Guinea Pig Peritoneal Exudate Macrophages Research Articles

CDNA cloning of guinea pig monocyte chemoattractant protein-1 and expression of the recombinant protein.

Monocyte chemoattractant protein-1 (MCP-1) cDNA was cloned from guinea pig spleen cells stimulated with Con A. The cDNA comprised 647 bp with an open reading frame that encoded a 120 amino acid protein. The sequence similarity of the first 99 amino acids to human MCP-1 is 56%. Recombinant guinea pig MCP-1 was expressed in COS-7 cells, then purified by a three step procedure with orange A-agarose, carboxymethyl-HPLC, and reversed phase-HPLC. The purified protein was found around 25 kDa as a broad band on a polyacrylamide gel under reducing conditions. Guinea pig MCP-1 attracted about 34% of input human monocytes at 5 x 10(-9) M. Guinea pig peritoneal exudate macrophages migrated toward guinea pig MCP-1 dose dependently, but only 1% of input cells responded to guinea pig MCP-1 at its optimal concentration of 5 x 10(-9) M. Human MCP-1 attracted 1% of input guinea pig peritoneal exudate macrophages at its highest concentration of 2.5 x 10(-8) M. Neither human nor guinea pig MCP-1 attracted guinea pig peritoneal resident macrophages. These results suggest that monocytes lose responsiveness to MCP-1 after differentiating to macrophages. Finally, intradermal injection of the recombinant protein into guinea pigs caused marked macrophage infiltration. Cloned and expressed guinea pig MCP-1 will help in studying the role of MCP-1 in vivo.

走化性因子刺激モルモット腹腔しん出マクロファージにおけるイノシトールりん脂質代謝とＧＴＰ結合蛋白質の連関性

The involvement of GTP-binding protein in inositol phospholipid metabolism in guinea pig peritoneal exudate macrophages stimulated with the chemoattractant N-formyl-Methionyl-Leucyl-Phenylalanine (fMLP) was examined. The GTP analog, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) caused a dose-dependent increase in the formation of inositol triphosphate in membranes of macrophages. This effect was specific for GTP and its analog. fMLP-induced inositol phospholipid turnover was markedly inhibited by the prior exposure of macrophages to 100 ng/ml of pertussis toxin (PT). Likewise, the pretreatment of macrophages with 100 ng/ml of PT evoked the inhibition of the increase in the intracellular free Ca2+ concentration and the spreading of macrophages induced by fMLP. These actions of PT were not associated with an alteration in the cellular concentration of cyclic AMP. Incubation of the membranes of macrophages with [32P]NAD and PT resulted in the ADP-ribosylation of a 41,000 Da protein. This ADP-ribosylation was diminished by the prior incubation of the membranes with 100 microM GTP gamma S plus 1 mM MgCl2, indicating that the 41,000 Da protein may be the alpha subunit of a GTP-binding protein. Moreover, there was a parallel between the time course of the ADP-ribosylation of intact macrophages by PT and the inhibition of the increase in intracellular free Ca2+ concentration as well as of the enhancement of the spreading of macrophages. These results suggest that the 41,000 Da protein, a GTP-binding protein, mediates the fMLP-stimulated inositol phospholipid metabolism.

Preferential loss of DNA polymerase alpha following suppression of replicative DNA synthesis of guinea pig macrophages by the immunostimulants muramyl dipeptide or lipopolysaccharide.

Oil-induced guinea pig peritoneal exudate macrophages were found to incorporate 3H-thymidine into trichloroacetic acid-insoluble fraction. In pulse-labeling experiments, the incorporated 3H-thymidine was detected in short fragments of DNA, which corresponded to the Okazaki fragments. These results indicate that the observed thymidine incorporation is due to nuclear DNA replication but not DNA repair. The observed DNA synthesis of the macrophages was remarkably suppressed when the cells were cultured in a presence of muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The significant decrease of DNA polymerase alpha activity was found in the cells treated with MDP or LPS. In contrast, the activity of polymerase beta was not at all affected by the same treatment.

Characterization of Ca 2+-activated phospholipid-dependent protein kinase C / protein kinase m in guinea pig peritoneal exudate macrophages

Protein kinase C (C-kinase) is shown to be present in the cytosolic and particulate fractions of mineral oil induced peritoneal macrophages of guinea pigs. By omission or use of a high concentration of leupeptin, three forms of the enzyme were obtained: stimulant/Ca2+/phospholipid-dependent C-kinase, eluted from DEAE 52 cellulose at 0.08-0.16 M NaCl; stimulant/Ca2+/phospholipid-independent protein kinase M (M-kinase), and Ca2+-inhibited & stimulant/phospholipid-dependent form of protein kinase, both eluted from DEAE 52 cellulose at 0.18-0.22 M NaCl. Phorbol ester or 1,2-diacylglycerol were used as stimulants. It is suggested that Ca2+-inhibited & stimulant/phospholipid-dependent protein kinase represents the in vivo form of the M-kinase in intact cells.

Enhanced binding and degradation of the C1q subcomponent of complement by thioglycollate-stimulated guinea pig peritoneal macrophages.

Expression of C1q receptors on the plasma membrane of thioglycollate-stimulated guinea pig peritoneal exudate macrophages increased 1.54 times as compared to unstimulated controls. A Scatchard plot of the binding of 125I-C1q to the cells revealed that the binding is a result of an increase in the number of receptors and not to an increased affinity of the receptors. Thioglycollate-activated macrophages were found to be 1.6 times more active than nonactivated macrophages in the binding of 125I-C1q at 4 degrees C. The enhanced binding of 125I-C1q by activated peritoneal macrophages was reflected in an increase in the amount of 125I-C1q degraded by these cells as compared to resident peritoneal macrophages. This suggests that stimulation of phagocytic cells leads to an increase in the expression of C1q receptors and to a concomitant increase in the uptake and degradation of C1q.

A thiol protease of peritoneal macrophages in the guinea pig.

Proteolytic enzymes of the guinea pig peritoneal exudate macrophages were investigated using synthetic fluorogenic peptide substrates. Among several enzymes, t-butyloxycarbonyl-phenylalanyl-seryl-arginine 4-methylcoumaryl-7-amide cleaving enzymes had the highest activity, and the activity in exudate macrophages was about 3 times stronger than that in resident macrophages. The molecular weight of the enzyme was around 35,000 and optimal pH around 6.5-7.0. It was inhibited by thiol-blocking reagents, suggesting a thiol protease.

Down-regulation of Fc receptor expression in guinea pig peritoneal exudate macrophages by muramyl dipeptide or lipopolysaccharide.

Expression of Fc receptors on the plasma membrane of guinea pig peritoneal exudate macrophages (PEM) was suppressed to almost one-half of that of the controls by long-term exposure to lipopolysaccharide (LPS) or muramyl dipeptide (MDP) in culture. The effect of the reagents was dose and time dependent, and as little as 0.5 ng/ml LPS or 5 ng/ml MDP was effective for the suppression. The expression of the Fc receptors decreased to 60 to 70% of the control level at 48 hr and to 45 to 50% at 72 hr after incubation of the cells in the presence of LPS or MDP. A Scatchard plot of the binding of 125I-soluble immune complexes (I.C.) to the cells revealed that the decrease in the binding of 125I-I.C. is due to a reduction in the number of Fc receptors on the cell membrane and not to a decreased affinity of the receptors. The membrane protein was radio-labeled with 125I, and the Fc receptors were purified by being bound to insoluble I.C. The specific binding of the 125I-labeled Fc receptors, from the LPS-treated macrophages, to the insoluble I.C. was almost one-half of that from the untreated control cells. SDS-PAGE analysis of the purified 125I-labeled Fc receptors revealed that the major peak of the m.w. 44,000 molecule in the LPS-treated cells was almost one-half of that of the control. Contrary to the effect of LPS or MDP, 72-hr incubation of macrophages with MIF-rich supernatant, cultured from lymph node cells, enhanced the expression of Fc receptors. Macrophages were treated with I.C. for 4 hr at 37 degrees C to remove the Fc receptors from the surface membrane. The reappearance of the receptors on the plasma membrane of the cells was significantly suppressed by LPS and MDP. The effect of LPS on the binding of five murine monoclonal antibodies (Ab) raised against PEM to the macrophage membrane and also that of 125I-wheat germ agglutinin (WGA) or 125I-insulin was studied. The monoclonal Ab were selected for their activity to induce superoxide anion generation in the macrophages, as do I.C., although the binding sites for the monoclonal Ab were not related to Fc receptors. The bindings of the five monoclonal Ab were not affected by exposure of the cells to LPS or MDP. Macrophages treated with the reagents bound as much 125I-insulin or WGA as did the untreated control cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of soluble aggregates of IgG on the binding, uptake and degradation of the C1q subcomponent of complement by adherent guinea pig peritoneal macrophages.

Earlier studies have indicated that C1q, the first subcomponent of complement component C1, is bound to lymphocytes via specific C1q receptor sites. We have recently shown that adherent guinea pig peritoneal exudate macrophages express specific receptors for C1q (Veerhuis, R. et al., Immunology 1985. 54: 801). The present studies were performed to determine whether binding of 125I-labeled human C1q (125I-C1qhu) to adherent guinea pig peritoneal exudate macrophages would also result in ingestion and subsequent degradation of 125I-C1qhu. The binding of 125I-C1qhu to adherent peritoneal macrophages at 4 degrees C is inhibited fully not only by C1qhu and guinea pig C1q (C1qgp) but also by pepsin fragments of C1qhu. The amount of trichloroacetic acid nonprecipitable radioactivity that appeared in the supernatant was used as a measure for the degradation of 125I-C1qhu. 125I-C1qhu is degraded initially into fragments of 25 kDa, after which it is degraded further into small molecular weight peptides. Ingestion of 125I-C1q by the macrophages occurs before the 125I-C1q is degraded. In the presence of limited amounts of soluble aggregates of guinea pig IgG2 (AIgG), a known activator of C1, part of the C1q is bound to the AIgG and all of the AIgG in turn is bound to the cellular Fc receptors leading to an enhanced binding of 125I-C1q to the cells, a binding that was maximal at near equimolar concentrations of 125I-C1qhu and 131I-AIgG. In the presence of a 30-fold excess of AIgG, however, only a small percentage of the AIgG binds to cellular Fc receptors and the interaction of C1q with its receptor is decreased due to competitive inhibition. The results presented in this report thus suggest that free C1q may be eliminated by specific interaction with C1q receptors present on circulating and tissue phagocytoses and, in addition, that in the presence of immune complexes modulation of elimination of C1q may be encountered.

Different mechanisms of macrophage activation with guinea pig macrophage activation factor, lipopolysaccharide and muramyl dipeptide.

The mechanism of macrophage activation was studied using three activating substances, guinea pig macrophage activation factor (MAF), lipopolysaccharide (LPS) and muramyl dipeptide (MDP). Guinea pig peritoneal exudate macrophages were activated to exhibit the accelerated glucose consumption in response to these activating substances. Calmodulin-specific inhibitors, trifluoperazine and No. 233, inhibited macrophage activation with MAF and LPS, while these inhibitors did not affect the activation with MDP. Ca2+ uptake into macrophages was enhanced in MAF-treated macrophages, but LPS and MDP did not affect the Ca2+ uptake. Methylamine and ethylamine, inhibitors of transglutaminase-dependent protein internalization into cells and/or of lysosomal enzymes, effectively inhibited the activating effect of LPS, but not those of MAF and MDP. These results suggest that Ca2+ and calmodulin play a role in macrophage activation with MAF, and neither transglutaminase-dependent internalization nor lysosomal enzymes participate in the activation process. In case of LPS, internalization into cells would be necessary for its activating effect. The processing of the contrary, since the activating effect of MDP was not affected by any of these inhibitors, the mechanism of activation with MDP remains obscure. Thus, the mechanisms of macrophage activation with MAF, LPS and MDP appear to be different from each other.

Inhibition of macrophage DNA synthesis by immunomodulators: II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide of [ 3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [ 3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [ 3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase α and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [ 3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [ 3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized “cold” thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [ 3H]thymidine. These results suggest that the suppression of [ 3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E 2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.

Enhancement of macrophage invasion of tumors by administration of chemotactic factor-antitumor antibody conjugates

The numbers of macrophages in peritoneal guinea pig hepatomas were significantly ( P < 0.005) elevated by the intraperitoneal injection of a covalent conjugate of the chemotactic peptide, formylmethionylleucylphenylalanine (fMLP), and IgG reactive with surface antigens on the hepatoma cells. These conjugates, which were previously shown to be chemotactic for guinea pig peritoneal exudate macrophages in vitro, increased the numbers of macrophages in the tumors approximately twofold when injected either in a single dose or in five doses. Although five injections of unconjugated fMLP were nearly as effective as the IgG-fMLP conjugates, free fMLP did not enhance the numbers of macrophages in tumors when injected as a single dose. Unconjugated IgG had no effect. The mean tumor weights were decreased in those groups of guinea pigs which received IgG-fMLP but statistical significance was not achieved due to tumor weight variability in all groups.

The mechanism of action of lymphokines VII. Modulation of the action of macrophage migration inhibitory factor by antioxidants and drugs affecting thromboxane synthesis

Guinea pig peritoneal exudate macrophages are active producers of oxygen radicals in response to membrane stimulation. We examined the involvement of oxygen radicals in the inhibition of macrophage migration caused by the lymphokine, macrophage migration inhibitory factor (MIF). It was found that the presence of scavergers of superoxide and hydrogen peroxide did not influence MIF action. Exogenous horseradish peroxidase, however, significantly enhanced the migration inhibitory effect of MIF. Among six scavengers of hydroxyl radicals and singlet oxygen that were tested, L-methionine (20–80 mM) and L-histidine (40–80 mM) were capable of preventing MIF action. Macrophage responsiveness to MIF was also blocked by the lipid antioxidant propyl gallate (0.125–0.25 mM). Lymphokine-induced inhibition of migration could not be prevented by inhibitors of phospholipase A2, cyclooxygenase, or lipoxygenase. The inhibition of thromboxane synthetase, however, by imidazole, 1-benzylimidazole and the prostaglandin endoperoxide analogs U-51605 and U-44069 effectively prevented MIF action. These findings are compatible with the hypothesis that the inhibition of macrophage migration by MIF is the result of reversible autotoxic damage by a yet unidentified product of the oxidative burst, possibly hypochlorous acid. Thromboxane also appears to be involved in MIF action probably by its adhesiveness increasing and proaggregatory actions. The intracellular target of the autotoxic attack and the relative importance of oxygen-derived products and thromboxane in the mediation of MIF-induced migration inhibition are unknown.

Unsaturated fatty acids as second messengers of superoxide generation by macrophages

Chemically elicited guinea pig peritoneal exudate macrophages respond by Superoxide (O 2 −) production to a large number of unrelated stimulants. It has been found that 8 out of 10 stimulants also induce arachidonic acid (20:4) liberation and thromboxane synthesis. The elicitation of O 2 − production by most stimulants was reduced or totally suppressed by three procedures that inhibit the activity of endogenous phospholipases: the use of drug p-bromophenacyl bromide, elevation of the cellular cyclic AMP level, and the removal of extracellular Ca 2+. O 2 − production in response to concanavalin A, wheat germ agglutinin, and fMet-Leu-Phe were exquisitely sensitive to inhibition of phospholipase activity. Exogenously applied 20:4 as well as other unsaturated fatty acids (linolenic, linoleic, and oleic) induced massive and instantaneous O 2 − production in a dose-dependent manner. Saturated fatty acids (stearic) and methyl esters of unsaturated acids were inactive. Lysophosphoglycerides were also inactive. Incubation of macrophages with inhibitors of cyclooxygenase or lipoxygenase did not prevent the elicitation of O 2 − production by stimulants or fatty acids. On the contrary, O 2 − formation was enhanced by indomethacin and indomethacin by itself was capable of evoking O 2 − generation. Treatment of 20:4 with soybean lipoxygenase did not abolish its capacity to induce O 2 − production; native and lipoxygenasetreated 20:4 exhibited similar dose-response ratios. Purified 15-hydroxyeicosatetraenoic acid also elicited O 2 − production by macrophages with a potency comparable to but not exceeding that of 20:4. Equimolar amounts of prostaglandin E 2 were inactive. These findings suggest that liberation of unsaturated fatty acid (principally, 20:4) from membrane phospholipids, as a consequence of phospholipase activation, is a necessary step in the elicitation of an oxidative burst in macrophages. O 2 − generation is stimulated by unesterified 20:4 and, possibly, by certain metabolites of 20:4. It appears that the lipoxygenase pathway may generate metabolites with stimulating capacity while the cyclooxygenase pathway is abortive.

Inhibition of macrophage DNA synthesis by immunomodulators. I. Suppression of [3H]thymidine incorporation into macrophages by MDP and LPS.

Oil-induced guinea pig peritoneal exudate macrophages were found to incorporate actively [3H]thymidine without any tissue fluids such as conditioned medium, lymphokines or inflammatory tissue exudates. The [3H]thymidine incorporation was markedly suppressed by macrophage stimulants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS), while glucosamine incorporation was simultaneously increased by these stimulants. The degree of suppression of thymidine incorporation depended on the cell density, the concentrations of the stimulants, and sera or culture media used. The exposure of macrophages to MDP for 30 min was sufficient to cause significant suppression.

In vitro effects of antitumor antibody-chemotactic factor complexes

Antitumor antibodies with in vitro chemotactic activity for guinea pig peritoneal exudate macrophages and polymorphonuclear leukocytes were obtained by covalent linkage of the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (fMLP) to rabbit IgG reactive with a strain 13 guinea pig hepatoma. In addition to its chemotactic properties, the fMLP coupled to antibody also retained its ability to stimulate the production of superoxide anions by macrophages. Neither the IgG nor the IgG treated with the carbodiimide coupling reagent exhibited any of these biological activities. The antibodies, which were adsorbed with sheep erythrocytes and normal strain 13 liver cells prior to the coupling procedure, reacted with surface antigens present on the hepatoma cells but not with those on fibrosarcoma cells or fibroblasts as determined by complement-dependent cytotoxicity. This complement-mediated function was, however, greatly diminished by coupling the IgG to fMLP although indirect immunofluorescent staining patterns established binding of the complexes to hepatoma cells but not to normal liver cells. When bound to tumor cells the antibody-peptide conjugates were also chemotactic, implying that the conjugated antibodies, conjugated antibody-tumor antigen complexes, or fMLP may be released and establish an effective chemotactic gradient for peritoneal exudate cells in vitro.

Effects of tumour cell culture supernatants on some biochemical activities of macrophages.

The following enzyme activities were measured in cell lysates and supernatants from mouse peritoneal macrophages incubated with products of cultured tumour and other cells: acid phosphatase, beta-D-glucuronidase, N-acetyl-D-glucosaminidase, muramidase (lysozyme), lactic dehydrogenase (supernatant only) and plasminogen activator (supernatant only). There were no noteworthy changes in enzyme activities. Hydrogen peroxide production by appropriately stimulated mouse and/or guinea-pig peritoneal exudate macrophages was variably inhibited by supernatants from some tumour cells and some normal cells. Changes in these biochemical activities of macrophages do not appear to be closely related to the anti-inflammatory activity of tumour cell products.

Ether phospholipids in guinea pig polymorphonuclear leukocytes and macrophages Occurrence of high levels of 1- O-alkyl-2-acyl- sn-glycero-3-phosphocholine

Significant proportions of the choline phosphoglycerides (CPG) were found to contain alkyl ether-type moieties (e.g., 1- O-alkyl-2-acyl-glycero-3-phosphocholine) in both guinea pig peritoneal exudate polymorphonuclear leukocytes (16.4%) and macrophages (13.5%). High proportions of the ethanolamine phosphoglycerides (EPG) contained alkenyl ether moieties in both cells (37.2 and 41.2%), while the proportions of the CPG containing alkenyl moieties and of the EPG containing alkyl moieties were shown to be small. The ether phospholipid composition as well as the fatty chain profiles of these two types of cells had relatively similar patterns. However, the fatty chains at the 1- and 2-positions for alkenyl ether, alkyl ether and diacyl phosphoglycerides showed considerable differences. The amount of 16:0 at the 1-position was higher in alkyl compounds than that in diacyl compounds of the CPG. This was also the case in ether-containing and diacyl EPG. The most predominant fatty acids at the 2-position was 18:2, in each lipid class, except for the alkenyl CPG. The amounts of 20:4 and other polyunsaturated fatty acids were low in every lipid class, though ether compounds contained higher amounts of 20:4 than diacyl compounds, particularly for EPG.

Superoxide anion and hydrogen peroxide production by chemically elicited peritoneal macrophages—Induction by multiple nonphagocytic stimuli

The ability of a number of stimulants to activate an oxidative burst (OB) in oil-elicited guinea pig peritoneal exudate macrophages (MPs) was examined. The parameters of the OB were the generation and extracellular release of Superoxide anions (O 2 −) and hydrogen peroxide (H 2O 2). We found that: (1) The cocarcinogen and skin irritant phorbol myristate acetate (PMA) was the most potent OB activator—The weak cocarcinogen 4- O-methyl PMA was a proportionally less effective OB activator; (2) The lectins concanavalin A (Con A) and wheat germ agglutinin (WGA), but not soybean, Lotus, and pokeweed lectins, were also quite effective OB activators—The ability of Con A to stimulate O 2 − production was abolished by succinylation and could be prevented by the presence of α-methyl- D-mannoside; (3) Other stimulators of an OB in MPs were: N-formyl-methionyl peptides, opsonized zymosan, the Ca 2+ ionophore A23187, phospholipase C, NaF, antimacrophage antibody, microtubule-disrupting drugs, and sodium nitroprusside—O 2 − generation induced by A23187 (but not that stimulated by PMA) was dependent on extracellular Ca 2+; (4) The amount of O 2 − produced per cell was higher at low cell densities; (5) The addition of Superoxide dismutase (SOD) to the medium totally prevented the detection of O 2 − and augmented twice the amount of H 2O 2 recovered; (6) Pretreatment of MPs with the SOD inhibitor sodium diethyldithiocarbamate had no effect on the release of O 2 − but blocked H 2O 2 release in a dose-dependent manner. These data were interpreted as indicating that the bulk of H 2O 2 was derived by enzymatic dismutation of O 2 −; (7) The common mechanism by which such a variety of stimuli provoke an OB in MPs was not elucidated. No evidence was found to suggest a role for a cyclic nucleotide messenger.

Cyclic GMP metabolism in macrophages: I. Regulation of cyclic GMP levels by calcium and stimulation of cyclic GMP synthesis by NO-generating agents

Levels of guanosine 3′,5′-cyclic monophosphate (cGMP) were determined by radioimmunoassay in adherence-purified, oil-induced guinea pig peritoneal exudate macrophages, after extraction of the cells with perchloric acid, purification on Dowex AG1-X8, and acetylation. We found that: (i) Basal cGMP levels were strictly dependent on the concentration of extracellular Ca 2+ (0.33 ± 0.03 pmol/mg macrophage protein in Ca 2+-free medium and 2.49 ± 0.42 pmol/mg in 1.8 m M Ca 2+). (ii) The stimulatory effect of Ca 2+ on cGMP levels was prevented by tetracaine. (iii) The cGMP content of macrophages was not elevated by incubation with the ionophore A23187 at extracellular Ca 2+ concentrations varying between 0 and 1.8 m M. (iv) Macrophage cGMP levels were increased markedly (up to 40-fold) by incubation of the cells with the nitric oxide (NO)-generating agents, sodium azide, hydroxylamine, sodium nitrite, and sodium nitroprusside. (v) Stimulation of cGMP accumulation by NO-generating agents occurred within 30 sec, was Ca 2+-independent, and developed in the presence and absence of the phosphodiesterase inhibitor, isobutyl-methylxanthine. (vi) A minimal elevation in the macrophage cGMP level (less than 2-fold) was induced by ascorbic acid but no significant increases were induced by the following agents, found effective in other cells: serotonin, acetylcholine, carbamylcholine, phorbol myristate acetate, arachidonic acid, Superoxide dismutase, and nitrate reductase.

Concanavalin A Receptor Proteins of Plasma Membranes of Guinea Pig Macrophages

The transmembrane organization of concanavalin A receptor proteins in purified plasma membranes from guinea pig peritoneal exudate macrophages was investigated. Inside-out vesicles prepared from the plasma membrane by affinity chro-matography on concanavalin A-Sepharose remained permeable to con-canavalin A and could be labelled from the outside only with immobilized lactoperoxidase-catalysed radio-iodination. After detergent solubliization, concanavalin A receptor proteins were isolated from the radio-iodinated inside-out vesicles and their mobilities were analyzed by electrophoresis in poly-acrylamide gels containing sodium dodecyl sulfate; this was followed by autoradiography. By these procedures, four iodinated proteins with apparent molecular weights of approximately 147, 000, 125, 000, 102, 000 and 77, 000 were specifically retained in the concanavalin A-Sepharose affinity columns, evidence that these glycoproteins span the plasma membrane.