Role of the sulfonylurea receptor in regulating human adipocyte metabolism.

A regulatory role for intracellular Ca2+ ([Ca2+]i) in adipocyte lipogenesis, lipolysis and triglyceride accumulation has been demonstrated. Compounds acting on the pancreatic sulfonylurea receptor (SUR) to increase (e.g., glibenclamide) or decrease (e.g., diazoxide) [Ca2+]i cause corresponding increases and decreases in weight gain. However, these weight gain and loss effects have been attributed to insulin release rather than to the primary effects of these compounds on the adipocyte SUR and its associated K(ATP) channel. Accordingly, we have evaluated the direct role of the human adipocyte SUR in regulating adipocyte metabolism. We used RT-PCR with primers for a highly conserved region of SUR1 to demonstrate that human adipocytes express SUR1. The PCR product was confirmed by sequence analysis and used as a probe to demonstrate adipocyte SUR1 expression by Northern blot analysis. Adipocytes exhibited glibenclamide dose-responsive (0-20 microM) increases in [Ca2+]i (P<0.05). Similarly, glibenclamide (10 microM) caused a 67% increase in adipocyte fatty acid synthase activity (P<0.001), a 48% increase in glycerol-3-phosphate dehydrogenase activity (P<0.01) and a 68% inhibition in lipolysis (P<0.01), whereas diazoxide (10 microM) completely prevented each of these effects. These data demonstrate that human adipocytes express a SUR that regulates [Ca2+]i and, consequently, exerts coordinate control over lipogenesis and lipolysis. Accordingly, the adipocyte SUR1 may represent an important target for the development of therapeutic interventions in obesity.     

Regulation of adiposity by dietary calcium.

Recent data from this laboratory demonstrate that increasing adipocyte intracellular Ca(2+) results in a coordinated stimulation of lipogenesis and inhibition of lipolysis. We have also noted that increasing dietary calcium of obese patients for 1 year resulted in a 4.9 kg loss of body fat (P<0.01). Accordingly, we tested the possibility that calcitrophic hormones may act on adipocytes to increase Ca(2+) and lipid metabolism by measuring the effects of 1, 25-(OH)(2)-D in primary cultures of human adipocytes, and found significant, sustained increases in intracellular Ca(2+) and a corresponding marked inhibition of lipolysis (EC(50) approximately 50 pM; P<0.001), suggesting that dietary calcium could reduce adipocyte mass by suppressing 1,25-(OH)(2)-D. To test this hypothesis, we placed transgenic mice expressing the agouti gene specifically in adipocytes on a low (0.4%) Ca/high fat/high sucrose diet either unsupplemented or with 25 or 50% of the protein replaced by non-fat dry milk or supplemented to 1.2% Ca with CaCO(3) for 6 wk. Weight gain and fat pad mass were reduced by 26-39% by the three high calcium diets (P<0.001). The high calcium diets exerted a corresponding 51% inhibition of adipocyte fatty acid synthase expression and activity (P<0.002) and stimulation of lipolysis by 3. 4- to 5.2-fold (P<0.015). This concept of calcium modulation of adiposity was further evaluated epidemiologically in the NHANES III data set. After controlling for energy intake, relative risk of being in the highest quartile of body fat was set to 1.00 for the lowest quartile of Ca intake and was reduced to 0.75, 0.40, and 0.16 for the second, third, and fourth quartiles, respectively, of calcium intake for women (n=380;P<0.0009); a similar inverse relationship was also noted in men (n=7114; P<0.0006). Thus, increasing dietary calcium suppresses adipocyte intracellular Ca(2+) and thereby modulates energy metabolism and attenuates obesity risk.     

Ca2+ priming during vitamin D-induced monocytic differentiation of a human leukemia cell line

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) induces monocytic differentiation of the human promyelocytic leukemia line, HL-60, and enhances Ca2+ transport in target cells of the mineral metabolism system. Hence, we determined whether the steroid's maturational effect on HL-60 involves alterations of intracellular calcium [( Ca2+]i). We found that, as detected by indo-1 fluorescence, [Ca2+]i increases in a slow tonic manner from 99 +/- 11 nM in virgin HL-60 to 182 +/- 19 nM (p less than 0.001) in those treated with 1,25-(OH)2D3 for 24 h. The first apparent rise in [Ca2+]i occurs at between 6 and 12 h and parallels expression of alpha-thrombin and N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptors. This increase in [Ca2+]i is derived from extracellular calcium as its reduction abolishes the effect. The increase in [Ca2+]i is associated with an increase in inositol trisphosphate-stimulated Ca2+ flux from intracellular stores. Interestingly, 1,25-(OH)2D3-mediated HL-60 differentiation as manifest by expression of the macrophage-specific antigen, 63D3, is not blocked by low extracellular calcium. In contrast, the fMLP-induced superoxide ion generation is diminished if the increase in [Ca2+]i is prevented. Furthermore, fMLP-stimulated signal transduction is also reduced by limiting the stimulation of [Ca2+]i during 1,25-(OH)2D3 treatment. Thus, although differentiation of HL-60 to the monocytic phenotype by 1,25-(OH)2D3 is Ca2+-independent, expression of response to regulatory stimuli requires priming of cellular Ca2+ stores. The latter appears to be induced by 1,25-(OH)2D3 via stimulated Ca2+ entry through the plasma membrane.     

Lead inhibits 1,25-dihydroxyvitamin D-3 regulation of calcium metabolism in osteoblastic osteosarcoma cells (ROS 17/2.8).

We have determined the dose-response of 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) on the intracellular free calcium-ion concentration ([Ca2+]i) in the osteoblastic osteosarcoma cells, ROS 17/2.8, using 19F-NMR and the intracellular divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5F-BAPTA). The dose-response demonstrated an inverted U-shaped relationship with maximal elevation of [Ca2+]i at doses of 1 to 10 nM 1,25-(OH)2D3. At 10 nM, 1,25-(OH)2D3 elevated the [Ca2+]i from a control level of 118 +/- 4 nM to a peak value of 237 +/- 8 nM within 40 min. 1,25-(OH)2D3 also increased the initial rate of Ca2+ influx into ROS 17/2.8 cells, measured by 45Ca uptake, with a dose-response relationship which paralleled its effect on [Ca2+]i. Treatment of ROS 17/2.8 cells with Pb2+ at 1 and 5 microM significantly increased [Ca2+]i but significantly reduced the 1,25-(OH)2D3-induced elevation of [Ca2+]i. Simultaneous treatment of naive cells with 1,25-(OH)2D3 and Pb2+ produce little reduction of 1,25-(OH)2D3-induced 45Ca uptake while 40 min treatment with Pb2+ before addition of 1,25-(OH)2D3 significantly reduced the 1,25-(OH)2D3-induced increase in 45Ca influx. These findings suggest that Pb2+ acts by inhibiting 1,25-(OH)2D3-activation of Ca2+ channels and interferes with 1,25-(OH)2D3 regulation of Ca2+ metabolism in osteoblastic bone cells.     

Effects of mitochondrial uncoupling on adipocyte intracellular Ca(2+) and lipid metabolism

Previous data from this laboratory demonstrate that increased intracellular Ca(2+) ([Ca(2+)]i) coordinately regulates human and murine adipocyte lipid metabolism by stimulating lipogenesis and inhibiting lipolysis. However, recent data demonstrate metabolic uncoupling increases [Ca(2+)]i but inhibits lipogenesis by suppressing fatty acid synthase (FAS) activity. Accordingly, we have evaluated the interaction between mitochondrial uncoupling, adipocyte [Ca(2+)]i, and adipocyte lipid metabolism. Pretreatment of 3T3-L1 cells with mitochondrial uncouplers (DNP or FCCP) amplified the [Ca(2+)]i response to depolarization with KCl by 2-4 fold (p <0.001), while this increase was prevented by [Ca(2+)]i channel antagonism with lanthanum. Mitochondrial uncouplers caused rapid (within 4hr) dose-dependent inhibition of FAS activity (p <0.001), while lanthanum caused a further additive inhibition. The suppression of FAS activity induced by uncoupling was reversed by addition of ATP. Mitochondrial uncouplers increased FAS expression significantly while [Ca(2+)]i antagonism with lanthanum decreased FAS expression (P <0.001). In contrast, mitochondrial uncouplers independently inhibited basal and isoproterenol-stimulated lipolysis (20-40%, p <0.001), while this inhibition was fully reversed by lanthanum. Thus, mitochondrial uncoupling exerted short-term regulatory effects on adipocyte [Ca(2+)]i and lipogenic and lipolytic systems, serving to suppress lipolysis via a Ca(2+) -dependent mechanism and FAS activity via a Ca(2+)-independent mechanism.     

Effects of vitamin D3 metabolites on cytosolic free calcium in confluent mouse osteoblasts

A fluorescent Ca2+ indicator, acetoxymethyl Quin2, was used to quantify changes in the cytosolic free calcium concentration ([Ca2+]i) of confluent mouse osteoblasts. 1,25 - Dihydroxycholecalciferol (1,25 - (OH)2D3, 10-100 pM), 25-hydroxycholecalciferol (25-(OH)D3, 10-100 nM), parathyroid hormone (PTH(1-84), 0.1-10 nM), and prostaglandin E2 (PGE2, 10-1000 nM) all induced immediate (t less than 15 s) transient increases in [Ca2+]i, from a basal level of 135 +/- 8 nM to levels of 179-224 nM. These increases rapidly returned to a plateau approximately 10% higher than the basal level. 24,25-Dihydroxycholecalciferol (24,25-(OH)2D2, 0.1-10 nM) induced a rapid increase in [Ca2+]i which remained elevated for 5 min before decreasing. The 1,25-(OH)2D3- and PTH-induced spikes were abolished by the prior addition of EGTA and Ca2+ entry blockers (verapamil, nifedipine, 1 microM) while the responses to 25-(OH)D3, 24,25-(OH)2D3, and PGE2 were unaffected. Addition of 1,25-(OH)2D3 + EGTA or PTH + EGTA caused enhanced Ca efflux. Addition of drugs which interfere with calcium sequestration by the endoplasmic reticulum (ER) (caffeine, 4 mM; 8-(diethyl-amino)-octyl 3,4,5-trimethoxybenzoate HCl, 0.5 mM) or mitochondria (antimycin, 10 microM; oligomycin, 5 microM) showed that 25-(OH)D3 and PGE2 mainly mobilized Ca2+ from ER. 1,25-(OH)2D3 and bovine PTH caused a transient increase in [Ca2+]i, 70% of which resulted from Ca2+ influx from outside the cells and 30% by release from the ER. The [Ca2+]i increase induced by 24,25-(OH)2D3 included a 30% contribution from the ER and 70% from the mitochondria.     

The effect of long-term caloric restriction on function of T-cell subsets in old mice

The effect of caloric restriction (from weaning to old age) on CD3-stimulated CD4+ and CD8+ lymphocyte proliferation and calcium mobilization was examined. Young ad libitum (ad lib) fed, old ad lib fed, old calorically restricted, and old calorically restricted mice which were fed ad lib during the last 6 weeks of their life (restricted/refed) were compared in both BDF1 [(C57BL/6 x DBA/2)F1] and C57BL/6 mice. Proliferation of CD4+ cells was lower in old ad lib animals than in young animals; this difference was not seen in CD8+ cells. Those CD4+ cells which did proliferate in old ad lib animals underwent similar cell cycle progression as young cells. In calorically restricted and calorically restricted/refed animals, CD4+ cell proliferation was similar to the young animals, and CD8+ cells showed a higher proliferative capacity than cells from either young or old ad lib mice. Differences in proliferative capacity were not correlated with alterations in transmembrane signaling efficiency as peak [Ca2+]i was reduced in both T-cell subsets in all groups of old mice relative to young mice. Additionally, reduced [Ca2+]i was observed in the CD8+ subset for which there was no deficit in proliferation, and the enhanced proliferation seen in old restricted and old restricted/refed mice did not manifest as increased [Ca2+]i mobilization. The percentage of CD4+ cells from both mouse strains was reduced in all groups of old mice compared with young mice, while the percentage of CD8+ cells was generally similar in young and all groups of old mice. Our studies would suggest that lifelong caloric restriction of mice prevents the age-associated decrease in T-cell proliferative capacity but that the enhanced proliferation of these cells is not due to increased efficiency of transmembrane signaling.     

1,25-Dihydroxyvitamin D3 increases the toxicity of hydrogen peroxide in the human monocytic line U937: the role of calcium and heat shock

1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) increases synthesis of heat shock proteins in monocytes and U937 cells and protects these cells from thermal injury. We examined whether 1,25-(OH)2D3 would also modulate the susceptibility of U937 cells to H2O2-induced oxidative stress. Cell viability was assessed by trypan blue exclusion and [3H]thymidine incorporation into DNA. Prior incubation for 24 h with 1,25-(OH)2D3 (25 pM or higher) unexpectedly increased H2O2 toxicity. Since cellular Ca2+ may be a mediator of cell injury we investigated effects of altering extracellular Ca2+ ([Ca2+]e) on 1,25-(OH)2D3-enhanced H2O2 toxicity as well as effects of 1,25-(OH)2D3 and H2O2 on cytosolic free Ca2+ concentration ([Ca2+]f). Basal [Ca2+]f in medium containing 1.5 mM Ca as determined by fura-2 fluorescence was higher in 1,25-(OH)2D3-pretreated cells than control cells (137 versus 112 nM, P less than 0.005). H2O2 induced a rapid increase in [Ca2+]f (to greater than 300 nM) in both 1,25-(OH)2D3-treated and control cells, which was prevented by a reduction in [Ca2+]e to less than basal [Ca2+]f. The 1,25(OH)2D3-induced increase in H2O2 toxicity was also prevented by preincubation with 1,25-(OH)2D3 in Ca2+-free medium or by exposing the cells to H2O2 in the presence of EGTA. Preexposure of cells to 45 degrees C for 20 min, 4 h earlier, partially prevented the toxic effects of H2O2 particularly in 1,25-(OH)2D3-treated cells, even in the presence of physiological levels of [Ca2+]e. Thus 1,25-(OH)2D3 potentiates H2O2-induced injury probably by increasing cellular Ca2+ stores. The 1,25-(OH)2D3-induced amplification of the heat shock response likely represents a mechanism for counteracting the Ca2+-associated enhanced susceptibility to oxidative injury due to 1,25-(OH)2D3.     

Proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures: effects of dexamethasone and calcitriol

During bone loss, osteoblast population can be replaced by adipose tissue. This apparent reciprocal relationship between decreased bone density and increased fat formation can be explained by an imbalance in the production of bone-forming and fat-forming cells in the marrow cavity. Thus, osteoblast and adipocyte pathways seem more closely and inversely related. In the present study, we investigated the effects of dexamethasone (dex) and calcitriol [1,25(OH)(2)D(3)] on proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures. Stromal cells were grown in primoculture in presence of dex and subcultivated in presence of dex and/or 1,25(OH)(2)D(3). Total cell proliferation, osteoblast and adipocyte-cells number, and -mRNA specific markers were used to study the effects of hormonal treatment on stromal cells. Total cell proliferation was stimulated by dex and inhibited by 1,25(OH)(2)D(3). Dex increased osteoblast and adipocyte cell population whereas calcitriol decreased bone-forming cell number and increased fat cell population. The presence of both hormones led to a strong decrease in osteoblastic cells and to a strong increase in adipocytic cell number. Dex induced mRNA osteoblastic markers expression like bone sialoprotein (BSP) and osteocalcin (OC) and an adipocyte marker expression, the fatty acid binding protein aP2. Calcitriol decreased the dex-induced BSP expression but stimulated slightly OC and aP2 mRNA. The effects of both hormones was to increase strongly OC and aP2 mRNA. These results support that, in rat bone marrow, adipocyte proliferation and differentiation are stimulated by glucocorticoids and calcitriol which act synergically, whereas osteoblastic cell proliferation and differentiation are increased by dex and inhibited by 1,25(OH)(2)D(3).     

Single cell analysis of changes in cytosolic calcium induced by vitamin D3 metabolites in cultured rat mesangial cells.

The acute effects of 1,25-Dihydroxy-vitamin D3 [1,25(OH)2D3] on the concentration of cytoplasmic ionized calcium [Ca2+] of cultured rat mesangial cells were studied at the single cell level by microspectrofluorometry of fura-2-loaded cells. Addition of 1,25(OH)2D3 produced an immediate increase of [Ca2]+. This rise in [Ca2+] was sustained and similar to that caused by the Ca2+ channel agonist BAY K 8644. Comparable changes were also observed in cultured human mesangial cells. The effects of the hormone (10 (-10)-10(-7) M) were dose-dependent (62% and 285%). Only 30-40% of the cells responded to stimulation with 1,25(OH)2D3. 25OHD3 also increased Ca2+ whereas 24,25(OH)2D3 and 1aOHD3 were inactive. Addition of 1 mM CoCl2 or 2-5 microM nifedipine largely blocked the effects of 1,25(OH)2D3 suggesting the involvement of Ca2+ channel activation in the rapid 1,25(OH)2D3-induced increase in mesangial cell [Ca2+]. 45Ca uptake studies are consistent with This interpretation.     

Characterization of a 1,25(OH)2-vitamin D3-responsive capacitative Ca2+ entry pathway in rat osteoblast-like cells

We investigated the existence of a capacitative Ca2+ entry (CCE) pathway in ROS 17/2.8 osteoblast-like cells and its responsiveness to 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3]. Depletion of inner Ca2+ stores with thapsigargin or 1,25(OH)2D3 in the absence of extracellular Ca2+ transiently elevated cytosolic Ca2+ ([Ca2+]i); after recovery of basal values, Ca2+ re-addition to the medium markedly increased Ca2+ entry, reflecting pre-activation of a CCE pathway. Recovery of the Ca2+ overshoot that followed the induced CCE was mainly mediated by the plasma membrane Ca2+-ATPase. Addition of 1,25(OH)2D3 to the declining phase of the thapsigargin-induced CCE did not modify further [Ca2+]i, indicating that steroid activation of CCE was dependent on store depletion. Pre-treatment with 1 microM Gd3+ inhibited 30% both thapsigargin- and 1,25(OH)2D3-stimulated CCE, whereas 2.5 microM Gd3+ was required for maximal inhibition ( approximately 85%). The activated CCE was permeable to both Mn2+ and Sr2+. Mn2+ entry sensitivity to Gd3+ was the same as that of the CCE. However, 1-microM Gd3+ completely prevented capacitative Sr2+ influx, whereas subsequent Ca2+ re-addition was reduced only 30%. These results suggest that in ROS 17/2.8 cells CCE induced by thapsigargin or 1,25(OH)2D3 is contributed by at least two cation entry pathways: a Ca2+/Mn2+ permeable route insensitive to very low micromolar (1 microM) Gd3+ accounting for most of the CCE and a minor Ca2+/Sr2+/Mn2+ permeable route highly sensitive to 1 microM Gd3+. The Ca2+-mobilizing agonist ATP also stimulated CCE resembling the Ca2+/Sr2+/Mn2+ permeable entry activated by 1,25(OH)2D3. The data demonstrates for the first time, the presence of a hormone-responsive CCE pathway in an osteoblast cell model, raising the possibility that it could be an alternative Ca2+ influx route through which osteotropic agents influence osteoblast Ca2+ homeostasis. Copyright Wiley-Liss, Inc.     

Effects of ovariectomy on duodenal calcium transport in the rat: altered ability to adapt to low-calcium diet

Studies were undertaken to determine whether ovariectomy (ovx) would alter the ability of female rats to adapt to low dietary Ca intake by exhibiting an in duodenal active Ca transport. Intact and ovx female rats were fed diets containing 1.5, 0.50, or 0.02% Ca prior to measuring active Ca transport using everted duodenal sacs in vitro. In some experiments, ovx animals were pair-fed to intact animals of the same age consuming the same diet. When ovx animals were allowed to eat ad lib, we found that both growth rate and duodenal active Ca transport increased relative to age-matched, intact controls. However, when growth of ovx animals was maintained at the control rate by pair-feeding, ovx per se did not affect intestinal active Ca transport. Ovx did not alter circulating levels of 1,25-dihydroxyvitamin D (1,25(OH)2D). We found that intact females responded to the low-Ca (0.02%) diet with increased circulating 1,25(OH)2D levels and increased intestinal active Ca transport. Ovx animals exhibited the same increase in circulating concentrations of 1,25(OH)2D in response to low-Ca diet, but did not demonstrate increased duodenal active Ca transport. When ovx animals consumed the diet ad lib, they became larger and exhibited higher Ca transport rates than intact animals fed the high-Ca diet, but there was no difference in Ca transport between ovx animals fed diets containing different Ca contents. The results of these experiments demonstrate that in female rats, the ability to adapt to altered dietary Ca intake is dependent on intact ovarian function and is not necessarily directly related to circulating concentrations of 1,25(OH)2D.     

Calcium supplementation does not alter lipid oxidation or lipolysis in overweight/obese women

Based on cell culture and studies in mice, increased dietary calcium appears to stimulate lipolysis and could possibly reduce body adiposity through hormonal influences on adipocyte calcium uptake. In this study, we investigated the effects of 1,500 mg supplemental calcium daily for 3 months on hormones regulating calcium and energy metabolism and rates of lipid oxidation and lipolysis in overweight women. Fifteen overweight (BMI > 25 kg/m(2)) premenopausal women were supplemented with 1,500 mg of calcium, as CaCO(3), per day for 3 months while maintaining their usual diets and activity levels. Baseline and endpoint measurements were obtained after the subjects consumed a standardized 25% fat diet for 4 days. Lipid oxidation was measured by indirect calorimetry, lipolysis by infusion of deuterated glycerol, and body fat by dual-energy X-ray absorptiometry. Urinary calcium, circulating levels of hormones involved in energy and lipid metabolism (insulin, leptin, and adiponectin) or calcium metabolism (25(OH)D, 1,25(OH)(2)D), and parathyroid hormone (PTH)) were also measured. Urinary levels of calcium (P = 0.005) increased and 1,25(OH)(2)D declined (P = 0.03). However other parameters, including body weight, body fat, PTH, insulin, leptin, adiponectin, 25(OH)D, as well as rates of lipid oxidation and lipolysis were not altered by calcium supplementation. Calcium supplementation for 3 months increased urinary calcium excretion, decreased circulating levels of 1,25(OH)(2)-D, but had no effect on rates of lipid oxidation or lipolysis, in these overweight women.     

Calcium signaling in cancer and vitamin D

Calcium signals induced by the Ca(2+) regulatory hormone 1,25(OH)(2)D(3) may determine the fate of the cancer cell. We have shown that, in breast cancer cell lines, 1,25(OH)(2)D(3) induces a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) by depleting the endoplasmic reticulum (ER) Ca(2+) stores via inositol 1,4,5-trisphosphate receptor/Ca(2+) release channel and activating Ca(2+) entry from the extracellular space via voltage-insensitive Ca(2+) channels. In normal cells, 1,25(OH)(2)D(3) triggered a transient Ca(2+) response via activation of voltage-dependent Ca(2+) channels, which were absent in breast cancer cells. The normal cells, but not breast cancer cells, expressed the Ca(2+) binding/buffering protein calbindin-D(28k) and were capable of buffering [Ca(2+)](i) increases induced by a mobilizer of the ER Ca(2+) stores, thapsigargin, or a Ca(2+) ionophore, ionomycin. The 1,25(OH)(2)D(3)-induced sustained increase in [Ca(2+)](i) in breast cancer cells was associated with induction of apoptotic cell death, whereas the transient [Ca(2+)](i) increase in normal cells was not. The forced expression of calbindin-D(28k) in cytosol or increase in the cytosolic Ca(2+) buffering capacity with the cell-permeant Ca(2+) buffer BAPTA prevented induction of apoptosis with 1,25(OH)(2)D(3) in cancer cells. The sustained increase in [Ca(2+)](i) in breast cancer cells was associated with activation of the Ca(2+)-dependent apoptotic proteases, mu-calpain and caspase-12, as evaluated with antibodies to active (cleaved) forms of the enzymes and the fluorogenic peptide substrates. Selective inhibition of the Ca(2+) binding sites of mu-calpain decreased apoptotic indices in the cancer cells treated with 1,25(OH)(2)D(3), thapsigargin, or ionomycin. The mu-calpain activation preceded expression/activation of caspase-12, and calpain was required for activation/cleavage of caspase-12. Certain non-calcemic vitamin D analogs (e.g., EB 1089) triggered a sustained [Ca(2+)](i) increase, activated Ca(2+)-dependent apoptotic proteases, and induced apoptosis in breast cancer cells in a fashion similar to that of 1,25(OH)(2)D(3). The 1,25(OH)(2)D(3)-induced transient Ca(2+) response in normal mammary epithelial cells was not accompanied by activation of mu-calpain and caspase-12. In conclusion, we have identified the novel apoptotic pathway in breast carcinoma cells treated with 1,25(OH)(2)D(3): increase in [Ca(2+)](i)-->mu-calpain activation-->caspase-12 activation-->apoptosis. Our results support the hypothesis that 1,25(OH)(2)D(3) directly activates this apoptotic pathway by inducing a sustained increase in [Ca(2+)](i). Differences of Ca(2+) regulatory mechanisms in cancer versus normal cells seem to allow 1,25(OH)(2)D(3) and vitamin D analogs to induce Ca(2+)-mediated apoptosis selectively in breast cancer cells. Thus, deltanoids may prove to be useful in the treatment of tumors susceptible to induction of Ca(2+)-mediated apoptosis.     

Rapid effects of calciotropic hormones on female rat enterocytes: combined actions of 1,25(OH)2-vitamin D3, PTH and 17beta-estradiol on intracellular Ca2+ regulation.

1,25(OH)(2)-Vitamin D(3) [1,25(OH)(2)D(3)], PTH and 17beta-estradiol increase intracellular Ca(2+) levels ([Ca(2+)](i)) in rat enterocytes by stimulating inner Ca(2+) store mobilization and voltage-dependent Ca(2+) channels through non-genomic activation of second-messenger cascades. The participation of store-operated Ca(2+) (SOC) channels in 17beta-estradiol regulation of enterocyte [Ca(2+)](i) has also been suggested. The aim of this work was to investigate whether PTH and/or 17beta-estradiol exert additive or synergistic effects acting in concert with the classic intestinal calciotropic hormone 1,25(OH)(2)D(3). Fura-2-loaded rat duodenal cells were stimulated using rPTH (10 nM), 17beta-estradiol (0.1 nM) or 1,25(OH)(2)D(3) (0.1 nM). The resulting Ca(2+) signal was characterized by an almost immediate rise in [Ca(2+)](i) (within 30 s) rapidly reaching peak levels, followed by a plateau phase that remained sustained as long as the cells were exposed to the stimulus. The addition of PTH at the sustained phase induced by 1,25(OH)(2)D(3) or, conversely, the addition of the secosteroid after the PTH-induced effect, did not induce additional increases in [Ca(2+)](i). Simultaneous treatment with both hormones resulted in an elevation of [Ca(2+)](i) equivalent to the maximal level caused by either agonist alone, suggesting common components for [Ca(2+)]i stimulation by PTH and 1,25(OH)(2)D(3). Treatment with 17beta-estradiol at the sustained phase induced by 1,25(OH)(2)D(3) or, conversely, treatment with the secosteroid after the 17beta-estradiol effect, induced additional increments in [Ca(2+)](i) (58 % and 63 %, respectively). Simultaneous treatment of enterocytes with both steroids potentiated their individual effects to the same extent as when added sequentially, also indicative of additive actions mediated by different sources of calcium signaling cascades. Moreover, 17beta-estradiol failed to further increase the 1,25(OH)(2)D(3)-induced initial Ca(2+) elevation in Ca(2+)-free medium, thus suggesting that extracellular influx mechanisms rather than intracellular Ca(2+) mobilization account for estrogen potentiation of 1,25(OH)(2)D(3) modulation of [Ca(2+)](i) in duodenal cells.     

Selective stimulation of in situ intermediary metabolism by free calcium in permeabilized rat adipocytes.

The hypothesis that ionized calcium [Ca2+]i may stimulate in situ rat adipocyte intermediary metabolism distal to glucose transport was tested. A metabolically active porous adipocyte model was employed in which pathway metabolism is exclusively pore-dependent using glucose 6-phosphate (G6P) as substrate. Cellular [Ca2+]i was, furthermore, directly adjusted to between 0-2.5 microM via the membrane pores. Three metabolic fluxes were examined, (1) glycolysis-Krebs ([6-14C]G6P oxidation), (2) glycolysis to lactate ([U-14C]G6P to [14C]lactate) and (3) pentose pathway ([1-14C]G6P oxidation). Glycolysis-Krebs oxidation was was found to be selectively (33% above basal P less than 0.001) stimulated by 0.625 microM free calcium. In contrast, there was no effect of [Ca2+]i on the other, exclusively cytoplasmic, pathways. The stimulation of glycolysis-Krebs by [Ca2+]i was inhibited by a mitochondrial calcium channel blocker (Ruthenium red) and persisted over a range of ATP/ADP ratios. Separate studies demonstrated that 2-[1-14C]ketoglutarate oxidation was also calcium-stimulated in the porous adipocytes (160% over baseline at 1 microM [Ca2+]i). These studies thus demonstrate that physiologically relevant increments in porous adipocyte [Ca2+]i enhance overall in situ glycolytic-Krebs pathway oxidation by a mechanism which entails mitochondrial calcium uptake. Methodologically, this metabolically active porous adipocyte model presents a novel experimental approach to investigations regarding the effects of ionized calcium on intermediary metabolism beyond glucose transport.     

Control of cytosolic free calcium in rat and chicken osteoclasts. The role of extracellular calcium and calcitonin

Single cell [Ca2+], studies were performed in chicken and rat osteoclasts loaded with fura-2 and exposed to a variety of treatments. Under resting conditions, basal [Ca2+]i, was 79.2 +/- 47.3 and 84.3 +/- 65.7 nM (averages +/- S.D.; n = 141 and 126) in the osteoclasts of the two species, respectively. Basal [Ca2+]i was stable in all rat and in approximately 80% of chicken osteoclasts. In the remaining 20%, spontaneous, irregular [Ca2+], fluctuations were observed (amplitude range: 50-200 nm over basal values). Increase of [Ca2+]o over the concentration of the Krebs-Ringer incubation medium (2 mM) induced rises of [Ca2+] in almost all cells investigated. [Ca2+] rises were already appreciable with 0.5 mM [Ca2+]o additions and reached high values with 4 mM additions: 390 +/- 113 and 364 +/- 214 nM [Ca2+], in rat and chicken osteoclasts, respectively (n = 122 and 101). Qualitatively, the responses to [Ca2+]o additions consisted of discrete [Ca2+]i transients, biphasic (an initial spike followed by a plateau), or monophasic (either the spike or the plateau). In a few chicken osteoclasts, the [Ca2+]i increase occurring after [Ca2+]o addition consisted of multiple, irregular fluctuations, similar to those observed in 20% of these cells under resting conditions. In individual osteoclasts subsequently exposed to multiple [Ca2+]o increase pulses, the type of the [Ca2+]i transient (mono- or biphasic) was maintained, and the size was dependent on the magnitude of the [Ca2+]o additions. Effects similar to those of [Ca2+]o were induced by the addition of Cd2+ or Ba2+ (but not La3+ or Mg2+) into the medium. The Cd2+ effect was maintained in part even in a Ca2+-free medium. Of various hormones and factors, parathormone, 1,25-dihydroxyvitamin D3, and prostaglandin E2 were inactive. In contrast, calcitonin was active in rat osteoclasts (which express numerous receptors). [Ca2+]i increases were small (19 +/- 17.9 nM; n = 21) when the hormone was administered alone; they were synergistic (severalfold potentiation) when the hormone was administered before or after [Ca2+]o. The [Ca2+]i effects of calcitonin were mimicked by 8Br-cAMP (31 +/- 26 nM; n = 12) when the nucleotide was administered alone; marked synergism when it was administered in combination with [Ca2+]o. This paper demonstrates for the first time that changes of [Ca2+]i are induced in osteoclasts by treatments with [Ca2+]o and calcitonin and can therefore be involved in intracellular mediation of the physiological effects of these two extracellular signals.     

Rapid effects of 1,25(OH)2 vitamin D3 on signal transduction systems in colonic cells.

Previous work from our laboratory demonstrated that 1,25(OH)2D3 rapidly stimulated hydrolysis of membrane polyphosphoinositides (PI) in rat colonocytes and in Caco-2 cells, generating the second messengers DAG and IP3. [Ca2+]i subsequently increased due to IP3-mediated release of intracellular Ca2+ stores, and to Ca2+ influx through a receptor-mediated Ca channel. Studies examining purified antipodal plasma membranes and experiments using Caco-2 cell monolayers found that 1,25(OH)2D3 influenced PI turnover only in the basolateral (BLM) and not brush border (BBM) membranes. Vitamin D analogues with poor affinity for the vitamin D receptor were found to effectively stimulate PI turnover, suggesting the presence of a unique vitamin D receptor in the BLM. Studies from our laboratory have demonstrated saturable, reversible binding of 1,25(OH)2 D3 to colonocyte BLM. Recently, we found that 1,25(OH)2D3 activated the tyrosine kinase c-src in colonocyte BLM by a heterotrimeric guanine nucleotide binding protein (G-protein)-dependent mechanism, with subsequent phosphorylation, translocation to the BLM, and activation of PI-specific phospholipase C gamma. Due to the rise in [Ca2+]i and DAG, two isoforms of protein kinase C (PKCalpha and PKCbeta2), but not other isoforms were activated by 1,25(OH)2D3 in rat colonocytes. Recent studies demonstrated that the seco-steroid translocated the beta2 isoform to the BLM, but not the BBM. In contrast, the alpha isoform did not translocate to either antipodal plasma membrane, but modulated IP3-mediated Ca2+ release from the endoplasmic reticulum. Preliminary studies have shown that 1,25(OH)2D3 also activated phosphatidylcholine phospholipase D (PLD) in Caco-2 cells, generating phosphatidic acid and contributing to the sustained rise in DAG. PLD stimulation occurred by both PKC-dependent and -independent mechanisms. Inhibitors of G-proteins, c-src, and PKC blunted the seco-steroid-mediated activation of PLD. Cells stably transfected with sense PKCalpha showed increased 1,25(OH)2D3-stimulated PLD activation, whereas transfectants with antisense PKCalpha had an attenuated response. In addition, 1,25(OH)2D3 also regulated PLD by activating the monomeric G-protein rho A by a mechanism independent of the G-protein/ c-src/PKC pathway.     

Cells from human bone giant cell tumors show a [Ca2+]o-sensing.

Giant cells from a human giant cell tumor of bone, showing several osteoclast features were tested for their capability of detecting the [Ca2+]o by a receptor like [Ca2+]o sensing. We found that cultured cells responded to elevation of [Ca2+]o, obtained adding 4 mM Ca2+ to the 2 mM Ca2+ containing buffer, by a transient increase of [Ca2+]i. Proliferative cells induced to differentiate by treatment with 10(-8) M 1,25 dihydroxyvitamin D3, were upregulated in their capability of responding to elevated [Ca2+]o. In fact, in this circumstance, the peak of [Ca2+]o-induced [Ca2+]i rise was increased compared to untreated cells. This suggests that 1,25 dihydroxyvitamin D3 induces a more efficient regulation of osteoclast activity.     

Heterogenous distribution of free calcium and propagation of calcium transient in gastric parietal cells revealed by digital imaging microscopy

Spatial and temporal changes of cytoplasmic free calcium concentration ([Ca2+]i) in single parietal cells of guinea pig were investigated with a digital imaging microscope equipped with a microspectrofluorometer, using a Ca2+-sensitive dye, fura-2. Intracellular distribution of [Ca2+]i was not homogeneous, but there were two kinds of [Ca2+]i gradient in the resting parietal cells, one a continuous gradient increasing towards the plasma membrane and a second discontinuous gradient (Ca2+ plateau) in some restricted regions of the cytoplasm. When treated with gastrin, only about 40% of parietal cells in the gastric gland responded with an almost twofold increase in the average resting [Ca2+]i of 52.4 +/- 7.1 nM. In the responding cells, the discontinuous plateaus transiently enlarged to the entire cytoplasm. In marked contrast, all of these cells responded to Ca2+ ionophore ionomycin. We also found that when provoked by gastrin Ca2+ transient in the parietal cells in the gastric gland often propagated to some adjacent cells, and occasionally spontaneous Ca2+ transient and oscillation were observed even in the resting state.