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Experimental Biology and Medicine 228:850-854 (2003)
© 2003 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Casein Binds to the Cell Membrane and Induces Intracellular Calcium Signals in the Enteroendocrine Cell: A Brief Communication

Tohru Hira, Hiroshi Hara1,, Fusao Tomita and Yoritaka Aoyama

Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Dietary protein but not amino acids stimulates cholecystokinin (CCK) secretion in rat mucosal cells. However, the dietary protein sensory mechanisms and the intracellular signal pathway in the enteroendocrine cells have not yet been clarified. The relationship between dietary protein binding to cell membrane and intracellular calcium responses were examined in the CCK-producing enteroendocrine cell line STC-1. The binding of solubilized STC-1 cell membrane to proteins was analyzed using a surface plasmon resonance sensor. Intracellular calcium concentrations of STC-1 cell suspensions loaded with Fura-2 AM were measured using a spectrafluorophotometer system with continuous stirring. Intracellular calcium concentrations in STC-1 cells were increased by exposure to {alpha}-casein or casein sodium, but not to bovine serum albumin. Solubilized STC-1 membranes bound to {alpha}-casein and casein sodium but did not bind to bovine serum albumin. {alpha}-Casein demonstrated higher membrane binding and intracellular calcium stimulating activities than casein sodium. Thus, protein binding to the STC-1 cell membrane and intracellular calcium responses were correlated. Intracellular calcium responses to {alpha}-casein were suppressed by an L-type calcium channel blocker. These results suggest that casein, a dietary protein, binds to a putative receptor on the CCK-producing enteroendocrine cell membrane and elicits the subsequent intracellular calcium response via an L-type calcium channel.

Key Words: dietary protein • casein • enteroendocrine cell • calcium


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Cholecystokinin (CCK) is a gut-brain hormone released from the enteroendocrine cell (I cell) lining in the intestinal epithelial cells in response to the ingestion of nutrients into the small intestine. CCK plays major roles in stimulating pancreatic enzyme secretion, gallbladder contraction, inhibition of gastric emptying, and appetite suppression. Dietary proteins and dietary fats are potent stimulators of CCK release in the gut (1–3). However, the sensory mechanisms by which these nutrients stimulate the enteroendocrine cells are still unclear (4).

Previous reports provided evidence suggesting that dietary proteins were able to stimulate CCK release and pancreatic enzyme secretion independently of the luminal protease-mediating feedback mechanism in rats and isolated intestinal mucosal cell suspensions (5–7). Recent studies have demonstrated that peptones (protein hydrolysates) stimulate CCK secretion in a CCK-producing murine enteroendocrine cell line STC-1 (8, 9). These studies indicate that intestinal CCK cells detect dietary proteins directly without mediation by endogenous CCK-releasing factors (10–12). However, the cellular surface and intracellular events initiated by dietary proteins in CCK-producing cells are not yet known.

As shown in several reports, the STC-1 cell is a suitable model for studying the physiology of CCK-producing enteroendocrine cells. This cell line responds to several physiological stimulants including nutrients, hormones and luminal CCK-releasing factors (8, 13–17). Recently, two groups studied the CCK release and the intracellular calcium response caused by fatty acids in STC-1 cells (18, 19). Fatty acids caused an increase in intracellular calcium concentrations and CCK release in STC-1 cells. The effects of added proteins on intracellular calcium have not been reported for intestinal or STC-1 cells.

Binding between dietary proteins and rat small intestinal brush-border membranes was recently demonstrated using a surface plasmon resonance sensor (SPR sensor) (20). In that study, the binding between dietary proteins and the brush-border membranes correlated with the physiological activities of the proteins in the rat small intestine. This finding suggested that the dietary protein binding to the intestinal apical cell membrane was an integral part of the sensory mechanism. The present study was designed to test whether selected dietary proteins bind to components of STC-1 cell membranes and whether this binding correlates with intracellular calcium signals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cell Culture.
STC-1 cells were kindly provided by Dr. D. Hanahan (University of California, San Francisco, CA). These cells were originally derived from an intestinal endocrine tumor obtained from double transgenic mice (21). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; 4.5 g/l glucose, with l-glutamine, without sodium pyruvate; GIBCO BRL 11965-092, Grand Island, NY) containing 10% fetal calf serum, 100 U/ml penicillin, and 100 mg/l streptomycin. A humidified, 5% CO2 atmosphere was maintained at 37°C. Cells between passage 30 and 40 were used at 80–90% confluence.

Measurement of Intracellular Calcium Concentration.
Intracellular calcium concentrations in STC-1 cells were evaluated by excitation fluorescence ratio (340 nm/380 nm) using a dual-wavelength spectrafluorophotometer (CAF-110; JASCO, Tokyo, Japan) in the cell suspensions loaded with Fura 2-AM. STC-1 cells in 75-cm2 tissue culture flasks were rinsed with DMEM and loaded with 10 µM Fura 2-AM (Molecular Probes, Eugene, OR) in the presence of 0.03% Pluronic F-127 (Molecular Probes) in 3 ml of DMEM at 37°C for 60 min. Cells were rinsed with 3 ml of phosphate-buffered saline for 30 sec and then exposed to 3 ml of a nonenzymatic cell dissociation solution (Sigma, St. Louis, MO) for 5–10 min (19). After incubation, cells were suspended with the addition of 3 ml of physiological saline solution (PSS). Cells suspensions were centrifuged (800g for 5 min) and resuspended in PSS at a concentration of 2 x 106 cells /ml. The PSS contained (in mM) 137 NaCl, 4.7 KCl, 0.56 MgCl2, 1.28 CaCl2, 1.0 NaH2PO4, 10.0 HEPES, MEM with essential amino acids (GIBCO BRL), 2.0 L-glutamate, and 5.5 D-glucose, and was adjusted to pH 7.4 (16). Cell suspensions were placed in a cuvette cell (400 µl for a single experiment) with continuous stirring. After baseline stabilization (approx. 60 sec), a test agent was injected. Fluorescence was monitored at excitations of 340 nm and 380 nm. Emissions were monitored at 510 nm. After exposure to the test agents, cells were lysed with 0.2% Triton X-100 and then exposed to 10 mM EGTA to estimate cytoplasmic calcium. The cytoplasmic calcium concentration was calculated as described by Grynkiewies et al. (22) using 224 nM as the dissociation constant (Kd) for the calcium complex of Fura 2.

Binding Analysis Between Proteins and STC-1 Membrane Extracts.
The membrane fraction of STC-1 cells was prepared using a previously described method (23) with minor modifications. STC-1 cells in culture flasks were washed once with HBS-E buffer (10 mM HEPES, 0.15 M NaCl, 3 mM EDTA, pH 7.4), and scraped into ice cold HBS-E. Cells were disrupted with a Polytron homogenizer (Kinematica, Lucerne, Switzerland) three times, for 6 sec each. The lysate was centrifuged at 20,000 g for 10 min (Hitachi 55P-72 ultracentrifuge; Hitachi-koki, Hitachinaka, Japan), and the supernatant was centrifuged at 35,000g for 30 min. The resultant pellet was resuspended in HBS-E containing 0.1% Triton X-100 and solubilized for 2 hr at 4°C with shaking (200 rpm, Vortex Shaker VR-36D; Taitec, Saitama, Japan). The solution was centrifuged at 100,000 g for 90 min, and the supernatant was collected as the solubilized STC-1 membrane. The supernatant was divided into aliquots and stored at -80°C. When the biosensor analysis was conducted, the aliquots were diluted 1:10 with HBS-E buffer before binding analysis (final concentration of Triton X-100 was 0.01%) (20).

Using a Surface plasmon resonance (SPR)-biosensor (BIACORE 3000 system; Biacore AB, Uppsala, Sweden), binding between immobilized molecules (proteins) on the sensor surface and soluble molecules (solubilized membranes) passed over the sensor surface was analyzed. Proteins (casein sodium, {alpha}-casein, bovine serum albumin [BSA]) were immobilized onto the sensor surface (flow-cell) of a Sensor chip CM5 using a mixture of N-ethyl-N'-dimethylaminopropyl-carbodiimide and N-hydroxysuccinimide in the amine coupling kit (Biacore AB, Uppsala, Sweden) according to previously described methods (24, 25). Casein sodium (40 µg/ml in 0.1 M sodium acetate buffer, pH 4.0), {alpha}-Casein (40 µg/ml in 0.1 M sodium acetate buffer, pH 4.0), or BSA (40 µg/ml in 0.1 M sodium acetate buffer, pH 5.0) were respectively injected over the activated sensor surfaces using the surface preparation software provided by the manufacture (BIACORE Control 3.0, Biacore AB, Uppsala, Sweden). After coupling, the activated surface was blocked by the injection of 0.1 M ethanolamine (pH 8.0). Blank flow-cells were prepared by immobilizing 0.1 M ethanolamine (pH 8.0) on the activated sensor surface without any protein exposure.

Solubilized STC-1 membrane was injected as an "analyte" and passed over the sensor surface on which the protein was immobilized. All analyses were conducted using HBS-E buffer containing 0.01% Triton X-100 as the running buffer. The flow rate was 10 µl/min at a temperature of 25°C. Analytes were delivered for 2 min, and the injections were repeated 4 times during each analysis. The amount of solubilized STC-1 membrane bound to the immobilized protein (resonance unit [RU]) was calculated by subtraction of the blank flow-cell response from the protein immobilized flow-cell response.

Statistical significance was assessed using one-way ANOVA and significant differences among mean values were evaluated by the least-significant difference method (P < 0.05).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
The interaction between proteins and CCK-producing enteroendocrine cells was examined to determine whether selected proteins could stimulate intracellular calcium signals within these cells and whether these proteins bound to membrane components of these same cells.

Figure 1Go shows changes in intracellular calcium concentrations in STC-1 cells exposed to three different proteins. Casein and BSA were selected respectively as proposed dietary and nondietary proteins. Exposure to casein sodium (0.05%) gradually increased intracellular calcium concentrations reaching a plateau after 10 sec. The increase in calcium was sustained for the following 20 sec. {alpha}-Casein (0.05%) produced a rapid and more intense increase in calcium concentration that slowly returned toward baseline during the subsequent 20 sec of observation. These results are the first evidence that a dietary protein causes an increase of the intracellular calcium level in CCK-producing enteroendocrine cells. In contrast, BSA (0.05%), the non-dietary protein, did not increase intracellular calcium concentration. In our previous study, casein sodium stimulated the CCK release from STC-1 cells in a dose-dependent manner, but BSA had only a small effect on CCK secretion (unpublished data). In another study, BSA was much less effective than casein, egg and meat hydrolysates (3). These results appear consistent with the current observation that casein but not BSA induced intracellular calcium signals in STC-1 cells. The induction of calcium signals by casein indicates that STC-1 cells recognize some but not all proteins, and this calcium response could trigger a subsequent CCK release.



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  		Figure 1. {alpha}-Casein and casein sodium but not BSA increased intracellular calcium in STC-1 cells Changes are illustrated for intracellular calcium concentrations in response to 0.05% {alpha}-casein (closed circles, n = 10 from approx. 2–3 measurements in four separate experiments), casein sodium (open circles, n = 4 from approx. 1–2 measurements in three separate experiments), and BSA (closed squares, n = 4 from approx. 1–2 measurements in three separate experiments). STC-1 cells loaded with Fura 2 were continuously stirred and kept at 37°C. The change of intracellular calcium concentrations was calculated from the monitored fluorescence ratios. Test solutions were injected at time 0 sec. A one-way analysis of variance indicates a P < 0.01 for {alpha}-casein and casein sodium. The effect of BSA was not significant. Values between 2 and 22 sec after {alpha}-casein injection and between 10 and 30 sec after casein sodium injection are significantly higher (P < 0.05 by least-significant difference) than the basal values for each treatment. Values are means ± SEM of repeated measurement.

 
{alpha}-Casein, a major subunit of casein, increased intracellular calcium levels more than casein sodium. This difference suggests that STC-1 cells are more sensitive to {alpha}-casein than to the whole casein complex with its more complicated structure. Putative active sites of {alpha}-casein might be exposed by its separation from the casein complex structure and enhance the ability to be detected by STC-1 cells.

Treatment of STC-1 cells (Fig. 2Go) with diltiazem chloride (10 µM, from 30 sec before the addition of {alpha}-casein) reduced the {alpha}-casein-induced intracellular calcium response. The reduction in the casein-induced increase in intracellular calcium by diltiazem suggests that L-type calcium channels were involved. L-type calcium channels have been proposed to participate in similar stimulatory effects of fatty acids (18, 19) and depolarization (26) on enteroendocrine cells. The persistence of a reduced calcium response in the presence of diltiazem suggests that either some L-type channel remain functional or other routes of calcium entry and/or release are involved.



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  		Figure 2. An L-type calcium channel is involved in Ca signaling in response to {alpha}-casein in STC-1 cells. Changes are illustrated in intracellular calcium concentration in response to {alpha}-casein in STC-1 cells treated with (open circles, n = 9 from approx. 2–4 measurements in three separate experiments) or without (closed circles, n = 10 from approx. 2–3 measurements in four separate experiments) 10 µM diltiazem, an L-type calcium channel antagonist, 30–50 sec before {alpha}-casein injection. STC-1 cells were continuously stirred and kept at 37°C. After diltiazem treatment, {alpha}-casein solutions were injected at time 0 sec. The change of intracellular calcium concentrations was calculated from monitored fluorescence ratios. Values are means ± SEM of repeated measurement.

 
Figure 3aGo shows overlaid sensorgrams (changes in the surface plasmon resonance signal) of an {alpha}-casein-immobilized surface in response to injections of various concentrations of solubilized STC-1 cell membrane. Membrane components of STC-1 cells injected onto immobilized {alpha}-casein showed binding signals with apparent association and dissociation curves. The amount (RU) bound to {alpha}-casein increased with increasing protein concentrations of the solubilized STC-1 membrane. Figure 3bGo shows the amount of solubilized STC-1 membrane bound to {alpha}-casein, casein sodium, and BSA immobilized onto the sensor surface. These values present the maximum amount of binding in the sensorgrams at the time just before the end of sample injections, and corrected by the amount of each ligand immobilized onto each sensor surface. Solubilized STC-1 membrane did not bind to the BSA immobilized sensor surface, but bound to both caseins in a dose-dependent manner. Binding to {alpha}-casein was apparently higher than that to casein sodium at all concentrations. These results suggest that casein and {alpha}-casein bind to STC-1 cell membrane components and that {alpha}-casein binds more readily than casein sodium. The difference between BSA and casein is consistent with previous observations with rat small intestinal brush-border membranes (20).



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  		Figure 3. Membrane components bound to casein sodium and {alpha}-casein, but not to BSA. (a) Overlaid sensorgrams are illustrated for an {alpha}-casein-immobilized surface in response to injections of various solubilized STC-1 membrane concentrations (protein concentrations approx. 1–7 µg/ml). (b) Binding responses (resonance unit; RU) are illustrated for the protein-immobilized surface (closed circles; {alpha}-casein, open circles; casein sodium, closed squares; BSA) in response to various concentrations of solubilized STC-1 membrane. Solubilized STC-1 membrane solutions (protein concentrations approx. 1–7 µg/ml) were injected over each protein-immobilized sensor chip for 120 sec at a flow rate 10 µl/min. Values are means ± SEM of four repeated measurements.

 
While some reports have demonstrated the direct effects of dietary proteins on CCK secretion from STC-1 cells and the rat small intestine (3, 7, 27, 28), there has been no study on the membrane binding and intracellular calcium signaling effects of dietary proteins in enteroendocrine cells. In the present study, immobilized dietary proteins bound to cell membrane components from STC-1 cells and these same proteins increased intracellular calcium in intact STC-1 cells. These findings suggest that the binding of dietary proteins to the cellular surface (receptor) activates intracellular calcium signals in CCK-producing enteroendocrine cells. Enteroendocrine cells may be equipped with novel receptors for dietary proteins similar to those proposed for fatty acids (18). Additional study will be required to support this hypothesis including the isolation of the putative receptor. Specific receptors have not yet been identified for endogenous luminal CCK-releasing factors or any of the nutrients described above (16, 17, 29).

In conclusion, casein sodium and {alpha}-casein but not BSA bind to membrane components extracted from CCK-producing enteroendocrine cells (STC-1 cells). Consistent with this result, casein and {alpha}-casein, but not BSA induced intracellular calcium signals in STC-1 cells. The intracellular calcium response to {alpha}-casein appears to employ L-type calcium channels. These collected results suggest that dietary proteins may bind to putative nutrient receptors on enteroendocrine cells, increase intracellular calcium and subsequently stimulate the release of CCK.


   Footnotes
 
Tohru Hira is a Research Fellow of the Japan Society for the Promotion of Science.

This work was supported by Research Fellowships of Japan Society for the Promotion of Science for Young Scientist.

1 To whom reprint requests should be addressed at the Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan. E-mail: hara{at}chem.agr.hokudai.ac.jp Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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Received for publication August 20, 2002. Accepted for publication March 4, 2003.




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