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.