Interleukin-1 inhibits PGE2 binding to macrophage-like P388D1 cells by a cyclic AMP-independent process.

The interaction between interleukin IL-1 alpha and PGE2 on P388D1 cells has been investigated. Preincubation of murine macrophage-like cells, P388D1, with IL-1 alpha (0-73 pM) reduced the binding of PGE2 to these cells in a concentration-dependent manner. Scatchard analysis showed that IL-1 alpha decreased the PGE2 binding by lowering both the high and low affinity receptor binding capacities (from 0.31 +/- 0.02 to 0.12 +/- 0.01 fmol/10(6) cells for the high affinity receptor binding sites and from 2.41 +/- 0.12 to 1.51 +/- 0.21 fmol/10(6) cells for the low affinity receptor binding sites). However, the dissociation constants of the receptors of the IL-1 alpha-treated cells remained unchanged. Inhibition of PGE2 binding by IL-1 alpha did not involve changes in either protein phosphorylation or intracellular cyclic AMP levels. Our data clearly show that IL-1 alpha inhibits the binding of PGE2 to monocytes/macrophages and may thereby counter the immunosuppressive actions of PGE2.     

Phospholipase activities of the P388D1 macrophage-like cell line

The murine macrophage (M phi) cell line, P388D1, was employed as a source of M phi phospholipases in order to characterize the enzymatic properties and subcellular localization of these enzymes because of their importance for prostaglandin biosynthesis. Phospholipase activity was assessed with dipalmitoylphosphatidylcholine (DPPC) as substrate. Phospholipases were characterized with respect to divalent cation dependence, pH optima, and localization in subcellular compartments using linear sucrose gradients. By these criteria a number of different phospholipases were identified. Most importantly, a single Ca2+-dependent activity with a pH optimum of 8.8 was identified in membrane-rich fractions (plasma membrane, mitochondria, and endoplasmic reticulum) and could be clearly separated from the remaining activities, which are Ca2+ independent and exhibit pH optima of 7.5, 5.1, and 4.2. The phospholipases with acidic pH optima may be associated with subcellular components containing lysosomal enzymes and both phospholipase A1 and phospholipase A2 activities are observed. In contrast, the phospholipase activity with a pH optimum of 7.5 sediments with the cytosolic proteins and is inhibited by 5 mM Ca2+. No significant phospholipase C activity was detected in assays performed with or without added Ca2+ at pH's 4.2, 5.1, 7.5, or 8.8 using DPPC as substrate. However, the P388D1 cells do contain a lysophospholipase that is at least 20 times more active than the phospholipase A activities identified. Its presence must be taken into account in evaluating the positional specificities and properties of the macrophage phospholipases.     

Localization of group V phospholipase A2 in caveolin-enriched granules in activated P388D1 macrophage-like cells

In murine P388D1 macrophages, the generation of prostaglandin E2 in response to long term stimulation by lipopolysaccharide involves the action of Group V secreted phospholipase A2 (PLA2), Group IV cytosolic PLA2 (cPLA2), and cyclooxygenase-2 (COX-2). There is an initial activation of cPLA2 that induces expression of Group V PLA2, which in turn induces both the expression of COX-2 and most of the arachidonic acid substrate for COX-2-dependent prostaglandin E2 generation. Because Group V PLA2 is a secreted enzyme, it has been assumed that after cellular stimulation, it must be released to the extracellular medium and re-associates with the outer membrane to release arachidonic acid from phospholipids. In the present study, confocal laser scanning microscopy experiments utilizing both immunofluorescence and green fluorescent protein-labeled Group V PLA2 shows that chronic exposure of the macrophages to lipopolysaccharide results in Group V PLA2 being associated with caveolin-2-containing granules close to the perinuclear region. Heparin, a cell-impermeable complex carbohydrate with high affinity for Group V PLA2, blocks that association, suggesting that the granules are formed by internalization of the Group V sPLA2 previously associated with the outer cellular surface. Localization of Group V PLA2 in perinuclear granules is not observed if the cells are treated with the Group IV PLA2 inhibitor methyl arachidonyl fluorophosphonate, confirming the important role for Group IV PLA2 in the activation process. Cellular staining with antibodies against COX-2 reveals the presence of COX-2-rich granules in close proximity to those containing Group V PLA2. Collectively, these results suggest that encapsulation of Group V PLA2 into granules brings the enzyme to the perinuclear envelope during cell activation where it may be closer to Group IV PLA2 and COX-2 for efficient prostaglandin synthesis.     

Intracellular calcium homeostasis during hydrogen peroxide injury to cultured P388D1 cells

The effects of exposure of cultured P388D1 cells to H2O2 on intracellular free calcium ([Ca++]i) was investigated utilizing the intracellular fluorescent calcium chelator "Quin 2." [Ca++]i rose from approximately 150 nM to greater than 2 microM over a time course that was strongly dependent on the concentration of H2O2 used (5 X 10(-5) to 5 X 10(-3) M). After exposure of P388D1 cells to 5 X 10(-3) M H2O2, Quin 2 was fully saturated between 15 and 30 min exposure. During this time, no apparent change in the rate of equilibration of 45Ca++ from the extracellular medium could be detected, whereas in cells preloaded with 45Ca, net 45Ca was lost from the cells at a greater rate than controls. Measurements of total cellular calcium by atomic absorption spectroscopy confirmed that there was a net loss of calcium from the cells during the first 30 min. At time points greater than 45 min after exposure to H2O2 the influx of extracellular 45Ca and net intracellular Ca++, Na+ and K+ rapidly increased. Half times for H2O2 catabolism by the cells varied from about 8 min at 5.0 X 10(-4) M H2O2 to 14.0 min at 5.0 X 10(-3) M. When the total [Ca++]i-buffering capacity of the Quin 2 pool was varied by increasing the loading of intracellular Quin 2 by 68-fold (1.1 X 10(2) - 7.6 X 10(3) amol per cell), the rate of rise of [Ca++]i was depressed by only 1.6-fold following exposure to 5 mM H2O2. During the rise of intracellular [Ca++]i, cell morphology was observed by both light and scanning electron microscopy and revealed that "surface blebs" appeared during this phase of injury. Both the rise in [Ca++]i and "blebbing" were observable before any loss in cell viability was detected by either loss of Trypan blue exclusion or loss of preloaded 51Cr from the cells. From these results we conclude the following, H2O2 exposure induces a dose-dependent disturbance of intracellular calcium homeostatis; the rise in [Ca++]i is mediated by exposure to H2O2 in the early phase of the injury, and is not dependent on the continuing presence of the oxidant; the rate of rise of [Ca++]i is largely independent of the quantity of calcium mobilized to the Quin 2 pool; during the early phase (less than 30 min) of rise of [Ca++]i, only intracellular calcium is involved in the response; these events occur concomitantly with gross morphological changes to the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)     

Plasma membrane depolarization reduces nitric oxide (NO) production in P388D.1 macrophage-like cells during Leishmania major infection

In the present study, we compare changes in host cell plasma membrane potential (V(m)), K(+) fluxes, and NO production during K(+) channel blockade with those changes that occur during infection with Leishmania major. Infection of P388D.1 cells with L. major promastigotes or treatment with K(+) channel blockers (either 1mM 4-AP, 10mM TEA, or 200 microM quinine) suppressed NO production. Inhibition of NO production correlated with depolarization of the P388D.1 cell V(m). Infection of P388D.1 cells with L. major increased the unidirectional influx of rubidium (86Rb), a tracer for K(+) flux, that was comparable to that induced by K(+) channel blockade by 1mM 4-AP. The similar effects of K(+) channel blockers and L. major on NO production, K(+) influx, and V(m) suggest that K(+) channel activity and the maintenance of V(m) is important for NO production in these cells. We suggest that intracellular parasites employ a strategy to inhibit NO production by disrupting V(m) during the invasion/infection process by altering host cell K(+) channel activity.     

Identification of a third pathway for arachidonic acid mobilization and prostaglandin production in activated P388D1 macrophage-like cells

Previous studies have demonstrated that P388D(1) macrophages are able to mobilize arachidonic acid (AA) and synthesize prostaglandins in two temporally distinct phases. The first phase is triggered by platelet-activating factor within minutes, but needs the cells to be previously exposed to bacterial lipopolysaccharide (LPS) for periods up to 1 h. It is thus a primed immediate phase. The second, delayed phase occurs in response to LPS alone over long incubation periods spanning several hours. Strikingly, the effector enzymes involved in both of these phases are the same, namely the cytosolic group IV phospholipase A(2) (cPLA(2)), the secretory group V phospholipase A(2), and cyclooxygenase-2, although the regulatory mechanisms differ. Here we report that P388D(1) macrophages mobilize AA and produce prostaglandins in response to zymosan particles in a manner that is clearly different from the two described above. Zymosan triggers an immediate AA mobilization response from the macrophages that neither involves the group v phospholipase A(2) nor requires the cells to be primed by LPS. The group VI Ca(2+)-independent phospholipase A(2) is also not involved. Zymosan appears to signal exclusively through activation of the cPLA(2), which is coupled to the cyclooxygenase-2. These results define a secretory PLA(2)-independent pathway for AA mobilization in the P388D(1) macrophages, and demonstrate that, under certain experimental settings, stimulation of the cPLA(2) is sufficient to generate a prostaglandin biosynthetic response in the P388D(1) macrophages.     

Purification and characterization of a lysophospholipase from a macrophage-like cell line P388D1

Two lysophospholipase activities (designated I and II) were identified in the macrophage-like cell line P388D1. Lysophospholipase I was purified (8,500-fold) to homogeneity by DEAE-Sephacel, Sephadex G-75, Blue-Sepharose, and chromatofocusing chromatography. Lysophospholipase II was separated from the lysophospholipase I in the Blue-Sepharose step. The apparent molecular mass of lysophospholipase I and II are 27,000 and 28,000 daltons, respectively, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their pI values were 4.4 and 6.1 respectively, as determined by isoelectric focusing. Lysophospholipase I exhibited a broad pH optimum between 7.5-9.0. The double-reciprocal plot of the substrate dependence curve of the purified lysophospholipase I showed a break around the critical micelle concentration of the substrate (1-palmitoyl-sn-glycerol-3-phosphorylcholine). The apparent Km, determined from substrate concentrations above 10 microM was 22 microM, and the apparent Vmax was 1.3 mumol min-1mg-1. The purified enzyme did not have phospholipase A1, phospholipase A2, acyltransferase, or lysophospholipase-transacylase activity. No activity was detected toward triacylglycerol, diacylglycerol, p-nitrophenol acetate, p-nitrophenol palmitate, or cholesterol ester. The enzyme did, however, hydrolyze monoacylglycerol although at a rate 20-fold less than lysophospholipid, 0.06 mumol min-1mg-1. The lysophospholipase I was inhibited by fatty acids but not by glycerol-3-phosphorylcholine, glycerol-3-phosphorylethanolamine, or glyc-fjerol-3-phosphorylserine. A synthetic manoalide analogue 3(cis,cis,-7,10)hexadecadienyl-4-hydroxy-2-butenolide inhibited the enzyme with half-inhibition (IC50) at about 160 microM. Triton X-100 decreased the enzymatic activity, although this apparent inhibition can be explained by a "surface dilution" effect. The pure lysophospholipase I was stable for at least 5 months at -20 degrees C in the presence of glycerol and beta-mercaptoethanol. Lysophospholipid also demonstrated a protective effect during the later stage of purification.     

Identification of platelet-activating factor receptors in P388D1 murine macrophages

Platelet-activating factor (PAF) binding and metabolism by eight murine and human cell lines was analyzed. Only the murine P388D1 macrophage line had specific, high affinity PAF binding sites. PAF binding reached saturation within 10 min at room temperature and was irreversible. Minimal PAF metabolism was observed at the time binding saturation was achieved. Scatchard analysis of PAF binding revealed a single class of PAF receptors (7872 +/- 1310/cell) which had a dissociation constant of 0.08 +/- 0.01 nM (mean +/- SEM, eta = 6). The dissociation constant was confirmed independently by quantifying the kinetics of initial specific PAF binding. PAF binding was stereospecific, required an sn-2 acetyl substituent, and was inhibited by structurally diverse PAF antagonists including kadsurenone, BN 52021, triazolam, and CV3988. The fact that the receptors are functionally active was shown by the observation that 1 to 100 pM PAF increased free intracellular calcium in P388D1 cells in a dose-related manner. These studies demonstrate that P388D1 macrophages have functional PAF receptors whose affinity and structural specificities are similar to PAF receptors in other cells. The availability of a stable cell line that binds but does not metabolize PAF will greatly facilitate studies of the PAF receptor.     

Ability of bisbenzylputrescine and propanediamine analogs to modulate the intracellular polyamine pool of P388D1 cells

The effects of a series of bisbenzyldiamine analogs have been tested on P388D1 cell line in vitro. Their effects on cell growth, polyamine oxidase (PAO) activity and intracellular polyamine content were determined. The cytotoxicity tests were performed in culture medium supplemented with 100 mol/L aminoguanidine (I), 100 mol/L aminoguanidine and 100 mol/L N,N-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72,527) (II), and finally 100 mol/L aminoguanidine and 200 mol/L D,L-difluoromethylornithine (DFMO) (III). The IC50 values under conditions I and III were similar, suggesting that inhibition of ornithine decarboxylase by DFMO did not affect the biological effect of our derivatives. Spermine and spermidine remained nontoxic in conditions I and III. However in the condition II, the toxicity of all tested compounds (excepted spermidine) was increased, suggesting that the inhibition of cellular PAO increased their toxicity.The enzymatic test of PAO showed that at high doses inhibition of this enzyme by putrescine analogs occurred, while the N-methylated propanediamine derivative increased the enzyme activity; however, these results do not correlate with cytotoxicity tests. When these derivatives were incubated for 48 h with the cells, all of them increased the cell content in putrescine (160%) and spermine (145%) and decreased the spermidine content (75%) without any modification of the total amount of polyamine.The correlation between the cytotoxic results and the intracellular polyamine determination shows that the increase in spermine content along with the inhibition of retroconverting PAO enzyme increases the toxic effect of tested compounds (including spermine), suggesting that spermine toxicity is more important in the absence of intracellular oxidation processes.     

Determination of binding strength and kinetics of binding initiation. A model study made on the adhesive properties of P388D1 macrophage-like cells

The adhesive properties of the mouse P388D1 macrophage-like line were explored. Cells were deposited in glass capillary tubes, and the kinetics of adhesion and spreading were studied. Binding involved the cell metabolism since it was decreased by cold, azide, or a divalent cation chelator. Glass-adherent cells were subjected to calibrated laminar shear flows with a highly viscous dextran solution. A tangential force of about 5 X 10(-3) dyn/cell was required to achieve substantial detachment. The duration of application of the shearing force strongly influenced cell-substrate separation when this was varied from 1-10 s. Further, this treatment resulted in marked cell deformation, with the appearance of an elongated shape. Hence, cell-substrate separation is a progressive process, and binding strength is expected to be influenced by cell deformability. The minimum time required for adhesion was also investigated by making cells adhere under flow conditions. The maximum flow rate compatible with adhesion was about 1000-fold lower than that required to detach glass-bound cells. A simple model was devised to provide a quantitative interpretation for the experimental results of kinetic studies. It is concluded that cell-to-glass adhesion required a cell-substrate contact longer than a few seconds. This first step of adhesion was rapidly followed by a large (about 1000-fold) increase of adhesion strength. It is therefore emphasized that adhesion is heavily dependent on the duration of cell-to-cell encounter, as well as the force used to remove so-called unbound cells.     

Properties of prostaglandin-sensitive adenylate cyclase system of a murine macrophage-like cell line (P388D1)

The properties of basal and prostaglandin (PG)-stimulated adenylate cyclase of membrane preparations of P388D1 cells were investigated. Three partially purified membrane fractions were obtained by sucrose density gradient centrifugation at the final step of purification from crude homogenate. About 96% of the basal and 89% of PGE2-stimulated adenylate cyclase activity in the homogenate were recovered in three membrane fractions. Two lighter membrane fractions (I and II), which were enriched 11-fold and 8.4-fold in adenylate cyclase activity over crude homogenate, were pooled and subjected to various studies. Results suggested that the basal activity of the membrane preparations has, as in many other cell types, a relatively broad pH optimum (pH 7.5 to 8.5), requires Mg2+, which must be present in excess ATP, and is inhibited by Ca2+. Highly reactive sulfhydryl group(s), which may be present in the lipid bilayer, is required for the adenylate cyclase activity. Because both fluoride ions and GTP augment the enzymatic activity, P388D1 cell membrane adenylate cyclase must possess stimulatory guanine nucleotide-binding protein. The membrane preparations respond to exogeneously added PG by 1.5-fold to 3-fold increase in adenosine 3'-5' cyclic monophosphate (cAMP) production. The magnitude of PG-responsiveness was dependent on the types of PG and the order of potency in stimulation was PGE1 greater than PGE2 greater than PGI2. PGA1, B1, B2, F1 alpha, and F2 alpha stimulated adenylate cyclase only at the highest concentration tested.     

Interferon-gamma down-regulates membrane adenylate cyclase activity of a murine macrophage-like cell line (P388D1)

Effects of recombinant murine interferon-gamma (rIFN-gamma) on the membrane adenylate cyclase of a murine macrophage cell line (P388D1) were investigated in order to explore the nature of a signal transmitted by IFN-gamma receptor. Following the incubation of P388D1 cells with 40 U/ml of rIFN-gamma, the intracellular level of cAMP gradually increased about twofold over the control level within 60 min, and then began to gradually decline to about half the control level by 24 h incubation. The initial rise in cAMP level appeared to be due to the modest activation of adenylate cyclase and not due to the inhibition of cAMP-phosphodiesterase. Later decrease of intracellular cAMP may be due to quantitative down-regulation of the adenylate cyclase system. The basal enzymatic activity of the membrane prepared from P388D1 cells exposed to IFN-gamma for 24 h was found to be reduced to about 20% of that of the control membrane. However, the quality of the adenylate cyclase system appeared unchanged, because the relative degree of the response of the down-regulated membrane adenylate cyclase to prostaglandin PGE2, NaF, guanylimidodiphosphate (GppNHp), choleara toxin (CT), or forskolin was found to remain unchanged. This quantitative down-regulation of adenylate cyclase must be due to the action of rIFN-gamma, since the prior treatment of rIFN-gamma with either acid (pH 2) or monoclonal anti-IFN-gamma antibody inhibited the ability of IFN-gamma to induce the down-regulation. The rIFN-gamma-induced down-regulation is a reversible process, since the adenylate cyclase activity of the membrane was found to be restored when the rIFN-gamma-exposed cells were cultured for 72 h in the absence of rIFN-gamma. In addition, the 48 h-incubation of P388D1 cells with rIFN-beta or IFN-alpha was found not to significantly affect the membrane adenylate cyclase system.     

Involvement of group VI Ca2+-independent phospholipase A2 in protein kinase C-dependent arachidonic acid liberation in zymosan-stimulated macrophage-like P388D1 cells.

We investigated the possible involvement of group VI Ca2+-independent phospholipase A2 (iPLA2) in arachidonic acid (AA) liberation in zymosan-stimulated macrophage-like P388D1 cells. Zymosan-induced AA liberation was markedly inhibited by methyl arachidonoyl fluorophosphonate, a dual inhibitor of group IV cytosolic phospholipase A2 (cPLA2) and iPLA2. We found that a relatively specific iPLA2 inhibitor, bromoenol lactone, significantly decreased the zymosan-induced AA liberation in parallel with the decrease in iPLA2 activity, without an effect on diacylglycerol formation. Consistent with this, attenuation of iPLA2 activity by a group VI iPLA2 antisense oligonucleotide resulted in a decrease in zymosan-induced prostaglandin D2 generation. These findings suggest that zymosan-induced AA liberation may be, at least in part, mediated by iPLA2. A protein kinase C (PKC) inhibitor diminished zymosan-induced AA liberation, while a PKC activator, phorbol 12-myristate 13-acetate (PMA), enhanced the liberation. Bromoenol lactone suppressed the PMA-enhanced AA liberation without any effect on PMA-induced PKC activation. Down-regulation of PKCalpha on prolonged exposure to PMA also decreased zymosan-induced AA liberation. Under these conditions, the remaining AA liberation was insensitive to bromoenol lactone. Furthermore, the PKC depletion suppressed increases in iPLA2 proteins and the activity in the membrane fraction of zymosan-stimulated cells. In contrast, the zymosan-induced increases in iPLA2 proteins and the activity in the fraction were facilitated by simultaneous addition of PMA. Although intracellular Ca2+ depletion prevented zymosan-induced AA liberation, the translocation of PKCalpha to membranes was also inhibited. Taken together, we propose that zymosan may stimulate iPLA2-mediated AA liberation, probably through a PKC-dependent mechanism.     

Involvement of histamine H1 and H2 receptors in hypothermia induced by ionizing radiation in guinea pigs

Radiation-induced hypothermia was examined in guinea pigs. Exposure to the head alone or whole-body irradiation induced hypothermia, whereas exposure of the body alone produced a small insignificant response. Systemic injection of disodium cromoglycate (a mast cell stabilizer) and cimetidine (H2-receptor antagonist) had no effect on radiation-induced hypothermia, whereas systemic and central administration of mepyramine (H1-receptor antagonist) or central administration of disodium cromoglycate or cimetidine attenuated it, indicating the involvement of central histamine through both H1 and H2 receptors in this response. Serotonin is not involved, since the serotonin antagonist methysergide had no effect on radiation-induced hypothermia. These results indicate that central histaminergic systems may be involved in radiation-induced hypothermia.     

PGE2-induced desensitization of adenylate cyclase of a murine macrophage-like cell line (P388D1)

The mode of PGE2-induced desensitization of the adenylate cyclase of a murine macrophage-like cell line, P388D1 was investigated. The exposure of cells to PGE2 for 60 min induced PGE2-specific desensitization of the adenylate cyclase system which still responded normally to other specific ligand such as isoproterenol, 5'-guanylimidodiphosphate (Gpp(NH)p), or forskolin. The exposure of the cells to PGE2 for 6 hr induced heterologous desensitization, as the responses of adenylate cyclase to PGE2 as well as to isoproterenol or Gpp(NH)p were significantly reduced. The lowest concentration of PGE2 to induce both early homologous and late heterologous desensitization was found to be about two-fold over the KD of the low affinity PGE2-binding sites of P388D1 cells. The early homologous desensitization appeared to be due in part to the reduction in number of PGE2 receptors from the cell surface. The late heterologous desensitization may involve functional and/or structural alteration of Gs proteins, in addition to the reduction of PGE2 receptors from the cell surface.     

Inductive effect of recombinant human interleukin-1 alpha and beta on differentiation of macrophage-like tumor cell line P388D1

We used the mouse monocyte/macrophage-like tumor cell line P388D1 to test whether or not interleukin-1 (IL-1) stimulates differentiation of monocyte/macrophage progenitors. Incubation of these cells with recombinant human interleukin-1 (rhIL-1) alpha and beta resulted in their increased adherence, stimulation of nonspecific esterase activity, and increased Fc rosette formation. rhIL-1s inhibited cell growth and stimulated Fc rosette formation in a dose-dependent fashion. The cell growth inhibition due to rhIL-1s depended on the concentration of serum in culture medium. Synergism between rhIL-1 and calcium ionophore A23187 was found for the cell growth inhibition and Fc rosette formation. The presence of ethylene glycol bis- (beta-aminoethyl ether) N,N,N,N,-tetraacetic acid(EGTA) in the medium abolished the stimulatory effect of rhIL-1 on Fc rosette formation of the cell line. These results demonstrate that rhIL-1s are a potent inducer of the differentiation of the macrophage-like tumor cell line P388D1.     

Intracellular elevations of free calcium induced by activation of histamine H1 receptors in interphase and mitotic HeLa cells: hormone signal transduction is altered during mitosis

A broad range of membrane functions, including endocytosis and exocytosis, are strongly inhibited during mitosis. The underlying mechanisms are unclear, however, but will probably be important in relation to the mitotic cycle and the regulation of surface phenomena generally. A major unanswered question is whether membrane signal transduction is altered during mitosis; suppression of an intracellular calcium [( Ca2+]i) transient could inhibit exocytosis; [Ca2+]i elevation could disassemble the mitotic spindle. Activation of the histamine H1 receptor interphase in HeLa cells is shown here by Indo-1 fluorescence to produce a transient elevation of [Ca2+]i. The [Ca2+]i transient consists of an initial sharp rise that is at least partially dependent on intracellular calcium followed by an elevated plateau that is absolutely dependent on extracellular calcium. The [Ca2+]i transient is completely suppressed by preincubation with the tumor promoter, phorbol myristate acetate, but is unaffected by preincubation with pertussis toxin (islet-activating protein). In mitotic (metaphase- arrested) HeLa cells, the [Ca2+]i transient is largely limited to the initial peak. Measurement of 45Ca2+ uptake shows that it is stimulated by histamine in interphase cells, but not in mitotics. We conclude that the histamine-stimulated generation of the second messenger, [Ca2+]i, in mitotic cells is limited by failure to activate a sustained calcium influx. The initial phase of calcium mobilization from intracellular stores is comparable to that in interphase cells. Hormone signal transduction thus appears to be altered during mitosis.     

Interleukin-1 inhibits PGE2 binding to macrophage-like P388D1 cells by a cyclic AMP-independent process

The interaction between interleukin IL-1α and PGE₂ on P388D₂ on cells has been investigated. Preincubation of murine macrophage-like cells, P388D₁, with IL-1α (0–73 pM) reduced the binding of PGE₂ to these cells in a concentration-dependent manner. Scatchard analysis showed that IL-1α decreased the PGE₂ binding by lowering both the high and low affinity receptor binding capacities (from 0.31 ± 0.02 to 0.12 ± 0.01 fmol/10⁶ cells for the high affinity receptor binding sites and from 2.41 ± 0.12 to 1.51 ± 0.21 fmol/10⁶ cells for the low affinity receptor binding sites). However, the dissociation constants of the receptor of the IL-1α-treated cells remained unchanged. Inhibition of PGE₂ binding IL-1α did not involve changes in either protein phosphorylation or intracellular cyclic AMP levels. Our data clearly show that IL-1α inhibits the binding of PGE₂ to monocytes/macrophages and may thereby counter the immunosuppressive actions of PGE₂.