Regulation of Plasma Membrane Expression of TRPV4 Channel by GSK1016790A and Hypotonic Stress in HeLa and M‐1 Cells

Claudins are a family of tight junction proteins that provide size and charge selectivity to solutes traversing the paracellular space. Thick ascending limbs (TALs) express numerous claudins, including claudin-19. Nitric oxide (NO), via cGMP, reduces dilution potentials in perfused TALs, a measure of paracellular permeability, but the role of claudin-19 is unknown. We hypothesized that claudin-19 mediates the effects of NO/cGMP on the paracellular pathway in TALs via increases in plasma membrane expression of this protein. We measured the effect of the NO donor spermine NONOate (SPM) on dilution potentials with and without blocking antibodies and plasma membrane expression of claudin-19. During the control period, the dilution potential was −18.2 ± 1.8 mV. After treatment with 200 μmol/l SPM, it was −14.7 ± 2.0 mV ( P < 0.04). In the presence of claudin-19 antibody, the dilution potential was −12.7 ± 2.1 mV. After SPM, it was −12.9 ± 2.4 mV, not significantly different. Claudin-19 antibody alone had no effect on dilution potentials. In the presence of Tamm-Horsfall protein antibody, SPM reduced the dilution potential from −9.7 ± 1.0 to −6.3 ± 1.1 mV ( P < 0.006). Dibutyryl-cGMP (500 µmol/l) reduced the dilution potential from −19.6 ± 2.6 to −17.2 ± 2.3 mV ( P < 0.002). Dibutyryl-cGMP increased expression of claudin-19 in the plasma membrane from 29.9 ± 3.8% to 65.9 ± 10.1% of total ( P < 0.011) but did not change total expression. We conclude that claudin-19 mediates the effects of the NO/cGMP signaling cascade on the paracellular pathway.

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Counterregulation of clathrin-mediated endocytosis by the actin and microtubular cytoskeleton in human neutrophils

AJP Cell Physiology ◽

10.1152/ajpcell.00454.2008 ◽

2009 ◽

Vol 296 (4) ◽

pp. C857-C867 ◽

Cited By ~ 20

Author(s):

Silvia M. Uriarte ◽

Neelakshi R. Jog ◽

Gregory C. Luerman ◽

Samrath Bhimani ◽

Richard A. Ward ◽

...

Keyword(s):

Plasma Membrane ◽

Actin Cytoskeleton ◽

Human Neutrophils ◽

Functional Responses ◽

Membrane Expression ◽

Granule Exocytosis ◽

Plasma Membrane Expression ◽

Microtubular Cytoskeleton ◽

Latrunculin A ◽

Azurophil Granule

We have recently reported that disruption of the actin cytoskeleton enhanced N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated granule exocytosis in human neutrophils but decreased plasma membrane expression of complement receptor 1 (CR1), a marker of secretory vesicles. The present study was initiated to determine if reduced CR1 expression was due to fMLP-stimulated endocytosis, to determine the mechanism of this endocytosis, and to examine its impact on neutrophil functional responses. Stimulation of neutrophils with fMLP or ionomycin in the presence of latrunculin A resulted in the uptake of Alexa fluor 488-labeled albumin and transferrin and reduced plasma membrane expression of CR1. These effects were prevented by preincubation of the cells with sucrose, chlorpromazine, or monodansylcadaverine (MDC), inhibitors of clathrin-mediated endocytosis. Sucrose, chlorpromazine, and MDC also significantly inhibited fMLP- and ionomycin-stimulated specific and azurophil granule exocytosis. Disruption of microtubules with nocodazole inhibited endocytosis and azurophil granule exocytosis stimulated by fMLP in the presence of latrunculin A. Pharmacological inhibition of phosphatidylinositol 3-kinase, ERK1/2, and PKC significantly reduced fMLP-stimulated transferrin uptake in the presence of latrunculin A. Blockade of clathrin-mediated endocytosis had no significant effect on fMLP-stimulated phosphorylation of ERK1/2 in neutrophils pretreated with latrunculin A. From these data, we conclude that the actin cytoskeleton functions to limit microtubule-dependent, clathrin-mediated endocytosis in stimulated human neutrophils. The limitation of clathrin-mediated endocytosis by actin regulates the extent of both specific and azurophilic granule exocytosis.

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Optimization and Application of a Biotinylation Method for Quantification of Plasma Membrane Expression of Transporters in Cells

The AAPS Journal ◽

10.1208/s12248-017-0121-5 ◽

2017 ◽

Vol 19 (5) ◽

pp. 1377-1386 ◽

Cited By ~ 13

Author(s):

Vineet Kumar ◽

Tot Bui Nguyen ◽

Beáta Tóth ◽

Viktoria Juhasz ◽

Jashvant D. Unadkat

Keyword(s):

Plasma Membrane ◽

Membrane Expression ◽

Plasma Membrane Expression ◽

In Cells

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Low Plasma Membrane Expression of the Miltefosine Transport Complex Renders Leishmania braziliensis Refractory to the Drug

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01694-08 ◽

2009 ◽

Vol 53 (4) ◽

pp. 1305-1313 ◽

Cited By ~ 54

Author(s):

María P. Sánchez-Cañete ◽

Luís Carvalho ◽

F. Javier Pérez-Victoria ◽

Francisco Gamarro ◽

Santiago Castanys

Keyword(s):

Plasma Membrane ◽

Cutaneous Leishmaniasis ◽

Oral Drug ◽

Leishmania Braziliensis ◽

Β Subunit ◽

Membrane Expression ◽

Plasma Membrane Expression ◽

Highly Sensitive ◽

P Type ◽

Transport Complex

ABSTRACT Miltefosine (hexadecylphosphocholine, MLF) is the first oral drug with recognized efficacy against both visceral and cutaneous leishmaniasis. However, some clinical studies have suggested that MLF shows significantly less efficiency against the cutaneous leishmaniasis caused by Leishmania braziliensis. In this work, we have determined the cellular and molecular basis for the natural MLF resistance observed in L. braziliensis. Four independent L. braziliensis clinical isolates showed a marked decrease in MLF sensitivity that was due to their inability to internalize the drug. MLF internalization in the highly sensitive L. donovani species requires at least two proteins in the plasma membrane, LdMT, a P-type ATPase involved in phospholipid translocation, and its β subunit, LdRos3. Strikingly, L. braziliensis parasites showed highly reduced levels of this MLF translocation machinery at the plasma membrane, mainly because of the low expression levels of the β subunit, LbRos3. Overexpression of LbRos3 induces increased MLF sensitivity not only in L. braziliensis promastigotes but also in intracellular amastigotes. These results further highlight the importance of the MLF translocation machinery in determining MLF potency and point toward the development of protocols to routinely monitor MLF susceptibility in geographic areas where L. braziliensis might be prevalent.

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