Changes in membrane potential induced by compound 48/80 in the peritoneal mast cells of rats

The changes in membrane potential induced by compound 48/80 were studied using rat peritoneal mast cells. The mean resting membrane potential of rat mast cells was -12.3 +/- 0.7 mM. When compound 48/80 was added to the mast cells, the cells were degranulated approximately 120 sec after the addition of the drug, after which immediate depolarization occurred. Degranulation of mast cells was not observed, even under the depolarization or hyperpolarization conditions caused by the replacement of a high K+ medium or the removal of K+ from the medium, respectively. Under both conditions, when compound 48/80 was added to the mast cells, degranulation was observed. Abrupt and marked depolarization was induced 30-60 sec after compound 48/80 was added. In addition, repolarization followed by gradual depolarization was observed without degranulation in mast cells treated with cytochalasin D after the addition of compound 48/80. These results suggest that the mast cells were depolarized by compound 48/80 independently of degranulation. It is also feasible that the gradual depolarization and repolarization induced by compound 48/80 in mast cells pretreated with cytochalasin D participated in the extracellular Na+ and Na+/K(+)-pump, respectively.     

Early kinetics of Ca2+ fluxes and histamine release in rat mast cells stimulated with compound 48/80

The kinetics of Ca2+ uptake and efflux have been measured in rat peritoneal mast cells stimulated with compound 48/80 using rapid mixing and a silicone oil centrifugation technique. Responses at one-second time intervals were resolved beginning as early as three seconds after initial stimulation. The results clearly demonstrate that Ca2+ uptake occurs after the initiation of histamine release. Ca2+ efflux occurs simultaneously with histamine release. The implications of these findings are discussed and the technique is described.     

Spectroscopic studies of rat mast cells, mouse mastocytoma cells, and compound 48/80-I. A spin-label study of membrane fluidities and the effect of 48/80

Purified rat mast cells, mixed peritoneal leukocytes and mouse mastocytoma cells take up spin-labeled membrane probes (5-doxyl, 12-doxyl and 16-doxyl stearic acids and their methyl stearate counterparts) in a manner similar to that of other cell types. The electron spin resonance spectra of these probes suggest that the membrane fluidities in the regions probed are similar in both mast cells and other normal leukocytes of the rat peritoneal cavity. However, the membranes of the mastocytoma cells were significantly more fluid in all of the regions probed. Membrane fluidity differences reported by the methyl stearate probes were greater than those reported by the corresponding fatty acid probes. This suggests that the chemical nature of the probe may contribute significantly to the apparent reported fluidity. The histamine liberator, compound 48/80, did not affect the spectra of any of the probes at concentrations that were magnetically dilute; however, it reduced the degree of spin exchange at high probe concentrations in mastocytoma cells. This suggests that 48/80 may increase the volume of the mastocytoma cell membranes, resulting in reduced intermolecular interactions among the probe molecules.     

Spectroscopic studies of rat mast cells,mouse mastocytoma cells,and compound 48/80—III: Evidence for a protein binding site for compound 48/80

The interaction of compound 48/80 with mast cells, mastocytoma cells, isolated membranes and liposomes has been studied using electron spin resonance (e.s.r.) and light-scattering techniques. Unlike spin-labeled stearic acid, spin-labeled 48/80 (SL-48/80) bound to mastocytoma cells in a manner that was unaffected by the non-ionic detergent, Triton X-100. In contrast, sodium dodecylsulfate (SDS), a protein denaturant, altered the SL-48/80 binding site(s), causing an apparent increase in the motion of the spin label. Treatment of SL-48/80-labeled mast cells with trypsin also increased the apparent motion of the bound label. Binding of 48/80 to mast cells, mastocytoma cells, erythrocyte ghosts, and isolated mastocytoma cell membranes increased their respective light-scattering properties. In contrast, the Rayleigh scatter of protein-free liposomes was unaffected by 48/80 treatment. Cationic, anionic, and non-ionic detergents also increased the light-scattering properties of mastocytoma cells, in contrast to the effect of several other cationic drugs, including histamine liberators. Increased light-scattering occurred after 48/80 treatment of inhibited mast cells (treated at 5°), indicating that the effect was not a consequence of degranulation. Untreated cells heated above 40° showed a similar increase in light scattering that became irreversible after reaching 45°. This indicates that cells treated with 48/80 resemble those in which the proteins have been denatured by heat. The light-scattering and the e.s.r. data, therefore, are consistent with the hypothesis that 48/80 binds to proteins in biological membranes.     

Binding of active components of compound 48/80 to rat peritoneal mast cells

The binding characteristics of compound 48/80 were examined using rat mast cells and fractionated 14C-labeled compound 48/80 components at 4 degrees C in vitro where no degranulation of the cells occurred. The binding potencies of these components in the presence of Ca2+ generally paralleled their histamine releasing activities, except in the case of fractions G (decamer) and H (nonamer), both Ca2+-independent releasers, for the binding of which Ca2+ was inhibitory. Scatchard analyses and displacement studies indicated that the mast cells had two types of binding sites with high and low affinities for fractions D (tridecamer, Ca2+-dependent releaser, Kd = 3.41 X 10(-8) M and 3.35 X 10(-6) M) and H (Ca2+-independent releaser, Kd = 1.11 X 10(-7) M and 9.11 X 10(-6) M), respectively. These sites partially overlapped each other, and also the fraction D site partially overlapped the IgE site and the fraction H site overlapped the neurotensin or substance P site.     

Glucose metabolism in rat mast cells. Stimulation of the pentose phosphate pathway by compound 48/80

The glycogen content of rat peritoneal mast cells (mean: 3 nmoles/10(6) cells) was increased 15% by incubation with glucose (1 mM) and reduced 35% when incubated without glucose at 37 degrees C for 15 min. The storage capacity for glycogen is thus low. Lactate production at 37 degrees C in a substrate-free medium was low (2.5-6.3 nmoles/10(6) cells in 40 min), but was stimulated 5-fold in the aerobic medium and 10-15 fold in the anaerobic medium by glucose. Both aerobic and anaerobic glycolysis in presence of glucose can thus provide energy for histamine secretion. The initial enzymes of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, have been demonstrated in mast cells. The enzyme activity in mast cells was, however, low compared to the high activity in the other peritoneal cells. The extent of the pentose cycle activity was determined from the conversion of 14C1- and 14C6-glucose to 14CO2, expressing the specific 14CO2 yields as fractions of the total glucose utilization. The normal pentose cycle activity with 1 mM glucose was 0.4% of the glucose metabolism. This was remarkably simulated by an electron acceptor, phenazine methosulfate. The pentose cycle was enhanced to 0.71% (80% stimulation) after exposure of the mast cells to compound 48/80, causing 68% histamine release. The stimulation of the pentose cycle by compound 48/80 seems to be due to the enhancement of biosynthetic processes during the regenerative phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic studies of rat mast cells, mouse mastocytoma cells, and compound 48/80-II. The synthesis and some binding properties of spin-labeled 48/80

Compound 48/80 has been spin labeled (SL-48/80), purified and its interaction with mastocytoma cells studied. The labeled drug was similar to the unlabeled compound with regard to biological activity and binding to biological membranes. The electron spin resonance (E.S.R.) spectra of membrane bound SL-48/80 were indicative of a bound probe in an environment which is more restricted than that of bound stearic acid spin labels, since the low field extremum was broad and the high field extremum was not discernible. Although ferricyanide ions broadened the E.S.R. signal of free SL-48/80, this agent did not affect the spectrum of SL-48/80 bound to mastocytoma cells. This would suggest that SL-48/80 binds in a region of the cell that is not in contact with the extracellular aqueous environment. Competition studies showed that SL-48/80 could be displaced by high concentrations of unlabeled 48/80 and polymyxin B.     

Inhibitory effects of steroidal anesthetics on histamine release from rat mast cells stimulated by concanavalin A, compound 48/80 and A23187

1. Alphaxalone and alphadolone acetate were found to inhibit histamine release from rat mast cells induced by concanavalin A by blocking the calcium channels of the cells. 2. They also both inhibited the release induced by A23187, but only alphaxalone inhibited the release induced by compound 48/80. 3. It is concluded that the inhibitory effects of these compounds were not due to their anesthetic properties, but may have been due to their inhibition of steps of the release cascade that open calcium channels and subsequent steps.     

The effect of concentration on the binding of compound 48/80 to rat mast cells: a fluorescence microscopy study

A fluorescent analog of the chemical histamine liberator, compound 48/80, has been synthesized by the covalent attachment of rhodamine to the 48/80 polymer (R-48/80). The histamine liberating characteristics of this analog were similar to those of the parent compound. The binding characteristics of R-48/80 to rat peritoneal mast cells were then studied using fluorescence microscopy. At concentrations that caused minimal secretory stimulation (less than 1.0 microgram/ml), R-48/80 bound to the mast cell surface in a diffuse manner, with no indication of patching or capping. When the cells were incubated at higher concentrations, where non-cytotoxic histamine secretion was stimulated, the drug bound heavily to the exposed granules, but not to unexposed granules or other cell organelles. At cytotoxic concentrations, R-48/80 caused extensive cell clumping, with the drug bound to masses of cell debris and released granules. Therefore, although R-48/80 binds initially to the cell membrane, its primary binding site at concentrations that induce secretion becomes the mast cell granule. The properties of these granules should thus be considered when studying the binding of compound 48/80 or other cationic drugs to rat peritoneal mast cells.     

Histamine release induced from mast cells by active components of compound 48/80

Compound 48/80 and 14C-labeled compound 48/80 were synthesized, and fractionated by thin-layer chromatography into 14 components (A-N) with various histamine releasing activities and different Ca2+ requirements for their actions. The histamine release induced from rat mast cells in vitro by the most active component, fraction D (molecular weight = 2280, a tridecamer composed of 13 monomer units), was greatly enhanced by extracellular Ca2+, and was partially reduced by pretreatment of the cells with dinitrophenylated ascaris antiserum, an IgE. In contrast, the histamine release induced by fraction H (molecular weight = 1580, a nonamer composed of 9 monomer units), was higher in Ca2+ -free medium than in Ca2+-containing medium, and was partially reduced by pretreatment of mast cells with neurotensin or substance P, Ca2+-independent releasers. Apparently both fractions D and H are useful reagents for investigating the role of Ca2+ in histamine release and releaser binding in mast cells.     

Broncho-Vaxom inhibits histamine release from rat mast cells induced by compound 48/80 and ionophore A23187

Broncho-Vaxom (BV) inhibited in dose-dependent manner the release of histamine from and degranulation of isolated rat peritoneal mast cells stimulated with compound 48/80 and the ionophore A23187. Inhibition persisted after removal of BV from the incubation medium before stimulation, but did not occur when bovine serum albumin (BSA) was used instead of BV. Binding of BV to mast cells was observed by electron microscopy on cells that had been incubated with colloidal-gold labelled BV. There was no significant difference between the binding of BV gold and BSA gold to the mast cells. Washing before fixation removed most of the BV gold from the cells. This study establishes BV as an in vitro histamine release inhibitor.     

Parallel secretion of endogenous 5-hydroxytryptamine and histamine from mast cells stimulated by vasoactive peptides and compound 48/80.

The peptides, neurotensin, substance P, somatostatin, and bombesin, several analogues and fragments of neurotensin and compound 48/80, all caused the secretion of both endogenous 5-hydroxytryptamine (5-HT) and histamine. There was no differential effect of any of the secretagogues tested on the secretion of 5-HT and histamine. Amitriptyline prevented the secretion of histamine in response to stimulation by neurotensin, substance P, somatostatin or compound 48/80 but was without effect on the secretion of endogenous 5-HT.     

Comparison of histamine release induced by synthetic polycations with that by compound 48/80 from rat mast cells.

We compared the histamine release induced by polyethylenimines and polyallylamines with that induced by compound 48/80. Lidocaine inhibited the histamine release induced by polyethylenimine with a molecular weight of 600 (PEI6), but disodium cromoglycate did not. The histamine releases induced by all polyethylenimines and polyallylamines tested were inhibited by lidocaine, but not by disodium cromoglycate. Islet activating protein inhibited the histamine release induced by PEI6. Its effects on the release by other polyethylenimines and polyallylamines were less than that on PEI6. It is likely that the inhibition of G proteins by islet activating protein resulted in a decrease of the histamine release. This possibility was supported by the finding that guanyl-5'-(beta, gamma-imino) triphosphate enhanced the histamine release. An inhibitor of polyphosphoinositide phosphodiesterase, neomycin, did not affect the histamine releases induced by these polymers. The effect of PEI6 seemed to resemble that of compound 48/80. After pretreatment of mast cells with wheat germ agglutinin and with Limax flavus agglutinin, releases of histamine induced by PEI6 and compound 48/80 decreased, suggesting that the binding sites of PEI6 and compound 48/80 had sialic acid and/or N-acetyl glucosamine residues. The binding site for PEI6 seemed to especially overlap those of compound 48/80.     

The utilization of adenosine triphosphate in rat mast cells during histamine release induced by anaphylactic reaction and compound 48/80

Summary The ATP content of rat peritoneal mast cells has been studied in relation to histamine release induced by compound 48/80 and antigen-antibody (anaphylactic) reaction in vitro. When the ATP content of actively sensitized mast cells was reduced to different levels by oligomycin, a good correlation was obtained between the ATP levels and the amounts of histamine released by the anaphylactic reaction. A similar linear relation has previously been demonstrated between the ATP levels of mast cells and histamine release induced by compound 48/80. The ATP content of mast cells was also studied at different intervals after the exposure of the cells to antigen or compound 48/80. No significant change in the ATP content was observed in untreated mast cells during the short period when histamine release occurs. If, however, the mast cells were preincubated with oligomycin or 2-deoxyglucose to reduce the rate of ATP synthesis while a large part of the histamine release remained unaffected—a decrease in the ATP content could be demonstrated in close time relation to both anaphylactic and compound 48/80-induced histamine release. The observations indicate an increased utilization of ATP in mast cells during the release process.     

The role of membrane bound sialic acid of rat mast cells in histamine release induced by compound 48/80 and derivatives as well as calcium

Histamine release induced by compound 48/80 from rat mast cells is not dependent on extracellular Ca2+. Preincubation of mast cells with trypsin has only little effects on histamine release induced by this polycation. This work also demonstrates that histamine release induced by compound 48/80 and its analogues in the absence of extracellular Ca2+ depends on membrane bound sialic acid of the mast cell. Neuraminidase treatment of the cells in the presence of extracellular Ca2+ leads to histamine liberation. These findings suggest that sialic acid residues of the mast cell membrane constitute the site at which polycations exert their stimulatory actions of histamine liberation.     

Dual effect of magnesium on compound 48/80-induced histamine secretion from rat peritoneal mast cells.

The effect of magnesium on the secretory response to compound 48/80 from rat peritoneal mast cells was studied. The decrease in secretion caused by calcium deprivation was enlarged by magnesium. Glucose partially counteracted the decrease caused by calcium deprivation but not the one caused by magnesium. The addition of calcium to the cells simultaneously with compound 48/80 completely restored the secretory response if magnesium was present. The response was only partially restored in a magnesium- and glucose-free medium, whereas it was almost completely restored if glucose was present. Magnesium had a considerable effect on the restoration of the secretory response of EGTA-treated cells, whereas the effect of glucose was minimal indicating that an effect on the energy metabolism was of minor importance. The secretory response could also be restored by an exposure of the cells to calcium prior to stimulation with compound 48/80. This was, however, only observed if magnesium was present and glucose had no effect. The influence of magnesium on the restoration of the secretory response may partly occur by an effect on the energy metabolism, partly by an effect on the stimulus-secretion coupling. We propose that insufficient supply of Mg2+ to the G-protein during activation by compound 48/80 might cause a suboptimal signal transduction.     

Effects of prolonged treatment with compound 48/80 on the gastric mucosa and mast cells in the rat

Effects of prolonged administration of compound 48/80 (48/80) on the gastric mucosa, serotonin and histamine levels in serum, and mast cells of rats were studied. Daily administration of 48/80 (0.75 mg/kg, i.p.) for 2 or 4 days produced widespread gastric lesions. Further administration of the agent for up to 12 days did not aggravate the lesions which had developed in the early period of administration of the drug. There were only a few visible lesions and numerous healed ones. Almost the same phenomenon was observed with the daily administration of serotonin plus histamine (10 mg/kg each, i.p.) for 2 to 12 days. While 48/80 given for 2 or 4 days increased serotonin and histamine levels in serum, it induced no appreciable increase of these amines after 8 or 12 days of treatment. Serotonin and histamine levels in peritoneal mast cells significantly decreased after the treatment with 48/80 over a 4 day period. The decrease in gastric lesions after prolonged treatment with 48/80 is due to both the depletion of serotonin and histamine from mast cells and an increased resistance of the gastric mucosa with healed lesions.