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ID naloge: 32 Letnik: 1998 Predmet: biokemija
Vpliv blokade p-glikoproteina na sekrecijo kortizola iz perfundiranih govejih nadledvicnic
Avtor: Sonja Vogric
Mentor: doc. dr. Tanja Cufer
Izhodišca: P-glikoprotein (P-gp) je 170 kD protein, ki je bil sprva identificiran v tumorskih celicah, kjer sodeluje pri aktivnem transportu citostatikov iz rakastih celic. Spada v družino transportnih proteinov s podobno gensko osnovo, ki jih najdemo v membrani normalnih celic vretencarjev in nekaterih bakterij. P-gp se nahaja v gastrointestinalnem traktu, v ledvicah in v jetrih, kjer sodeluje pri aktivnem transportu metabolitov in toksicnih substanc iz telesa. Nahaja se tudi v hematoencefalni barieri in v žilju okoli testisa, kjer verjetno sodeluje pri vzdrževanju "cistega" okolja. P-gp je prisoten tudi v nekaterih endokrinih žlezah, najvec v skorji nadledvicnice. Funkcija P-gp v tem organu do sedaj še ni dognana. In vitro je bilo ugotovljeno, da je sposoben aktivno transportirati razlicne endogene substance, tudi steroide. Poskusi na celicnih linijah mu pripisujejo vlogo v fiziološkem izlocanju kortizola, cemur pa nasprotuje raziskava na kuncih, pri katerih se serumska koncentracija kortizola po aplikaciji P-gp blokatorja valspodarja ni znižala, ampak celo zvišala.
Namen: V tej nalogi smo se namenili podrobneje preuciti vlogo P-glikoproteina v nadledvicni žlezi. Predpostavili smo, da P-gp ne sodeluje v fiziološkem izlocanju kortizola, ampak da je njegova funkcija v nadledvicni žlezi zašcita celic pred ksenobiotiki iz okolja. Namenili smo se opazovati izlocanje kortizola ob blokadi P-gp, nato pa še ob izpostavitvi žleze razlicnim ksenobiotikom in oceniti funkcionalno okvaro žlez brez in po dodatku P-gp blokatorja.
Metode: Izolirane goveje nadledvicne žleze smo retrogradno perfundirali z Medijem 199, ki smo mu dodali kortikotropin. Posamezne poskusne skupine smo dodatno perfundirali s selektivnim P-gp blokatorjem valspodarjem, z dvema ksenobiotikoma mitotanom in doksorubicinom ter kombinacijo ksenobiotik - P-gp blokator. Vzorce zbranega perfuzata smo analizirali z radioimunskim testom za kvantitativno dolocanje kortizola. Spremembe v izlocanju kortizola smo statisticno ovrednotili s Studentovim t-testom za dva neodvisna vzorca.
Rezultati: Povprecna kolicina kortizola izlocenega iz žlez perfundiranih s P-gp blokatorjem se ni statisticno znacilno razlikovala od kontrolne skupine. Po perfuziji s ksenobiotikoma je kolicina izlocenega kortizola v primerjavi s kontrolno skupino padla: za 59% v primeru mitotana in 39% v primeru doksorubicina. Po perfuziji s ksenobiotikom in P-gp blokatorjem pa je bil padec kolicine izlocenega kortizola napram kontrolni skupini še vecji (90,5% v primeru mitotana in 68% v primeru doksorubicina). Razlike v kolicini izlocenega kortizola v primeru perfuzije s ksenobiotikom in perfuzije s kombinacijo ksenobiotika in P-gp blokatorja, so tako pri mitotanu, kot tudi pri doksorubicinu, dosegle prag statisticne znacilnosti (p < 0,05). Dolocili smo tudi dinamiko izlocanja kortizola. Razlike v izlocanju kortizola pri perfuziji s ksenobiotikom in perfuziji s kombinacijo ksenobiotik - P-gp blokator so bile pri mitotanu statisticno znacilne že od prvega polurnega intervala dalje, pri doksorubicinu pa od tretjega intervala dalje (p < 0,05).
Zakljucki: Izlocanje kortizola iz perfundiranih nadledvicnic se ob blokadi P-gp z visoko selektivnim blokatorjem valspodarjem ne zmanjša. Blokada P-gp z valspodarjem poveca toksicnost lipofilnih ksenobiotikov mitotana in doksorubicina. Izlocanje kortizola, ki je merilo funkcionalnosti žleze, se ob socasni aplikaciji ksenobiotika in selektivnega P-gp blokatorja bolj zniža kot ob aplikaciji samega ksenobiotika
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[Abstract / English version]
The effects of p-glycoprotein blocade on cortisol secretion from perfused bovine adrenal gland
Author: Sonja Vogric
Mentor: doc. dr. Tanja Cufer
Background:P-glycoprotein (P-gp) is 170 kD protein, which was first identified in tumor cells where it is involved in active transport of cytotoxic agents from cancer cells. It belongs to the group of transport proteins with a similar genetic basis, which can be found in the membranes of normal cells of the vertebrates and of some bacteria. P-gp occurs in the gastrointestinal tract, kidneys and liver, where it participates in the active transport of metabolites and toxic substances from the body. It is also found in the hematoencephalic barrier and in testicular blood vessels, where it presumably plays a role in the maintenance of pure environment. P-gp is also present in some endocrine glands, and particularly in the adrenal cortex. However, the function of P-gp in this organ has not been proved yet. In vitro investigations have shown that P-gp has the capacity to actively transport different endogenous substances, among them also steroids. Experiments on cell lines have pointed out its possible role in physiological cortisol secretion. This observation, however, has been contradicted by the results of a study on rabbits, according to which the serum cortisol levels after application of valspodar, a P-gp blocker, were found to have increased rather than decreased.
Aim:This study was aimed to further investigate the role of P-glycoprotein in the adrenal gland. It has been presumed that P-gp, rather than being involved in the physiological cortisol excretion, it plays a role in protecting the adrenal gland cells against xenobiotics from the environment. First, cortisol excretion was studied during P-gp blocking. Afterwards, the glands were exposed to different xenobiotics, and the functional damage of the gland in the presence or absence of a P-gp blocker was evaluated.
Methods:Isolated bovine adrenal glands were perfused retrogradely with Medium 199 supplemented with corticotrophin. Individual test groups were additionally perfused with a selective P-gp blocker, valspodar, as well as with two xenobiotics (mitotane and doxorubicin), and a xenobiotic-P-gp blocker combination. Samples of the collected perfusate were analyzed with radioimmune assay for quantitative cortisol determination. Changes in cortisol excretion were statistically evaluated using two-tail Student's t-test.
Results:The mean quantity of cortisol excreted from the glands perfused with a P-gp blocker was not statistically significantly different from that of the control group. After perfusion with xenobiotics, the quantity of excreted cortisol has decreased ( by 59% with mitotane and by 39% with doxorubicin) as compared to the control group. After perfusion with a xenobiotic and P-gp blocker, the decrease in the quantity of excreted cortisol was even more obvious (90.5% with mitotane, and 68% with doxorubicin) in comparison with the controls. With either mitotane or doxorubicin, the differences in the quantity of excreted cortisol in the case of perfusion with a xenobiotic alone, or with a combination of xenobiotic - P-gp blocker, have reached the threshold of statistical significance (p<0.05). The rate (algorithm) of cortisol excretion was determined as well. In mitotane, the differences in cortisol excretion in perfusion with xenobiotics and perfusion with a combination of xenobiotic - P-gp blocker were statistically significant starting already with the first half-hour interval, while in the case of doxorubicin this was apparent from the third half-hour interval on (p<0.05).
Conclusions:When p-gp is blocked with a highly selective blocker, valspodar, cortisol excretion from perfused adrenal glands does not decrease. The valspodar blockage of P-gp increases the toxicity of the lipophilic xenobiotic mitotane and doxorubicin. When xenobiotic and selective P-gp blocker are applied simultaneously, the cortisol secretion, which is the measure of the glands function, decreases more than when only xenobiotic is applied.
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