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Articles

Casein Phosphopeptides Influence Calcium Uptake by Cultured Human Intestinal HT-29 Tumor Cells^{1 ,2}

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.

	ABSTRACT

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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.

	INTRODUCTION

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Milk and dairy products are excellent sources of bioavailable calcium because they contain high amounts of calcium and substances that may enhance the fraction of ingested calcium and that can be used for normal physiologic functions and storage (1 2 3) . This beneficial effect is attributed to the association of calcium with the casein fraction of milk, particularly with some phosphorylated serine residues contained therein (4 5 6 7) . The casein fragments carrying these residues, called casein phoshopeptides (CPP),⁴ are peptides of different length; they are characterized by the presence of the sequence "ser(P)-ser(P)-ser(P)-glu-glu," serving as the binding site for di- or trivalent minerals and making CPP more resistant to further proteolysis (8 9 10 11) . These phosphopeptides are released into the gastrointestinal tract during normal digestion of _s1-, _s2- and ß-casein by the action of digestive proteolytic enzymes (12 ,13) . Compounds very similar to those isolated from in vivo digests can be obtained in purified form from the tryptic or chymotryptic digestion of caseins in vitro, and have been widely employed for experimental purposes (3 ,5 ,14 ,15) .

In vitro studies demonstrated that CPP can prevent the precipitation of calcium ions as insoluble salts such as calcium phosphate (16 ,17) . This suggested the possibility that CPP enhance the amount of soluble calcium in the intestinal lumen, thereby increasing the mineral availability for absorption in the small intestine (3 4 5 6 7) . Experiments performed on intestinal preparations (everted sacs, loops) provided evidence supporting this possibility (18 ,19) . However, in vivo investigations performed on whole animals designed to ascertain a role of CPP in both absorption and bioavailability of calcium, generated some controversial results. In fact, studies on growing pigs, as well as on weaning and adult (female) rats, showed that diets supplemented with CPP influenced neither calcium absorption nor bone mineralization (20 21 22) . On the contrary, rats fed a CPP-supplemented soybean protein diet had significantly greater calcium absorption than controls fed soybean alone (23) . Moreover, the bioavailability of calcium appeared to be increased by CPP-enriched infant formula in rat pups (24) , and the presence of CPP in the diet prevented mineral density decline in old ovariectomized female rats (25) . Finally, CPP were shown to enhance calcium absorption in both rachitic and normal chicks (26) . Interestingly, CPP also induced Ca²⁺ uptake by boar spermatozoa, facilitating sperm penetration into pig oocytes; the effect was reduced by dephosphorylation of CPP (27) .

In all of these investigations, CPP were generally viewed as agents capable of maintaining intestinal calcium in its "soluble" form, thus facilitating the mineral flux through the membranes. However, the presence or absence of substances in the diet such as phosphate or phytate, that are capable of forming insoluble calcium salts or complexes, was not accurately assessed. This may be the basis for the conflicting results in in vivo studies. No determination was made of the direct interactions of CPP with the plasma membrane (particularly that of intestinal cells), which might affect calcium flux through the same membrane, regardless of any calcium-solubilizing action. The present work was designed to explore the possibility of a direct CPP influence on calcium uptake, using as a study model the human intestinal tumor cell line, HT-29, which tends to undergo an enterocytically oriented differentiation in culture (28) . Calcium uptake was monitored as a rise in free cytosolic calcium concentration due to calcium ion movement through the plasma membrane (29 30 31) .

	MATERIALS AND METHODS

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Materials.

Fura-2 acetoxymethyl ester (Fura-2/AM), Fura-2 pentasodium salt, thapsigargin (Tg) and Ionomycin (used only for calibration purposes) were obtained from Calbiochem (La Jolla, CA); cell culture media, fetal calf serum, ATP, Pluronic F-127 and EGTA were from Sigma Chemical (St. Louis, MO). All other chemicals, supplied by Merck (Darmstadt, Germany), were of the highest available purity.

Cell cultures.

The colon carcinoma cell line HT-29 was obtained from the Istituto Zooprofilattico Sperimentale di Brescia (Brescia, Italy). Cells were routinely grown in 25 cm² plastic flasks (Costar, Concorezzo, Italy) in low D-glucose RPMI-1640 medium, supplemented with 100 mL/L fetal calf serum, 2 mmol/L L-glutamine, 0.1 mg/L streptomycin, 1 x 10⁵ U/L penicillin and 0.25 mg/L amphotericin-B (32 ,33) . Cultures, kept at 37°C in a 5% CO₂/95% air atmosphere, were periodically checked for the presence of mycoplasma and were free of contamination. The measured pH was 7.4. The culture medium was changed daily and the cells used in the experiments underwent from 155 to 165 passages. Under these culture conditions, cells are reported to reach a seemingly terminal degree of enterocytic differentiation, although morphofunctionally heterogeneous (features of absorptive- and mucous-like cells) (33) . Cell viability, assessed by the trypan blue exclusion test, and cell morphology, examined by optical microscope, remained unaffected by CPP treatment up to 40 mmol/L for 24 h.

Casein phosphopeptides (CPP).

Casein phosphopeptides from two different sources were employed, i.e., CPP DMV (DMV International, Veghel, The Netherlands) and a purified peptide, ß-CN(1–25)4P, kindly provided by Prof. Hans Meisel (University of Kiel, Kiel, Germany). CPP DMV is a mixture [5 main components, all of them containing the ser(P)-ser(P)-ser(P)-glu-glu motif] of casein phosphopeptides (CPP content: 93.8% as dry matter; 96% pure; total nitrogen content, 10.8%; phosphorous content, 3.7%; nitrogen/phosphorous ratio, 3.1; P/ser ratio, 0.85 mol/mol; average molecular weight 2500). ß-CN(1–25)4P (95.3% dry matter, > 98% homogeneous; nitrogen/phosphorous ratio, 3.6; P/ser ratio, 0.80 mol/mol) has the amino acid sequence RELEELNVPGEIVELEESITR (where is serine-phosphate), and a molecular weight of 3125 (34) . In the text, CPP refers to CPP DMV, unless otherwise specified.

The determination of calcium content in CPP was carried out using a specific o-cresolphthalein complexone calcium detection reagent (Sigma), or spectrofluorimetrically with Fura-2 (35) . After demineralization through ionic exchange chromatography, both CPP DMV and ß-CN(1–25)4P were assessed to be free of calcium.

For the cell calcium measurement experiments, CPP were dissolved in doubly distilled water in stock solutions (1000X concentrated, with respect to the final concentration) and stored at -20°C.

Measurement of [Ca²⁺]_i at a single-cell level.

Cells grown at confluency in plastic flasks were suspended by trypsin/EDTA (final concentration 0.5 g/L/0.2 g/L) treatment, washed several times with the culture medium (these washings also removed EDTA) and seeded onto a glass coverslip (24 mm diameter, thickness 0.13–0.17 mm) in petri dishes (35 mm diameter) at 2.6 x 10⁴ cells/cm². The experiments were done on d 4, 5 and 6 after seeding, when the cells were still in a subconfluent state. Cytoplasmic calcium was measured according to the procedure described by Tsien and Poenie (36) on the basis of the changes in the excitation spectrum of the fluorescent probe Fura-2 when complexed with calcium ions in the cytosol. Briefly, cells on glass coverslip were loaded with 5 µmol/L Fura-2/AM and 2.5 µmol/L Pluronic F-127 in Krebs-Ringer-HEPES solution (KRH) containing (mmol/L) NaCl 125.0, KCl 5.0, KH₂PO₄ 1.2, CaCl₂ 2.0, MgSO₄ 1.2, glucose 6.0 and HEPES 25.0, and adjusted to pH 7.4. After an incubation period of 30 min at 37°C, the cells were rinsed extensively with KRH and maintained for an additional 30 min at room temperature to allow deesterification of the fluorescent probe. Then, the coverslip was mounted in a thermostatted (TC-202 A, Medical System Corporation, Harvard Apparatus, Holliston, MA) perfusion chamber (PDMI-2, Medical System Corporation) and placed on the stage of a microscope (TE 200, Nikon, Tokyo, Japan). The cells, incubated in 2 mL of KRH, were alternately excited at 340–380 nm through a 40X oil immersion objective (numerical aperture = 1.3, Nikon, Tokyo, Japan). The emitted fluorescence at 510 nm was measured at 0.7- to 1-s intervals by a CCD intensified camera (Extended Isis, Photonic Science, Millham, UK), and ratio images of single cells, averaged over 4 frames, within a chosen window of at least 50 cells, were collected and analyzed after background subtraction, using a Fluorescence image acquisition and data analysis system, which was supplied by Applied Imaging (High Speed Dynamic Video Imaging Systems, Quanticell 700, Sunderland, UK). The amount of intracellular free calcium, [Ca²⁺]_i, within the cells was calculated from the 340/380 nm images by means of a calibration performed with external standards of calcium and Fura-2, according to the equation of Grynkiewicz et al. (37) . For experiments requiring calcium-free solutions, CaCl₂ was omitted from KRH and 1 mmol/L EGTA was added to complex any traces of contaminating Ca²⁺. CPP were added to reach the final desired concentration. Under the experimental conditions described, the duration of each experiment never exceeded 2.0 min, a period of time in which the cells appeared to maintain full viability. In this case, viability was assessed by a metabolic assay, based upon stimulation with ATP at the end of each experiment and recording the consequent [Ca²⁺]_i increase known to be evoked in cells by ATP-induced production of inositol (1,4,5,)-triphosphate (38) .

Measurement of [Ca²⁺]_i in cell population.

Cells grown as a monolayer in a 25 cm² flask were detached with trypsin-EDTA (see above); after several washings with KRH, 4 x 10⁶ cells were loaded for 30 min at 37°C with 5 µmol/L Fura-2/AM and 2.5 µmol/L Pluronic F-127 in KRH. The loaded cell suspension was rinsed extensively with KRH and then divided into different aliquots, each containing 0.5 x 10⁶ cells. Each aliquot was suspended by gentle swirling in 2 mL of KRH and transferred to a thermostatted cuvette in a Perkin-Elmer LS-50B spectrofluorimeter (Perkin-Elmer, Beaconsfield, UK). During the experiment, the cells were stirred continuously; at the end, calibration was performed according to Mc Cormack and Cobbold (39) . Fluorescence emission at 485 nm of Fura-2/AM loaded cells, excited at 343 nm, was followed. Because Fura-2 fluorescence increases with increasing [Ca²⁺]_i at these wavelength settings, the changes in fluorescence intensity reflected the changes in [Ca²⁺]_i concentration. Under these conditions, the duration of the experiments did not exceed 20 min, a period of time in which cells maintained full viability, assessed as specified for free calcium measurements at the single-cell level. For experiments without extracellular calcium, the cells were loaded as described, washed in KRH without CaCl₂ and, after transferring to a cuvette, EGTA was added to reach a final 3 mmol/L concentration. Cells were eventually allowed to equilibrate in the final medium before starting measurements.

Aliquots containing different amounts of cells (0.25, 0.50, 0.75 and 1.00 x 10⁶) were employed in some experiments and processed as specified above. Results in each case were expressed as [Ca²⁺]_i peak (percentage of basal) after conversion of fluorescence intensity units in calcium concentration by the use of a calibration performed "in situ" for each experiment.

Rationale of the experimental design.

Because of the action of calcium pumps, ubiquitously located on the plasma membrane of mammalian cells, the concentration of extracellular calcium ions is in the mmol/L range, and the cytosolic concentration, [Ca²⁺]_i, is 0.1 µmol/L under resting conditions (38) . Calcium ions are also trapped into intracellular stores (part of the endoplasmic reticulum) by the action of endoplasmic pumps (38) . Therefore a transient enhancement of [Ca²⁺]_i can be due to a calcium influx from the extracellular compartment or to a release by the intracellular stores or to both events. In the case of intestinal absorption after a meal, the concentration of calcium in the lumen reaches the mmol/L range (up to 3–4 mmol/L in rats, 7–8mmol/L calculated in humans) (40) . Calcium flows through the apical membrane of enterocytes by active and passive mechanisms, driven by the high extracellular/intracellular concentration gradient and sustained by the calcium pump (located in the basal membrane), which pushes calcium into the capillary compartment. Therefore, the calcium flux from the intestinal lumen to the interior of enterocytes (the first step of absorption) mimics the flux of calcium from the extracellular milieu to the cytosol of the HT-29 cells, under the culture conditions described. To establish whether CPP are able to influence calcium uptake by HT-29 cells from the medium, CPP were added to the medium in the presence or absence of CaCl₂ (2 mmol/L, i.e., the extracellular calcium concentration used in this type of experiment, 38 ), and in the presence or absence of ATP (100 µmol/L), which promotes calcium efflux from intracellular stores (41) . Finally, the possible action of CPP on the pump mechanisms controlling calcium storage into intracellular vesicles was inspected by the addition of Tg (1 µmol/L), which blocks the endoplasmic calcium-ATPase family of calcium pumps (42) , leading to complete and irreversible depletion of the intracellular calcium stores.

Statistical analysis.

Results are reported as means ± SD. Student’s t test was used to determine significant differences between two mean values. (An independent two-population t test was performed with Origin 4.1.) Differences with a P-value < 0.05 were considered significant.

	RESULTS

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The possible influence of CPP on calcium uptake by cultured HT-29 tumor cells was assessed by directly measuring the intracellular free calcium concentration, [Ca²⁺]_i, after exposure to different CPP preparations, i.e., CPP DMV, a mixture of CPP resembling the CPP formed in the intestinal tract under physiologic conditions, and CPP ß-CN(1–25)4P, a single CPP of 25 amino acids isolated in pure form.

The [Ca²⁺]_i changes recorded in individual HT-29 cells stimulated with the two preparations of CPP at 8 µmol/L are reported in Figure 1 . During these experiments, the extracellular calcium concentration, [Ca²⁺]_o, was kept at 2 mmol/L and the cells, from a minimum of 30 to a maximum of 70, were followed concomitantly before and after the addition of CPP. As shown, the addition of both preparations of CPP was followed by a prompt and transient rise of [Ca²⁺]_i, with the response occurring within 30–40 s. This response was regularly shared and exhibited by at least 90% of cells. However, it was not fully uniform in shape, amplitude or rapidity, probably reflecting some expected heterogeneity of the HT-29 cell line, in terms of different modes of differentiation (28) . Moreover, both CPP caused a monophasic rise in [Ca²⁺]_i followed by a rapid return to the basal level, with only a very few cells exhibiting a biphasic response. Cell exposure to KRH, before CPP addition, had no influence on [Ca²⁺]_i, (see Fig. 1 ), indicating that the effect of CPP was not a cell response to a merely mechanical stimulation. The first signs of [Ca²⁺]_i increase were detected at 1 µmol/L CPP. At the end of each experiment, the pH of the medium was measured; no appreciable changes were evident.

View larger version (30K): [in this window] [in a new window]   Figure 1. Effect of casein phosphopeptides (CPP) on intracellular calcium in HT-29 cells. Panel A: effect of the administration of 8 µmol/L mixture of casein phosphopeptides from a commercial source (CPP DMV, solid arrow) in single cells incubated at 37°C in Krebs-Ringer-HEPES (KRH) containing 2 mmol/L extracellular calcium. Panel B: effect of 8 µmol/L purified phosphopeptide ß-CN(1–25)4P (solid arrow), in single cells incubated as in panel A. Dotted arrows represent cell stimulation with KRH. Each line in both panels refers to the behavior of a single cell from a chosen field of 50 cells. The graphs are representative of at least three experiments.

View larger version (25K): [in this window] [in a new window]   Figure 2. Effect of casein phosphopeptides (CPP) and ATP administration on intracellular calcium in HT-29 cells. Panels A and C: HT-29 cells incubated in Krebs-Ringer-HEPES (KRH) without extracellular calcium and containing 1 mmol/L EGTA were stimulated (dotted arrow) with 8 µmol/L casein phosphopeptides from a commercial source [CPP DMV (A)], or purified phosphopeptide ß-CN(1–25)4P (C) and subsequently (solid arrow) with 100 µmol/L ATP. Panels B and D: HT-29 cells incubated in KRH containing 2 mmol/L extracellular calcium were stimulated (dotted arrow) with 8 µmol/L mixture of casein phosphopeptides from a commercial source [CPP DMV (B)] or purified phosphopeptide ß-CN(1–25)4P (D) and subsequently with 100 µmol/L ATP. In all panels, each trace corresponds to the behavior of a single cell within a chosen window of 50 cells. The traces are representative of three experiments.

View larger version (19K): [in this window] [in a new window]   Figure 3. Influence of extracellular calcium on the casein phosphopeptide (CPP)-induced rise in intracellular calcium in HT-29 cells: cell population study. The intracellular Fura-2 fluorescence from a loaded cell population of 0.5 x 106 cells was recorded. Vertical scale indicates fluorescent intensity at 485 nm emission wavelength after excitation at 343 nm. Panel A: Fura-2 fluorescence variations were recorded from control cells (dotted line) incubated in Krebs-Ringer-HEPES (KRH) without extracellular calcium; where indicated (arrows), 3mmol/L EGTA and 5 mmol/L CaCl2 were added. Solid line refers to cells incubated in the same experimental conditions as control cells but, where indicated (arrows), also stimulated with 320 µmol/L mixture of casein phosphopeptides from a commercial source (CPP DMV). Panel B: cells were incubated as reported in panel A, stimulated with 320 µmol/L CPP DMV, 100 µmol/L ATP and finally treated with 1 µmol/L thapsigargin (Tg). Panel C: cells incubated in KRH containing 2 mmol/L Ca2+ were stimulated with 1 µmol/L Tg, 320 µmol/L CPP DMV and 100 µmol/L ATP; Fura-2 fluorescence was recorded continuously. Each panel is representative of 3–5 separate experiments. The effects of EGTA on Fura-2 fluorescence (see inset Panel A), caused primarily by a small fraction of free dye in the incubation medium and determined in unstimulated cells, were subtracted graphically from the traces in panels A and B (//). Cell autofluorescence, accounting for 3% of the emitted signal, was not subtracted. Preliminary experiments showed that casein phosphopeptides were unable, per se, to affect fluorescence under the established experimental conditions. Similar results were obtained with the purified phosphopeptide ß-CN(1–25)4P.

View larger version (10K): [in this window] [in a new window]   Figure 4. Concentration dependence of casein phosphopeptide (CPP)-induced intracellular calcium ([Ca2+]i) changes in HT-29 cells. Changes in intracellular calcium were analyzed using the cell population model in which the fluorescence signal recorded at 343 nm excitation and 485 emission wavelengths was averaged from 0.5 x 106 Fura-2/acetoxymethyl ester–loaded cells incubated in Krebs-Ringer-HEPES (KRH) containing 2 mmol/L Ca2+. At the end of each experiment, a calibration was performed to convert fluorescence intensity in [Ca2+]i nmol/L (see Materials and Methods). Data are expressed as [Ca2+]i peak (percentage of basal), where the increase was calculated as the difference between the [Ca2+]i measured at the top of the peak reached after CPP addition and the basal [Ca2+]i. Values are means ± SD, n = 3. *Significantly different from 160 µmol/L casein phosphopeptide dose-response value (P < 0.05). The casein phosphopeptide preparation was CPP DMV.

View larger version (14K): [in this window] [in a new window]   Figure 5. Relationship between the casein phosphopeptide (CPP)-induced intracellular calcium increase and the number of HT-29 cells. Changes in intracellular calcium concentration were investigated in dependence of different cell numbers after administration of 320 µmol/L mixture of casein phosphopeptides from a commercial source (CPP DMV). Values are means ± SD, n = 3. *Significantly different from 0.25 x 106 cells (P < 0.01).

View larger version (23K): [in this window] [in a new window]   Figure 6. Effect of repeated stimulation of HT-29 cells with casein phosphopeptides (CPP). Panel A: Fura-2/acetoxymethyl ester–loaded 0.5 x 106 cells were incubated in Krebs-Ringer-HEPES (KRH) containing 2 mmol/L extracellular calcium; at the indicated times (arrows), 320 µmol/L mixture of casein phosphopeptides from a commercial source (CPP DMV) was added. The trace in the graph represents the fluorescence recorded at 485 nm after excitation at 343 nm. Panel B: single-cell behavior of HT-29 cells incubated in KRH containing 2 mmol/L Ca2+ is shown when 8 µmol/L mixture of casein phosphopeptides from a commercial source (CPP DMV) was repeatedly added (arrows). The data presented are typical of three experiments. Similar results were obtained with the purified phosphopeptide ß-CN(1–25)4P.

	DISCUSSION

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It is noteworthy that no substantial differences were observed in the HT-29 cell response to the two different CPP preparations employed. One was a mixture of different CPP, closely resembling those formed physiologically during intestinal digestion of caseins; the other was a single pure CPP. This suggests that a chemical feature, shared by the two products, is instrumental in the effect. It is tempting to speculate that this feature is the motif ser(P)- ser(P)- ser(P)-glu-glu because Nagai et al. (27) reported the crucial importance of this domain for the CPP effect, although in a completely different cell model. Of course, this point must be investigated thoroughly, and it must be determined whether serine phosphorylation is required for the calcium flux–inducing effect.

The seemingly linear relationship between the CPP dose and the [Ca²⁺]_i increase in the HT-29 cell population studies, as well as the data showing lack of desensitization, obtained with both single cells and cell population, favors the hypothesis that the process influenced by CPP is nonsaturating. Therefore, a direct interaction of CPP with a membrane receptor or an endogenous ion channel seems to be excluded. This line of interpretation also fits the evidence provided by cell population studies that the efficacy of CPP action (i.e., the response/dose ratio) is greater the lower the cell number. A challenging observation is that the CPP concentration range eliciting a rise in [Ca²⁺]_i was quantitatively much higher (40-fold) in the cell population study than in the single-cell experiments. This could be related to the different experimental conditions. In the Video-Imaging experiments, the cells were grown attached to a glass coverslip, a situation resembling the physiologic intestinal monolayer, whereas in the spectrofluorimetry experiments, the cells were suspended in a cuvette and continuously stirred. However, the cell numbers in the two experiments were very different, with much larger (0.5 x 10⁶ vs. 2.6 x 10⁴) cell numbers used in the experiments on cell populations than on single cells. This observation parallels the above discussion and also supports the hypothesis that CPP interact directly with the plasma membrane but do not influence receptors or ion channels present therein.

A suggestion concerning the mode of CPP action on the transmembrane flux of calcium is that CPP might insert themselves into the plasma membrane and form their own calcium-selective channels or act as calcium-carrier peptides rapidly internalized via endocytosis or other processes, and eventually provide ionized calcium in the cytosol. Consistent with an internalization mechanism is the evidence that the [Ca²⁺]_i increase elicited by CPP is transient and lacking desensitization. Of interest is the recent report (43) that peptides such as the Alzheimer’s ß-amyloid, human islet amylin and prion protein fragment, all featuring a ß-pleated sheet structure, can interact spontaneously with the plasma membrane of susceptible cells, forming unregulated Ca²⁺ channels. Because of their small size, the presence of ß-pleated sheet structures in CPP seems unlikely. However, it was reported (44) that CPP can aggregate in oligomers. This may enable them to display the physicochemical characteristics suitable for insertion into the plasma membrane.

In conclusion, the present work provides evidence that CPP favor the flux of extracellular calcium into HT-29 cells, causing a transient rise of [Ca²⁺]_i without affecting the intracellular calcium stores. This evidence constitutes the first direct proof that CPP, although in a particular cellular system, enhance calcium uptake from the extracellular medium by a direct action on the plasma membrane and presumably regardless of their calcium-solubilizing capacity. It should now be established whether this mechanism also operates in the intestinal tract under physiologic conditions. In this case, summation of the direct effect on calcium uptake and calcium-solubilizing capacity may be beneficial to enhance calcium absorption, especially when substances forming insoluble complexes with calcium are present in the lumen. If so, the concept that CPP play a role in calcium absorption and bioavailability in animals would receive further attention. The molecular aspects of the CPP-induced [Ca²⁺]_i rise are presently under investigation.

	FOOTNOTES

 
¹ Presented in part at the 44th Congress of The Italian Society for Biochemistry and Molecular Biology [Ferraretto A., Signorile A., Fiorilli A. & Tettamanti G. (1999) CPP influence on calcium uptake by the tumoral cells of intestinal origin HT-29 and Caco-2 in culture, p. 95] and at the 45th Congress of The Italian Society for Biochemistry and Molecular Biology [ Ferraretto, A., Signorile, A., Gravaghi, C., Fiorilli, A. & Tettamanti, G. (2000) Casein phosphopeptides-induced [Ca⁺⁺]_i changes in individual tumor cells of intestinal origin HT-29 cultured in vitro, p. 149].

² Supported in part by the EU FAIR Programme Project CT98–3077 entitled: Casein phosphopeptide (CPP): nutraceutical/functional food ingredients for food and pharmaceutical applications.

⁴ Abbreviations used: [Ca²⁺]_i, intracellular free calcium concentration; [Ca²⁺]_o, extracellular free calcium concentration; CPP, casein phoshopeptides; Fura-2/AM, Fura-2 acetoxymethyl ester; KRH, Krebs-Ringer-HEPES; Tg, thapsigargin.

Manuscript received November 15, 2000. Initial review completed January 4, 2001. Revision accepted March 5, 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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