D. Vazifeh et al., CELLULAR ACCUMULATION OF THE NEW KETOLIDE RU-64004 BY HUMAN NEUTROPHILS - COMPARISON WITH THAT OF AZITHROMYCIN AND ROXITHROMYCIN, Antimicrobial agents and chemotherapy, 41(10), 1997, pp. 2099-2107
We analyzed the uptake of RU 64004 by human neutrophils (polymorphonuc
lear leukocytes [PMNs]) relative to those of azithromycin and roxithro
mycin, RU 64004 was strongly and rapidly accumulated by PMNs, with a c
ellular concentration/extracellular concentration ratio (C/E) of great
er than 200 in the first 5 min, and this was followed by a plateau at
120 to 180 min, with a C/E of 461 +/- 14.8 (10 experiments) at 180 min
. RU 64004 uptake was moderately sensitive to external pH, and activat
ion energy was also moderate (63 +/- 3.8 kJ/mol), RU 64004 was mainly
located in PMN granules (about 70%) and egressed slowly from loaded ce
lls, owing to avid reuptake, The possibility that PMN uptake of RU 640
04 and other macrolides occurs through a carrier-mediated system was s
uggested by three key results. First, there existed a strong interindi
vidual variability in uptake kinetics, suggesting variability in the n
umbers or activity of a transport protein, Second, macrolide uptake di
splayed saturation kinetics characteristic of that of a carrier-mediat
ed transport system: RU 64004 had the highest V-max value (3,846 ng/2.
5 x 10(6) PMNs/5 min) and the lowest K-m value (about 28 mu M), indica
ting a high affinity for the transporter, Third, as observed previousl
y with other erythromycin A derivatives, Ni2+ (a blocker of the Na+/Ca
2+ exchanger which mediates Ca2+ influx in resting neutrophils) impair
ed RU 64004 uptake by PMNs, with a 50% inhibitory concentration of abo
ut 3.5 mM, In addition, me found that an active process is also involv
ed in macrolide efflux, because verapamil significantly potentiated th
e release of all three macrolides tested, This effect of verapamil doe
s not seem to be related to an inhibition of Ca2+ influx, because neit
her EGTA [ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraa
cetic acid] nor Ni2+ modified macrolide efflux. The nature and charact
eristics of the entry-and efflux-mediating carrier systems are under i
nvestigation.