engleski

P-glycoprotein in adrenals of rabbits treated with cyclosporin A

P-glycoprotein (P-gp) is a membrane transport protein in many normal and neoplastic tissues which functions as an energy dependent efflux pump of many cytotoxic drugs, a phenomenon known as multidrug resistance. Although P-gp is highly expressed in the adrenal cortex of humans and animals its function has not been defined yet. The function of P-gp can be modified by several modulator drugs that bind competitively to P-gp. Cyclosporin A (CsA) is one of the potent P-gp modulators. The aim of this study was to determine whether the blockade of P-gp by cyclosporin A, a potent immunosupressive drug, inhibits P-gp activity and cortisol secretion in rabbit adrenals. Eighteen New Zealand White adult male rabbits housed in laboratory conditions on standard pellet diet and top water were divided into two groups. Seven rabbits received s.c. injections of 30 mg/kg CsA dissolved in Cremophor EL(Sandoz) for five days and seven rabbits 60 mg/kg CsA for two days, respectively. Four animals served as control. Animals were sacrified by decapitation after mild narcosis. Freshly removed adrenals were fixed in 4% buffered formaldehyde (pH 7, 4), freshly prepared methacarn and ModAMeX and embedded in paraplast. Deparaffinized sections were submitted to PAP, APAAP and ABC immunohistochemical procedures using mouse monoclonal C219 antibody ("Centocor", USA) at the concentration of 5 and 10 µg/ml PBS and incubated for 1-2 hours at room temperature. The staining result s have clearly shown that the optimal preservation of morphology and P-gp antigenicity was achieved with methacarn fixation and APAAP procedure, expressed in sharply delineated blue precipitate. The adrenal glands of normal and CsA treated animals showed P-gp immunoreactivity in plasma membranes of the cortical cells while the medulla was negative. The strongest reactivity was shown in the cells of zona fasciculata, a bit less in zona reticularis, while zona glomerulosa showed only partly slight reactivity. Sinusoidal linning cells were negative. In CsA treated animals zona fasciculata showed stronger staining of P-gp. The immunohistochemical evidence of the strong expression of P-gp in the adrenal cortex of normal and CsA treated rabbits suggests that the primary role of P-gp may be related to the normal secretion of cortical hormones. It is already known that some steroid hormones can be transported across the cell membrane by P-gp. Our recent study clearly showed that serum cortisol levels, both baseline and ACTH stimulated, significantly increased after low and high dose CsA treatment. The increase of cortisol, determined by radioimmunoassay, was dose dependent, e.g. the mean basel ine cortisol levels increased from 7.7 (SD=4.9) to 44.9 nmol/l (SD= 34.5) in the high dose group. Contrary to our expectations from in vi tro studies, CsA treatment of rabbits apparently increases serum cortisol levels, however, the possible mechanisms for the inhanced both cortisol levels and P-gp activity after CsA treatment remains obscure.

p-gycoprotein; adrenal gland; rabbit; cyclosporin A

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