inositol-1-4-5-trisphosphate and Osteosarcoma

Otopetrin1 (Otop1) is a multitransmembrane domain protein required for the formation of otoconia in the vertebrate inner ear. Otoconia are complex calcium carbonate (CaCO(3)) biominerals that are required for the sensation of gravity. Examination of the phenotypes of animals with mutations or deficiencies in Otop1 suggests a direct role for Otop1 in the initiation of extracellular biomineralization, possibly through the regulation of intracellular Ca(2+). Here, we demonstrate that Otop1 overexpression can modulate purinergic-mediated Ca(2+) homeostasis in transfected cell lines. These experiments define a unique set of biochemical activities of Otop1, including depletion of endoplasmic reticulum Ca(2+) stores, specific inhibition of the purinergic receptor P2Y, and regulation of the influx of extracellular Ca(2+) in response to ATP, ADP, and UDP. These activities can be inhibited by the polyanion suramin in a rapidly reversible manner. This first characterization of the consequences of Otop1 overexpression indicates a profound effect on cellular Ca(2+) regulation. In a physiologic setting, these activities could direct the formation and growth of otoconia and regulate other biomineralization processes.

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium Signaling; Cell Line; Chlorocebus aethiops; COS Cells; Endoplasmic Reticulum; Green Fluorescent Proteins; Humans; Inositol 1,4,5-Trisphosphate; Membrane Proteins; Nucleotides; Osteosarcoma; Rats; Receptors, Purinergic P2; Suramin; Uridine Triphosphate

The effect of miconazole, an anti-fungal drug, on cytoplasmic free Ca2+ concentrations ([Ca2+]i) in human osteosarcoma cells (MG63) was explored by using the Ca2+-sensitive dye fura-2. Miconazole acted in a concentration-dependent manner with an EC50 of 75 microM. The Ca2+ signal comprised a gradual rise and a sustained elevation. Removal of extracellular Ca2+ reduced 50% of the signal. In Ca2+-free medium, the [Ca2+]i rise induced by 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) was completely inhibited by pretreatment with 20 microM miconazole. Pretreatment with thapsigargin partly inhibited miconazole-induced Ca2+ release. The miconazole-induced Ca2+ release was not changed by inhibition of phospholipase C with 2 microM U73122. By using tetrazolium as a fluorescent probe, it was shown that 10-100 microM miconazole decreased cell proliferation rate in a concentration-dependent manner. Collectively, this study shows that miconazole induces [Ca2+]i rises in human osteosarcoma cells via releasing Ca2+ mainly from the endoplasmic reticulum in a manner independent of phospholipase C activity, and by causing Ca2+ influx. Furthermore, miconazole may be cytotoxic to the cells at higher concentrations.

Topics: Bone Neoplasms; Calcium; Calcium Channel Blockers; Cell Proliferation; Humans; Inositol 1,4,5-Trisphosphate; Miconazole; Osteoblasts; Osteosarcoma; Tumor Cells, Cultured

Carvedilol is a useful cardiovascular drug for treating heart failure, however, the in vitro effect on many cell types is unclear. In human MG63 osteosarcoma cells, the effect of carvedilol on intracellular Ca2+ concentrations ([Ca2+]i) and cytotoxicity was explored by using fura-2 and tetrazolium, respectively. Carvedilol at concentrations greater than 1 microM caused a rapid rise in [Ca2+]i in a concentration-dependent manner (EC50=15 microM). Carvedilol-induced [Ca2+]i rise was reduced by 60% by removal of extracellular Ca2+. Carvedilol-induced Mn2+-associated quench of intracellular fura-2 fluorescence also suggests that carvedilol induced extracellular Ca2+ influx. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of carvedilol on [Ca2+]i was inhibited by 50%. Conversely, pretreatment with carvedilol to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+. U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca2+ mobilizer)-induced, but not carvedilol-induced, [Ca2+]i rise. Pretreatment with phorbol 12-myristate 13-acetate and forskolin to activate protein kinase C and adenylate cyclase, respectively, did not alter carvedilol-induced [Ca2+]i rise. Separately, overnight treatment with 0.1-30 microM carvedilol inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human MG63 osteosarcoma cells, carvedilol increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum and other stores via a phospholipase C-independent manner. Carvedilol may be cytotoxic to osteoblasts.

Topics: Adenylyl Cyclases; Calcium; Calcium Channels; Calcium Signaling; Carbazoles; Carvedilol; Cell Compartmentation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colforsin; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Estrenes; Fluorescence; Fura-2; Histamine; Histamine Antagonists; Humans; Inositol 1,4,5-Trisphosphate; Manganese; Osteosarcoma; Phorbol Esters; Propanolamines; Protein Kinase C; Pyrrolidinones; Tetrazolium Salts; Thapsigargin; Time Factors; Type C Phospholipases

In human MG63 osteosarcoma cells, the effect of calmidazolium on [Ca(2+)](i) and proliferation was explored using fura-2 and ELISA, respectively. Calmidazolium, at concentrations greater than 0.1 micromol/L, caused a rapid increase in [Ca(2+)](i) in a concentration-dependent manner (EC(50) = 0.5 micromol/L). The calmidazolium-induced [Ca(2+)](i) increase was reduced by 66% by removal of extracellular Ca(2+). In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic increase in [Ca(2+)](i), after which the effect of calmidazolium to increase [Ca(2+)](i) was completely inhibited. U73122, an inhibitor of phospholipase C (PLC), abolished histamine (but not calmidazolium)-induced increases in [Ca(2+)](i). Pretreatment with phorbol 12-myristate 13-acetate to activate protein kinase C inhibited the calmidazolium-induced increase in [Ca(2+)](i) in Ca(2+)-containing medium by 47%. Separately, it was found that overnight treatment with 2-10 micromol/L calmidazolium inhibited cell proliferation in a concentration-dependent manner. These results suggest that calmidazolium increases [Ca(2+)](i) by stimulating extracellular Ca(2+) influx and also by causing release of intracellular Ca(2+) from the endoplasmic reticulum in a PLC-independent manner. Calmidazolium may be cytotoxic to osteosarcoma cells.

Topics: Bone Neoplasms; Calcium; Calcium Signaling; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Fluorescent Dyes; Fura-2; Humans; Imidazoles; Inositol 1,4,5-Trisphosphate; Kinetics; Osteoblasts; Osteosarcoma; Type C Phospholipases

Bone morphogenetic protein-7 (BMP-7) affects differentiation of preosteoblasts enabling the resultant cells to respond optimally to acutely acting regulators. As the phosphoinositide cascade and, particularly, the calcium-mobilizing inositol 1,4,5-trisphosphate (InsP3) receptor are integral to stimulus-secretion coupling in osteoblasts, the hypothesis that BMP-7 affects InsP3 receptor expression was examined in the G-292 human osteosarcoma cell line and in primary cultures of human osteoblasts. G-292 osteosarcoma cells were found to be a valid experimental model for primary human osteoblasts, expressing osteoblastic mRNAs encoding osteocalcin, bone sialoprotein, alkaline phosphatase, alpha1-collagen, epidermal growth-factor receptor, and BMP type II receptor. When cultured long term in the presence of ascorbic acid and beta-glycerophosphate, G-292 cells underwent further osteoblastic differentiation, forming nodules and exhibiting restricted mineralization. G-292 cells responded to BMP-7 with an increase in InsP3 receptor density. Ligand-binding studies established that BMP-7 (50 ng/ml) treatment of G-292 cells increased InsP3 receptor density 2.4-fold with no apparent change in affinity. Immunoblot analysis with antibodies specific for type I, type II, and type III InsP3 receptors revealed that BMP-7 (50 ng/ml) treatment resulted in a specific increase (206+/-8%) in the type I receptor. Reverse transcription-polymerase chain reaction and Northern blot analyses of G-292 and primary human osteoblasts confirmed an increase in type I InsP3 receptor mRNA upon BMP-7 treatment. These results demonstrate that G-292 cells respond to BMP-7 with an increase InsP3 receptor density, consistent with the enhanced capacity of these cells to respond to Ca2+-mobilizing secretory hormones during osteoblast differentiation.

Topics: Alkaline Phosphatase; Ascorbic Acid; Blotting, Northern; Bone Morphogenetic Protein 7; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Proteins; Calcification, Physiologic; Calcium Channels; Cell Differentiation; Cells, Cultured; Collagen; ErbB Receptors; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Glycerophosphates; Humans; Immunoblotting; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Integrin-Binding Sialoprotein; Osteoblasts; Osteocalcin; Osteosarcoma; Phosphatidylinositols; Polymerase Chain Reaction; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Receptors, Growth Factor; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factor beta; Tumor Cells, Cultured

To study the effects of 17 beta-estradiol (E2) on intracellular free calcium ([Ca2+]i), inositol-1,4,5-trisphophate (IP3) and calmodulin (CaM) in human osteoblast-like cell line TE85.. Using Fluo-3/AM as fluorescent indicator, the [Ca2+]i was measured by laser confocal microscopy system. The IP3 content was determined by anion-exchange chromatography. CaM content was detected by a high sensitive assay based on stimulation of Ca(2+)-dependent phosphodiesterase activity.. E2 at dose of 0.1 and 1.0 nmol/L increased fluorescent level by 4.7 and 6.1 times. Pretreatment with thapsigargin (100 nmol/L), the E2 caused only 1.5 times elevation in fluorescence. E2 induced a concommitant bi-peak increase in IP3 content. At the presence of E2(1.0 nmol/L), the CaM content increased by 85.2%. Tamoxifen did not affect the effect of E2 on [Ca2+]i, IP3 and CaM content. But, the inhibitor of phospholipase C (neomycin) and pertusis toxin depressed them partly or completely.. E2 regulate bone cells function by way of Ca2+/CaM activation.

Topics: Adolescent; Bone Neoplasms; Calcium; Calmodulin; Estradiol; Female; Humans; Inositol 1,4,5-Trisphosphate; Osteoblasts; Osteosarcoma; Tumor Cells, Cultured

The effect of extracellular calcium on human osteoblast-like cells (HOS) has been demonstrated. An experimental setup was used for applying defined rates of change in the extracellular calcium concentration. The intracellular calcium concentration was monitored using the fluorescence dye fura-2. HOS cells showed qualitatively different responses of the intracellular calcium concentration to changes of the extracellular calcium concentration depending on its changing rate. A small rate caused only a small and slow increase of the intracellular calcium concentration, whereas faster changes are able to cause a rapid transient increase followed by a sustained elevation of the internal calcium level. Surprisingly, both an increasing as well as a decreasing external calcium concentration is able to cause cellular responses. These signals could be reduced by the IP3-inhibitor neomycin. We propose that the G-protein dependent signalling pathway of HOS cells can not only sense the extracellular calcium concentration but also its time derivative.

Topics: Biophysical Phenomena; Biophysics; Bone Remodeling; Calcium; Enzyme Inhibitors; Extracellular Space; GTP-Binding Proteins; Humans; Inositol 1,4,5-Trisphosphate; Intracellular Fluid; Kinetics; Neomycin; Osteoblasts; Osteosarcoma; Tumor Cells, Cultured; Type C Phospholipases

The effects of parathyroid hormone (PTH) on 1,4,5-inositol triphosphate (1,4,5-IP3) and intracellular free calcium (Cai2+) 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 Cai2+ remains unclear. Recent studies indicate that protein kinase C (PKC) inhibits PTH-stimulated Cai2+ 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 Cai2+ changes. In suspended cells loaded with the calcium indicator dye fura-2, pretreatment with PKC inhibitors calphostin C (100 nM x 1 h) and H-7 (30 microM x 18 h) potentiated the effects of 1 microgram/ml bPTH (1-84) on Cai2+ (83% increase over basal) by 1.4- and 1.65-fold, respectively. In comparison, PTH (10 ng-1 micrograms/ml) was without significant effect on adherent cell Cai2+ 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 (approximately lambda 510 nm) detected and optimized at lambda 348/374 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 Cai2+ 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 Cai2+ 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 microM x 30 min). PTH-induced Cai2+ 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

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenylyl Cyclases; Animals; Calcium; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Inositol 1,4,5-Trisphosphate; Naphthalenes; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Protein Kinase C; Rats; Tumor Cells, Cultured

The enzymes and substrates involved in phosphoinositide signal transduction which have been detected in the nucleus of several cell types have been demonstrated to be responsive to agonists. The complexity of this aspect of inositide function has been previously analyzed in some cell models characterized by a mitogenic or differentiating response to specific factors. An interesting experimental model is represented by human derived osteosarcoma Saos-2 cells, characterized by the expression of high affinity receptors for interleukin 1 alpha (IL-1 alpha), which is one of the most potent stimulators of bone resorption. In particular, we investigated the earliest intracellular events following the binding of IL-1 alpha to its receptor, involving the inositide signal transduction pathway. Saos-2 cells present a partitioning of the phosphoinositidase (PLC) isoforms; in fact, the nucleus contains both PLC beta 1 and gamma 1, while the cytoplasm contains almost exclusively the gamma 1 isoform. IL-1 alpha evokes a rapid and transient increase of the PLC beta 1 activity in the nucleus, which causes the hydrolysis of phosphatidylinositol mono- and bis-phosphate. In response to IL-1 alpha, not only the canonical inositol lipid pathway appears to be involved; also the 3'-phosphorylated lipids generated by phosphatidylinositol 3-kinase (PI 3-K), which may act as second messengers, appear to be affected. In fact, Saos-2 cells present a nuclear PI 3-K activity which can be enhanced by the IL-1 alpha treatment. Among the possible targets of the second messengers released by the nuclear PLC beta 1 activation, we found that some protein kinase C isoforms, namely the epsilon and zeta, which are present within the nucleus, are activated after IL-1 alpha exposure. These activated PKC isoforms, in turn, could modulate the activity of the transcription factor NFkB, which, 5 min after IL-1 alpha treatment, has already translocated to the nucleus and bound to DNA to promote gene activation. The actual role of the inositide pathway in the Saos-2 cell function has also been investigated by utilizing cell clones transfected with the mouse sequence of the PLC beta 1.

Topics: Animals; Cell Nucleus; Humans; Inositol 1,4,5-Trisphosphate; Interleukin-1; Isoenzymes; Lipid Metabolism; NF-kappa B; Osteosarcoma; Phosphatidylinositol 3-Kinases; Phosphatidylinositols; Protein Kinase C; Rats; Receptors, Interleukin-1; Signal Transduction; Tumor Cells, Cultured; Type C Phospholipases

While calcium release from intracellular stores is a signaling mechanism used universally by cells responding to hormones and growth factors, the compartmentalization and regulated release of calcium is cell type-specific. We employed thapsigargin and 2,5,-di-(tert-butyl)-1,4-benzohydroquinone (tBuHQ), two inhibitors of endoplasmic reticulum (ER) Ca(2+)-ATPase activity which block the transport of Ca2+ into intracellular stores, to characterize free Ca2+ compartmentalization in UMR 106-01 osteoblastic osteosarcoma cells. Each drug elicited transient increases in cytosolic free Ca2+ ([Ca2+]i), followed by a stable plateau phase which was elevated above the control [Ca2+]i. The release of Ca2+ from intracellular stores was coupled to an increased plasma membrane Ca2+ permeability which was not due to L-type Ca2+ channels. Thapsigargin and tBuHQ emptied the intracellular calcium pool which was released in response to either ATP or thrombin, identifying it as the inositol 1,4,5-trisphosphate-sensitive calcium store. The results of sequential and simultaneous additions of thapsigargin and tBuHQ indicate that both drugs depleted the same Ca2+ store and inhibited the same Ca(2+)-ATPase activity.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Bone Neoplasms; Calcium; Calcium-Transporting ATPases; Cell Membrane; Cell Membrane Permeability; Cytosol; Egtazic Acid; Endoplasmic Reticulum; Fura-2; Hydroquinones; Inositol 1,4,5-Trisphosphate; Nifedipine; Osteoblasts; Osteosarcoma; Terpenes; Thapsigargin; Thrombin; Tumor Cells, Cultured

1 alpha,25-Dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) has been shown to increase cytosolic calcium and inositol triphosphate levels in rat osteosarcoma cells (ROS 17/2.8) and to increase nuclear calcium in these cells. To determine the mechanism(s) of 1 alpha,25-(OH)2D3-induced changes in nuclear calcium, the effect of the hormone on phospholipid metabolism in isolated osteoblast nuclei was assessed. 1 alpha,25(OH)2D3, 20 nM, increased inositol triphosphate levels in the nuclei after 5 min of treatment. The biologically inactive epimer, 1 beta,25-(OH)2D3, had no significant effect on inositol triphosphate levels. ATP, 1 mM, also increased inositol triphosphate levels in the isolated nuclei after 5 min. 1 alpha,25-(OH)2D3, 20 nM, increased calcium in the isolated nuclei in the presence but not in the absence of extranuclear calcium within 5 min. Nuclear calcium was also increased within 5 min by ATP, 1 mM, and inositol triphosphate, 1 mM. The effect of ATP on nuclear calcium was not additive with 1 alpha,25-(OH)2D3, suggesting that these two agents increase nuclear calcium in these osteoblast-like cells by similar mechanisms. In summary, 1 alpha,25-(OH)2D3 and ATP rapidly increase inositol triphosphate levels in nuclei isolated from ROS 17/2.8 cells. The hormone, the nucleotide, and the inositol phospholipid increase nuclear calcium. Thus, the 1 alpha,25-(OH)2D3 and ATP effects on nuclear calcium may be mediated by changes in phospholipid metabolism in the nuclei of these osteoblast-like cells.

Topics: Adenosine Triphosphate; Animals; Calcitriol; Calcium; Cell Nucleus; Inositol 1,4,5-Trisphosphate; Nuclear Envelope; Osteoblasts; Osteosarcoma; Rats; Spectrometry, Fluorescence; Time Factors; Tumor Cells, Cultured

UMR-106 rat osteogenic sarcoma cells express two calcium signaling P2 purinergic receptors. One is a P2U receptor with EC50's for adenosine triphosphate (ATP) and uridine triphosphate (UTP) of 2.6 and 2.4 microM, respectively. The other is a novel P2T receptor for adenosine diphosphate (ADP) (EC50 0.4 microM), adenosine 5'-O-(2-thiodiphosphate). (EC50 5 microM), 2-methylthio ATP (EC50 0.2 microM), and 2-methylthio ADP (EC50 0.04 microgram M). Responses to these ligands are desensitized by ADP but not by UTP. Responses to UTP and ATP are desensitized by UTP but not by ADP or adenosine 5'-O-(2-thiodiphosphate). 2-ChloroATP interacts with both receptors at high concentrations but with only the P2T receptor at low concentrations (EC50 0.05 microM). The weak platelet P2T receptor antagonist AMP blocks this P2T and not the P2U receptor. Addition of ATP after UTP desensitization of P2U receptors inhibits subsequent responsiveness to ADP but evidence for rapid conversion of ATP to ADP complicates interpretation of this apparent antagonism of P2T receptors by ATP. A subpassage of UMR-106.P135 cells lose P2U but retain P2T ligand responsiveness. Activation of either P2 receptor increases cellular IP3 concentrations in UMR-106 cells. Neither receptor can activate divalent cation entry as evidenced by their lack of effect on Mn++ quenching of fura-2 fluorescence. Neither receptor can modify parathyroid hormone receptor-mediated elevation of cellular cyclic AMP. This receptor for ADP demonstrates many of the same characteristics of the ADP receptor which we have previously reported as a P2T receptor expressed in K562 and Dami cells.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Calcium; Cyclic AMP; Dose-Response Relationship, Drug; Inositol 1,4,5-Trisphosphate; Osteoblasts; Osteosarcoma; Phenotype; Rats; Receptors, Purinergic P2; Tumor Cells, Cultured; Uridine Triphosphate

The effect of inositol 1,4,5 trisphosphate (IP3) on calcium mobilization was studied in human osteosarcoma lines, Saos-2 and G292, as well as isolated rat osteoblastic and osteoclastic cells. Cells were permeabilized with saponin and calcium mobilization was studied with the fluorescent dye, fura-2 in a recording spectrofluorometer. IP3 (10 microM) increased calcium release in all cell types studied. The effect was dependent on ATP and occurred in the presence of mitochondrial inhibitors. The effect was not seen with inositol 1-phosphate (IP) or inositol 1,4-diphosphate (IP2). Inositol 1,3,4,5 tetrakisphosphate (IP4) appeared to elicit a decrease in the calcium released. Depletion of the intracellular pool with the calcium ionophore, ionomycin, as well as incubation with the inhibitor of intracellular calcium mobilization, TMB-8, obliterated the IP3 effect. The results are consistent with the hypothesis that increases in IP3 can cause a rapid elevation of bone cell cytosolic calcium.

Topics: Animals; Calcium; Cell Membrane Permeability; Cells, Cultured; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Osteoblasts; Osteoclasts; Osteosarcoma; Rats; Rats, Inbred Strains; Tumor Cells, Cultured

Epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and heparin-binding growth factor-1 (HBGF-1) stimulated the proliferation of a variant of the human osteosarcoma cell line, MG-63-LS (LS = low serum). Transforming growth factor beta (TGF-beta) completely inhibited cell growth in basal medium supplemented with 2% fetal calf serum (FCS), blocked PDGF- and EGF-stimulated cell proliferation, and modulated that of HBGF-1. PDGF, but not EGF or HBGF-1, activated the inositol trisphosphate/diacylglycerol (IP3/DAG) second message system in a dose-dependent manner. EGF inhibited phosphoinositol lipid turnover and HBGF-1 and TGF-beta stimulated phosphatidylinositol hydrolysis to produce inositol phosphate (IP) but not IP3. Preincubation of quiescent cells with TGF-beta for 30-40 minutes prior to the addition of PDGF resulted in an inhibition of PDGF-induced production of IP3. This suggested that TGF-beta was an indirect inhibitor and blocked PDGF-stimulated cell growth in part by interfering with the generation of the second messenger, IP3.

Topics: Cell Division; Cell Line; Fibroblast Growth Factor 1; Humans; Hydrolysis; Inositol; Inositol 1,4,5-Trisphosphate; Kinetics; Osteosarcoma; Phosphatidylinositols; Platelet-Derived Growth Factor; Transforming Growth Factor beta

Permeabilized and intact UMR-106-01 cells attached to culture plates or coverslips were used to evaluate compartmentalized generation and the effective concentration of inositol 1,4,5-trisphosphate (In-1,4,5-P3) during agonist-mediated Ca2+ release. In permeabilized cells, Ca2+ release had the following characteristics. In-1,4,5-P3 released approximately 65% of the Ca2+ incorporated into intracellular stores. Prostaglandin F2 alpha (PGF2 alpha), endothelin, or GTP(gamma S) alone released a small amount or no Ca2+. However, the agonists together with GTP(gamma S) were as effective as In-1,4,5-P3 in releasing Ca2+. Both agonist- and In-1,4,5-P3-mediated Ca2+ release required the presence of permeable ion. Agonists, like In-1,4,5-P3, stimulated 45Ca uptake from low Ca2+ medium devoid of permeable ions into Ca2(+)-loaded intracellular stores. The permeabilized cell system was then used to evaluate compartmentalized generation and action of In-1,4,5-P3 during agonist stimulation. Mass measurement shows that in intact resting cells In-1,4,5-P3 concentration was 1.4 microM and was reduced to 0.05 microM following permeabilization. Stimulation with agonists increases In-1,4,5-P3 concentration from 0.05 to 0.34 microM. Ca2+ release by this concentration of In-1,4,5-P3 evenly distributed in the cytosol can account for only part of the agonist-mediated Ca2+ release. However, the effects of saturating In-1,4,5-P3 concentration and agonists were blocked by the specific inhibitor heparin. Measurement of heparin dependency of In-1,4,5-P3-mediated Ca2+ release was used to calculate an affinity for In-1,4,5-P3 of 0.39 microM. Similar measurements with agonists show that In-1,4,5-P3 concentration at the site of Ca2+ release during agonist stimulation is 11.2 microM. Hence, the total increase in In-1,4,5-P3 is reflected in considerably higher localized concentrations. This is interpreted to suggest compartmentalized generation and action of In-1,4,5-P3 during agonist stimulation.

Topics: Animals; Biological Transport; Calcium; Cell Line; Cell Membrane Permeability; Dinoprostone; Endothelins; Endothelium, Vascular; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Heparin; Inositol 1,4,5-Trisphosphate; Kinetics; Osteosarcoma; Peptides; Thionucleotides; Tumor Cells, Cultured

Parathyroid hormone (PTH)-stimulated signal transduction through mechanisms alternate to adenosine 3',5'-cyclic monophosphate (cAMP) production were studied in UMR 106-01 cells, a cell line with an osteoblastic phenotype. PTH produced transient, dose-related increases in cytosolic calcium [( Ca2+]i), inositol trisphosphates, and diacylglycerol (DAG). Both inositol 1,4,5-trisphosphate (Ins-1,4,5P3) and inositol 1,3,4-trisphosphate (Ins-1,3,4P3) production were rapidly stimulated by PTH. Consistent with the production of Ins-1,3,4P3, rapid stimulation of late eluting inositol tetrakisphosphate was observed. The effects on the inositol phosphates were induced rapidly, consistent with roles as signals for changes in [Ca2+]i. In saponin-permeabilized UMR 106-01 cells, Ins-1,4,5P3 stimulated 45Ca release from a nonmitochondrial intracellular pool. Thus the hypothesis that PTH-stimulated Ins-1,4,5P3 production initiates Ca2+ release and contributes to transient elevations of [Ca2+]i is supported. Pretreatment of UMR 106-01 cells with pertussis toxin had no effect on PTH stimulation of inositol phosphates. Pertussis toxin reduced PTH-stimulated elevations of [Ca2+]i, but cAMP analogues had an even greater effect than pertussis toxin. These data suggest that stimulation of cAMP production during PTH stimulation may negatively affect production of rises in [Ca2+]i during PTH stimulation. The inactivation of the inhibitory G protein of adenylate cyclase by pertussis toxin could explain its action similar to cAMP analogues. Cyclic nucleotides diminish the effects of PTH on [Ca2+]i, probably interacting on a biochemical step subsequent to or independent of Ins-1,4,5P3 release.

Topics: Adenylate Cyclase Toxin; Animals; Calcium; Cell Line; Cytosol; Diglycerides; Glycerides; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Pertussis Toxin; Rats; Signal Transduction; Stimulation, Chemical; Sugar Phosphates; Virulence Factors, Bordetella