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Dear all,
I'm working with caco-2 cells and I want to study if some derivatives
of beta-lactams antibiotics are substrate of hPEPT1 transporter. This
transporter is well-known to be expressed in caco-2 cells and I also
verify it by RT-PCR. I also performed the uptake and transport of
gly-sar, a dipeptide recognized by PEPT1. I try to inhibit the uptake
and transport of gly-sar by an other dipeptide (gly-leu) or by
gly-sar itself. I also modified the extracellular pH (pH 7.4 in place
of pH 6.0) because the transporter's driving force is provided by an
inwardly directed proton gradient.
My experimental condition are : Caco-2 cells came from ATCC. They are
cultured for 21 days on Falcon insert and the medium is change every
48-72h and 24h before experiment The day of experiment, the medium is
removed and cells are washed 2 times with modified krebs buffer and
cells are preincubated 30 min with 1ml of buffer pH6.0 at apical side
et 2ml of buffer pH 7.4 at basolateral side. I begin the experiment
by adding 1 ml of buffer pH 6.0 containing 0.4 mM of a mixture of
radiolabelled and non radiolabbelled gly-sar in pH 6.0 (control) or
1ml of 0.4 mM of mixture gly-sar in pH6.0 + 20 mM of Gly-leu or Non
radiolabelled Glysar (inhibitor). After 15 minutes, I wash the cells
2 times with ice-cold PBS and the cells are scrapped and sonicated.
For the experiment of pH, the apical medium consist of 1 ml of buffer
pH 7.4 (30 min preincubation) and I begin by adding 1 ml of 0.4 mM
glysar in pH 7.4.
With this expperimental condition, I don't have the effect of the 2
inhibitors and of the pH 7.4 whereas the PEPT1 is expressed.
I would greatly appreciate if someone could help me to understandi
these problem.
Thank you very much
Hugues Chanteux
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Dear Mr. Chanteux,
You are modifying conditions for conducting transport studies of peptides
for unclear reasons. Many papers have been published in this area and there
are a few things to be careful of:
1. Cell monolayers should not be washed and incubated with pH6 buffer
because it can slightly change the intracellular pH.
2. Cell monolayers should not be exposed to antibiotics such as penicillin
for at least the last 2-3 feeding period. We obtained better results when
they are not exposed to antibiotics at all for 21 days. However, it is
quite demanding of one's cell culture technique.
3. You probably can benefit from time course study of gly-sar uptake to
determine the best time for your uptake study too.
4. Peptide transporter is underexpressed in Caco-2 cells, hence, you may see
uptake slower than you expected.
Hope this help.
Ming Hu, Ph.D.
Washington State University
College of Pharmacy
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Dear Mr. Chanteux,
I have noticed that the effects of pH gradient were more pronounced on the
the transport across the cells rather than the accumulation in the cells. A
pH gradient of pH 6 to pH 7.4 resulted in almost a factor 2 increase in
permeability whereas the effects on the accumulation were only a factor 1.2
to 1.4 (in Environmental Toxicology and Pharmacology 2002).Furthermore, I
have noticed that inhibition by other peptides and beta-lactams was time
dependent; the effects were less at short incubation time points. May be
inbuation for a longer period will show the pH dependcy and inhibition on
the transport and accumulation in the cells.
greetings
carolien versantvoort
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