David S. Miller: Scientist, Mentor, Friend-a tribute and thank you.

David S. Miller was Acting Scientific Director of the Division of Intramural Research at the National Institute of Environmental Health Sciences, National Institutes of Health, and Head of the Intracellular Regulation Group in the Laboratory of Toxicology and Pharmacology before he retired in 2016. David received his Ph.D. in biochemistry from the University of Maine in 1973. David was a Group Leader at the Michigan Cancer Foundation before joining the NIEHS in 1985. His research covered a wide range from renal excretory transport mechanisms to regulation of transporters at the blood-CSF and blood-brain barriers, from fish, amphibians and birds to mammals. David was an outstanding scientist with irresistible enthusiasm for science and an incredible ability to think outside the box while being an exceptional mentor and friend.

Tyrosine phosphorylation and dissociation of occludin-ZO-1 and E-cadherin-beta-catenin complexes from the cytoskeleton by oxidative stress.

The oxidative-stress-induced alteration in paracellular junctional complexes was analysed in Caco-2 cell monolayer. Oxidative stress induced a rapid increase in tyrosine phosphorylation of occludin, zonula occludens (ZO)-1, E-cadherin and beta-catenin. An oxidative-stress-induced decrease in transepithelial electrical resistance was associated with a redistribution of occludin-ZO-1 and E-cadherin-beta-catenin complexes from the intercellular junctions. Genistein, a tyrosine kinase inhibitor, prevented the oxidative-stress-induced decrease in resistance and redistribution of protein complexes. Occludin, ZO-1, E-cadherin and beta-catenin in the Triton-insoluble cytoskeletal fraction were reduced by oxidative stress, which was prevented by genistein. Oxidative stress also reduced the co-immunoprecipitation of ZO-1 with occludin, which was prevented by genistein. Co-immunoprecipitation of beta-catenin with E-cadherin was unaffected by oxidative stress or genistein. ZO-1, E-cadherin and beta-catenin in the plasma membrane or membrane-cytoskeleton were either slightly reduced or unaffected by oxidative stress or genistein. These results show that oxidative stress induces tyrosine phosphorylation and cellular redistribution of occludin-ZO-1 and E-cadherin-beta-catenin complexes by a tyrosine-kinase-dependent mechanism.

Regulation of MRP2-mediated transport in shark rectal salt gland tubules.

We examined endothelin-1 (ET-1) regulation of the xenobiotic efflux pump, multidrug resistance-associated protein isoform 2 (MRP2), in intact dogfish shark rectal salt gland tubules using a fluorescent substrate sulforhodamine 101 and confocal microscopy. Subnanomolar to nanomolar concentrations of ET-1 rapidly reduced the cell-to-lumen transport of sulforhodamine 101. These effects were prevented by an ET(B) receptor antagonist but not by an ET(A) receptor antagonist. Immunostaining with an antibody to mammalian ET(B) receptors showed specific localization to the basolateral membrane of the shark rectal gland epithelial cells. ET-1 effects on transport were blocked by a protein kinase C (PKC)-selective inhibitor, implicating PKC in ET-1 signaling. A protein kinase A (PKA)-selective inhibitor had no effect. Forskolin reduced luminal accumulation of sulforhodamine 101, but inhibition of PKA did not block the forskolin effect. Consistent with this observation, a cAMP analog that does not activate PKA reduced luminal accumulation of sulforhodamine 101. These results indicate that shark rectal gland transport on MRP2 is regulated by ET acting through an ET(B) receptor and PKC. In addition, cAMP affects transporter function through a PKA-independent mechanism, possibly by competition for transport.