Modulation of L-type Ca2+ channels in UMR 106 cells by parathyroid hormone-related protein.

The effects of rat parathyroid hormone-related protein (rPTHrP) and bovine and rat parathyroid hormone (bPTH and rPTH) on L-type Ca2+ channels in UMR 106 cells were investigated using the patch clamp technique. rPTHrP increased the whole cell L-type Ca2+ channel currents and the increase was concentration dependent. rPTHrP, at a concentration of 62.5 nM, increased the L-type Ca2+ channel current by 122+/-25%. bPTH was less potent. A concentration of 7.5 microM bPTH increased the current by 99+/-24%. Results obtained with rPTH were similar to those obtained using bPTH. Single channel measurements, using the cell-attached version of the patch clamp technique, showed an increase in both the number of channel openings and the mean open time when the cells were exposed to rPTHrP. This suggested that rPTHrP affected the gating of L-type Ca2+ channels in UMR 106 cells. This study demonstrates that the actions of bPTH and rPTHrP in UMR cells are mediated in part by extracellular Ca2+ entry. PTHrP, a paracrine agent important in development, is more potent in regulating Ca2+ entry than PTH.     

Possible involvement of protein kinase C in parathyroid hormone degradation by osteoblast-like rat osteosarcoma cell line UMR106

The effects of 12-O-tetraadecanoyl phorbol-13-acetate (TPA), 1-oleoyl-2-acetyl-glycerol (OAG), and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) on the parathyroid hormone (PTH) degrading activity in a PTH-responsive osteoblast-like rat osteosarcoma cell line UMR106 were investigated to assess the role of Ca2+-activated. Phospholipid dependent protein kinase (protein kinase C) on the degradation of hormones. TPA and OAG, activators of protein kinase C, enhanced the PTH degrading activity dose-dependently, whereas H-7, an inhibitor of protein kinase C, exhibited a dose-dependent inhibition on this activity. These data suggest that protein kinase C activation may enhance PTH degrading activity by UMR106 cells as a possible regulator of PTH degradation.     

Vagotonic effects of enkephalin are not mediated by sympatholytic mechanisms

This study examined the hypothesis that vagotonic and sympatholytic effects of cardiac enkephalins are independently mediated by different receptors. A dose-response was constructed by administering the delta-receptor opioid methionine-enkephalin-arginine-phenylalanine (MEAP) by microdialysis into the interstitium of the canine sinoatrial node during vagal and sympathetic stimulation. The right cardiac sympathetic nerves were stimulated as they exited the stellate ganglion at frequencies selected to increase heart rate approximately 35 bpm. The right cervical vagus was stimulated at frequencies selected to produce a two-step decline in heart rate of 25 and 50 bpm. A six-step dose-response was constructed by recording heart rates during nerve stimulation as the dose of MEAP was increased between 0.05 pmol/min and 1.5 nmol/min. Vagal transmission improved during MEAP at 0.5 pmol/min. However, sympathetically mediated tachycardia was unaltered with any dose of MEAP. In Study 2, a similar dose-response was constructed with the kappa-opioid receptor agonist trans(+/-)-3-4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide-HCl (U-50488H) to illustrate an independent sympatholytic effect and to verify its kappa-receptor character. U-50488H gradually suppressed the sympathetic tachycardia, with a significant effect obtained only at the highest dose (1.5 nmol/min). U-50488H had no effect on vagally mediated bradycardia. Surprisingly, the sympatholytic effect was not reversed by withdrawing U-50488H or by the subsequent addition of the kappa-antagonist 17,17'-(dichloropropylmethyl)-6,6',7,7'-6,6'-imino-7,7'-binorphinan-3,4',14,14'-tetroldi-hydrochloride (norBNI). Study 3 was conducted to determine whether the sympatholytic effect of U-50488H could be prevented by norBNI. NorBNI blocked the sympatholytic effect of the U50488H for 90 mins. When norBNI was discontinued afterward and U-50488H was continued alone, a sympatholytic effect emerged within 30 mins. Collectively these observations support the hypothesis that the vagotonic influence of MEAP is not dependent on a sympatholytic influence. Furthermore, the sympatholytic effect is mediated independently by kappa-receptors. The sympatholytic effect of sustained kappa-receptor stimulation appears to evolve gradually into a functional state not easily reversed.     

Up-regulation of connexin 43 and gap junctional intercellular communication by Coleusin Factor is associated with growth inhibition in rat osteosarcoma UMR106 cells

Gap junctions, formed by connexin (Cx) family proteins, permit direct exchange of regulatory ions and small signal molecules between neighbouring cells. Gap junctional intercellular communication (GJIC) plays an important role in maintaining the homeostasis and preventing cell transformation. Most of the tumour cells feature deficient or aberrant connexin expression and GJIC level, and restoration of connexin expression and GJIC is correlated with cell growth control. Numerous researches has suggested the possibility of connexins as potential anti-tumour targets for chemoprevention and chemotherapy. We investigated the ability of Coleusin Factor (CF, also named FSK88) to regulate the Cx43 expression and GJIC level in rat osteosarcoma UMR106 cells. The results have demonstrated that CF increased the mRNA and protein expression of Cx43 in both in a dose- and timedependent manner, and concomitant with up-regulation of Cx43, CF treatment up-regulated the diminished GJIC level in UMR106 cells as assayed by dye transfer experiments. In addition, Cx43 distribution at the plasma membrane was also enhanced dramatically by CF treatment. Furthermore, we discovered that CF was potent to inhibit the growth and proliferation of UMR106 cells. These results provide the first evidence that CF can regulate connexin and GJIC, indicating that Cx43 may be a target of CF to exert its anti-tumour effects.     

Protein kinase C modulator effects on parathyroid hormone-induced intracellular calcium and morphologic changes in UMR 106-H5 osteoblastic cells

The effects of parathyroid hormone (PTH) on 1,4,5-inositol triphosphate (1,4,5-IP₃) and intracellular free calcium (Ca_i²⁺) in osteoblasts are variable, whereas adenylate cyclase activity is consistently stimulated. Cyclic AMP is considered a mediator in the contractile effects of PTH on osteoblasts, but the regulation and role of Ca_i²⁺ remains unclear. Recent studies indicate that protein kinase C (PKC) inhibits PTH-stimulated Ca_i²⁺ increases in osteoblastic cells. Therefore, the objectives of this study were to determine the effects of PKC modulators and PTH on UMR 106-H5 rat osteoblastic cell morphology, and the relationship between cell shape and PTH-induced Ca_i²⁺ changes. In suspended cells loaded with the calcium indicator dye fura-2, pretreatment with PKC inhibitors calphostin C (100 nM × 1 h) and H-7 (30 μM × 18 h) potentiated the effects of 1 μg/ml bPTH(1–84) on Ca_i²⁺ (83% increase over basal) by 1.4- and 1.65-fold, respectively. In comparison, PTH (10 ng-1 μg/ml) was without significant effect on adherent cell Ca_i²⁺ as measured by single-cell image analysis, although another in vitro bone resorbing agent, thrombin (10 U/ml), produced an acute 3-fold increase in the ratio (R) of emission (∼ λ510 nm) detected and optimized at λ348/374 nm (i.e., Ca-bound dye/free dye) in control cells. Phase-contrast microscopy revealed PKC inhibitor-treated cells changed from a spread configuration to a stellate form with retracting processes or cell rounding and a collapse of actin stress fibers. Within 1 h of PTH addition, PKC inhibitor-treated cells continually became extended/respread up to 3 h with an associated increase in actin stress fibers that was preceded by an acute 1.6-fold Ca_i²⁺ increase. In contrast, control or PKC activator-treated cells (phorbol 12,13-dibutyrate or 12-O-tetradecanoylphorbol-13-acetate; TPA) contracted/retracted within 5 min in response to PTH. A role for Ca_i²⁺ in PTH-induced cell spreading was further indicated by a contractile response to PTH when PKC-inhibitor-treated cells were loaded with the intracellular calcium chelator dimethyl BAPTA (3 μM × 30 min). PTH-induced Ca_i²⁺ increases in adherent PKC inhibitor-treated cells were also associated with a 1.8-fold 1,4,5-IP₃ increase as measured by mass assay. The data suggest PKC contributes to UMR 106-H5 cell morphology and selectively regulates signal pathways activated by PTH to promote either cell contraction (cAMP) or extension (1,4,5-IP₃/Ca_i²⁺). J. Cell. Biochem. 65:276–285. © 1997 Wiley-Liss, Inc.     

Membrane potential and cation content of osteoblast-like cells (UMR 106) assessed by fluorescent dyes

A number of cellular functions have recently been associated with alterations of the membrane potential in non-excitable cells. To assess the electrophysiologic regulation of osteoblast function, a method for measuring the membrane potential (Em) of a rat osteogenic sarcoma cell line (UMR 106) by the voltage-sensitive oxonol dye di-BA-C4(3) was developed. The fluorescent signal of di-BA-C4(3) was calibrated through a null point method using the protonophore FCCP. At null point, Em is equivalent to H+ equilibrium potential, and may be calculated by the Nernst equation. Intracellular pH (pHi) changes induced by the protonophore were monitored using BCECF, a pH-sensitive fluorescent probe. In the presence of FCCP, intracellular pH was found to be linearly correlated to extracellular pH (pHo). Therefore, the value of pHi at null point was extrapolated as well. With this technique, we estimated the plasma membrane potential of the "putative" rat osteoblasts (UMR 106) as -28.3 +/- 4.0 mV (n = 10). This method corrected the 16% overestimation of Em derived from the assumption that pHi does not change during the calibration procedure, as described in previous studies employing pH null point techniques. With null point methods, using BCECF and the carboxylic ionophores nigericin and monensin, intracellular concentrations of potassium and sodium were also measured and found to be 125 +/- 0.7 mM (n = 3) and 24 +/- 5.3 mM (n = 3), respectively. Although the Em of UMR 106 cells was dependent on extracellular potassium concentration, these cells did not behave as a potassium electrode. The sodium/potassium permeability ratio, calculated by the Goldman equation, was estimated at 0.317. This high membrane permeability to sodium may contribute to the genesis of the low plasma membrane potential of UMR 106 cells.     

Exercise training-induced adaptations of immune response are mediated by beta-adrenergic receptors in aged but not young mice.

Beta-adrenergic blockade was used to determine whether the exercise training-induced adaptations of immune response to viral infection were mediated by catecholamines in young and old mice. Young (2 mo) and older (16 mo) male BALB/c mice were randomly assigned to an exercise or control group, and half of the mice in each group received the beta-adrenergic receptor antagonist nadolol. After 8 wk of moderate exercise training, mice were challenged with herpes simplex virus (HSV) 24 h postexercise. The results showed that exercise treatment increased anti-HSV IgM antibody, enhanced IL-10, and altered the kinetics of IFN-gamma and IL-2 production in young and old mice. Unique to older mice, exercise decreased mitogen-induced proliferation, increased splenocytes, and tended to decrease memory cells (CD44(hi+)). In contrast, exercise increased mitogen-induced proliferation but decreased the number of splenic lymphocyte and CD4+ cells in young mice. beta-Adrenergic blockade blunted the exercise-induced changes in anti-HSV IgM, IL-2, IFNgamma, and mitogen-induced proliferation in old but not young mice. The findings suggest that some of the immunomodulatory effects of chronic exercise are mediated via beta-adrenergic receptors and that the role of beta-adrenergic receptors is age dependent.     

BFA effects are tissue and not just plant specific

Brefeldin A (BFA) is one of the most popular drugs used by researchers for studies on secretion and endocytosis because it interferes with specific vesicle coat proteins via action on a guanine nucleotide exchange factor. Due to its range of morphological effects on the Golgi apparatus in a variety of plant tissues, we believe that there is more to the BFA response than the primary molecular targets so far identified.     

Vitamin D compounds exert anti-apoptotic effects in human osteosarcoma cells in vitro

Several studies have demonstrated that vitamin D regulates growth and differentiation in bone cells in vitro. In addition, in vivo studies have shown that vitamin D stimulates bone formation, increases the number of osteoblast precursor cells and prevents bone mineral loss. These observations indicate that vitamin D may have anabolic effects on bone, and thus therapeutic potential in the treatment of osteoporosis. However, little is known about the effects of vitamin D on apoptosis in bone cells and about the contribution of this process to the effect of vitamin D on bone mineral loss. To investigate this aspect in more detail, we studied the effect of 1alpha,25(OH)(2)D(3) and a series of analogues on apoptosis in human osteosarcoma cells. No significant induction of apoptosis was observed with any of the compounds after a 5 day treatment period. In contrast, some of the analogues showed a tendency to protect the cells from undergoing apoptosis. This anti-apoptotic effect of vitamin D was further confirmed by the ability of 1alpha,25(OH)(2)D(3) to suppress camptothecin- and staurosporin-induced DNA fragmentation in the cells. In cultures treated simultaneously with 1alpha,25(OH)(2)D(3) in combination with camptothecin or staurosporin, the level of DNA fragmentation was markedly reduced compared with cultures treated with camptothecin or staurosporin alone. On the basis of the present results, it is therefore concluded that vitamin D displays anti-apoptotic effects in human osteoblast-like osteosarcoma cells in vitro. This observation suggests that besides regulating growth and differentiation, vitamin D exerts its anabolic effects on bone by protecting osteoblastic cells from undergoing apoptosis.     

Differential effects of interferon-gamma on metabolism of lipoprotein immune complexes mediated by specific human macrophage Fcgamma receptors

The objectives were to determine whether there are differences in the mechanisms of lipoprotein metabolism associated with different FcgammaRs and how metabolism associated with FcgammaRs compares to that mediated by scavenger receptors (SRA). To analyze lipoprotein metabolism in a receptor-specific manner, bispecific antibodies were used to target low density lipoproteins (LDL) labeled with (125)I or [(3)H]cholesterol linoleate to FcgammaRI or FcgammaRIIA in human macrophages. Interferon-gamma (IFN-gamma), which stimulates expression of FcgammaRI while inhibiting expression of SRA, was used to help delineate differences in metabolism between each receptor. For each receptor, the total amount of lipoprotein degradation paralleled changes in receptor expression induced by IFN-gamma. In particular, while SRA-mediated degradation typically exceeded degradation mediated by FcgammaRI, in IFN-gamma-treated cells degradation associated with FcgammaRI and SRA was similar. Assay of [(3)H]cholesterol linoleate-labeled lipoproteins indicated that total uptake and hydrolysis of [(3)H]cholesterol linoleate was similar for each class of receptor, and inhibited by IFN-gamma. For FcgammaRI versus FcgammaRIIA, in the presence or absence of IFN-gamma, the [(3)H]cholesterol derived from FcgammaRIIA-mediated uptake was preferentially targeted for re-esterification to [(3)H]cholesterol oleate, in comparison to that resulting from hydrolysis of [(3)H]cholesterol linoleate incorporated by selective uptake. For SRA, the formation of [(3)H]cholesterol oleate, which was substantial in control cells, was significantly inhibited in the presence of IFN-gamma. We conclude that there may be differences in cholesterol trafficking with respect to lipoprotein immune complex metabolism mediated by different classes of FcgammaRs.     

Macrophage hydrogen peroxide production and phagocytic function are decreased following phagocytosis mediated by Fc receptors but not complement receptors

Previous in vivo and in vitro studies have shown that the phagocytosis of IgG-coated erythrocytes results in a depression of macrophage function. The present study compared the effect of phagocytosis mediated by Fc receptors with that mediated by complement receptors. The phagocytosis of IgG-coated erythrocytes by elicited peritoneal macrophages depressed their capacity to produce hydrogen peroxide as well as phagocytic function. Phagocytosis of erythrocytes coated with IgM and complement had neither of these effects. These results implicate the intracellular signaling that results from Fc receptor mediated phagocytosis in the depression of macrophage function that is caused by phagocytosis.     

Acetylcholine receptors are not functionally independent.

Analysis of current recordings from acetylcholine-activated channels has largely rested so far on the hypothesis of independence, which states that the opening of one channel does not influence that of its neighbors. We have submitted this assumption to several tests, using as experimental material single channel currents from rat myotubes. We found that, even though the distribution of multiple channel openings may be approximated by the Poisson law, openings are strongly coupled. This conclusion is derived from the analysis of two-time properties associated with patch-clamp data. We show how these properties, which contain more detailed information than the stationary probabilities, can be calculated in practice and why a Poisson analysis is misleading in the present case. The implications of our findings are finally discussed in terms of channel structure and function.     

Synergistic effects of cytokine and hyperthermia on cytotoxicity in HT-29 cells are not mediated by alteration of induced protein levels

In this study, we investigated the mechanism of synergistic effects of cytokine and hyperthermia on cytotoxicity in HT-29. When cells were heated at 42°C in the presence of recombinant human tumor necrosis factor (rhTNF-α), recombinant interferon-gamma (rhIFN-γ), or in a combination of both, a synergistic increase in the cytotoxic effects of the respective drugs was observed. We hypothesized that alteration of cytokine or heat-induced polypeptides synthesis was responsible for a synergistic interaction between heat and cytokine. Five heat shock proteins (HSPs, M_r 110,000, 100,000, 90,000, 70,000, and 28,000) were preferentially synthesized during chronic heating at 42°C. In contrast, the synthesis of two proteins (M_r 60,000 and 29,000) was induced by treatment with rhIFN-γ (1,000 U/ml). Although the combination of chronic hyperthermia (42°C) with TNF-α, IFN-γ, or TNF-α + IFN-γ increased cytotoxicity, alteration/induction of polypeptides was not correlated with the synergistical cytotoxic effects of cytokine and heat. Thus, the synergistic effects of cytokine and hyperthermia are not mediated through an induction of polypeptides. © 1993 Wiley-Liss, Inc.     

Effects of parathyroid hormone on cyclic AMP-formation and cytoplasmic free Ca2+ in the osteosarcoma cell line UMR 106-01

The effects of parathyroid hormone (PTH) on cytoplasmic free CA²⁺ (Ca _i ²⁺ ) and cAMP-formation were investigated in the rat osteosarcoma cell line UMR 106-01.In fura-2 loaded adherent single cells bPTH 1-34 (10 nM–1M) induced a rapid transient increase in Ca _i ²⁺ in 11% of the studied cells. In fura-2 tracings from UMR 106-01 cells in suspension, bPTH 1-34 (0.1 M) induced a transient increase in Ca _i ²⁺ in 20% of the experiments. The transient increase in Ca _i ²⁺ seen in suspensions of cells was not abolished by addition of EGTA (2.5 mM) prior to challenge with PTH, suggesting that the increase in Ca _i ²⁺ was derived from intracellular stores.A marked rapid increase in cAMP-formation was observed in all experiments with cells in suspension, also in the experiments where PTH did not affect Ca _i ²⁺ .These data show that PTH causes a release of Ca²⁺ from intracellular stores in a small percentage of osteosarcoma UMR 106-01 cells, and that PTH is capable of inducing an increase in cAMP-formation without affecting Ca _i ²⁺ in osteoblasts.     

Apoptotic effects of a progesterone receptor antagonist on rat granulosa cells are not mediated via reduced protein isoprenylation

Progesterone is a survival factor in rat periovulatory granulosa cells. The mechanisms involved are unclear but progesterone receptor (PGR) antagonists have been shown to inhibit cholesterol synthesis and induce apoptosis. Furthermore, reports suggest that statins induce apoptosis by inhibition of protein isoprenylation. Statins inhibit the rate-limiting step of the cholesterol synthesis, thereby reducing availability of intermediates used for the post-translational isoprenylation process. It has been suggested that PGR antagonists in a similar manner induce apoptosis by decreasing cholesterol synthesis and thereby protein isoprenylation. In this study we hypothesized that the mechanism by which the nuclear PGR antagonist Org 31,710 induces apoptosis in rat periovulatory granulosa cells, is by decreasing cholesterol synthesis and thereby general cell protein isoprenylation. Incubation of isolated granulosa cells with Org 31,710 or simvastatin for 22 hr resulted in increased apoptosis and reduced cholesterol synthesis. However, simvastatin caused a substantial inhibition of cholesterol synthesis after 6 hr in culture without inducing apoptosis. In contrast, Org 31,710 had only a modest effect on cholesterol synthesis after 6 hr while it significantly induced apoptosis. Addition of isoprenylation substrates partially reversed apoptosis induced by simvastatin and to a lesser extent apoptosis induced by Org 31,710. In addition, and in contrast to Org 31,710, simvastatin caused a decrease in isoprenylation of a selected isoprenylation marker protein, the Ras-related protein RAB11. In conclusion, we demonstrate that the PGR antagonist inhibits cholesterol synthesis in granulosa cells but reduced protein isoprenylation is not the mediating mechanism of increased apoptosis as previously hypothesized.