Transepithelial transport of fluorescent p-glycoprotein and MRP2 substrates by insect Malpighian tubules: confocal microscopic analysis of secreted fluid droplets

Leader, John P.; O’Donnell, MJ

doi:10.1242/jeb.01911

Cited by 47 publications

(36 citation statements)

References 38 publications

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“…Moreover, an increase in saline osmolality was as- The bath concentration required for half maximal transport in control saline is 7.1 mM (Leader and ODonnell, 2005). However, at higher dye concen- Thus, the release of diuretic factors in response to insecticides (Maddrell and Casida, 1971) (Self et al, 1964) so release of diuretic factors may aid elimination of such compounds in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…Transepithelial dye flux is calculated as the product of fluid secretion rate (measured in the Ramsay assay) and fluorochrome concentration. The detection limit for lumenal concentrations of MRP2 and P-gp substrates using this method is greater than that achievable with radiolabelled compounds (Leader and ODonnell, 2005). Stimulants of fluid secretion might be expected to alter net transepithelial transport of P-gp and MRP2 substrates by one or more of several mechanisms.…”

mentioning

confidence: 90%

“…Confocal microscopic studies of iso- (Leader and ODonnell, 2005). Transepithelial dye flux is calculated as the product of fluid secretion rate (measured in the Ramsay assay) and fluorochrome concentration.…”

mentioning

confidence: 99%

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Changes in fluid secretion rate alter net transepithelial transport of MRP2 and P‐glycoprotein substrates in Malpighian tubules of Drosophila melanogaster

O’Donnell

Leader

2006

Arch Insect Biochem Physiol

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The effects of stimulants of fluid secretion on net transepithelial transport of the MRP2 substrate Texas Red and the p-glycoprotein substrate daunorubicin were examined in Malpighian tubules of Drosophila melanogaster. Fluid secretion rates were determined using the Ramsay assay and secreted fluid concentrations of Texas Red and daunorubicin were determined using a microfluorometric technique. Nanoliter droplets of secreted fluid were collected in optically flat glass capillaries and dye concentration was determined from fluorescence intensity measured by confocal laser scanning microscopy. Net transepithelial flux of each compound was then calculated as the product of its concentration in the secreted fluid and the fluid secretion rate. Net transepithelial flux of Texas Red increased when fluid secretion was stimulated by tyramine, cyclic AMP or hypoosmotic saline. Net flux decreased when fluid secretion rate of cAMP-stimulated tubules was reduced by elevating saline osmolality with sucrose. Net transepithelial flux of daunorubicin increased when fluid secretion was stimulated by cAMP. Significant increases in dye flux were seen only when the dyes were present at concentrations close to or greater than the concentration required for half maximal transport. Regression analyses showed that 57- 88% of the change in dye flux was attributable to the change in fluid secretion rate when tubules were stimulated with cAMP, cGMP, or tyramine. The results do not suggest that the effects of tyramine and cAMP are mediated through changes in transepithelial potential, nor do they indicate the direct effects of the stimulants on MRP2-like or p-glycoprotein-like transporters (e.g., via protein kinases). Instead, the results suggest that increases in fluid secretion rate minimize diffusive backflux of these dyes and, thus, facilitate higher rates of net transepithelial transport indirectly.

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Section: Discussionmentioning

confidence: 99%

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confidence: 90%

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Changes in fluid secretion rate alter net transepithelial transport of MRP2 and P‐glycoprotein substrates in Malpighian tubules of Drosophila melanogaster

O’Donnell

Leader

2006

Arch Insect Biochem Physiol

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“…Studying movement of compounds into the brain by looking through the eye is limited to flies containing the white mutation, as the pigment in red eyes masks visualization of fluorophore content. In this study we have used the confocal microscope as a sensitive fluorometer (32) to study permeation of the fluorescent anion fluorescein into the Drosophila brain.…”

Section: Resultsmentioning

confidence: 99%

Oatp58Dc contributes to blood-brain barrier function by excluding organic anions from the Drosophila brain

Seabrooke

O’Donnell

2013

American Journal of Physiology-Cell Physiology

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The blood-brain barrier (BBB) physiologically isolates the brain from the blood and, thus, plays a vital role in brain homeostasis. Ion transporters play a critical role in this process by effectively regulating access of chemicals to the brain. Organic anion-transporting polypeptides (Oatps) transport a wide range of amphipathic substrates and are involved in efflux of chemicals across the vertebrate BBB. The anatomic complexity of the vascularized vertebrate BBB, however, creates challenges for experimental analysis of these processes. The less complex structure of the Drosophila BBB facilitates measurement of solute transport. Here we investigate a physiological function for Oatp58Dc in transporting small organic anions across the BBB. We used genetic manipulation, immunocytochemistry, and molecular techniques to supplement a whole animal approach to study the BBB. For this whole animal approach, the traceable small organic anion fluorescein was injected into the hemolymph. This research shows that Oatp58Dc is involved in maintaining a chemical barrier against fluorescein permeation into the brain. Oatp58Dc expression was found in the perineurial and subperineurial glia, as well as in postmitotic neurons. We specifically targeted knockdown of Oatp58Dc expression in the perineurial and subperineurial glia to reveal that Oatp58Dc expression in the perineurial glia is necessary to maintain the barrier against fluorescein influx into the brain. Our results show that Oatp58Dc contributes to maintenance of a functional barrier against fluorescein influx past the BBB into the brain.

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“…Due to structural similarity, many MRPs show overlap in their substrate and inhibitor specificity. Whereas combination of Texas Red and MK-571 is used specifically to monitor activity of MRP2 [49], MK-571 possesses inhibitory activity against both MRP1 and MRP2 [47]. Hypericin content was increased in HT-29, but also in MCF-7 cells with low MRP1 activity, suggesting that MRP2 also contributes to regulation of hypericin intracellular content.…”

Section: Discussionmentioning

confidence: 99%

Potentiation of hypericin-mediated photodynamic therapy cytotoxicity by MK-886: Focus on ABC transporters, GDF-15 and redox status

Kuchárová

Mikeš

Jendželovský

et al. 2015

Photodiagnosis and Photodynamic Therapy

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Transepithelial transport of fluorescent p-glycoprotein and MRP2 substrates by insect Malpighian tubules: confocal microscopic analysis of secreted fluid droplets

Cited by 47 publications

References 38 publications

Changes in fluid secretion rate alter net transepithelial transport of MRP2 and P‐glycoprotein substrates in Malpighian tubules of Drosophila melanogaster

Changes in fluid secretion rate alter net transepithelial transport of MRP2 and P‐glycoprotein substrates in Malpighian tubules of Drosophila melanogaster

Oatp58Dc contributes to blood-brain barrier function by excluding organic anions from the Drosophila brain

Potentiation of hypericin-mediated photodynamic therapy cytotoxicity by MK-886: Focus on ABC transporters, GDF-15 and redox status

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