engleski

Immunohistochemical demonstration of P-glycoprotein in organ s of normal male rabbits

P-glycoprotein (P-gp) is a pleiotropic membrane transport protein (170 kD) in many normal and neoplastic tissues which functions as an energy dependent efflux purnp with broad substrate specifity. Several studies have linked the presence of P-gp with resistence to a wide range of lipophilic cytotoxic chemotherapeutic drugs, a phenomenon known as multidrug resistance (MDR). The purpose of this study was to use more accurate irnmunohistochemical techniques to localize in paraffine sections the P-gp in rabbit adrenals, liver, kidney and gut using mouse monoclonal C219 antibody. This work is a part of amore comprehensive study on the effect of cyclosporin, an immunosuppressant- cirug, and ACTH on rabbit adrenal gland and other organs. Eight male rabbits weighing 3.5 to 4.0 kgs were sacrified by decapitation after mild narcosis. Freshly removed tissue specimens of 5 mm thickness were immersed at room temperature in a) phosphate buffered formaldehyde (pH 7.4) for 18 hours ; b) freshly prepared mixture of methanol, chloroform and glaciai acetic acid (methacarn) (6:3:1 v/v) for 6 hours ; c) mixture of corrosive sublimate (2.5 gm) and trichloroacetic acid (2.5 gm) dissolved in methanol (80 ml) and formaldehyde sol. (20 ml) for 4 to 6 hours ; and d) in AMeX modification by Schleifer et al.which includes stepwise fixation in cold acetone of different temperatures for 18 hours. After fixation tissue samples were gradually brought to a non watery state and embedded in paraffin. For dehydration, before transferring into paraffin, isopropyl alcohol (100%) was used after c) the fixation procedure. Paraffin sectiones (6µm) of blocks fixed in the above mentioned solutions were deparaffinized and submitted to PAP (peroxidase-antiperoxidase), APAAP (alkaline phosphatase-antialkaline phosphatase) and ABC procedures.The staining results of five organs in male rabbits have clearly shown that with methacarn fixation and APAAP technique two goals postulated histochemistry were achieved: a good preservation of antigenicity of P-gp and optimal preservation of morphology of the tissue exarnined. The APAAP procedure was superior to the PAP and ABC technicques according to the intensity of final reaction product expressed in sharply delineated blue chromogen precipitate at P-gp localization. The adrenal gland showed strong immunoreactivity of P-gp on plasma membranes of the majority of cells in the cortex, but the medulla was negative. The strongest reactivity was shown by the cells of zona fasciculata and zona reticularis, while zona glomerulosa showed a slight reactivity. Sinusoidal linning cells were negative. In the liver P-gp was found solely on the biliary surfaces of hepatocytes with individual zonal differences. In the normal liver, biliary canaliculi were clearly labeled in the periportal zone. In the kidney P-glycoprotein was restricted to the brush border of proximal tubules (Pl and P2) with stronger immunoreactivity in the straight portion (P2). The epithelium of billary ductules and endothelium of blood vessels were negative. In jejunum and colon high levels of P-glycoprotein occured on the apical surfaces of superficial columnar epithelial cells. The strong expression of P-glycoprotein observed by us, in the adrenal gland, kidney, liver and intestine of untreated male rabbits, suggests that the primary role of P-gp is normal secretion of adrenal hormones, physiological metabolites and ingested chemicals as in the normal human and hamster tissues. P-gp is a smali group of three isoforms in rodents and two in humans, which appear similar in structures but are functionaliy distinct. Since only two of the three members of MDR genes in the mouse have been found to be related to multidrug resistance it is likely that different P-gp isoforms perform distinct but related transport functions. In this context one should recali the study of Bradley et al. in normal chinese hamster tissues. In a competitive immunohistochemical assay the authors registered three P-gp isoforms in several organs, but showed also striking sex differences in P-gp content in the adrenal cortex. Only in male adrenals were the cells of zona fasciculata and zona reticularis labeled with C219 antibody, which is in agreement with our results.

immunohistochemical; P-glycoprotein; organs; rabbit

nije evidentirano