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Department of Medical Chemistry and Biochemistry, The Medical Faculty, University of Milan, L.I.T.A., 20090 Segrate, Milan, Italy
3To whom correspondence should be addressed. E-mail: guido.tettamanti{at}unimi.it.
We investigated the direct effects of casein phosphopeptides (CPP),
which are formed by the proteolytic degradation of
- and
ß-caseins, on calcium uptake by human HT-29 intestinal tumor cells,
which undergo an enterocytically oriented differentiation in culture. A
commercial preparation containing a mixture of purified CPP and an
individual CPP of 25 amino acids, both containing the characteristic
Ca2+ binding motif, ser(P)-ser(P)-ser(P)-glu-glu, were
employed. The study was performed at the single-cell level and on a
cell population and measured the changes in cytosolic calcium
concentration before and after CPP addition. In the presence of 2
mmol/L extracellular calcium, both CPP preparations induced a transient
rise of free intracellular calcium ions, which did not influence
ATP-induced release of calcium from intracellular stores, and which
disappeared completely in the absence of extracellular calcium.
Pretreatment of these cells with thapsigargin, which completely empties
the intracellular calcium stores, did not abolish the cell responses to
CPP. Repetitive stimulation of HT-29 cells with CPP always elicited a
transient calcium rise, suggesting a lack of desensitization. The
CPP-stimulated cytosolic calcium rise was dependent on CPP dose, in
a seemingly nonsaturating mode, and on cell numbers. All of this is
consistent with the hypothesis that CPP do not influence
membrane-bound receptors or ion channels, but may act as calcium
ionophores or calcium carriers across the membrane. The reported
findings provide a new basis on which to assess the possibility that
CPP enhance calcium absorption and bioavailability in animals.
KEY WORDS: casein phosphopeptides calcium HT-29 cells Fura-2.
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