Occurrence of selenoprotein enzyme activities and mRNA in bovine mammary tissue.

To elucidate the possible role of selenoproteins for milk formation and mammary gland physiology, the activities of selenoprotein enzymes and the expression of selenoprotein genes were studied in the bovine mammary gland. Messenger RNA was demonstrated for selenoprotein P, thioredoxin reductase 1, and for glutathione peroxidase (GPx) 1, 3, and 4. Significant differences in mRNA expression between the cows were seen for GPx 1 and GPx 3. The enzyme activity of glutathione peroxidase varied approximately 16-fold among cows, and the activity of thioredoxin reductase and the concentration of soluble Se varied approximately 6-fold among cows. There were positive correlations between glutathione peroxidase activity, thioredoxin reductase activity, and soluble Se, the correlation between glutathione peroxidase activity and soluble Se being the strongest. Furthermore, selenoprotein P expression correlated with GPx 1 mRNA expression and with soluble Se. There was also a correlation between glutathione peroxidase activity and the mRNA expression of GPx 1. The general conclusion from the data was that the activity of glutathione peroxidase and thioredoxin reductase and the mRNA expression of selenoprotein P and GPx 1 and 3 were influenced by Se status, but the expression of GPx 4 and thioredoxin reductase 1 were not. These results indicate that the Se status in mammary tissue is an important regulator of selenoprotein activity and expression, but that other factors are also in operation.

Studies on the effective size of phospholipid headgroups in bilayer vesicles using lectin-glycolipid interaction as a steric probe.

An experimental approach is described which provides information about the relative, effective size of phospholipid headgroups in bilayer vesicles. It is based on determination of the binding of lectins (Ricinus communis agglutinin or concanavalin A) to synthetic glycolipids inserted in such vesicles, using a vesicle agglutination assay. It is shown that the ability of a glycolipid containing a shorter (4-member) spacer arm to bind the appropriate lectin is highly sensitive to the headgroup structure of the surrounding phospholipid in mixed glycolipid-phospholipid vesicles. Furthermore, when the phospholipid was phosphatidate a change in protonation or in monovalent counter-ion species (Li+, NH+4, N(CH3)+4 or Na+) significantly influenced lectin binding. The interference with lectin binding described above was reduced when the glycolipid spacer arm was extended from a 4- to a 6-member length. Furthermore, the sensitivity to phospholipid headgroup structure or to changes in the ionic environment was completely eliminated when the glycolipid contained a longer (10- or 12-member) spacer arm between the hydrophobic part and the lectin-binding group. It is concluded that the modulation of lectin binding in the former case is due to steric inhibition determined by the effective (hydrated) size of the various phospholipid headgroups.

Promotion of acid-induced membrane fusion by basic peptides. Amino acid and phospholipid specificities.

The ability of oligo- and polymers of the basic amino acids L-lysine, L-arginine, L-histidine and L-ornithine to induce lipid intermixing and membrane fusion among vesicles containing various anionic phospholipids has been investigated. Among vesicle consisting of either phosphatidylinositol or mixtures of phosphatidic acid and phosphatidylethanolamine rapid and extensive lipid intermixing, but not complete fusion, was induced at neutral pH by poly-L-ornithine or L-lysine peptides of five or more residues. When phosphatidylcholine was included in the vesicles, the lipid intermixing was severely inhibited. Such lipid intermixing was also much less pronounced among phosphatidylserine vesicles. Poly-L-arginine provoked considerable leakage from the various anionic vesicles and caused significantly less lipid intermixing than L-lysine peptides at neutral pH. When the addition of basic amino acid polymer was followed by acidification to pH 5-6, vesicle fusion was induced. Fusion was more pronounced among vesicles containing phosphatidylserine or phosphatidic acid than among those containing phosphatidylinositol, and occurred also with vesicles whose composition resembles that of cellular membranes (i.e., phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine, 50:30:20, by mol). Liposomes with this composition are resistant to fusion by Ca2+ or by acidification after lectin-mediated contact. The tight interaction among vesicles at neutral pH, resulting in lipid intermixing, does not seem to be necessary for the fusion occurring after acidification, but the basic peptides nevertheless appear to play a more active role in the fusion process than simply bringing the vesicles in contact. However, protonation of the polymer side chains and transformation of the polymer into a polycation does not explain the need for acidification, since the pH-dependence was quite similar for poly(L-histidine)- and poly(L-lysine)-mediated fusion.

Studies on the enzymatic pathways of calcium ionophore-induced phospholipid degradation and arachidonic acid mobilization in peritoneal macrophages.

Exposure of mouse peritoneal macrophages to ionophore A23187 caused a rapid and extensive Ca2+-dependent phospholipid degradation and mobilization of arachidonic acid. Phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine all contributed to the arachidonic acid release, although the ethanolamine phospholipids incorporated [3H]arachidonic acid more slowly during the prelabeling period, particularly the plasmalogen form. Several enzymatic pathways could be positively identified as contributing to the ionophore-induced phospholipid degradation by the use of several different radiolabeled phospholipid precursors: (i) a phospholipase A-mediated deacylation, (ii) a phosphodiesterase (phospholipase C) reaction, rapidly generating diacylglycerol units from inositol phospholipids, and (iii) enzymatic processes generating diacylglycerol and CDP- and phosphocholine/ethanolamine from phosphatidylcholine/ethanolamine. The diacylglycerol formed was in part phosphorylated and in part hydrolyzed to monoacylglycerol, with retention of its arachidonic acid. These, and other, results indicate that the Ca2+-ionophore activates several apparently distinct phospholipid-degrading processes, in contrast to stimuli acting via cellular receptors.

Evidence for a catalytic role of phospholipase A in phorbol diester- and zymosan-induced mobilization of arachidonic acid in mouse peritoneal macrophages.

Inositol phospholipid degradation and release of phospholipid-bound arachidonic acid was induced in intact peritoneal macrophages by exposure to phorbol myristate acetate (PMA) or zymosan particles. PMA, known to activate protein kinase C, selectively enhanced the deacylation of phosphatidylinositol (i.e., degradation by phospholipase A), while zymosan particles enhanced degradation via both phospholipase A and inositol lipid phosphodiesterase (phospholipase C). The release of arachidonic acid was found to correlate with the degradation of phosphatidylinositol by the phospholipase A pathway and could be dissociated from the phospholipase C-catalyzed cleavage of inositol phospholipids in several experimental situations: (i) when PMA was the stimulus, (ii) by the difference in Ca2+ dependence between the two enzymatic processes when zymosan was the stimulus and (iii) by the parallel inhibition by chlorpromazine of the phospholipase A pathway and arachidonic acid release, but not inositol phospholipid phosphodiesterase. In addition, phloretin, a reported inhibitor of protein kinase C, was found to inhibit arachidonic acid release and the deacylation of phosphatidylinositol. The results are consistent with a model in which arachidonic acid release is mediated by phospholipase(s) A and in which PMA or the phosphodiesterase-catalyzed degradation of phosphoinositides causes activation of the phospholipase A pathway via protein kinase C.

Phosphatidylethanol counteracts calcium-induced membrane fusion but promotes proton-induced fusion.

The susceptibility of phosphatidylethanol-containing lipid vesicles towards Ca2+- and proton-induced fusion has been investigated, using a system of interacting vesicles. The results show that phosphatidylethanol-rich vesicles are quite resistant to Ca2+-induced fusion while being highly sensitive to proton-induced fusion. Inclusion of phosphatidylethanol was also found to promote and inhibit, respectively, the proton-induced and Ca2+-induced fusion of bilayer vesicles containing also phosphatidylethanolamine and either phosphatidylserine or phosphatidic acid. Thus, phosphatidylethanol affected Ca2+- and proton-induced fusion in opposite directions, in contrast to the naturally occurring anionic phospholipids phosphatidic acid, phosphatidylserine and phosphatidylinositol, which affect the sensitivity to Ca2+- and H+-induced fusion in the same direction. However, the fusion competence of phosphatidylethanol vesicles in response to both Ca2+ and H+ was inversely related to the apparent thickness of the polar headgroup layer, determined by using lectin-glycolipid interaction as a steric probe, as previously found for vesicles containing naturally occurring anionic phospholipids.

A role for protein kinase C-mediated phosphorylation in the mobilization of arachidonic acid in mouse macrophages.

Mouse peritoneal macrophages respond to activators of protein kinase C and to zymosan particles and calcium ionophore by rapid enhancement of a phospholipase A pathway and mobilization of arachidonic acid. The pattern of protein phosphorylation induced in these cells by 4 beta-phorbol 12-myristate 13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol, exogenous phospholipase C and by zymosan and ionophore A23187 was found to be virtually identical. The time course of phosphorylation differed among the phosphoprotein bands and in only some of those identified (i.e., those of 45 and 65 kDa) was the phosphorylation sufficiently rapid to be involved in the activation of the phospholipase A pathway. Phosphorylation of lipocortin I or II could not be detected. Down-regulation of kinase C by a 24-h pretreatment with PMA resulted in extensive inhibition of both protein phosphorylation and the mobilization of arachidonic acid in response to PMA or dioctanoylglycerol. The phosphorylation of the 45 kDa protein in response to zymosan and A23187 was also inhibited by pretreatment with PMA, while only arachidonic acid release induced by zymosan was inhibited by this pretreatment. Depletion of intracellular calcium had little effect on kinase C-dependent phosphorylation, although arachidonic acid mobilization is severely inhibited under these conditions. Bacterial lipopolysaccharide and lipid A induced a phosphorylation pattern different from that induced by PMA, and down-regulation of protein kinase C did not affect lipopolysaccharide-induced protein phosphorylation. The results indicate (i) that protein kinase C plays a critical role also in zymosan-induced activation of the phospholipase A pathway mobilizing arachidonic acid; (ii) that such activation requires calcium at some step distal to kinase C-mediated phosphorylation and (iii) that phosphorylation of lipocortins does not explain the kinase C-dependent activation.

Protein-mediated intermembrane contact specifically enhances Ca2+-induced fusion of phosphatidate-containing membranes.

Ca2+-induced fusion of glycolipid-phospholipid vesicles containing several different anionic phospholipids was investigated, with and without lectin-mediated intervesicle contact. In vesicles containing phosphatidylserine, phosphatidylinositol or its mono- or diphosphate as the anionic phospholipid fusion was induced only at 1-10 mM Ca2+ both in the absence and presence of lectin. In contrast, the Ca2+-threshold for fusion of phosphatidate-containing vesicles was reduced to less than or equal to 0.1 mM Ca2+ by lectin-mediated intermembrane contact.

Control of membrane fusion by phospholipid head groups. I. Phosphatidate/phosphatidylinositol specificity.

We have studied the characteristics of fusion of large unilamellar vesicles composed of phosphatidate and phosphatidylinositol alone and in mixtures with other naturally occurring phospholipids. Fusion was induced by the addition of Ca2+ or Mg2+ and was monitored by detecting the mixing of aqueous vesicle contents. Release of vesicle contents was measured by dequenching of carboxyfluorescein fluorescence. Aggregation was monitored by 90 degrees light scattering. The results indicated striking differences with respect to the fusion capacity of the different vesicles. Phosphatidate vesicles fuse in the presence of both Ca2+ and Mg2+ at threshold concentration ranges of 0.03-0.1 mM (Ca2+) and 0.07-0.15 mM (Mg2+) depending on the pH of the medium, 8.5-6.0, respectively. In contrast, phosphatidylinositol vesicles do not fuse with either Ca2+ or Mg2+ even at 50 mM concentrations, in spite of aggregation induced by both cations in the range of 5-10 mM. A large difference in terms of fusion capacity is retained even when these two phospholipids are mixed with phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in 2 : 2 : 4 : 2 molar ratios. The results are discussed in terms of the molecular mechanism of membrane fusion and the possible role of the metabolic interconversion of phosphatidylinositol to phosphatidate as an on-off control system for membrane fusion phenomena involved in secretion.

Metabolism of different monoacylphospholipids in isolated hepatocytes and the intact rat.

The 1-[3H] palmitoyl, 2-[3H] oleoyl, and 2-[14C] linoleoyl derivatives of sn-glycero-3-phosphoethanolamine and the corresponding derivatives of sn-glycero-3-phosphocholine were injected intraportally to rats and their incorporation into liver lipids was studied 15 min thereafter. Both the uptake by the liver and the degree of acylation was higher for the unsaturated compounds. The uptake of lysophosphatidylethanolamine was higher than that of lysophosphatidlycholine. The metabolism of 1-lysophosphatidylethanolamine was also studied in isolated hepatocytes. The degree of hydrolysis was much more prominent than in vivo. After injecting 2-[14C] linoleoyl derivatives, a large part of the 14C was recovered in the dienoic phospholipids. Subfractionation by reversed-phase partition chromatography showed that the isotope was located in the palmitoyllinoleoyl and stearoyl-linoleoyl fraction. The 100 X stearoly/(palmitoyl + stearoyl) ratio was 84 in dienoic phosphatidylethanolamine and 59 in dienoic phosphatidylcholine. This preference for stearic acid is significantly larger than in other pathways yielding dienoic phospholipids. It can be concluded that the monoacylphospholipid acyltransferase reactions operating at positions 1 or 2 yield different saturated acyl chain profiles in phosphatidylethanolamine and phosphatidylcholine of a specific unsaturation. This may be important in the regulation of the fatty acid composition of the membrane phospholipids.

Proton-induced membrane fusion. Role of phospholipid composition and protein-mediated intermembrane contact.

Glycolipid-phospholipid vesicles containing phosphatidate and phosphatidylethanolamine were found to undergo proton-induced fusion upon acidification of the suspending medium from pH 7.4 to pH 6.5 or lower, as determined by an assay for lipid intermixing based on fluorescence resonance energy transfer. Lectin-mediated contact between the vesicles was required for fusion. Incorporation of phosphatidylcholine in the vesicles inhibited proton-induced fusion. Vesicles in which phosphatidate was replaced by phosphatidylserine underwent fusion only when pH was reduced below 4.5, while no significant fusion occurred (pH greater than or equal to 3.5) when the anionic phospholipid was phosphatidylinositol. It is suggested that partial protonation of the polar headgroup of phosphatidate and phosphatidylserine, respectively, causes a sufficient reduction in the polarity and hydration of the vesicle surface to trigger fusion at sites of intermembrane contact.

Activation of phosphatidylinositol-4-phosphate kinase in rat liver plasma membranes by polyamines.

The formation of phosphatidylinositol 4,5-bisphosphate (PIP2) from endogenous substrate in rat liver plasma membranes was stimulated approximately 3-fold by 1 mM spermine, with half-maximal effect at 0.2 mM polyamine. This effect of spermine was due to enhancement of phosphatidylinositol-4-phosphate kinase activity rather than to a decrease in degradation of PIP2 formed or the substrate phosphatidylinositol 4-phosphate (PIP). The stimulation of phosphatidylinositol-4-phosphate kinase by spermine decreased to half at physiological ionic strength, and was not affected appreciably by variations in the concentration of ATP and MgCl2. Among several di- and polyamines only spermine and spermidine were effective. Although spermine may cause aggregation of membrane vesicles, thereby potentially increasing substrate availability for phosphatidylinositol-4-phosphate kinase, our results do not support such an explanation for the enhancement in enzyme activity. Phosphatidylinositol kinase activity, contrary to phosphatidylinositol-4-phosphate kinase, was not stimulated appreciably by spermine.

Phosphatidylinositol 3-kinase in zymosan- and bacteria-induced signalling to mobilisation of arachidonic acid in macrophages.

Stimulation of mouse peritoneal macrophages with zymosan or bacteria results in activation of 85-kDa cytosolic phospholipase A(2) (cPLA(2)) and release of arachidonate. We have investigated the role of phosphatidylinositol 3-kinase (PtdIns 3-kinase) in the signalling leading to activation of cPLA(2) and release of arachidonate in response to zymosan and the bacterium Prevotella intermedia. The specific PtdIns 3-kinase inhibitor wortmannin completely inhibited zymosan- and bacteria-induced release of arachidonate with an IC(50) value of 10-20 nM. Wortmannin also completely inhibited the zymosan-induced activation of cPLA(2), while the cPLA(2) activation by bacteria was partially inhibited by about 50%. Further experiments showed that zymosan-induced activation of extracellular signal-regulated kinase was inhibited, and bacteria-induced activation of the kinase strongly reduced, in the presence of wortmannin. Also zymosan-induced activation of p38 mitogen-activated protein kinase was inhibited by wortmannin, while p38 activation induced by bacteria was not. The zymosan- and bacteria-induced activation of phospholipase C, as determined by the generation of inositol phosphates, was also inhibited by wortmannin. Moreover, zymosan caused activation of PtdIns 3-kinase, which was totally inhibited by wortmannin. In contrast to zymosan and bacteria, arachidonate release induced by calcium ionophore alone, or further amplified by phorbol ester, was not sensitive to wortmannin. These results suggest that PtdIns 3-kinase constitutes a critical component in the zymosan- and bacteria-induced signalling leading to release of arachidonate and that PtdIns 3-kinase is positioned upstream of phospholipase C in this pathway.

Subcellular localization and enzymatic properties of rat liver phosphatidylinositol-4-phosphate kinase.

The phosphatidylinositol-4-phosphate kinase activity in rat liver showed a subcellular distribution different from that of phosphatidylinositol kinase. It was preferentially associated with plasma membrane-rich subcellular fractions, while no or minimal activity could be ascribed to mitochondria, lysosomes, Golgi membranes or the endoplasmic reticulum. The plasma membrane enzyme phosphorylated endogenous and exogenously added phosphatidylinositol 4-phosphate at comparable initial rates. The phosphorylation of endogenous substrate was strongly inhibited by Triton X-100, while the phosphorylation of added substrate was enhanced, suggesting that endogenous phosphatidylinositol 4-phosphate was readily available to the enzyme in unperturbed plasma membranes. The total activity of phosphatidylinositol-4-phosphate kinase in rat liver was only 1/20 that of phosphatidylinositol kinase. The enzyme activity showed an unusually broad pH-optimum in the neutral range. Mg2+ was the preferred divalent cation and Km towards ATP was about 3-fold higher than the corresponding value for phosphatidylinositol kinase.

Activation of arachidonate release and cytosolic phospholipase A2 via extracellular signal-regulated kinase and p38 mitogen-activated protein kinase in macrophages stimulated by bacteria or zymosan.

The mitogen-activated protein kinases (MAP kinases), extracellular signal-regulated kinase (ERK) and p38, can both contribute to the activation of cytosolic phospholipase A2 (cPLA2). We have investigated the hypothesis that ERK and p38 together or independent of one another play roles in the regulation of cPLA2 in macrophages responding to the oral bacterium Prevotella intermedia or zymosan. Stimulation with bacteria or zymosan beads caused arachidonate release and enhanced in vitro cPLA2 activity of cell lysate by 1.5- and 1.7-fold, respectively, as well as activation of ERK and p38. The specific inhibitor of MAP kinase kinase, PD 98059, and the inhibitor of p38, SB 203580, both partially inhibited cPLA2 activation and arachidonate release induced by bacteria and zymosan. Together, the two inhibitors had additive effects and completely blocked cPLA2 activation and arachidonate release. The present results demonstrate that ERK and p38 both have important roles in the regulation of cPLA2 and together account for its activation in P. intermedia and zymosan-stimulated mouse macrophages.

Dexamethasone differentially regulates cytokine transcription and translation in macrophages responding to bacteria or okadaic acid.

Many microorganisms and microbial products induce expression of pro-inflammatory cytokines such as interleukin-1 (IL-1alpha/beta) and tumour necrosis factor-alpha (TNF-alpha) in macrophages, primarily by transcriptional activation. We show here, by using mouse macrophages in primary culture, that pre-treatment with dexamethasone inhibits bacteria-induced IL-1beta expression as mRNA and cellular pro-IL-1beta in parallel, consistent with an effect primarily on transcriptional activation. In contrast, the expression of TNF-alpha mRNA was only partly inhibited despite virtually complete inhibition of TNF-alpha protein formation. Furthermore, the selective induction of primarily cell-associated 26,000 M, pro-TNF-alpha by the protein phosphatase inhibitor okadaic acid also was partly inhibited at the mRNA level by dexamethasone, whereas additional translational inhibition appeared to be lacking. This latter finding is reminiscent of earlier findings regarding signalling to activation of cytosolic phospholipase A2, which is sensitive to dexamethasone when elicited by bacteria, but not when elicited by okadaic acid. The present results raise the possibility that the inhibitory effect of dexamethasone on TNF-alpha translation, but not on transcriptional activation, is mediated by one or more okadaic acid-sensitive protein phosphatases.

Protein kinase C and intracellular pH regulate zymosan-induced lysosomal enzyme secretion in macrophages.

Binding of zymosan particles to macrophage beta-glucan receptors has previously been shown to trigger exocytosis of preformed lysosomal contents. In the present study, the involvement of Ca(2+)-, PKC-, and pH-dependent processes in the signaling to macrophage lysosomal secretion by zymosan was investigated. Also, the PKC dependence of lysosomal secretion in response to some soluble agents that directly alters intracellular pH was considered. Signaling to macrophage lysosomal secretion differs from that of many other secretory systems, because an elevation of cytosolic Ca2+ did not trigger a large secretory response, nor did attempts to reduce cytosolic Ca2+ affect the lysosomal secretory response to other stimuli. PKC activation by phorbol diester was also a poor stimulus of lysosomal secretion. However, when triggered by zymosan or by soluble stimuli raising lysosomal pH, the secretory response could be down-regulated by a prior prolonged incubation with phorbol diester. Such treatment also had marked effects on the binding and uptake of zymosan particles, the study of which was made possible by a novel approach. Furthermore, a synergistic effect on lysosomal secretion was obtained when stimuli that elevated lysosomal pH and stimuli that activated PKC were combined. This is of likely relevance for the secretory response to zymosan particles, a stimulus that both activates PKC and elevates lysosomal pH. The secretory response to zymosan was furthermore shown to be inhibited by a reduction of extracellular pH or [Na+], conditions that impair macrophage extrusion of acid equivalents. Earlier studies using soluble stimuli have shown a sensitivity of the secretory response to changes in cytosolic pH. We suggest a model in which the lysosomal secretory response to an elevation of lysosomal pH (1) is dependent on basal PKC activity and (2) can be enhanced further by activation of PKC. We consider PKC activity and elevation of lysosomal pH as independent and necessary signals, while cytosolic pH has a modulatory effect on some component(s) in the signal transduction pathway or in the secretory apparatus itself.

Antirheumatic gold compounds and penicillamine enhance protein kinase C-mediated activation of the arachidonate-mobilizing phospholipase A2 in mouse macrophages.

The effects of antirheumatic gold compounds and D-penicillamine on protein kinase C- and Ca(2+)-mediated activation of arachidonate mobilization and the formation of eicosanoids in mouse macrophages have been investigated. Auranofin (0.2-2 microM) enhanced the response to phorbol ester two- to three-fold, and similar enhancement was caused by aurothiomalate, aurothioglucose, and penicillamine, but only after pretreatment for 1-4 h. The enhanced mobilization of arachidonate was accompanied by increased formation and release of prostaglandin E2 and 6-keto prostaglandin F1 alpha, but not of lipoxygenase metabolites. No such enhancement occurred when the arachidonate-mobilizing phospholipase A2 was activated directly (calcium ionophore A23187). Instead, auranofin caused selective inhibition of calcium ionophore-induced formation of leukotriene C4. Treatment of macrophages with 4 beta-phorbol 12-myristate 13-acetate causes a rapid increase in the phosphorylation and a 1.4-1.8-fold increase in the activity of the 85-kd arachidonate-mobilizing phospholipase A2 as determined in an in vitro assay. The increase in activity was further enhanced by both the gold compounds and penicillamine. These findings indicate that the target for the enhancing effect of the antirheumatic drugs is located between protein kinase C and phospholipase A2 in the signal chain leading to activation of the latter enzyme.

Dexamethasone lowers cytosolic pH in macrophages by altering alkalinizing pH-regulatory mechanisms.

The effect of dexamethasone on cytosolic pH (pHc) in resident mouse peritoneal macrophages was investigated using the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein tetra-acetoxymethyl ester (BCECF-AM). Dexamethasone was found to significantly lower pHc and this reduction of pHc evolved gradually with time, was near maximal at 10 nM dexamethasone, and could be prevented by the glucocorticoid receptor antagonist RU-38486. The lower pHc of dexamethasone-treated cells was neither due to a reduction of cellular buffer capacity nor to an altered regulation of pHc by Na+/H+-exchange or by acidifying Na+-independent Cl-/HCO3- exchange, as assessed by studies of pH recovery after acute acid and alkali loads, respectively. Instead, an impaired pHc recovery by both the H+-ATPase and the alkalinizing Na+-dependent Cl-/HCO3- exchange was observed. This impairment was most likely not caused by an altered expression or localization of the 39-kDa subunit of the proton pump. Dexamethasone treatment caused a reduction of pHc also in a HCO3--containing solution, suggesting that acid extrusion by both the H+-ATPase and Na+-dependent Cl-/HCO3- exchange is important for maintenance and regulation of macrophage resting pHc. The lowering of macrophage pHc might be one mechanism whereby glucocorticoids exert their anti-inflammatory effects.

Cyclosporin-sensitive expression of cytokine mRNA in mouse macrophages responding to bacteria.

Characteristics of the cytokine response in resident mouse macrophages to certain Gram-positive and Gram-negative bacteria have been investigated by monitoring the expression of mRNA encoding interleukin-1 alpha and -beta (IL-1 alpha/beta) and tumor necrosis factor-alpha (TNF-alpha). Expression of these cytokine mRNAs occurred within 30-60 min. Both the flavonoid quercetin and phloretin inhibited the expression of IL-1 alpha/beta as well as TNF-alpha mRNA, with quercetin being more potent than phloretin and TNF-alpha expression somewhat more sensitive than that of IL-1 alpha/beta. Expression of all three cytokine mRNAs was also inhibited by prostaglandin E2, with an IC50 of > 1 microM, but not by the phosphodiesterase inhibitor pentoxifylline, although lipopolysaccharide-induced expression of TNF-alpha mRNA was inhibited. Down-regulation of phorbol ester-sensitive isoforms of protein kinase C had virtually no effect on the cytokine response to bacteria, and treatment of resting macrophages with phorbol ester did not cause expression of any of the cytokine mRNAs investigated. Among protein phosphatase inhibitors, cyclosporin A caused extensive inhibition of bacteria-induced expression of both IL-1 alpha/beta and TNF-alpha mRNA, while okadaic acid in itself caused selective induction of TNF-alpha, but not IL-1 alpha/beta mRNA, with a sharp peak at 0.3 microM concentration. At higher concentrations of okadaic acid, at which protein/phosphatase 2B/calcineurin would also be inhibited, the induction was completely reversed. This suggests that critical phosphorylation events, counteracted by one or more okadaic acid-sensitive protein phosphatase(s), and a dephosphorylation event carried out by a cyclosporin-sensitive protein phosphatase are both necessary for transcriptional activation of the TNF-alpha gene.