Role of extracellular calcium in in vitro uptake and intraphagocytic location of macrolides.

We compared the uptakes and intracellular locations of four 14-membered-ring macrolides (roxithromycin, dirithromycin, erythromycin, and erythromycylamine) in human polymorphonuclear neutrophils (PMNs) in vitro. Intracellular location was assessed by cell fractionation and uptake kinetics in cytoplasts (granule-poor PMNs). Trapping of dirithromycin within PMN granules (up to 80% at 30 min) was significantly more marked than the intracellular trapping of the other drugs (erythromycylamine, 45% +/- 5.1%; erythromycin, 42% +/- 3.7%; roxithromycin, 35% +/- 3.0%). A new finding was that, in the absence of extracellular calcium, the uptakes of all of the macrolides by PMNs and cytoplasts were significantly impaired, by about 50% (PMN) and 90% (cytoplasts). Furthermore, inorganic Ca2+ channel blockers inhibited macrolide uptake in a concentration-dependent manner, with 50% inhibitory concentrations of 1.6 to 2.0 mM and 29 to 35 microM, respectively, for Ni2+ and La3+. The intracellular distributions of the drugs were unchanged in the presence of Ni2+ and La3+ and in Ca(2+)-free medium supplemented with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The organic Ca2+ channel blocker nifedipine had no effect on macrolide uptake, whereas verapamil inhibited it in a time- and concentration-dependent manner. These data show the importance of extracellular Ca2+ in macrolide uptake by phagocytes and suggest a link with Ca2+ channels or a Ca2+ channel-operated mechanism.

Effects of dirithromycin and erythromycylamine on human neutrophil degranulation.

Dirithromycin and, to a lesser extent, erythromycylamine and erythromycin directly induced the release of three intragranular enzymes (lysozyme, lactoferrin, and beta-glucuronidase) from unstimulated human neutrophils. Macrolide-induced enzyme release was dependent upon the incubation time (30 to 180 min) and drug concentration. Dirithromycin was the most effective. At 120 min, release of lysozyme, beta-glucuronidase, and lactoferrin by macrolide (100 micrograms/ml)-treated cells, expressed as a percentage of total enzyme content, was, respectively, 58% +/- 8.3%, 52% +/- 10.7%, and 35% +/- 5.1% (dirithromycin); 42% +/- 3.9%, 28% +/- 5.8%, and 10% +/- 2.2% (erythromycylamine); and 35% +/- 4.0%, 19% +/- 4.3%, and 10% +/- 5.2% (erythromycin) (mean +/- standard error of the mean of three to eight experiments). The lowest macrolide concentrations which induced significant enzyme release were 10, 100, and 25 micrograms/ml, respectively, for dirithromycin, erythromycylamine, and erythromycin. Furthermore, we obtained evidence of a link between the prodegranulation effects of dirithromycin and erythromycylamine and the intragranular location of these drugs. Indeed, cell-associated drug levels increased for up to 60 min and then plateaued and declined substantially. Increasing the pH from 7 to 9 resulted in a parallel increase in drug uptake and the prodegranulation effect. Finally, when macrolide-treated neutrophils were disrupted by sonication and centrifuged, a correlation was found between lysozyme and beta-glucuronidase activities (both granule markers) and pellet-associated macrolide levels. Taken together, our results suggest that dirithromycin and erythromycylamine concentrate within neutrophil granules and then induce degranulation.

Investigation of dirithromycin and erythromycylamine uptake by human neutrophils in vitro.

Dirithromycin, a new semisynthetic 14-membered-ring macrolide was avidly concentrated by human neutrophils in a time- but not concentration-dependent manner with mean cellular/extracellular, concentration ratios (C/E) of 9 within the first 5 min and up to 47 at 120 min. Erythromycylamine, the hydrolysis product of dirithromycin, was concentrated significantly less by neutrophils, reaching C/E values of 4 and 19 (at 5 and 120 min). A point of interest was the interindividual variability in the antibiotic uptake kinetics; in particular, 7 out of 47 neutrophil samples from different healthy volunteers displayed very slow uptake of both drugs (C/E values at 30 min: dirithromycin, 5.8; erythromycylamine, 4.6). The reason(s) for this is unknown. The uptake of both drugs was decreased at acidic pH and increased at basic pH. Chloroquine, an antimalarial drug which is concentrated in and alkalinizes azurophilic granules, reduced uptake by half. Metabolic inhibitors (2-4 dinitrophenol, sodium fluoride, potassium cyanide and sodium azide) did not impair the uptake of either drug but, interestingly, ouabain, an inhibitor of membrane Na+/K+ ATPase activity, impaired uptake by about 30%. Competitive inhibitors of some transport systems identified on neutrophil membrane (nucleosides, D-glucose and various aminoacids) did not alter the uptake of either drug. Dirithromycin and to a lesser extent, erythromycylamine, reached intracellular concentrations much higher than those required to inhibit the growth of sensitive microorganisms. Although the mechanism of uptake is not clear, one interesting hypothesis involves trapping by protonation into acidic compartments of neutrophils.