Isolation of cellular membranes from rat mast cells.

Large amounts of membranes enriched either in perigranular membranes or in plasma membranes have been successfully isolated from rat peritoneal mast cells. A cycle consisting of a single sonication pulse to disrupt the mast cells followed by centrifugation to separate the released granules was repeated until 90% of the mast cells were disrupted. This technique resulted in a high yield of intact granules since the released granules were only exposed to the single sonication pulse. The intact granules were separated from plasma membrane fragments by centrifugation through a Percoll gradient. The perigranular membranes were then obtained by osmotic lysis of the purified intact granules. The plasma membrane fraction was enriched 4.5-fold (range, 4.1-6.1) in 5'-nucleotidase activity, a plasma membrane marker enzyme. No suitable marker enzyme activity was found for the perigranular membrane fraction. An important aspect of this procedure is its potential for obtaining both a plasma and perigranular membrane preparation in high yield and purity from the same mast cell preparation.

Purification and biochemical characterization of an 11 000-dalton beta-bungarotoxin.

The chromatographic separation and biochemical characterization of a beta-bungarotoxin is described. This toxin is isolated as the most basic eluting protein of Bungarus multicinctus venom when separated by column chromatography on CM-Sephadex C-25. The protein migrated as a single band on pH 4.3 and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight of this toxin was estimated to be 10 000 +/- 1000 by analytical sedimentation analysis. This value was consistent with the electrophoretic mobility of the toxin in SDS-polyacrylamide gels. The amino acid composition of this 11 000-dalton beta-bungarotoxin was similar to that of the 22 000-dalton beta-bungarotoxin previously reported (Lee et al. (1972) J. Chromatogr. 72, 71--82; Kelly, R.B. and Brown, III, F.R. (1974) J. Neurobiol. 5, 135--150; Kondo et al. (1978) J. Biochem. Tokyo 83, 91--99), suggesting that the 11 000-dalton toxin may be one of the polypeptide chains of the larger toxin. The 11 000-dalton beta-bungarotoxin was toxic to mice when injected intravenously. Animals that received lethal doses exhibited hyperexcitability followed by ataxia, convulsions, and death. The minimum lethal dose was 0.12 microgram/g body weight. This beta-bungarotoxin exhibited Ca2+-dependent phospholipase A activity comparable to that of the 22 000-dalton beta-bungarotoxin. The enzyme exhibited phospholipid substrate specificity in the rank order of phosphatidyl-choline, phosphatidylserine, phosphatidylethanolamine, and phosphatidyl-inositol. The enzyme activity was destroyed by boiling for 3 min at pH 8.6. In addition, an enzymatically inactive quantity of the 11 000-dalton toxin, equivalent to five times the minimum lethal dose of enzymatically active toxin, was not lethal when injected into mice. To test whether phospholipase A activity is responsible for lethality, bee venom phospholipase A2 was injected into mice at similar and greater concentrations with no toxic effect. Thus, while phospholipase A activity may be required for the lethal effect of the 11 000-dalton beta-bungarotoxin, the specificity of action of the toxin is not determined by its enzyme activity.

The effect of calmodulin on histamine release in the rat peritoneal mast cell.

To investigate the role of the Ca2+-binding protein calmodulin on histamine release in the rat peritoneal mast cell, we exposed cells to exogenous calmodulin in the presence of a variety of histamine secretagogues. Histamine release stimulated by compound 48/80, polymyxin B and ionophore A23187 was inhibited while concanavalin A-stimulated release was not affected. Calmodulin in the presence of the secretagogues did not affect cell viability and calmodulin alone had no effect on histamine release. No direct interaction between calmodulin and the secretagogues was observed. Exogenous calmodulin does not appear to be incorporated into the cell. The inhibition of histamine release by calmodulin can be explained as a labile interaction between the protein and the cell that requires externally-bound Ca2+. These experiments demonstrate the use of exogenous calmodulin as a probe in the study of the mechanism of histamine release.

The mast cell.

The mast cell is the cellular basis for immediate hypersensitivity reactions. The specificity of the immediate hypersensitivity reaction is attributable to IgE molecules fixed to specific membrane receptors which, when stimulated by specific antigen, initiates the process of degranulation of the mast cell. The granules provide three separate sources of biologic activity: performed or primary mediators, newly generated or secondary mediators, and activities associated with the granular matrix. A number of biologic consequences are generated in response to these mediators and these include: increased vascular permeability, vasodilation, smooth muscle spasm, polymorphonuclear leukocyte chemotaxis, stimulation of adenylate and guanylate cyclase, superoxide radical generation, prostaglandin formation, mucous and gastric acid secretion, hypotension, tissue destruction, and mononuclear leukocyte infiltration. This pharmacopia of activities accounts for the clinical aspects of allergic diseases, suggests that the mast cell granule may be involved in the host's defense against parasitic infections, and is compatible with a suggested role of the mast cell as a widely distributed, monocellular endocrine system.

Effect of proteases on the beta-thromboglobulin radioimmunoassay.

Rat peritoneal mast cells and mast cell granules were evaluated by radioimmunoassay for the presence of beta-thromboglobulin and platelet factor 4. The initial assays indicated that a beta-thromboglobulin cross reacting material was released from mast cells by compound 48/80 in a similar dose-dependent manner as histamine release. The material was also found to be associated with purified granules. However, the use of protease inhibitors in the buffers completely abolished the positive assays. Further evaluation of the effects of various proteases on the beta-thromboglobulin assay indicated that elastase would also generate a false positive assay which could then be neutralized by the use of alpha 1-antitrypsin as a protease inhibitor. There was no protease effect on the platelet factor 4 radioimmunoassay which always showed no detectable amounts with mast cells, granules or proteases. These results clearly indicate the artifactual positive assays which can arise when using certain radioimmunoassay tests in the presence of cell proteases. The use of protease inhibitors is a necessary control when applying a radioimmunoassay to a system with potentially active proteases.

Canine postradiation histamine levels and subsequent response to Compound 48/80.

Radiation-induced hypotension in the beagle is accompanied by increased intestinal blood flow (IBF) and hematocrit (HCT). This study was performed to correlate these radiation-induced changes with plasma histamine (PH) levels following radiation. The histamine (H) levels were monitored in the systemic arterial circulation (SA) and the hepatic portal vein (HPV) before and after radiation. To examine the effect of radiation on the mobilization of total body H stores, Compound 48/80 was given I.V., and H responses were monitored in both control and radiated animals. Data obtained indicated that 100 Gy, whole-body, gamma-radiation produced a decrease in systemic mean blood pressure (BP), an increase in IBF and an increase in HCT. Concurrently, the mean PH/SA values increased and the PH/HPV levels decreased. Compound 48/80 produced a marked increase in PH levels in both control and radiated animals; however, the levels found in the radiated animals were consistently lower than those in the controls, although not statistically different. This implies that H may mediate these observed intestinal responses and that the mobility of histamine is decreased in radiated animals.

Adenyl cyclase activity of mouse liver membranes after incubation with endotoxin and epinephrine.

Adenyl cyclase activity in isolated mouse liver cell membranes was stimulated two-fold by endotoxin. Furthermore, endotoxin inhibited epinephrine induction of adenyl cyclase activity, apparently through interruption of the phospholipid moiety of the enzyme complex.

Presence of a high-affinity Ca2+- and Mg2+-dependent ATPase in rat peritoneal mast-cell membranes.

Purified perigranular and plasma membranes isolated from rat peritoneal mast cells were examined for Ca2+- and Mg2+-dependent ATPase activity. Isolated perigranular membranes contained only a low-affinity Ca2+- or Mg2+-dependent ATPase (Km greater than 0.5 mM). The plasma membranes contained both a low-affinity Ca2+- or Mg2+-dependent ATPase (Km = 0.4 mM, Vmax. = 20 nmol of Pi/min per mg), as well as a high-affinity Ca2+- and Mg2+-dependent ATPase (Km = 0.2 microM, Vmax. = 6 nmol of Pi/min per mg).

Plasma histamine and catecholamine levels during hypotension induced by morphine and compound 48/80.

Histamine receptors are present in adrenergic terminals, and histamine is reported to inhibit release of the neurotransmitter norepinephrine (NE) at certain neuroeffector junctions. However, a physiological role for histamine in modifying adrenergic neurotransmission has not been established. To examine the interaction of elevated plasma histamine and catecholamine release, two compounds that release histamine, morphine (3 mg/kg), and compound 48/80 (0.5 mg/kg), were administered intravenously (i.v.). Plasma norepinephrine (NE) levels were used to monitor sympathetic nervous system activity, and plasma epinephrine (Epi) levels were used to monitor adrenal activity. Both morphine and compound 48/80 caused an immediate and marked increase in plasma histamine. Simultaneous with this increase, a marked decrease in mean arterial pressure occurred. Plasma NE levels increased in animals administered compound 48/80, but in morphine-treated animals, plasma NE levels did not change from pretreatment values. Plasma Epi levels increased in both groups, but the magnitude and duration of the responses differed. The results indicate that elevated plasma catecholamines can increase in response to histamine-induced hypotension but this effect can be suppressed by the central actions of morphine.

Plasma histamine and hemodynamic responses following administration of nalbuphine and morphine.

A comparative study of plasma histamine levels following administration or morphine and nalbuphine in pentobarbital anesthetized dogs was performed. Two concentrations, 3 mg/kg and 0.3 mg/kg of these drugs were investigated. High dose morphine caused an immediate marked increase in plasma histamine from 5.0 +/- 0.4 to 340 +/- 72 ng/ml. Simultaneous with this increase in plasma histamine was a marked decrease in mean arterial blood pressure within the first minute. In contrast significant alterations in plasma histamine levels were not observed with high or low doses of nalbuphine. A low dose of morphine (0.3 mg/kg) did not increase plasma histamine levels. Heart rate was not changed by any drug treatment. The use of compound 48/80 a specific mast cell degranulating agent allowed for the identification of a specific pool of mast cells capable of responding to morphine. In vitro exposure of purified dog leukocytes to high doses of morphine did not result in histamine release. These results indicate that nalbuphine does not increase plasma histamine, while morphine does, and that the source of the increase in plasma histamine is from tissue mast cells.

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.

Recognition of antigenic differences among neurons using antiserums to clonal neural retina hybrid cells.

Antiserums prepared against hybrid cell strains formed between freshly isolated rodent neural retina cells and a human fibroblast cell line W.I. 18, VA-2 recognize cell surface antigens that are 1) restricted to the neural retina, 2) present in both embryonic and adult retina, and 3) localized to groups of cells within the retina. Normal segregants (i.e., those lines that had retained rodent chromosomes and lost human chromosomes) were used as immunogens in rabbits to produce the antiserums. Antiserums against both whole cells and purified plasma membranes were adsorbed with human parent VA-2 cells and nonneural and neural rodent tissue to remove cross-reacting specificities. All six antiserums studied continued to react with embryonic retina after brain cross-reactivity was removed.

Antihistamines block radiation-induced increased intestinal blood flow in canines.

Radiation-induced systemic hypotension is accompanied by increased intestinal blood flow (IBF) and an increased hematocrit (HCT) in dogs. Histamine infusion leads to increased IBF and intestinal edema with consequent secretion of fluid into the intestinal lumen. This study was performed to determine whether these effects could be diminished by prior administration of H1 and H2 histamine blockers. Dogs were given an iv infusion of mepyramine (0.5 mg/min) and cimetidine (0.25 mg/min) for 1 hr before and for 1 hr after radiation (H1 and H2 blockers, respectively). Mean systemic arterial blood pressure (MBP), IBF, and HCT were monitored for 2 hr. Systemic plasma histamine levels were determined simultaneously. Data obtained indicated that the H1 and H2 blockers, given simultaneously, were successful in blocking the increased IBF and the increased HCT seen after 100 Gy, whole-body, gamma radiation. However, the postradiation hypotension was only somewhat affected, with the MBP falling to a level 28% below the preradiation level. Plasma histamine levels reached a sharp peak, as much as 20% above baseline, at 4 min postradiation. These findings implicate histamine in the radiation-induced increase in IBF and HCT but not for the gradual decrease in postradiation blood pressure.

Blockade of neuromuscular transmission by enzymatically active and inactive beta-bungarotoxin.

beta-Bungarotoxins have been shown to be presynaptic blockers of neuromuscular transmission. This paper reports experiments using the most positively charged beta-bungarotoxin that elutes from a CM-Sephadex C-25 column. The toxin is shown to be a single polypeptide with a molecular weight of approximately 11,000 and has phospholipase A2 activity. The application of the enzymatically active toxin to the frog sciatic nerve-sartorius muscle preparation results in an initial decrease in the average endplate potential amplitude followed by a temporary rebound in endplate potential amplitude, and finally a complete inhibition of endplate potentials. Similarly, minature endplate potential frequency is initially reduced upon toxin application but then increases dramatically. After the phospholipase A2 of the toxin is inactivated, treatment with the toxin results in only the initial decrease in transmitter release. There results suggest that this beta-bungarotoxin acts in two functionally separate steps: (i) by binding to a specific presynaptic site possibly associated with calcium entry, and (ii) by perturbing the presynaptic membrane by its enzyme action, which results in an increase and then a failure in transmitter release.

Organophosphate-induced histamine release from mast cells.

To examine the hypothesis that soman intoxication leads to degranulation of mast cells with the release of histamine, we studied the effects of soman on rat peritoneal mast cells (RPMC) in vitro and in vivo. In vitro studies were performed with RPMC harvested from Edgewood rats, and challenged with soman (10(-8)-3 X 10(-3) M) in Tyrode's buffer. The RPMC exhibited a dose-dependent release of histamine, with maximal release of 50% at 3 to 6 X 10(-4) M. The release process is an active, secretory, noncytotoxic event, which is calcium and temperature dependent, requires metabolic energy and is influenced by intracellular levels of cyclic AMP. We next studied the in vivo effects of disodium cromoglycate (DSCG, 10(-4) M) on soman and Compound 48/80-induced histamine release. In vivo studies were performed by the i.p. injection of 5 ml of Tyrode's buffer containing soman (O-1 LD50), or Compound 48/80 as a positive control, with or without DSCG. The fluid recovered after approximately 10 min in the peritoneal cavity was examined for percentage of histamine release. In vivo, Compound 48/80 induced 49 +/- 1% histamine release, with no inhibition by DSCG (Compound 48/80 plus DSCG induced 49 +/- 0.4% histamine release). On the other hand, soman (1 LD50) induced 17 +/- 5% extracellular histamine release, with complete inhibition by DSCG (soman plus DSCG induced 3 +/- 1% histamine release). The data indicate that soman induced a dose-dependent release of histamine from RPMC, and provide evidence that histamine is a potentially important mediator of the pathophysiological response to organophosphate intoxication.(ABSTRACT TRUNCATED AT 250 WORDS)