Presence Of Polymyxin Research Articles

Characterization of bone-resorbing activity in human periapical lesions

Extracts of human periapical granulomas were tested for the presence of bone-resorbing activity. All granulomas (10 of 10) contained low but significant levels of bone-resorbing activity, ranging from 2.1 to 4.9% treatment-% control/mg specific 45Ca release, as determined by the fetal rat long bone assay. Healthy periodontal ligament and dental pulp had no significant resorbing activity. In characterization studies, the resorbing activity in an extract pool was unaffected by the presence of polymyxin B, indicating an active moiety distinct from lipopolysaccharide. Resorbing activity was also unaffected by heating to 56 degrees C for 30 min, but was completely abolished by proteinase K treatment or heating to 70 degrees C, indicating that activity was largely protein mediated. Fast performance liquid chromatography gel filtration studies demonstrated that activity could be resolved to two major peaks, of M(r) 30,000 to 60,000 (I), and 15,000 to 20,000 (II), with a minor peak present at < 1,000 (III). Peak III was identified as prostaglandin E2 by radioimmunoassay. In inhibition studies, virtually all of the resorbing activity present was inhibited by anti-interleukin 1 beta (69%) and anti-tumor necrosis factor beta (66%) antisera, whereas anti-interleukin 1 alpha and antitumor necrosis factor alpha had no effect. Treatment with the cyclooxygenase inhibitor indomethacin also reduced activity by 74%. Taken together, these data demonstrate that most bone-resorbing activity present in chronic human periapical lesions is attributable to the action of resorptive cytokines interleukin 1 beta and tumor necrosis factor beta, acting via both indomethacin-dependent and independent pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

Interdigitated gel phase bilayers formed by unsaturated synthetic and bacterial glycerolipids in the presence of polymyxin B and glycerol

The ability of synthetic phosphoglycerolipids with a cis mono-unsaturated acyl chain in the 2-position and a saturated chain in the 1-position of glycerol to form interdigitated gel phase bilayers in the presence of amphipathic substances was monitored using a fatty acid spin label, 16-doxylstearic acid, and phosphatidylglycerol spin label containing 16-doxylstcaric acid. These spin labels become significantly more motionally restricted in an interdigitated gel phase bilayer than in a non-interdigitated gel phase bilayer. The results indicated that polymyxin B and polymyxin B nonapeptide caused interdigitation of 1-palmitoyl,2-oleoyl-phosphatidylglycerol (POPG) and glycerol caused interdigitation of 1-stearoyl,2-olcoyl-phosphatidylcholine (SOPC), similar to their effects on disaturated lipids. The fluidity gradient present in non-interdigitated gel phase bilayers was abolished. However, glycerol did not cause POPG to become interdigitated, in contrast to SOPC. We reported earlier that there is a kinetic barrier to interdigitation of saturated PG in the presence of glycerol, in contrast to saturated PC. This barrier is even greater for the unsaturated species of PG. Furthermore, these compounds lowered the gel to liquid-crystalline phase transition temperatures of the unsaturated lipids more than of saturated lipids suggesting that the interdigitated bilayer of the former may be less ordered or less stable than that of the latter. Since polymyxin B is an antibiotic we also examined its effect on a lipid extract from the Gram-negative bacteria Pseudomonas aeruginosa in order to assess whether interdigitation might be involved in its mechanism of bactericidal or bacteriostatic effect. Polymyxin B and polymyxin B nonapeptide also caused motional restriction of a small percentage (about 13% at −2°C and 25% at −14°C for polymyxin B) of the spin label in the lipid extract at low temperatures, where the lipid is in the gel phase, consistent with formation of a small domain of interdigitated bilayer lipid. However, the degree of immobilization was less than that in the interdigitated bilayers of the synthetic unsaturated lipids. This may be a result of the heterogeneous nature of the lipids in the extract. However, it cannot be ruled out that the motional restriction of the spin label in this extract may be caused by something other than interdigitation. Thus the results with the lipid extract are less conclusive of interdigitation than for the synthetic lipids. A motionally restricted population was not detectable at higher temperatures. This study shows that a cis-double bond in the acyl chain of phosphoglycerolipids does not prevent formation of an interdigitated gel phase bilayer. However, such lipids are in the liquid-crystalline phase at physiological temperature and thus will not form interdigitated domains in biological membranes.

Biological properties and spectrum of activity of tolaasin, a lipodepsipeptide toxin produced by the mushroom pathogen Pseudomonas tolaasii

The biological properties and spectrum of activity of tolaasin, a lipodepsipeptide toxin produced by the mushroom pathogen Pseudomonas tolaasii, were investigated. An erythrocyte lysis bioassay was developed and used to assay tolaasin activity. Tolaasin-induced haemolysis was dose dependent, maximal between pH 6·0 and 7·0, increased with temperature and was inhibited by the addition of divalent metal ions. Tolaasin production commenced during exponential growth of P. tolaasii and continued into stationary phase. Culture filtrate from wild type P. tolaasii was able to pit mushroom tissue and lyse erythrocytes, whereas culture filtrate from a tolaasin defective mutant had no lytic activity. Ultrastructural studies showed disruption of the Agaricus bisporus plasma membrane and vacuole membranes by both P. tolaasii and tolaasin, but not by a tolaasin defective mutant. Pre-incubation of tolaasin with multilamellar liposomes resulted in loss of lytic activity and osmotic protectants prevented tolaasin-induced haemolysis, demonstrating that tolaasin partitions into membranes, forms pores in erythrocyte membranes and causes lysis by a colloid osmotic mechanism. Tolaasin was phytotoxic when infiltrated into leaves of Nicotiana tabacum and was shown to be active against a range of basidiomycetes and Gram-positive bacteria. Gram-negative bacteria were resistant to tolaasin, but became susceptible when treated concomitantly with sub-minimal inhibitory concentrations of polymyxin B. P. tolaasii was resistant to tolaasin even in the presence of polymyxin B.

Influence of phorbol esters on stimulation-induced noradrenaline release and contractile force of isolated guinea-pig atria

Phorbol-12,13-dibutyrate (PDB) and phorbol-12-myristate-13-acetate (PMA) increased the field-stimulation-induced efflux of radioactivity from guinea-pig atria preloaded with 7-[3H]-noradrenaline. The efflux was more than doubled by 10(-7) mol/l of either compound. Phorbol-12-myristate-13-acetate-4-O-methylether (PME), which has no effect on the protein kinase C (PKC), did not modify the stimulation-induced efflux of radioactivity, while a slight reduction was seen with 70 microM of the PKC inhibitor polymyxin B. In the presence of polymyxin B, the effects of PMA and PDB were greatly attenuated. In addition, PDB had a concentration-dependent negative inotropic effect (EC50 7.0 x 10(-10) mol/l). Pretreatment with polymyxin B shifted the concentration-response curve for PDB to the right (EC50 4.6 x 10(-9) mol/l). No negative inotropic effect was seen with PMA or PME. The results suggest that all effects of the phorbol esters were mediated by a stimulation of PKC. The different lipophilicity of PMA and PDB or a different influence on the various isozymes of PKC may account for the diverging postjunctional effects of the two compounds.

Lactoferrin-lipopolysaccharide interactions. Effect on lactoferrin binding to monocyte/macrophage-differentiated HL-60 cells.

Lactoferrin (LF) has been implicated in a number of functions including the negative regulation of myelopoiesis in vitro and in vivo, an effect mediated by suppression of cytokine release from monocytes/macrophages. This suppression is abrogated by bacterial LPS. In the present study, HL-60 cells were induced to differentiate to monocytes/macrophages by 12-O-tetradecanoyl phorbol-13-acetate, and LF-binding assays were performed. After differentiation, HL-60 cells showed a twofold increase of LF-binding sites with no difference in the specificity or affinity of LF between pre- and post-differentiated cells. CD11a, CD11b, and CD11c Ag, which have been associated with specific binding sites for LPS on monocytes/macrophages, were also increased three- to fourfold after differentiation. With the use of this system, the effect of LPS on LF binding was studied. At 37 degrees C, LPS enhanced LF binding on HL-60 cells, especially after differentiation. Conversely, at 4 degrees C, LPS inhibited LF binding. There was little effect of temperature on LF binding in the absence of LPS. In the presence of polymyxin B sulfate, the enhanced LF binding by LPS was abrogated. Also, pretreatment with mAbCD11 and/or mAb5D3, which are associated with or directed against candidate LPS receptors, reduced LF binding. Cross-linking studies using an iodinated, photoactivatable LPS derivative ([125I]ASD-LPS) demonstrated directly the specific binding of LPS to LF. These data indicate a dichotomous nature of LF binding on monocyte/macrophage-differentiated HL-60 cells--one being mediated by specific LF receptors whereas the other is apparently mainly via LPS receptors after formation of an LF-LPS complex. These interactions, for which a model is proposed, help to explain the mechanism behind LPS abrogation of the myelopoietic suppressive effects of LF, and a situation that probably occurs during bacterial infection.

Delta pH-dependent accumulation of tetracycline in Escherichia coli

The effects of ionophores on tetracycline accumulation in Escherichia coli cells were investigated in the presence of polymyxin B nonapeptide. Accumulation was inhibited by nigericin but not by valinomycin. Tetracycline accumulation was stimulated by decreasing the pH of the medium and inhibited by the addition of magnesium ions. These results indicated that tetracycline enters cells through diffusion as a protonated form (TH2) and is accumulated as a membrane-impermeable magnesium-tetracycline chelate complex (THMg+). This noncarrier diffusion hypothesis was confirmed by the fact that tetracycline accumulated in protein-free liposomes through an artificially imposed pH difference.

Leishmania major amastigotes initiate the L-arginine-dependent killing mechanism in IFN-gamma-stimulated macrophages by induction of tumor necrosis factor-alpha.

Macrophages exposed to IFN-gamma and infected with amastigotes of Leishmania major develop the capacity to eliminate the intracellular pathogen. This antimicrobial activity of activated macrophages correlates with the initiation of nitrogen oxidation of L-arginine, yet other reports suggest that two signals are required for induction of this biochemical pathway for effector activity. In the present studies, macrophages treated with up to 100 U/ml IFN-gamma, or 100 ng LPS, or 10(7) amastigotes produced minimal quantities (less than 9 microM) of NO2- and failed to develop cytotoxic effector activities. In contrast, the combination of IFN-gamma and either LPS (greater than 0.1 ng) or amastigotes (10(6) induced high concentrations (much greater than 30 microM) of NO2- and macrophage cytotoxicity against intra- and extracellular targets. The induction of nitrogen oxidation by amastigotes could be dissociated from LPS-induced events by 1) performing the assays in the presence of polymyxin B (which blocked LPS effects, but not amastigote effects), 2) determining the threshold of IFN-gamma required to prime cells for subsequent trigger (1 U/ml for LPS trigger effects; 10-fold higher for amastigotes), and 3) determining the heat sensitivity of the two trigger agents (amastigote effects abolished at 100 degrees C; LPS effects unaffected at this temperature). Further, culture fluids from amastigote-infected macrophages did not contain detectable LPS (less than 6 pg/ml). Possible parasite and cell-associated factors that could contribute to the induction of nitrogen oxidation and cytotoxic activity of IFN-gamma treated macrophages were examined: only certain intact microorganisms, LPS from a variety of bacteria, and the cytokine TNF alpha were effective. Both NO2- production and intracellular killing were abolished by the addition of anti-TNF-alpha mAb in the assay. TNF-alpha was produced by amastigote-infected macrophages and IFN-gamma dramatically enhanced secretion of this cytokine; IFN-gamma alone had no effect. Endogenous TNF-alpha produced during infection of macrophages with L. major acted in an autocrine fashion to trigger the production of L-arginine-derived toxic nitrogen intermediates that killed the intracellular parasites.

Cell envelope impermeability to daptomycin inPseudomonas aeruginosa andPasteurella multocida

The exclusively gram-positive antibacterial spectrum of the lipopeptide daptomycin (LY146032) suggests that the underlying basis for intrinsic resistance in gram-negative organisms involves envelope impermeability. This study was undertaken to determine whether the outer membranes ofPseudomonas aeruginosa andPasteurella multocida can be rendered permeable to daptomycin by experimental modifications that result in susceptibility of gram-negative bacteria to lipophilic molecules. Turbidimetric growth assays revealed sublethal concentrations of polymyxin B or ethylenediaminetetraacetate (EDTA) sensitized all strains examined to the hydrophobic antibiotic novobiocin. Neither permeabilizer renderedPs. aeruginosa or a hydrophilicPa. multocida variant susceptible to daptomycin; however, polymyxin B sensitized a hydrophobicPa. multocida variant, whereas EDTA did not. Cells cultured with sublethal concentrations of polymyxin B or EDTA retained negatively charged cell surfaces comparable to those of control cells. Growth ofPa. multocida strains in the presence of polymyxin B did not result in modification of cell envelope lipid composition. These findings indicate that the ability of the outer membrane to retard the diffusion of daptomycin does not require normally intact structure, thereby suggesting that the residual negative charge of the cell surface may preclude interaction with the acidic antibiotic owing to electrostatic repulsion.

Protein kinases, nitric oxide and long-term depression of synapses in the cerebellum

We have analysed the effects of polymyxin B, a potent inhibitor of calcium-dependent protein kinases, of L-N-monomethylarginine, an inhibitor of nitric oxide synthesis, and of methylene blue which prevents activation of soluble guanylate cyclase, on long-term depression of parallel fibre-mediated EPSPs of rat cerebellar Purkinje cells in slices maintained in-vitro. In control conditions, a long-term depression of parallel fibre-mediated EPSPs was consistently induced by their pairing with calcium spikes directly elicited in the postsynaptic cells. This long-term change in synaptic strength was not observed in the presence of polymyxin B, of L-N-monomethylarginine, or of methylene blue, suggesting that calcium-dependent protein kinases and nitric oxide are both involved.

Adenosine alters the vascular effects of a diacylglycerol analogue and polymyxin B

Endothelium-denuded rat aorta rings were used to study the possible relationship between protein kinase C and the mechanism of adenosine-induced smooth muscle relaxation. Adenosine (5 × 10 −4M) partially relaxed the aortic rings contracted by either a depolarising amount of KCl (4 × 10 −2 M) or activation of protein kinase C with l-oleoyl-2- acetyl-sn-glycerol (10 −6 M). The same amount of adenosine blocked the further relaxation obtained in the presence of polymyxin B (5 × 10 −5 M), a protein kinase C blocking agent. These results suggest a possible interaction in vascular smooth muscle between adenosine and protein kinase C.

Antimicrobial susceptibility of Salmonella typhimurium carrying the outer membrane permeability mutation SS-B

The antibiotic susceptibility profile of Salmonella typhimurium SS-B, a mutant susceptible to some antimicrobial agents, was studied in detail. Twenty-eight agents were tested, and eleven of these had MICs significantly lower (32- to greater than 250-fold) for the SS-B strain than for its parent. The drugs were generally hydrophobic or amphiphilic. Polymyxin B nonapeptide, which has a known outer membrane permeabilizing action, further reduced the MIC of several of these agents for the SS-B strain by a factor of approximately 10 to 30. In most cases, the resulting MICs were lower than the corresponding MICs for the parent strain grown in the presence of polymyxin B nonapeptide. In addition, the hydrophobic fluorescent probe N-phenyl naphthylamine was rapidly embedded in the membranes of the SS-B strain but was poorly embedded in those of the parent strain.

Induction of IL-1 release through stimulation of the C3b/C4b complement receptor type one (CR1, CD35) on human monocytes.

Soluble polymerized human C3b and C3b bound to Sepharose induced the production of cell-associated IL-1 and the extracellular release of IL-1 activity from cultured human adherent monocytes. Cultures were performed in serum-free conditions in the presence of polymyxin B and of the PG synthesis inhibitor indomethacin. Induction of intracellular IL-1 activity was associated with that of IL-1 alpha and IL-1 beta Ag. Biologically active released IL-1 was IL-1 beta. Monomeric C3b induced cell-associated IL-1 but not the release of IL-1 from monocytes. IL-1 production was also induced by stimulation of CR1 on adherent monocytes with anti-C3b receptor (CR1) antibody. The ability of multivalent C3b to induce IL-1 production could be dissociated from that of possibly contaminating LPS by several criteria including: the lack of detectable LPS in purified C3b; functional inhibition of LPS in cultures with polymyxin B; the lack of induction of extracellular IL-1 by C3b monomer; a correlation between the expression of CR1 on monocytes and the monocytic response to C3b; a dissociation between the expression of CR1, and the expression of LPS binding sites and the IL-1 response of monocytes to endotoxin. Induction of IL-1 represents a novel pathway by which C3b-CR1 interactions may modulate the immune response.

Effects of polymyxin B on the sarcoplasmic reticulum membrane

Polymyxin B, a cyclic peptide antibiotic, inhibits Ca2+-ATPase, p-nitrophenyl phosphatase and phosphorylase kinase activities associated with rabbit skeletal muscle sarcoplasmic reticulum membranes; 50% inhibition is induced by 100 microM, 130 microM and 550 microM of polymyxin respectively. The fluorescence intensity of fluorescein isothiocyanate-labeled Ca2+-ATPase, decreases in the presence of polymyxin (50% of the total decrease at 70 microM polymyxin). On the other hand, the polypeptide inhibits calmodulin-dependent endogenous phosphorylation of 60 kDa, 20 kDa and 14 kDa membrane proteins, while an increase of calmodulin-dependent phosphorylation is observed in 132 kDa and 86 kDa proteins.

ＴＧＰ‐３（遺伝子組換え型ヒトインターロイキン２）の発熱機序 ｉｎｔｅｒｌｅｕｋｉｎ １βの関与の可能性について

Because recombinant human interleukin 2 was reported to induce fever on clinical trials, the involvement of interleukin 1 (IL-1) in inducing fever was studied in human blood mononuclear cells and in rabbits.1. When the mononuclear cells were cultured with 10-3, 000 ng/ml of TGP-3 in the presence of polymyxin B at 37°C for 24 hr, 2-5 ng/106 cells of IL-1β was detected in the culture extract (intracellular and culture supernatant) as determined by a radioimmunoassay. However, the level of IL-1β fluctuated depending on the lot of cell preparations.2. When the culture extract of the mononuclear cells containing a relatively large amount of IL-1β (over 10 ng/ml) were injected intravenously into the rabbits, the rectal temperature was raised with a peak 50-60 min after the injection; the fever pattern was similar to that typically induced by endogenous pyrogens.These results suggest that the fever observed on clinical trials of TGP-3 might be due to IL-1β production.

Expression of tumor necrosis factor in vitro by human mononuclear phagocytes stimulated with whole Mycobacterium bovis BCG and mycobacterial antigens.

Four mycobacterial preparations directly stimulated human blood monocytes and alveolar macrophages to produce tumor necrosis factor (TNF). Monoclonal antibody against TNF blocked (99%) TNF activity. Lipopolysaccharide was not responsible for the TNF activity generated; activity was unaffected in the presence of polymyxin B.

Interaction of polymyxin B 1 and polymyxin B 1 nonapeptide with phosphatidic acid monolayer and bilayer membranes

The interactions of the antibiotic polymyxin B 1 and its enzymatic cleavage product polymyxin B 1 nonapeptide with phosphatidic acid monolayers and with bilayer membranes were investigated. Temperature-dependent pressure-area analysis of the monolayer reveals a linear increase of the lipid mean molecular area in the liquid condensed state for polymyxin concentrations between 10 −6 and 4 × 10 −2 M. Depending on the surface pressure, the area increase amounts to 30–70 Å 2. A linear dependence was also observed in the liquid expanded state but saturation is reached already at 10 −7 M polymyxin. The adsorption of polymyxin to phosphatidic acid bilayers is also linear and of a Langmuir type. Saturation is reached at a 1:4 polymyxin/lipid molar ratio. Polymyxin induces a phase separation in phosphatidic acid monolayers which was concluded from the thermotropic phase transition curves. In agreement with earlier bilayer experiments a second lowered phase transition appears in the presence of polymyxin. These fluidized domains again exhibit a linear polymyxin uptake comparable to the one of the liquid expanded monolayer at a temperature, where the undisturbed lipid is still in the condensed state. Polymyxin nonapeptide also causes an expansion of phosphatidic acid monolayers but only by maximally 10 Å 2. The thermotropic phase transition of the monolayer is reduced and considerably broadened by the nonapeptide. In phosphatidic acid bilayers we observed a decrease of the lipid phase transition temperature by 24°C. The lateral chain packing is considerably disturbed by the peptide part of polymyxin. The branched fatty acid tail may only serve to anchor the peptide within the matrix but does not contribute considerably to the formation of less ordered subcells within the membranes. For polymyxin B 1 nonapeptide, the binding curve of peptide to bilayers is linear but saturation was not reached.

Interleukin-1 production by monocytes from patients with allergic asthma after stimulation in vitro with lipopolysaccharide and Dermatophagoides pteronyssinus mite allergen.

Monocytes from 6 patients with asthma and positive bronchial challenge with extract from the house dust mite Dermatophagoides pteronyssinus (Dp) were stimulated with lipopolysaccharide (LPS) and allergen extract from Dp. In order to neutralize putative endotoxin contamination of the allergen extract, some cultures were incubated in the presence of polymyxin B. Interleukin-1 (IL-1) activity was assayed by the comitogenic activity of the crude monocyte supernatants on phytohemagglutinin-stimulated murine thymocytes. Our results suggest that, upon stimulation with LPS and allergen, monocytes from atopic patients possess a normal capacity to produce IL-1. Like monocytes from healthy controls, patient monocytes do not produce IL-1 spontaneously. The specificity of the allergen stimulus is not well-defined, and further characterization of the IL-1-inducing property awaits the availability of endotoxin-free allergen preparations.

LPS augments human B-cell differentiation by direct stimulation of PWM-responsive B cells

Bacterial lipopolysaccharide (LPS) augments production of IgM and IgG by two- to seven-fold in cultures of peripheral blood lymphocytes (PBL) stimulated by pokeweed mitogen (PWM), but only if monocytes are rigorously depleted. When PBL were separated into adherent cell (AC), B-cell-enriched, and T-cell-enriched fractions, pulsed with LPS, and recombined in culture with PWM, increased generation of plasma cells was seen only in cultures containing LPS-treated B cells. This effect of LPS appears to be independent of soluble factors. Supernatants from LPS-stimulated B cells or AC did not consistently increase PWM responses when cultured with fresh B cells in the presence of polymyxin B. Furthermore, puising of B cells with purified interleukin 1 from two different commercial sources failed to augment PWM-induced differentiation. When B cells were depleted of surface IgD (sIgD)-bearing cells by panning, no effect on LPS-mediated augmentation of PWM-driven differentiation was seen. B cells were also fractionated by rosetting with mouse erythrocytes. Treatment of BMR + cells with LPS did not induce them to respond to PWM, while treatment of BMR − cells with LPS augmented generation of plasma cells. These results indicate that LPS acts directly to augment differentiation of PWM-responsive B cells, rather than recruiting sIgD +, BMR + cells to become PWM responsive.

Polymyxin B, a selective inhibitor of protein kinase C, diminishes the release of noradrenaline and the enhancement of release caused by phorbol 12,13-butyrate.

Slices of the rabbit hippocampus were labelled with 3H-noradrenaline, superfused continuously with a modified Krebs-Henseleit medium containing the uptake inhibitor cocaine and stimulated electrically (2 ms, 3 Hz, 24 mA, 5 V/cm). Phorbol 12,13-dibutyrate (PDB), a potent activator of protein kinase C (PKC), strongly enhanced the electrically-evoked overflow of tritium. In contrast, polymyxin B, a relatively selective inhibitor of PKC, diminished the evoked tritium overflow in a time- and concentration-dependent manner. The enhancement of the evoked overflow of tritium caused by PDB was strongly reduced in the presence of polymyxin B (100 mumol/l). These results suggest 1. that PKC may be involved in the physiological mechanism of action-potential-induced noradrenaline release from noradrenergic nerve terminals and 2. that the PDB-induced enhancement of noradrenaline release may be due to a direct activation of PKC.

Inhibition of T3 mediated T-cell proliferation by Ca2+-channel blockers and inhibitors of Ca2+/phospholipid-dependent kinase.

The potential roles of Ca2+ ions in the response of T lymphocytes to stimulation with monoclonal antisera to the T3 antigen were investigated by means of pharmacological agents that predominantly inhibit the flux of Ca2+ ions into cells (verapamil, nifedipine) or the activity of Ca2+-dependent kinases (trifluoperazine, polymyxin B). As assessed by uptake of [3H]thymidine, proliferation induced with anti-T3 +/- recombinant IL-2 at 72 h was inhibited by greater than 80% in the presence of nifedipine at 50 microM, and almost completely arrested (greater than 95% inhibition) with the other agents at the same concentration. Further quantitative assays of the effects of polymyxin B and trifluoperazine on C-kinase labelling of exogenous substrate showed a major reduction with both agents, but inhibition was substantially greater with polymyxin B that with trifluoperazine (IC50 = 14 and 70 microM respectively). These results were confirmed by qualitative assessment of Ca2+/phospholipid-dependent phosphorylation of endogenous substrates, which demonstrated major phosphoproteins of MW 56,000, 52,000, 43,000, and 20,000, and dose-dependent reduction in labelling in the presence of polymyxin B. Similar results were obtained under more physiological conditions in intact cells labelled with 32P orthophosphate. These findings indicate several possible roles for Ca2+ in T-cell activation, and several possible levels of activity, including modulation of calmodulin-dependent kinases and effects on Ca2+/phospholipid-dependent kinases and Ca2+ channels.