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

2005 ◽  
Vol 208 (23) ◽  
pp. 4363-4376 ◽  
Author(s):  
J. P. Leader
Keyword(s):  
Microscopic Analysis ◽  
Malpighian Tubules ◽  
Transepithelial Transport ◽  
P Glycoprotein

scholarly journals Transepithelial transport of P-glycoprotein substrate by the Malpighian tubules of the desert locust

PLoS ONE ◽  
2019 ◽  
Vol 14 (10) ◽  
pp. e0223569 ◽  
Author(s):  
Marta Rossi ◽  
Davide De Battisti ◽  
Jeremy Edward Niven
Keyword(s):  
Malpighian Tubules ◽  
Transepithelial Transport ◽  
Desert Locust ◽  
P Glycoprotein

Transepithelial transport of nicotine and vinblastine in isolated malpighian tubules of the tobacco hornworm (Manduca sexta) suggests a P-glycoprotein-like mechanism.

1998 ◽  
Vol 201 (18) ◽  
pp. 2637-2645 ◽  
Author(s):  
L S Gaertner ◽  
C L Murray ◽  
C E Morris
Keyword(s):  
Manduca Sexta ◽  
Electrical Potential ◽  
Tobacco Hornworm ◽  
Malpighian Tubules ◽  
Transepithelial Transport ◽  
Multidrug Transporter ◽  
Basal Surface ◽  
P Glycoprotein ◽  
Apical Side ◽  
Vectorial Transport

We have examined the accumulative transport properties of the Malpighian (excretory) tubules of the tobacco hornworm Manduca sexta to test the hypothesis that a P-glycoprotein-like multidrug transporter is active and is responsible for the excretion of dietary nicotine in this tissue. Isolated tubules were cannulated and exposed to radiolabelled forms of either nicotine (5 min exposure) or the P-glycoprotein substrate vinblastine (60 min exposure) in the bathing (basal surface) fluid. The luminal (apical) contents were then flushed, and lumen-to-bath ratios were measured. Although these ratios provide conservative estimates of the physiological ability of Malpighian tubules to move compounds from blood to lumen, tubules concentrated nicotine 10-fold from an initial bath concentration of 0.5 mmol l-1 and vinblastine threefold (from an initial concentration of 1 micromol l-1). Vectorial transport of vinblastine and nicotine was eliminated by 25 micromol l-1 verapamil (a P-glycoprotein inhibitor) and was not dependent on the presence of a transepithelial electrical potential. Nicotine transport was inhibited by atropine (3 mmol l-1), while nicotine (> or = 50 micromol l-1) significantly reduced vinblastine transport. Verapamil was effective at reducing vinblastine transport when applied to the basal side alone, but not when applied to the apical side alone. Taken together, these results are consistent with the idea that the active excretion of nicotine and other alkaloids by the tobacco hornworm is mediated by a P-glycoprotein-like mechanism.

Identification of the synthetic surfactant nonylphenol ethoxylate: a P-glycoprotein substrate in human urine

1998 ◽  
Vol 274 (6) ◽  
pp. F1127-F1139 ◽  
Author(s):  
Jeffrey H. M. Charuk ◽  
Arthur A. Grey ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Cyclosporin A ◽  
Drug Transport ◽  
Mdck Cells ◽  
Chinese Hamster ◽  
Multidrug Resistant ◽  
The Body ◽  
Transepithelial Transport ◽  
Synthetic Surfactant ◽  
Nonylphenol Ethoxylate ◽  
P Glycoprotein

P-glycoprotein (Mdr1p) is an ATP-dependent drug efflux pump that is overexpressed in multidrug-resistant cells and some cancers. Mdr1p is also expressed in normal tissues like the kidney, where it can mediate transepithelial drug transport. A human urinary compound that reverses multidrug resistance and blocks [3H]azidopine photolabeling of P-glycoprotein was purified to homogeneity and identified by 1H-NMR and mass spectrometry as the synthetic surfactant nonylphenol ethoxylate (NPE). Multidrug-resistant Chinese hamster ovary (CHO) C5 cells accumulated less [3H]NPE than parental drug-sensitive Aux-B1 cells, and Mdr1p substrates, verapamil and cyclosporin A, increased this surfactant’s accumulation in C5 cells. NPE blocked the net transepithelial transport (basolateral to apical) of [3H]cyclosporin A in epithelia formed by Madin-Darby canine kidney (MDCK) cells. Net transepithelial transport (basal to apical) of [3H]NPE was demonstrated in MDCK cells and was inhibited by cyclosporin A. These findings show NPE is a Mdr1p substrate excreted into urine by kidney P-glycoprotein. NPE is a widely used surfactant and a known hormone disrupter that is readily absorbed orally or topically. The current findings indicate the function of kidney Mdr1p may be to eliminate exogenous compounds from the body.

scholarly journals Intestinal P Glycoprotein Acts as a Natural Defense Mechanism against Listeria monocytogenes

2004 ◽  
Vol 72 (7) ◽  
pp. 3849-3854 ◽  
Author(s):  
Brien L. Neudeck ◽  
Jennifer M. Loeb ◽  
Nancy G. Faith ◽  
Charles J. Czuprynski
Keyword(s):  
Listeria Monocytogenes ◽  
Defense Mechanism ◽  
Transepithelial Transport ◽  
Bacterial Invasion ◽  
Wild Type ◽  
In Vivo Experiments ◽  
Natural Defense ◽  
P Glycoprotein

ABSTRACT Mechanisms by which the intestinal epithelium resists invasion by food-borne pathogens such as Listeria monocytogenes are an evolving area of research. Intestinal P glycoprotein is well known to limit the absorption of xenobiotics and is believed to act as a cytotoxic defense mechanism. The aim of this study was to determine if intestinal P glycoprotein is involved in host defense against L. monocytogenes. Caco-2 cells and a P-glycoprotein-overexpressing subclone (Caco-2/MDR) were employed in addition to mdr1a−/− mice and wild-type controls. In vitro invasion assays and in vivo experiments were employed to measure bacterial invasion and dissemination. In addition, L. monocytogenes proteins were labeled with [35S]methionine, and the transepithelial transport across Caco-2 monolayers was characterized in both directions. Overexpression of P glycoprotein in Caco-2/MDR cells led to increased resistance to L. monocytogenes invasion, whereas P-glycoprotein inhibition led to increased invasion. Flux of [35S]methionine-labeled L. monocytogenes proteins was significantly greater in the basolateral-to-apical direction than in the apical-to-basolateral direction, indicating dependence on an apically located efflux transporter. Moreover, inhibiting P glycoprotein reduced the basolateral-to-apical flux of the proteins. Early dissemination of L. monocytogenes from the gastrointestinal tract was significantly greater in the mdr1a−/− mice than in wild-type controls. Expression and function of intestinal P glycoprotein is an important determinant in resistance to early invasion of L. monocytogenes.

Central role of the apical membrane H+-ATPase in electrogenesis and epithelial transport in Malpighian tubules

2000 ◽  
Vol 203 (9) ◽  
pp. 1459-1468 ◽  
Author(s):  
K.W. Beyenbach ◽  
T.L. Pannabecker ◽  
W. Nagel
Keyword(s):  
Epithelial Transport ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Coupling ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Malpighian Tubules ◽  
Proton Channel ◽  
Transepithelial Transport

The effects of bafilomycin A(1), a blocker of V-type H(+)-ATPases, were investigated in Malpighian tubules of Aedes aegypti. Bafilomycin A(1) reduced rates of transepithelial fluid secretion and the virtual short-circuit current (vI(sc)) with an IC(50) of approximately 5 micromol l(−)(1). As vI(sc) decreased, the electrical resistance increased across the whole epithelium and across the apical membrane, indicating effects on electroconductive pathways. Bafilomycin A(1) had no effect when applied from the tubule lumen, pointing to the relative impermeability of the apical membrane to bafilomycin A(1). Thus, bafilomycin A(1) must take a cytoplasmic route to its blocking site in the proton channel of the H(+)-ATPase located in the apical membrane of principal cells. The inhibitory effects of bafilomycin A(1) were qualitatively similar to those of dinitrophenol in that voltages across the epithelium (V(t)), the basolateral membrane (V(bl)) and the apical membrane (V(a)) depolarized towards zero in parallel. Moreover, V(bl)always tracked V(a), indicating electrical coupling between the two membranes through the shunt. Electrical coupling allows the H(+)-ATPase to energize not only the apical membrane, but also the basolateral membrane. Furthermore, electrical coupling offers a balance between electroconductive entry of cations across the basolateral membrane and extrusion across the apical membrane to support steady-state conditions during transepithelial transport.

scholarly journals P-glycoprotein Detoxification by the Malpighian Tubules of the Desert Locust

2019 ◽  
Author(s):  
Marta Rossi ◽  
Davide De Battisti ◽  
Jeremy E. Niven
Keyword(s):  
Surface Area ◽  
Rhodamine B ◽  
Ex Vivo ◽  
Fluid Secretion ◽  
Malpighian Tubules ◽  
Secretion Rate ◽  
Desert Locust ◽  
P Glycoprotein ◽  
Transporter Family ◽  
Wide Range

ABSTRACTDetoxification is essential for allowing animals to remove toxic substances present in their diet or generated as a biproduct of their metabolism. By transporting a wide range of potentially noxious substrates, active transporters of the ABC transporter family play an important role in detoxification. One such class of transporters are the multidrug resistance P-glycoprotein transporters. Here, we investigated P-glycoprotein transport in the Malpighian tubules of the desert locust (Schistocerca gregaria), a species whose diet includes plants that contain toxic secondary metabolites. To this end, we studied transporter physiology using a modified Ramsay assay in which ex vivo Malpighian tubules are incubated in different solutions containing the P-glycoprotein substrate dye rhodamine B in combination with different concentrations of the P-glycoprotein inhibitor verapamil. Our evidence shows that: (i) the Malpighian tubules contain a P-glycoprotein; (ii) tubule surface area is positively correlated with the tubule fluid secretion rate; and (iii) as the fluid secretion rate increases so too does the net extrusion of rhodamine B. We were able to quantify precisely the relationships between the fluid secretion, surface area, and net extrusion. We interpret these results in the context of the life history and foraging ecology of desert locusts. We argue that P-glycoproteins play an important role in the detoxification by contributing to the removal of xenobiotic substances from the haemolymph, thereby enabling gregarious desert locusts to maintain toxicity through the ingestion of toxic plants without suffering the deleterious effects themselves.

scholarly journals Malpighamoeba infection compromises fluid secretion and P-glycoprotein detoxification in Malpighian tubules

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Marta Rossi ◽  
Swidbert R. Ott ◽  
Jeremy E. Niven
Keyword(s):  
Water Stress ◽  
Surface Area ◽  
Fluid Secretion ◽  
Malpighian Tubules ◽  
Secretion Rate ◽  
Energy Costs ◽  
Natural Environments ◽  
Desert Locust ◽  
P Glycoprotein ◽  
The Impact

Abstract Malpighian tubules, analogous to vertebrate nephrons, play a key role in insect osmoregulation and detoxification. Tubules can become infected with a protozoan, Malpighamoeba, which damages their epithelial cells, potentially compromising their function. Here we used a modified Ramsay assay to quantify the impact of Malpighamoeba infection on fluid secretion and P-glycoprotein-dependent detoxification by desert locust Malpighian tubules. Infected tubules have a greater surface area and a higher fluid secretion rate than uninfected tubules. Infection also impairs P-glycoprotein-dependent detoxification by reducing the net rhodamine extrusion per surface area. However, due to the increased surface area and fluid secretion rate, infected tubules have similar total net extrusion per tubule to uninfected tubules. Increased fluid secretion rate of infected tubules likely exposes locusts to greater water stress and increased energy costs. Coupled with reduced efficiency of P-glycoprotein detoxification per surface area, Malpighamoeba infection is likely to reduce insect survival in natural environments.

Quantitative Assessment of P-Glycoprotein Expression and Function Using Confocal Image Analysis

2014 ◽  
Vol 20 (5) ◽  
pp. 1329-1339 ◽  
Author(s):  
Zahra Hamrang ◽  
Yamini Arthanari ◽  
David Clarke ◽  
Alain Pluen
Keyword(s):  
Image Analysis ◽  
Cell Lines ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Quantitative Imaging ◽  
Microscopic Analysis ◽  
Live Cell ◽  
Distribution Analysis ◽  
Spatial Intensity Distribution ◽  
P Glycoprotein

AbstractP-glycoprotein is implicated in clinical drug resistance; thus, rapid quantitative analysis of its expression and activity is of paramout importance to the design and success of novel therapeutics. The scope for the application of quantitative imaging and image analysis tools in this field is reported here at “proof of concept” level. P-glycoprotein expression was utilized as a model for quantitative immunofluorescence and subsequent spatial intensity distribution analysis (SpIDA). Following expression studies, p-glycoprotein inhibition as a function of verapamil concentration was assessed in two cell lines using live cell imaging of intracellular Calcein retention and a routine monolayer fluorescence assay. Intercellular and sub-cellular distributions in the expression of the p-glycoprotein transporter between parent and MDR1-transfected Madin–Derby Canine Kidney cell lines were examined. We have demonstrated that quantitative imaging can provide dose–response parameters while permitting direct microscopic analysis of intracellular fluorophore distributions in live and fixed samples. Analysis with SpIDA offers the ability to detect heterogeniety in the distribution of labeled species, and in conjunction with live cell imaging and immunofluorescence staining may be applied to the determination of pharmacological parameters or analysis of biopsies providing a rapid prognostic tool.

Heat-shock-stimulated transepithelial daunomycin secretion by flounder renal proximal tubule primary cultures

1995 ◽  
Vol 268 (1) ◽  
pp. F135-F144 ◽  
Author(s):  
C. Sussman-Turner ◽  
J. L. Renfro
Keyword(s):  
Heat Shock ◽  
Proximal Tubule ◽  
Organic Cation ◽  
Organic Anion ◽  
Primary Cultures ◽  
Renal Proximal Tubule ◽  
Transepithelial Transport ◽  
Protein Synthesis Inhibitor ◽  
Mild Heat ◽  
P Glycoprotein

Primary monolayer cultures of winter flounder renal proximal tubule epithelium mounted in Ussing chambers were used to characterize transepithelial transport of daunomycin (Dau). Control tissues performed active net secretion of Dau (0.064 +/- 0.027 nmol.cm-2.h-1). Mild heat shock (5 degrees C elevation for 6-8 h followed by return to normal temperature) almost doubled Dau secretion (0.114 +/- 0.026 nmol.cm-2.h-1). This response was inhibited approximately 40% by addition of the protein synthesis inhibitor, cycloheximide. Dau secretion was inhibited by verapamil, vinblastine, cyclosporin A, and to a lesser degree by the organic cation, tetraethylammonium. In addition, tetraethylammonium secretion was inhibited by vinblastine. Dau secretion was not inhibited by the organic anion, p-aminohippurate, and p-aminohippurate secretion was not inhibited by vinblastine. The transepithelial reabsorptive flux of Dau and the electrical characteristics of the tissues, including rheogenic glucose transport, were unaffected by any of the above treatments. Reaction of tissues with a monoclonal antibody to P-glycoprotein (C219) revealed the presence of this transporter on only apical microvilli. The data indicate that flounder possess an active mechanism for the renal excretion of Dau that is stimulated by mild heat shock. This mechanism is distinct from organic anion, but not organic cation, transport and has characteristics consistent with transport by an apical P-glycoprotein.

Sign in / Sign up

Export Citation Format

Share Document