Yashwant Deshpande

The Role of Prostanoids in the Production of Acute Acalculous Cholecystitis by Platelet-activating Factor

Gallbladder tissue from patients with acute acalculous cholecystitis contains increased amounts of prostanoids when compared to normal gallbladder tissue. Platelet-activating factor (PAF) is a potent stimulus of eicosanoid formation. It has been implicated as a mediator of acute inflammatory processes and systemic responses to shock. In this study the role of PAF in acute acalculous cholecystitis was evaluated. Anesthetized cats underwent gallbladder perfusion with a physiologic buffer solution containing [14C]polyethylene glycol as a nonabsorbable tracer to quantitate mucosal water absorption. Platelet-activating factor was infused into the hepatic artery for 2 hours. Control experiments were performed when vehicle alone was infused. Experiments also were performed when indomethacin was administered intravenously and when indomethacin and PAF were administered. Gallbladder mucosal absorption/secretion and perfusate and tissue prostaglandin E (PGE) and 6 keto prostaglandin F1 alpha (6-keto PGF1 alpha) levels were evaluated. Gallbladder inflammation was evaluated by beta-glucuronidase and myeloperoxidase tissue concentrations and by a histologic scoring system. Platelet-activating factor eliminated gallbladder absorption and produced net fluid secretion associated with dose-related increases in perfusate PGE concentrations and gallbladder tissue PGE and 6 keto PGF1 alpha levels when compared to control values. Platelet-activating factor produced significant inflammation in the gallbladder with increases in the histologic score of inflammation and tissue lysosomal enzyme activities. Indomethacin significantly decreased the fluid secretion, prostanoid levels, and inflammation produced by PAF. The results suggest that PAF may induce acute gallbladder inflammation associated with systemic stress through a prostanoid-mediated mechanism.

Role of calcium in adaptive cytoprotection and cell injury induced by deoxycholate in human gastric cells

We have developed an in vitro model of adaptive cytoprotection induced by deoxycholate (DC) in human gastric cells and have shown that pretreatment with a low concentration of DC (mild irritant, 50 μM) significantly attenuates injury induced by a damaging concentration of DC (250 μM). This study was undertaken to assess the effect of the mild irritant on changes in intracellular Ca2+ and to determine if these perturbations account for its protective action. Protection conferred by the mild irritant was lost when any of its effects on intracellular Ca2+ were prevented: internal Ca2+ store release via phospholipase C and inositol 1,4,5-trisphosphate sustained Ca2+ influx through store-operated Ca2+ channels or eventual Ca2+ efflux. We also investigated the relationship between Ca2+accumulation and cellular injury induced by damaging concentrations of DC. In cells exposed to high concentrations of DC, sustained Ca2+ accumulation as a result of extracellular Ca2+ influx, but not transient changes in intracellular Ca2+ content, appeared to precede and induce cellular injury. We propose that the mild irritant disrupts normal Ca2+ homeostasis and that this perturbation elicits a cellular response (involving active Ca2+ efflux) that subsequently provides a protective action by limiting the magnitude of intracellular Ca2+ accumulation.We have developed an in vitro model of adaptive cytoprotection induced by deoxycholate (DC) in human gastric cells and have shown that pretreatment with a low concentration of DC (mild irritant, 50 microM) significantly attenuates injury induced by a damaging concentration of DC (250 microM). This study was undertaken to assess the effect of the mild irritant on changes in intracellular Ca2+ and to determine if these perturbations account for its protective action. Protection conferred by the mild irritant was lost when any of its effects on intracellular Ca2+ were prevented: internal Ca2+ store release via phospholipase C and inositol 1,4, 5-trisphosphate sustained Ca2+ influx through store-operated Ca2+ channels or eventual Ca2+ efflux. We also investigated the relationship between Ca2+ accumulation and cellular injury induced by damaging concentrations of DC. In cells exposed to high concentrations of DC, sustained Ca2+ accumulation as a result of extracellular Ca2+ influx, but not transient changes in intracellular Ca2+ content, appeared to precede and induce cellular injury. We propose that the mild irritant disrupts normal Ca2+ homeostasis and that this perturbation elicits a cellular response (involving active Ca2+ efflux) that subsequently provides a protective action by limiting the magnitude of intracellular Ca2+ accumulation.

Prostaglandins Protect Human Intestinal Cells Against Ethanol Injury by Stabilizing Microtubules (Role of Protein Kinase C and Enhanced Calcium Efflux)

Prostaglandins (PG) protect gastrointestinalcells against damage induced by ethanol (EtOH) and othernoxious agents, a process termed cytoprotection. Thepresent study investigated the relationships between microtubule (MT) stability, protein kinase C(PKC) activation, and calcium efflux as a possiblemechanism of PGs protective action using a humancolonic cell line (Caco-2) exposed to known damagingconcentrations of EtOH (7.5% and 10% ). Preincubation ofCaco-2 cells with 16,16-dimethyl-PGE2 (PG,2.6 μM) significantly increased PKC activity in thesecells. Pretreatment of Caco-2 cells with 50 μM OAG (asynthetic diacylglycerol and PKC activator) or 30 nM TPA(a direct PKC activator) prior to exposure to 7.5% or10% EtOH for 5 min significantly reduced cell injury, asdetermined by trypan blue exclusion, and increased MT stability, as confirmed by confocalmicroscopy. Pretreatment of Caco-2 cells with 4alpha-PDD (an inactive phorbol ester, 20 nM) failed toprevent cell injury and disruption of the MTcytoskeleton. Preincubation with staurosporine (a PKC inhibitor, 3 nM)abolished the protective effects of PG in cells exposedto 7.5% and 10% EtOH. Incubation of Caco-2 cells withA23187 (a Ca2+ ionophore), similar to 10%EtOH, caused a significant reduction in cell viability andMT stability. Preincubation with A23187 in combinationwith PG or OAG prior to subsequent exposure to EtOHsignificantly abolished the protective effects of PG or OAG pretreatment. Finally, pretreatmentwith OAG, TPA, or PG resulted in significant increasesin calcium-45 efflux, which correlated with increasedstability of the MT cytoskeleton. These data suggest that PG possesses direct protective effectsagainst EtOH injury in Caco-2 cells and may act bystabilizing MT through the PKC signal transductionpathway and/or stimulation of calcium efflux from thecells.

Adaptive cytoprotection against deoxycholate-induced injury in human gastric cells in vitro : Is there a role for endogenous prostaglandins?

The majority of previous work investigatingadaptive cytoprotection has involved in vivo studies,which have suggested that this protective response is inlarge part mediated by endogenous prostaglandins (PGs). The aim of this study was to investigateadaptive cytoprotection under in vitro conditions inhuman gastric cells and to better delineate the role ofendogenous PGs in this protective response. AGS cells (a human gastric carcinoma cell line)were characterized morphologically and subsequently usedfor all experiments. Sodium deoxycholate was used asboth the mild irritant and the damaging agent, and cell injury was quantified using both acommercial viability/cytotoxicity kit as well astransepithelial permeability studies. Finally,endogenous PG synthesis in response to varyingconcentrations of deoxycholate was determined. AGS cells were determined to bemorphologically similar to gastric mucous cells.Pretreatment of cells with low-dose deoxycholatesignificantly attenuated injury upon subsequent exposure to damaging concentrations of deoxycholate, andthis protection was determined to be dependent upon bothconcentration and duration of mild irritant exposure.Preincubation of AGS cells with indomethacin reversed protection induced by mild irritantpretreatment and also significantly increased cellularsusceptibility to injury. Results of the permeabilitystudies closely paralleled those assessing cell mortality. While deoxycholate exposureincreased PG synthesis, the concentrations required weremuch higher than those needed to initiate protection.Adaptive cytoprotection exists in AGS cells under in vitro conditions independent of intact bloodflow, neural innervation, or circulating humoralmediators. While this protection is reversed byindomethacin, it appears that this reversal results fromincreased cellular injury secondary to diminished basalPGs, rather than inhibition of endogenous PGsynthesis.

Indomethacin increases susceptibility to injury in human gastric cells independent of PG synthesis inhibition

Indomethacin and other nonsteroidal anti-inflammatory drugs are commonly used to indirectly deduce the possible role of PGs in a process being studied. The objective of this study was to determine if indomethacin, at concentrations comparable to plasma and tissue levels obtained in humans taking therapeutic doses, predisposes human gastric cells to injury through inhibition of PGs or acts through an alternate mechanism. The role of intracellular Ca2+ in this damaging process was also assessed. Indomethacin pretreatment, although by itself nondamaging, was associated with elevated intracellular Ca2+ concentrations and an increased cellular permeability, an effect that was dependent on extracellular Ca2+. Furthermore, indomethacin pretreatment significantly predisposed AGS cells to injury induced by two dissimilar agents (deoxycholate and A-23187), both of which are associated with intracellular Ca2+ accumulation. The addition of exogenous PGs did not reverse the predisposition to injury induced by indomethacin. The observed effects of indomethacin were dependent on concentration and not on ability to inhibit PG synthesis. Similar effects were not observed with equipotent concentrations of ibuprofen or aspirin. Finally, the exacerbation of deoxycholate-induced injury induced by indomethacin was not observed when extracellular Ca2+ was removed. Indomethacin, by disturbing intracellular Ca2+homeostasis, predisposes human gastric cells to injury through mechanisms independent of PG synthesis. The current study suggests that data resulting from studies employing only indomethacin as a PG synthesis inhibitor should be interpreted with caution.

Calcium accentuates injury induced by ethanol in human gastric cells.

The mechanism(s) whereby ethanol induces cellular injury remains poorly understood. Furthermore, the role of calcium in gastric mucosal injury under in vitro conditions is poorly defined. The major objectives of this study were to (1) define the temporal relationship between intracellular calcium accumulation induced by ethanol and cellular injury, (2) characterize the mechanising) whereby ethanol increases cellular calcium content, and (3) determine whether calcium removal would attenuate ethanol-induced cellular injury. Human gastric cells (AGS) were used for all experiments. Sustained intracellular calcium accumulation induced by ethanol, but not transient changes, preceded and directly correlated with cellular injury. Cells exposed to damaging concentrations of ethanol demonstrated an initial calcium surge that appeared to be a consequence of inositol 1,4,5-triphosphate (IP3) generation and subsequent internal store release followed by a sustained plateau resulting from extracellular calcium influx through store-operated calcium channels. Finally, both morphologic (cellular injury) and functional (clearance of bovine serum albumin) changes induced by ethanol were significantly attenuated when extracellular Ca++ influx was prevented, and further decreased when intracellular Ca++ stores were depleted. These data indicate that calcium plays a significant role in cellular injury induced by ethanol.

Studies on the etiology of acute acalculous cholecystitis: The effect of lipopolysaccharide on human gallbladder mucosal cells

Previous studies in animals have shown that lipopolysaccharide produces experimental cholecystitis possibly through a platelet-activating factor-prostanoid mediated process. In this study it was intended to evaluate the effect of LPS on primary cultures of human gallbladder mucosal cells. Gallbladder mucosal cells were isolated from gallbladders removed during routine cholecystectomies or other operations. The cells were cultured for 24 h before treatment. Unstimulated cells produced low levels of prostanoids and significant basal levels of PAF. LPS produced stimulation of eicosanoid and PAF secretion. The increased prostanoid formation was not enhanced when LPS and PAF were administered together. Prostanoid synthesis was inhibited by the administration of a cyclooxygenase inhibitor while administration of a PAF receptor antagonist significantly increased prostanoid formation, suggesting that increased PAF levels function as a negative control mechanism to decrease prostanoid synthesis. The results suggest that endotoxemia may produce a cascade of inflammatory processes in human gallbladder mucosal cells resulting in the development of acute acalculous cholecystitis.

Evaluation of the role of the F prostaglandins in canine bile flow

Prostaglandin F2 alpha (PGF2 alpha) has been shown to be an effective stimulant of hepatic bile flow producing a specific chloride rich bile. Subsequent evaluation by radioimmunoassay has shown that prostaglandin F compounds are present in relatively large amounts in canine hepatic bile. This study evaluates the effect of PGF2 alpha administration and of prostaglandin synthetase inhibition by aspirin and indomethacin on bile flow and radioimmunoassayable prostaglandin F (iPGF) secretion. Chronic, canine bile fistula preparations were utilized and the enterohepatic circulation was maintained by intravenous bile salts. Bile volume and composition were evaluated by standard techniques as well as bile PGF concentration by radioimmunoassay during bile salt infusion and during bile salt and PGF2 alpha, aspirin and indomethacin infusion in varying doses. Both aspirin and PGF2 alpha were potent stimulants of hepatic bile flow with aspirin producing a chloride rich bile similar to that produced by PGF2 alpha. PGF2 alpha produced dose related increases in bile iPGF concentration and output indicating that as the systemic concentration increases during infusion of PGF2 alpha the lipid appears in bile. Aspirin in the highest dose administered, decreased iPGF concentration in bile while output was unchanged. Indomethacin was ineffectual in consistently altering bile flow or iPGF secretion. This study demonstrates that iPGF is present in canine bile, that its concentration can be altered by prostaglandin infusion while prostaglandin synthetase inhibition has minimal effects on bile iPGF secretion.

The effect of prostaglandin F2α on canine hepatic bile flow and biliary cyclic AMP secretion

Abstract This study evaluates the effects of prostaglandin F 2α (PGF 2α ) on canine hepatic bile flow and gastric hydrogen ion output and on biliary cyclic AMP excretion. During the experiments the enterohepatic circulation was maintained by intravenous bile salt infusion. PGF 2α was administered through either a separate intravenous or intra-arterial line in doses of 1, 2, 4, and 8 μ/kg/min. PGF 2α produced progressive increases in bile output and gastric hydrogen ion output. The choleresis was characterized by increased bile salt and chloride secretion suggesting that PGF 2α altered bile flow at the canalicular level. There was no greater response produced when the prostaglandins were administered intra-arterially compared to the response produced by intravenous PGF 2α . Bile cyclic AMP and cyclic GMP concentrations were measured by radioimmunoassay. Cyclic AMP concentrations were decreased by all doses of PGF 2α administered while cyclic AMP output was significantly decreased only by 8 μ/kg/min of PGF 2α . Cyclic GMP levels were consistently less than 0.8 pmol/ml and were not changed by PGF 2α . The results indicate that PFG 2α stimulates hepatic bile flow and gastric hydrogen ion output in a dose-related manner, the choleresis may be canalicular in nature, and metabolic degradation of PGF 2α by the lungs in these studies did not greatly influence the results obtained. Biliary cyclic AMP levels were decreased, suggesting that PGF 2α does not produce its response by stimulating the adenyl cyclase-cyclic AMP system.

THE EFFECT OF CLOSTRIDIUM DIFFICILE TOXIN ON COLONOCYTE PROSTANOID ACTIVITY

Antibiotic-associated colitis is caused by Clostridium difficile toxin. However, the pathophysiology of this entity is poorly understood. The aim of this study was to determine the effects of C. difficile toxin on colonocyte cyclooxygenase and phospholipase A2 (PLA2) activity. A transformed colonocyte cell line (Caco-2) was grown to confluency on 6 well plates. The cells were stimulated with graded concentrations of C. difficile toxin. In separate experiments, the cells were pretreated for one hour prior to stimulation with the cyclooxygenase inhibitor, indomethacin, or the glucocorticoid, dexamethasone. The culture media was collected one hour following C. difficile stimulation. Prostaglandin E2 (PGE2), 6-keto prostaglandin F1 alpha (6KPGF), thromboxane B2 (TxB2) and leukotriene B4 (LTB4) levels were determined in the media by an ELISA. Platelet activating factor (PAF) concentration was determined by a RIA. C. difficile toxin stimulated PGE2 and 6KPGF levels in a dose dependent fashion but failed to stimulate TxB2, LTB4 or PAF. Prostanoid production was inhibited by indomethacin dose dependently but was not inhibited by dexamethasone. The presence of indomethacin resulted in production of PAF. Our results show that the effects of C. difficile toxin on colonocytes are mediated by cyclooxygenase activity. The increase in PAF formation associated with indomethacin administration suggests that the prostanoids modulate PLA2 activity and inhibit PAF formation.