Vitamin D and intestinal cell membranes.
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Animals↗
Biomedical subjects
I Nemere
Publications and source records attributed to I Nemere.
Intestinal calcium transport: pleiotropic effects mediated by vitamin D.
The primary biological response to vitamin D is an increase in intestinal calcium transport. Efforts to elucidate the molecular mechanism by which this increased transport occurs has revealed that vitamin D mediates pleiotropic effects that are most likely a result of both genomic and nongenomic mechanisms of action of vitamin D. The spectrum of intestinal responses to vitamin D must now include an alteration in the topography of intestinal epithelial cells' brush border membrane (BBM). Initial indications of such an event were obtained with the polyene antibiotic filipin. Filipin induces active calcium transport in vitro in the small intestine of vitamin D-deficient chicks by specifically interacting with cholesterol and thereby altering membrane organization. Alterations in BBM topography in response to vitamin D were detected with the use of membrane-impermeable protein-labeling reagents and limited proteolysis. Administration of vitamin D to vitamin D-deficient chicks induces specific changes in accessibility of BBM proteins to labeling reagents. In addition, an alteration is induced in the rate of solubilization of BBM hydrolases during limited proteolysis. These vitamin D-mediated alterations in membrane topography offer new possibilities for regulation of intestinal calcium transport.
Animals↗
Early actions of parathyroid hormone and 1,25-dihydroxycholecalciferol on isolated epithelial cells from rat intestine: I. Limited lysosomal enzyme release and calcium uptake.
Epithelial cells isolated from rat intestine were analyzed for their responsiveness in vitro to parathyroid hormone (PTH) and to 1,25-dihydroxycholecalciferol [1,25-(OH)2D3]. Criteria included determination of whether the agonists promoted extracellular liberation of lysosomal enzyme activities above control values during incubation in Ringer's solution at 22 C. PTH-augmented release of the representative hydrolase activities, cathepsin B and acid phosphatase, to the particle-free supernatant fraction of the medium was evident within 5 min of hormone treatment and was sustained in statistically significant degree for at least 30 min to greater than 20% above control levels. Basal release rarely exceeded 15% of the total cellular content of these enzymic activities. Lactate and succinate dehydrogenase activities were undetectable in the particle-free supernatant fraction under conditions of maximal hormone effect, indicating integrity of the cells and selectivity of the organellar response. Treatment of corresponding cells with 1,25-(OH)2D3 resulted in similar time of onset, magnitude, and duration of response. The most sensitive indicator of limited lysosomal labilization by either hormone was beta-N-acetyl-D-glucosaminidase activity, which underwent accentuated extracellular liberation in the presence of as little as 10(-16) M PTH or 10(-11) M 1,25-(OH)2D3, the latter eliciting a response of greater than 40% above control levels. Parathyroidectomy diminished basal release of the hydrolase activities and sensitized the intestinal cells to the action of PTH vs. preparations from intact or sham-operated animals, as judged by excess liberation of the glycosidase. Nontarget lung cells failed to respond to supramaximal levels of either hormone by the criterion of reduced latency of lysosomal hydrolases. In additional acute experiments with intestinal cells, both PTH and 1,25-(OH)2D3 promoted enhanced 45Ca2+ accumulation above control values. Collectively, these data indicate that PTH is capable of provoking direct effects on intestinal cells, similar in onset and extent to those elicited by 1,25-(OH)2D3.
Acetylglucosaminidase↗
Early actions of parathyroid hormone and 1,25-dihydroxycholecalciferol on isolated epithelial cells from rat intestine: II. Analyses of additivity, contribution of calcium, and modulatory influence of indomethacin.
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Animals↗
Putative basal lateral membrane receptors for 24,25-dihydroxyvitamin D(3) in carp and Atlantic cod enterocytes: characterization of binding and effects on intracellular calcium regulation.
The vitamin D metabolite, 24R,25-dihydroxyvitamin D(3) (24R,25(OH)(2)D(3)), was tested for its ability to specifically bind to basal lateral membranes isolated from intestinal epithelium of Atlantic cod (a seawater fish), carp (a freshwater fish), and chicken. Specific saturable binding was demonstrated in membranes from all three species. Membranes from Atlantic cod, carp, and chicken revealed K(d)'s of 7.3 +/- 0.9, 12.5 +/- 0.9 and 7.8 +/- 0.1 nM, and a B(max) for each species estimated to 57.9 +/- 2.9, 195.1 +/- 8.4 and 175 +/- 0.8 fmol/mg protein, respectively. Scatchard analyses indicated a convex curvature and Hill analyses revealed apparent Hill coefficients of 1.84 +/- 0.28, 1.80 +/- 0.29, and 1.78 +/- 0.27 for Atlantic cod, carp and chicken, suggesting a positive cooperative binding in all three species. Basal lateral membranes from Atlantic cod and carp were used to further characterize the binding moiety. In competition studies, basal lateral membranes from Atlantic cod or carp did not discriminate between 24R,25(OH)(2)D(3) and the 24S,25(OH)(2)D(3) isomer, whereas, 1,25(OH)(2)D(3) and 25(OH)D(3), were less effective in competing with [(3)H]24R,25(OH)(2)D(3) for binding to basal lateral membranes in Atlantic cod and carp. In both the Atlantic cod and carp enterocyte basal lateral membranes, the binding activity could be extracted equally well with high salt as with detergent, indicating a peripheral membrane protein rather than an integral membrane binding protein. Finally, isolated Atlantic cod and carp enterocytes were chosen for analyses of signal transduction events mediated by the putative receptor. In both species, 24R,25(OH)(2)D(3) but not 24S,25(OH)(2)D(3), suppressed Ca(2+)-uptake by enterocytes in a dose-dependent manner. Enterocytes from Atlantic cod and carp, acclimated to Ca(2+)-free media, responded by an intracellular Ca(2+)-release within seconds after addition of 24R,25(OH)(2)D(3) or 24S,25(OH)(2)D(3). The effects on intracellular Ca(2+)-release were dose-dependent for both metabolites. 24S,25(OH)(2)D(3) was effective at lower concentrations and triggered a higher response compared to 24R,25(OH)(2)D(3). These results suggest that the binding molecule(s) for 24R,25(OH)(2)D(3) and 24S,25(OH)(2)D(3) is/are capable of acting as a receptor, mediating rapid, non-genomic responses in intestinal cells.
24,25-Dihydroxyvitamin D 3↗
1,25-(OH)2D3 modulates growth plate chondrocytes via membrane receptor-mediated protein kinase C by a mechanism that involves changes in phospholipid metabolism and the action of arachidonic acid and PGE2.
1,25-(OH)2D3 (1,25) exerts its effects on growth plate chondrocytes through classical vitamin D (VDR) receptor-dependent mechanisms, resulting in mineralization of the extracellular matrix. Recent studies have shown that membrane-mediated mechanisms are involved as well. 1,25 targets cells in the prehypertrophic and upper hypertrophic zones of the costochondral cartilage growth plate (GC cells), resulting in increased specific activity of alkaline phosphatase (ALP), phospholipase A2 (PLA2), and matrix metalloproteinases (MMPs). At the cellular level, 1,25 action results in rapid changes in arachidonic acid (AA) release and re-incorporation, alterations in membrane fluidity and Ca ion flux, and increased prostaglandin E1 and E2 (PGE2) production. Protein kinase C (PKC) is activated in a phospholipase C (PLC) dependent-mechanism, due in part to the increased production of diacylglycerol (DAG). In addition, AA acts directly on the cell to increase PKC specific activity. AA also provides a substrate for cyclooxygenase (COX), resulting in PGE2 production. 1,25 mediates its effects through COX-1, the constitutive enzyme, but not COX-2, the inducible enzyme. Time course studies using specific inhibitors of COX-1 show that AA stimulates PKC activity and PKC then stimulates PGE2 production. PGE2 acts as a mediator of 1,25 action on the cells, also stimulating PKC activity. The rapid effects of 1,25 on PKC are nongenomic, occurring within 3 min and reaching maximal activation by 9 min. It promotes translocation of PKC to the plasma membrane. When 1,25 is incubated directly with isolated plasma membranes, PKCalpha is stimulated although PKCzeta is also present. In contrast, when isolated matrix vesicles (MVs) are incubated with 1,25, PKCzeta is inhibited and PKCalpha is unaffected. These membrane-mediated effects are due to the presence of a specific membrane vitamin D receptor (mVDR) that is distinct from the classical cytosolic VDR. Studies using 1,25 analogs with reduced binding affinity for the classical VDR, confirm that rapid activation of PKC by 1,25 is not VDR dependent. The membrane-mediated effects of 1,25 are critical to the regulation of events in the extracellular matrix produced by the chondrocytes. MVs are extracellular organelles associated with maturation of the matrix, preparing it for mineralization. MV composition is under genomic control, involving VDR-mechanisms. In the matrix, no new gene expression or protein synthesis can occur, however. Differential distribution of PKC isoforms and their nongenomic regulation by 1,25 is one way for the chondrocyte to control events at sites distant from the cell. GC cells contain 1a-hydroxylase and produce 1,25; this production is regulated by 1,25, 24,25, and dexamethasone. 1,25 stimulates MMPs in the MVs, resulting in increased proteoglycan degradation in mineralization gels, and increased activation of latent transforming growth factor-beta 1 (TGF-beta1).
Animals↗