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Biomedical subjects

R Boland

Publications and source records attributed to R Boland.

At least 73 records · Page 4Linked to original sources
PubMed1991

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.

Adenylate Cyclase Toxin↗
PubMed1991

Evidence on the participation of the 3',5'-cyclic AMP pathway in the non-genomic action of 1,25-dihydroxy-vitamin D3 in cardiac muscle.

Several studies have suggested that vitamin D plays a role in cardiovascular function. It has been recently shown that in vitro treatment of vitamin D-deficient chick cardiac muscle with physiological concentrations of 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) induces a rapid (1-10 min) increase of tissue 45Ca uptake which can be suppressed by Ca channel blockers. The hormone simultaneously stimulated heart microsomal membrane protein phosphorylation. Experiments were performed to investigate the existence of a relationship between these changes and to obtain information about the mechanism involved in 1,25(OH)2D3-induced modifications in cardiac protein phosphorylation. Dibutyryl cyclic AMP (10 microM) and forskolin (10 microM), known activators of the cAMP pathway, produced time courses of changes in 45Ca uptake by chick heart tissue similar to 1,25(OH)2D3 (10(-10) M). Analogously to the hormone, the effects of both compounds were abolished by nifedipine (30 microM) and verapamil (10 microM). In agreement with these observations, 1,25(OH)2D3 significantly increased (34-70%) heart muscle cAMP levels within 1-10 min of treatment. In addition, 1,25(OH)2D3 and forskolin caused similar changes in cardiac microsomal membrane protein phosphorylation (e.g. stimulation in 43 kDa and 55 kDa proteins). These changes were also evidenced by direct exposure of isolated heart microsomes to 1,25(OH)2D3, suggesting a direct membrane action of the hormone. The fast effects of 1,25(OH)2D3 on dihydropyridine-sensitive cardiac muscle Ca uptake could be reproduced in primary-cultured myocytes isolated from chick embryonic heart. Furthermore, the effects of the hormone could be suppressed by a specific protein kinase A inhibitor. These results suggest that 1,25(OH)2D3 affects heart cell calcium metabolism through regulation of Ca channel activity mediated by the cAMP pathway.

Animals↗
PubMed1991

Rapid stimulation of calcium uptake and protein phosphorylation in isolated cardiac muscle by 1,25-dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D3 has been shown to induce rapid changes in calcium fluxes in skeletal muscle and other target tissues independently of gene activation. The possibility that the hormone would produce similar effects in heart where 1,25-dihydroxyvitamin D3 receptors and activities have been shown, was studied. A significant increase of 45Ca uptake by left ventricular slices from vitamin D-deficient chicks was observed upon incubation for 1-10 min with physiological doses of 1,25-dihydroxyvitamin D3. This stimulation was dose-dependent and specific for the hormone when compared with vitamin D3, 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 and could not be associated to changes in lipid synthesis as assessed by measurements of [3H]glycerol incorporation into cardiac tissue lipids. The Ca channel blockers nifedipine (30 microM) and verapamil (10 microM) abolished the increase in Ca uptake produced by 1,25-dihydroxyvitamin D3. The rapid effects of the hormone on heart Ca influx were accompanied by a stimulation of the phosphorylation of two microsomal proteins of 43 kDa and 55 kDa. These results further support a direct action of 1,25-dihydroxyvitamin D3 in the regulation of cardiac muscle Ca metabolism which may involve activation of Ca channels.

Animals↗
PubMed1991

Sequences near the CCAAT region and putative 1,25-dihydroxyvitamin D3-response element and further upstream novel regulatory sequences of calbindin-D28k promoter show DNase I footprinting protection.

1,25-Dihydroxyvitamin D3, the hormonally active form of vitamin D (1,25(OH)2D3), plays a major role in the transcriptional regulation of the vitamin D-induced calcium binding protein calbindin-D28k in the chick intestine. Sequence-specific protein-DNA interactions within the promoter of the calbindin-D28k gene were studied by DNAse I footprinting analysis to obtain information on the mechanism by which the 1,25(OH)2D3 receptor and other transcription factors regulate its expression. Restriction fragments spanning nucleotides -679 to +44 of the calbindin-D28k gene were used as probes Intestinal nuclear extracts prepared from vitamin D-deficient chicks generated several protected regions. Two prominent areas of protection against DNase I digestion were located at nucleotides -595 to -572 (21 bp) and -372 to -337 (36 bp). The -372 to -337 protected segment includes a CACCC sequence motif. Additional protection regions (-333/-328, -319/-315 and -308/-304) were observed within and near the candidate chicken calbindin-D28k 1,25(OH)2D3-response element (-329/-313) and the CCAAT box (-326/-322). DNase I digestion patterns obtained with liver nuclear extracts, containing low levels of 1,25(OH)2D3 receptor, revealed weaker protein-DNA interactions in these regions.

Animals↗
PubMed1991

Effects of 1,25-dihydroxy-vitamin D3 on phosphate accumulation by myoblasts.

The effects of 1,25-dihydroxy-Vitamin D3 on phosphate uptake by cultured chick embryonic muscle cells were investigated. Preincubation of primary myoblast cultures during 4-24 hours with physiological levels of 1,25(OH)2D3 resulted in a significant stimulation of velocity and total capacity of phosphate accumulation by the cells. Maximal responses were obtained at 8 hours of treatment with the sterol. In agreement with previous studies, 25-hydroxy-Vitamin D3 also stimulated myoblast phosphate uptake. 24,25-dihydroxy-Vitamin D3 and vitamin D3 were ineffective. Evidence was obtained indicating that 1,25(OH)2D3 affects the Na(+)-linked component of muscle cell phosphate uptake through a mechanism dependent on "de movo" protein and RNA synthesis.

Animals↗
PubMed1990

Stimulation of calmodulin synthesis in proliferating myoblasts by 1,25-dihydroxy-vitamin D3.

Myoblasts contain a receptor specific for 1,25-dihydroxy-vitamin D3. Morphological data have indicated that the hormone stimulates both myoblast proliferation and fusion. The synthesis of myoblast proteins in response to the sterol was studied during the proliferating stage of the cells. Chick embryo myoblast primary cultures (precultured for 24 h in the presence of low levels of 1,25-dihydroxy-vitamin D3 after isolation) were used. Labelling (2 h) of cells incubated in the absence and presence of 1,25-dihydroxy-vitamin D3 (10(-10) M) for 1-12 h with [14C]leucine and [3H]leucine, respectively, followed by coelectrophoresis of double-labelled proteins on sodium dodecyl sulfate polyacrylamide gels showed that the sterol initially stimulates the synthesis of proteins of 60 kDa (1.2 h), 70 kDa (2.4 h) and 80 kDa (4 h). These changes were transient and between 6 and 12 h a protein of 19 kDa was induced. This protein was identified as calmodulin on the basis of its isoelectric point (pI 4.1), Ca2(+)-dependent electrophoretic mobility, ability to bind 45Ca and to interact with an immobilized phenothiazine in a Ca2(+)-dependent manner, and by means of immunoblotting with a specific anti-calmodulin antibody and 3',5'-cyclic AMP phosphodiesterase activation assays. In agreement with these results, hybridization analysis with a specific cDNA probe showed increased calmodulin mRNA levels in myoblasts treated for 4-12 h with 1,25-dihydroxy-vitamin D3. These changes were paralleled by a stimulation of [3H]thymidine incorporation into DNA suggesting that they may be involved in the mitogenic action of the hormone.

Animals↗
PubMed1989

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.

Animals↗
PubMed1987

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.

Animals↗
PubMed1987

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.

Acetates↗
PubMed1987

Phosphate accumulation by muscle in vitro and the influence of vitamin D3 metabolites.

Phosphate accumulation by muscle in vitro and the effects of vitamin D3 metabolites thereupon were studied in cultures of chick embryo skeletal muscle myoblasts and intact chick soleus muscles. A significant proportion of phosphate accumulation by the cells was Na+-dependent, saturable with respect to phosphate, energy-dependent and inhibited by ouabain and arsenate, in agreement with the operation of a Na+-phosphate cotransport system in the muscle cell plasma membrane as has been described for intestine and kidney. This was further supported by the demonstration of substrate-saturable phosphate uptake in sarcolemma vesicles isolated from chick skeletal muscle. Preincubation of myoblast and soleus muscle cultures with physiological levels of 25-hydroxy-vitamin D3 resulted in a significant stimulation of phosphate accumulation by cultures. 1,25-dihydroxy-vitamin D3 had no effects on the differentiated tissue whereas it markedly increased phosphate accumulation by embryonic muscle cells. In addition, it could be shown that 25-hydroxy-vitamin D3 affects the Na+-linked component of cell phosphate uptake through a mechanism dependent on de novo protein synthesis.

24,25-Dihydroxyvitamin D 3↗
PubMed1985

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.

Animals↗
PubMed1985

Presence of a 1,25-dihydroxy-vitamin D3 receptor in chick skeletal muscle myoblasts.

The presence of a specific receptor for 1,25-dihydroxy-vitamin D3 was investigated in myoblasts released from chick embryo skeletal muscle by trypsin and collagenase treatment. Density gradient analysis of the cytosol obtained from these muscle cell preparations showed that 1,25-dihydroxy-vitamin D3 binds specifically to a 3.7 S macromolecule. Scatchard analysis yielded an equilibrium dissociation constant of 2.46 x 10(-10) M and a Nmax of 74 fmol/mg of cytosol protein. The data is in agreement with previous evidence which indicates that the action of the vitamin D metabolite on muscle Ca uptake is mediated by de novo protein and RNA synthesis, and supports the concept that muscle is a target organ for 1,25-dihydroxy-vitamin D3.

Animals↗
PubMed1983

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.

Animals↗