Ca2+ requirement of prostanoid but not of superoxide production by rat Kupffer cells

The release of the prostanoids prostaglandin D2 (PGD2), prostaglandin E2 (PGE2) and thromboxane induced by zymosan and phorbol ester in cultured rat Kupffer cells was found to depend on the extracellular concentration of Ca2+ to some extent. Prostanoid formation following the addition of the calcium ionophore A 23187 was totally inhibited when calcium ions were withdrawn from the medium whereas the prostanoid synthesis from added arachidonic acid was independent of Ca2+. A half-maximal rate of PGE2 release by cells treated with zymosan, phorbol ester or A23187 was obtained at 0.6-0.7 microM free extracellular Ca2+ and greater than or equal to 100 microM free Ca2+ was required to stimulate PGE2 formation maximally. The calmodulin antagonist R24571 partially inhibited the release of PGE2 elicited by zymosan and A23187 but not by phorbol ester or arachidonic acid. Verapamil and nifedipine, two calcium channel blockers, had no effect on the formation of PGE2 irrespective of the stimulus. TMB 8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester] an intracellular calcium antagonist, inhibited the synthesis of PGE2 induced by zymosan and phorbol ester. The superoxide formation following the addition of zymosan and phorbol ester was not influenced by removal of calcium ions from the medium or by addition of the various calcium antagonists. The data presented here suggest that Ca2+-dependent reactions are involved in the synthesis of prostanoids induced by zymosan and phorbol ester and that both extracellular Ca2+ and mobilization of Ca2+ from intracellular stores are needed to induce maximally the production of prostanoids in cultured rat Kupffer cells.     

Activation of phospholipase C is not correlated to the formation of prostaglandins and superoxide in cultured rat liver macrophages.

This study evaluates the role of inositol phosphates as possible mediators of the activation of phospholipase A2 and NADPH oxidase in cultured rat liver macrophages. Inositol phosphate formation was achieved by zymosan, immune complexes, latex particles and calcium ionophore while the release of arachidonic acid and the formation of prostaglandin E2 was also elicited by phorbol ester and NaF, but not by latex particles; generation of superoxide was obtained by zymosan and phorbol ester only. The kinetics of the formation of inositol phosphates revealed that within the first few minutes after zymosan addition inositol trisphosphate was formed, followed by inositol bisphosphate and inositol monophosphate. Pre-treatment of the cells with dexamethasone or removal of extracellular calcium led to an inhibition of the zymosan-induced formation of inositol phosphates and prostaglandin E2 but had no effect on the generation of superoxide; inhibition of the Na+/H+ exchanger by removal of extracellular sodium ions led to a decrease of the zymosan-induced synthesis of prostaglandin E2, but did not affect the formation of inositol phosphates and superoxide. Pre-treatment of the cells with phorbol ester decreased the zymosan-induced synthesis of prostaglandin E2 and superoxide, but even enhanced the zymosan-induced formation of inositol phosphates. These data indicate that in cultured rat liver macrophages the formation of prostaglandins and superoxide cannot be correlated to an activation of phospholipase C.     

Differential inhibition of prostaglandin and superoxide production by dexamethasone in primary cultures of rat Kupffer cells

Dexamethasone inhibited the stimulus-induced prostaglandin E2 formation by rat Kupffer cells in primary culture, e.g. after treatment with zymosan, phorbol ester, calcium ionophore A23187, platelet-activating factor or lipopolysaccharide. Prostaglandin E2 production from added free arachidonic acid was not influenced by the hormone. The time course, as well as the partial inhibition of the hormone effect by actinomycin D and cycloheximide, point to the hormone-induced formation of a protein which regulates phospholipase A2. The hormone did not affect the phagocytotic activity of the Kupffer cells. The quantity of [3H]arachidonic acid incorporated into phospholipids was also not altered by dexamethasone. After stimulation with zymosan, [3H]arachidonic acid was liberated from phosphatidylcholine only. Superoxide generation by rat Kupffer cells was induced by zymosan, phorbol ester and, to a much smaller extent, by platelet-activating factor. A23187 and lipopolysaccharide were without effect. In contrast to prostaglandin formation, the generation of superoxide was not influenced by dexamethasone. These results indicate that in cultured rat Kupffer cells prostaglandin formation and superoxide generation are independently triggered processes.     

Signal transduction in endotoxin-stimulated synthesis of TNF-alpha and prostaglandin E2 by rat Kupffer cells. Role of extracellular calcium ions and protein kinase C.

Endotoxin is a well established elicitor of cytokine production in mononuclear cells. Nevertheless, the path of signal transduction between the crucial contact of the cells with endotoxin (lipopolysaccharide) and the synthesis and release of the mediators is yet poorly understood. In particular, the involvement of Ca2+ and protein kinase C in this process is still a matter of controversy. Here, it will be demonstrated that removal of extracellular Ca2+ by EGTA does not have a significant effect on the endotoxin-stimulated production of tumor necrosis factor-alpha (TNF-alpha) and on total protein synthesis in rat Kupffer cells. However, the release of prostaglandin E2 could not be raised above the basal level under these conditions. Treatment with inhibitors of protein kinase C such as the isoquinoline derivative, H-7, or staurosporin is without influence on TNF-alpha synthesis. The depletion of protein kinase C through preincubation of rat Kupffer cells with phorbol 12-myristate 13-acetate for 24 h was also without effect on TNF-alpha production. The effectiveness of these inhibitors under the conditions used was ascertained by measurement of the O2- release from the same cell batches. Superoxide production known as protein kinase C-dependent in Kupffer cells (Dieter et al. (1986) Eur. J. Biochem. 86, 451-457) was suppressed in a dose-dependent manner by staurosporin or after prolonged pretreatment with the phorbol ester. H-7 decreased superoxide production only slightly in high doses that severely harm the Kupffer cells. Prostaglandin E2 release, although clearly protein-kinase C-dependent in phagocytosing rat Kupffer cells, is not decreased following exposure to lipopolysaccharide in the presence of protein kinase C inhibitors.     

Protein kinase C involved in zymosan-induced release of arachidonic acid and superoxide but not in calcium ionophore-elicited arachidonic acid release or formation of prostaglandin E2 from added arachidonate.

Zymosan and phorbol ester induced in liver macrophages the release of arachidonic acid, prostaglandin E2, and superoxide; the calcium ionophore A 23187 elicited a release of arachidonic acid and prostaglandin E2 but not of superoxide, and exogenously added arachidonic acid led to the formation of prostaglandin E2 only. The zymosan- and phorbol-ester-induced release of arachidonic acid, prostaglandin E2, and superoxide was dose-dependently inhibited by staurosporine and K252a, two inhibitors of protein kinase C, and by pretreatment of the cells with phorbol ester which desensitized protein kinase C. The release of arachidonic acid or prostaglandin E2 following the addition of A 23187 or arachidonic acid was not affected by these treatments. Zymosan and phorbol ester but not A 23187 or arachidonic acid induced a translocation of protein kinase C from the cytosol to membranes in intact cells. These results demonstrate an involvement of protein kinase C in the zymosan- and phorbol-ester-induced release of arachidonic acid, prostaglandin E2, and superoxide; the release of arachidonic acid and prostaglandin E2 elicited by A 23187 and the formation of prostaglandin E2 from exogenously added arachidonic acid, however, is independent of an activation of protein kinase C.     

A selective defect in arachidonic acid release from macrophage membranes in high potassium media

Murine peritoneal macrophages cultured in minimal essential medium (alpha-MEM; 118 mM Na+, 5 mM K+) released arachidonic acid (20:4) from phospholipids on encountering a phagocytic stimulus of unopsonized zymosan. In high concentrations of extracellular K+ (118 mM), 3H release from cells prelabeled with [3H]20:4 was inhibited 80% with minimal reduction (18%) in phagocytosis. The inhibitory effect of K+ on 20:4 release was fully reversed on returning cells to medium containing Na+ (118 mM). Preingestion of zymosan particles by macrophages maintained in high K+ medium resulted in cells being "primed" for 20:4 release, which was only effected (without the further addition of particles) by changing the medium to one containing Na+. In contrast, 20:4 release from cells stimulated with the calcium ionophore A23187 was unimpaired by the elevated K+ medium, suggesting no direct effect of high K+ on the phospholipase. Macrophages stimulated with zymosan in alpha-MEM metabolized the released 20:4 to prostacyclin, prostaglandin E2 (PGE2), and leukotriene C (LTC). The smaller quantity of released 20:4 in high K+ medium was recovered as 6-Keto-PGF1 alpha, the breakdown product of prostacyclin, and PGE2. No LTC was synthesized. In high K+, resting (no zymosan) macrophages synthesized hydroxyeicosatetraenoic acids from exogeneously supplied 20:4 in proportions similar to cells maintained in alpha-MEM. These findings and the similarity of products (including LTC) produced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 stimulated cells in alpha-MEM and high K+ medium indicated that the cyclooxygenase and lipoxygenase pathway enzymes were not directly inhibited by high extracellular K+. We conclude that high concentrations of extracellular K+ uncouple phagocytosis of unopsonized zymosan from the induction of the phospholipase responsible for the 20:4 cascade and suggest that the lesion is at the level of signal transduction between the receptor-ligand complex and the phospholipase.     

Effect of RNA and protein synthesis inhibitors on the release of inflammatory mediators by macrophages responding to phorbol myristate acetate

The interaction of phorbol myristate acetate with resident populations of mouse peritoneal macrophages causes an increased release of arachidonic acid followed by increased synthesis and secretion of prostaglandin E2 and 6-keto-prostaglandin F1 alpha. In addition, phorbol myristate acetate causes the selective release of lysosomal acid hydrolases from resident and elicited macrophages. These effects of phorbol myristate acetate on macrophages do not cause lactate dehydrogenase to leak into the culture media. The phorbol myristate acetate-induced release of arachidonic acid and increased synthesis and secretion of prostaglandins by macrophages can be inhibited by RNA and protein synthesis inhibitors, whereas the release of lysosomal hydrolases is unaffected. 0.1 microgram/ml actinomycin D blocked the increased prostaglandin production due to this inflammatory agent by more than 80%, and 3 microgram/ml cycloheximide blocked prostaglandin production by 78%. Similar results with these metabolic inhibitors were found with another stimulator of prostaglandin production, zymosan. However, these inhibitors do not interfere with lysosomal hydrolase releases caused by zymosan or phorbol myristate acetate. It appears that one of the results of the interaction of macrophages with inflammatory stimuli is the synthesis of a rapidly turning-over protein which regulates the production of prostaglandins. It is also clear that the secretion of prostaglandins and lysosomal hydrolases are independently regulated.     

Superoxide Generation by Kupffer Cells and Priming of Neutrophils During Reperfusion After Hepatic Ischemia

The objective of this study was to identify the cellular source of the vascular oxidant stress in hepatic ischemia-reperfusion injury in male Fischer rats. Nonparenchymal cells (Kupffer cells, endothelial cells) and neutrophils were isolated from postischemic liver lobes by collagenase-pronase digestion followed by centrifugal elutriation. The spontaneous and stimulated generation of superoxide by these cells were subsequently quantified in vitro. Large Kupffer cells from the postischemic lobes spontaneously generated 300% more superoxide than similar cells from control animals. No difference in spontaneous superoxide formation was found when the small Kupffer cells were compared. No other cells isolated from the postischemic lobes or control liver including neutrophils released any detectable superoxide spontaneously. In contrast, small Kupffer cells and neutrophils from the postischemic liver generated significantly more superoxide after stimulation with phorbol ester or opsonized zymosan than the controls. The considerably higher response with zymosan stimulation compared to phorbol ester indicates a particular priming for a receptor-mediated signal transduction pathway during reperfusion. These studies demonstrate that Kupffer cells are the principal source of the oxidant stress during the initial reperfusion phase after hepatic ischcmia. The priming of neutrophils during this time may be an important factor for the later neutrophil-induced injury phase.     

Synthesis of prostanoids and cyclic nucleotides by phagocytosing rat Kupffer cells

Rat Kupffer cells in monolayer culture were allowed to phagocytose unopsonized zymosan granules. They responded with a strongly stimulated synthesis and release of prostanoids, mainly the immunologically determined prostaglandins PGE2 and PGF2 alpha. The same response could be obtained by treatment with the calcium ionophore A23187. The effects of the ionophore and the zymosan particles were of the same magnitude but not additive. The rapid uptake of Ca2+ after contact with phagocytosable material recently described by us [(1983) Eur. J. Biochem. 131, 539-543] appears to mediate the enhanced prostaglandin synthesis. That response was suppressed not only by indomethacin but also by trifluoperazine which does not inhibit Ca2+ entry in the Kupffer cells. Similar effects by R24571 and 4-bromophenacyl bromide support the participation of calcium-calmodulin and of phospholipase A2. The calcium channel blocker Verapamil did not influence the zymosan-provoked production of prostaglandin PGE2 nor were any indications obtained for a feedback inhibition by PGE1 or PGE2. Contact with zymosan resulted in a rapid but transient rise of the intracellular levels of cAMP and cGMP: 10 nM indomethacin completely blocked the increase of both cyclic nucleotides while trifluoperazine elicited different responses in the cAMP and cGMP levels. The stimulated release of prostaglandin E2 was inhibited in a dose-dependent manner by nordihydroguaiaretic acid, an inhibitor of 5-lipoxygenase and by FPL 55712, known as a receptor antagonist for some leukotrienes. This suggests a regulatory role for its metabolites on prostaglandin synthesis.     

Synergistic Effect of Prostaglandin E2 and Ouabain on Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells

We recently reported that prostaglandin E2 (PGE2) stimulated phosphoinositide metabolism in cultured bovine adrenal chromaffin cells and that PGE2 and ouabain, an inhibitor of Na+,K+-ATPase, synergistically induced a gradual secretion of catecholamines from the cells. The effect on catecholamine release was specific for prostaglandin E1 (PGE1) and PGE2 among prostaglandins tested (E1 = E2 greater than F2 alpha greater than D2). The release evoked by PGE2 plus ouabain was greatly reduced in Na+-depleted medium and not observed in Ca2+-free medium. Here we examined the synergistic effect of PGE2 and ouabain on the release with specific reference to ion fluxes. Regardless of the presence of PGE2, ouabain stimulated the release in a dose-dependent manner with half-maximal stimulation at 1 microM, and omission of K+ from the medium, a condition which suppresses the Na+,K+-ATPase activity, also enhanced the release from chromaffin cells exposed to PGE2. Ouabain induced a continuous accumulation of 22Na+ and 45Ca2+, as well as secretion of catecholamines. Although PGE2 itself showed hardly any effects on these cellular responses, PGE2 potentiated all of them induced by ouabain. The time course of catecholamine release was correlated with that of accumulation of 45Ca2+ rather than with that of 22Na+. The release evoked by PGE2 and ouabain was inhibited in a dose-dependent manner by amiloride and the analogue ethylisopropylamiloride, inhibitors of the Na+,H+-antiport, but not by the Na+-channel inhibitor tetrodotoxin nor by the nicotinic receptor antagonist hexamethonium. Ethylisopropylamiloride at 1 microM inhibited PGE2-enhanced accumulation of 22Na+ and 45Ca2+ and release of catecholamine by 40, 83, and 71%, respectively. Activation of the Na+,H+-antiport by elevation of the extracellular pH from 6.6 to 8.0 increased the release of catecholamines linearly. Furthermore, PGE2 induced a sustained increase in intracellular pH by about 0.1 pH unit above the resting value, which was abolished by amiloride or in Na+-free medium. These results taken together indicate that PGE2 activates the Na+,H+-antiport by stimulating phosphoinositide metabolism and that the increase in intracellular Na+ by both inhibition of Na+,K+-ATPase and activation of Na+,H+-antiport may lead to the redistribution of Ca2+, which is the initial trigger of catecholamine release.     

Effect of fluoride, pertussis and cholera toxin on the release of arachidonic acid and the formation of prostaglandin E2, D2, superoxide and inositol phosphates in rat liver macrophages.

Fluoride elicited in liver macrophages a release of arachidonic acid and prostaglandins but not formation of inositol phosphates or superoxide. The effects of fluoride required extracellular calcium and were inhibited by staurosporine and by phorbol ester treatment of the cells. Furthermore, fluoride led to a translocation of protein kinase C from the cytosol to membranes. This indicates that the calcium-dependent protein kinase C is involved in the action of fluoride. Cholera toxin decreased the zymosan-induced release of arachidonic acid and prostaglandins but not of inositol phosphates or superoxide. Pertussis toxin ADP-ribosylated a 41,000 molecular weight membrane protein; enhanced specifically the zymosan-induced formation of prostaglandin(PG)E₂ but did not affect the zymosan-induced release of arachidonic acid, PGD₂, inositol phosphates or superoxide. These data suggest that activation of phospholipase (PL)A₂, phosphoinositide (PI)-specific PLC and NADPH oxidase in liver macrophages is most probably not mediated by activation of guanine nucleotide binding (G)-proteins coupled directly to these enzymes.     

Effect of RNA and protein synthesis inhibitors on the release of inflammatory mediators by macrophages responding to phorbol myristate acetate

The interaction of phorbol myristate acetate with resident populations of mouse peritoneal macrophages causes an increased release of arachidonic acid followed by increased synthesis and secretion of prostaglandin E₂ and 6-keto-prostaglandin F_1α. In addition, phorbol myristate acetate causes the selective release of lysosomal acid hydrolases from resident and elicited macrophages. These effects of phorbol myristate acetate on macrophages do not cause lactate dehydrogenase to leak into the culture media. The phorbol myristate acetate-induced release of arachidonic acid and increased synthesis and secretion of prostaglandins by macrophages can be inhibited by RNA and protein synthesis inhibitors, whereas the release of lysosomal hydrolases is unaffected. 0.1 μg/ml actinomycin D blocked the increased prostaglandin production due to this inflammatory agent by more than 80%, and 3 μg/ml cycloheximide blocked prostaglandin production by 78%. Similar results with these metabolic inhibitors were found with another stimulator of prostaglandin production, zymosan. However, these inhibitors do not interfere with lysosomal hydrolase releases caused by zymosan or phorbol myristate acetate. It appears that one of the results of the interaction of macrophages with inflammatory stimuli is the synthesis of a rapidly turning-over protein which regulates the production of prostaglandins. It is also clear that the secretion of prostaglandins and lysosomal hydrolyses are independently regulated.     

Effect of fluoride, pertussis and cholera toxin on the release of arachidonic acid and the formation of prostaglandin E2, D2, superoxide and inositol phosphates in rat liver macrophages

Fluoride elicited in liver macrophages a release of arachidonic acid and prostaglandins but not formation of inositol phosphates or superoxide. The effects of fluoride required extracellular calcium and were inhibited by staurosporine and by phorbol ester treatment of the cells. Furthermore, fluoride led to a translocation of protein kinase C from the cytosol to membranes. This indicates that the calcium-dependent protein kinase C is involved in the action of fluoride. Cholera toxin decreased the zymosan-induced release of arachidonic acid and prostaglandins but not of inositol phosphates or superoxide. Pertussis toxin ADP-ribosylated a 41,000 molecular weight membrane protein; enhanced specifically the zymosan-induced formation of prostaglandin(PG)E₂ but did not affect the zymosan-induced release of arachidonic acid, PGD₂, inositol phosphates or superoxide. These data suggest that activation of phospholipase (PL)A₂, phosphoinositide (PI)-specific PLC and NADPH oxidase in liver macrophages is most probably not mediated by activation of guanine nucleotide binding (G)-proteins coupled directly to these enzymes.     

Prostaglandin responses in isolated perfused rat liver: Ca2+ and K+ fluxes, hemodynamic and metabolic effects

Addition of prostaglandin F2 alpha and prostaglandin E2 to isolated perfused rat liver led to a dose-dependent, transient net Ca2+ release, which was completed within 3 min. Withdrawal of the prostaglandins resulted in a Ca2+ re-uptake over a period of about 10 min. Simultaneously, these prostaglandins induced an increase of portal pressure, stimulated hepatic glucose output and 14CO2 production from [1-14C]glutamate and led to K+ movements across the hepatocyte plasma membrane similar to those observed with other Ca2+-mobilizing agents. With prostaglandin F2 alpha there was a close correlation between the net Ca2+ release and the maximal rate of initial net K+ uptake by the liver (linear regression coefficient r = 0.902; n = 20). Prostaglandin F2 alpha was more effective than prostaglandin E2 or D2. Because prostaglandins are known to be produced by hepatic non-parenchymal cells during stimulation by phagocytosis or by addition of extracellular ATP or UTP, these data suggest an interaction between non-parenchymal and parenchymal liver cells and point to a modulating role of prostaglandins in hepatic metabolism and microcirculation, which is mediated by Ca2+-mobilizing mechanisms.     

4-Bromophenacyl bromide inhibits prostaglandin D2 synthesis from arachidonic acid rather than phospholipase A2 activity in liver macrophages

4-Bromophenacyl bromide at a concentration of 50 microM does not inhibit phospholipase A2 activity in liver macrophages. Rather, this compound increases the amount of radioactivity released from [3H]arachidonate-prelabeled Kupffer cells and leads to the formation of small amounts of thromboxane, prostaglandin D2 and prostaglandin E2. Also the zymosan-induced formation of thromboxane and prostaglandin E2 from endogenous sources which is thought to involve phospholipase A2 remains unaffected in the presence of this compound. The generation of superoxide and the formation of prostaglandin D2 from arachidonate and after stimulation of the cells with zymosan, however, are blocked by 4-bromophenacyl bromide. Furthermore, this compound suppresses the incorporation of externally added arachidonate into membrane lipids of the cells. 4-Bromophenacyl bromide seems, therefore, not to be a useful tool to demonstrate the involvement of phospholipase A2 in complex biological systems.     

RO 31-8220 and RO 31-7549 show improved selectivity for protein kinase C over staurosporine in macrophages

Two new potent protein kinase C inhibitors, RO 31-8220 and RO 31-7549, and staurosporine were found to inhibit dose-dependently the phorbol ester-induced formation of prostaglandin E2 and superoxide in cultured liver macrophages. Prostaglandin E2 formation from exogenously added arachidonate was not affected by these compounds. The zymosan-induced formation of inositol phosphates was decreased by simultaneous addition of phorbol ester and was enhanced by prior desensitization of protein kinase C indicating that protein kinase C negatively modulates phospholipase C activation in these cells. While staurosporine suppressed almost totally the zymosan-induced formation of inositol RO 31-8220 and RO 31-7549 inhibited the protein kinase C-mediated effect on inositol phosphate formation, only. Phagocytosis of zymosan was not affected by RO 31-8220 and RO 31-7549 but was decreased by staurosporine. These results demonstrate that two new potent protein kinase C inhibitors, RO 31-8220 and RO 31-7549, are more selective in their actions as staurosporine and are useful tools to determine an involvement of protein kinase C in cellular systems.     

Relationship of prostaglandin secretion by rabbit alveolar macrophages to phagocytosis and lysosomal enzyme release

The phospholipids of rabbit alveolar macrophages were pulse-labelled with [(14)C]-arachidonic acid, and the subsequent release of labelled prostaglandins was measured. Resting macrophages released measurable amounts of arachidonic acid, the prostaglandins E(2), D(2) and F(2alpha) and 6-oxoprostaglandin F(1alpha). Phagocytosis of zymosan increased the release of arachidonic acid and prostaglandins to 2.5 times the control value. In contrast, phagocytosis of inert latex particles had no effect on prostaglandin release. Indomethacin inhibited the release of prostaglandin, and, at high doses (20mug/ml), increased arachidonic acid release. Analysis of the cellular lipids showed that after zymosan stimulation the proportion of label was decreased in phosphatidylcholine, but not in other phospholipids or neutral lipids. Cytochalasin B, at a dose of 2mug/ml, inhibited the phagocytosis induced by zymosan but increased prostaglandin synthesis to 3.4 times the control. These data suggest that the stimulation of prostaglandin synthesis by zymosan is not dependent on phagocytosis. Exposure to zymosan also resulted in the release of the lysosomal enzyme, acid phosphatase. Furthermore, cytochalasin B augmented the zymosan-stimulated release of acid phosphatase at the same dose that stimulated prostaglandin synthesis. However, indomethacin, at a dose that completely inhibited prostaglandin synthesis, failed to block the lysosomal enzyme release. Thus despite some parallels between the release of prostaglandins and lysosomal enzymes, endogenous prostaglandins do not appear to mediate the release of lysosomal enzymes. The prostaglandins released from the macrophages may function as humoral substances affecting other cells.     

Hormonal control of glycogenolysis in parenchymal liver cells by Kupffer and endothelial liver cells

Conditioned media of isolated Kupffer and endothelial liver cells were added to incubations of parenchymal liver cells, in order to test whether secretory products of Kupffer and endothelial liver cells could influence parenchymal liver cell metabolism. With Kupffer cell medium an average stimulation of glucose production by parenchymal liver cells of 140% was obtained, while endothelial liver cell medium stimulated with an average of 127%. The separation of the secretory products of Kupffer and endothelial liver cells in a low and a high molecular weight fraction indicated that the active factor(s) had a low molecular weight. Media, obtained from aspirin-pretreated Kupffer and endothelial liver cells, had no effect on the glucose production by parenchymal liver cells. Because aspirin blocks prostaglandin synthesis, it was tested if prostaglandins could be responsible for the effect of media on parenchymal liver cells. It was found that prostaglandin (PG) E1, E2, and D2 all stimulated the glucose production by parenchymal liver cells, PGD2 being the most potent. Kupffer and endothelial liver cell media as well as prostaglandins E1, E2, and D2 stimulated the activity of phosphorylase, the regulatory enzyme in glycogenolysis. The data indicate that prostaglandins, present in media from Kupffer and endothelial liver cells, may stimulate glycogenolysis in parenchymal liver cells. This implies that products of Kupffer and endothelial liver cells may play a role in the regulation of glucose homeostasis by the liver.     

Platelet Activating Factor Stimulates and Primes the Liver, Kupffer Cells and Neutrophils to Release Superoxide Anion

Platelet activating factor (PAF) is considered a key mediator in eliciting the immunologic and metabolic consequences of endotoxic shock and sepsis. Release of oxygen-derived radicals is one of the important and relevant actions of PAF. This study examines the direct and priming effects of PAF on superoxide anion release by perfused liver, isolated Kupffer cells and blood neutrophils. One hour after PAF infusion at a dose of 2.2 μ/kg body weight a significant amount of superoxide release (0.71 ± 0.01 nmol/min/g liver) was measured in the perfused liver compared with the control livers (0.2 ± 0.01). In the in vitro presence of either phorbol ester or opsonized zymosan, superoxide release following PAF treatment in vivo was significantly increased to 1.36 ± 0.2 and 4.29 ± 0.36, respectively. The administration of PAF receptor antagonist (SDZ 63-441) almost completely inhibited the release of this radical. Kupffer cells (KC1, KC2, KC3) and blood neutrophils isolated from PAF-treated rats were also primed for increased production when these cells were challenged in vitro by the activator of protein kinase C, opsonin-coated zymosan as well as the chemotactic factors, complement 5a and F-met-leu-phe. PAF added in vitro to the perfused livers, isolated Kupffer cells or neutrophils from normal animals stimulated the release of superoxide with or without the above agonists. The direct stimulatory effect of PAF on superoxide release was inhibited by the PAF receptor antagonist in vitro. The role of PAF in the LPS-induced superoxide release by the perfused liver was also examined by the administration of PAF antagonist in endotoxic rats. The antagonist inhibited the LPS-mediated superoxide release at 1 hr, but not at 3 hr post-treatment. These results indicate that PAF stimulates and primes the hepatic elements to release superoxide. PAF may be an important factor during the early phase of endotoxemia, while other bioactive substances may take over at a later phase. Therefore, PAF is a key mediator that can directly enhance the release of toxic oxygen-derived radicals which may contribute to organ failure during endotoxemia or sepsis.     

Calcium regulation of tissue plasminogen activator and prostaglandin biosynthesis in HeLa cells

Phorbol 12-myristate 13-acetate, 1-20 nM, induced the synthesis in HeLa cells of a 65 200 Mr tissue-type plasminogen activator, and of prostaglandin E2. Omission of Ca2+ from the incubation medium inhibited the induction of plasminogen activator synthesis by 40-60% and abolished the induction of prostaglandin E2 synthesis. Maximal plasminogen activator synthesis could be maintained at extracellular Ca2+ concentrations of approx. 0.1 mM, while maximal prostaglandin synthesis required at least 0.45-0.9 mM Ca2+. The induction of each factor was inhibited by 10-100 microM 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular C2+ mobilization. Prostaglandin synthesis, but not plasminogen activator synthesis, was also inhibited by 10-100 microM verapamil and nifedipine, which inhibit intracellular Ca2+ uptake via the so-called 'slow-channels' and by 0.5-10 microM trifluoperazine, an inhibitor of calmodulin. Neither plasminogen activator synthesis nor prostaglandin synthesis were stimulated by 5-50 microM 1-oleoyl-2-acetylglycerol or 1-250 microM 1,2-dioctanoylglycerol, alone and in combination with 50 nM-1 microM ionophore A23187. These results indicate that the synthesis of plasminogen activator and prostaglandins in HeLa cells is Ca2+-dependent, and that the Ca2+ requirements for each process are not identical. Thus, Ca2+ regulation of the production of tissue plasminogen activator and prostaglandin E2 occurs at multiple points in their biosynthetic pathways.