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R Boland

Publications and source records attributed to R Boland.

At least 37 records · Page 2Linked to original sources
PubMed2000

Involvement of tyrosine kinase activity in 1alpha,25(OH)2-vitamin D3 signal transduction in skeletal muscle cells.

In cultured chick skeletal muscle cells loaded with Fura-2, the tyrosine kinase inhibitors herbimycin A and genistein abolished both the fast inositol 1,4,5-trisphosphatedependent Ca(2+) release from internal stores and extracellular Ca(2+) influx induced by 1alpha, 25(OH)(2)-vitamin D(3) (1alpha,25(OH)(2)D(3)). Daidzein, an inactive analog of genistein, was without effects. Tyrosine phosphatase inhibition by orthovanadate increased cytosolic Ca(2+). Anti-phosphotyrosine immunoblot analysis revealed that 1alpha, 25(OH)(2)D(3) rapidly (0.5-10 min) stimulates in a concentrationdependent fashion (0.1-10 nm) tyrosine phosphorylation of several myoblast proteins, among which the major targets of the hormone could be immunochemically identified as phospholipase Cgamma (127 kDa), which mediates intracellular store Ca(2+) mobilization and external Ca(2+) influx, and the growth-related proteins mitogen-activated protein (MAP) kinase (42/44 kDa) and c-myc (65 kDa). Genistein suppressed the increase in phosphorylation and concomitant elevation of MAPK activity elicited by the sterol. Both genistein and the MAPK kinase (MEK) inhibitor PD98059 abolished stimulation of DNA synthesis by 1alpha,25(OH)(2)D(3). The sterol-induced increase in tyrosine phosphorylation of c-myc, a finding not reported before for cell growth regulators, was totally suppressed by the specific Src inhibitor PP1. These results demonstrate that tyrosine phosphorylation is a previously unrecognized mechanism involved in 1alpha,25(OH)(2)D(3) regulation of Ca(2+) homeostasis in hormone target cells. In addition, the data involve tyrosine kinase cascades in the mitogenic effects of 1alpha, 25(OH)(2)D(3) on skeletal muscle cells.

Animals↗
PubMed2000

1,25(OH)(2)-vitamin D(3) affects the subcellular distribution of protein kinase C isoenzymes in rat duodenum: influence of aging.

We have previously shown that the steroid hormone 1, 25-dihydroxy-vitamin D(3) [1,25(OH)(2)D(3)] stimulates total cell protein kinase C (PKC) activity in rat duodenum, an effect that is severely impaired in old animals. We further examined the role of 1, 25(OH)(2)D(3) on PKC as it relates to aging by measuring hormone-induced changes in subcellular localization of PKC activity and isoenzymes in duodenal mucosae from young (three-month-old) and aged (24-month-old) rats. Short treatment of duodenum with 1, 25(OH)(2)D(3) (0.1 nM, 1 min) increased membrane-associated PKC activity, whereas it decreased the activity in the cytosol of young rats but was without significant effect in aged animals. Furthermore, the ability to translocate was present in young animals after a short treatment with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA; 100 nM) or dioctanoyl-glycerol (50 microM), whereas the ability was absent in aged rats, suggesting that PKC function was impaired with aging independent of agonist stimulation. The expression of specific PKC isoenzymes and changes in their subcellular distribution after short exposure of the duodenum to the hormone were determined. Western blot analysis of total homogenates using antibodies to various PKC isoforms allowed detection of PKC alpha, beta, and delta. The expression of the straight theta and the zeta isoforms was in addition demonstrated by reverse transcription-polymerase chain reaction. The pattern of isoenzymes present in the duodenum was unaffected by aging. In young rats, 1, 25(OH)(2)D(3) translocates PKC alpha, beta, and delta to the membrane and nucleus; however, no translocation of PKC isoforms was observed in 24-month-old animals in response to the hormone. In summary, in rat duodenum, 1,25(OH)(2)D(3) modulation of PKC activity and isoenzyme subcellular distribution are impaired with aging and may explain age-induced alterations in the intestinal processes under the control of the hormone.

Aging↗
PubMed2000

Nicotiana glauca: another plant species containing vitamin D(3) metabolites.

Vitamin D(3)-related compounds have been detected in various plant species, mostly belonging to the Solanaceae. In this work we show that Nicotiana glauca, a widespread member of this taxonomic family, contains 7-dehydrocholesterol, vitamin D(3) and hydroxylated derivatives bearing precursor-product metabolic relationships in vertebrates. Leaves collected in the field and callus cultures were used. By means of specific radioreceptor binding assays and mass spectrometry of purified fractions obtained from plant tissue lipid extracts by Sephadex LH-20 and Sep-Pak C18 chromatography followed by HPLC, we established the presence of 7-dehydrocholesterol, vitamin D(3), 25(OH)-vitamin D(3) and 1alpha,25(OH)(2)-vitamin D(3) (1alpha,25(OH)(2)D(3)), the latter being a hormonally relevant metabolite in animals. These results indicate that N. glauca may represent a useful species in which to characterize the biosynthetic pathway and physiological functions of vitamin D(3) compounds in plants. In addition, tissue culture of N. glauca could become a significant tool for biotechnological production of 1alpha,25(OH)(2)D(3).

Journal Article↗
PubMed2000

Effect of aging on the mechanisms of PTH-induced calcium influx in rat intestinal cells.

We have investigated the effects of aging on parathyroid hormone (PTH) modulation of intracellular calcium homeostasis and their relationship to signal transduction pathways in isolated rat duodenal cells (enterocytes). PTH (10(-8)-10(-9) M) increased enterocyte (45)Ca(2+) influx and intracellular Ca(2+) concentration ([Ca(2+)](i)) to a greater extent (twofold and 50%, respectively) in aged (24 months) than in young (3 months) animals. The [Ca(2+)](i) response of old cells to the hormone was slower, lacking the early phase of changes in cytosolic Ca(2+). Ca(2+) influx induced by PTH was prevented by the protein kinase A antagonist Rp-cAMPS in both young and aged enterocytes, whereas neomycin and compound U73122, inhibitors of PLC-catalyzed phosphoinositide hydrolysis, abolished hormone-dependent Ca(2+) influx in young but had no effect on aged cells. Higher basal adenylyl cyclase (AC) activity and cAMP content were detected in old enterocytes. PTH increased the absolute levels of cAMP in aged cells and AC activity of microsomes isolated therefrom to a greater extent (>/= twofold) than in young enterocytes/membranes. In young cells, the hormone also induced a rapid and transient release of inositoltrisphosphate (IP(3)) and diacylglycerol (neomycin-sensitive) at 45 sec, and a delayed phase of DAG at 5 min (neomycin-insensitive). The early formation of IP(3) and DAG was blunted in aged animals. These results suggest that both the PLC and adenylyl cyclase cascades are involved in PTH stimulation of Ca(2+) influx in duodenal cells. During aging, however, only the cAMP pathway is operative, mediating a potentiation of the effects of the hormone. Additional studies are required to establish the relative role of PTH-dependent messenger systems in the regulation of intestinal calcium absorption and age-related abnormalities.

Adenylyl Cyclases↗
PubMed2000

Lumbar spine traction: evaluation of effects and recommended application for treatment.

Despite the widespread use of traction, little is known of the mode of effect, and application remains largely anecdotal. The efficacy of traction is also unclear because of generally poor design of the clinical trials to date, and because subgroups of patients most likely to benefit have not been specifically studied. These observations prompted this review, the purposes of which are to evaluate the mechanisms by which traction may provide benefit and to provide rational guidelines for the clinical application of traction. Traction has been shown to separate the vertebrae and it appears that large forces are not required. Vertebral separation could provide relief from radicular symptoms by removing direct pressure or contact forces from sensitised neural tissue. Other mechanisms proposed to explain the effects of traction (e.g. reduction of disc protrusion or altered intradiscal pressure) have been shown not to occur. We conclude that traction is most likely to benefit patients with acute (less than 6 weeks' duration) radicular pain with concomitant neurological deficit. The apparent lack of a dose-response relationship suggests that low doses are probably sufficient to achieve benefit.

Animals↗
PubMed2000

Hospitalization in the Program of All-Inclusive Care for the Elderly (PACE): rates, concomitants, and predictors.

BACKGROUND: The Program of All-Inclusive Care for the Elderly (PACE) replicates the model of comprehensive, community-based geriatric care pioneered by On Lok, that enrolls frail older adults who meet states' criteria for nursing home care, and that uses interdisciplinary teams to assess the participants and to deliver care in appropriate settings. As managed care, PACE receives capitated payment from Medicare and Medicaid. Thus, PACE's fiscal incentives are thought to be aligned with the goals of optimizing health, function, and quality of life through the delivery of effective primary, preventive, restorative, supportive, and palliative care and through the avoidance of inappropriate and expensive hospital and nursing home utilization. OBJECTIVES: To describe short-term hospital utilization, hospital discharge diagnoses, time from enrollment to first hospitalization and its clinical predictors, and hospitalization in relation to mortality among PACE participants. METHODS: Data on short-term hospitalization and participants were recovered from PACE's minimum data set. Bed use was evaluated in annual cross-sections of current participants. Primary hospital discharge diagnoses were available for discharges from September 1, 1993 through March 31, 1997. The time from enrollment to hospitalization was calculated for the participants (n = 5478) who were admitted between January 1, 1990 and March 31, 1997. The characteristics of this inception cohort were used to develop a Cox regression model of hospitalization. All PACE deaths were identified and the place of death was recovered, together with the medical records used in the hospital during PACE enrollment or 6 months before death. RESULTS: Bed-days per 1,000 PACE participants per year were comparable with the general Medicare (fee-for-service) population, at 2,046 (in 1998) versus 2014 (in 1997) despite the greater morbidity and disability for PACE participants, as reflected in their enrollment characteristics and primary hospital discharge diagnoses. The time to hospitalization was 773 days (median); 95% confidence interval, 725, 814, and was predicted by disease, treatment, social and demographic factors. Whereas 8% of PACE deaths occurred in acute hospitals, less than one-third of the decedents spent any time in the hospital in the 6-month interval before death. CONCLUSIONS: Overall, short-term hospital utilization among PACE participants is low in contrast with that for other older and disabled populations. Participant predictors of hospitalization in PACE are generally consistent with other studies in older clinical and community populations. Both utilization and risk vary considerably across PACE sites, independent of participant-level risk factors, hence suggesting that further investigation is required to study PACE's management of acute illness and hospitalization decisions. Critical to maintaining PACE's success is an understanding of the independent impact of the organization and the environment of health care on this management.

Activities of Daily Living↗
PubMed1999

17beta-oestradiol increases intracellular Ca2+ concentration in rat enterocytes. Potential role of phospholipase C-dependent store-operated Ca2+ influx.

The involvement of the phospholipase C (PLC) pathway in the non-genomic regulation of duodenal cell Ca2+ concentration by 17beta-oestradiol was investigated. The PLC inhibitors neomycin (0.5 mM) and U-73122 (2 microM) suppressed the stimulatory effect of 0.1 nM 17beta-oestradiol on the 45Ca2+ influx into enterocytes isolated from rat duodenum. The hormone (1 pM to 10 nM) increased the formation of 1,2-diacylglycerol in a biphasic pattern, characterized by an early peak at 45 s (+82%) and a later peak at 5 min (+46%). Both PLC inhibitors suppressed the first peak but were unable to block the 17beta-oestradiol effect at 5 min. 17beta-Oestradiol also increased the generation of inositol 1,4,5-trisphosphate within 15 s, with maximal stimulation at 30 s. 17beta-Oestradiol induced a rapid (30 s) and sustained (up to 5 min) increase in the intracellular Ca2+ concentration ([Ca2+]i) of fura 2-loaded enterocytes. The fast rise in [Ca2+]i was specific because other sex steroid hormones were without effect and could be blocked to a great extent by U-73122 (by 86% at 1 min). The effects of 17beta-oestradiol on enterocyte [Ca2+]i were decreased significantly (by 75%) in a Ca2+-free extracellular medium but a pronounced increase in [Ca2+]i was obtained after readmission of Ca2+ to the medium. The latter change was suppressed by 10 microM La3+, whereas nitrendipine (1 microM) and verapamil (10 microM) separately were without effect. The permeability of the 17beta-oestradiol-induced Ca2+ influx pathway to Mn2+ was increased 2.8-fold by treatment with oestrogen. These results suggest the operation of a PLC-dependent store-operated Ca2+ channel mechanism in 17beta-oestradiol regulation of enterocyte extracellular Ca2+ influx.

Animals↗
PubMed1999

Characterization of membrane estrogen binding proteins from rabbit uterus.

Estrogens exert fast non-genomic actions in their target tissues which may involve the participation of receptors located at the cell membrane. Studies were performed to identify and characterize membrane-associated 17beta-estradiol binding proteins in rabbit uterus. Specific and saturable [3H]17beta-estradiol binding sites of high affinity (Kd = 0.36 nM) were detected in uterine microsomes at higher concentration than in cytosol (370 +/- 98 vs. 270 +/- 87 fmol/mg protein, respectively). Various other steroid hormones, the stereoisomer 17alpha-estradiol and the antiestrogen tamoxifen were significantly less effective than 17beta-estradiol to compete with the radioactive ligand for binding to the membranes. The microsome binding sites were trypsin-sensitive and could be extracted to a great extent (80-90%) with 0.4/0.6 M KCl. Assays of the marker enzyme glucose-6-P dehydrogenase excluded membrane contamination with cytosolic soluble components. Immunoblot analysis of particulate and soluble fractions using monoclonal antibodies against the transactivation, heat shock protein recognition, and steroid binding domains of the nuclear estrogen receptor (ER; 67 kDa), revealed lower concentrations of the ER in membranes and the presence of five additional immunoreactive proteins of 57, 50, 32, 28, and 11 kDa which were absent in cytosol. Moreover, the antibody against the steroid binding domain was as effective as an inhibitor for cytosolic and membrane specific radioligand binding. Extraction of microsomes with the nondenaturing detergent CHAPS allowed a 2-fold enrichment of ER-like binding proteins as shown by antibody labeling and [3H]17beta-estradiol binding analysis. The results of this work are consistent with the existence of novel 17beta-estradiol membrane binding proteins structurally related to the intracellular ER. Future studies should investigate whether any of these proteins are involved in the primary events (e.g. receptor function) mediating nongenomic estrogen effects.

Animals↗
PubMed1999

Effect of 1,25(OH)(2)-vitamin D(3) on the activation of natural killer cells: role of protein kinase C and extracellular calcium.

As a first approach for studying the implication of PKC and the steroid hormone 1,25(OH)(2)-vitamin D(3) [1,25(OH)(2)D(3)] on natural killer cell (NK) activity, we analyzed in the YT NK cell line the expression of PKC isoforms and the effects of 1, 25(OH)(2)D(3) on BLT-esterase (a marker of NK lytic granules) activity. Western blot and RT-PCR showed a greater extent of PKC alpha, beta, delta, zeta, epsilon, theta, and lambda and lower levels of PKC mu and eta. In a dose-dependent manner 1, 25(OH)(2)D(3) induced significant increases in BLT-esterase and PKC activities and the stimulatory effect on BLT-esterase activity was mimicked and blocked, respectively, by the PKC activator phorbol ester PMA and PKC inhibitors (H7, PKC(19-36), and N-myristoylated PKC(19-31) peptides). Moreover, the effects of 1,25(OH)(2)D(3) on BLT-esterase could be blocked in a Ca(2+)-free (+EGTA) medium and mimicked by the Ca2+ ionophore A23187. The results suggest that 1, 25(OH)(2)D(3) is a stimulatory factor of NK activity acting through a mechanism involving PKC and extracellular Ca2+.

Base Sequence↗
PubMed1999

In vivo treatment with calcitriol (1,25(OH)2D3) reverses age-dependent alterations of intestinal calcium uptake in rat enterocytes.

The vitamin D endocrine system has been involved in the impairment of intestinal calcium absorption during aging. Alterations in the nongenomic mechanism of calcitriol (1,25-dihydroxy-vitamin D3; [1, 25(OH)2D3] have been recently evidenced. In enterocytes isolated from aged rats, 1,25(OH)2D3 stimulation of Ca2+ channels through the cAMP/PKA pathway is blunted. We have now investigated whether in vivo administration of calcitriol to senescent rats reverses the absence of hormonal effects in isolated intestinal cells. In enterocytes from 20-24-month-old rats given 1,25(OH)2D3 for 3 days (30 ng/100 g bw/day), calcitriol (10(-10) M, 3-5 minutes) stimulated Ca2&plus uptake and intracellular cAMP to the same degree and protein quinase A (PKA) activity to a lesser degree than in enterocytes from young animals. Significantly higher basal levels of cAMP and PKA detected in enterocytes from old rats were not affected by prior injection of animals with 1,25(OH)2D3. When the aged rats were injected with 25(OH)D3, similar Ca2+ influx, cAMP, and PKA responses to in vitro stimulation with calcitriol were obtained. 1, 25(OH)2D3-dependent changes in Ca2+ uptake by enterocytes from both young and old rats treated with calcitriol were totally suppressed by the cAMP antagonist Rp-cAMPS, whereas the response to the agonist Sp-cAMPS was markedly depressed in aged animals. These results suggest that intestinal resistance to nongenomic 1,25(OH)2D3 stimulation of duodenal cell Ca2+ uptake develops in rats upon aging and show that in vivo administration of 1,25(OH)2D3 or its precursor to senescent rats restores the ability of the hormone to stimulate duodenal cell calcium influx through the cAMP messenger system.

Aging↗
PubMed1999

Rapid actions of calcitriol and its side chain analogues CB1093 and GS1500 on intracellular calcium levels in skeletal muscle cells: a comparative study.

1. The ability of synthetic analogues of the secosteroid hormone 1alpha,25-dihydroxy-vitamin-D3 [calcitriol, CT; 1,25(OH)2D3] to exert non-genomic (rapid) effects on target cells has been scarcely studied. To evaluate the pharmacological potential of the CT side-chain analogues CB1093 and GS1500, we compared their fast effects on intracellular calcium concentration ([Ca2+]i) in chick skeletal muscle cells with those elicited by the natural hormone. 2. Both analogues, similarly to CT, specifically induced rapid (30-60 s) and sustained rises in [Ca2+]i levels. CB1093 and GS1500 were more potent than the natural hormone at concentrations as low as 10(-13) M (4.5 fold stimulation) and 10(-12) M (2.5 fold), respectively, whereas higher concentrations (10(-9)- 10(-8) M) of CT were more effective than the analogues in elevating [Ca2+]i. Cyclic AMP was markedly increased by both analogues pointing for a role of this messenger in the fast actions of the synthetic compounds. 3. In Ca2+ free medium CT and analogues elicited a transient elevation in [Ca2+]i. The PLC inhibitors U73122 (2 microM) and neomycin (0.5 mM), as well as depletion of intracellular stores with thapsigargin (1 microM), completely prevented CB1093/GS1500-dependent changes in [Ca2+]i suggesting that, similarly to CT, these analogues mobilized Ca2+ from an IP3/thapsigargin-sensitive store. 4. The voltage-dependent calcium channel (VDCC) blocker nifedipine (2 microM) reduced by 50-60% the influx phase of the [Ca2+]i response to CB1093 and GS1500, indicating that VDCC contributed partially to Ca2+ entry. The Ca2+ readdition protocol suggested that analogue-dependent activation of a SOC entry pathway accounted, to the same extent as for CT, for the remaining non-VDCC mediated Ca2+ influx.

Animals↗
PubMed1998

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.

Argentina↗
PubMed1998

Age-related loss of calcitriol stimulation of phosphoinositide hydrolysis in rat skeletal muscle.

We have examined the effects in vitro of calcitriol [1,25(OH)2D3], the hormonal form of vitamin D3, on the breakdown of membrane phosphoinositides in skeletal muscle from young (3 months) and aged (24 months) rats. Calcitriol (10(-9) M) induced a rapid and transient release of IP3/inositol phosphates and diacylglycerol (DAG) from muscle slices/membranes prelabeled with [3H]myo-inositol and [3H]arachidonate, respectively. Inositol phosphate release was maximal at 15 s and then declined. The effects of hormone specificity exhibited as the closely related derivatives of vitamin D3, 25OHD3, 1alphaOHD3 and 24,25(OH)2D3 did not alter muscle inositol phosphate levels. The stimulation of DAG was biphasic, the early phase (15 s) being abolished by neomycin (0.5 mM), an inhibitor of phosphoinositide hydrolysis, similar to IP3 formation and consistent with a role of phospholipase C (PLC) in intracellular signal generation. Neomycin had no effect on the second DAG peak (2 min) induced by calcitriol, suggesting that the late phase of DAG formation is independent from the hydrolysis of phosphoinositides. Higher basal inositol phosphate and DAG levels were detected in muscle from aged rats thereby reducing the effects of the hormone on second messenger generation ( -80 and -60% for IP3 and DAG, respectively). Calcitriol stimulation of PLC was mimicked, in both young and old rats, by GTPgammaS, a non-hydrolyzable analogue of GTP, while GDPbetaS, a G protein inhibitor, suppressed the effect of the hormone. The early effects of calcitriol and GTPgammaS were not additive. Bordetella pertussis toxin abolished by 85% the effects of calcitriol on inositol phosphate release in young rats but was without effect in aged animals. These results demonstrate that calcitriol activates phosphoinositide-PLC in rat skeletal muscle by a mechanism which involves a pertussis-sensitive G protein and that the effects of the hormone are altered with ageing.

Aging↗
PubMed1998

1,25(OH)2-vitamin D3 affects the subcellular distribution of protein kinase C isoenzymes in muscle cells.

Previous studies have shown the involvement of protein kinase C (PKC) in 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] regulation of DNA synthesis (long-term effect) and Ca2+ channel activity (short-term effect) in cultured myoblasts. Both events mediate stimulation of myoblast cell proliferation and growth by 1,25(OH)2D3. To characterise further the role of PKC in the hormone mode of action in muscle cells, the presence of PKC isoenzymes in chicken embryo myoblasts and changes in their total cell and subcellular levels after treatment (72 h and 5 min) with 1,25(OH)2D3 (1 nM), 12-O-tetradecanoyl phorbol 13-acetate (TPA; 100 nM) and 1,2-dioctanoyl-rac-glycerol (DOG; 50 microM) were investigated. Western blot analysis provided evidence on the expression of PKC alpha, beta and delta isoforms in avian myoblasts. Two immunoreactive bands of 80 kDa (intact molecule) and 50 kDa (catalytic fragment) were detected for each isoenzyme. 1,25(OH)2D3 and DOG, which increased myoblast PKC activity parallel with the stimulation of DNA synthesis and culture growth and the phorbol ester TPA which induced the opposite changes, exerted differential effects on PKC isoenzymes. Long-term (72 h) treatment with 1,25(OH)2D3 and DOG did not change total PKC isoform levels but decreased the 80 kDa species and increased the release of the catalytic fragment of PKC delta and beta, whereas TPA augmented the total amounts of the three PKC isoforms, increasing the band of 80 kDa of PKC beta and delta and the 50 kDa species for PKC alpha. Subcellular distribution studies showed that the 80 kDa molecule is only present in the cytosolic fraction whereas in the particulate fractions the 50 kDa fragments are detected. Increased amounts of the catalytic fragments of PKC beta and delta both in the nucleus and membranes were observed after 72 h treatment with DOG while 1,25(OH)2D3 increases PKC beta in the nucleus and PKC delta in membranes. TPA induced the appearance of the 50 kDa species of PKC alpha in the nuclear and membrane fractions. The phorbol ester also decreased the catalytic fragments of PKC beta and delta in membranes. Increased levels of PKC beta, and to a lesser extent of PKC delta, in membranes and cytosol could be detected after short exposure (5 min) of myoblasts to 1,25(OH)2D3, DOG and TPA. In conclusion, the data indicate the operation in myoblasts of PKC signal transduction pathways mediated by the Ca(2+)-dependent PKCs alpha and beta and the Ca(2+)-independent PKC delta. Moreover, the results suggest that the beta and delta isoforms of PKC could play a role in the regulation of muscle cell metabolism by 1,25(OH)2D3.

Animals↗
PubMed1998

Calcitriol transmembrane signalling: regulation of rat muscle phospholipase D activity.

In rat skeletal muscle, calcitriol, the hormonal form of vitamin D3, rapidly stimulates the biphasic formation of diacylglycerol (DAG), the second phase being independent of phosphoinositide hydrolysis driven by phospholipase C. In this work we showed that the effect of calcitriol on the second phase of DAG formation was totally inhibited in the absence of extracellular Ca2+ and by the Ca2+-channel blockers nifedipine and verapamil, whereas the Ca2+ ionophore A23184, similar to calcitriol, increased DAG formation by 100%. GTPgammaS, which activates G protein-mediated signals, mimicked the effects of the hormone while GDPbetaS, an inhibitor of G proteins, suppressed calcitriol-induced DAG formation. To elucidate the metabolic pathway of the late phase of DAG production, we examined the contribution of phospholipase D (PLD), which acts on phosphatidylcholine (PC) generating phosphatidic acid that is converted to DAG by a phosphatidate phosphohydrolase. In [3H]arachidonate-labeled muscle, calcitriol increased [3H]phosphatidylethanol (PEt) formation in the presence of ethanol, a reaction specific for PLD. The effects of the hormone were time- and dose-dependent with maximum PEt levels achieved at 10(-9) M. The phorbol ester TPA also stimulated PEt formation. The combination of calcitriol and TPA was more effective than either compound alone. In rat muscle, calcitriol increased PKC activity in a time-dependent fashion. Bisindolymaleimide, a selective inhibitor of the enzyme, completely suppressed TPA-induced PEt and attenuated the effects of the hormone. These results provide the first evidence concerning calcitriol stimulation of the hydrolysis of PC in a mammalian tissue through a phospholipase D catalyzed mechanism involving Ca2+, protein kinase C, and G proteins.

Animals↗
PubMed1997

Stimulation of Ca2+ release-activated Ca2+ channels as a potential mechanism involved in non-genomic 1,25(OH)2-vitamin D3-induced Ca2+ entry in skeletal muscle cells.

As in other vitamin D target cells, activation of voltage-dependent Ca2+ channels (VDCC) mediates the fast, non-genomic, 1,25(OH)2D3 stimulation of Ca2+ influx in skeletal muscle cells (SMC). 1,25(OH)2D3 has also been shown to rapidly induce the release of Ins(1,4,5)P3 in SMC. Experiments were performed to investigate whether Ca2+ release-activated Ca2+ channels (CRAC) also participate in the mechanism by which 1,25(OH)2D3 regulates Ca2+ entry into these cells. In cultured chick SMC loaded with Fura-2/AM the hormone (10(-12) - 10(-8) M) induced a rapid (30 sec) followed by a sustained (up to 5 min) increase in intracellular Ca2+ concentration ([Ca2+]i) associated to Ca2+ mobilization from internal stores and influx of extracellular Ca2+, respectively. Thus, the initial, transient, 1,25(OH)2D3-dependent increment in [Ca2+]i could be observed in Ca2+-free medium and was abolished by the PLC inhibitor U73122. Readdition of Ca2+ to cells that had undergone the initial 1,25(OH)2D3-induced [Ca2+]i rise in Ca2+ free medium resulted in a fast increment in [Ca2+]i indicating the existence of a hormone-activated CRAC entry pathway. The sustained phase of the Ca2+ response to 1,25(OH)2D3 was only partially (60%) suppressed by nifedipine, whereas lanthanum (10 microM) completely abolished the hormone effects. Accordingly, depletion of intracellular Ca2+ stores by thapsigargin reproduced 1,25(OH)2D3-induced Ca2+ influx, inhibiting any further response to the sterol. 1,25(OH)2D3 increased the rate of quenching of Fura-2 fluorescence by Mn2+, indicating activation of Mn2+ permeable channels. Altogether, these results provide the first evidence involving CRAC channels in the rapid modulation of Ca2+ entry in animal cells by 1,25(OH)2D3.

Animals↗
PubMed1997

Effects of calcitriol and its analogues, calcipotriol (MC 903) and 20-epi-1alpha,25-dihydroxyvitamin D3 (MC 1288), on calcium influx and DNA synthesis in cultured muscle cells.

The fast actions of the secosteroid hormone 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3; calcitriol] and the synthetic analogues calcipotriol (MC 903) and 20-epi-1alpha,25(OH)2D3 (MC 1288) on cell calcium influx were compared in rat duodenum enterocytes as well as in cells from chick embryo skeletal muscle (myoblasts) and heart (myocytes), at various concentrations (10(-12) to 10(-8) M) and treatment intervals (1-10 min). In enterocytes, at a concentration of 10(-11) M, MC 1288 was significantly more active than 1,25(OH)2D3 in rapidly stimulating 45Ca2+ uptake by enterocytes (80 vs 38% above controls, respectively), whereas MC 903 was devoid of activity. However, calcipotriol increased Ca2+ influx in myocytes and myoblasts to a greater extent than the natural hormone, whereas MC 1288 was more active only in myoblasts. Analogously to 1,25(OH)2D3, the fast MC 903- and MC 1288-induced stimulation of 45Ca2+ uptake in enterocytes and muscle cells could be blocked by both verapamil and nifedipine. In addition, MC 903 and MC 1288 were more effective than 1,25(OH)2D3 in stimulating DNA synthesis in proliferating myoblasts and in inhibiting DNA synthesis in differentiating myoblasts. The results suggest, therefore, that modifications in the side-chain of the 1,25(OH)2D3 molecule increase its ability to modulate muscle cell Ca2+ metabolism and growth. These findings are potentially relevant for the development of analogues for the treatment of vitamin D-dependent myopathies.

Animals↗