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Aminoglycoside antibiotics inhibit adriamycin uptake in rat peritoneal mast cells
Pharmacological Research, Vol . 25, Supplement 2, 1992
30 0
AMINOGLYCOSIDE ANTIBIOTICS INHIBIT ADRIAMYCIN UPTAKE IN RAT PERITONEAL MAST CELLS. G . Decorti, F . Bartoll Klugmann, L. Candussio, L . Baldini . Institute of Pharmacology, University of Trieste, Via A. Valerio n'32, 34100 Trieste, Italy. Key words: adriamycin, aminoglycosides, mast cells, exocytosis, uptake . The anthracyclines induce an important non cytotoxic histamine release from rat peritoneal mast cells and exhibit a very high affinity for mast cells in comparison with other normal or neoplastic cells (1) . Anthracycline-induced exocytosis is similar to that of basic secretagogues: indeed, it is very rapid, is independent from extracellular calcium, and is inhibited by a pretreatment with pertussis toxin (2) . This last characteristic suggests that the activation of GTP-binding proteins (G proteins) plays a role in the cascade of events leading to adriamycin-induced histamine secretion. It has been suggested that two different G proteins are involved in stimulus-secretion coupling in mast cells, one connected to phospholipase C activation (GP) and the other coupled directly to exocytosis (GE) (3) . It has been recently shown that adriamycin binds specifically to inositol phospholipids and in particular to PIP2 (phosphatidylinositol-4,5-diphosphate) in model systems and it has been suggested that this specific binding takes place also in biological membranes (4) . For this binding adriamycin competes efficiently with the nonpenetrating antibiotic neomycin . It is well known that the aminoglycoside binds strongly to PIP2 and other inositol phospholipids and that this leads to inhibition of phospholipase C and to decreased cellular response after stimulation of GP. Aim of this study was therefore to evaluate the effect of neomycin and other aminoglycosides on adriamycin-induced histamine release and uptake in rat peritoneal mast cells . Purified rat peritoneal mast cells were incubated in quadruplicate at 37 ' C for predetermined experimental times with adriamycin (100 pg/ml) and various concentrations of aminoglycoside antibiotics . The amount of histamine released was assayed by the fluorimetric method of Shore et al . (1); adriamycin concentration was measured by the fluorimetric method of Bachur et al. (1) . A 10 min preincubation of mast cells with neomycin (0.1-10 mM) inhibited adriamycin uptake in a dose dependent manner; on the contrary histamine release was not affected . Similar effects were obtained with gentamicin and amikacin (fig . 1) . However, the aminoglycosides showed differences in inhibition, with the order of potency being neomycin>gentamicin>amikacin; this ranking correlates with the cationic structure of the antibiotics; indeed neomycin has six ionizable amino groups, gentamicin five and amikacin only four. If added 10 min after adriamycin, neomycin displaced the antineoplastic agent from its mast cell binding sites, and adriamycin uptake progressively decreased during a period of 10 min . When the mast cells were disrupted by sonication and granular material became therefore directly accessible to adriamycin, its uptake was comparable with that of intact cells . Once more the aminoglycosides, with the same order of potency as above, were active in inhibiting adriamycin uptake. In conclusion, the efficacy of the aminoglycosides in inhibiting adriamycin uptake in intact and sonicated cells, suggests that adriamycin and the aminoglycosides compete for the same binding sites (probably PIP2) on mast cell plasma and granular membranes . On the contrary, the lack of effect of neomycin on adriamycin-induced exocytosis suggests that, similarly to other basic secretagogues (5), the antineoplastic agent is capable of inducing secretion by 1043-6618/92/25110300-02/$03 .00/0
© 1992 The Italian Pharmacological Society
Pharmacological Research, Vol. 25, Supplement 2, 1992
30 1
binding to other anionic phospholipids and hence activating exocytosis .
80
that is directly coupled to
7i
U,
70
• 6-
N
U
• 60
In
0 5
a,
50 • ~ 40 E
N
GE
30
C 0 0 C V
rn 4v
E 20
20 10
*
E
E
>,e
a,
C 0 a
-0 0
0 0 *
v 0
0
0 + N + G + A
-
I I + G + A
Fig. I .Effect of a 10 min pretreatment with I mM neomycin (N), gentamicin (G) and amikacin (A) on adriamycin (100 Mg/ml)-induced histamine release and uptake . * : p < 0 .001, Student's t test for independent samples .
Acknowledgements: this work was supported by grants from the M .U .R.S.T. 40 and 60% . References I. Decorti G, Bartoli Klugmann F, Candussio L, Furlani A, Scarcia V, Baldini L Uptake of adriamycin by rat and mouse mast cells and correlation with histamine release . Cancer Res 1989, 49: 1921-1926 . 2. Bartoli Klugmann F. Decord G . Candussio L, Baldini L A pertussis toxin-sensitive Gprotein is involved in adriamycin-induced histamine release and uptake in rat peritoneal mast cells. Eur J Cancer 1991, 27: S5 I . 3. Cockcroft S. Stutchfield J . G proteins, the inositol lipid signalling pathway and secretion . Phil Trans R Soc Lond 1988, B320 : 247-265 . 4. de Woff FA, Demel RA, Bets D, van Kats C. de Kruijff B. Characterization of the interaction of doxorubicin with (poly)phosphoinositides in model systems . FEBS Letters 1991, 288: 237-240 . 5. Aridor M, Traub LM, Sagi-Eisenmerg R. Exocytosis in mast cells by basic secretagogues : evidence for direct activation of GTP-binding proteins . J Cell Biol 1990, 111 : 909-917 .
30 0
AMINOGLYCOSIDE ANTIBIOTICS INHIBIT ADRIAMYCIN UPTAKE IN RAT PERITONEAL MAST CELLS. G . Decorti, F . Bartoll Klugmann, L. Candussio, L . Baldini . Institute of Pharmacology, University of Trieste, Via A. Valerio n'32, 34100 Trieste, Italy. Key words: adriamycin, aminoglycosides, mast cells, exocytosis, uptake . The anthracyclines induce an important non cytotoxic histamine release from rat peritoneal mast cells and exhibit a very high affinity for mast cells in comparison with other normal or neoplastic cells (1) . Anthracycline-induced exocytosis is similar to that of basic secretagogues: indeed, it is very rapid, is independent from extracellular calcium, and is inhibited by a pretreatment with pertussis toxin (2) . This last characteristic suggests that the activation of GTP-binding proteins (G proteins) plays a role in the cascade of events leading to adriamycin-induced histamine secretion. It has been suggested that two different G proteins are involved in stimulus-secretion coupling in mast cells, one connected to phospholipase C activation (GP) and the other coupled directly to exocytosis (GE) (3) . It has been recently shown that adriamycin binds specifically to inositol phospholipids and in particular to PIP2 (phosphatidylinositol-4,5-diphosphate) in model systems and it has been suggested that this specific binding takes place also in biological membranes (4) . For this binding adriamycin competes efficiently with the nonpenetrating antibiotic neomycin . It is well known that the aminoglycoside binds strongly to PIP2 and other inositol phospholipids and that this leads to inhibition of phospholipase C and to decreased cellular response after stimulation of GP. Aim of this study was therefore to evaluate the effect of neomycin and other aminoglycosides on adriamycin-induced histamine release and uptake in rat peritoneal mast cells . Purified rat peritoneal mast cells were incubated in quadruplicate at 37 ' C for predetermined experimental times with adriamycin (100 pg/ml) and various concentrations of aminoglycoside antibiotics . The amount of histamine released was assayed by the fluorimetric method of Shore et al . (1); adriamycin concentration was measured by the fluorimetric method of Bachur et al. (1) . A 10 min preincubation of mast cells with neomycin (0.1-10 mM) inhibited adriamycin uptake in a dose dependent manner; on the contrary histamine release was not affected . Similar effects were obtained with gentamicin and amikacin (fig . 1) . However, the aminoglycosides showed differences in inhibition, with the order of potency being neomycin>gentamicin>amikacin; this ranking correlates with the cationic structure of the antibiotics; indeed neomycin has six ionizable amino groups, gentamicin five and amikacin only four. If added 10 min after adriamycin, neomycin displaced the antineoplastic agent from its mast cell binding sites, and adriamycin uptake progressively decreased during a period of 10 min . When the mast cells were disrupted by sonication and granular material became therefore directly accessible to adriamycin, its uptake was comparable with that of intact cells . Once more the aminoglycosides, with the same order of potency as above, were active in inhibiting adriamycin uptake. In conclusion, the efficacy of the aminoglycosides in inhibiting adriamycin uptake in intact and sonicated cells, suggests that adriamycin and the aminoglycosides compete for the same binding sites (probably PIP2) on mast cell plasma and granular membranes . On the contrary, the lack of effect of neomycin on adriamycin-induced exocytosis suggests that, similarly to other basic secretagogues (5), the antineoplastic agent is capable of inducing secretion by 1043-6618/92/25110300-02/$03 .00/0
© 1992 The Italian Pharmacological Society
Pharmacological Research, Vol. 25, Supplement 2, 1992
30 1
binding to other anionic phospholipids and hence activating exocytosis .
80
that is directly coupled to
7i
U,
70
• 6-
N
U
• 60
In
0 5
a,
50 • ~ 40 E
N
GE
30
C 0 0 C V
rn 4v
E 20
20 10
*
E
E
>,e
a,
C 0 a
-0 0
0 0 *
v 0
0
0 + N + G + A
-
I I + G + A
Fig. I .Effect of a 10 min pretreatment with I mM neomycin (N), gentamicin (G) and amikacin (A) on adriamycin (100 Mg/ml)-induced histamine release and uptake . * : p < 0 .001, Student's t test for independent samples .
Acknowledgements: this work was supported by grants from the M .U .R.S.T. 40 and 60% . References I. Decorti G, Bartoli Klugmann F, Candussio L, Furlani A, Scarcia V, Baldini L Uptake of adriamycin by rat and mouse mast cells and correlation with histamine release . Cancer Res 1989, 49: 1921-1926 . 2. Bartoli Klugmann F. Decord G . Candussio L, Baldini L A pertussis toxin-sensitive Gprotein is involved in adriamycin-induced histamine release and uptake in rat peritoneal mast cells. Eur J Cancer 1991, 27: S5 I . 3. Cockcroft S. Stutchfield J . G proteins, the inositol lipid signalling pathway and secretion . Phil Trans R Soc Lond 1988, B320 : 247-265 . 4. de Woff FA, Demel RA, Bets D, van Kats C. de Kruijff B. Characterization of the interaction of doxorubicin with (poly)phosphoinositides in model systems . FEBS Letters 1991, 288: 237-240 . 5. Aridor M, Traub LM, Sagi-Eisenmerg R. Exocytosis in mast cells by basic secretagogues : evidence for direct activation of GTP-binding proteins . J Cell Biol 1990, 111 : 909-917 .