Superoxide anion production in glass-adherent polymorphonuclear leukocytes and its relationship to calcium movement.

The production of superoxide anion (O2-.) was measured in relation to 45Ca movement in glass-adherent polymorphonuclear leukocytes (PMNs), and the results were compared with those obtained by ourselves and others on PMNs in suspension. In adherent PMNs, O2-. production stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP) was of rapid onset and short duration; this was also true of PMNs in suspension. However, O2-. production was insensitive to the concentration of extracellular calcium. Both in adherent and non-adherent PMNs, O2-. production stimulated with phorbol myristate acetate (PMA) had a latency time and was of long duration. In adherent PMNs, pretreatment with PMA potentiated the FMLP-induced O2-. production by lengthening its duration without changing its initial rate. In adherent PMNs (10(-10)-10(-7) M) FMLP induced a fast but transient dose-dependent increase in 45Ca within 1 min, whereas PMA only released 45Ca about 5 min after its addition to the cell culture medium. Pretreatment of PMNs with 10 or 100 ng/ml PMA for 3 min before stimulation by 10(-7) M FMLP reduced the 45Ca efflux observed with FMLP alone. We conclude that O2-. production by adherent PMNs cannot simply be related to Ca2+ movement. In comparison with non-adherent cells, adherence seemed to interfere with the characteristics of both calcium and O2-. generation, probably by modifying the cytoskeleton.

C cell activity during the prenatal and postnatal periods in the rat.

The high plasma calcitonin (CT) level found in suckling newborns and baby rats led us to hypothesize that thyroid C cells might be exhausted during the postnatal period. This prompted us to evaluate the CT concentration of the thyroid C cells during the prenatal and postnatal periods in the rat as the CT content to number ratio of C cells. The CT content of the thyroid gland increased exponentially from 17.5 (0.03 ng) to 21.5 (1.72 ng) days of gestation in the rat fetus; C cells were detected by immunofluorescence and counted from 19.5 days of gestation until term. A value of 600 + 90 C cells was obtained at 19.5 days, 1557 +/- 239 at 20.5 days, and 2602 +/- 536 at 21.5 days of gestation. Plasma CT concentrations were undetectable (less than 150 pg/ml) before 20.5 days of gestation, but increased to approximately 500 microgram/ml in 20.5- and 21.5-day-old fetuses. After birth, both the thyroid CT content and the number of C cells increased progressively. In 3-day-old suckling newborns, 4298 +/- 412 C cells were found; 9679 +/- 1114 were found 7 days after birth, and 12202 +/- 1280 were observed 15 days after birth; at the same stages, the CT contents of the thyroid gland were 6.54 +/- 0.18, 8.59 +/- 0.19, and 19.49 +/- 0.79 ng, respectively. Thus, the CT concentrations of the C cells (approximately 1.0 pg/cell) were roughly similar during the prenatal and postnatal periods in the rat. These results suggest the presence of active C cells during fetal life in the rat. They also indicate a capacity of C cells during the prenatal and postnatal periods to increase their secretion of CT while maintaining their hormone content, since the CT concentration of the C cell remains unaltered in spite of the high CT secretion in suckling rats.

Uterine cells other than stromal decidual cells are required for 1,25-dihydroxyvitamin D3 production during early human pregnancy.

Human decidual cells are known to produce 1,25-(OH)2D3 at the end of pregnancy, the present study evaluates this capacity, and the part played by stromal decidual cells, in early pregnancy. Cells were obtained from nine human decidua by aspiration or curettage during early pregnancy (7-10 weeks), separated on Ficoll-Paque and plastic adherence, and incubated for 1 h with 25-(OH)D3. Incubation medium and cells were extracted and chromatographed on two successive HPLC systems. The cells examined were of both physiological and pathological (ectopic pregnancy) origin. Endometrial cells obtained in four non-pregnant situations (myomas) were also studied to determine whether the 1,25-(OH)2D3 synthesis by the uterus is associated with the appearance of decidual cells. Results show that human decidual cells from early pregnancy convert 25(OH)D3 (2.5 nM or 2.5 microM) into a metabolite with the physicochemical characteristics of synthetic 1,25-(OH)2D3. This ability is shared by cells isolated during early pregnancy, whether physiological or ectopic (tubal pregnancy). Non-adherent cells, which include mainly stromal decidual cells, are less able to produce 1,25-(OH)2D3 than are the adherent cells, suggesting that macrophages, granulocytes or as yet unidentified cell types are required for the 1,25-(OH)2D3 production by decidual tissue during early human pregnancy. In addition, one out of four experiments with non-pregnant endometrial cells could produce 1,25-(OH)2D3 suggesting that, although not the rule in the non-pregnant state, in vitro production of 1,25-(OH)2D3 by uterine cells can be found in the absence of decidual cells.

Basal bone resorption in the rat fetus related to the hormonal status of the mother.

Fetal bone resorption was measured by an organ culture technique using fetuses from thyroparathyroidectomized (TPTX) pregnant rats to investigate the roles of fetal and maternal hormones in the regulation of basal bone resorption in utero. Thyroparathyroidectomized female rats were treated with thyroxine and/or metabolites of vitamin D3. Basal bone resorption was greatly increased in fetuses from TPTX mothers. Administration of 1,25-dihydroxycholecalciferol or 1,24,25-trihydroxycholecalciferol to TPTX female rats normalized the basal bone resorption of the fetus whereas injection of 24,25-dihydroxycholecalciferol was without effect. Treatment of the TPTX mothers with thyroxine was also shown to normalize basal bone resorption in the fetus.

Effect of calcitonin in pregnant rats on bone resorption in fetuses.

Fetal bone resorption was measured by an organ culture technique using fetuses from intact or thyroparathyroidectomized pregnant rats. These experiments were performed to investigate the effects of 1,25-dihydroxycholecalciferol (1,25-DHCC) and salmon calcitonin (SCT) in pregnant rats, on both fetal growth and fetal bone resorption. Pregnant rats were given 0.1-0.5 microgram 1,25-DHCC per day from day 17 of gestation: in intact rats bone resorption was increased and fetal growth decreased; 1,25-DHCC probably modified fetal bone resorption in the absence of fetal parathyroid secretion. Infusion of SCT in minipumps (30 mu./h) did not modify plasma calcium levels in either the mother or fetuses, neither was bone resorption altered. In 1,25-DHCC-treated rats, SCT infusion resulted in an increase in fetal weight and a decrease in fetal bone resorption. On the other hand, SCT infusion was found to facilitate phosphate accumulation in fetuses. At the end of the SCT infusion the SCT concentration was 450 ng/l in maternal plasma and 553 +/- 60 ng/l in fetal plasma. Salmon calcitonin was shown to cross the placental barrier in the rats; it may interact with the effects of 1,25-DHCC in the fetus.

Nuclear retinoic acid receptor characterization in cultured human trophoblast cells: effect of retinoic acid on epidermal growth factor receptor expression.

Vitamin A is an important factor during gestation and its metabolite, retinoic acid (RA), is a potent teratogen. However, RA action on the placenta is still poorly understood. In this study we analysed the presence of RARs and RXRs in human trophoblastic cells. We determined that RAR alpha was the more expressed form in term placenta, and that RAR beta was induced by RA treatment. Then we analysed RA effects on endocrine activities and on epidermal growth factor (EGF) receptor expression. We found that RA decreased 125I-labeled EGF binding and EGF-dependent phosphorylation. Furthermore, RA treatment led to a concentration-dependent decrease in the amount of EGFR protein expression. This treatment also decreased EGF receptor mRNA levels, suggesting transcriptional regulation of the EGF receptor. Thus we demonstrated that RA could interact with feto-placental development by modulating trophoblast EGF receptors expression, probably via its nuclear receptors.

Effects of calcitonin on human trophoblastic cells in culture: absence of autocrine control.

The purpose of this work was to investigate the effects of calcitonin (CT) on trophoblastic cells with respect to cAMP levels and human chorionic gonadotrophin (hCG) secretion in cultured cells from first-trimester and term placentas and in a choriocarcinoma cell line (JEG-3). The expression of the CT gene was investigated to elucidate a putative autocrine control of CT during pregnancy. The addition of salmon CT (10(-10) M and above) resulted in concentration-dependent increases in cAMP secretion by normal trophoblastic cells from term and first-trimester placentas. Moreover, CT was found to increase cAMP secretion preferentially in completely differentiated cells, i.e. after 4-7 days in culture. Addition to the culture medium of JEG-3 cells slightly increased cAMP secretion only at a concentration of 10(-8) M. The basal level of hCG in the medium was found to be higher in the first-trimester than in the term trophoblast culture, but salmon CT induced an increase in hCG secretion by term placenta cells only. CT gene expression in our experimental model was investigated to elucidate a putative autocrine control of CT action during pregnancy. It was not found to be expressed in syncytiotrophoblast cells from either first-trimester or term placenta cells by the method used. Our data demonstrate the absence of autocrine control of CT effects in trophoblastic cells, and suggest that CT is likely to exert its effect preferentially on differentiated cells.

Effect of calcitriol on peripheral blood lymphocyte cytotoxicity.

To clarify the biochemical mechanism responsible for the inhibition by calcitriol (1,25-dihydroxyvitamin D3) of cytotoxicity in peripheral blood lymphocytes (PBL), the human NK sensitive K562 cell line and the human tumor necrosis factor-sensitive murine L929 cell line were used as targets and subsequently compared. The cytotoxicity of PBLs for K562 cells was not changed by preincubation for 4 h with 10 ng/ml phorbol myristate acetate (PMA), but was reduced after an overnight preincubation with 10(-9) or 10(-8) M calcitriol. Using L929 cells, preincubation of PBLs with 10 ng/ml PMA for 4 h increased their cytotoxicity. Overnight incubation with calcitriol significantly reduced PBL cytotoxicity for L929 cells in a dose related manner and suppressed the enhancement of this cytotoxicity by PMA. Pretreatment of PBLs with cycloheximide reduced their cytolysis for L929 cells but did not change their cytotoxicity towards K562 cells. Consequently, the natural cytotoxicity of PBLs for K562 cells does not involve the same mechanism as their cytotoxicity for L929 cells and is therefore subject to different forms of regulation. However, calcitriol reduced PBL cytotoxicity towards both target cells.

[Metabolism of bone in culture (author's transl)]. / Etude du métabolisme de l'os en culture.

A method for studying in vitro bone matrix resorption by the use of 35S injection was investigated. Various hormones were tested and their effects on 35S and 45Ca metabolism were compared.

Effects of experimental diabetes on the vitamin D metabolism of pregnant rats and their fetuses.

The effects of streptozotocin-induced diabetes on the vitamin D metabolism of pregnant rats were investigated in mothers and their fetuses, 11 and 14 days after streptozotocin (SZ) injection, i.e., on days 18 and 21 of gestation. In the mothers' plasma, the levels of 25-hydroxycholecalciferol (25OHD) and 1,25-dihydroxycholecalciferol (1,25(OH)2 D) were not different from control levels on day 18, but on day 21, 25OHD had increased, 1,25 (OH)2 D had diminished, and significant hypercalcemia was noted (10.1 +/- 0.27 mg/dl vs. 9.47 +/- 0.19 mg/dl, mean +/- SD). In hyperglycemic fetuses from the diabetic mothers, plasma insulin levels were reduced at day 18 but enhanced at day 21. 25OHD levels were not different from those of the controls at day 18, but were lower at day 21 (2.12 +/- 0.70 ng/g BW, n = 13, vs. 3.75 +/- 1.40 ng/g BW n = 29 controls, means +/- SD). Fetal body levels of 1,25 (OH)2 D were lower than that in the controls at day 18 (16.6 +/- 2.9 pg/g BW, n = 9 x 2, vs. 28.7 +/- 6.3 pg/g BW, n = 7 x 2, mean +/- SD P less than 0.001), but identical to control levels on day 21. The role of fetal or placental enzymes in the regulation of vitamin D metabolism in fetuses is discussed.

Comparative effects of the ionophore A23187 and vitamin D metabolites on 45Ca desaturation curves derived from bone cells: interaction of a calcium channel channel inhibitor diltiazem.

The effects of diltiazem, a calcium channel inhibitor, on the cellular transport of calcium were studied in isolated heterogenous rat bone cells. Efflux was measured after equilibrating the cells with 45Ca and adding the vitamin D metabolite (1,25dihydroxycholecalciferol-1,25(OH)2D3 or 24,25dihydrocholecalciferol-24,25(OH)2D3), the ionophore A23187 and/or diltiazem. Results were analysed by fitting the desaturation curve to a model of two exponential terms. Kinetic analyses of curve indicated the presence of 2 exchangeable pools with different rate constants of exchange between the medium and cells (expressed by K.). After incubation of bone cells with diltiazem (20 nmol/10(6) cells) the following changes were recorded: a marked decrease in the rate constant of efflux from the fast turnover calcium pool (K12) and a reduction of the calcium pool sizes. Incubation of 10(6) cells with 0.5 ng 1,25(OH)2D3 plus diltiazem significantly reduced K12 compared to incubation with 1,25(OH)2D3 alone. In presence of 24,25(OH)2D3, diltiazem did not significantly alter K12 which was raised by incubation with the metabolite alone. Ionophore A23187 (0.5 micrograms/10(6) cells) increased the value of slow turnover constants of efflux whose values were affected by diltiazem. The possible involvement of Ca movements in bone resorption does not seem confirmed in the present experiment since in vitro effects of diltiazem in organ culture (observed in an initial previous experiment) were not reflected in the calcium 45 desaturation kinetics in heterogenous bone cells.

[Effects of 1,25-dihydroxyvitamin D3 on bone resorption and 45Ca2+ efflux in bone cells]. / Effets de la 1.25-dihydroxyvitamine D3 sur la résorption osseuse et l'efflux du 45Ca2+ dans les cellules osseuses.

1,25-dihydroxyvitamin D3[1,25(OH)2D3] effects on bone resorption in organ culture and on 45Ca2+ efflux rates in bone cells were measured in presence of a calcium channel inhibitor, diltiazem. Though, diltiazem reduced the 45Ca release from mice calvaria it did not act at the same Ca compartment as 1,25(OH)2D3 to alter Ca2+ flux parameters. It therefore seems difficult to hypothesize a simple relationship between bone resorption and Ca2+ movements in bone cells.

[Effect of experimental diabetes on levels of 25-hydroxycholecalciferol in pregnant rats and fetuses]. / Effet du diabète expérimental sur les taux de 25-hydroxycholécalciférol chez la rate gestante et le foetus.

Induction of diabetes in female rats by streptozotocin administration 7 days before mating led to an increase in maternal and fetal calcemia at day 21 of gestation. The plasma levels of 25-hydroxycholecalciferol (25 OH D3) were increased in the diabetic mother whereas the 25 OHD3 contents in entire fetuses were greatly decreased in comparison with control values obtained in both normal pregnant rat and normal fetuses. Our results obtained in pregnant rat were different from those found in the literature concerning non pregnant animals in which only 1,25-dihydroxycholecalciferol was affected by diabetes.

Regulation of 24-hydroxylase activity in mouse skin fibroblast by cholecalciferol derivatives, triamcinolone acetonide and a calcium modulating agent, nicardipine.

Mouse skin fibroblasts in culture were used to study the regulation of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) induced 24 hydroxylase (24-OH-ase) under the influence of 3 agents: (1) 24,25-Dihydroxycholecalciferol (24,25(OH)2D3), 62.5 10(-9) M, which led to a significant decrease in the 1,25(OH)2D3-induced 24-OH-ase, probably acted through a nuclear effect mediated by the 1,25(OH)2D3 receptor protein. (2) Triamcinolone acetonide (10(-6)M) which was found to increase the 24-OH-ase enhancement induced by 1.25 and 6.25 nM 1,25(OH)2D3 whereas it did not alter the effect of 31.2 nM 1,25(OH)2D3. (3) A factor which is likely to induce changes in the cell calcium transport or in the Ca pool sizes, i.e. a calcium channel blocker, nicardipine. The effect of 1.25 nM 1,25(OH)2D3 on 24-OH-ase activity was increased by nicardipine (20 microM) which was found to reduce the effect of 6.25 nM 1,25(OH)2D3. The rate of DNA synthesis (measured by [3H]thymidine incorporation) was increased after incubation of fibroblasts with 1,25(OH)2D3 (1.25 nM) plus triamcinolone acetonide (10(-6) M), although it was reduced by nicardipine in comparison with 1,25(OH)2D3 alone. So the effects of these agents on the 1,25(OH)2D3 induced 24-hydroxylase were shown to be independent of the rate of DNA synthesis.

Effect of maternal insulin deficiency on vitamin D metabolite concentrations in normoglycemic pregnant rats and their fetuses.

The effect on vitamin D metabolite concentrations of insulin deficiency, not accompanied by hyperglycemia, were investigated in pregnant rats and in their fetuses injected with 75 mg/kg BW streptozotocin (SZ). These concentrations were measured in maternal plasma and whole fetal body. In the insulinopenic mothers, the 25OHD concentration was found to rise compared with that of control pregnant rats (7.00 +/- 1.66 ng/ml, n = 16, versus control 4.50 +/- 1.60, n = 10, 0.001 less than P less than 0.01). The concentration of 1,25(OH)2D, which was previously found to decrease in pregnant rats that were both hypoinsulinic and hyperglycemic, was previously found to decrease in pregnant rats that were both hypoinsulinic and hyperglycemic, was not different in our control and insulinopenic rats (107.36 +/- 38.25 pg/ml, n = 11, versus control 122.90 +/- 18.20, n = 18.20, n = 8). In fetuses from our SZ-injected rats, the 24,25(OH)2D level diminished compared with the control level (2.12 +/- 0.70 ng/g, n = 11, versus control 5.23 +/- 0.95 ng/g, n = 13, P less than 0.001). The Ca/P ratio in fetal body also decreased (0.68 versus control 1.12). It is suggested that the placental metabolism is an important determinant or normal fetal growth.

Effects of 1,25-dihydroxyvitamin D3 on in vitro lymphocyte reactions: arguments for a role at the maternofetal interface.

Calcitriol or 1,25-dihydroxyvitamin D3 is synthesized by successive hydroxylations of vitamin D in the liver and kidney and during pregnancy in the placenta and the decidua. The aim of the present study was to clarify the immunoregulatory role of calcitriol, if any, during pregnancy. Calcitriol concentrations of 10(-10) to 10(-8) M was shown to reduce the proliferation of allogeneically stimulated lymphocytes and cytotoxic cell generation in a dose-dependent manner. However, calcitriol did not inhibit IL-2-dependent proliferation of CTLL-2 cell line. Calcitriol reduced non-MHC restricted cytotoxicity. Calcitriol, therefore, might be involved in the successful engraftment and growth of the fetoplacental unit possibly synergized with other products of placental or decidual origin.

Induction and inhibition of bone matrix resorption.

A method is described by which the action of hormones on cartilage and bone matrix in mice is ascertained by measuring 35S release from bone explants. Parathyroid extract was found to increase the rate of 35S release in vitro as well as in samples from pretreated animals, whereas calcitonin and 25-hydroxycholecalciferol were effective only in vitro.

Phorbol myristate acetate enhances adenylate cyclase activity in human polymorphonuclear leukocytes.

The putative role of protein phosphorylation in modulating adenylate cyclase activity in polymorphonuclear neutrophil membranes was assessed using phorbol myristate acetate (PMA) to stimulate the activity of protein kinase C. PMA was demonstrated to enhance the adenylate cyclase activity stimulated by isoproterenol.

Calcium-transport in quiescent and 1,25-dihydroxycholecalciferol-stimulated human osteosarcoma cells. Role in 24 hydroxylase enhancement.

The present study evaluates in osteosarcoma cells, the effects of a calcium channel inhibitor nicardipine in 24-hydroxylase activity and 45Ca desaturation curve in presence of 1,25-dihydroxycholecalciferol (1,25(OH)2D3). This sterol induced an increase in 24-OHase activity and 45Ca fluxes. Nicardipine reversed the effect of 1,25(OH)2D3 on 45Ca fluxes but reinforced the enhancement of the 24-OHase activity. The fact that the effects of 1,25(OH)2D3 were reduced by cycloheximide support the hypothesis of a de novo protein synthesis. Our study has allowed us to dissociate the effects of 1,25(OH)2D3 on 24-OHase enhancement from those on Ca2+ transport.

Effect of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 maternal loads on maternal and fetal vitamin D metabolite levels in the rat.

Two groups of female rats were used to investigate vitamin D metabolism in the pregnant animals and in their fetuses. In the first group, 3 micrograms of 25-hydroxyvitamin D3 (25-OH-D3) per kg of body weight were injected into intact or nephrectomized (NX) pregnant rats 3 h before sacrifice on day 21 of pregnancy; in the second group, 2 and 6 ng, respectively, of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) per day were infused continuously into pregnant rats between days 17 and 21 of pregnancy. The findings in the fetuses were obtained by quantitative analysis of extracts (Extrelut) of total fetal body lipids; the extracts were purified on Sep Pak and vitamin D sterols were further separated by high-pressure liquid chromatography. Three hours after the dams were injected with 25-OH-D3, the maternal plasma concentration (mean +/- SD) of 1,25(OH)2D3 was 221 +/- 84 pg/ml. In NX pregnant rats, the 1,25(OH)2D3 levels were still elevated: 95.6 +/- 49.0 pg/ml vs 45 +/- 22 pg/ml in control rats. In fetuses from intact or NX dams, the levels of 25-OH-D3 and 1,25(OH)2D3 were not different from the results obtained in the control fetuses but 24,25(OH)2D3 concentrations were increased (6.7 +/- 1.2 ng vs 2.2 +/- 0.7 ng/g body weight). After maternal infusion of 2 or 6 ng/day of 1,25(OH)2D3 (n = 8), plasma concentrations (mean +/- SD) of the metabolite were 64 +/- 31 and 517 +/- 356 pg/ml, respectively, the second being significantly higher than that of the control rats; 25-OH-D3 and 24,25(OH)2D3 levels did not change. 1,25(OH)2D3 contents (mean +/- SD) in fetuses from the treated dams were not different from those of control fetuses (10 +/- 2 pg/g body weight). Our results suggest that pregnant rats and their fetuses were protected against an excessive increase of 1,25(OH)2D3 concentrations in the maternal plasma; although there was some individual hypercalcemia, no significant increase in mean calcemia was detected in the dams, and 1,25(OH)2D3 either did not cross the placental barrier or was rapidly metabolized because we did not find any changes in the fetal content. As in intact or NX pregnant rats, 25-OH-D3 was metabolized into 1,25(OH)2D3, the increase of 24,25(OH)2D3 in the fetuses might be associated with a protective mechanism.