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Journal of Endocrinology (2005) 185, 81-91    DOI: 10.1677/joe.1.05945
© 2005 Society for Endocrinology
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1,25-dihydroxyvitamin D3 stimulates vesicular transport within 5 s in polarized intestinal epithelial cells

Tremaine M Sterling and Ilka Nemere

Department of Nutrition and Food Sciences and the Center for Integrated BioSystems, Utah State University, Logan, Utah 84322, USA

(Requests for offprints should be addressed to I Nemere; Email: nemere{at}cc.usu.edu)


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Controversy remains regarding whether the seco-steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) enhances calcium and phosphate movement across the intestinal epithelial cell by facilitated diffusion or a vesicular transport mechanism. In this study we investigated whether membrane trafficking, as judged by confocal microscopy, was sufficiently rapid in comparison to hormone-stimulated uptake of phosphate (32P). Primary cultures of chick intestinal cells were established overnight either in Petri dishes (uptake studies) or chambered coverslips (confocal microscopy). Addition of 130 pM 1,25(OH)2D3 resulted in an apparent increase in 32P uptake within 1 min, relative to controls, that was statistically significant from 3–10 min of incubation. Using the endocytic marker dye, FM1–43, confocal microscopy revealed a profound decrease in membrane-associated fluorescence (apical> basal) within 10 s of hormone treatment, a return of fluorescence at 15–65 s, followed by another round of decreasing and increasing fluorescence. Between 3–9 min of incubation, fluorescence intensity increased 50% (apical region) and 20% (basal region) over control conditions. An antibody (Ab 099) directed against a putative membrane receptor for 1,25(OH)2D3 (1,25D3-MARRS) inhibited both 32P uptake, and changes in fluorescence. In addition, the protein kinase C (PKC) inhibitor, calphostin C, inhibited both 32P uptake and the observed 1,25(OH)2D3-mediated changes in fluorescence. At the microscopic level, calphostin C pretreatment abolished the very rapid redistribution of the endocytic marker dye, although a slight increase in fluorescence was still observed. We conclude that 1,25(OH)2D3-stimulated vesicular trafficking is mediated by the 1,25D3-MARRS protein, implicates a PKC signaling mechanism, and occurs in a time frame that is commensurate with a role in ion transport.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In intestinal cells from young (3–7 weeks), vitamin D replete chicks, the primary effect of the steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) appears to be on phosphate uptake (Zhao & Nemere 2002) and transport (Nemere 1996a), mediated in part by protein kinase C (PKC; Zhao & Nemere 2002, Larsson & Nemere 2003) rather than calcium uptake (Nemere & Campbell 2000) and transport (Nemere 1996a).

Phosphate uptake at the apical region of the enterocytes is meditated by a Na/Pi cotransporter (Murer et al. 2001). However, phosphate accumulation has been reported to occur in lysosomes of fibroblasts (Pisoni 1991), as well as intestinal epithelial cells exposed to transport conditions (Nemere 1996b), suggesting that vectorial movement of the anion occurs in vesicles. Moreover, the transporter itself has been found to be internalized under low uptake conditions (Murer et al. 2001) and is presumably reinserted into the plasma membrane when phosphate transport is increased.

In the current study, we use primary cultures of chick intestinal epithelial cells to assess the time course of 32P uptake as stimulated by 1,25(OH)2D3, and compare that to the time course of vesicular trafficking with confocal microscopy. Our results indicate that 1,25(OH)2D3-mediated vesicular trafficking is sufficiently rapid to play a role in phosphate uptake or transport.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Surgical procedures and cell isolation

All surgical procedures were approved by the Institutional Animal Use and Care Committee at Utah State University (Logan, UT, USA). White leghorn cockerels (Privett Hatchery, Portales, NM, USA) were obtained on the day of hatch and raised for 3–7 weeks on a commercially available vitamin D-replete diet (Nutrena Feeds, Murray, UT, USA). On the day of use, chicks were anesthetized with chloropent (0.3 ml/100 g body weight), the duodenal loop was surgically removed to ice-cold 0.9% saline solution and chilled for 15 min. The pancreas was excised from the duodenal loop and discarded. The duodenal loop was everted and rinsed in chilled saline solution.

The chick enterocytes were isolated with citrate chelation media (Nemere & Campbell 2000, Zhao & Nemere 2002) adjusted to pH 5.0 to promote viability and retention of morphology (Caldwell et al. 1993). The cells were collected by low speed centrifugation (500 g, 5 min, 4 °C) and resuspended in a small volume of Gey’s balanced salt solution (GBSS; containing 119 mM NaCl, 4.96 mM KCl, 0.22 mM KH2PO4, 0.84 mM NaHPO4, 1.03 mM MgCl2.6H2O, 0.28 mM MgSO4.7H2O, 0.9 mM CaCl2, pH 7.3). Aliquots of the cell suspension (1 ml) were pipetted into 35 mm plastic Petri dishes (Falcon, Fisher Scientific, Houston, TX, USA) containing 3 ml of RPMI-1640 medium (Hyclone, Logan, UT, USA) and antibiotics (100 units/ml penicillin, 100 mg/ml streptomycin (both from Sigma)). The cells were incubated for 24 h (in the absence of serum) at 37 °C with 5% CO2/95% air to promote cell adhesion.

Time course of phosphate uptake

After cells were cultured as described above, the media were aspirated and replaced with GBSS containing radio-nuclide (2 µCi/ml H332PO4; New England Nuclear, Boston, MA, USA) at 23 °C. After each dish was exposed to GBSS with radionuclide for 10 min, dishes for the zero time point were washed three times each with 4 ml of ice cold GBSS, and treated with lysis buffer (10 mM Tris, 1.25 mM EDTA, 2 mM dithiothreitol, pH 7.4 (TED) containing 0.1% Tritonx-100). The remaining dishes were treated with 0.01% ethanol (final concentration) for vehicle controls or 130 pM 1,25(OH)2D3 for hormone treated cells. At designated time intervals after hormone addition, the media were aspirated and the cells washed and then lysed in preparation for protein determination by the Bradford Assay (BioRad, Hercules, CA, USA) against bovine {gamma}-globulin as standard (Sigma) and radionuclide measurement by liquid scintillation spectrophotometry. The c.p.m. in each sample was related to protein levels in the corresponding sample to yield specific activity. For time course studies, basal uptake levels were used to normalize sample values obtained during the treated phase.

Statistical comparisons between controls and treated samples were performed using the Student’s t-test for unpaired observations or SAS 9.1 for ANOVA; significant differences were determined with a 95% probability.

Effect of inhibitors on phosphate uptake

The role of the 1,25D3-MARRS protein (membrane associated, rapid response steroid-binding protein – a putative membrane receptor for 1,25(OH)2D3) was analyzed using a highly specific polyclonal antibody (Ab 099; Nemere et al., 2000, 2004) generated by the multiple antigenic peptide format to the N-terminal sequence of the protein (Center for Integrated BioSystems, Logan, UT, USA). After cells were cultured as described above, the media in each dish were aspirated and replaced with GBSS/0.1% BSA with or without 1/500 dilution of Ab 099 and preincubated for 5 min prior to experimentation. After cell cultures were overlayed with GBSS containing radionuclide (2 µCi/ml H332PO4) for 10 min, each dish was exposed to vehicle or hormone for 7 min. The media were aspirated and the cells washed and then lysed in preparation for analysis as described above.

To assess the role of PKC, adherent cells were preincubated with or without 500 nM calphostin C (Alexis Biochemicals, San Diego, CA, USA) for 3–3.5 h (Kobayashi et al. 1989, Bruns et al. 1991) and then incubated with radionuclide with or without hormone as described for the time course of phosphate uptake.

Confocal microscopy

Vesicular movement was examined using confocal microscopy with the probe FM 1–43, which is nonfluorescent in the aqueous environment of the medium but fluoresces once it inserts into the plasma membrane. A MRC 1024 laser-scanning confocal microscope system mounted in the Keller position and attached to a Nikon TE-200 microscope (BioRad) was used for confocal imaging. The krypton-argon laser produced 3 excitation lines of 488 nm, 568 nm, and 647 nm. The emission filter consisted of a 522/32 bandpass filter that collected all light between 506–538 nm. Images were collected with BioRad LASERSHARP acquisition software (BioRad), using a 60x oil immersion objective and analyzed for pixel intensity using Adobe Photoshop 6.0 (Adobe Systems, Inc, San Jose, CA, USA).

Cells resuspended in RPMI were pipetted into German borosilicate, chambered glass coverslips (Lab-Tek, Fisher Scientific) containing 3–5 ml of RPMI-1640 media with antibiotics and cultured overnight at 37 °C with 5% CO2/95% air. FM 1–43 (Molecular Probes, Eugene, OR, USA) was used to label membranes; this probe was excited by the 488 nm laser line with maximum emission spectra at 625 nm. Lysotracker-red (Molecular Probes) was used for lysosomal visualization; this label was excited at 568 nm laser line with maximum emission spectra at 590 nm. The dyes were diluted into GBSS-0.1% BSA and the solution was then added to the cells. After a 15- to 30-min incubation with the dye, the medium was overlayed with GBSS-0.1% BSA only for vehicle or GBSS-0.1% BSA with hormone for treated cells. For inhibitor studies, cells were preincubated with 1/500 dilution Ab 099 (5 min at 23 °C) or 500 nM calphostin C (3–3.5 h at 23 °C under fluorescent light) prior to addition of fluorescent label.

The images were then captured at 5-s intervals for 10 min using a 60x oil immersion objective and analyzed for pixel intensity as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of 1,25(OH)2D3 on phosphate uptake

Figure 1Go illustrates the results of time course studies on 32P uptake. In controls, a time dependent decrease in 32P levels was observed. Hormone-treated cells also exhibited a time dependent decline in 32P levels, but were consistently higher than the corresponding controls. We surmise that this increase represents steroid-mediated uptake. Thus, 130 pM 1,25(OH)2D3 stimulated an apparent increase in 32P uptake within 1 min when compared with controls. In the controls, the corresponding 32P values (c.p.m./µg protein compared to basal levels, means± S.E.M.) for T=1, 3, 5, 7 and 10 min were 0.71±0.14, 0.45±0.09, 0.50±0.12, 0.47±0.11, and 0.49±0.11 respectively. After addition of 1,25(OH)2D3, 32P uptake increased by approximately 203%, 216%, 204%, 204% and 214% of controls at T=1, 3, 5, 7 and 10 min respectively (n=5, P< 0.05 for T=3–10 min).



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  		Figure 1 Time course of 1,25(OH)2D3-mediated phophate (32P) uptake in isolated intestinal epithelial cells. Enterocytes were isolated by a chelation protocol, collected by low speed centrifugation, and resuspended in GBSS. One milliliter aliquots of cell suspensions were seeded into 35 mm Petri dishes containing 3 ml RPMI-1640 medium and incubated overnight (37 °C with 5% CO2/95% air) to allow cells to adhere. For uptake studies, media were aspirated and replaced with 2 µCi 32H3PO4/1 ml of GBSS. Cells were treated with 130 pM 1,25(OH)2D3 (•) or vehicle (0.01% ethanol, final concentration, {circ}), and washed with GBSS at the indicated times. After lyses in buffer containing 0.1% Triton X-100, aliquots were taken for determination of protein using the Bradford assay and radioactivity analysis by liquid scintillation spectrophotometry. Data were calculated as c.p.m./µg protein and then related to corresponding basal levels. Values represent means±S.E.M. (n=6). Significant differences (*P< 0.05), determined using the student t-test, are relative to corresponding controls.

 
Effect of 1,25(OH)2D3 on distribution of FM 1–43

An equivalent time course was used in studies with confocal microscopy of cells labeled with FM 1–43. This dye, which is non-fluorescent in an aqueous environment, fluoresces when associated with a membraneous environment, and thus can be used to monitor endocytosis. Images of control cells revealed bright fluorescence throughout the length of the cells, including the brush border (Fig. 2AGo). Solid areas of fluorescence appeared to be associated with the plasma membrane, and largely obscured punctate fluorescence (Fig. 2AGo). In pilot studies, fluorescent labeling remained unchanged for 10 min under control conditions. In subsequent studies, control conditions were applied for 50 s of image capture after which the steroid was added.





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  		Figure 2 Effect of 1,25(OH)2D3 on endocytic distribution of FM 1–43 (n =2). Confocal images are presented of cells labeled with FM 1–43, then treated with 1,25(OH)2D3 Cells were plated overnight in chambered coverslips. The dye was diluted into GBSS, which was then overlayed onto the cells. Since no changes in fluorescence distribution occurred in controls over a 10 min period (not shown), subsequent experiments used a 1 min control period for image capture, after which GBSS containing hormone was added to the medium and additional images were captured at 5 s intervals for 10 min using a 60x oil immersion objective. Outlined areas were analyzed for pixel intensity using Adobe Photoshop 6.0. The emission and excitation wavelengths were 488 and 522 nm, respectively. (A) control; (B-AA) every 5 s after hormone addition; (BB) 3 min; (CC) 5 min; (DD) 7 min; (EE) 9 min. The arrow in this and subsequent micrographs indicates the apical surface.

 
Within 5 s of 1,25(OH)2D3 addition, a decrease in apical fluorescence was observed (Fig. 2BGo) with an even more dramatic decrease at 10 s (Fig. 2CGo). The intensity of punctate fluorescence increased markedly after 15 s (Fig. 2DGo). These very rapid effects were observed in three independent experiments. In subsequent frames (Figs 2F–MGo), apical fluorescence appeared punctate while basal staining appeared continuous. Between 1 min and 1 min 25 s after 1,25(OH)2D3 addition, the staining pattern became more punctate at supranuclear localizations (Figs 2M–RGo). A loss of overall fluorescence was again observed between 1 min 30 s and 2 min 5 s after hormone addition, followed by a dramatic increase in fluorescence 5 s later (Fig. 2AAGo). A similar loss and return of fluorescence was observed around 3 min after hormone addition (data not shown). In pilot studies, at low resolution microscopy and in real time, this increase and decrease in fluorescent intensity occurred so rapidly that cells treated with 1,25(OH)2D3 appeared to flicker. For the remainder of the time course (3–9 min after 1,25(OH)2D3 addition), an overall increase in fluorescence was observed (Figs 2BB–EEGo). The observed changes in fluorescence at the apical (Fig. 3Go; diamond symbols) and basal regions (Fig. 3Go; circles) are quantitatively verified by pixel intensity for each confocal image.



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  		Figure 3 Pixel intensities for confocal images of representative epithelial cells treated with 1,25(OH)2D3 Outlined areas in Fig. 2Go were analyzed for pixel intensity using Adobe Photoshop 6.0.Values for apical (•) and basal ({diamondsuit}) intensities are expressed as means±S.D. (n=4 for each).

 
A second dye, Lyso-Tracker Red, was also tested in time course studies using confocal microscopy. This dye, which is non-fluorescent at a neutral pH, fluoresces when incorporated into acidic organelles. Studies with Lyso-Tracker Red suggested increased movement of labeled organelles after hormone addition (data not shown), but definitive patterns of movement were not evident.

Effect of Ab 099 on 1,25(OH)2D3 -mediated 32P uptake

To assess the role of a putative membrane receptor, 1,25D3 MARRS protein, on phosphate uptake, cultured cells were preincubated in the absence or presence of Ab 099. Radionuclide and either vehicle or hormone was then added and incubation continued an additional 7 min. Figure 4Go illustrates that 130 pM 1,25(OH)2D3 increased 32P uptake to 170% of controls (1,25D; solid bar). Pre-incubation of cells with Ab 099 (Ab only; open bar) showed no significant difference in 32P uptake when compared with controls without Ab 099 but completely inhibited hormone stimulated uptake (Ab1,25D; striped bar) when compared with cells treated with 1,25(OH)2D3 alone.



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  		Figure 4 Comparisons of the effects of 1,25(OH)2D3 and Ab 099, alone or in combination, on phosphate (32P) uptake in isolated chick enterocytes. Cells were pretreated with 1/500 dilution of Ab 099 (Ab) for 5 min at 23°C prior to addition of radionuclide and either 130 pM 1,25(OH)2D3 (1,25D) or vehicle (0.01% ethanol, final concentration) for a total of 7 min. Experimental procedures for 32P uptake experiments are described in Fig. 1Go. Data were calculated as c.p.m. and related to corresponding basal values. All values were normalized to % control for final comparisons and are represented at means± S.E.M. (n=9).

 
Effect of Ab 099 and 1,25(OH)2D3 on distribution of FM 1–43.

Preincubation of cells with Ab 099 also abolished rapid (5–60 s) 1,25(OH)2D3-mediated changes in FM 1–43 distribution (data not shown), but as indicated in Fig. 5Go some relocalization of dye occurred as judged by the disappearance of nuclear outlines in the two cells depicted between 1–9 min after hormone addition (Figs 5C–GGo). The pixel intensities given in Fig. 6Go indicate that changes observed in the nuclear (triangles) and basal (circles) regions of the confocal images were subtle.



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  		Figure 5 Effect of Ab 099 and 1,25(OH)2D3 on endocytic distribution of FM 1–43 labeling (n =2). Confocal images were obtained of cells preincubated with Ab 099 at 1/500 dilution, labeled with FM1–43, then treated with 130 pM 1,25(OH)2D3 Cells were plated overnight in chambered coverslips. The dye was diluted into GBSS, which was then overlayed onto the cells. After a 1-min incubation with the dye alone GBSS containing hormone was added and images were captured and analyzed as described in Fig. 2Go. (A) control; (B) 10 s; (C) 1 min; (D) 3 min; (E) 5 min; (F) 7 min; (G) 9 min.

 


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  		Figure 6 Pixel intensities for confocal images of representative epithelial cells preincubated with Ab 099 and treated with 1,25(OH)2D3 Outlined areas in Fig 5Go. were analyzed for pixel intensity using Adobe Photoshop 6.0. Values for the basal (•) and nuclear ({blacktriangleup}) regions are expressed as means±S.D. (n=2, for each).

 
Effect of PKC inhibitor calphostin C on 1,25(OH)2D3-mediated 32P uptake

To identify the involvement of PKC in 1,25(OH)2D3-mediated phosphate uptake, cultured cells were preincubated with or without calphostin C before exposure to hormone (Fig. 7Go). Figure 7Go illustrates the results of time course studies on 32P uptake in the presence of inhibitor. Whereas the steroid hormone (closed circles) stimulated phosphate uptake relative to controls (open circles), pre-treatment of cells with calphostin C abolished such 1,25(OH)2D3-mediated enhancement (closed triangles). Statistical comparison by ANOVA indicated that 1,25(OH)2D3-treated cells exhibited significantly greater uptake relative to both controls and calphostin-pretreated cells (n=5, P< 0.05 for T=1–5 and 10 min).



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  		Figure 7 Inhibition of 1,25(OH)2D3-mediated 32P uptake by calphostin C in isolated intestinal epithelial cells. Enterocytes were isolated by a chelation protocol, collected by low speed centrifugation, and resuspended in GBSS. Cells were pretreated with vehicle ({circ}), 500 nM calphostin C followed by hormone ({blacktriangleup}), or with 130 pM 1,25(OH)2D3 only (•). Experimental procedures for 32P uptake experiments were described in Fig. 1Go. Values represent means± S.E.M. (n=7). Significant differences (*P< 0.05) were determined using SAS 9.1 for ANOVA and are relative to cells treated with 130 pM 1,25(OH)2D3 only (•).

 
Effect of calphostin C on distribution of FM 1–43

Pretreatment of cells with calphostin C effectively blocked the very rapid (5–60 s) 1,25(OH)2D3-mediated changes in FM 1–43 fluorescence (data not shown). Instead, a gradual increase in cellular fluorescence became noticeable at the apical and basal regions of the cells after 10 min (compare Figs. 8A–8GGo). Although these slight changes in fluorescence were observed, quantitative analysis of the pixel intensities indicated that these effects were not significantly different (Fig. 9Go).



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  		Figure 8 Effect of calphostin C and 1,25(OH)2D3 on endocytic distribution of FM 1–43 labeling (n =2). Confocal images are presented of cells preincubated for 3 h with a PKC inhibitor (500 nM calphostin C), labeled with FM 1–43, then treated with 130 pM 1,25(OH)2D3. Cells were plated overnight in chambered coverslips. Procedures were as indicated in the legend to Fig. 2Go for addition of the dye and hormone, and image capture. (A) control; (B) 10 s; (C) 1 min; (D) 3 min; (E) 5 min; (F) 7 min; (G) 9 min.

 


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  		Figure 9 Pixel intensities for confocal images of representative epithelial cells preincubated with calphostin C and treated with 1,25(OH)2D3. Outlined areas in Fig. 8Go were analyzed for pixel intensity using Adobe Photoshop 6.0. Values for apical (•) and basal ({diamondsuit}) intensities are expressed as means±S.D. (n=2).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous experiments with isolated chick enterocytes in suspension have demonstrated that 1,25(OH)2D3 increases phosphate uptake as early as 5 min (Zhao & Nemere 2002). In the present study with cultured chick enterocytes, an increase in 1,25(OH)2D3-mediated phosphate uptake occurs as early as 1 min and is significantly higher than controls at 3 min. Although the treated/average basal values decreased with time, in both hormone-treated cells when compared with basal, these levels remain closer to basal values than those reported for cells in suspension. Perhaps as a consequence, the increase in 32P uptake is sustained at an average of 208% of controls from 3 to 10 min compared with 151% of controls observed at 10 min with cells in suspension (Zhao & Nemere 2002).

Confocal imaging of these cells indicates that membrane trafficking occurs as early as 5 s after hormone addition. The extremely rapid decrease in apical fluorescence that is observed within 10 s of 1,25(OH)2D3 treatment has, however, two interpretations. The results could suggest an endocytotic mechanism that is consistent with the findings of Warner & Coleman (1975). This study followed calcium uptake in vitamin 1,25(OH)2D3-mediated D-deficient chicks using electron probe analysis, and suggested calcium may be endocytosed just below the brush border, appearing as discreet localizations in the apical region, followed by extrusion at the lateral membrane. Our observations are also consistent with delivery of unlabeled, Golgi-derived vesicles, perhaps containing the Na/Pi transporter. In kidney cells, Murer et al.(2001) have reported internalization of the transporter when parathyroid hormone (PTH) is withdrawn. Such sequestration mechanisms are known to be rapidly reversible and would be amenable to testing with an antibody against the transporter. The sustained increase in fluorescence intensity between 1–9 min after 1,25(OH)2D3 addition suggests repeated endocytotic/exocytotic events, and can be attributed to labeling of existing membrane, new membrane, and probe trapped within vesicles (Ryan et al. 1996). To determine the signal transduction events that may facilitate the vectorial transport of phosphate ions, Ab 099 (against the 1,25D3 MARRS protein) and a PKC inhibitor (calphostin C), were employed in uptake experiments for the current study.

Using Ab 099, Nemere et al.(2000) have immunochemically characterized a putative plasmalemmal receptor, 1,25D3 MARRS protein. In addition to these findings, several studies have demonstrated that 1,25D3 MARRS protein is responsible for binding 1,25(OH)2D3 in the baso-lateral membrane (Nemere et al. 2000, 2004). In the current work, preincubation of isolated intestinal epithelial cells with Ab 099 completely inhibited 1,25(OH)2D3-mediated phosphate uptake. Using equivalent conditions for confocal microscopy, no hormone-mediated increase in fluorescence or redistribution of perinuclear fluorescence occurred. In essence, pretreatment of cells with Ab 099 inhibits 1,25(OH)2D3-induced changes.

Earlier studies have indicated that PKC is most likely involved in phosphate uptake: phorbol ester, but not forskolin, stimulates 32P uptake in enterocytes (Zhao & Nemere 2002); and the dose–response curves for 1,25(OH)2D3-stimulated phosphate transport parallels PKC activation (Larsson & Nemere 2003). In the current study, pretreatment of cultured cells with a PKC inhibitor (calphostin C) abolished steroid-enhanced uptake of phosphate. Although slight increases in fluorescence are observed in these confocal images, the presence of calphostin C significantly inhibited the membrane trafficking that is apparently involved with 1,25(OH)2D3-mediated phosphate uptake.

In conclusion, 1,25(OH)2D3 stimulated exceedingly rapid membrane trafficking that is directly involved in phosphate uptake and transport. The 1,25D3-MARRS protein mediates hormone-induced signal transduction, including activation of PKC (Nemere et al. 2000, 2004). Since phosphate transport is critical in young growing animals to support rapid bone development, the 1,25D3-MARRS protein (Nemere et al. 2004) may represent a new therapeutic target for amelioration of bone loss.


   Acknowledgements
 
This work was supported by NRI-CSREES-USDA grant 2004–35206–14134 (IN), and the Utah Agricultural Experiment Station. Approved as journal paper no. 7638. A portion of these results were presented by TMS in partial fulfillment for the degree of Masters of Science. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bruns RF, Miller FD, Merriman RL, Howbert JJ, Heath WF, Koboyashi E, Takahashi I, Tamaoki T & Nakano H 1991 Inhibition of protein kinase C by calphostin C is light-dependent. Biochemical and Biophysical Research Communications 176 288–293.[CrossRef][ISI][Medline]

Caldwell DJ, Droleskey RE, Elissalde MH, Kogut MH, DeLoach JR & Hargis BM 1993 Isolation and primary culture of chicken intestinal epithelial cells retaining normal in vivo-like morphology. Journal of Tissue Culture Methods 15 15–18.

Kobayashi E, Nakano H, Morimoto M & Tamaoki T 1989 Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C. Biochemical and Biophysical Research Communications 159 548–553.[CrossRef][ISI][Medline]

Larsson B & Nemere I 2003 Effect of growth and maturation on membrane-initiated actions of 1,25-Dihydroxyvitamin D3-II Calcium transport, receptor kinetics, and signal transduction in intestine of female chickens. Journal of Cellular Biochemistry 90 901–913.[CrossRef][ISI][Medline]

Murer H, Hernando N, Forster I & Biber J 2001 Molecular mechanisms in proximal tubular and small intestinal phosphate reabsorption. Molecular and Membrane Biology 18 3–11.

Nemere I 1996a Apparent non-nuclear regulation of intestinal phosphate transport Effects of 1,25-dihydroxyvitamin D3, and 25-hydroxyvitamin D3. Endocrinology 137 3750–3757.[Abstract]

Nemere I 1996b Apparent non-nuclear regulation of intestinal phosphate transport Effects of 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, and 25-hydroxyvitamin D3. Endocrinology 137 2254–2261.[Abstract]

Nemere I & Campbell K 2000 Immunochemical studies on the plasmalemmal membrane receptor for 1,25-dihydroxyvitamin D3 Effect of vitamin D status. Steroids 65 451–457.[CrossRef][ISI][Medline]

Nemere I, Ray R & McManus W 2000 Immunochemical studies on the putative plasmalemmal receptor for 1, 25(OH)2D3. I. Chick intestine. American Journal of Physiology – Endocrinology and Metabolism 278 E1104–E1114.

Nemere I, Farach-Carson MC, Rohe B, Sterling TM, Norman AW, Boyan BD & Safford SE 2004 Ribozyme knockdown functionally links a 1,25(OH)2D3 membrane binding protein (1,25D3-MARRS) and phosphate uptake in intestinal cells. PNAS 101 7392–7397.[Abstract/Free Full Text]

Pisoni RL 1991 Characterization of a phosphate transport system in human fibroblast lysosomes. Journal of Biological Chemistry 266 979–985.[Abstract/Free Full Text]

Ryan TA, Smith SJ & Reuter H 1996 The timing of synaptic vesicle endocytosis. PNAS 93 5567–5571.[Abstract/Free Full Text]

Warner RR & Coleman JR 1975 Electron probe analysis of calcium transport by small intestine. Journal of Cell Biology 6454–6474.

Zhao B & Nemere I 2002 1,25(OH)2D3-mediated phosphate uptake in isolated chick intestinal cells Effect of 24,25(OH)2D3, signal transduction activators and age. Journal of Cellular Biochemistry 86 497–508.[CrossRef][ISI][Medline]

Received 26 January 2005
Accepted 27 January 2005
Made available online as an Accepted Preprint 7 February 2005




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