Biomedical subjects

R Dziak

Publications and source records attributed to R Dziak.

At least 19 recordsLinked to original sources

PubMed1995

Role of epidermal growth factor-induced membrane depolarization and resulting calcium influx in osteoblastic cell proliferation.

This study investigated the effects of epidermal growth factor (EGF) on the membrane potential of rat calvarial osteoblasts, in order to understand the mechanism responsible for calcium influx and the role these EGF-induced events have in osteoblastic cell proliferation. Changes in plasma membrane potential were measured using patch clamp techniques in isolated cells. EGF induced changes in plasma membrane potential only after cells had been in culture for at least 6 days. EGF induced membrane depolarization in 55% of rat calvarial osteoblasts studied after 6 to 8 days in culture. This membrane event was dependent on extracellular calcium, therefore, one or more calcium conductances were involved. Nifedipine, a voltage-activated calcium channel blocker, significantly reduced membrane depolarization, and demonstrated the existence of a nifedipine-insensitive conductance. Osteoblastic cell proliferation was measured by cell count. The EGF-dependent increase in cell proliferation was blocked by addition of 10 microM nifedipine. Therefore, it appears that the mechanism of action of EGF-induced osteoblastic cell proliferation is mediated by changes in plasma membrane potential which result in extracellular calcium influx.

Animals↗

PubMed1995

Role of extracellular calcium influx in EGF-induced osteoblastic cell proliferation.

This study investigated the effects of epidermal growth factor (EGF) on cytosolic calcium ([Ca++]i) levels in rat calvarial osteoblasts, the nature of the regulation of this event, and the role these EGF-induced [Ca++]i changes have in osteoblastic cell proliferation. EGF significantly increased [Ca++]i measured in fura-2-loaded, individual cells. This increase was related to extracellular calcium influx. Activation of protein kinase C(PKC) by pretreating the cells with phorbol esters blocked the EGF-induced increase in [Ca++]i. EGF failed to increase inositol trisphosphate levels measured by high performance liquid chromatographic analysis. However, it did increase inositol bisphosphate and inositol tetrakisphosphate production. The EGF-dependent increase in DNA synthesis was partially blocked by the addition of calcium channel blockers. Therefore, it appears that the mechanism of action of EGF-induced osteoblastic cell proliferation is mediated by changes in [Ca++]i primarily due to extracellular calcium influx.

Analysis of Variance↗

PubMed1992

Effects of interleukin-1, tumor necrosis factor -beta, and forskolin on tissue plasminogen activator activity in human osteoblastic osteosarcoma cells.

The effects of interleukin-1 (IL-1), forskolin, and tumor necrosis factor beta (TNF-beta) on tissue plasminogen activator (t-PA) activity were studied in the human osteoblastic osteosarcoma cell line, G292. t-PA activity was measured in the cell media using the chromogenic substrate, S-2251. After a 24 hour incubation period, IL-1 increased t-PA in a dose-dependent manner. The effect of IL-1 at 10.0 U/ml was partially inhibited in the presence of indomethacin. Forskolin (1.0 microM) increased t-PA activity after 24 hours with the effects of combined treatment of IL-1 (1.0 U/ml, 10.0 U/ml) and forskolin being apparently additive in nature. TNF-beta (10(-8)-10(-7)M) also produced increased t-PA activity in the cell media after a 24 hour incubation period. These results suggest that the cytokines, IL-1 and TNF-beta, can increase t-PA activity in G292 cells and that there is both a cAMP-dependent as well as a cAMP-independent pathway involved in the regulation of this osteoblastic cell function.

Colforsin↗

PubMed1992

Regulation of C-myc protooncogene expression in osteoblastic cells by arachidonic acid metabolites: relationship to proliferation.

Prostaglandin E2 and leukotriene B4 are metabolites of arachidonic acid with well-characterized effects on osteoblastic cells. Prostaglandin E2 has been shown to be a potent bone-resorbing agent and to stimulate as well as inhibit osteoblastic cell proliferation. Leukotriene B4 has also been demonstrated to stimulate or inhibit osteoblastic cell proliferation, depending on the cell type tested. In the present study, the potential relationship of the effects of prostaglandin E2 and leukotriene B4 on osteoblastic cell proliferation to c-myc protooncogene expression was investigated. Prostaglandin E2 has been shown previously to inhibit normal rat osteoblastic cell proliferation. The present studies show that prostaglandin E2 at 10(-6) M decreased c-myc expression in these cells. In the human osteoblastic osteosarcoma cell line, G292, prostaglandin E2 increased c-myc expression and inhibited proliferation. In contrast, epidermal growth factor increased DNA synthesis as well as c-myc expression. Prostaglandin E2 also inhibited proliferation of another human osteoblastic osteosarcoma cell line, Saos-2, but it did not produce any changes in c-myc expression. In these cells, epidermal growth factor did not affect either DNA synthesis or c-myc expression. Leukotriene B4 did not show any effects on c-myc expression in any of the osteoblastic cells tested.

Animals↗

PubMed1992

Second messenger systems stimulated by bradykinin in osteoblastic cells: evidence for B2 receptors.

The effects of bradykinin, analogs and inhibitors on the human osteoblastic osteosarcoma cell lines Saos-2 and G292 and on normal rat calvarial osteoblastic cells were investigated. In all cell types, bradykinin (1 nM-100 microM) caused significant time- and dose-dependent changes in the levels of inositol phosphates. Neomycin inhibited the inositol phosphate response to bradykinin, while indomethacin had no effect. Bradykinin also elicited a dose-dependent increase in free cytosolic calcium concentration. Bradykinin and T-kinin did not affect cyclic AMP levels in these cells. Doses of des-Arg9-bradykinin, a B1 receptor agonist, up to 100 nM did not stimulate the osteoblastic inositol phosphate response. In addition, the bradykinin-stimulated inositol phosphate response was unaffected by des-Arg9-[Leu8]-bradykinin, a B1 receptor antagonist, while it was inhibited by D-Arg-[Hyp3-[beta-(2-thienyl)-Ala]5,8-D-Phe7]-bradykinin, a B2 receptor antagonist. These results suggest that in osteoblastic cells the mechanism of action of bradykinin involves stimulation of the phosphoinositide metabolism and increases in cytosolic calcium levels through activation of B2 receptors.

Amino Acid Sequence↗

PubMed1991

Thrombin effects on osteoblastic cells. II. Structure-function relationships.

Thrombin has been shown to cause in vitro bone resorption and to stimulate osteoblastic cell proliferation, phosphoinositide turnover and cytosolic calcium levels. In the present study, the role of the active site of thrombin in its action on osteoblastic cells was investigated. Either hirudin or (4-amidinophenyl)methanesulfonyl fluoride inhibited, in a dose-dependent manner, the effects of thrombin on human osteoblast-like osteosarcoma cells (G292 and Saos-2 cell lines) and on normal rat calvarial osteoblastic cells. Thrombin-induced stimulation of cell proliferation, cytosolic calcium increases, and stimulation of phosphoinositide metabolism were concomitantly, and to a proportionally similar extent, inhibited. The inhibitors, when present in the absence of thrombin, did not affect the basal levels of cell functions. Both zeta-thrombin and gamma-thrombin, forms resulting from proteolytic cleavage of alpha-thrombin, were capable of stimulating the osteoblastic cells. These data indicate that thrombin's actions on osteoblast-like cells are dependent on the availability of its catalytic site.

Animals↗

PubMed1991

Effects of leukotrienes on osteoblastic cell proliferation.

Leukotriene B4 (LTB4) is an arachidonic acid lipoxygenase metabolite with well-characterized effects on leukocytes. LTB4 has been implicated in acute inflammatory reactions and in bone resorption. In the present study, the effect of LTB4 on osteoblastic cells was examined. LTB4 inhibited cell proliferation in normal osteoblastic rat calvaria cells in a dose-dependent manner and had biphasic effects in the human osteoblast-like osteosarcoma cell lines Saos-2 and G292. Indomethacin did not modulate the nature of these LTB4 effects in any of the cells. However, it potentiated the high LTB4 dose effects in normal cells and G292 cells. AA861, an inhibitor of lipoxygenase, did not modulate the LTB4 effects in these two cell lines. These results suggest that LTB4 is involved in the regulation of osteoblastic cell proliferation and may interact with prostaglandins to modulate these effects.

Animals↗

PubMed1991

Effects of inositol trisphosphate on calcium mobilization in bone cells.

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.

Animals↗

PubMed1991

Prostaglandin-induced changes in calcium uptake and cAMP production in osteoblast-like cells: role of protein kinase C.

Phorbol esters were used to evaluate the putative effect of protein kinase C (PKC) activation on prostaglandin E2 (PGE2)-induced increases in calcium uptake and cAMP production in the human osteoblastic osteosarcoma cell line, Saos-2. The cells were pretreated for 15 min with phorbol myristate acetate (PMA) followed by a 5 min incubation with PGE2. Calcium uptake was measured with 45Ca and cAMP by radioimmunoassay. A significant increase in calcium uptake was noted in the PGE-treated cells compared with controls and preincubation with the PMA caused a significant decrease in this response. Preincubation with PMA also inhibited the PGE2-induced increase in cAMP under identical conditions. The effect of PMA on the cAMP response was not influenced by the addition of a phosphodiesterase inhibitor. PMA had no effect on the basal levels of either calcium uptake or cAMP production. Likewise, the inactive phorbol esters, phorbol 12,13-didecanoate (PDD) and 4 alpha-phorbol 12-myristate, 13-acetate (4 alpha), had no effect on either basal levels of these parameters or on the PGE2-induced increases. These results suggest that PKC is involved in the down-regulation of PGE2-induced increases in calcium uptake and cAMP production in the Saos-2 osteoblastic cell line.

Calcium↗

PubMed1991

Recombinant interleukin-1 (IL-1) stimulates prostaglandin E2 production by osteoblastic cells: role of calcium, calmodulin, and cAMP.

Interleukin-1 (IL-1) has been previously shown to stimulate prostaglandin E2 (PGE2) production by osteoblastic cells. This IL-1 effect has also been shown to be potentiated by parathyroid hormone (PTH), which activates both the calcium and the cAMP signal transduction pathways in osteoblastic cells. In the present study, the role of calcium, calmodulin, and cAMP in potentiating the IL-1 effect was examined. The calcium channel blocker verapamil (100 microM) completely inhibited the IL-1 effect. Similarly, the calmodulin antagonist W-7 (50 microM) inhibited the IL-1-induced stimulation. Conversely, the calcium ionophore A23187 (0.1 microM) potentiated the IL-1 effect. The phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX; 100 microM), which elevates cAMP levels in the cells, had a strong potentiating effect on the IL-1-induced PGE2 production. These results suggest that both the calcium and the cAMP second messenger systems can modulate the IL-1 effect on osteoblastic cells.

1-Methyl-3-isobutylxanthine↗

PubMed1990

Effects of tumor necrosis factor alpha on parathyroid hormone-induced increases in osteoblastic cell cyclic AMP.

Tumor necrosis factor alpha (10(-10) - 10(-8) M) had no effects on cyclic AMP production by the osteoblastic osteosarcomal cells, Saos-2 and G292, or normal rat calvarial cells. The cytokine did, however, inhibit the parathyroid hormone (PTH)-induced effect on cyclic AMP in the Saos-2 and normal rat osteoblastic cells. This inhibitory effect did not occur on prostaglandin E2-induced cyclic AMP increases in the osteoblastic cells. Interleukin-1 (10 U/ml-100 U/ml) did not produce any effect on basal levels or PTH-induced cyclic AMP increases in these cells.

Animals↗

PubMed1989

Thrombin's effects on osteoblastic cells. I. Cytosolic calcium and phosphoinositides.

Thrombin, a blood coagulation factor, has been shown to be a very effective in vitro bone resorbing agent whose mechanism of action on osteoblastic cells remains to be elucidated. In the present study, the effects of highly purified human thrombin on Saos-2 and G292 cells, two human osteoblast-like osteosarcoma cell lines, were investigated. Thrombin (0.6-16 U/ml) caused a significant, dose-dependent increase in osteoblastic cell proliferation. Thrombin also elicited a dose-dependent increase in cytosolic calcium concentration in both Saos-2 and G292 cells (maximal increases were 38% and 200% over baseline, respectively). Addition of thrombin to the osteoblast-like cells resulted in significant time- and dose-dependent changes in phosphoinositide levels: the percentage of inositol monophosphate levels were decreased, whereas the percentage of inositol bisphosphate, inositol trisphosphate and inositol tetrakisphosphate levels were increased. The relative magnitude of the changes in phosphoinositide levels was similar to the changes in cytosolic calcium concentration. These results suggest that thrombin's mechanism of action on bone cells may involve increases in cytosolic calcium levels and in phosphoinositide metabolism.

Bone Resorption↗

PubMed1989

Recombinant human lymphotoxin effects on osteoblastic cells.

Lymphotoxin, or tumor necrosis factor beta, has been shown to be a potent bone resorbing cytokine. In the present study, the effect of recombinant human lymphotoxin on osteoblastic cell proliferation and prostaglandin synthesis was investigated. Lymphotoxin (10(-10)-10(-7) M) caused a significant, dose-dependent decrease of rat osteoblastic cell proliferation. This appeared to be an indirect, prostaglandin-dependent action, since in the presence of indomethacin (1 microM) the lymphotoxin effect was reversed. Subsequently, prostaglandin E2 and prostacyclin (assayed as 6-keto-prostaglandin F1 alpha) levels produced by the osteoblastic cells in response to lymphotoxin were measured. The cytokine caused a dose-dependent increase of these arachidonic acid metabolites, with the maximum effect at 10(-8) M. These results suggest that lymphotoxin's mechanism of action on bone may involve increases in arachidonic acid metabolite synthesis and an indirect, prostanoid-mediated decrease in the proliferation rate of osteoblastic cells.

Animals↗

PubMed1989

Leukotrienes in orthodontic tooth movement.

Prostaglandins (PGs) and leukotrienes (LTs) are products of arachidonic acid conversion. PGs have an established role in mediating orthodontic tooth movement. The role of LTs in modulating or mediating orthodontic tooth movement was investigated in this study. One hundred thirty-two Sprague-Dawley rats were used; the animals weighed 300 to 400 gm with equal numbers of male and female rats. They were divided into five main groups of 24 animals each and a sham group of 12 animals. An orthodontic appliance was placed and activated on all the animals except the sham group; in this group the appliances were not active. Each main group was given one of the following treatments daily: distilled water, 5% gum arabic solution, PG synthesis inhibitor indomethacin, LT synthesis inhibitor AA861, and a combination of both drugs. Each group was divided into six subgroups of four animals; the animals were killed at either 1, 3, 5, 7, 10, or 14 days, and tooth movement measured. The three sham subgroups received distilled water and were killed at 1, 7, or 10 days. The first maxillary molar (the moved tooth) and surrounding tissues were removed from all animals in the sham group and the subgroups killed at 1, 7, and 10 days in the gum arabic solution group and the LT synthesis inhibitor group. Prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) were extracted, measured with radioimmunoassay (RIA), and standardized per milligram of protein in the sample. A significant inhibition of tooth movement occurred beginning on day 7 in the indomethacin, AA861, and combination groups; there was no significant difference among these groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Regulation of bone cell metabolism.

Bone formation and resorption are normal physiologic processes. In pathologic states such as in periodontal disease or osteoporosis a shift in the balance of these two processes occurs, resulting in a net loss of mineralized tissue. Osteoclasts have historically been considered to be the primary bone resorbing cells, but current research has lead to the hypothesis that osteoblastic cells play an integral role in bone resorption as well. It appears that osteoblasts respond to bone resorbing agents via a series of intracellular responses after interactions with specific surface receptors. Two basic pathways involving different "second messengers" have been identified. The first pathway involves cyclic 3',5' adenosine monophosphate (cAMP) production and the second involves membrane phospholipids, diacylglycerol and calcium. A cytosolic enzyme, protein kinase C (PKC), has been shown to affect both cAMP as well as calcium fluxes and may act to regulate both these pathways. It is the purpose of this paper to discuss current studies and hypotheses concerning the nature of mechanisms involved in regulation of bone metabolism with emphasis on second messenger systems. Information of this nature is critical to the development of rationale regarding diagnosis, treatment and management of systemic and local pathoses of bone.

Animals↗

PubMed1988

The effects of phenytoin on calcium uptake in osteoblastic cells.

The effects of phenytoin (diphenylhydantoin, DPH) on calcium uptake in osteoblastic cells were studied to elucidate the potential mechanism of action of this antiepileptic drug on bone metabolism. Preincubation of the human osteoblastic osteosarcomal cell line, SaOS-2, and normal rat osteoblastic cells with DPH decreased basal calcium uptake. This inhibition occurred at DPH doses from 0.1 to 50 micrograms/ml. Parathyroid hormone (PTH) and prostaglandin E2 (PGE2) increased calcium uptake in the SaOS-2 cell line. Following preincubation with DPH, calcium uptake in cells treated with PTH or PGE2 did not exceed control levels. However, significant increases in the PTH- or PGE2-treated + DPH-pretreated cells compared to DPH pretreatment alone were still observed. These studies indicate that DPH induces decreases in osteoblastic calcium influx and they add further information on the possible mode of action of this drug on bone.

Animals↗

PubMed1988

Recombinant interleukin-1 stimulates prostaglandin E2 production by osteoblastic cells: synergy with parathyroid hormone.

Recombinant mouse IL-1 (Interleukin-1) has been shown to be capable of stimulating prostaglandin E2 (PGE2) production by isolated rat osteoblastic cells in a dose-dependent manner. The rapidity of the effect (1 hour) and the potency of IL-1 (5 x 10(-12) M) in producing this effect suggest that IL-1 may exert some of its effects on bone via PGE2. Parathyroid hormone (PTH) appears to have a strong synergistic effect with IL-1. These data further substantiate the role of IL-1 in bone physiology.

Animals↗

PubMed1986

The effects of calmodulin antagonists on prostaglandin E2-induced responses in rat calvarial bone cells.

Osteoclastic (OC) and osteoblastic (OB) cells were isolated by sequential collagenase digestions of new-born rat calvaria. Prostaglandin E2(PGE2) did not alter total calmodulin levels after a 5 or 60 min incubation. The calmodulin antagonists, trifluoperazine (TFP) at 10-50 microM and W-7 (50 microM) inhibited PGE2-induced increases in calcium uptake by OC cells, but had no effect on control OC or OB calcium levels. W-5 (50 microM), a chlorine-deficient analogue of W-7 with weak anti-calmodulin activity, had no effect. Compound 48/80 (100-500 micrograms/ml), a highly effective calmodulin antagonist in other systems, had no effect on PGE2-induced calcium levels or control calcium uptake. There was inhibition of PGE2-induced increases in cyclic AMP by compound 48/80 (100 micrograms/ml) in both OC and OB cells but no effect on control levels. TFP at 50 microM inhibited both control and PGE2-induced increases in cyclic AMP but at 10 microM it lessened only the hormone-induced effect. W-7 (100 microM) inhibited PGE2-induced increases in OC and OB cyclic AMP but had no effect on control levels; W-5 (50 microM) had no effect on either of these. Dibutyryl cyclic AMP had no effect on control calcium uptake, PGE2-induced increases or W-7 inhibition of the PGE-2 effect on calcium uptake. The calmodulin antagonists, at doses which had affected only PGE2-induced increases in calcium uptake and/or cyclic AMP production, had no effect on leucine uptake by OC or OB cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗