Effect of HEPES Buffer on the Uptake and Transport of P-Glycoprotein Substrates and Large Neutral Amino Acids

Luo, Shuanghui; Pal, Dhananjay; Shah, Sujay; Kwatra, Deep; Paturi, K.; Mitra, Ashim K.

doi:10.1021/mp900193e

Cited by 30 publications

(35 citation statements)

References 44 publications

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“…Luo et al showed that ATP production increased with 70% in MDCK-MDR-1 cells grown in culture medium with 20 mM HEPES for 6-7 days compared to the same cell line in medium without HEPES. This caused an increase in MDR-1 efflux activity, which could be a relevant effect in our model (26). Care should be taken when interpreting data from artificially buffered cell cultures, as the buffer compounds may exert an influence, independent of the buffering effect.…”

Section: The Main Effect Of the Additionally Buffered Media Was Most mentioning

confidence: 98%

“…HEPES, MOPS, and TES have all been found to exert a wide range of different adverse effects in different cell culture systems (24)(25)(26)(27). Luo et al showed that ATP production increased with 70% in MDCK-MDR-1 cells grown in culture medium with 20 mM HEPES for 6-7 days compared to the same cell line in medium without HEPES.…”

Section: The Main Effect Of the Additionally Buffered Media Was Most mentioning

confidence: 99%

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Paracellular Tightness and Claudin-5 Expression is Increased in the BCEC/Astrocyte Blood–Brain Barrier Model by Increasing Media Buffer Capacity During Growth

Helms

Waagepetersen

Nielsen

et al. 2010

AAPS J

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Abstract. Most attempts to develop in vitro models of the blood-brain barrier (BBB) have resulted in models with low transendothelial electrical resistances (TEER), as compared to the native endothelium. The aim of the present study was to investigate the impact of culture pH and buffer concentration on paracellular tightness of an established in vitro model of the BBB consisting of bovine brain capillary endothelial cells (BCEC) co-cultured with rat astrocytes. BCEC and rat astrocytes were isolated and cocultured using astrocyte-conditioned media with cAMP increasing agonists and dexamethasone. The coculture had average TEER values from 261±26 Ωcm 2 to 760±46 Ωcm 2 dependent on BCEC isolation batches. Furthermore, mRNA of occludin, claudin-1, claudin-5, JAM-1, and ZO-1 were detected. Increased buffer concentration by addition of HEPES, MOPS, or TES to the media during differentiation increased the TEER up to 1,638±256 Ωcm 2 independent of the type of buffer. This correlated with increased expression of claudin-5, while expression of the other tight junction proteins remained unchanged. Thus, we show for the first time that increased buffer capacity of the medium during differentiation significantly increases tightness of the BCEC/astrocyte in vitro BBB model. This regulation may be mediated by increased claudin-5 expression. The observations have practical implications for generating tighter BBB cell culture models, and may also have physiological implications, if similar sensitivity to pH-changes can be demonstrated in vivo.

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Section: The Main Effect Of the Additionally Buffered Media Was Most mentioning

confidence: 98%

Section: The Main Effect Of the Additionally Buffered Media Was Most mentioning

confidence: 99%

Paracellular Tightness and Claudin-5 Expression is Increased in the BCEC/Astrocyte Blood–Brain Barrier Model by Increasing Media Buffer Capacity During Growth

Helms

Waagepetersen

Nielsen

et al. 2010

AAPS J

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“…Digoxin is not a substrate for Bcrp [58], but other studies have indicated that the poor BBB permeability of digoxin is not explained by P-gp alone [11,38]. Verapamil has been used as a P-gp inhibitor in concentrations from 5 to 150 μM [23,[59][60][61], but it has also been shown to inhibit Mrp activity in concentrations of 5-10 μM [45,62] as well as Bcrp activity, although not in the concentration range applied here [63,64]. In our study, 50 μM verapamil lowered the efflux ratio of digoxin to 1.1, whereas the Bcrp inhibitor, Ko 143, had no effect on the digoxin transport indicating that the verapamil inhibition of A-L digoxin transport we observed was due to inhibition of P-gp or Mrp's.…”

Section: Efflux Transporter Activity In Bovine Blood-brain Barrier Momentioning

confidence: 99%

An Electrically Tight In Vitro Blood–Brain Barrier Model Displays Net Brain-to-Blood Efflux of Substrates for the ABC Transporters, P-gp, Bcrp and Mrp-1

Helms

Hersom

Kuhlmann

et al. 2014

AAPS J

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Abstract. Efflux transporters of the ATP-binding cassette superfamily including breast cancer resistance protein (Bcrp/Abcg2), P-glycoprotein (P-gp/Abcb1) and multidrug resistance-associated proteins (Mrp's/ Abcc's) are expressed in the blood-brain barrier (BBB). The aim of this study was to investigate if a bovine endothelial/rat astrocyte in vitro BBB co-culture model displayed polarized transport of known efflux transporter substrates. The co-culture model displayed low mannitol permeabilities of 0.95±0.1·10 H-etoposide revealed polarized transport favouring the brain-to-blood direction for all substrates. Steady state efflux ratios of 2.5±0.2 for digoxin, 4.4±0.5 for estrone-3-sulphate and 2.4±0.1 for etoposide were observed. These were reduced to 1.1±0.08, 1.4±0.2 and 1.5±0.1, by addition of verapamil (digoxin), Ko143 (estrone-3-sulphate) or zosuquidar+reversan (etoposide), respectively. Brain-toblood permeability of all substrates was investigated in the presence of the efflux transporter inhibitors verapamil, Ko143, zosuquidar, reversan and MK 571 alone or in combinations. Digoxin was mainly transported via P-gp, estrone-3-sulphate via Bcrp and Mrp's and etoposide via P-gp and Mrp's. The expression of P-gp, Bcrp and Mrp-1 was confirmed using immunocytochemistry. The findings indicate that P-gp, Bcrp and at least one isoform of Mrp are functionally expressed in our bovine/rat co-culture model and that the model is suitable for investigations of small molecule transport.KEY WORDS: blood-brain barrier; breast cancer resistance protein; multidrug resistance-associated protein; p-glycoprotein; polarized small molecule transport.

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“…[32] ] of the buffer. The HEPES buffer system is extensively used for physicochemical and biological investigations; [33] therefore, all of the experiments were run in this buffer. Figure 1 shows the adsorption kinetics of DiC 8 -PC dissolved in the HEPES buffer at the interfaces with air, and with air saturated with PFH, for three selected concentrations of DiC 8 -PC.…”

Section: Adsorption Of Dic8-pc In the Presence Or Absence Of Perfluormentioning

confidence: 99%

A Nonpolar, Nonamphiphilic Molecule Can Accelerate Adsorption of Phospholipids and Lower Their Surface Tension at the Air/Water Interface

et al. 2011

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The adsorption dynamics of a series of phospholipids (PLs) at the interface between an aqueous solution or dispersion of the PL and a gas phase containing the nonpolar, nonamphiphilic linear perfluorocarbon perfluorohexane (PFH) was studied by bubble profile analysis tensiometry. The PLs investigated were dioctanoylphosphatidylcholine (DiC(8)-PC), dilaurylphosphatidylcholine, dimyristoylphosphatidylcholine, and dipalmitoylphosphatidylcholine. The gas phase consisted of air or air saturated with PFH. The perfluorocarbon gas was found to have an unexpected, strong effect on both the adsorption rate and the equilibrium interfacial tension (γ(eq)) of the PLs. First, for all of the PLs, and at all concentrations investigated, the γ(eq) values were significantly lower (by up to 10 mN m(-1)) when PFH was present in the gas phase. The efficacy of PFH in decreasing γ(eq) depends on the ability of PLs to form micelles or vesicles in water. For vesicles, it also depends on the gel or fluid state of the membranes. Second, the adsorption rates of all the PLs at the interface (as assessed by the time required for the initial interfacial tension to be reduced by 30%) are significantly accelerated (by up to fivefold) by the presence of PFH for the lower PL concentrations. Both the surface-tension reducing effect and the adsorption rate increasing effect establish that PFH has a strong interaction with the PL monolayer and acts as a cosurfactant at the interface, despite the absence of any amphiphilic character. Fitting the adsorption profiles of DiC(8)-PC at the PFH-saturated air/aqueous solution interface with the modified Frumkin model indicated that the PFH molecule lay horizontally at the interface.

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Effect of HEPES Buffer on the Uptake and Transport of P-Glycoprotein Substrates and Large Neutral Amino Acids

Cited by 30 publications

References 44 publications

Paracellular Tightness and Claudin-5 Expression is Increased in the BCEC/Astrocyte Blood–Brain Barrier Model by Increasing Media Buffer Capacity During Growth

Paracellular Tightness and Claudin-5 Expression is Increased in the BCEC/Astrocyte Blood–Brain Barrier Model by Increasing Media Buffer Capacity During Growth

An Electrically Tight In Vitro Blood–Brain Barrier Model Displays Net Brain-to-Blood Efflux of Substrates for the ABC Transporters, P-gp, Bcrp and Mrp-1

A Nonpolar, Nonamphiphilic Molecule Can Accelerate Adsorption of Phospholipids and Lower Their Surface Tension at the Air/Water Interface

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