Calcium signals are affected by ciprofloxacin as a consequence of reduction of mitochondrial DNA content in Jurkat cells

Abstract

The effects of ciprofloxacin on mitochondrial DNA (mtDNA) content, oxygen consumption, mitochondrial membrane potential, cellular ATP formation, and capacitative Ca(2+) entry into Jurkat cells were investigated. In cells incubated for several days with 25 mug/ml ciprofloxacin, a 60% reduction of mtDNA content, inhibition of the respiratory chain, and a significant decrease in mitochondrial membrane potential were observed. These changes led to a decrease in the calcium buffering capacity of mitochondria which, in turn, resulted in a gradual inhibition of the capacitative Ca(2+) entry. On days 4, 7, and 11 of incubation with ciprofloxacin, the initial rates of Ca(2+) entry were reduced by 33%, 50%, and 50%, respectively. Ciprofloxacin caused a transient decrease in the cellular capability for ATP formation. In cells incubated for 15 min with glucose, pyruvate, and glutamine as exogenous fuel, ciprofloxacin reduced ATP content by 16% and 35% on days 4 and 7, respectively, of incubation with the drug. However, on day 11 of incubation with ciprofloxacin, a recovery of cellular ATP formation was observed. In conclusion, long-term exposure of Jurkat cells to ciprofloxacin at a concentration of 25 mug/ml seriously affects cellular energy metabolism and calcium homeostasis.

FIG. 1.

Effect of ciprofloxacin on mtDNA content. Cells were grown in the absence (control) or in the presence of ciprofloxacin (25 μg/ml) for 4, 7, or 11 days. (A) Southern blots with visualized mtDNA fragment (a, left blot) and nuclear DNA fragment (b, right blot) used as a reference. (B) Collected data from three independent experiments expressed as ratios between the intensity of the radioactivity of bands a and b shown in panel A. The a/b value for control cells is taken as 1. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 2.

Effect of ciprofloxacin on oxygen consumption by permeabilized cells. Cells were grown in the presence of ciprofloxacin (25 μg/ml) for 4, 7, or 11 days. Oxygen consumption was measured in digitonin-permeabilized cells suspended in BSS supplemented with 5 mM malate and 5 mM glutamate. At times indicated by arrows, rotenone (rot) or 5 mM succinate (succ) was added. The traces show results of one typical experiment out of six.

FIG. 3.

Effect of ciprofloxacin on mitochondrial membrane potential (ΔΨ) in Jurkat cells. Cells were grown in the presence of ciprofloxacin (25 μg/ml) for 4, 7, or 11 days. ΔΨ was measured in intact cells by flow cytometry with a JC-1 probe. The cells were suspended in PBS supplemented with 1 mM pyruvate and 5 mM glucose. (A) Control cells. The shift of cellular population caused by the addition of valinomycin reflects a complete dissipation of ΔΨ. Oligomycin did not affect ΔΨ in control cells. (B) Ciprofloxacin-treated cells. Exposure to ciprofloxacin led to a gradual decrease in ΔΨ (left-hand column). The addition of oligomycin to the cell suspension shortly before the measurement resulted in a further decrease in ΔΨ (right-hand column). Numbers in each panel express the proportion of cells with dissipated ΔΨ. Axes FL1-H and FL2-H correspond to the intensity levels of green and red fluorescence, respectively, of the JC-1 probe. The figure presents results of one typical experiment out of six.

FIG. 4.

Effect of ciprofloxacin on mitochondrial membrane potential (ΔΨ) in permeabilized cells incubated with succinate. Cells were grown in the presence of ciprofloxacin (25 μg/ml) for 4, 7, or 11 days. ΔΨ was measured with 5 μM safranine O in digitonin (DIG)-permeabilized cells suspended in BSS and incubated in the presence of 5 mM glutamate and 5 mM malate. After inhibition of mitochondrial respiration by 1 μM rotenone (rot), 5 mM succinate (succ) was added. Bars a, b, c, and d show changes in fluorescence after complete mitochondrial uncoupling by carbonyl cyanide m-chlorophenylhydrazone (CCCP), corresponding to increases in ΔΨ due to respiration with succinate as a sole substrate. The figure presents results of one typical experiment out of three.

FIG. 5.

Effect of ciprofloxacin and oligomycin on cellular ATP formation. Cells were incubated for 15 min in PBS containing 2 mM glutamine and 1 mM pyruvate, with or without 5 mM glucose and with or without oligomycin (oligo; 1 mg/ml). Then, incubation was terminated, and ATP content in neutralized acidic cellular extracts was determined. (A) Effect of oligomycin in control cells with or without glucose. (B) Effect of oligomycin in the absence of glucose in cells pretreated with ciprofloxacin for 4, 7, or 11 days. (C) Effect of oligomycin in cells pretreated with ciprofloxacin for 4, 7, or 11 days in the presence of 5 mM glucose. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 6.

Effect of ciprofloxacin on Ca²⁺ entry into Jurkat cells. Cells suspended in nominally Ca²⁺-free BSS were preincubated with oligomycin (0.1 mg/ml). After depletion of intracellular calcium stores by the addition of 0.1 μM thapsigargin, the cell suspension was supplemented with 3 mM CaCl₂. The rate of Ca²⁺ entry was estimated as a tangent of the initial part of the curve. (A) A typical experiment. (B) Data collected from several experiments, as indicated. ***, P < 0.001.