Assessment of Ileal Epithelial P-Glycoprotein Dysfunction Induced by Ischemia/Reperfusion using in vivo Animal Model

Biological & Pharmaceutical Bulletin

Kitazato

et al. 2011

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ATP binding cassette (ABC) transporters form one of the largest families of membrane transport proteins and are expressed in all organisms. They have been studied extensively and play a vital role in many cellular processes. ABC transporters are responsible for the multidrug resistance of cancer cells 1) but may also be capable of transporting several substrates such as metal ions, peptides, amino acids, sugars, and a large number of hydrophobic compounds and metabolites across the plasma membrane as well as intracellular membranes.2) ABC transporters play important roles in the defense mechanisms of several tissues through the excretion of toxic compounds and their metabolites. 3,4) In addition, the expression levels of the transporters are tightly regulated during tissue defense. 5)P-Glycoprotein (P-gp) is a typical ABC transporter. P-gp, which plays a very important role in drug therapy, was first discovered in 1976 as an efflux pump in a colchicine-resistant cell line, and 10 years later, the gene for P-gp was encoded. It is the most extensively studied of all of the ABC transporters.During small intestine grafting, rejection and ischemia/ reperfusion (I/R) injury are important problems. Immunosuppressive therapy is always required after transplantation. Since transplantation depends on a delicate balance between immunosuppression and rejection, the maintenance of adequate blood concentrations of immunosuppressants is critical. Cyclosporine A and tacrolimus are representative P-gp substrates. Therefore, P-gp controls the efficacies of these immunosuppressants. Information about the changes in P-gp induced by intestinal I/R would be useful for clinicians performing immunosuppressive therapy. We previously reported in in vitro 6) and in vivo 7-9) studies that I/R influences the function and expression of P-gp. In these reports, a correlation was found between the function of P-gp 1 h after reperfusion and the expression level of P-gp, but no such relationship was recognized 24 h after reperfusion. 7,8) It was suggested that this was due to functional abnormalities or abnormal localization of P-gp.In the present study, we examined whether abnormal P-gp localization is induced by intestinal I/R. Moreover, the mechanism and factors responsible for this phenomenon were also examined. MATERIALS AND METHODS MaterialsAll reagents were of analytical grade or better. Animals and Experimental Design Male Wistar/ST rats (8 weeks old) were purchased from Japan SLC Ltd. (Shizuoka, Japan). All animal experiments were performed according to the guidelines of Tokyo University of Pharmacy and Life Sciences. The animals were fasted for 18 h before the start of the experiment. Water was given freely during fasting. We have previously reported an in vivo intestinal I/R model that was produced using a spring scale and surgical sutures.7-9) Briefly, the superior mesenteric artery and vein in rats were occluded by lifting them using surgical-sutures (Natsume No. 3) connected to a spring balance for 1 h (ischemia condition), before r...

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Changes in the Localization of Ileal P-Glycoprotein Induced by Intestinal Ischemia/Reperfusion

Biological & Pharmaceutical Bulletin

Kitazato

et al. 2011

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Effects of antioxidants on drug absorption in in vivo intestinal ischemia/reperfusion

Kitazato

Eur J Drug Metab Pharmacokinet

et al. 2010

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Ischemia/reperfusion (I/R) injury must be overcome in order to succeed in small intestinal transplantation. Reactive oxygen species (ROS) are generated by I/R, and they induce lipid peroxidation which is one of the causes of mucosal lesion. We previously reported the protection effects of antioxidants to I/R injury in the in vitro study. In the present study, we examined the inhibitive effect of antioxidants on intestinal I/R injury in the in vivo study. Intestinal ischemia was induced in Wistar/ST rats using the spring scale and the surgical suture for 1 h, followed by reperfusion for 1 h. We used 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron), astaxanthin (ATX) and epigallocatechin gallate (EGCG) as antioxidants. The inhibitive effects on mucosal lesion, opening of TJ and decrease in protein expression level of P-gp by in vivo intestinal I/R were admitted by three kinds of antioxidant. Tiron and EGCG inhibited P-gp function but ATX did not. Therefore, for the use of P-gp substrate like immunosuppressants after the intestinal transplantation, ATX, which does not inhibit P-gp is considered to be effective for intestinal I/R injury.

Changes in the expression levels of tight junction components during reconstruction of tight junction from mucosal lesion by intestinal ischemia/reperfusion

Eur J Drug Metab Pharmacokinet

Tomita

et al. 2013

Tight junction (TJ) is composed of the most apical components of the intercellular junctional complex in epithelial cells; TJ has cell polarity and functions as a major determinant of epithelial barrier function. In this study, to clarify the components of TJ required for its reconstruction and functional acquisition, we examined the changes in intestinal mucosal structure that depended on mucosal lesion by intestinal I/R, that is, the changes in mRNA and protein expression of the claudin family and scaffold proteins. We used an in vivo intestinal I/R model made using the spring scale and surgical sutures, and examined the mRNA and protein expression levels of TJ components by real-time RT-PCR and Western blotting, respectively. Changes in mRNA and protein expression levels of TJ components by intestinal I/R were observed. Among them, characteristic changes were observed in claudin-2 and claudin-4. In addition, the expression behavior of multi-PDZ domain protein (MPDP) mRNA was similar to that of claudin-4. In conclusion, in the recovery process of TJ from mucosal lesion by intestinal I/R, it was suggested that claudin-2 and claudin-4 strongly participate in the reconstruction and functional acquisition of TJ, respectively. Furthermore, it was suggested that MPDZ, which is scaffold protein, also has an important role in these processes.