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

Abstract

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 Ca 2؉ channel blockers inhibited macrolide uptake in a concentrationdependent manner, with 50% inhibitory concentrations of 1.6 to 2.0 mM and 29 to 35 M, respectively, for Ni The organic Ca 2؉ 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 Ca 2؉ in macrolide uptake by phagocytes and suggest a link with Ca 2؉ channels or a Ca 2؉ channel-operated mechanism.Infections caused by organisms which survive and multiply within host cells, such as Legionella spp., Chlamydia spp., and Mycobacteria spp., are difficult to cure, partly because of the intracellular locations of these pathogens, which are protected from the antimicrobial activities of non-cell-penetrating agents (20). However, the abilities of antibiotics to enter and concentrate within host cells, generally estimated from the intracellular concentration-to-extracellular concentration ratio, may not actually reflect their overall intracellular bactericidal activities (17). Another factor of importance is the respective intracellular locations of the drug and the pathogen. Possible interactions between the drug and host cell factors (inactivation, binding, synergy) may also result from the particular location of the drug. In addition, intracellular accumulation of antibacterial agents or the mechanism used for this cellular transport may in turn affect host cell metabolism and functions, ultimately modifying the overall course of infectious diseases. Among those cell-penetrating agents, macrolide antibiotics have been much studied (14,27). Many publications examine their intracellular (particularly intraphagocytic) accumulation (for a review, see reference 29) and their possible interference with phagocyte activities (for a review, see reference 28). Despite abundant data, the mechanism underlying macrolide uptake by host cells is poorly understood. Various hypotheses have been put forward. These hypotheses involve liposolubility and active transport systems, particularly those used by nucleosides (18, 21). A likely mechanism for their intracellular accumulation involves the weakly basic nature of macrolides and the possibility of trapping by protonation within ...

Influence of P-Glycoprotein Inhibitors on Accumulationof Macrolides in J774 MurineMacrophages

“…The present experiments have been limited to J774 macrophages, which were used as a model, and generalization of our conclusions to other phagocytic cells therefore remains unwarranted. In particular, the presence of a functional P-glycoprotein and its potential role in macrolide handling in polymorphonuclear leukocytes are controversial (4,14,21,38). It is interesting, however, that human KB and G-185 cells overexpressing P-glycoprotein show decreased levels of macrolide accumulation and correspondingly reduced levels of antimicrobial activity against intracellular forms of Listeria monocyto- genes (22).…”

Role of Leukocyte Elastase in Preventing Cellular Re-Colonization of the Mural Thrombus

“…23 Cells were maintained in HBSS. The viability, measured by the release of lactate dehydrogenase (LDH) 29 and purity of the final preparation were greater than 99 and 95%, respectively. PMN degranulation was obtained by addition of formyl-methionyl-leucyl-phenylalanine (fMLP) at 10 Ϫ6 M for 10 minutes, at 37°C (500 l of conditioned medium corresponding to the release of 5 ϫ 10 5 activated PMN were used throughout the experimental procedure).…”

Cellular Uptake of Two Fluoroketolides, HMR 3562 and HMR 3787, by Human Polymorphonuclear Neutrophils In Vitro

“…Aliquots of, 2.5 ϫ 10 6 PMN were loaded with the drugs at 10 g/ml (30 min at 37°C) and were centrifuged through the oil cushion. The cell pellet was sonicated in the presence of 0.5% Triton X-100 or 0.73 M sucrose to protect granules (17,23,24). After centrifugation, the amounts of lysozyme (a granule marker) and radiolabeled drugs in the pellet and the supernatant were determined as previously described (18).…”

“…The following experimental conditions were varied to study the mechanism of uptake: pretreatment of PMN with 10% formaldehyde followed with two washes in HBSS (for cell viability); pH from 6.5 to 8.5; temperatures of 0, 20, 37, and 40°C; extracellular concentrations of 2.5 to 100 g/ml; pretreatment for 15 min with various metabolic inhibitors (NaF, KCN, or 2,4-dinitrophenol; 1 mM) or with various activators and/or inhibitors of PMN functions which have been reported to interfere with macrolide uptake (17,23,24), namely Ni 2ϩ , a blocker of the Na ϩ -Ca 2ϩ exchanger, at 1.25 to 5 mM; addition of phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, at 100 and 10 ng/ml; addition of H89, a PKA inhibitor, at 50 M; and addition of verapamil, a Ca 2ϩ channel blocker, at 125 and 250 M. We also studied the inhibitory effect of various macrolides and ketolides; the concentrations chosen were the mean K m (23,24). PMN were incubated at 37°C for 5 min with unlabeled azithromycin (51 g/ml), HMR 3004 (22 g/ml), HMR 3647 (117 g/ml), or HBSS.…”

“…110-018, 1996]) and HMR 3004 uptake, whereas it increases that of azithromycin and HMR 3647 (M. T. Labro and D. Vazifeh, unpublished data). The Ca 2ϩ channel inhibitor verapamil displays similar activating or inhibitory properties according to the macrolide-ketolide subgroup (17,23,24), whereas Ni 2ϩ , which blocks the Na ϩ -Ca 2ϩ exchanger on the PMN membrane, impairs macrolide uptake whatever the subgroup (17,23,24). We tested various PMN activators and inhibitors on the uptake of the new fluoroketolides (Table 1).…”