Safety profile of 1,25-dihydroxyvitamin D3 of herbal origin in broiler chicken.

INTRODUCTION: The safety of supplementing broiler feed with a standardised herbal extract, Solanum Glaucophyllum Standardised Leaves (SGSL) containing glycosylated 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3) and standardised to contain 10 µg/g 1,25(OH)2D3 equivalent, was examined in two studies. In a first study, we examined the potential of SGSL to substitute vitamin D3 (VD3) and the tolerated dose range of SGSL when applied without concomitant VD3 by analyzing performance and blood chemical parameters after 14, 25 and 38 days on diets containing two doses of SGSL (1 and 10 g/kg feed) as source of 1,25(OH)2D3. In the second study, the no adverse effect level of SGSL was determined by analyzing the same parameters after 35 days on diets containing basic VD3 supply and in addition 0.2, 1.0, 2.0 and 4.0 g of SGSL/kg feed. We showed that SGSL was able to substitute VD3 in broilers as far as the performance parameters were concerned. Also, we found that the no adverse effect level is at least 4 g SGSL/kg feed when used with moderate doses of VD3. This is 20 times higher than the upper limit of the commercially recommended dose. We concluded that SGSL is a safe feed additive to use in broiler chicken.

INTRODUCTION: Dans la cadre de deux études, on a examiné la sécurité de l'extrait de plante standardisé Solanum Glaucophyllum Standardised Leaves (SGSL) comme complément alimentaire chez les poulets d'engraissement. Le SGSL contient de façon standardisée 10 µg/g de 1,25(OH)2D3 sous forme glycolysée. Dans la première étude, on a examiné le potentiel d'action en tant que remplaçant de la vitamine D3 (VD3) et le domaine de dose de SGSL toléré, ceci en ne donnant que du SGSL sans addition de VD3 . On a examiné la performance et les paramètres de chimie sanguine après 14, 25 et 38 jours d'affouragement de deux doses différentes (1 et 10 g/kg d'aliment) de SGSL comme source de 1,25(OH)2D3. Dans la seconde étude, on a recherché le No Adverse Effect Level sur la base des mêmes paramètres après 35 jours avec une alimentation contenant, outre une quantité modérée de VD3, 0.2, 1.0, 2.0 et 4.0 g de SGSL/kg. On a pu démontrer que le SGSL peut remplacer la vitamine D3 chez les poulets d'engraissement en ce qui concerne les performances étudiées. Le No Adverse Effect Level se situait aux environs d'au moins 4g de SGSL/kg d'aliment lorsqu'il était associé avec des quantités modérées de Vitamine D3. Cette dose est vingt fois supérieure à la dose maximale recommandée par le fabriquant. Nous en déduisons que le SGSL est un complément alimentaire sûr pour les poulets d'engraissement.

Osteoblastic protein tyrosine phosphatases inhibition and connexin 43 phosphorylation by alendronate.

Bisphosphonates (BPs), potent inhibitors of bone resorption which inhibit osteoclasts, have also been shown to act on osteocytes and osteoblasts preventing apoptosis via connexin (Cx) 43 hemichannels and activating the extracellular signal regulated kinases ERKs. We previously demonstrated the presence of a saturable, specific and high affinity binding site for alendronate (ALN) in osteoblastic cells which express Cx43. However, cells lacking Cx43 also bound BPs. Herein we show that bound [(3)H]-alendronate is displaced by phosphatase substrates. Moreover, similar to Na3VO4, ALN inhibited the activity of transmembrane and cytoplasmic PTPs, pointing out the catalytic domain of phosphatases as a putative BP target. In addition, anti-phospho-tyrosine immunoblot analysis revealed that ALN stimulates tyrosine phosphorylation of several proteins of whole cell lysates, among which the major targets of the BP could be immunochemically identified as Cx43. Additionally, the transmembrane receptor-like PTPs, RPTPµ and RPTPα, as well as the cytoplasmic PTP1B, are highly expressed in ROS 17/2.8 cells. Furthermore, we evidenced that Cx43 interacts with RPTPµ in ROS 17/2.8 and ALN decreases their association. These results support the hypothesis that BPs bind and inhibit PTPs associated to Cx43 or not, which would lead to the activation of signaling pathways in osteoblasts.

Role of connexin 43 in the mechanism of action of alendronate: dissociation of anti-apoptotic and proliferative signaling pathways.

Bisphosphonates (BPs) inhibit osteocyte and osteoblast apoptosis via opening of connexin (Cx) 43 hemichannels and activating the extracellular signal regulated kinases ERKs. Previously, we hypothesized that intracellular survival signaling is initiated by interaction of BPs with Cx43. However, using whole cell binding assays with [(3)H]-alendronate, herein we demonstrated the presence of saturable, specific and high affinity binding sites in the Cx43-expressing ROS 17/2.8 osteoblastic cells, authentic osteoblasts and MLO-Y4 cells expressing Cx43 or not, as well as in HeLa cells lacking Cx43 expression and ROS 17/2.8 cells pretreated with agents that disassemble Cx channels. In addition, both BPs and the PTP inhibitor Na(3)VO(4) increased proliferation of cells expressing Cx43 or not. Furthermore, although BPs are internalized and inhibit intracellular enzymes in osteoclasts, whether the drugs penetrate non-resorptive bone cells is not known. To clarify this, we evaluated the osteoblastic uptake of AF-ALN, a fluorescently labeled analog of alendronate. AF-ALN was rapidly internalized in cells expressing Cx43 or not indicating that this process is not mediated via Cx43 hemichannels. Altogether, these findings suggest that although required for triggering intracellular survival signaling by BPs, Cx43 is dispensable for cellular BP binding, its uptake, as well as the proliferative effects of these agents.

Modulation of ERK 1/2 and p38 MAPK signaling pathways by ATP in osteoblasts: involvement of mechanical stress-activated calcium influx, PKC and Src activation.

There is evidence that extracellular nucleotides, acting through multiple P2 receptors, may play an important role in the regulation of bone metabolism by activating intracellular signaling cascades. We have studied the modulation of mitogen-activated protein kinase (MAPK) signaling pathways and its relationship to changes in intracellular calcium concentration ([Ca(2+)](i)) induced by ATP in ROS-A 17/2.8 osteoblastic cells. ATP and UTP (10 microM) increased [Ca(2+)](i) by cation release from intracellular stores. We have found that when the cells are subsequently subjected to mechanical stress (medium perturbation), a transient calcium influx occurs. This mechanical stress-activated calcium influx (MSACI) was not observed after ADP stimulation, indicating that P2Y(2) receptor activation is required for MSACI. In addition, ERK 1/2 and p38 MAPK were activated by ATP in a dose- and time-dependent manner. This activation was almost completely blocked using neomycin (2.5mM), an inhibitor of phosphoinositide-phospholipase C (PI-PLC), Ro 318220 (1 microM), a protein kinase C (PKC) inhibitor, and PP1 (50 microM), a potent and selective inhibitor of the Src-family tyrosine kinases. Ca(2+)-free extracellular medium (containing 0.5mM EGTA) and the use of gadolinium (5 microM), which suppressed MSACI, prevented ERK 1/2 and p38 phosphorylation by ATP. Altogether, these results represent the first evidence to date suggesting that P2Y(2) receptor stimulation by ATP in osteoblasts sensitizes mechanical stress activated calcium channels leading to calcium influx and a fast activation of the ERK 1/2 and p38 MAPK pathways. This effect also involves upstream mediators such as PI-PLC, PKC and Src family kinases.

Suppression of 1,25-dihydroxy-vitamin D3-dependent calcium transport by protein synthesis inhibitors and changes in phospholipids in skeletal muscle.

The effects of protein synthesis inhibitors on 1,25-dihydroxy-vitamin D3-dependent Ca uptake were evaluated in vitamin D-deficient chick soleus muscle and chick embryo myoblast cultures in order to obtain information about the mechanism by which 1,25-dihydroxy-vitamin D3 affects muscle calcium transport. Puromycin (50 microM, 5 h) and cycloheximide (50 microM, 24 h) blocked the increase in Ca uptake induced by the metabolite in soleus muscle and myoblasts, respectively. Actinomycin D (1.6 microM, 12 h) was also effective in inhibiting 1,25-dihydroxy-vitamin D3-dependent Ca uptake in myoblasts. These results suggest that the effects of 1,25-dihydroxy-vitamin D3 on muscle Ca uptake are mediated by de novo protein and RNA synthesis. In addition, it could be observed that myoblasts treated with 1,25-dihydroxy-vitamin D3 had increased lipid phosphorus, phosphatidylcholine and sphingomyelin contents. These changes may be the consequence of the nuclear action of the sterol or, alternatively, represent an independent effect as has been proposed for intestine.

Identification of 7-dehydrocholesterol, vitamin D3, 25(OH)-vitamin D3 and 1,25(OH)2-vitamin D3 in Solanum glaucophyllum cultures grown in absence of light.

Solanum glaucophyllum contains the calciotropic hormone 1, 25-dihydroxy-vitamin D3 (1,25(OH)2D3). The metabolic pathway leading to the formation of 1,25(OH)2D3 in the plant is largely unknown. Specifically, there is controversy about the participation of a photolytic reaction in the generation of vitamin D3 and its metabolites. To investigate the requirement for light, S. glaucophyllum tissue (callus) and cell suspension cultures grown under strict conditions of darkness were extracted with chloroform/methanol (1:2, v/v) followed by purification of the lipidic fraction by Sephadex LH-20 and high-performance liquid chromatography. HPLC peaks with elution times similar to those of authentic samples of 7-dehydrocholesterol, vitamin D3, 25(OH)D3 and 1,25(OH)2D3 were detected. The presence of 1,25(OH)2D3 was also evidenced by [3H]1,25(OH)2D3 competitive binding analysis using the chick hormone intestinal receptor. Furthermore, 7-dehydrocholesterol, vitamin D3, 25(OH)D3 and 1,25(OH)2D3 were unequivocally identified by mass spectrometry. Incubation of control samples of 7-dehydrocholesterol under the same conditions as S. glaucophyllum cultures did not result in vitamin D3 formation, excluding the influence of light in these experiments. The results suggest that a synthetic route of vitamin D3 compounds independent of light operates in Solanum glaucophyllum cultured in vitro.

Changes in muscle lipid metabolism induced in vitro by 1,25-dihydroxy-vitamin D-3.

1,25-Dihydroxy-vitamin D-3 has been shown to increase phosphatidylcholine and decrease phosphatidylethanolamine levels in skeletal muscle in vitro. To elucidate the metabolic pathway and mechanism involved in the effect of the sterol, chick embryo myoblast and vitamin D-deficient chick soleus muscle cultures were treated with 1,25-dihydroxy-vitamin D-3 (5.0 X 10(-10)-3.6 X 10(-11) M, total concentration) for 12-14 h and 1 h, respectively, in the absence and presence of protein and RNA synthesis inhibitors. Lipids were then labelled with [3H]glycerol and [14C]acetate. A significant increase in phosphatidylcholine and triacylglycerol labelling and a decrease in phosphatidylethanolamine labelling were observed in response to the hormone. Cycloheximide suppressed these changes in both types of preparations. Puromycin and actinomycin D were also effective blockers in cultured muscle cells. In addition, double-labelling of control and 1,25-dihydroxy-vitamin D-3-treated myoblasts with [3H]choline and [14C]ethanolamine were performed. The sterol did not affect [3H]choline labelling of total cell lipid extracts and phosphatidylcholine. However, the total lipid fraction of treated cells was labelled to a greater extent with [14C]ethanolamine. In addition, an increased incorporation of this precursor into phosphatidylcholine accompanied by a proportional decrease in phosphatidylethanolamine could be shown in cells pretreated with the metabolite. These changes were abolished by cycloheximide and actinomycin D. The results suggest that 1,25-dihydroxy-vitamin D-3 stimulates methylation of phosphatidylethanolamine into phosphatidylcholine in myoblasts by a nuclear mechanism. The data are consistent with the presence in the cells of a receptor specific for the hormone.

Modulation of DNA synthesis in cultured muscle cells by 1,25-dihydroxyvitamin D-3.

Biphasic effects of 1,25-dihydroxyvitamin D-3 on DNA synthesis were shown in primary cultured (24 h) chick embryo myoblasts exposed to physiological concentrations of the hormone. The sterol stimulated [3H]thymidine incorporation into DNA in proliferating myoblasts, e.g., at early stages of culture prior to cell fusion or in high serum-treated cells. The opposite effects were observed during the subsequent stage of myoblast differentiation in low-serum media. The mitogenic effect of 1,25-dihydroxyvitamin D-3 was correlated with an increase in c-myc mRNA and a decrease in c-fos mRNA levels, whereas its inhibitory action on DNA synthesis was accompanied by increased myofibrillar and microsomal protein synthesis and an elevation of creatine kinase activity, the latter suggesting a stimulation of muscle cell differentiation by the sterol. These data are in agreement with the results of previous morphological studies. Treatment of myoblasts with the calcium ionophore X-537 A or the phorbol ester TPA caused only a transient stimulation of [3H]thymidine incorporation into DNA, which occurred earlier than the response elicited by 1,25-dihydroxyvitamin D-3, suggesting that changes in intracellular Ca2+ and kinase C activity are not major mediators of the hormone effects. A similar temporal profile of changes in calmodulin mRNA levels as that of [3H]thymidine incorporation into DNA was observed after treatment of myoblasts with the sterol, in accordance with the role of calmodulin in the regulation of cell proliferation. 1,25-dihydroxyvitamin D-3 may play a function in embryonic muscle growth and differentiation.

Synthesis of the calcium transport ATPase of sarcoplasmic reticulum and other muscle proteins during development of muscles cells in vivo and in vitro.

The effect of medium Ca2+ concentration upon the concentration and the rate of synthesis of muscle proteins was investigated in chicken pectoralis muscle cultures. There is an easily identifiable class of muscle protein which includes the Ca2+-ATPase of sarcoplasmic reticulum, myosin, troponin C, ATP : creatine phosphotransferase, muscle specific actin, tropomysin 1 and 2, and muscle hemagglutinin, which show a large increase in concentration during normal development. The increased synthesis of these proteins was inhibited, without inhibition of cell proliferation, in culture media of relatively low Ca2+ concentration, 0.05--0.3 mM, where fusion was prevented. Similar medium Ca2+ concentration was required for the expression of all these proteins, suggesting their coordinate regulation. The proteins are denoted as 'calcium-modulated proteins'. The increased Ca2+ transport activity of sarcoplasmic reticulum in cultured chicken pectoralis muscle cells during development at 1.8 mM medium calcium concentration represents de novo synthesis of the Ca2+ transport ATPase, as shown by immunoprecipitation, active site labeling and direct identification of the Ca2+ transport ATPase on two-dimensional gel electropherograms of whole muscle homogenates. The concentration and the turnover rate of the majority of the muscle proteins is not affected significantly by medium Ca2+ concentration between 0.06 and 1.8 mM. It is proposed that increase in cytoplasmic free Ca2+ concentration during fusion plays a central role in the regulation of the synthesis of calcium-modulated proteins.

Preparation of sarcoplasmic reticulum with high calcium-sensitive ATPase and stable calcium transport function from rat skeletal muscle.

A method was developed for the isolation from rat skeletal muscle of sarcoplasmic reticulum vesicles in which the calcium transport function does not decay during storage. High initial and maximum uptake of calcium and calcium-dependent ATPase activity were obtained for membranes isolated from mixed muscles or pure red fibers. Unstable vesicles resulted when 2 mM EDTA was included in the isolation medium. The calcium uptake activity was lost upon ageing at 0 degrees C, probably due to conversion of the calcium-dependent ATPase to a calcium-independent form. Addition of Ca2+ counteracted the affects of EDTA, suggesting their involvement in maintaining the structure of the calcium transport system. This is supported by the fact that different structural states of the ATPase in stable and unstable vesicles were detected by DEAE-cellulose column chromatography.

Effects of vitamin D-3 on phosphate and calcium transport across and composition of skeletal muscle plasma cell membranes.

The effects of vitamin D-3 on calcium and phosphate transport in skeletal muscle plasma membranes were studied. Sarcolemma vesicles were isolated from vitamin D-deficient and vitamin D-treated (one week) chicks by sucrose density gradient centrifugation of a crude muscle plasma membrane fraction. Measurement of (Na+ + K+)-ATPase activity, cholesterol to phospholipid molar ratios and levels of intracellular marker enzymes showed a high degree of purification of the preparations. Administration of vitamin D-3 significantly increased active Ca2+ and phosphate uptake into the vesicles. The efflux of both ions from preloaded vesicles was only slightly altered by the sterol. Ca2+-ATPase activity was higher in sarcolemma from treated animals. This confirms that the effects of vitamin D-3 on calcium transport are related to the Ca2+ pump and not to the passive permeability properties of the membrane. No changes in the protein composition of vesicles from both experimental groups were observed. However, treatment with vitamin D-3 increased sphingomyelin and phosphatidylcholine concentrations. These changes in lipid structure may play a role in the effects of vitamin D-3 on transport characteristics of sarcolemma.

Modulation by 1,25(OH)2-vitamin D3 of the adenylyl cyclase/cyclic AMP pathway in rat and chick myoblasts.

We have previously reported that the calciotropic hormone 1,25(OH)2-vitamin D3 stimulates influx of Ca2+ into cultured rat and embryonic chick myoblasts via voltage sensitive Ca(2+)-channels. In the present study, we show that this effect of 1,25(OH)2D3 requires the mediation of the adenylylcyclase signalling system since the hormone-dependent Ca2+ influx is abolished by specific inhibitors of adenylylcyclase and protein kinase A and mimicked by forskolin and dibutyryl cAMP. 1,25(OH)2D3-stimulated elevations in cellular cAMP paralleled increases in Ca2+ uptake, further suggesting a coupling of adenylylcyclase activation and calcium influx. Fluoride and GTP gamma S mimicked 1,25(OH)2D3-stimulation of calcium influx while GDP beta S suppressed the effect of the hormone. Cholera toxin and Bordetella pertussis toxin both increased 45Ca2+ uptake in rat and chick myoblasts. The hormone further increased cholera toxin actions, but was unable to modify pertussis toxin-induced 45Ca2+ uptake, suggesting a similar target of action for pertussis toxin and 1,25(OH)2D3. Incubation of microsomal membranes with the sterol (10 nM, 2 min) markedly displaces (-32%) [35S]GTP gamma S binding to the membranes. ADP-ribosylation of the pertussis toxin-sensitive 41 kDa substrate was significantly increased (+40%) in 1,25(OH)2D3-pretreated cells. These results suggest that 1,25(OH)2D3-stimulated influx of Ca2+ into rat and embryonic chick cultured myoblasts sequentially requires inhibition of a pertussis toxin-sensitive G protein, accumulation of cAMP and activation of dihydropyridine-sensitive Ca(2+)-channels through PKA-mediated phosphorylation events.

1,25(OH)2-vitamin D-3 stimulates phospholipase A2 activity via a guanine nucleotide-binding protein in chick myoblasts.

The steroid hormone 1,25(OH)2-vitamin D-3 [1,25(OH)2D3] stimulated phospholipase A2 (PLA2) activity in embryonic chick myoblasts releasing [3H]arachidonic acid from the sn-2 position of phospholipids. GTP-binding protein mediation of 1,25(OH)2D3-dependent PLA2 activity was investigated in cells prelabeled with [3H]arachidonic acid. AIF4-, a G-protein activator, mimicked 1,25(OH)2D3-stimulated arachidonic acid release from myoblasts in a dose-dependent manner. Consistent with the involvement of a G-protein in the activation of PLA2 by the hormone, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a stable GTP analogue which activates G-protein mediated signals, strongly enhanced arachidonic acid release in myoblasts. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which competitively inhibits G-protein activation by GTP and its analogues, abolished 1,25(OH)2D3-dependent arachidonic acid release. Bordetella pertussis toxin pretreatment significantly suppressed the hormone action whereas cholera toxin had minor effects on 1,25(OH)2D3 action. Hormone-induced activation of PLA2 was mimicked by the Ca2+ ionophore A23187 and blocked by nifedipine, but was unaffected by neomycin, a phospholipase C inhibitor, ruling out the contribution of phosphoinositide metabolism to arachidonic acid release. These results suggest that 1,25(OH)2D3-stimulation of PLA2 activity in embryonic chick myoblasts is mediated by a pertussis toxin-sensitive GTP-binding protein coupled to influx of extracellular calcium.

1,25-Dihydroxyvitamin D-3 induces arachidonate mobilization in embryonic chick myoblasts.

1,25-Dihydroxyvitamin D-3 (1,25(OH)2D3) which activates the phospholipase C (PLC)-protein kinase C (PKC) signalling pathway, induces within 1 min a dose-dependent (10(-11)-10(-7) M) increase in the release of [3H]arachidonic acid ([3H]AA) from prelabeled embryonic chick myoblasts. The response is dependent on extracellular calcium, since it is suppressed by EGTA and nifedipine, a Ca(2+)-channel blocker, and is mimicked by the calcium ionophore A23187. 1,25(OH)2D3-induced release of [3H]AA is not affected by neomycin (0.5 mM), an inhibitor of phosphoinositide hydrolysis. 12-o-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, induces an extracellular Ca(2+)-independent release of [3H]AA and amplifies the release of AA stimulated by 1,25(OH)2D3. 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H7), a PKC inhibitor, markedly suppressed TPA as well as 1,25(OH)2D3-induced [3H]AA release. Down-regulation of cellular PKC abolishes the effect of the phorbol ester, and partially inhibits 1,25(OH)2D3-induced [3H]AA release. Temporally correlated with AA liberation, the hormone increases the formation of lysophosphatidylcholine (lysoPC) and lysophosphatidylethanolamine (lysoPE) and decreases the cellular content of PC and PE. These results indicate that part of AA release by 1,25(OH)2D3 derives from PLA2 activation and that the effects of the hormone are mediated by PKC in a mode independent of phosphoinositide hydrolysis by PLC.

Synthesis and composition of vitamin D-3 metabolites in Solanum malacoxylon.

The synthesis of vitamin D-3 hydroxylated metabolites in Solanum malacoxylon was investigated. When crude leaf homogenates and subcellular fractions were incubated with [3H]vitamin D-3 and [3H]25-hydroxy-vitamin D-3 under conditions described for animal vitamin D-3-25-hydroxylase and 25-hydroxy-vitamin D-3-1 alpha-hydroxylase, respectively, labelled metabolites identified on the basis of their chromatographic properties as 25-hydroxy-vitamin D-3 and 1,25-dihydroxy-vitamin D-3 were formed. Other unidentified product metabolites were also detected. Vitamin D-3-25-hydroxylase activity was localized in microsomes and 25-hydroxy-vitamin D-3-1 alpha-hydroxylase in mitochondria and microsomes. Chromatography of sterols isolated from leaf extracts preincubated with beta-glucosidase on Sephadex LH-20 columns permitted the isolation of three biologically active fractions with elution properties similar to vitamin D-3, 25-hydroxy-vitamin D-3 and 1,25-dihydroxy-vitamin D-3, respectively. Ultraviolet spectra characteristic of vitamin D-3 and its metabolites were obtained after purification of the fractions by TLC. Co-chromatography of individual fractions with authentic metabolites on TLC provided further evidence that the plant contains vitamin D-3, 25-hydroxy-vitamin D-3 and 1,25-dihydroxy-vitamin D-3 as glucoside derivatives. These results suggest that a similar pathway of vitamin D-3 hydroxylation as in animals may be operative in S. malacoxylon.

The tyrosine kinase c-Src is required for 1,25(OH)2-vitamin D3 signalling to the nucleus in muscle cells.

We have recently shown that the hormonal form of vitamin D3, 1,25(OH)2-vitamin D3 (1,25(OH)2D3), stimulates the enzymatic activity of the non-receptor protein tyrosine kinase c-Src in skeletal muscle cells. In this study we show that intracellular and extracellular Ca2+ chelation with BAPTA and EGTA, respectively, blocked hormone stimulation of c-Src activity/dephosphorylation, indicating that the calcium messenger system is an upstream activator of c-Src. Tyrosine phosphorylation and stimulation of the growth-related mitogen-activated protein kinase (MAPK) by 1,25(OH)2D3 was shown to be dependent on activation of c-Src, since pretreatment with the c-Src specific inhibitor PP1 or muscle cell transfection with an antisense oligodeoxynucleotide directed against c-Src mRNA markedly reduced hormone stimulation of MAPK phosphorylation. Evidence was obtained indicating that MAPK is then translocated to the cell nucleus in active phosphorylated form and induces the expression of c-myc oncoprotein, as the MAPK kinase (MEK) inhibitor PD98059 abolished stimulation of c-myc synthesis by 1,25(OH)2D3. In addition, the hormone rapidly stimulated tyrosine phosphorylation of c-myc. In cells pretreated with PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-D3,4-pyrimidine), the 1,25(OH)2D3-induced increase in tyrosine phosphorylation of c-myc was suppressed. Taken together, these results demonstrate that 1,25(OH)2D3 stimulates proliferation-associated signalling pathways in skeletal muscle cells and implicate c-Src kinase as mediator of this response.

The phospholipid and fatty acid composition of skeletal muscle cells during culture in the presence of vitamin D-3 metabolites.

The phospholipid and fatty acid composition of primary cultures (24 h) of chick embryo skeletal muscle myoblasts treated for 4-24 h with physiological concentrations of 1,25-dihydroxyvitamin D-3 and 25-hydroxyvitamin D-3 were analyzed. 25-Hydroxyvitamin D-3 did not alter the relative amounts of individual muscle cell phospholipids whereas 1,25-dihydroxyvitamin D-3 significantly increased phosphatidylcholine content, mainly at the expense of a decrease in phosphatidylethanolamine concentration. The increase in phosphatidylcholine occurred at a faster rate during the first 8 h than in the subsequent 8-24 h treatment period. A similar time course in 1,25-dihydroxyvitamin D3-dependent changes in myoblast calcium uptake has been observe. In addition, this metabolite markedly increased (100%) the arachidonate content of myoblast phosphatidylcholine near the fusion stage of the cells (24 h of treatment). The levels of docosahexaenoate, a minor polyunsaturated fatty acid, in phosphatidylcholine and phosphatidylethanolamine were also substantially elevated by 1,25-dihydroxyvitamin D-3. No significant changes in fatty acid composition in response to 25-hydroxyvitamin D-3 were observed. Modifications in phospholipids and polyunsaturated fatty acids may play a role in the effects of 1,25-dihydroxyvitamin D-3 on muscle cell calcium transport and differentiation.

Effects of 1,25-dihydroxyvitamin D-3 on phospholipid metabolism in chick myoblasts.

1,25-Dihydroxyvitamin D-3 has been shown to increase phosphatidylcholine and decrease phosphatidylethanolamine levels of myoblasts. Recent studies have suggested that the metabolite stimulates the methylation of phosphatidylethanolamine into phosphatidylcholine. In addition, the sterol increases the arachidonate content of phosphatidylcholine. Experiments were carried out to identify the steps of muscle cell lipid metabolism affected by 1,25-dihydroxyvitamin D-3. Primary cultures of chick embryo myoblasts pretreated with physiological concentrations of 1,25-dihydroxyvitamin D-3 were labelled with [14C]ethanolamine. The sterol increased the incorporation of precursor into dimethylphosphatidylethanolamine and phosphatidylcholine, whereas it decreases the labelling of phosphatidylethanolamine. Prior treatment with cycloheximide and actinomycin D blocked these changes. 1,25-Dihydroxyvitamin D-3 also stimulated the incorporation of [14C]ethanolamine into CDP-ethanolamine. In addition, the sterol increased the incorporation of [3H]arachidonic acid into the phosphatidylcholine fraction but did not affect the incorporation of [14C]palmitic acid. The incorporation of labelled fatty acids into diacylglycerol was not changed by the sterol, whereas it stimulated incorporation of both precursors into triacylglycerol. The data indicate that 1,25-dihydroxyvitamin D-3 enhances the synthesis of phosphatidylcholine through a stimulation of de novo synthesis and methylation of phosphatidylethanolamine via a nuclear mechanism. The sterol may also increase the polyunsaturated fatty acid content of phosphatidylcholine by means of an activation of its deacylation-reacylation cycle.

A guanine nucleotide-binding protein mediates 1,25-dihydroxy-vitamin D-3-dependent rapid stimulation of Ca2+ uptake in skeletal muscle.

1,25-Dihydroxyvitamin D-3 (1,25(OH)2D3) has been shown to increase Ca2+ uptake readily in skeletal muscle through a dihydropyridine-sensitive pathway, cAMP levels and adenylate cyclase activity. In the present study, fluoride (F-), a potent guanine nucleotide binding protein (G protein) stimulator, rapidly increases vitamin D-deficient skeletal muscle Ca2+ uptake in a dose-dependent manner and with a similar time-course as 1,25(OH)2D3. The increment is detected within 1 min (15%) and steadily increases up to 15 min (60%). The effects of 1,25(OH)2D3 and F- are also observed in muscle from normal, vitamin D-replete chicks. AlCl3, which is required for G protein stimulation by F-, potentiates the effects of F-, Ca2+ uptake in 1,25(OH)2D3-dependent muscle is potentiated by F- and, analogous to the hormone, the effects of F- can be suppressed by Ca(2+)-channel antagonists. Direct exposure of microsomal membranes to 1,25(OH)2D3 reduces the specific binding of [gamma-35S]GTP to the membranes 40%. Pretreatment of muscle with Bordetella pertussis toxin (PTX), known to inhibit Gi, or with cholera toxin (CTX), known to stimulate Gs, produces an acute elevation of muscle Ca2+ uptake. 1,25(OH)2D3 potentiates CTX, but has no additional effect on PTX-dependent Ca2+ uptake. These results indicate that an interaction with an inhibitory G protein coupled to adenylate cyclase may be part of the mechanism by which 1,25(OH)2D3 increase Ca2+ uptake through regulation of Ca(2+)-channel gating by a cAMP-dependent pathway in skeletal muscle.