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I Nemere

Publications and source records attributed to I Nemere.

At least 37 records · Page 2Linked to original sources
PubMed1996

Parathyroid hormone rapidly stimulates phosphate transport in perfused duodenal loops of chicks: lack of modulation by vitamin D metabolites.

Intestinal tissue of normal (vitamin D-replete) chicks was analyzed for responsiveness to PTH by the criteria of phosphate transport in perfused duodenal loops and binding of iodinated hormone to isolated basal lateral membranes. Using the N-terminal fragment of the bovine hormone [bPTH-(1-34)], the peptide agonist was found to stimulate phosphate transport within 4 min of perfusion (P < 0.05), reaching 152% of the control level after 40 min of exposure to 65 pM hormone. Dose-response analyses revealed an apparent optimum between 65-650 pM bPTH-(1-34), with decreased responsiveness at 6.5 nM hormone. Perfusion of duodenal loops with 65 pM 1,25-dihydroxyvitamin D3[1,25-(OH)2D3] also stimulated phosphate transport, although a combination of both hormones did not have additive effects. Augmented phosphate transport mediated by bPTH-(1-34) was not attenuated by simultaneous perfusion with 6.5 nM 24,25-(OH)2D3, in contrast to earlier findings with 1,25-(OH)2D3-stimulated phosphate transport. Subcellular fractionation of duodena vascularly perfused with control medium or 65 pM bPTH-(1-34) for 5 min revealed hormone-augmented levels of 32P in lysosomal and mitochondrial fractions and decreased soluble 32P relative to control levels. In parallel studies, 65 pM 1,25-(OH)2D3 resulted in qualitatively similar subcellular distributions of radionuclide, whereas simultaneous perfusion with 65 pM 1,25-(OH)2D3 and 6.5 nM 24,25-(OH)2D3 abolished the increase in lysosomal 32P, but not augmented mitochondrial, levels. These findings suggest that lysosomes are vesicular phosphate carriers. Finally, preparation of isolated basal lateral membranes for binding studies with 125I-labeled [Nle8.18,Tyr34]bPTH-(1-34) amide indicated specific saturable binding with a Kd of 0.2 nM. At 1000-, 100-, and 10-fold excesses of unlabeled competitor, bPTH-(1-34) was 90%, 80%, and 33%, respectively, as effective as the analog in displacing labeled peptide. The combined data suggest that PTH may play a significant physiological role in regulating intestinal phosphate absorption.

Animals↗
PubMed1995

Chemistry and conformation of vitamin D molecules.

1 alpha,25-Dihydroxyvitamin D3 (1,25) is a structurally unique steroid hormone because it not only possesses the complete 25-hydroxycholesterol side chain, but most notably, it possesses a seco-B triene structure (it lacks a B-ring and is usually depicted in a non-steroidal, extended conformation). In contrast, the classical steroid hormones possess a truncated side chain (progesterone, cortisol, and aldosterone) or no side chain (estradiol and testosterone) and they all possess the fully intact ABCD steroid rings. These structural differences render the seco-B-steroid 1,25 considerably more conformationally flexible. Since 1,25 is now known to target a myriad of tissues where specific interactions occur to produce an array of biological responses, it is of interest to determine whether different topologies of 1,25 (resulting from different conformational orientations of 1,25) are necessary to interact effectively at the different target sites. The array of biological responses include both non-genomic and genomic effects and there is considerable promise for the efficacy of 1,25 analogs as chemotherapeutic agents in a variety of human disease states. For the non-genomic calcium transport response of transcaltachia, the finding that two 6-s-cis locked analogs, 1 alpha,25-dihydroxyprevitamin D3 (pre-1,25) and 1 alpha,25-dihydroxylumisterol3 (1,25-Lumi), are equipotent to 1,25, points strongly to the involvement of the 6-s-cis conformer of 1,25 as the biologically active conformer. Since there is a continuum of easily interconvertible 6,7-single bond conformers of the seco-B ring available to 1,25, conformational minima (either local or global) may have little to do with the manner in which 1,25 is bound to receptor. For the genomic calcium transport response, and for other genomic (or non-genomic) effects, there is no clear evidence whether the steroidal (s-cis) or non-steroidal (s-trans) conformer of 1,25 is involved. In order to address this matter further, efforts are underway to evaluate other conformationally locked analogs of 1,25 which might mimic either the planar 6-s-trans-1,25 or some intermediate conformer between it and the planar-6-s-cis form.

Animals↗
PubMed1995

Nongenomic effects of 1,25-dihydroxyvitamin D3: potential relation of a plasmalemmal receptor to the acute enhancement of intestinal calcium transport in chick.

A number of model systems now exist for studying the nonnuclear actions of the seco-steroid hormone 1,25(OH)2D3. The perfused duodenal loop of vitamin D-replete chicks has provided the best correlation between nonnuclear actions and a physiological end point, namely enhanced calcium transport. Recent progress has been made in identifying and purifying an integral protein of the basal lateral membrane that may be a receptor for 1,25(OH)2D3. Studies with analogues (particularly 1,25(OH)2-7-dehydrocholesterol and 1,25(OH)2-lumisterol3) have provided definite correlations between binding to the solubilized membrane receptor and the ability to initiate transcaltachia (the rapid hormonal stimulation of calcium transport).

Animals↗
PubMed1994

Identification of a specific binding protein for 1 alpha,25-dihydroxyvitamin D3 in basal-lateral membranes of chick intestinal epithelium and relationship to transcaltachia.

The steroid hormone 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) elicits biological responses by both genomic and nongenomic pathways. This report describes purification of a receptor for 1 alpha,25-(OH)2D3 (VDR) located in the basal-lateral membrane (BLM) of vitamin D-replete chick intestinal epithelium, which is implicated in the nongenomic stimulation of calcium transport (transcaltachia). The BLM-VDR exhibited saturable binding for [3H]1,25-(OH)2D3 (KD = 0.72 x 10(-9)M, Bmax = 0.24 pmol/mg of protein). A 4500-fold purification of the BLM-VDR receptor was achieved. In addition, saturable binding was observed for [3H]24R,25-(OH)2D3 at physiologically relevant levels (KD = 19 x 10(-9) M, Bmax = 2.4 pmol/mg of protein) to a component apparently distinct from the 1 alpha,25-(OH)2D3 BLM-VDR. A functional correlation between the BLM-VDR and transcaltachia was observed in three experimental situations: (i) vitamin D deficiency, which suppresses transcaltachia, resulted in reduced specific [3H]1 alpha,25-(OH)2D3 binding in the BLM-VDR, relative to corresponding fractions from vitamin D-replete chicks; (ii) the BLM-VDR exhibited down-regulation of specific [3H]1 alpha,25-(OH)2D3 binding following exposure to nonradioactive 1 alpha,25-(OH)2D3; and (iii) the relative potencies of two "6-s-cis" analogs of 1 alpha,25-(OH)2D3 to initiate transcaltachia and their ability to compete with [3H]1 alpha,25-(OH)2D3 for binding to the BLM-VDR were parallel. The combined results support the existence of a plasmalemal 1 alpha,25-(OH)2D3 receptor which is a prime candidate for signal transduction in transcaltachia.

Animals↗
PubMed1994

Nonnuclear effects of the steroid hormone 1 alpha,25(OH)2-vitamin D3: analogs are able to functionally differentiate between nuclear and membrane receptors.

The steroid hormone 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] stimulates biological responses via both genomic mechanisms and nongenomic mechanisms (opening of voltage-gated Ca2+ channels). We report here that the two closed B-ring steroid analogs of 1 alpha,25(OH)2D3, 1 alpha,25(OH)2-7-dehydrocholesterol and 1 alpha,25(OH)2-lumisterol3, are able to generate the nongenomic response, transcaltachia, without the ability to compete with the natural metabolite for binding to its nuclear receptor. We propose that the nongenomic membrane associated receptor can accept the ligand in its closed "6-s-cis" conformation whereas the nuclear receptor prefers the extended "6-s-trans" conformer.

Animals↗
PubMed1993

Demonstration that 1 beta,25-dihydroxyvitamin D3 is an antagonist of the nongenomic but not genomic biological responses and biological profile of the three A-ring diastereomers of 1 alpha,25-dihydroxyvitamin D3.

The steroid hormone 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) generates biological responses via both genomic and nongenomic mechanisms. This article reports the biological profile of four A-ring diastereomers of this secosteroid (results are expressed as percentage of the response of 1 alpha,25-(OH)2D3. The activity of the compounds, 1 alpha,25-(OH)2D3, 1 alpha,25-(OH)2-3-epivitamin D3, 1 beta,25-(OH)2D3, 1 beta,25-(OH)2-3-epivitamin D3, for in vivo intestinal Ca2+ absorption and bone Ca2+ mobilization and in vitro binding to the nuclear receptor (genomic responses) was, respectively, 100, 2.8, < 0.1, and < 0.1% (intestinal Ca2+ absorption); 100, 1.5, < 0.1, and < 0.1% (bone Ca2+ mobilization); 100, 24, 0.2, and 0.8% (receptor binding). In the in vivo nongenomic transcaltachia assay the results were 100, 80, 0, and 20-30%, and in ROS 17/2.8 cells (45Ca2+ uptake through voltage-gated Ca2+ channels) 1 alpha,25-(OH)2D3 had 100% activity and 1 beta,25-(OH)2D3 (the only diastereomer evaluated) had no agonist activity. Keratinocyte proliferation was inhibited in the order 1 alpha,25-(OH)2D3 > 1 alpha,25-(OH)2-3-epivitamin D3 > 1 beta,25-(OH)2D3 > 1 beta,25-(OH)2-3-epivitamin D3. 1 beta,25-(OH)2D3 was a potent antagonist of 1 alpha,25-(OH)2D3-mediated transcaltachia and 45Ca2+ uptake in ROS 17/2.8 cells but was unable to block the genomic 1 alpha,25-(OH)2D3 induction of chick calbindin-D28k (in vivo), induction of MG-63 cell osteocalcin, and HL-60 cell differentiation. These results suggest that analogs of 1 alpha,25-(OH)2D3 may be synthesized which are selective agonists or antagonists of genomic or nongenomic responses in the vitamin D endocrine system.

Animals↗
PubMed1993

Structure-function studies of 1,25-dihydroxyvitamin D3 and the vitamin D endocrine system. 1,25-dihydroxy-pentadeuterio-previtamin D3 (as a 6-s-cis analog) stimulates nongenomic but not genomic biological responses.

The hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) generates biological responses via both genomic and nongenomic mechanisms. This article addresses activity differences between the 6-s-trans (extended) and the 6-s-cis (steroid-like) conformation of 1,25-(OH)2D3 to initiate these responses. Because of facile interconversion of the 6-s-trans and 6-s-cis conformers of 1,25-(OH)2D3, kinetically competent amounts of both conformers exist to interact with any potential receptors for 1,25-(OH)2D3. We have chemically synthesized 1,25-(OH)2-9,14,19,19,19-pentadeuterio-pre-D3 (1,25-(OH)2-d5-pre-D3), an analog of the 6-s-cis conformation of 1,25-(OH)2D3. We found that 1,25-(OH)2-d5-pre-D3 and 1,25-(OH)2D3 were equivalently active in two nongenomic systems (transcaltachia as measured in the perfused chick intestine and 45Ca2+ uptake through voltage-gated Ca2+ channels in ROS 17/2.8 cells). 1,25-(OH)2-d5-pre-D3 was significantly less active both in binding in vitro to the plasma vitamin D-binding protein (7%) and to the chick (10%) and pig (4%) intestinal nuclear 1,25-(OH)2D3 receptors generating genomic biological responses in vivo (induction of plasma levels of osteocalcin, < 5%) or in cultured cells (inhibition of HL-60 cell differentiation, < 5%; inhibition of MG-63 proliferation, < 2%; and induction of osteocalcin, < 2%). These results suggest that the genomic and nongenomic responses are mediated by separate receptors. Further, the 6-s-cis form (steroid-like conformation) of the natural hormone, 1,25-(OH)2D3, may be selectively responsible for its nongenomic function(s).

Animals↗
PubMed1993

Nontranscriptional effects of steroid hormones.

A growing body of literature exists documenting the nonnuclear effects of steroid hormones, and in particular, binding of the lipophillic ligands to specific plasmalemmal recognition sites. This review compiles evidence supporting the presence of membrane receptors for extradiol 17 beta, progesterone, corticosteroids, thyroid hormone, and 1,25-dihydroxyvitamin D3. In addition, 1,25-dihydroxyvitamin D3-mediated nonnuclear signal transduction mechanisms and physiological responses are discussed.

Cell Membrane↗
PubMed1992

1 beta, 25 (OH)2-vitamin D3 is an antagonist of 1 alpha,25 (OH)2-vitamin D3 stimulated transcaltachia (the rapid hormonal stimulation of intestinal calcium transport).

The steroid hormone 1 alpha,25-dihydroxyvitamin-D3 [1 alpha,25(OH)2D3] stimulates biological responses via both genomic mechanisms and nongenomic mechanisms (opening of voltage-gated Ca2+ channels). We report here that 1 beta, 25(OH)2-vitamin-D3 (a) is devoid of activity as an agonist for transcaltachia, (b) is a potent stereospecific antagonist of 1 alpha,25 (OH)2D3 stimulation of the nongenomic transcaltachia response and also (c) has less than 1% the ability of 1 alpha,25(OH)2D3 to bind to the chick intestinal nuclear 1 beta,25(OH)2D3 receptor. We conclude that the membrane response element(s) which generates the nongenomic response of transcaltachia has a different ligand specificity than the classic nuclear 1 alpha, 25(OH)2D3 receptor.

Animals↗
PubMed1992

A parathyroid-related peptide induces transcaltachia (the rapid, hormonal stimulation of intestinal Ca2+ transport).

PTH-related peptide (PTHrP) has been shown to be responsible for the hormonal hypercalcemia of malignancy. Previously, we demonstrated that both 1,25(OH)2-vitamin D3 and the N-terminal fragment of PTH (1-34) stimulates the rapid transport of Ca2+ (transcaltachia) in the perfused chick intestine. Since there is a sequence homology between these two hormones in the n-terminal fragment, in this study we examined the effect of PTHrP(1-40) on stimulation of transcaltachia in the perfused chick duodenum. The results indicate that the maximal stimulation of transcaltachia occurs at 50 pM PTHrP(1-40), and that the dose-response curve is biphasic in nature. Perfusion with 25 pM, 50 pM, 100 pM or 200 pM PTHrP(1-40) for 40 min increases the transport of Ca2+ in perfused intestine 1.8-, 3.0-, 2.4- and 1.6-fold, respectively. The response is rapid, occurring within 10 min of introduction of the PTHrP. The Ca2+ channel inhibitor nifedipine, which is known to abolish the transcaltachic effect elicited by 1,25(OH)2 vitamin D3, also inhibited the rapid transport of Ca2+ stimulated by PTHrP(1-40). The transcaltachic effect of PTHrP(1-40) may be mediated by a signal transduction pathway in which Ca2+ channels are activated.

Animals↗
PubMed1992

1,25-Dihydroxyvitamin D3 analog structure-function assessment of the rapid stimulation of intestinal calcium absorption (transcaltachia).

The possibility is now emerging that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] can mediate biologic responses via both genomic and nongenomic pathways. To understand the molecular basis of the nongenomic response of transcaltachia, defined as the 1,25-(OH)2D3-mediated rapid (2-10 minutes) stimulation of calcium transport from the brush border to the basal lateral membrane of the epithelial cell in vitamin D-replete chick intestine, and to address the issue of whether the same receptor for the secosteroid serves as the signal transducer for both genomic and nongenomic pathways, we carried out structure-function studies using seven analogs of 1,25-(OH)2D3 with different affinities for the classic nuclear 1,25-(OH)2D3 receptor as measured by determination in a steroid competition assay of the relative competitive index (RCI). The RCI of 1,25-(OH)2D3 is by definition 100. 1,25-(OH)2D3 initiates transcaltachia within 2-10 minutes of vascular perfusion and yields a biphase response curve. Dose-dependent stimulations of Ca2+ transport by the seven analogs indicates that different structural features are essential for initiating the transcaltachic response as contrasted with binding to the classic nuclear receptor. Vascular perfusion with analogs AT (25-OH-16-ene-23-yne-D3) and Y (25-OH-23-yne-D3), which are known to activate Ca2+ channels but bind very poorly to the classic receptor (RCI less than 0.5), are efficient in stimulating Ca2+ transport. By comparison, compounds BT [1 alpha,24S-(OH)2-22-en-26,27-dehydrovitamin D3] and V (1,25-(OH)2-16-ene-23-yne-D3], which bind very well to the classic nuclear receptor (RCI 75-111) but do not activate Ca2+ channels, are inefficient in stimulating Ca2+ transport. These results indicate that the membrane components that respond to the analogs of 1,25-(OH)2D3 with activation of Ca2+ channels have a different ligand specificity than the classic nuclear receptor.

Animals↗
PubMed1992

Rapid actions of vitamin D compounds.

The rapid actions of vitamin D compounds are surveyed in a variety of target tissues, including intestine, muscle, bone, hepatocytes, fibroblasts, HL-60 cells, kidney, mammary gland, and parathyroid. Evidence for non-nuclear receptors vs. membranophilic effects is discussed, followed by a consideration of signal transduction mechanisms including steroid hormone activated Ca2+ channels, phospholipid metabolism, protein kinases, and the role of G-proteins.

Animals↗
PubMed1992

Noncytoplasmic and filamentous appearance of calbindin-D28k and tubulin in double, indirect immunofluorescent staining of embryonic chick tissue.

Double indirect immunofluorescent labeling of embryonic chick tissue was undertaken for the vitamin D-induced calcium binding protein, calbindin-D28k, and microtubules. Immunoreactivities for both calbindin-D28k and tubulin were found to exhibit a filamentous staining pattern in mesonephros, metanephros, intestine, and brain. In the intestine, staining was absent at 19 days, while immunolabeling of tubulin became evident at 20 days, and both antigens were present in 21-day tissue. In intestinal epithelium, as well as in 10-day metanephros, it was strikingly evident that cells either stained for both antigens or were negative for both calbindin-D28k and tubulin. In 11-12-day metanephros, an increased number of cells with both immunoreactivities were found. In 15-17-day brain, tubulin was evident within all cells but stained most intensely in Purkinje cells which were also positive for calbindin-D28k. Mesonephros from 4-5-day embryos revealed immunolabeling of both tubulin and the calcium binding protein. A statistical analysis of the various cell types revealed that the vast majority contained either both antigens or neither of the immunoreactivities. Of the more than 600 cells scored, none were found to be positive for calbindin-D28k, while at the same time negative for tubulin. It is concluded that calbindin-D28k exhibits a noncytoplasmic distribution in all tissues tested and that the filamentous appearance may reflect localization of the antigen in tubulo-vesicular organelles associated with cytoskeleton.

Animals↗
PubMed1992

1,25(OH)2-vitamin D3, a steroid hormone that produces biologic effects via both genomic and nongenomic pathways.

The hormonally active form of vitamin D is 1,25(OH)2-vitamin D3 [1,25(OH)2D3]. This seco-steroid is the key mediator of the vitamin D endocrine system which produces biological effects in over 28 target tissues. In these target tissues, the biological responses may be generated both by a signal transduction mechanism which involves a nuclear receptor for 1,25(OH)2D3 that modulates gene transcription or a signal transduction pathway which involves rapid opening of Ca2+ channels which are externally located on the plasma membrane. This paper reviews the evidence in support of the pleiotropic effects of this steroid hormone and presents evidence that the receptor of the genomic effects is likely to be separate from the receptor/membrane recognition element which initiates the rapid nongenomic biological effects.

Animals↗
PubMed1992

Vesicular calcium transport in chick intestine.

A critical question in transcellular calcium transport relates to the identity of the calcium carrier. Such a carrier is needed if for no other reason than to minimize toxicity due to a high intracellular concentration of the calcium ion. After briefly reviewing several of the carriers that have been proposed in the past, attention is directed to the endosomal-lysosomal transport pathway. New findings are presented in support of the rapidity with which lysosomes might be mobilized to transport calcium. New data demonstrate that these organelles can take up calcium even under conditions of relatively low luminal levels of the divalent cation (0.9 mmol/L of CaCl2). They may therefore play a role in the facilitated movement of calcium through the cell.

Animals↗
PubMed1991

1,25-Dihydroxyvitamin D3-mediated alterations in microtubule proteins isolated from chick intestinal epithelium: analyses by isoelectric focusing.

Recent work has indicated that vectorial Ca2+ transport across the intestinal epithelium occurs in vesicles and may involve the participation of microtubules [Nemere et al., 1986]. Since 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) stimulates this Ca2+ transport process, microtubule (MT) isotypes were studied as a potential regulatory point. The effect of 1,25(OH)2D3 status on tubulin isotypes was analyzed by isoelectric focusing (IEF) gels of taxol stabilized MTs prepared from intestinal epithelium of vitamin D-deficient chicks dosed with vehicle (-D) or 1.3 nmoles of 1,25(OH)2D3 (+D) 2.5, 5, 10, 15, or 43 h prior to sacrifice. Four bands, one of which was identified as alpha-tubulin on the basis of Western analysis, increased in Coomassie Blue staining intensity 5-15 h after 1,25(OH)2D3, corresponding to the time course of augmented vesicular Ca2+ transport. Dose-response studies revealed similar changes in tubulin isotype profiles in IEF gels, again corresponding to doses known to elicit enhanced Ca2+ absorption (52-6,500 pmoles of hormone). The role of Ca2+ transport was also examined. Isoelectrically focused intestinal epithelial tubulin from -D chicks allowed to transport Ca2+ for 30 min revealed increased staining of bands relative to nonabsorbing -D controls. By comparison, Ca2+ transport in +D chicks resulted in fainter bands relative to nonabsorbing, +D controls. MTs prepared from fasted or fed chicks revealed similar changes upon IEF, but of much smaller magnitude. Enhanced phosphorylation did not account for the appearance of the more acidic bands, although 1,25(OH)2D3 treatment resulted in decreased 32P content of a presumptive non-tubulin component, relative to preparations from -D controls. Glucocorticoids, which are known to suppress 1,25(OH)2D3-stimulated Ca2+ transport, led to severely diminished levels of total tubulin, as judged by SDS-PAGE, rather than altered tubulin isotypes. Thus, MTs of intestine are subject to regulation by hormonal status, as well as by the amount of Ca2+ available for transepithelial transport.

Animals↗