Mechanism of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP+) by phospholipid vesicles

Sedgwick, Edward G.; Bragg, Philip D.

doi:10.1016/0005-2736(93)90345-z

Cited by 7 publications

(4 citation statements)

References 16 publications

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“…fluorescence intensity in the presence of cells is attributed to movement of the dye molecules from the head group region of the cytoplasmic membrane bilayer into the region of the fatty acyl chains (24). An instantaneous rise in fluorescence emission at 567 nm upon addition of the probe to the cell suspension was followed by a slower phase in the ⌬OprM, ⌬MexA, or ⌬MexB ( Fig.…”

Section: Dmp Fluorescence Response In the Wild Type Strain And Mexa-mentioning

confidence: 99%

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Use of Fluorescence Probes to Monitor Function of the Subunit Proteins of the MexA-MexB-OprM Drug Extrusion Machinery inPseudomonas aeruginosa

Ocaktan¹,

Yoneyama

Nakae

1997

Journal of Biological Chemistry

119

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The MexA-MexB-OprM efflux pump of Pseudomonas aeruginosa consists of two inner membrane proteins, MexA and MexB, and one outer membrane protein, OprM. We investigated the role of the components of this drug extrusion system by evaluating the repercussions of deleting these subunit components on the accumulation of several fluorescent probes. Fluorescence intensities of positively charged 2-(4-dimethylaminostyryl)-1-ethylpyridinium and uncharged N-phenyl-1-naphtylamine were 7 and 4 times higher, respectively, in the mutant lacking OprM and 4 and 1.7 times higher, respectively, in the mutants lacking MexA or MexB than in the wild type strain. This order of fluorescence intensity was fully consistent with a previously reported minimum inhibitory concentration of antibiotics such as tetracycline, chloramphenicol, and fluoroquinolones. Ethidium bromide accumulation in all the Mex mutants proceeded at about 5 times faster than the rate in the wild type cells. This result is in accord with the minimum inhibitory concentration of ␤-lactam antibiotics. These results suggest that the fluorescence probes could be successfully used in real time monitoring of the function of the drug extrusion machinery in Gram-negative bacteria. The downhill extrusion kinetics of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, which orients perpendicular to the inner leaflet of the cytoplasmic membrane, from preloaded cells lacking the extrusion pump was preceded by a slow increase in fluorescence intensity, whereas the wild type cell immediately released the dye. This observation was explained by a slow trans-cytoplasmic membrane crossing of intracellular dye in the mutants. These results reflected higher accumulation of the probe in the cytoplasmic membrane in the mutants and strengthened the hypothesis that extrusion of hydrophobic substrate mediated by MexA-MexB-OprM mainly takes place from the interior of the cytoplasmic membrane.Multiple antibiotic resistance in bacteria has been associated with the overexpression of endogenous efflux genes (for reviews see Refs. 1 and 2). In Pseudomonas aeruginosa, active extrusion (3, 4) combined with a tight diffusion barrier at the outer membrane (5, 6) are the main reasons for low specific resistance against diverse antibiotics and other toxic elements (7).Multidrug resistance in P. aeruginosa is now attributed mainly to the overexpression of three sets of operons. Overexpression of the operon mexA-mexB-oprM is characteristic for nalB type multidrug-resistant mutants, (8, 9), whereas overexpression of the operon mexC-mexD-oprJ (10) is responsible for multidrug resistance in nfxB type mutants (11). A third type designated as nfxC has also been associated with a diminished intracellular drug accumulation (12). The genes coding for the proteins involved in the drug extrusion have been characterized recently (13) and named mexE-mexF-oprN.A common characteristic of multiantibiotic-resistant mutants in P. aeruginosa is their broad resistance to quinolones, and hence one method generally used t...

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Section: Dmp Fluorescence Response In the Wild Type Strain And Mexa-mentioning

confidence: 99%

“…Cell suspension at a higher cell density leads to rapid oxygen depletion in the uptake medium, resulting in respiration-related artifacts, especially during NPN and DMP accumulation experiments (23,24). Experimental measurements were generally performed within 2 h after cell preparation.…”

Section: Methodsmentioning

confidence: 99%

Use of Fluorescence Probes to Monitor Function of the Subunit Proteins of the MexA-MexB-OprM Drug Extrusion Machinery inPseudomonas aeruginosa

Ocaktan¹,

Yoneyama

Nakae

1997

Journal of Biological Chemistry

119

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“…Indeed, the negative surface charge of lipid bilayers has been shown to support exponential binding of cationic dyes. For example, increasing the negative charge of artificial liposomes markedly increases the surface binding of the cationic styryl dye, 2-(4-dimethylaminostyryl)-1-ethylpyridinium [19], by artificial liposomes. Consequently, we modeled the time course (from t=3-70 s) of the fluorescence increase of tubules following exposure to NBD-TMA assuming that the increase involves three components (subsequently abbreviated cmp): cmp I, step-like chamber exchange; cmp II, rapid exponential binding; and cmp III, linear uptake of NBD-TMA.…”

Section: Nbd-tma Transportmentioning

confidence: 99%

“…However, under control conditions, 4-Di-ASP + undergoes net renal reabsorption, rather than net secretion [14], suggesting that its predominant interaction may not be with the renal transport process directly associated with the net secretion of organic cations such as cimetidine and procainamide [15]. In addition, the amphipathic structure of styryl dyes can lead to partitioning into lipid bilayers resulting in a substantial change in the optical properties of the fluorophore [19], and thus introducing another potential problem to the interpretation of uptake data.…”

Section: Introductionmentioning

confidence: 95%

NBD-TMA: a novel fluorescent substrate of the peritubular organic cation transporter of renal proximal tubules

Bednarczyk

Mash

Aavula

et al. 2000

Pflugers Arch - Eur J Physiol

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Traditionally, the measurement of transport activity has employed radiolabeled compounds. The resulting experimental procedures do not measure transport in real time and are limited in temporal and spatial resolution. The use of epifluorescence microscopy provides the ability to measure transport activity in real time with high temporal and spatial resolution. Using epifluorescence microscopy we characterized the transport of the fluorescent organic cation, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD-TMA+, MW 266). NBD-TMA+ has structural characteristics common to other secreted organic cations and is fluorescent (lambda(ex)=458 nm; lambda(em)=530 nm). The excitation and emission spectra are insensitive to changes in [Cl-] and minimally sensitive to pH in the physiologically relevant range (pH 5.0-7.4). A microscope equipped with a photon-detection system was used to measure accumulation of NBD-TMA+ by isolated rabbit renal proximal tubules. Accumulation of NBD-TMA+ by proximal tubules was time dependent and saturable (Michaelis-Menten constant Km 12 microM). Proximal tubule accumulation of NBD-TMA+ was inhibited by the organic cations tetraethylammonium (TEA+) (apparent inhibitory constant K(app)TEA 134 microM), cimetidine, and N1-methylnicotinamide (NMN). Our experimental results provide strong evidence that NBD-TMA+ is transported by one or more of the basolateral organic cation transporters involved in the renal secretion of this chemical class of compound. This fluorescent substrate provides a sensitive means of investigating organic cation transport.

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D

2010

Handbook of Biological Dyes and Stains

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Mechanism of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP+) by phospholipid vesicles

Cited by 7 publications

References 16 publications

Use of Fluorescence Probes to Monitor Function of the Subunit Proteins of the MexA-MexB-OprM Drug Extrusion Machinery inPseudomonas aeruginosa

Use of Fluorescence Probes to Monitor Function of the Subunit Proteins of the MexA-MexB-OprM Drug Extrusion Machinery inPseudomonas aeruginosa

NBD-TMA: a novel fluorescent substrate of the peritubular organic cation transporter of renal proximal tubules

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