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Redistribution of Calbindin-D_28k in Chick Intestine in Response to Calcium Transport^*

Division of Biomedical Sciences and Department of Biochemistry, University of California Riverside, California 92521;
The Molecular Cytology Laboratory, Jerry L. Pettis Memorial Veterans Administration Hospital Loma Linda, California 92357

Address all correspondence and requests for reprints to: Dr. Ilka Nemere, Department of Biochemistry, University of California, Riverside, California 92521.

Abstract

Vitamin D and its hormonally active metabolite 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] are known to alter several parameters associated with stimulated intestinal Ca²⁺ transport: levels of calbindin-D_28K, tubulin, and endosomal-lysosomal organelles containing Ca²⁺, and calbindin-D_28K. In the present study the as yet unexamined relationship among Ca²⁺ transport, calbindin-D_28K, and microtubules was studied by immunofluorescence microscopy.

In vitamin D₃-treated or l,25-(OH)₂D₃-treated chicks, in the absence of Ca²⁺ transport, immunofluorescence microscopy of intestinal tissue fixed at 25 C indicated a colocalization of calbindin-D_28K and tubulin along epithelial cell brush border and basal-lateral membranes. Initiation of in situ Ca²⁺ absorption for 10, 20, or 30 min before tissue fixation resulted first in increased punctate calbindin-D_28K staining and then in a progressive decrease in intestinal cell- and microtubule-associated calbindin-D_28K, with a concomitant increase in calbindin-D_28K labeling in the villus core. When intestinal tissue from 1,25- (OH)₂D₃-treated chicks was chilled to 4 C before fixation (a procedure shown by others to cause microtubule depolymerization), evaluation by immunofluorescence microscopy revealed diffuse cytoplasmic staining of both the immunoreactive tubulin and its associated calbindin-D_28K- These results indicate the possible involvement of calbindin-D_28K with tubulin during the process of Ca²⁺ transport and the secretion of the calbindin-D_28K as a consequence of the overall transport process.

Electron microscopy with immunogold labeling revealed intestinal epithelial calbindin-D_28K to be localized inside of small vesicles and lysosome-like structures, with sparse cytoplasmic labeling. Subsequent electron microscopic analysis of intestinal epithelial microtubules prepared by polymerization and depolymerization revealed immunogold labeling in coprecipitated vesicular remnants, with consistently light staining of filaments traversing segments of the microtubules. In biochemical studies, isolation of intestinal microtubules or tubulin by three distinct procedures revealed increasing levels of associated calbindin- D_28K as a function of time after 1,25-(OH)₂D₃ repletion of vitamin D-deficient chicks. Addition of calbindin-D_28K to intestinal microtubules isolated from vitamin D-deficient chicks exhibited saturable binding when exogenous calbindin-D_28K reached levels comparable to those present in vitamin D-replete chick intestine.

Collectively, these results suggest that calbindin-D_28K is predominantly located in membrane-delimited vesicles, with a very minor component associated with filamentous elements that can be isolated with tubulin and microtubules. Additionally, calbindin-D_28K is dynamically involved in Ca²⁺ transport in the intestine. (Endocrinology 129: 2972-2984,1991)

Footnotes

^* This work was supported in part by USPHS Grants DK-09012-027 (to A.W.N.) and HD-21318 (to W.H.F.).

Received April 12, 1991.

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