Biomedical subjects

I Nemere

Publications and source records attributed to I Nemere.

At least 19 recordsLinked to original sources

PubMed2006

Phosphate uptake in chick kidney cells: effects of 1,25(OH)2D3 and 24,25(OH)2D3.

Phosphate homeostasis is controlled in part by absorption from the intestine, and reabsorption in the kidney. While the effect of Vitamin D metabolites on enterocytes is well documented, in the current study we assess selected responses in primary cultures of kidney cells. Time course studies revealed a rapid stimulation of phosphate uptake in cells treated with 1,25(OH)(2)D(3), relative to controls. Dose-response studies indicated a biphasic curve with optimal stimulation at 300 pM 1,25(OH)(2)D(3) and inhibition at 600 pM seco-steroid. Antibody 099--against the 1,25D(3)-MARRS receptor - abolished stimulation by the steroid hormone. Moreover, phosphate uptake was mediated by the protein kinase C pathway. The metabolite 24,25(OH)(2)D(3), which was found to inhibit the rapid stimulation of phosphate uptake in intestinal cells, had a parallel effect in cultured kidney cells. Finally, the 24,25(OH)(2)D(3) binding protein, catalase, was assessed for longer term down regulation. In both intestinal epithelial cells and kidney cells incubated with 24,25(OH)(2)D(3) for 5-24h, both the specific activity of the enzyme and protein levels were decreased relative to controls, while 1,25(OH)(2)D(3) increased both parameters over the same time periods. We conclude that the Vitamin D metabolites have similar effects in both kidney and intestine, and that 24,25(OH)(2)D(3) may have effects at the level of gene expression.

24,25-Dihydroxyvitamin D 3↗

PubMed2005

Regulation of parathyroid hypertensive factor secretion by vitamin D3 analogs in parathyroid cells derived from spontaneously hypertensive rats.

Parathyroid hypertensive factor (PHF) is a novel substance secreted by the parathyroid gland (PTG), which is elevated in 30-40% of all hypertensive patients; specifically, the low-renin subset. However, very little is known about the regulation of PHF secretion. Since the classical parathyroid regulator, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), may be elevated concurrent with or preceding the development of low-renin hypertension and elevated plasma PHF, we hypothesized that 1,25-(OH)2D3 would stimulate PHF release. To test this hypothesis, PTG organ and cell cultures, derived from spontaneously hypertensive rats (SHR) and the normotensive genetic control Wistar Kyoto (WKY) rats, were exposed to various vitamin D3 metabolites and PHF release measured by ELISA. 1,25-(OH)2D3 rapidly stimulated PHF release with enhanced sensitivity in SHR versus WKY cultures indicated by a leftward shift in the dose-response curve, whereas 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) had the converse effect. Vitamin D3 analog "BT," an agonist for the classical nuclear vitamin D receptor (1,25VDR(nuc)), was without effect suggesting a 1,25VDR(nuc)-independent mechanism and potential involvement of the plasma membrane-bound vitamin D receptor (1,25 D3-MARRS). Interestingly, protein expression of the 1,25 D3-MARRS was increased in SHR versus WKY parathyroid cells. In conclusion, these results support the idea that 1,25-(OH)2D3 may contribute to elevated plasma PHF in the SHR.

24,25-Dihydroxyvitamin D 3↗

PubMed2004

Ribozyme knockdown functionally links a 1,25(OH)2D3 membrane binding protein (1,25D3-MARRS) and phosphate uptake in intestinal cells.

We used a ribozyme loss-of-function approach to demonstrate that the protein product of a cDNA encoding a multifunctional membrane-associated protein binds the seco-steroid 1,25(OH)(2)D(3) and transduces its stimulatory effects on phosphate uptake. These results are paralleled by studies in which the ability of the hormone to stimulate phosphate uptake in isolated chick intestinal epithelial cells is abolished by preincubation with Ab099 directed against the amino terminus of the protein. We now report the complete sequence of the cloned chicken cDNA for the 1,25D(3)-MARRS (membrane-associated, rapid-response steroid-binding) protein and reveal it to be identical to the multifunctional protein ERp57. Functional studies showed that active ribozyme, but not a scrambled control, decreased specific membrane-associated 1,25(OH)(2)D(3) binding, but did not affect binding to the nuclear receptor for 1,25(OH)(2)D(3). Seco-steroid-dependent stimulation of protein kinase C activity was diminished as 1,25D(3)-MARRS protein levels were reduced in the presence of the ribozyme, as judged by Western blot analyses. Phosphate uptake in isolated cells is an index of intestinal phosphate transport that occurs during growth and maturation. Whereas cells and perfused duodena robustly responded to 1,25(OH)(2)D(3) in preparations from young birds, older animals no longer responded with stimulated phosphate uptake or transport. The age-related decline was accompanied by a decrease in 1,25D(3)-MARRS mRNA that was apparent up to 1 year of age. Together, these studies functionally link phosphate transport in the chick duodenum with the 1,25D(3)-MARRS protein and point to a previously uncharacterized role for this multifunctional protein class.

Amino Acid Sequence↗

PubMed2004

Receptors for steroid hormones: membrane-associated and nuclear forms.

Steroid hormones are now recognized to act through both nuclear and membrane-associated receptors. In this review we focus on receptors for estrogen and the vitamin D metabolite 1,25-dihydroxyvitamin D(3). While the nuclear receptors are part of a 'superfamily' with common structural elements, membrane receptors are more diverse, ranging from variants of the nuclear forms to unrelated proteins. We conclude that both rapid (membrane-initiated) actions, as well as regulation of gene transcription, are necessary to explain the complex actions of steroid hormones on target cells.

Animals↗

PubMed2003

Membrane receptors for vitamin D steroid hormones: potential new drug targets.

There is increasing evidence that steroid hormones derived from vitamin D act through classical nuclear receptors (nVDR), as well as specific binding sites on the plasma membrane of target cells that are coupled to signal transduction systems. These sites are referred to as Membrane Associated, Rapid Response Steroid (MARRS) binding proteins or complexes. In the case of the seco-steroid 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the two 'receptors' appear to be different proteins with distinct affinities for vitamin D analogs. These differences may be useful in a number of clinical situations. In treating leukemias, it would be useful to promote the actions of the nVDR for differentiation to macrophages while blocking the 1,25D3-MARRS in intestine, which might contribute to the undesirable side effect of hypercalcemia. In contrast, stimulation of the intestinal 1,25D3-MARRS would be desirable in the elderly, since this signalling system appears to decline with age in model systems, potentially contributing to diminished intestinal absorption of calcium and associated bone loss. Bone itself is known to have osteoblasts that respond to 1,25(OH)2D3 through both nVDR and 1,25D3-MARRS mechanisms. Both systems are required for bone-building activities. Osteoclasts lack the nVDR, but may become activated through the 1,25D3-MARRS, offering another site of drug intervention in the treatment of osteoporosis. Finally, during tooth mineralization, immunohistochemical studies reveal an absence of the nVDR and a marked appearance of the 1,25D3-MARRS. In addition to our growing knowledge of 1,25(OH)2D3, the physiological actions of a lesser studied metabolite of vitamin D, 24,25(OH)2D3, are coming to light and may offer additional targets for pharmaceutical modulation.

24,25-Dihydroxyvitamin D 3↗

PubMed2003

Putative membrane receptor for 1,25(OH)2 vitamin D3 in human mineralized tissues during prenatal development.

The calciotropic hormone, 1,25(OH)2vitamin D3[1,25(OH)2D3], controls the formation of dental and bone mineralized tissues. The role of nuclear 1,25(OH)2D3 receptor has been extensively studied in the diverse secretory cells, i.e., osteoblasts, chondrocytes, ameloblasts, and odontoblasts. A nongenomic pathway also has been characterized and related to the interactions of 1,25(OH)2D3 ligand with a putative cell membrane receptor. This recognition moiety called 1,25(OH)2vitamin D3 membrane-associated, rapid-response steroid-binding [1,25D3-MARRS] protein is investigated here in the craniofacial skeleton of human embryos and fetuses. Immunolocalization using a specific Ab099 against chick intestinal basolateral 1,25D3-MARRS protein was performed. The data show a complementary expression pattern of the membrane receptor when compared with published data on the nuclear receptor, notably during amelogenesis. In mandible, membrane receptors for 1,25(OH)2D3 were identified in the heterogenous bone cell population, including osteoblasts and osteoclasts. Differential 1,25D3-MARRS protein levels were observed in distinct developmental stages and anatomical sites of tooth and bone, suggesting the existence of cross-talk between local factors and 1,25D3-MARRS protein expression.

Gestational Age↗

PubMed2002

Does PTH have a direct effect on intestine?

Dogma for the past three decades has dictated that parathyroid hormone (PTH) has no direct effect on intestine with regard to calcium or phosphate absorption, but rather that PTH acts to promote the synthesis of a hormonally active form of vitamin D, namely 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. However, diverse laboratories have each provided some evidence to suggest PTH does indeed have a direct effect on intestine. We will briefly review the evidence for biological effects, biochemical effects, and the presence of intestinal receptors for PTH, and conclude with the implications for biomedical research.

Animals↗

PubMed2002

Biochemical characterization and purification of a binding protein for 24,25-dihydroxyvitamin D3 from chick intestine.

An earlier study revealed that 24R,25-dihydroxyvitamin D(3) (24R,25(OH)(2)D(3)) inhibits the rapid actions of 1,25(OH)(2)D(3) on stimulation of calcium transport in perfused duodena, as well as activation of protein kinases C and A. In the present work, a specific binding protein (24,25-BP) has been identified and partially characterized. Percoll-gradient resolution of differential centrifugation fractions from mucosal homogenates revealed the highest levels of specific [(3)H]24R,25(OH)(2)D(3) binding to be in lysosomes (approximately eight to tenfold greater than in basal lateral membrane fractions). Incubation of isolated enterocytes with 6.5 nM [(3)H]24R,25(OH)(2)D(3) for 10 s also demonstrated targeting of the steroid to lysosomal fractions. Using freshly isolated lysosomal fractions, time course studies indicated maximal specific binding after a 2-h incubation on ice. Western analyses revealed that the serum transport protein, DBP (vitamin D binding protein), was absent from both lysosomal and basal lateral membrane fractions. Protein dependence studies demonstrated linear binding between 0.05 and 0.155 mg of lysosomal protein. Saturation analyses yielded K(d)=7.4+/- 1.8 nM, B(max)=142+/-16 fmol/mg protein for lysosomes, and K(d)=8.5 nM, B(max)=149+/-25 fmol/mg protein for basal lateral membranes. Hill analyses of lysosomal binding yielded a Hill coefficient of 0.57+/-0.11, indicative of negative cooperativity. Studies with lysosomal proteins revealed a 81%+/-7% competition of 24S,25(OH)(2)D(3) with [(3)H]24R,25(OH)(2)D(3) for binding (P>0.05, relative to competition with 24R,25(OH)(2)D(3)), while 25(OH)D(3) and 1,25(OH)(2)D(3) yielded 53%+/-13% and 39%+/-11% competition respectively (each, P<0.05, relative to competition with 24R,25(OH)(2)D(3)). The apparent affinity of 24S,25(OH)(2)D(3) for 24,25-BP led to testing of the metabolites effectiveness in the perfused duodenal loop system. Vascular perfusion with 130 pM 1,25(OH)(2)D(3) stimulated (45)Ca transport to 2.5-fold above control levels after 40 min, while simultaneous perfusion with 6.5 nM 24S,25(OH)(2)D(3) and 130 pM 1,25(OH)(2)D(3) abolished the stimulatory activity completely. Purification of the 24,25-BP by chromatography revealed a single protein band upon SDS-PAGE and silver staining of 66 kDa. The combined results suggest that 24R,25(OH)(2)D(3) may mediate its hormonal activities through a specific binding protein.

24,25-Dihydroxyvitamin D 3↗

PubMed2001

Steroid hormone receptors in target cell membranes.

Numerous reports of rapid steroid hormone effects in diverse cell types cannot be explained by the generally prevailing theory that centers on the activity of hormone receptors located exclusively in the nucleus. Cell membrane forms of steroid hormone receptors coupled to intracellular signaling pathways may also play an important role in hormone action. Membrane-initiated signals appear to be the primary response of the target cell to steroid hormones and may be prerequisite to subsequent genomic activation. Recent dramatic advances in this area have intensified efforts to delineate the nature and biologic roles of all receptor molecules that function in steroid hormone-signaling pathways. This work has profound implications for our understanding of the physiology and pathophysiology of hormone actions in responsive cells and may lead to development of novel approaches for the treatment of many cell proliferative, metabolic, inflammatory, reproductive, cardiovascular, and neurologic defects.

Animals↗

PubMed2000

Immunochemical studies on the putative plasmalemmal receptor for 1, 25-dihydroxyvitamin D(3). III. Vitamin D status.

The effect of vitamin D status on levels of the putative 1, 25(OH)(2)D(3) membrane receptor (pmVDR) was studied in chick intestine, kidney, and brain. Western analyses and assays for specific [(3)H]1,25(OH)(2)D(3) binding indicated that, in intestine, pmVDR levels were greatest in -D chicks relative to +1,25D and +D animals (P < 0.05). In kidney, protein levels and specific binding followed the order +D > +1,25D, -D. In brain, vitamin D status did not affect protein levels or specific binding levels. In tissue from normal chicks, both protein and specific binding followed the order of intestine > kidney > brain membranes. Intestinal cells were further evaluated for the effect of 1,25(OH)(2)D(3) on selected "rapid responses." Extrusion of (45)Ca in response to 130 pM 1, 25(OH)(2)D(3) in vitro was greater in cells from -D chicks than from +1,25D or normal birds. Analyses of signal transduction events revealed diminished hormone-induced intracellular calcium oscillations (as assessed by fura-2 fluorescence), and lack of steroid-enhanced protein kinase (PK) A activity in intestinal epithelial cells from -D chicks relative to +D chicks. PK C activation by 130 pM 1,25(OH)(2)D(3) was approximately twofold in cells from +D or -D chicks. The combined results indicate that vitamin D status differentially affects the pmVDR in intestine, kidney, and brain. In intestine, vitamin D deficiency differentially affects (45)Ca handling, intracellular calcium oscillations, PK A and PK C activities in response to 1,25(OH)(2)D(3).

Animals↗

PubMed2000

Immunochemical studies on the putative plasmalemmal receptor for 1, 25(OH)(2)D(3). I. Chick intestine.

Antisera were raised against the NH(2)-terminus of the putative basal lateral membrane (BLM) receptor for 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3); BLM-VDR]. In Western analyses of BLM proteins, antibody (Ab) 099 was monospecific for a 64.5-kDa band. A protein of 64.5 kDa was also labeled by the affinity ligand [(14)C]1, 25(OH)(2)D(3)-bromoacetate; label was diminished in the presence of excess unlabeled secosteroid. The monoclonal antibody against the nuclear VDR (9A7) failed to detect an appropriate band in BLM fractions. Preincubation of isolated intestinal cells with Ab 099, but not 9A7, affected the following two 1,25(OH)(2)D(3)-mediated signal transduction events: augmented intracellular calcium and protein kinase C activity. Subcellular distribution of Ab 099 reactivity by Western analyses and fluorescence microscopy revealed the highest concentrations in BLM followed by the endoplasmic reticulum. Exposure of isolated intestinal cells to 1,25(OH)(2)D(3) for 10 s or vascular perfusion of duodena for 5 min resulted in a time-dependent increase in nuclear localization of the BLM-VDR antigen, as judged by electron microscopy, whereas 24, 25-dihydroxyvitamin D(3) failed to increase antigenic labeling in nuclei. Densitometric quantitation of Western blots of subcellular fractions prepared from isolated intestinal cells treated with vehicle or 1,25(OH)(2)D(3) confirmed a hormone-induced increase of putative BLM-VDR in the nucleus. It is concluded that a novel cell surface binding protein for 1,25(OH)(2)D(3) has been identified.

Affinity Labels↗

PubMed2000

A specific binding moiety for 1,25-dihydroxyvitamin D(3) in basal lateral membranes of carp enterocytes.

Carp (Cyprinus carpio), a freshwater fish that lives in a low-calcium environment, and Atlantic cod (Gadus morhua), a seawater fish that lives in a high-calcium environment, were studied for the presence of a novel membrane binding protein ("receptor") for the vitamin D metabolite, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. Basal lateral membranes from enterocytes of either species were prepared and analyzed for specific saturable binding. Membranes from carp revealed a dissociation constant of 1.23 nM with a maximal binding capacity of 212 fmol/mg protein. In comparison, membranes from Atlantic cod enterocytes revealed very low and nonsignificant levels of specific binding. The [(3)H]1,25(OH)(2)D(3) binding activity in carp was further characterized for protein dependence, detergent extractability, and competition for binding with the metabolites 25(OH)D(3) and 24R,25(OH)(2)D(3). Finally, introduction of 1,25(OH)(2)D(3) to isolated carp enterocytes enhanced protein kinase C activity within 5 min, whereas intracellular Ca(2+) concentrations were unaffected by a range of 1,25(OH)(2)D(3) concentrations, as judged by fura 2 fluorescence. Thus the binding moiety may be a putative plasma membrane receptor for vitamin D, because it is functionally coupled to at least one signal transduction pathway.

Animals↗

PubMed1999

Immunochemical studies on the putative plasmalemmal receptor for 1,25-dihydroxyvitamin D3 II. Chick kidney and brain.

Chick kidney and brain were analyzed for the subcellular distribution (if any) of a putative plasma membrane receptor for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Fractionation protocols were found to be based not only on differential centrifugation conditions, but also gentleness of resuspension procedures, and sufficiently dense Percoll gradients. The postnuclear pellets were resolved on 21.85% Percoll gradients overlayed on 2.4 M sucrose cushions. For both kidney and brain, fraction 1 (bottom of tube) was found to be enriched over whole homogenate 5.4- and 1.6-fold, respectively, in acid phosphatase activity, fractions 2 through 5 were enriched four- and eightfold, respectively, in succinate dehydrogenase activity, fraction 8 contained Golgi, as judged by a small peak of alpha-mannosidase activity, and fraction 9 was enriched sevenfold (for each tissue) in Na+,K+-ATPase activity. Western analyses, using a characterized antibody to the putative chick intestinal plasma membrane vitamin D receptor, revealed the highest levels of antigenicity in both chick kidney and brain in plasma membrane and Golgi fractions, followed by unidentified membranes in fractions 6 and 7 of Percoll gradients. Distribution of specific binding of [3H]1,25(OH)2D3 in Percoll gradient fractions paralleled that of antigenicity. Qualitatively, kidney plasma membrane contained more antigen than brain plasma membrane after Western blot analyses; these results were mirrored by differences in specific binding of the tritiated secosteroid (65 +/- 14.5 and 34 +/- 11.9 fmol/mg of protein, respectively).

Acid Phosphatase↗

PubMed1999

Physiological importance of the 1,25(OH)2D3 membrane receptor and evidence for a membrane receptor specific for 24,25(OH)2D3.

We have recently identified a membrane vitamin D receptor (mVDR) specific for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and shown that it mediates the rapid activation of protein kinase C (PKC) in growth zone chondrocytes (GCs). In this study, we examine the role of the 1, 25(OH)2D3-mVDR in chondrocyte physiology and provide evidence for the existence of a specific membrane receptor for 24, 25-dihydroxyvitamin D3 (24,25(OH)2D3-mVDR). Fourth-passage cultures of growth plate chondrocytes at two distinct stages of endochondral development, resting zone (RC) and growth zone (GC) cells, were used to assess the role of the mVDR in cell proliferation, PKC activation, and proteoglycan sulfation. To preclude the involvement of the nuclear vitamin D receptor (nVDR), we used hybrid analogs of 1, 25(OH)2D3 with <0.1% affinity for the nVDR (2a, 1alpha-CH2OH-3beta-25D3; 3a, 1alpha-CH2OH-3beta-20-epi-22-oxa-25D3; and 3b, 1beta-CH2OH-3alpha-20-epi-22-oxa-25D3). To determine the involvement of the mVDR, we used an antibody generated against the highly purified 1,25(OH)2D3 binding protein from chick intestinal basolateral membranes (Ab99). Analog binding to the mVDR was demonstrated by competition with [3H]1,25(OH)2D3 using matrix vesicles (MVs) isolated from cultures of RC and GC cells. Specific recognition sites for 24,25(OH)2D3 in RC MVs were demonstrated by saturation binding analysis. Specific binding of 24,25(OH)2D3 was also investigated in plasma membranes (PMs) from RC and GC cells and GC MVs. In addition, we examined the ability of Ab99 to block the stimulation of PKC by analog 2a in isolated RC PMs as well as the inhibition of PKC by analog 2a in GC MVs. Like 1,25(OH)2D3, analogs 2a, 3a, and 3b inhibit RC and GC cell proliferation. The effect was dose dependent and could be blocked by Ab99. In GC cells, PKC activity was stimulated maximally by analogs 2a and 3a and very modestly by 3b. The effect of 2a and 3a was similar to that of 1, 25(OH)2D3 and was blocked by Ab99, whereas the effect of 3b was unaffected by antibody. In contrast, 2a was the only analog that increased PKC activity in RC cells, and this effect was unaffected by Ab99. Analog 2a had no effect on proteoglycan sulfation in RC cells, whereas analogs 3a and 3b stimulated it and this was not blocked by Ab99. Binding of [3H]1,25(OH)2D3 to GC MVs was displaced completely with 1,25(OH)2D3 and analogs 2a, 3a, and 3b, but 24, 25(OH)2D3 only displaced 51% of the bound ligand. 24,25(OH)2D3 displaced 50% of [3H]1,25(OH)2D3 bound to RC MVs, but 2a, 3a, and 3b displaced <50%. Scatchard analysis indicated specific binding of 24, 25(OH)2D3 to recognition sites in RC MVs with a Kd of 69.2 fmol/ml and a Bmax of 52.6 fmol/mg of protein. Specific binding for 24, 25(OH)2D3 was also found in RC and GC PMs and GC MVs. GC membranes exhibited lower specific binding than RC membranes; MVs had greater specific binding than PMs in both cell types. 2a caused a dose-dependent increase in PKC activity of RC PMs that was unaffected by Ab99; it inhibited PKC activity in GC MVs, and this effect was blocked by Ab99. The results indicate that the 1, 25(OH)2D3 mVDR mediates the antiproliferative effect of 1,25(OH)2D3 on chondrocytes. It also mediates the 1,25(OH)2D3-dependent stimulation of PKC in GC cells, but not the 2a-dependent increase in RC PKC activity, indicating that 24,25(OH)2D3 mediates its effects through a separate receptor. This is supported by the failure of Ab99 to block 2a-dependent stimulation of PKC in isolated PMs. The data demonstrate for the first time the presence of a specific 24, 25(OH)2D3 mVDR in endochondral chondrocytes and show that, although both cell types express mVDRs for 1,25(OH)2D3 and 24,25(OH)2D3, their relative distribution is cell maturation-dependent.

Animals↗

PubMed1999

24,25-dihydroxyvitamin D3 suppresses the rapid actions of 1, 25-dihydroxyvitamin D3 and parathyroid hormone on calcium transport in chick intestine.

Studies were undertaken to determine whether 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) modulates the rapid effects of 1, 25-dihydroxyvitamin D3 (1,25(OH)2D3) and parathyroid hormone (PTH) on calcium transport in the perfused chick intestine. Perfusion with control media resulted in a transport ratio (treated/average basal) of 1.07 +/- 0.06 at t = 40 minutes, while perfusion with 65, 130, 300, or 650 pM 1,25(OH)2D3 yielded ratios of 1.92 +/- 0.23, 2.6 +/- 0.4, 2.8 +/- 0.08, and 3.34 +/- 0.37, respectively. Simultaneous perfusion with each of these doses and 6.5 nM 24,25(OH)2D3 reduced treated/average basal ratios to approximately 1.4 after 40 minutes of perfusion. Vascular perfusion with 65 pM bovine PTH [bPTH(1-34)] stimulated intestinal calcium transport ratios to 3.0 +/- 0.5 after 40 minutes, while the inclusion of 6.5 nM 24,25(OH)2D3 reduced ratios at this time point to 0.56 +/- 0.19. To investigate the effect of these agents on signal transduction, isolated intestinal cells were monitored for intracellular calcium changes using the indicator dye fura-2. After establishing a stable baseline, addition of 130 pM 1,25(OH)2D3 induced rapid calcium oscillations. Intestinal cells exposed to 6.5 nM 24,25(OH)2D3 also exhibited rapid oscillations in fluorescence, which were not further altered by subsequent addition of 1,25(OH)2D3. Incubation of isolated cells with 130 pM 1,25(OH)2D3 was found to increase protein kinase C (PKC) activity within 5 minutes, and protein kinase A (PKA) activity within 7 minutes. Exposure of cells to 65 pM bPTH(1-34) had minimal effect on PKC activity, but resulted in pronounced increases in PKA activity. Stimulation of protein kinases by either secosteroid or peptide hormone was inhibited in the presence of 6.5 nM 24,25(OH)2D3. It is concluded that 24,25(OH)2D3 may exert endocrine actions on intestine.

24,25-Dihydroxyvitamin D 3↗

PubMed1998

Membrane receptors for steroid hormones: a case for specific cell surface binding sites for vitamin D metabolites and estrogens.

Steroid hormones, including vitamin D metabolites and estrogens, activate target cells through specific receptors that discriminate among ligands based upon recognition of distinct structural features. For both classes of ligands, cell surface and nuclear receptors co-exist in many target cells. Upon ligand binding, these receptors generate both rapid and long lasting responses. While the structure of the nuclear receptors and their function as transcriptional activators of specific target genes is generally understood, the identity of the membrane receptors remains elusive. Using pharmacological, functional and biochemical approaches, new insights are being gained into nature of the cell surface receptors for both vitamin D metabolites and estrogens.

Animals↗

PubMed1998

Identification of a membrane receptor for 1,25-dihydroxyvitamin D3 which mediates rapid activation of protein kinase C.

This paper is the first definitive report demonstrating a unique membrane receptor for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) which mediates the rapid and nongenomic regulation of protein kinase C (PKC). Previous studies have shown that 1,25(OH)2D3 exerts rapid effects on chondrocyte membranes which are cell maturation-specific, do not require new gene expression, and do not appear to act via the traditional vitamin D receptor. We used antiserum generated to a [3H]1,25(OH)2D3 binding protein isolated from the basal lateral membrane of chick intestinal epithelium (Ab99) to determine if rat costochondral resting zone (RC) or growth zone (GC) cartilage cells contain a similar protein and if cell maturation-dependent differences exist. Immunohistochemistry demonstrated that both RC and GC cells express the protein, but levels are highest in GC. The binding protein is present in both plasma membranes and matrix vesicles and has a molecular weight of 66,000 Da. The 66 kDa protein in GC matrix vesicles has a Kd of 17.2 fmol/ml and Bmax of 124 fmol/mg of protein for [3H]1,25(OH)2D3. In contrast, the 66 kDa protein in RC matrix vesicles has a Kd of 27.7 fmol/ml and a Bmax of 100 fmol/mg of protein. Ab99 blocks the 1,25(OH)2D3-dependent increase in PKC activity in GC chondrocytes, indicating that the 1,25(OH)2D3-binding protein is indeed a receptor, linking ligand recognition to biologic function.

Animals↗

PubMed1996

Apparent nonnuclear regulation of intestinal phosphate transport: effects of 1,25-dihydroxyvitamin D3,24,25-dihydroxyvitamin D3, and 25-hydroxyvitamin D3.

The steroid hormone 1,25-(OH)2D3 modulates a number of cellular functions through nonnuclear pathways, particularly the stimulation of intestinal calcium transport (transcaltachia). The present studies determined whether 1,25-(OH)2D3 rapidly enhanced phosphate transport in the perfused duodenal loop of normal chicks. Vascular perfusion with 65 pM 1,25-(OH)2D3 significantly stimulated the appearance of 33P in the venous effluent within 2-8 min, reaching an average of 160% of control levels by 40 min. Lumenal perfusion with hormone failed to augment levels of 33P in the venous effluent. Vascular perfusion with a range of 1,25-(OH)2D3 concentrations yielded an apparent biphasic dose-response curve. Two additional vitamin D metabolites, 24,25(OH)2D3 and 25(OH)D3, which initiate transcaltachia, were tested for their effect on phosphate transport. Neither 6.5 nM 24,25(OH)2D3 nor 100 nM 25(OH)D3 stimulated 33P movement from the lumen to the venous effluent. When duodena were vascularly perfused with 65 pM 1,25-(OH)2D3 and either 6.5 nM 24,25(OH)2D3 or 100 nM 25(OH)D3, enhanced phosphate transport was attentuated or abolished. Phosphate transport was also analyzed at metabolite levels 5-fold lower or higher than those in normal chicks. For 24,25(OH)2D3, 1.3 nM metabolite did not augment phosphate transport, although stimulation did occur at 32.5 nM steroid (180 +/- 0.2% of controls). For 25(OH)D3, no stimulation was observed at 500 nM metabolite, whereas 20 nM steroid resulted in transport that was 160 +/- 0.14% of controls. The presence or absence of lumenal Ca2+ did not influence phosphate transport in duodena vascularly perfused with control medium, 65 pM 1,25-(OH)2D3, or 6.5 nM 24,25(OH)2D3. In contrast, vascular perfusion with 100 nM 25(OH)D3 stimulated phosphate transport when lumenal Ca2+ was present but not when it was absent. The influence of lumenal P(i) on transcaltachia was then studied. Although 65 pM 1,25-(OH)2D3, 6.5 nM 24,25(OH)2D3, and 100 nM 25(OH)D3, initiate transcaltachia in the presence of lumenal P(i), the absence of the anion abolished the stimulatory effects of 1,25-(OH)2D3 and 24,25(OH)2D3 on calcium transport but not those induced by 25(OH)D3. These data suggest a complex regulation of both calcium and phosphate transport based on relative levels of circulating vitamin D metabolites as well as the ionic content of the lumen.

24,25-Dihydroxyvitamin D 3↗