Active microtubule-dependent secretion of a fluorescent bile salt derivative in skate hepatocyte clusters

David S. Miller, Gert Fricker, Ursula Schramm, John H. Henson, David N. Hager, Surajit Nundy, Nazzareno Ballatori, James L. Boyer

Research output: Contribution to journalArticlepeer-review

Abstract

Fluorescence microscopy and video image analysis were used to study the transport of a fluorescent bile acid derivative [N-[7-(4-nitrobenzo-2-oxa- 1,3-diazol)]-7β-amino-3α,12α-dihydroxy-5β-cholan-24-oyl-2- aminoethanesulfonate (NBD-TC)] in isolated clusters of hepatocytes from the little skate Raja erinacea. Analysis of images of hepatocyte clusters that were incubated in medium with 0.5-1 μM NBD-TC showed that the fluorescent derivative accumulated in the cells and that the clusters retained a patent canalicular lumen as well as the ability to actively transport the bile acid derivative from the cells into the lumen; i.e., the lumen-to-cell fluorescence ratio greatly exceeded unity. NBD-TC uptake by hepatocytes was inhibited by several organic anions, of which taurocholate was the most effective. Uptake was also blocked by metabolic inhibitors and by incubation in the cold. Neither Na replacement nor increased medium K, which depolarizes the membrane electrical potential [potential difference (PD)], affected NBD- TC accumulation by hepatocytes. Transport of NBD-TC into the canalicular lumen was inhibited by incubation in the cold and was substantially reduced by high-K medium; these blocks were removed by warming and transfer to normal-K medium, respectively. Treatment of hepatocytes with 20-40 μM nocodazole, a drug that reversibly depolymerizes microtubules, reduced cellular NBD-TC accumulation and blocked its secretion into the canalicular lumen; nocodazole effects were reversed by washing the hepatocyte clusters in drug-free medium. Thus uptake of NBD-TC by skate hepatocytes is active and carrier mediated but not dependent on the PD or Na gradient. NBD-TC secretion from cell to canalicular lumen also appears to be active and carrier mediated. Canalicular secretion appears to be driven at least in part by the PD and is highly dependent on an intact microtubular system in this marine species.

Original languageEnglish (US)
Pages (from-to)G887-G896
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume270
Issue number6 33-6
DOIs
StatePublished - Jun 1996
Externally publishedYes

Keywords

  • bile canaliculus
  • elasmobranch
  • fluorescence microscopy
  • hepatic transport
  • little skate
  • liver
  • nocodazole
  • taurocholate

ASJC Scopus subject areas

  • Physiology
  • Hepatology
  • Gastroenterology
  • Physiology (medical)

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