. Enzymatic cleavage of GP2 and amylase release from pancreatic acini varied directly as a function of pH of the acinar human. Alkali-induced GP2 release could be correlated with ultrastructural and biochemical evidence for stimulated retrieval (endocytosis) of exocytic membranes at the acinar lumen. Our study defines functional roles for ductal bicarbonate in acinar cell and lumen physiology and provides a potential explanation for the biological significance of enzyme-mediated cleavage of GP2 from the apical plasma membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗