Antimicrobial action of compound 48/80--II mechanism of action.

The mixture of compounds called compound 48/80 had been shown to have antimicrobial activity against a wide variety of microorganisms. In this paper it is shown that its primary site of attack appears to be on the membrane of the cell. In its presence, Tetrahymena became much more sensitive to osmotic stress, and alpha-methylglucose was rapidly released from preloaded Escherichia coli cells. The drug also had effects on cell viability, respiration, cell division, and the release of material absorbing at 260 nm. In general, its effects paralleled those of polymyxin B, although its structure is quite different except for the presence of amino groups and hydrophobic regions in both molecules. The activity of 48/80 was not due to detergent-like, surface-active properties and was antagonized by magnesium and other cations and by phosphatidylserine. Purification of the active principle might provide a relatively simple and readily modifiable probe of membrane function and possibly a new family of useful antimicrobial compounds.

Relationship between phosphatidylserine and cromolyn in histamine release.

The opposing actions of phosphatidylserine (PS) and cromolyn on histamine release were studied in rat peritoneal mast cells in vitro. Histamine-releasing drugs could be separated into 2 groups on the basis of whether they were potentiated by PS and inhibited by muM cromolyn (dextran, antigen (BSA), concanavalin A) or neither potentiated by PS nor inhibited by muM cromolyn (48/80, polymyxin, phospholipase C and ATP). Compound 48/80 and polymyxin chemically combined with PS, but this could be circumvented by preincubating the mast cells in PS for one hour, and then washing them free of PS in solution prior to addition of the drugs. These data define at least 2 pathways for induction of histamine release: (1) a PS-potentiated pathway inhibited by muM cromolyn, and (2) a PS-independent pathway unaffected by muM cromolyn. Dose-response curves of the muM cromolyn inhibition of PS-potentiated release revealed a parallel shift, suggesting that cromolyn may compete with PS.

Antimicrobial action of compound 48/80 against protozoa, bacteria, and fungi.

Compound 48/80 inhibited the growth of protozoa, bacteria, and fungi but had no effect on the multiplication of viruses. All susceptible organisms were inhibited by 10 microgram/ml of crude compound 48/80, and some were inhibited by as little as 0.1 microgram/ml. Against Tetrahymena pyriformis, this drug was seven times more potent than quinine. Separation of compound 48/80 into different fractions indicated that some antimicrobial activity could be separated from the histamine-liberating activity. It was found that compound 48/80 is not surface active at 500 microgram/ml.

Benzalkonium chloride: selective inhibitor of histamine release induced by compound 48/80 and other polyamines.

Benzalkonium chloride (BAC) is a mixture of quaternary benzyldimethylalkylammonium chlorides which was found to inhibit histamine release induced by polyamines (48/80, ATP, bradykinin, curare, guanethidine, polylysine, polymyxin B, poly-THIQ, protamine, stilbamidine or substance P), but not that caused by antigens, concanavalin A, dextran, lonophores (A23187 or X-537A), enzymes (chymotrypsin or phospholipase C), monoamines (dextromethorphan, meperidine or chlorpromazine) or detergents (decylamine, Triton X-100 or a fire ant venom alkylpiperidine). Inhibition by 1.5 and 3 microgram of BAC per ml caused parallel shifts of the 48/80 dose-response curves to the right with no loss of efficacy, indicating that the antagonism was surmountable. Phospholipase C was partially inhibited by BAC, but Triton X-100 also inhibited phospholipase C (but not 48/80), indicating that the inhibition of phospholipase C by BAC was probably a nonspecific, detergent effect. BAC caused histamine release by itself at concentrations over 5 microgram/ml. Heat inactivation (50 degrees C for 15 min) of the mast cells did not prevent this release, suggesting a lytic mechanism for this action. Structure-activity relations studies on various members of the BAC family for their ability to inhibit 48/80-induced histamine release indicated that benzyldimethyltridecylammonium chloride was the most potent.

Antagonism of the final common pathway of mast cell histamine secretion by arylalkylamines.

The antagonism of histamine secretion from rat peritoneal mast cells by a range of drugs (e.g., antihistamines, local anesthetics, tranquilizers, antidepressants, adrenergics and antiadrenergics) and arylalkylamines was studied. Simple arylalkylamines, those without quaternary ammonium, phenolic or anionic groups, uniformly inhibited histamine secretion induced via three major secretory pathways of mast cells (compound 48/80, A23187 and concanavalin A). That is, each simple arylalkylamine nonselectively inhibited all three secretagogues at about the same concentration (avg. range = 2.2). This nonselective inhibition was rapid in onset (seconds), readily reversible by washing and not related to a decrease in ATP. It is likely that these agents act nonspecifically at a step in the final common pathway in the cell membrane, possibly to inhibit fusion of the secretory granules with the plasma membrane. Other, more complex arylalkylamines were selective in their antagonism of histamine secretion. Quaternary arylalkylammonium compounds selectively antagonized polyamine secretagogues (48/80 and polymyxin B), whereas arylalkylamines, with other hydrophilic groups, selectively inhibited one or another of the secretory pathways. These complex arylalkylamines must act before the final common pathway because of their selective inhibition. At higher concentrations most of the arylalkylamines caused disruption of the mast cells and liberated histamine.

The site of action of the histamine releaser compound 45/80 in causing mast cell degranulation.

A primary amine analog of compound 48/80 (nor-48/80) was purified and attached to Sepharose beads through an albumin link. Suspensions of rat peritoneal cells were passed over the beads in an affinity chromatography column, and the proportion of the mast cells that was retained by the beads was determined. Sixty-seven percent of the mast cells were found to be retained by the column, indicating the existence of a binding site for nor-48/80 on the exterior of mast cells. The beads were larger than the cells, and hence precluded the entry of the nor-48/80 into the cells. Neither the Sepharose beads nor the albumin link was responsible for this amount of binding because control beads without nor-48-80 retained only 22% of the mast cells. Mast cells incubated with large quantities of the beads in a batch procedure released up to 53% of the mast cell histamine, whereas control beads without the polymer released only 18%. This release could not be attributable to soluble nor-48/80 because only trace amounts of radioactive nor-48/80 were released from beads soaked overnight, and these supernatants released insignificant amounts of histamine when incubated with mast cells. These studies indicate the presence of a receptor for 48/80 on the mast cell membrane which can trigger histamine release.

Competitive inhibition of 48/80-induced histamine release by benzalkonium chloride and its analogs and the polyamine receptor in mast cells.

Benzalkonium Cl is a family of benzyldimethylalkylammonium compounds which are selective inhibitors of histamine release induced by 48/80 and many other polyamines, but do not inhibit histamine release caused by antigen-antibody reactions, ionophores, enzymes or detergents. In this study, additional histamine-releasing poly- and monoamines found to be antagonized by benzalkonium Cl in rat mast cells were cadaverine, hexamethonium, decamethonium, lysine, dilysine, trilysine, pentalysine, mellitin, pentamidine, spermidine, spermine, morphine, norepinephrine and tyramine. Benzyldimethyltetradecylammonium Cl, one of the safest and most potent members of the benzalkonium Cl family, was found to antagonize 48/80-induced histamine release by a competitive mechanism. Benzyldimethyltetradecylammonium Cl was also found to inhibit 48/80-induced histamine release from mast cells of the hamster in vitro and in the rat, cat and mongoose in vivo. Structure-activity relations studies revealed that: 1) substitution of alkyl, cycloalkyl or other aryl groups for the benzyl group of benzyldimethyltetradecylammonium Cl reduced activity only slightly; 2) demethylation to form the tertiary or secondary amines dramatically reduced activity; 3) optimal length of the alkyl side chain was usually 12 to 14 carbons; and 4) replacement of the nitrogen with phosphorus or sulfur did not significantly alter activity. It appears that the polyamine receptor on mast cells is: 1) widespread in mammals; 2) stimulated by a broad range of polyamines and some monoamines; 3) responsive both in vitro and in vivo; and 4) competitively antagonized by a fairly diverse family of inhibitors possessing a permanent positive charge attached to a substantial but limited hydrophobic moiety.