Gentamicin 및 tobramycin이 적혈구막을 통한 Ca++이동에 미치는 영향

Abstract

[한글]

Aminoglycoside 항생제중 gentamicin과 tobramycin은 그람 음성균 감염치료에 흔히 사용되는 약물이나 큰 단점은 nephrotoxicity를 유발시키는 것이다(Bennett등, 1976; Gary등, 1976: Kaloyanides 및 Pastoriza-Munoz, 1980). 이와 같은 독성유발의 기전은 이들항생제의 poly-cationic group이 Ca**++ 대신 세포막의 polar lipid에 결합하므로써(Hostetler 및 Hall, 1982) 세포막의 정상기능과 integrity가 변화되어 (Johansson등, 1982)양이온의 투과도가 달라지기 때문에 세포가 손상되는 것이라고 보고되었다(Trump등, 1973; Schanne등, 1979: Humes 및 Weinberg, 1980). 그러므로 항생제 사용시 Ca**++ 이나 Mg**++을 병행해서 투여하면 nephrotoxicity를 경감시킬 수 있다고 하는데, 그 기전은 이들이 신세뇨관 세포막에서 항생제의 결합과 항생제의 uptake론 경쟁적으로 억제하기 때문이라고 하였다(Humes등, 1984: Quarum등, 1984).

따라서 저자는 이들 항생제가 세포막의 기능을 변화시키고 막 효소의 활성도를 억제한다면 세포막을 통한 물질이동도 변화시킬 가능성이 있을 것으로 생각되어 이들 항생제가1. 적혈구의 삼투성 취약성, 2. 적혈구막에서 Ca**++ 결합과 3. 적혈구막을 통한 Ca**++의

능동적 및 수동적 이동에 미치는 영향을 관찰하여 다음과 같은 결론을 얻었다.

1. Gentamicin 및 tobramycin은 공히 적혈구의 삼투성 허약성을 증가시켰으며, 그 증가정도는 약물의 농도에 비례하였다. 그러나 이와 같은 삼투성 취약성의 증가는 incubation용액에 Ca**++ 또는 Mg**++ 을 첨가하므로써 억제되었으며 그 억제효과는 Ca**++ 이 같은 농도의 Mg**++ 보다 더 컸다.

2. Gentamicin 및 tobramycin은 적혈구막 소편에서 Ca**++ 결합을 상경적으로 억제시켰다.

3. Resealed RBC막을 통한 능동적인 (45)**Ca 유출은 낮은 농도의 gentamicin 및 tobramycin에 의해서는 영향을 받지 않았으나 높은 농도에서는 억제되었다. 그러나 이들 항생제는 수동적인 (45)**Ca 유입에는 별 영향을 미치지 않았다.

4. Gentamicin 및 tobramycin은 적혈구막 소편에서 Ca**++ -ATPase의 활성도를 억제시켰다.

이상의 성적으로 미루어 볼 때 gentamicin 및 tobramycin으로 야기된 적혈구의 삼투성취약성의 증가는 이들 항생제가 적혈구막에 결합된 Ca**++ 의 양을 변화시켜 나타난 것으로 생각되며 resealed R8C막을 통한 능동적 (45)**Ca 유출의 감소는 막효소인 Ca**++ -AT

Pase 활성도의 감소에 의한 것이라고 사료되는 바이다.

[영문]

Gentamicin and tobramycin are aminoglycoside antibiotics used frequently for the treatment of gram-negative infections. Nephrotoxicity is a side effect of these agents(Kalo-yanides and Pastoriza-Munoz, 1987). There is no simple explanation of

the mechanism for this side effect, but the first step in the pathogenesis is thought to alter a variety of membrane events. Polycations of aminoglycosides as a group are recognized as potential nephrotoxins(Schanne 등 1979: Humes 및 Weinberg, 1980) and these compounds are capable of binding to negative charge along the membrane(Humes 및 Weinberg, 1980). Cations interacting with Ca**++ in the membrane promotes disturbance of normal membrane function and integrity(Johansson 등, 1982).

A recent study demonstrated that the divalent cations, Ca**++ and Mg**++, inhibit a large number of aminoglrcoside-membrane interaction(Bennett et at., 1982). Ca**++ prevents the nephrotoxic effect by the action of Ca**++ to inhibit the effect of gentamicin at subcellular membrane sites of the renal proximal tubular cells.

It huts been reported that aminoglycosides reduce Na**+ -K**+ ATPase activity in the human erythrocyte ghost, and the microsomal fractions of the cortex and the outer medulla of the guinea pig kidney(Chahwala and Harpur, 1982).

The present study was undertaken to elucidate the mechanisms of action of gentamicin and tobramycin on the human erythrocyte by examining their effects on osmotic fragility, Ca**++ binding to the membrane fragments and calcium transport across red blood cell membrane.

The results are summarized as follows:

1) Gentamicin and tobramycin increased osmotic fragility at all concentrations tested, the effects being similar for both antibiotics. The concentration of NaCl solution for 100% hemolysis was 1/13∼1/16M in the presence of 0.2mg/ml of the antibiotios and 1/18M in the absence of the antibiotics(control).

2) When erythrocytes were suspended in 1/14 M NaCl solution. 62±3.5% of the cells were hemolyzed. The degree of hemolysis was enhanced by the addition of bott antibiotics.

3) When, Ca**++ and Mg**++ were added to the suspending medium, the degree of hemolysis was inhibited. Calcium was more effective than magnesium in protecting against hemolysis.

4) In the Presence of gentamicin and tobramycin the passive binding of Ca**++ to RBCMF exponentially inhibited. Lineweaver-Burk plot of theme inhibitory effects demonstrated that both antibiotics were competitive inhibitors of the binding

reactions.

5) Gentamicin and tobramycin did not affect active calcium extrusion across resealed RBC at concentrations below 0.1 mg/ml but inhibited it by about 40% at concentrations above 0.5mg/ml. When gentamicin and totramrcin were added to inside and outside red blood cell simultaneously, the inhibitory effect of active (45)**Ca extrusion was more pronounced. This suggests that these antibiotics inhibit not only active outward Ca**++ transport but also inhibit passive (45)**Ca-Ca**++ exchange diffusion through the red blood cell membrane.

6) Ca**++ -activates ATPase activity of RBCMF was inhibited significantly by both antibiotics at concentrations above 0.5 mg/ml but baa not influenced by these agents at concentration below 0.25 mg/ml.

From the above results, it mar be speculated that aminoglycoside antibiotics induced hemolysis is related to alterations of Ca**++ content in the membrane. The inhibition of Ca**++ transport across red blood cells by these agents appears to be the blocking Ca**++ -activated ATPase activity which was been known to he linked with Ca**++ transport.