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An in vivo role of Mrp2 in the rat hepatocytes by immunocytochemistry for amoxicillin using the transporter-deficient EHBR

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Abstract

An in vivo role of the multidrug resistant-associated protein (Mrp2) in rat hepatocytes was examined by immunocytochemistry (ICC) for amoxicillin (AMPC) by the use of the transporter-deficient Eisai hyperbilirubinemic rats (EHBR). The ICC revealed that in the liver of EHBR at 3-h post-administration, amoxicillin accumulated in the cytoplasmic pools and nuclei of the hepatocytes in a characteristic granular morphology on the bile capillaries. However, no amoxicillin was observed on the surface of the lumina ranging from the bile capillaries to the interlobular bile ducts. The drug persisted at least for 6-h after administration. In contrast, in the control rat liver at 3-h post-administration, AMPC-adsorption occurred on such luminal surface, while AMPC accumulated to a less level in both the cytoplasm and nuclei of the hepatocytes. The drug completely disappeared in the hepatocytes at 6-h post-administration. These results strongly suggest that AMPC taken up into the cytoplasm of the hepatocytes excretes via Mrp2 into the bile flow. Furthermore, electron microscopy demonstrated that the lower electron density areas in large sizes, corresponding to the cytoplasmic pools in ICC for AMPC, occurred in the cytoplasm peripheral to the nuclei of the hepatocytes in EHBR at 3-h post-administration, and then disappeared 24 h after administration.

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Abbreviations

EHBR:

Eisai hyperbilirubinemic rats

AMPC:

Amoxicillin

ABPC:

Ampicillin

GM:

Gentamicin

ICC:

Immunocytochemistry

GA:

Glutaraldehyde

ELISA:

Enzyme-linked immunosorbent assay

HRP:

Horseradish peroxidase

BSA:

Bovine serum albumin

AMPC-GA-BSA:

Amoxicillin-glutaraldehyde-bovine serum albumin conjugate

RT:

Room temperature

TBS:

50 mM Tris–HCl buffer, pH 7.4, containing 0.86 % NaCl

TBST:

TBS supplemented with 0.1 % Triton X-100

CEM:

Conventional electron microscopy

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Acknowledgments

We are grateful to Kazumi Nishiyama and Eityu Shin for technical assistance throughout this study. This study was supported in part by a grant the Japanese Society for the Promotion of Science 15590148.

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Authors and Affiliations

  1. Department of Applied Life Science, Faculty of Biotechnology and Life Science, Sojo University, Ikeda 4-22-1, Kumamoto, 860-0082, Japan

    Kunio Fujiwara, Masashi Shin, Yohei Yoshizaki, Tsubasa Miyazaki & Tetsuya Saita

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  1. Kunio Fujiwara
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  2. Masashi Shin
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  3. Yohei Yoshizaki
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  4. Tsubasa Miyazaki
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  5. Tetsuya Saita
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Correspondence to Kunio Fujiwara.

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Fujiwara, K., Shin, M., Yoshizaki, Y. et al. An in vivo role of Mrp2 in the rat hepatocytes by immunocytochemistry for amoxicillin using the transporter-deficient EHBR. J Mol Hist 43, 371–378 (2012). https://doi.org/10.1007/s10735-012-9406-2

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  • DOI: https://doi.org/10.1007/s10735-012-9406-2

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