Co-Variation of Serum Osteoprotegerin and Pigment-Epithelial Derived Factor as Biomarker of Pancreatic Cancer.

Pancreatic cancer is one the most lethal cancers. Currently, there are reliable predictive markers to assess cancer development. Widely used CA19-9 molecular marker has been less effective in the diagnosis of early stages of cancer. Objective: To study if the soluble Osteoprotegerin (OPG) and pigment-epithelial derived factor (PEDF) levels in serum will be an indicator of cancer progression. Methods: Soluble OPG and PEDF were measured from human pancreatic cancer patients by ELISA. Results: We show that while OPG has been less predictive features, PEDF is more sensitive than CA19-9 in cancer detection. More importantly, PEDF and CA19-9 as combined markers showed higher sensitivity in stratifying early stages of pancreatic cancer. Conclusion: Results from the pilot studies suggest that PEDF is useful biomarker for pancreatic cancer.

Growth hormone-releasing factor stimulates pancreatic enzyme secretion.

Growth hormone-releasing factors (GRF's) from two human pancreatic tumors (hpGRF's) that caused acromegaly and from the rat hypothalamus ( rhGRF ) were recently isolated and characterized. Although these peptides are potent growth hormone secretagogues, they have not until now been described to have actions outside the pituitary. These GRF's were shown to stimulate digestive enzyme secretion from an exocrine pancreatic preparation in vitro, rhGRF being more than 100 times as potent as hpGRF. Adenosine 3',5'-monophosphate mediates this action of the GRF's.

Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line.

Serosal application of carbachol to T84 cell monolayers mounted in an Ussing chamber caused an immediate increase in short circuit current (Isc) that peaked within 5 min and declined rapidly thereafter, although a small increase in Isc persisted for approximately 30 min. The increase in Isc was detectable with 1 microM carbachol; half-maximal with 10 microM carbachol; and maximal with 100 microM carbachol. Unidirectional Na+ and Cl- flux measurements indicated that the increase in Isc was due to net Cl- secretion. Carbachol did not alter cellular cAMP, but caused a transient increase in free cytosolic Ca2+ ([Ca2+]i) from 117 +/- 7 nM to 160 +/- 15 nM. The carbachol-induced increase in Isc was potentiated by either prostaglandin E1 (PGE1) or vasoactive intestinal polypeptide (VIP), agents that act by increasing cAMP. Measurements of cAMP and [Ca2+]i indicated that the potentiated response was not due to changes in these second messengers. Studies of the effects of these agents on ion transport pathways indicated that carbachol, PGE1, or VIP each increased basolateral K+ efflux by activating two different K+ transport pathways on the basolateral membrane. The pathway activated by carbachol was not sensitive to barium, while that activated by PGE1 or VIP was; furthermore, their action on K+ efflux are additive. Our study indicates that carbachol causes Cl- secretion, and that this action may result from its ability to increase [Ca2+]i and basolateral K+ efflux. Carbachol's effect on Cl- secretion is greatly augmented in the presence of VIP or PGE1, which open a cAMP-sensitive Cl- channel on the apical membrane, accounting for a potentiated response.

Effects of caerulein on the apical cytoskeleton of the pancreatic acinar cell.

In this study experiments were performed to correlate the rate of digestive enzyme secretion to morphologic observations of the apical cytoskeleton using dispersed rat pancreatic acini with various concentrations of caerulein. Caerulein at concentrations of 10 pM to 0.1 nM stimulated increasing rates of secretion of amylase, a digestive enzyme. Greater concentrations of caerulein caused progressively less amylase secretion. Transmission electron microscopy demonstrated several characteristics of the apical cytoskeleton in untreated acini that were altered with the "inhibitory" concentrations of caerulein. In control acini and acini stimulated with concentrations of caerulein up to 0.1 nM, the micrographs reveal an apical actin network extending into microvilli, an intermediate filament band, and electron-dense structures contained in both the actin filament network and the intermediate filament band. With concentrations of caerulein greater than 0.1 nM, these structures were progressively ablated. The findings with respect to the actin filament network were confirmed with light microscopic observations of dispersed acini stained with rhodamine-phalloidin. These results indicate that caerulein has marked morphologic effects on the pancreatic acinar cell cytoskeleton and that the cytoskeletal changes may modulate the secretory response.

Cl- secretion induced by bile salts. A study of the mechanism of action based on a cultured colonic epithelial cell line.

When applied to the basolateral (serosal) side of the T84 colonic epithelial monolayer, taurodeoxycholate caused net Cl- secretion in a dose-dependent manner with a threshold effect observed at 0.2 mM. In contrast, when applied to the apical (luminal) surface, concentrations of taurodeoxycholate below 1 mM had little or no effect. Only when the concentration of taurodeoxycholate present on the apical side was greater than or equal to 1 mM did apical addition results in an electrolyte transport effect. This apical effect on electrolyte transport was associated with an abrupt increase in the permeability of the monolayer. Cyclic AMP and cyclic GMP in the T84 monolayers were not increased by the bile salt, but in the presence of extracellular Ca2+, free cytosolic Ca2+ increased with a graded dose effect and time course that corresponded approximately to the changes in short circuit current (Isc). The results suggest that luminal bile salts at a relatively high concentration (greater than or equal to 1 mM) increase tight junction permeability. Once tight junction permeability increases, luminal bile salts could reach the basolateral membrane of the epithelial cells where they act to increase free cytosolic Ca2+ from extracellular sources. The resulting increases in free cytosolic Ca2+, rather than in cyclic nucleotides, appear to be involved in transcellular Cl- secretion.

Pancreatic acinar cells produce, release, and respond to tumor necrosis factor-alpha. Role in regulating cell death and pancreatitis.

The aim of this study was to determine whether tumor necrosis factor-alpha (TNFalpha) and receptors for TNFalpha are expressed in the exocrine pancreas, and whether pancreatic acinar cells release and respond to TNFalpha. Reverse transcription PCR, immunoprecipitation, and Western blot analysis demonstrated the presence of TNFalpha and 55- and 75-kD TNFalpha receptors in pancreas from control rats, rats with experimental pancreatitis induced by supramaximal doses of cerulein, and in isolated pancreatic acini. Immunohistochemistry showed TNFalpha presence in pancreatic acinar cells. ELISA and bioassay measurements of TNFalpha indicated its release from pancreatic acinar cells during incubation in primary culture. Acinar cells responded to TNFalpha. TNFalpha potentiated NF-kappaB translocation into the nucleus and stimulated apoptosis in isolated acini while not affecting LDH release. In vivo studies demonstrated that neutralization of TNFalpha with an antibody produced a mild improvement in the parameters of cerulein-induced pancreatitis. However, TNFalpha neutralization greatly inhibited apoptosis in a modification of the cerulein model of pancreatitis which is associated with a high percentage of apoptotic cell death. The results indicate that pancreatic acinar cells produce, release, and respond to TNFalpha. This cytokine regulates apoptosis in both isolated pancreatic acini and experimental pancreatitis.

Cross-talk between calcium and cAMP-dependent intracellular signaling pathways. Implications for synergistic secretion in T84 colonic epithelial cells and rat pancreatic acinar cells.

Treatment of various cells with combinations of agents that increase either cAMP or cytosolic calcium can lead to synergistic responses. This study examined interactions, or cross-talk, between these two intracellular messengers and its implication for signaling in two secretory cell types, T84 human colonic epithelial cells and rat pancreatic acinar cells. T84 cell chloride secretion was measured in Ussing chambers. Acinar cell activation was monitored as amylase secretion. Cytosolic calcium was assessed via fura-2 microfluorimetry. A cell-permeant analogue of cAMP synergistically enhanced secretory responses to calcium-mobilizing hormones in both cell types, but paradoxically reduced overall calcium mobilization. The reduction in calcium mobilization could be attributed to an inhibition of calcium influx in T84 cells, although a different mechanism likely operates in acinar cells. The effects of the cAMP analogue were reproduced by other agents that increase cAMP. Furthermore, econazole, an inhibitor of calcium influx, potentiated secretory responses to calcium-dependent stimulation in T84 cells without itself inducing secretion. We conclude that there is cross-talk between calcium and cAMP-dependent signaling pathways at the level of second messenger generation in two secretory cell types. This cross-talk appears to regulate the extent of secretory responses.

Cholecystokinin-induced changes in polysome structure regulate protein synthesis in pancreas.

Translational regulation of digestive enzyme synthesis during short-term stimulation by cholecystokinin-octapeptide (CCK-OP), was examined in minced rabbit pancreas by measuring protein synthesis and monitoring alterations in the size of polysomes attached to the rough endoplasmic reticulum (RER). The effect of CCK-OP on protein synthesis was determined by measuring [3H]leucine incorporation into trichloroacetic-acid-precipitable proteins. Concentrations of CCK-OP that caused maximal enzyme secretion (10 and 30 nM) decreased protein synthesis by approx. 50% compared to control. Protein synthesis returned to the control level 60 min after terminating the action of CCK-OP. Autoradiography of [35S]methionine-labeled proteins separated by one-dimensional SDS-polyacrylamide gel electrophoresis demonstrated that CCK-OP reversibly inhibited the synthesis of all of the major groups of digestive enzymes. Northern blot analysis revealed that CCK-OP did not alter the cellular content of amylase and elastase mRNA. Incubation with CCK-OP caused a decrease in the size distribution of RER-bound polysomes. Polysome profiles returned to the control pattern 60 min following termination of the stimulus. These results suggest that the inhibitory effects of CCK-OP on the synthesis of digestive enzymes is regulated at translation by decreasing the number of RER-bound ribosomes that are actively translating digestive enzyme mRNA.

Intracellular mechanisms involved in short-term regulation of net protein synthesis in pancreatic acini.

The mechanisms regulating the net synthesis of digestive enzymes during short-term stimulation by agonists were examined in pancreatic acini isolated from the rat. Dispersed pancreatic acini were stimulated for up to 60 min with various concentrations of cholecystokinin octapeptide (CCK-OP), carbachol, A23187, 4 beta-phorbol 12-myristate 13-acetate (PMA). The effects of these agonists on net protein synthesis was determined by measuring the incorporation of [3H]leucine or [35S]methionine into protein. Carbachol, PMA, A23187 and concentrations of CCK-OP of 100 pM and greater caused inhibition of protein synthesis. Fluorography of [35S]methionine labeled acinar cell proteins separated by one-dimensional SDS-polyacrylamide gel electrophoresis demonstrated that the agonists inhibited the synthesis of the digestive enzymes. Northern blot analysis using cDNA probes revealed that CCK-OP, carbachol and PMA did not alter the cellular content of amylase, lipase and elastase mRNA. The protein kinase C inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and staurosporine failed to reverse the inhibitory effects of CCK-OP, carbachol and PMA on protein synthesis. CCK-OP and PMA activated phospholipase A (PLA) which liberated lysophosphatidylcholine (LPC) and free fatty acids from membrane phosphatidylcholine. Exogenously added PLA2 (Naja naja venom) inhibited protein synthesis and increased LPC to a similar extent as CCK and PMA. The results suggest that the inhibitory effects of CCK and carbachol on net protein synthesis are due to their effects on intracellular calcium and PLA-mediated breakdown of phosphatidylcholine rather than protein kinase C activation.

Sphingosine regulates Ca(2+)-ATPase and reloading of intracellular Ca2+ stores in the pancreatic acinar cell.

The purpose of present study was to examine the effects of sphingosine on cellular Ca2+ transports using dispersed rat pancreatic acini. The results demonstrated that sphingosine had a specific effect to inhibit Ca2+ uptake into the cell's agonist-sensitive pool as well as inhibiting microsomal Ca(2+)-ATPase. The ability of sphingosine to inhibit Ca2+ uptake resulted in both augmentation of Ca2+ release from the pool by inositol 1,4,5-trisphosphate (IP3) and conversion of the Ca2+ release by inositol 1,4,5-trisphosphate from a transient response to a sustained response. Furthermore, by preventing Ca2+ pool refilling sphingosine mimicked the effect of the agonist, carbachol, to maintain an increased [Ca2+]i during sustained stimulation. These results suggest that regulation of Ca(2+)-ATPase by sphingosine or a sphingosine-like agent mediates some of the effects of agonist on cell Ca2+ transports.

Cyclic GMP regulates free cytosolic calcium in the pancreatic acinar cell.

The present studies were performed in order to measure the effects of cyclic GMP (cGMP) on the regulation of free cytosolic calcium [( Ca2+]i) in the pancreatic acinar cell. In guinea pig dispersed pancreatic acini the findings demonstrated that the Ca2+ ionophore, Br A23187, caused a sustained increase in [Ca2+]i in the presence of 3 mM CaCl2 in the media and a transient 20 fold rise in cellular cGMP followed by a sustained 3-4 fold rise in cellular cGMP. Increasing cellular cGMP with nitroprusside, hydroxylamine or dibutyryl cGMP had no effect on resting [Ca2+]i. However, these agents attenuated the increase in [Ca2+]i resulting from Br A23187-induced Ca2+ influx. Nitroprusside also attenuated the carbachol-induced sustained rise in [Ca2+]i that resulted from Ca2+ influx. The nitroprusside effect on carbachol-stimulated acini occurred without decreasing Ca2+ influx across the plasma membrane or alteration in the mobilization of Ca2+ from the intracellular agonist-sensitive pool. Inhibition of the increase in cellular cGMP caused by Br A23187 by the guanylate cyclase inhibitor, 6-anilino-5,8-quinolinedione (LY83583), resulted in augmentation of the increase in [Ca2+]i. This augmentation was reversed with dibutyryl cGMP. These results indicated that cGMP regulated [Ca2+]i in the pancreatic acinar cell. The mechanism involves the removal of Ca2+ from the cytoplasm.

Characteristics of intracellular calcium changes required for augmentation of phorbol ester-stimulated pancreatic enzyme secretion.

Previous studies demonstrated that Ca2+ ionophores augment the pancreatic enzyme secretion caused by phorbol esters. The present study was performed to determine the nature of the cellular Ca2+ effects responsible for the augmentation. Relatively low concentrations (0.3-1.0 microM) of the nonfluorescent Ca2+ ionophore, 4-bromo-A23187 (Br-A23187), did not measurably increase free cytosolic Ca2+ ([Ca2+]i) and caused little or no enzyme release from guinea pig pancreatic acini. However, these concentrations of Br-A23187 augmented the amylase release caused by the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA). This augmentation occurred in the absence of extracellular Ca2+ as long as the intracellular agonist-sensitive pool contained Ca2+. Greater concentrations of Br-A23187 (3-10 microM) alone caused transient increases in [Ca2+]i and transient increases in amylase release. Although not resulting in an increase in [Ca2+]i, the low concentrations of Br-A23187 caused release of Ca2+ from the intracellular agonist-sensitive pool. These results suggest that Ca2+ mediates enzyme release by two distinct mechanisms in the pancreatic acinar cell. First, an increase in [Ca2+]i alone mediates enzyme release. Second, Ca2+ release from the agonist-sensitive pool not resulting in a measurable increase in [Ca2+]i augments enzyme release stimulated by a phorbol ester. The second effect of Ca2+ may be due to a small localized change in cell Ca2+ or an induction of cytosolic Ca2+ oscillations.

Isolation of subcellular agonist-sensitive calcium stores from the pancreatic acinar cell.

The purpose of the present study was to develop a technique to identify, isolate and partially purify these membrane bound compartments for further characterizations of their Ca2+ transport and storage mechanisms. We 45Ca(2+)-loaded the agonist-sensitive Ca2+ stores in rat pancreatic acini. The loading was accomplished by first depleting the stores with carbachol stimulation followed by the addition of 45Ca2+ and atropine to the extracellular media. After homogenization of the 45Ca(2+)-loaded acini, subcellular fractions were resolved on sucrose and Nycodenz gradients. 45Ca2+ fluxes were minimized during these procedures by inclusion in the media of LaCl3. Five subcellular fractions were identified that specifically accumulated 45Ca2+ after carbachol stimulation. Electron microscopic observations of the fractions demonstrated that three of the fractions consisted of rough membrane vesicles; that one consisted of a mixture of rough and smooth membrane vesicles; and that one consisted of smooth membrane vesicles. All fractions were enriched in glucose-6-phosphatase. All 5 fractions demonstrated ATP dependent 45Ca2+ uptake. By Western blot analysis, all fractions contained calnexin, p58, sarcoplasmic reticulum type Ca(2+)-ATPase, and IP3 receptor. These results demonstrated that the 45Ca(2+)-loading technique can be used to isolate and characterize distinct compartments of the agonist-sensitive Ca2+ store in the pancreatic acinar cell.

nNOS and Ca2+ influx in rat pancreatic acinar and submandibular salivary gland cells.

Regulation of agonist-activated Ca2+ influx by the NOS pathway through generation of cGMP is being found in an increasing number of cell types. In the present work, we examined the role of the NOS pathway in agonist-evoked [Ca2+]i oscillations and attempted to identify the NOS isoform most likely to regulate Ca2+ influx. For this, we first show that two Ca(2+)-mobilizing agonists acting on pancreatic acinar cells, bombesin (BS) and the cholecystokinin analog CCK-JMV-180 (CCKJ), evokes different type of [Ca2+]i oscillations. The BS-evoked [Ca2+]i oscillations rapidly became acutely dependent on the presence of extracellular Ca2+, whereas the CCKJ-evoked oscillations continue for long periods of time in the absence of Ca2+ influx. This differential behavior allowed us to isolate Ca2+ influx and study its regulation while controlling for non specific effects on all other Ca2+ transporting events involved in generating [Ca2+]i oscillations. Inhibitors of selective steps in the NOS pathway inhibited agonist-induced cGMP production. The inhibitors were then used to show that scavenging NO with reduced hemoglobin, inhibition of guanylyl cyclase with 1H-[1,2,4] oxadiazolo[4,3-a] quinoxaline-1-one (ODQ) and inhibition of protein kinase G with Rp-8-pCPT-cGMPS inhibited [Ca2+]i oscillations evoked by BS but not those evoked by CCKJ. These findings were extended to duct and acinar cells of the SMG. In these cells, Ca(2+)-mobilizing agonists stimulate large Ca2+ influx, which was inhibited by all inhibitors of the NOS pathway. Western blot analysis and immunolocalization revealed that the cells did not express iNOS, eNOS was expressed only in blood vessels and capillaries whereas nNOS was expressed at high levels next to the plasma membrane of all cells. Accordingly, the nNOS inhibitor 7-nitroindazole (7-NI) inhibited BS- but not CCKJ-evoked [Ca2+]i oscillations and Ca2+ influx into SMG acinar and duct cells. Thus, together, our findings favor nNOS as the isoform activated by the Ca2+ released from internal stores to generate cGMP and regulate Ca2+ influx.

Calcium-stimulated parathyroid hormone-like protein secretion: potentiation through a protein kinase-C pathway.

The effect of altering intracellular calcium levels on PTH-like protein (PLP) secretion was assessed in the NCI-H727 cell line. Ionomycin stimulated PLP secretion in a dose- and time-dependent manner, with a maximum rate of secretion occurring within minutes. Phorbol esters (phorbol 12-myristate 13-acetate and phorbol 12,13-didecanoate) stimulated PLP secretion at 10(-7)-10(-5) M, whereas forskolin treatment had no effect. When cells were treated initially with phorbol esters, ionomycin-stimulated PLP secretion was potentiated. The kinetics of this effect were rapid (t1/2, less than 1 min), and pretreatment with cycloheximide or actinomycin-D before phorbol ester and ionomycin treatment did not alter PLP secretion. Pretreatment for 24 h with phorbol 12-myristate 13-acetate to desensitize the cells or use of inactive phorbol ester isomer 4 alpha-phorbol 12,13-didecanoate did not potentiate the ionomycin-stimulated PLP secretion. Treatment with exogenously added phospholipase-C caused a dose-dependent increase in PLP secretion. These data indicate that PLP secretion can be stimulated through a phospholipase-C-mediated mechanism.

Calcium metabolism and bombesin-stimulated pancreatic enzyme secretion.

In dispersed guinea pig pancreatic acini, bombesin stimulates a rapid increase in the concentration of free cytosolic Ca2+ ([Ca2+]i) by releasing Ca2+ from an intracellular agonist-sensitive pool. This increase is transient, and it mediates the initial secretion of digestive enzymes from the acinar cell. During sustained bombesin stimulation, [Ca2+]i returns to the resting or near-resting level, but bombesin-stimulated enzyme secretion continues. Although [Ca2+]i is at the resting level during continued stimulation, 45Ca2+ flux measurements indicate that bombesin continues to release Ca2+ from an intracellular store that is probably the agonist-sensitive pool. Low concentrations (0.3 microM) of the non-fluorescent Ca2+ ionophore, 4-bromo-A23187 (Br-A23187), release Ca2+ from the agonist-sensitive pool without measurably increasing [Ca2+]i. This ionophore-induced Ca2+ release without production of an increase in [Ca2+]i does not by itself stimulate enzyme secretion, but it does augment the secretion caused by the phorbol ester, 4 beta-phorbol, 12-myristate, 13-acetate (PMA). These results suggest a role for intracellular Ca2+ release in bombesin-stimulated sustained secretion.

Cellular mechanisms mediating agonist-stimulated calcium influx in the pancreatic acinar cell.

Figure 1 summarizes our current concept of a signaling mechanism to explain agonist-induced Ca2+ entry in the pancreatic acinar cell. We propose that cGMP can modulate Ca2+ entry under conditions of internal Ca2+ store depletion and that the NO signaling system may be involved in coupling Ca2+ depletion to cGMP formation. The finding that Ca2+ entry after Ca2+ store depletion can occur with no elevation in [Ca2+]i37 raises the possibility that alternative signaling pathways may converge to stimulate cGMP formation or that additional messengers may activate plasmalemmal Ca2+ entry mechanisms in parallel.

Free cytosolic calcium and secretagogue-stimulated initial pancreatic exocrine secretion.

In order to establish the role of secretagogue-induced changes in free cytosolic Ca2+ ([Ca2+]i) in pancreatic enzyme secretion, we measured the effects of carbachol, cholecystokinin-octapeptide (CCK-OP), bombesin, substance P, and bromo-A23187 on amylase release and [Ca2+]i in guinea pig pancreatic acini loaded with the Ca2+-selective fluorescent indicator, fura-2. Evaluation of time courses and dose-response curves indicated that carbachol, CCK-OP, bombesin, and substance P cause extracellular Ca2+-independent transient increases in [Ca2+]i and transient bursts in amylase release (initial secretion). The potencies for the secretagogues to increase [Ca2+]i and initial amylase release were similar. Bromo-A23187 also caused an extracellular Ca2+-independent transient increase in [Ca2+]i and amylase release. In the absence of extracellular Ca2+, sequential additions of substance P followed by carbachol caused transient increases in [Ca2+]i correlating with transient bursts in amylase release. In contrast, in acini first treated with carbachol, the ability of substance P to increase [Ca2+]i and amylase release was blocked. Sustained secretion caused by the secretagogues was dependent on extracellular Ca2+ but occurred at basal [Ca2+]i. Increasing [Ca2+]i during the sustained phase of stimulation by increasing the extracellular Ca2+ concentration or with bromo-A23187 did not increase the rate of sustained secretion.

Differing effects of ethanol on in vitro stimulated pancreatic enzyme secretion in ethanol-fed and control rats.

The single most important risk factor for chronic and acute pancreatitis is the abuse of ethanol, which, theoretically, could affect the pancreas by interacting either with some of its cell receptors or with any of its intracellular signal transduction mechanisms. Therefore, we determined in isolated pancreatic acini from 3 month ethanol-fed rats and controls the dose-response effects of secretagogues on enzyme secretion, both in the presence and absence of ethanol in the incubation medium. In ethanol-fed rats, pancreatic amylase activity was decreased by 40%, compared to controls (with identical carbohydrates intakes), whereas lipase and trypsinogen activities were unaffected. With no ethanol in the incubation medium, basal enzyme releases and enzyme dose-response curves to CCK-8, VIP, secretin, bombesin, and bethanechol were essentially unchanged in ethanol-fed rats compared to controls. In contrast, with 0.1 M ethanol present in the medium, enzyme responses to VIP, secretin, and CCK-8 were inhibited and that to CCK-8 also shifted to the right in ethanol-fed rats compared to controls. Hence, if rechallenged to ethanol, acini from ethanol-fed rats show inhibited secretions, in response to two secretagogues acting through the release of cyclic AMP, and an inhibited and right-shifted secretory response to CCK.