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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.sub.B] receptor antagonist but not by an [ET.sub.A] receptor antagonist. Immunostaining with an antibody to mammalian [ET.sub.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.sub.B] receptor and PKC. In addition, cAMP affects transporter function through a PKA-independent mechanism, possibly by competition for transport. endothelin-1; multidrug resistance-associated protein isoform 2; protein kinase C

Xenobiotic efflux pumps in isolated fish brain capillaries. Am J Physiol Regulatory Integrative Comp Physiol 282: R191-R198, 2002; 10.1152/ ajpregu.00305.2001.--To identify specific transporters that drive xenobiotics from the central nervous system to blood, the accumulation of fluorescent drugs was studied in isolated capillaries from killifish and dogfish shark brain using confocal microscopy and quantitative image analysis. In killifish brain capillaries, luminal accumulation of fluorescent derivatives of cyclosporin A and verapamil was concentrative, specific, and energy dependent (inhibition by KCN). Transport was reduced by PSC-833, but not by leukotriene [C.sub.4], indicating the involvement of P-glycoprotein. The ability of capillaries to transport the cyclosporin A derivative was unchanged over 20 h, demonstrating the long-term viability of the preparation. Luminal accumulation of the fluorescent organic anions sulforhodamine 101 and fluorescein-methotrexate was also concentrative, specific, and energy dependent. Transport of these compounds was reduced by leukotriene [C.sub.4], but not by PSC-833, indicating the involvement of a multidrug resistance-associated protein (Mrp). Similar results were obtained for isolated capillaries from dogfish shark. Immunostaining localized P-glycoprotein and Mrp2 to the luminal surface of the killifish brain capillary endothelium. These findings validate a new and long-lived comparative model for studying drug transport across the bloodbrain barrier and, as in mammals, implicate P-glycoprotein and Mrp2 in transport from the central nervous system to blood in fish.

The purpose of this study was to investigate the effect of wearing earplugs on classical musicians' pitch perception across three experimental conditions: no earplugs, foam earplugs, and Etymotic earplugs. Participants were graduate and undergraduate music majors attending a large school of music in the southeastern United States (N = 72). Participants adjusted the pitch of five complex stimulus tones (C#4, C#5, C#3, G#4, and F#3) using a continuous response digital interface until they believed the interval was in tune with an oboe (C#4) reference tone. Participants tended to tune flat when the stimulus tone was presented flat and to tune sharp when it was presented sharp across all three earplug conditions. Overall cent deviation in tuning responses showed that in both directional and absolute deviation analyses, listeners were most accurate when tuning without earplugs, then when using Etymotic earplugs, and least accurate with foam earplugs. Significant differences, however, were limited to specific intervals and in magnitudes not likely to be perceived. Although more research is needed, the use of Etymotic earplugs may provide valuable protection against noise-induced hearing loss with negligible effects on pitch perception. Implications for musicians and recommendations for future research are discussed. [ABSTRACT FROM AUTHOR]

The function of protein kinase C (PKC) in the regulation of organic anion (fluorescein, FL) transport in killifish renal proximal tubules was examined through fluorescence microscopy and digital image analysis. Phorbol ester decreased cellular and luminal accumulation of FL and protein kinase inhibitors increased cellular and luminal accumulation. The phorbol ester induced PKC translocation from cytoplasm to the plasma membrane. Results showed that renal organic anion transport is negatively correlated with PKC activity and that PKC directly or indirectly regulates the basolateral step in the transport.

The mechanisms of organic anion transport in killifish renal proximal tubules were analyzed by laser scanning-confocal microscopy. Microscopic analysis of killifish proximal tubules indicated the presence of specific fluorescein-methotrexate (FL-MTX) transport from the cell to the lumen. Furthermore, the luminal transport of FL-MTX was mediated by cellular metabolism and the accumulation of Fl-MTX was reduced by p-aminohippurate, folate and probenecid.

The mechanism of transport of organic anions across the luminal membrane of proximal tubular cells was investigated in killifishes by conventional fluorescence microscopy. At steady state, luminal fluorescence intensities were higher than cellular fluorescence intensities. Both intensities were markedly reduced when fluorescein was used as substrate, but was only slightly reduced when carboxyfluorescein diacetate or monochlorobimane was used as substrate. Depolarization of the cells had no effect on the cell-to-lumen distribution of the anions.

The transcellular translocation of bile salt derivative (N-(7-(4-nitrobenzo-2-oxa-1,3-diazol-))-7beta-amino-3alpha,12aplha-dihydroxy -5beta-cholan-24-oyl-2-aminoethanesulfonate (NBD-TC)) in skate hepatocytes is microtubule dependent. This dependence is essential for canalicular secretion. The uptake of NBD-TC is active and carrier mediated, and independent of the sodium or the potential difference gradient.