Biomedical subjects

R Dziak

Publications and source records attributed to R Dziak.

At least 19 recordsLinked to original sources

PubMed2003

Effects of sphingosine-1-phosphate and lysophosphatidic acid on human osteoblastic cells.

The effects of the lysophospholipids, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) were studied in human primary osteoblastic cells and the human osteosarcomal cell lines, G292 and MG-63. The studies focused on the role of the Gi protein in the regulation of S1P and LPA-induced proliferation, the effects of the phospholipids on alkaline phosphatase, an early marker of osteoblastic cell proliferation, and the presence of edg receptors. Proliferation was assessed by 3H-thymidine incorporation. Short-term incubation with S1P or LPA induced increases in proliferation that were attenuated in the presence of the Gi inhibitor, pertussis toxin. Alkaline phosphatase activity was measured with a spectrophotometric assay. Biphasic effects of S1P and LPA were observed with the nature of the response dependent upon the cell type, concentration of test agent and the time period of incubation. RTPCR studies revealed that edg-1,2,4,5 receptors are present in the primary normal osteoblastic cells, the MG63 and G292 cells. Only the G292 cells expressed the edg-3 receptor to any significant extent.

Alkaline Phosphatase↗

PubMed2002

Role of protein kinase C alpha in primary human osteoblast proliferation.

Protein kinase C (PKC) isoforms have been shown to have specific expression profiles and individual isoforms are believed to play distinct roles in the cells in which they are found. The goal here was to determine which specific isoform(s) is involved in proliferation of primary human osteoblasts. In primary human osteoblasts, 10 microM of acute sphingosine-1-phosphate (S1P) treatment induced an increase in proliferation that correlated with an increase in PKCalpha and PKCiota expression. To further delineate which isoforms are involved in osteoblastic cell proliferation, the effect of low versus high serum culture conditions on PKC isoform expression was determined. Likewise, the effect of antisense oligodeoxynucleotides (ODNs) to specific PKC isoforms on proliferation and MAPK activation was studied. The effect of S1P on intracellular translocation of activated PKC isoforms was also evaluated. The results indicated that in primary human osteoblasts, PKCalpha was not expressed under conditions of low proliferative rate while PKCdelta and PKCiota expression was not affected. The specific inhibition of PKCalpha by antisense ODNs resulted in inhibition of MAPK activity leading to a significant decrease in proliferation. S1P up-regulated antisense ODN inhibited PKCalpha expression and MAPK activity and led to an increase in proliferation. Subsequent experiments using platelet-derived growth factor (PDGF) as an additional mitogen generated similar data. PDGF stimulation resulted in a significant increase in proliferation that correlated with an up-regulation of inhibited PKCalpha expression in antisense ODN-treated cells. Immunofluorescence methods showed that mitogenic stimulation of PKCa resulted in nuclear translocation. Our findings present original data that PKCalpha is the isoform specifically involved in the proliferation of primary human osteoblasts.

Cell Compartmentation↗

PubMed2001

Sphingosine-1-phosphate effects on PKC isoform expression in human osteoblastic cells.

Sphingosine-1-phosphate (S1P) has been shown to participate in the proliferative process in human osteoblasts.(1) The mitogenic effect of S1P has been postulated to involve two signaling pathways, the Gi linked protein receptor pathway and the PKC pathway. To define the possible role of PKC isoforms in osteoblastic cell proliferation, the effects of S1P on PKC isoform expression was determined. While PKC lambda was minimally detected, the isoforms alpha, delta and iota were all found to be highly expressed by the human osteoblast. In human osteoblastic cells, S1P induced a 25% increase in the expression of PKC alpha and approximately a 30% increase in the expression of PKC iota. S1P did not have an effect on PKC delta expression. Pretreatment with pertussis toxin (PT) led to an inhibition of the observed S1P effects on the expression of the alpha and iota isoforms.

Blotting, Western↗

PubMed2000

Changes in sphingolipid levels induced by epidermal growth factor in osteoblastic cells. Effects of these metabolites on cytosolic calcium levels.

Sphingolipids mediate a number of cellular functions in a variety of cell systems. The role they play in osteoblast signaling is yet unknown. This study investigated the effects of epidermal growth factor (EGF) on the levels of ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P) in rat calvariae osteoblastic cells, and whether these metabolites mediated cytosolic calcium ([Ca2+]i) mobilization in these cells. EGF significantly (P<0.05) increased the levels of all three sphingolipids, and the phorbol ester PMA partially inhibited these effects. SPH and S1P markedly increased [Ca2+]i levels, with thapsigargin (depletes [Ca2+]i pools) decreasing the response by 60%. Verapamil (calcium channel blocker) only inhibited ceramide's effects on [Ca2+]i. Furthermore, SPH enhanced the EGF' induced increase in [Ca2+]i. This study demonstrates that ceramide, SPH and S1P mediate [Ca2+]i mobilization in rat calvarial osteoblastic cells, and that EGF induces changes in the levels of these metabolites with PKC playing an important role in the mechanisms regulating these events.

Animals↗

PubMed2000

Platelet-derived growth factor enhancement of a mineral-collagen bone substitute.

BACKGROUND: Anorganic bovine bone-collagen matrix is commercially available for bone regeneration procedures. Platelet-derived growth factor-BB (PDGF-BB) has been demonstrated to stimulate bone formation in vivo and in vitro. It was the aim of these studies to examine 1) the interaction of this mineral-collagen matrix with PDGF-BB and 2) determine if the adsorption of PDGF-BB to the mineral-collagen matrix stimulates osteoblastic cell proliferation above that of the untreated matrix. METHODS: Measurement of PDGF-BB adsorption and release was accomplished using 125I radiolabeled growth factor. The PDGF-BB was incubated with the anorganic bovine bone-collagen matrix and the amount which adsorbed was determined. In the release studies, radiolabeled PDGF-BB was adsorbed to the matrix material, then the samples were incubated in buffer for various time periods. The amount of PDGF-BB retained on the matrix was measured and the percent of growth factor released calculated. The biological activity was tested in an in vitro assay with primary culture neonatal rat osteoblastic cells. Osteoblastic cells were cultured on bone mineral-collagen matrix with known amounts of adsorbed PDGF-BB. Proliferation of the cells was assessed by 3H-thymidine incorporation and cell attachment measured by prelabeling cells with 3H-leucine. RESULTS: PDGF-BB adsorbed to the mineralized-collagen matrix material in a rapid, concentration-dependent fashion. The growth factor was slowly released from the matrix such that approximately 30% of the adsorbed protein was liberated over 10 days. PDGF-BB treated mineralized-collagen matrix displayed significantly (P < 0.05, ANOVA) enhanced proliferation of cultured osteoblastic cells compared to the mineralized-collagen matrix alone. CONCLUSIONS: These results suggest that PDGF-BB is rapidly adsorbed then slowly released from the anorganic bovine bone-collagen matrix. PDGF-BB adsorbed to this material is able to stimulate proliferation of the attached osteoblastic cells. These data suggest that it may be clinically feasible to adsorb PDGF to this bone-collagen matrix and that this combination of bone growth factor and mineral-collagen matrix has the potential for clinical applications.

Adsorption↗

PubMed1999

Sphingolipids stimulate cell growth via MAP kinase activation in osteoblastic cells.

Ceramide, ceramide-1-phosphate (C1P) sphingosine (SPH) and sphingosine-1-phosphate (S1P) effects on proliferation and extracellular-signal regulated kinases, ERKs (also known as MAPKs), activation were investigated in human and rat osteoblastic cells. MAPK activation was sphingolipid-specific in cells from both species. In human osteoblastic cells, S1P and C1P markedly stimulated ERK2 phosphorylation with a slight increase in phosphorylation of ERK1. SPH nor ceramide induced phosphorylation of either ERK isoform. In rat osteoblastic cells, SIP, ceramide and SPH stimulated phosphorylation of both isoforms. C1P did not induce phosphorylation of ERK1 but produced a mild increase in phosphorylation of ERK2. In human cells, only S1P significantly (P<0.05) increased osteoblastic cell proliferation, while in the rat cells all four sphingolipids significantly (P<0.05) induced proliferation. The calcium channel blocker verapamil blocked (P<0.05) these effects in both cell types. The MAPK inhibitor, PD98059, inhibited (P<0.05) the mitogenic effect of SIP in human cells. In rat cells, PD98059 effects were less substantial but significant for S1P and C1P. This study demonstrates that sphingolipids are mitogens for both human and rat osteoblastic cells with the MAPK pathway and calcium mediating in part these effects in a species specific manner.

Animals↗

PubMed1999

Molecular characterization of the alpha1 subunit of the L type voltage calcium channel expressed in rat calvarial osteoblasts.

Voltage-activated calcium channels (VACCs) regulate extracellular calcium influx in many cells. VACCs are composed of five subunits. The alpha1 subunit is considered the most important in regulating channel function. Three isoforms of this subunit have been described: skeletal, cardiac, and neuroendocrine. It was the purpose of the present study to determine the molecular identity of the alpha1 subunit of the VACCs in rat calvarial osteoblasts and to study the nature of the regulation of these channels as a function of cellular growth. We also attempted to identify which isoform of the alpha1 subunit of the VACCs mediates the effects of epidermal growth factor (EGF) on osteoblastic cell proliferation. Reverse transcription-polymerase chain reaction was used to detect the isoforms of the VACCs that are expressed in osteoblastic cells. These analyses showed that the proliferative state of the cell and the time in culture influence RNA expression. The only alpha1 subunit detected in osteoblasts corresponds to the cardiac isoform. In additional experiments, the effects of EGF on cytosolic calcium and osteoblast proliferation were determined. For these experiments, the synthesis of the different isoforms of the VACCs was selectively blocked by antisense oligonucleotides prior to EGF stimulation. These studies showed that the cardiac isoform mediates the effects of EGF on cytosolic calcium and cellular proliferation in rat calvarial osteoblasts.

Alkaline Phosphatase↗

PubMed1999

Anorganic bovine bone supports osteoblastic cell attachment and proliferation.

BACKGROUND: It was the aim of these studies to examine the ability of an anorganic bovine bone matrix material as an alternative to autogenous bone grafts and demineralized cadaver bone to support the attachment, spreading, and proliferation of isolated osteoblastic cells. METHODS: Primary culture osteoblastic cells were isolated from neonatal rat calvaria by sequential collagenase digestion. In the attachment studies, cells which had been labeled with 3H-leucine were incubated with the matrix material in sterile microfuge tubes for 15, 90, or 180 minutes or 24 hours. The attached cells were released and the radioactivity measured by liquid scintillation spectrometry. In the proliferation experiments, the cells were cultured with the matrix material for 24 hours and 3H-thymidine was added during the last 2 hours of the incubation. The cells were released and the radioactivity measured by liquid scintillation spectrometry. Scanning electron microscopy (SEM) was employed to observe osteoblastic cell interaction with the anorganic bone matrix. In these studies the cells were seeded on the bone graft material, then the material was removed and processed for SEM after 30, 60 or 120 minutes, or 24 or 48 hours. RESULTS: The cells attached to the matrix material in a time-dependent manner. There were significantly (P<0.05) more cells attached after 180 minutes than after the 15 and 90 minute incubations. The matrix material also supported proliferation of the attached osteoblastic cells. Cells seeded onto 100 mg of anorganic bovine bone resulted in significantly (P<0.05) more measurable proliferation than cells seeded onto 10 mg of material. The cells appeared to be round as they attached, then flatten and spread over time. There was also evidence of cellular processes extending into the pores of the material. CONCLUSIONS: These results demonstrate that this anorganic bovine bone graft material is able to support the attachment and proliferation of osteoblastic cells.

Animals↗

PubMed1999

Modification of an osteoconductive anorganic bovine bone mineral matrix with growth factors.

BACKGROUND: Osteoconductive anorganic bovine bone mineral matrix material has been used clinically in bone regeneration procedures. Platelet-derived growth factor-BB (PDGF-BB) and insulin-like growth factor (IGF-I) are important anabolic growth factors for bone. It was the aim of these studies to 1) examine the interaction of this bone graft material with PDGF-BB and IGF-I and 2) determine if the combination of growth factors with the matrix could stimulate osteoblastic cell proliferation. METHODS: Adsorption of PDGF-BB and IGF-I was done using 125I radio-labeled growth factors. The PDGF-BB or IGF-I was incubated with the anorganic bovine bone matrix, and the amount of adsorbed growth factor was measured. In the desorption studies, radiolabeled growth factors were adsorbed to the matrix material. The samples were incubated in buffer for various time periods, and the amount remaining on the matrix was measured to calculate the percentage of released growth factor. The biological activity was tested in an in vitro assay with primary culture neonatal rat osteoblastic cells. Porous bone matrix with known amounts of adsorbed PDGF-BB or IGF-I was produced. The osteoblastic cells were cultured on the bone mineral matrix, with and without adsorbed growth factor, and proliferation was assessed by 3H-thymidine incorporation. RESULTS: Both PDGF-BB and IGF-I adsorbed to bone mineral matrix in a concentration-dependent fashion. The affinity of IGF-I for the material was 10-fold greater than PDGF-BB. In the experiments that measured the release of the initially adsorbed growth factors, approximately 50% of the PDGF-BB and 10% of the IGF-I were released after 10 days. PDGF-BB adsorbed to the matrix material significantly (P <0.05, ANOVA) enhanced the proliferation of cultured osteoblastic cells compared to the mineralized matrix alone. However, IGF-I adsorbed to the matrix material did not significantly enhance cell proliferation. CONCLUSIONS: These results suggest that PDGF-BB can be adsorbed to the anorganic bovine bone mineral matrix and that this growth factor subsequently enhances the osteogenic properties of this bone graft material. IGF-I also adsorbed to the graft material; however, it was not readily released and it did not produce significant effects in the biologic assay. It appears that it may be clinically feasible to adsorb PDGF to anorganic bovine bone and that this combination of bone growth factor and mineral matrix has the potential for clinical applications.

Adsorption↗

PubMed1998

Phosphatidic acid effects on cytosolic calcium and proliferation in osteoblastic cells.

Our previous studies show that epidermal growth factor (EGF) stimulates phospholipase D (PLD)-induced phosphatidic acid (PA) formation in rat calvarial osteoblastic cells. This study investigated the effects of PA on cytosolic calcium ([Ca2+]i) and proliferation, and the possible involvement of the PLD pathway in EGF effects on [Ca2+]i and proliferation in rat calvarial osteoblastic cells. PA markedly increased [Ca2+]i. This response was unaffected by thapsigargin, which depletes [Ca2+]i pools, blocked by verapamil, a calcium channel blocker, and enhanced by propanolol, an inhibitor of PA-phosphohydrolase. PA also reduced the EGF dependent-[Ca2+]i increase by 60%, while a PLD inhibitor blocked these effects. Furthermore, PA significantly increased cell proliferation (P < 0.05) which was inhibited by verapamil and enhanced by H-7 (PKC inhibitor). The PLD inhibitor significantly (P < 0.05) reduced the EGF-induced increase in proliferation. In summary, PA stimulates rat calvarial osteoblastic cell proliferation and mobilization of [Ca2+]i using extracellular pools, and EGF's mitogenic effect on these cells requires activation of PLD.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1998

Activation of phospholipase D signaling pathway by epidermal growth factor in osteoblastic cells.

The receptor-mediated activation of phospholipase D (PLD) is a major signaling pathway in several cell systems. This study determined the effects of epidermal growth factor (EGF) on PLD activity in normal rat osteoblastic cells. Primary cultures were obtained from fetal rat calvaria by sequential collagenase digestion and seeded in BGJb media supplemented with 10% fetal calf serum. PLD activity was assayed by the transphosphatidylation reaction in [H3]myristic acid (5 microCi/ml)-labeled cells treated with EGF in the presence of 5% ethanol and measuring the production of phosphatidylethanol (PEtOH). Lipids were extracted and separated by thin-layer chromatography, detected by iodine staining, and the areas of interest were scraped off and transferred to vials for scintillation counting. EGF significantly increased PEtOH production in a dose-dependent manner and at short (10-60 s) and long (up to 30 minutes) incubation periods (p < 0.05). Phosphatidic acid levels were also significantly increased (p < 0.05) compared with unstimulated controls, but the levels were approximately 60% less than those of PEtOH. 4b-phorbol 12-myristate, 13-acetate (PMA) also produced a significant increase in PEtOH levels when compared with unstimulated control cultures, but when PMA was added together with EGF, the production of PEtOH was reduced about 30%. Pretreatment of cells with the protein kinase C (PKC) inhibitor H-7 caused a significant increase in PEtOH levels, compared with cells stimulated with EGF alone. Preincubation of cells with pertussis toxin produced a partial decrease in PEtOH levels. This study demonstrates that EGF activates the PLD signaling cascade in normal rat osteoblastic cells and that the pathway appears to involve, at least in part, a PKC- and Gi protein-dependent mechanism.

Analysis of Variance↗

PubMed1996

Inositol trisphosphate receptor gene expression and hormonal regulation in osteoblast-like cell lines and primary osteoblastic cell cultures.

The inositol trisphosphate receptor (IP3R) is an intracellular calcium channel that mediates the cellular actions of a wide variety of hormones, growth factors, and cytokines. In osteoblastic cell cultures, many bone resorbing hormones increase phosphoinositide turnover, inositol trisphosphate production, mobilization of intracellular calcium, and the secretion of osteoclast recruitment and activating factors. In this study, the effects of 17 beta-estradiol, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), phrobol ester, and serum on IP3R mRNA levels were evaluated in osteogenic-osteosarcoma cells and in primary osteoblastic cultures derived from neonatal rat calvaria. Type-specific reverse transcription polymerase chain reaction (RT-PCR) indicated that all cell types evaluated (G-292, U-2 OS, Saos-2, MC3T3-E1, UMR-106, and calvarial osteoblastic cells) express IP3R mRNA type I; G-292, U-2 OS, MC3T3-E1, and calvarial osteoblastic cells also express type II IP3R mRNA; and UMR-106 and the calvarial osteoblastic cells express type III IP3R mRNA. Northern blot and RT-PCR analyses of human G-292 osteosarcoma cells and rat calvarial osteoblastic cells showed that phorbol ester and serum increase IP3R mRNA levels, whereas 17 beta-estradiol and 1,25(OH)2D3 decrease these levels. In G-292 cells, the effect of 17 beta-estradiol was not due to accelerated IP3R mRNA degradation and required continued protein synthesis. The results show that multiple IP3R types are expressed in osteoblasts and osteoblastic osteosarcoma cells and that this expression is regulated by 17 beta-estradiol and other osteoporotic and antiosteoporotic hormones. These findings indicate that hormonal control of IP3R expression may be relevant in the chronic regulation of osteoblast secretory activity.

Animals↗

PubMed1996

Immediate early-gene induction in rat osteoblastic cells after mechanical deformation.

Previous studies have reported changes in proliferation, second-messenger generation and activation of various cellular processes when osteoblasts have been mechanically stimulated. Recent evidence suggests that mechanical loading of long bones induces immediate early-gene expression. Immediate early genes, such as Egr-1, are genes that control cell proliferation, are involved in signal transduction, and share properties of transcription factors. The purpose of this study was to examine how mechanical deformation of osteoblasts affects cellular proliferation and Egr-1 mRNA induction. Osteoblasts were isolated from collagenase digestion of newborn rat calvariae, cultured in Petri dishes with flexible bottoms and then constantly stretched, producing an increase of 3 or 7% in surface area. A mechanical stretch of 7% for 0.5 or 24 h resulted in a doubling of [3H]thymidine incorporation, while 50 nM of epidermal growth factor resulted in a 4-fold increase. A time-course experiment showed that a 7% stretch induced Egr-1 mRNA as early as 15 mm, reaching maximum levels by 60 min and returning to baseline by 120 min. Epidermal growth factor at 50 nM for 60 min resulted in a 3.8-fold Egr-1 mRNA induction. A mechanical stretch of 3% for 30 min also produced an Egr-1 mRNA induction. No induction of Egr-1 mRNA was seen in osteoblasts that were exposed to conditioned media from deformed cells. It is concluded that the immediate early gene, Egr-1, may be directly involved in the signal-transduction pathway of mechanical stimuli in osteoblasts.

Animals↗

PubMed1996

Tumor necrosis factor alpha stimulates arachidonic acid metabolism in human osteoblastic osteosarcomal cells.

The effects of tumor necrosis factor alpha (TNF-alpha) on arachidonic acid (AA) metabolism were investigated by prelabeling the human osteoblastic osteosarcoma cell line, G292, with [3H]AA. TNF-alpha differentially stimulates cyclooxygenase and lipoxygenase pathways of AA metabolism in a dose response manner in the cells. The highest concentration of TNF-alpha (10(-8)M) significantly increased the cyclooxygenase pathway, with prostaglandin E2 (PGE2) being a major product. However, at the lowest concentration (10(-10)M) of TNF-alpha, 15-hydroxyeicosatetraenoic acid (HETE) production was significantly increased, with no significant effects on the other identifiable products. When the concentration of TNF-alpha was increased to 10(-9) M leukotriene B4 (LTB4), 15-, 12-, and 5-HETE were significantly increased. The calcium ionophore A23187 (10(-6) M) significantly increased 15-HETE production, without significantly affecting cyclooxygenase metabolites. However, a combination of TNF-alpha (10(-8)M) and A23187 (10(-6)M) caused an inhibitory effect on each agent-induced PGE2 or 15-HETE production.

Arachidonic Acid↗

PubMed1996

Osteoporosis and its relationship to oral bone loss.

Osteoporosis, an age-related condition, is classified into primary and secondary types. Primary osteoporosis encompasses the postmenopausal and senile types; secondary osteoporosis occurs "secondary" to endocrine and renal diseases. Subjects affected by osteoporosis have an overall reduced bone mass and become highly susceptible to bone fractures. Dual energy x-ray absorptiometry is the method most often used to determine the risk for osteoporotic fractures. In the past decade, a number of studies have suggested a possible correlation between systemic osteoporosis and alveolar bone loss in periodontal disease pathogenesis. It appears that a clear correlation between periodontal health and the general mineral status of the skeleton is still lacking. This review addresses the pathogenesis of osteoporosis and emphasizes the multifactorial nature of bone loss. The current concepts in alveolar bone loss resulting from osteoporosis and its implications as a risk factor in periodontal disease development are also presented.

Aged↗

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↗

PubMed1994

Calcium currents in osteoblastic cells: dependence upon cellular growth stage.

Patch clamp physiological techniques were used to characterize the voltage-activated calcium currents (VACC) expressed in the plasma membrane of osteoblastic cells as a function of time in culture and proliferative state of the cell. Osteoblast-enriched preparations were isolated by collagenase digestions of newborn rat calvaria and cultured under different conditions which affected cell proliferation (i.e., low serum in the media to arrest proliferation). VACC were isolated by replacing the intracellular potassium with cesium, and adding 1 microM tetrodotoxin to the bath. Under conditions that favored cell proliferation, low cell density, and media supplemented with 10% fetal calf serum (FCS), a transient calcium current was not expressed until day 3 in culture. There was a statistically significant relationship between the percentage of cells expressing this current and the time in culture. The magnitude of the current significantly increased as days in culture increased. Under the same conditions, the sustained VACC was detected after 7 or 8 days in culture. However, arresting cell proliferation after 2 days in culture by reducing the FCS concentration to 0.01% induced the expression of the sustained VACC the next day. The data suggest that the expression of VACC in the plasma membrane of rat calvarial osteoblasts depends on the time in culture and the state of proliferation of the cells. These results should prove to be valuable in studying the functional significance of VACC in osteoblastic cells and their regulation by various bone regulatory agents.

Animals↗