Phenols present in olive oil may contribute to the health effects of the Mediterranean lifestyle. Olive oil antioxidants increase the resistance of low-density lipoproteins (LDL) against oxidation in vitro, but human intervention studies have failed to demonstrate similar consistent effects. To better mimic the in vivo situation, plasma was incubated with either individual olive oil phenols or olive oil extracts with different phenolic compositions, and LDL was subsequently isolated and challenged for its resistance to oxidation. The results show that the ortho-dihydroxy phenols (hydroxytyrosol and oleuropein-aglycone) are more efficient than their mono-hydroxy counterparts (tyrosol and ligstroside-aglycone) in increasing the resistance of LDL to oxidation. However, the concentration of antioxidants required to inhibit LDL oxidation when added to whole plasma was substantially higher as compared to previous data where antioxidants are directly added to isolated LDL. In conclusion, this study supports the hypothesis that extra virgin olive oil phenols protect LDL in plasma against oxidation. The explanation that in vitro studies show protective effects in contrast to the lack of effect in the majority of human studies may be that the dose of the phenols and thus their plasma concentration in humans was too low to influence ex vivo LDL oxidizability. Further studies are required to gain a better understanding of the potential health benefits that extra virgin olive oil may provide.

(Kcccived 2 June128 Augusl 1995) -ESR 95 0889/6 The putative phospholipase C inhibitor, U73122, transiently increascs the cytosolic free Ca' ' concentration in rabbit pancreatic acinar cells by stimulating the release of CaZt froin intracellular stores [Willems, Van de Put, Engbersen, Bosch, Van Hoof & De Pont (1994) Pfliigers Arch. 427, 233-2431. In order to elucidate the exact mechanism of action or U73122 we studied its cffects on both Ca' ' -stimulated Mg"-dependent ATPase activity and Ca2 ' -sti tnulated ATP-dependent Ca2+ uptake in rat liver microsotnes. In addition, we studied its effects on Ca' ' release from steady-state loaded microsoincs. The effects of U73122 were compared with those or thimerosal, describcd in the literature as inhibiting CaZ+-ATPases and scnsitizing inositol 1,4,5-trisphosphate-operated Ca2+ release channels, and thapsigargin, a specific inhibitor of sarcnplasmic and endoplasmic reticuluni C$+-ATPases. Both U73122 (lC5,, = 9 pM) and thimerosal (1C5() = 11 pM) dose-dependently inhibited Ca''-stimulated Mg' ' -dependent ATPase activity, without significantly affecting Mg"-stimulated ATPasc activity. Similarly, both U73122 (IC<,, = 9 pM) and thimerosal (IC5,, = 14 pM) dose-dependently inhibited ATP-dependent Ca2+ uptake. At concentrations beyond 20 pM, U73122 stimulatcd Ca'+ release from stcady-stale loaded microsomcs at a rate considerably higher than obtained with a maximally inhibitory concentration of thapsigargin ('I pM). This observation, which was not reached with equally inhibitory concentrations of thimerosal, demonstrates that higher U73 122 concentrations cause an additional increase of the passive Ca2+ lcak. The data presented demonstratc that U73122 stimulates the release of actively stored Ca2+ primarily through inhibition of the internal Ca2+ pump.Keywords: U73122 ; thimerosal ; Ca2+-ATPase; endoplasmic reticulum ; hepatocytes. In order to explorc the involverncnt of phospholipase C in the mechanism of action of Ca2+-mobilizing stimuli, recent studies havc used the aininosteroid U73 122 [ S -101. lJ731 22 was originally described as a potent inhibitor of platelet aggregation induced by a variety of agoni ts 151. The inhibitory action of U73122 was paralleled by inhib ion of the agonist-stimulated

Stimulation of pancreatic acinar cells raises [Intracellular Ca 2ϩ stores play a dominant role in Ca 2ϩ signaling in pancreatic acinar cells. Indeed, the Ca 2ϩ -mobilizing action of the intracellular messenger inositol-1,4,5-trisphosphate (InsP 3 ) 1 was first demonstrated using a permeabilized pancreatic acinar cell preparation (1). This initial work also identified the endoplasmic reticulum (ER) as the intracellular Ca 2ϩ store responsible for agonist-induced increases in [Ca 2ϩ ] i (1). Recently, however, zymogen granules have been proposed to act as a Ca 2ϩ store in pancreatic acinar cells. (2). This hypothesis stemmed initially from the observation that stimulation of pancreatic (and other) acinar cells with acetylcholine results in a polarized rise in cytosolic free [Ca 2ϩ ], with the [Ca 2ϩ ] i increase being initiated at the apical pole of the cell where zymogen granules are clustered (3-6). Subsequently the rise in [Ca 2ϩ ] i spreads to the basal pole of the acinar cell (3-6). The role of the proposed zymogen granule Ca 2ϩ store would be to act as the releasable Ca 2ϩ store responsible for the initiation of the intracellular Ca 2ϩ signal at the apical pole (2, 7). Propagation of the increase in [Ca 2ϩ ] i toward other regions of the cell could then be mediated by the ER Ca 2ϩ stores, since the rough endoplasmic reticulum is found throughout the acinar cell (see e.g. Ref. 8). This zymogen granule Ca 2ϩ store model is attractive since it can explain the restriction of the [Ca 2ϩ ] i signal to only the luminal (apical) region when low physiological doses of cholinergic agonists are applied (7, 9). However, there is as yet no conclusive evidence on whether zymogen granules are equipped with intracellular messenger-triggered Ca 2ϩ release. Indeed, recent evidence suggests that the report of InsP 3 -sensitive Ca 2ϩ release from granules (2) may be an artifact produced by the impurity of the zymogen granule preparation employed (10).A further problem with the report of zymogen granule Ca 2ϩ stores (2) is that no evidence was found for a Ca 2ϩ uptake mechanism in the proposed zymogen granule Ca 2ϩ store. However, an active Ca 2ϩ -sequestering mechanism is essential to explain the refilling of Ca 2ϩ stores and hence the repetitive nature of the agonist-induced [Ca 2ϩ ] i transients. These conflicting results have prompted us to re-evaluate the suggestion that the endoplasmic reticulum can act as a functional Ca 2ϩ store in all subcellular regions of the pancreatic acinar cell. Our results demonstrate that the endoplasmic reticulum can indeed act as a Ca 2ϩ store in all subcellular regions, including the apical pole. EXPERIMENTAL PROCEDURESCell Preparation-Small clusters of acinar cells were prepared from the pancreas of one 200-g male Sprague-Dawley rat by the same enzymatic digestion procedure described in Ref. 11. After isolation, cells were resuspended in a HEPES/Tris-buffered physiological saline containing 133 mM NaCl, 4.2 mM KCl, 1.0 mM CaCl 2 , 1.0 MgCl 2 , 5.8 mM glucose, 0.2 mg/ml soybean trypsin ...

The effect of the putative inhibitor of phospholipase C activity, U73122, on the Ca2+ sequestering and releasing properties of internal Ca2+ stores was studied in both permeabilized and intact rabbit pancreatic acinar cells. U73122 dose dependently inhibited ATP-dependent Ca2+ uptake in the inositol (1,4,5)-trisphosphate-[Ins(1,4,5)P3]-sensitive, but not the Ins(1,4,5)P3-insensitive, Ca2+ store in acinar cells permeabilized by saponin treatment. In a suspension of intact acinar cells, loaded with the fluorescent Ca2+ indicator, Fura-2, U73122 alone evoked a transient increase in average free cytosolic Ca2+ concentration ([Ca2+]i,av), which was largely independent of external Ca2+. Addition of U73122 to cell suspensions prestimulated with either cholecystokinin octapeptide or JMV-180 revealed an inverse relationship in size between the U73122- and the agonist-evoked [Ca2+]i,av transient. Moreover, thapsigargin-induced inhibition of intracellular Ca(2+)-ATPase activity resulted in a [Ca2+]i,av transient, the size of which was not different following maximal prestimulation with either U73122 or agonist. These observations suggest that U73122 selectively affects the Ins(1,4,5)P3- casu quo agonist-sensitive internal Ca2+ store, whereas thapsigargin affects both the Ins(1,4,5)P3-sensitive and -insensitive Ca2+ store. Digital-imaging microscopy of Fura-2-loaded acinar cells demonstrated that U73122, in contrast to thapsigargin, evoked sustained oscillatory changes in [Ca2+]i. The U73122-evoked oscillations were abolished in the absence of external Ca2+. The ability of U73122 to generate external Ca(2+)-dependent Ca2+ oscillations suggests that depletion of the agonist-sensitive store leads to an increase in Ca2+ permeability of the plasma membrane and that the Ins(1,4,5)P3-insensitive Ca2+ pool is necessary for the Ca2+ oscillations.

2؉ release in a dose-dependent, "quantal" fashion. The kinetics of this release were similar to those reported for suspensions of permeabilized pancreatic acinar cells. Interestingly, the permeabilized acinar cells showed no intercellular variation in Ins(1,4,5)P 3 sensitivity. Although SLO treatment is known to result in a considerable loss of cytosolic factors, permeabilization did not result in a redistribution of zymogen granules, as judged by electron microscope analysis. These results suggest that Ins(1,4,5)P 3 -sensitive Ca 2؉ stores are unlikely to be redistributed as a result of SLO treatment. The effects of Ins(1,4,5)P 3 were therefore subsequently studied at the subcellular level. Detailed analysis demonstrated that no regional differences in Ins(1,4,5)P 3 sensitivity exist in this permeabilized cell system. Therefore, we propose that additional cytosolic factors and/or the involvement of ryanodine receptors underlie the polarized pattern of agonist-induced Ca 2؉ signaling in intact pancreatic acinar cells.