Gender difference in tooth autotransplantation with complete root formation: a retrospective survey.

Gender-related risk factors in the survival of transplanted teeth with complete root formation have not yet been identified. The purpose of this study was to investigate gender differences in tooth autotransplantation at dental clinics. We asked participating dentists to provide information on transplantations they had undertaken from 1 January 1990 to 1931 December 2010. The data were screened to exclude patients who underwent more than one transplantation, smokers or those whose smoking habits were unknown, patients under 30 or who were 70 years old and over, cases where the transplanted teeth had incomplete root formation or multiple roots and those with fewer than 20 present teeth post-operation. We analysed 73 teeth of 73 males (mean age, 47.2 years) and 106 teeth of 106 females (mean age, 45.3 years) in this study. The cumulative survival rate and mean survival time were calculated using the Kaplan-Meier method. The cumulative survival rate for males was 88.3% at the 5-year mark, 64.8% at 10 years and 48.6% at 15 years; for females, it was 97.2% at the 5-year mark, 85.9% at 10 years and 85.9% at 15 years. A log-rank test indicated the difference between males and females to be significant (P = 0.011). There was also a significant difference in the main causes for the loss of transplanted teeth: males lost more transplanted teeth due to attachment loss than females (P < 0.05). These results indicate that males require more attention during the autotransplantation process, particularly at the stage of pre-operation evaluation and that of follow-up maintenance.

Comparison of prognosis of separated and non-separated tooth autotransplantation.

The aim of this study was to compare the prognosis of separated and non-separated tooth autotransplantation of the upper first and second molars with complete root formation undertaken at dental clinics. The participating dentists were requested to provide information on transplantations they had undertaken from 1 January 1990 to 31 December 2010. Data on a total of 708 teeth from 637 patients were collected. This study analysed 35 separated teeth and 22 non-separated teeth of 47 participants ranging from 27 to 76 years of age (mean age: 55·0 years) after data screening and elimination. The cumulative post-transplantation survival rate at 10 years was 77·1% for separated teeth and 63·6% for non-separated teeth as calculated with the Kaplan-Meier method. There were no significant differences between separated teeth and non-separated teeth in a log rank test (P = 0·687). Separated-tooth autotransplantation can help fill narrow recipient sites and increase occlusal supporting zones, but the clinical success rate was only 48·6%. Although transplantation of teeth with complete root formation has limited prognosis, transplantation of upper first and second molars, whether separated or non-separated, is a viable option to replace missing teeth.

Influence of age on tooth autotransplantation with complete root formation.

The aim of this study was to investigate risk factors with age in the long-term prognosis of autotransplantation of teeth with complete root formation at dental clinics. Participating dentists were asked to provide information on transplantations they had undertaken from 1 January 1990 to 31 December 2010. Data on a total of 708 teeth from 637 patients were collected. The data were screened to exclude patients who were under 25 or 70 years of age and over, those who were smokers or whose smoking habits were unknown, those whose transplanted teeth had incomplete root formation or multiple roots and those with fewer than 25 present teeth post-operation. The participants in this study were 71 men (74 teeth) and 100 women (107 teeth) ranging from 25 to 69 years of age. Third molars were used as donor teeth in 89·0% of the cases. The participants were divided into three age groups of 25-39, 40-54 and 55-69. Survival analysis was conducted using the Kaplan-Meier method, and a log-rank test revealed that there were no significant differences in age groups for men or women. Cox regression analysis indicated that the survival of transplanted teeth was not influenced by age. However, although not statistically significant, the clinical success rate was lower in the 55-69-year-old group than that in the younger groups. These results indicate that if suitable donor teeth are available and the conditions are right, autotransplantation is a viable treatment for missing teeth regardless of the age of the patient.

Risk factors affecting third molar autotransplantation in males: a retrospective survey in dental clinics.

The aim of this study was to investigate the risk factors affecting long-term prognosis of autotransplantation of third molars with complete root formation in males at dental clinics. Participating dentists were requested to provide information on transplantations they had undertaken from 1 January 1990 to 31 December 2010. Data on a total of 708 teeth from 637 patients were collected. After data screening and elimination, participants of this study consisted of 183 teeth of 171 males ranging from 20 to 72 years of age (mean age, 44·8 years). The cumulative survival rate was 86·0% at the 5-year mark, 59·1% at 10 years and 28·0% at 15 years. The mean survival time was 134·5 months, as calculated by the Kaplan-Meier method. Single factor analysis using the log-rank test showed that the following factors had significant influence (P < 0·05) on survival of transplanted teeth: periodontal disease as the reason for recipient site tooth extraction, fewer than 25 present teeth and Eichner index Groups B1 to C. Cox regression analysis examined five factors: age, smoking habit, recipient site extraction caused by periodontal disease, fewer than 25 present teeth and Eichner index. This analysis showed that two of these factors were significant: fewer than 25 present teeth was 2·63 (95% CI, 1·03-6·69) and recipient site extraction caused by periodontal disease was 3·80 (95% CI, 1·61-9·01). The results of this study suggest that long-term survival of transplanted teeth in males is influenced not only by oral bacterium but also by occlusal status.

A retrospective survey of autotransplantation of teeth in dental clinics.

The aim of this study was to investigate the usage of tooth autotransplantation in dental clinics which offer the treatment and evaluate its practicality. Participating dentists were requested to provide information on transplantations they had undertaken from 1 January 1990 to 31 December 2010. A total of 614 teeth from 552 patients (37 dentists) ranging in age from 17 to 79 (mean age: 44·1) were examined. Cumulative survival rate and mean survival time were calculated using the Kaplan-Meier method, and log rank test was used for analysis of factors. The mean number of autotransplantation patients per clinic per year was 1·4. Upper third molars constituted 36·8% of donor teeth, while 37·1% were lower third molars. The lower first molar region was the most common recipient site at 32·6%, followed by the lower second molar region (28·0%). Prosthodontic treatment of transplanted teeth involved coverage with a single crown (72·5%) and abutment of bridge (18·9%). A total of 102 transplanted teeth were lost owing to complications such as attachment loss (54·9%) and root resorption (25·7%). The cumulative survival rate in cases where donor teeth had complete root formation was 90·1% at 5 years, 70·5% at 10 years and 55·6% at 15 years. The mean survival time was 165·6 months. Older age was a significant risk factor (P < 0·05) for survival. In cases where suitable donor teeth are available, autotransplantation of teeth may be a plausible treatment option for dealing with missing teeth in dental clinics.

Relatively decreased 11C-methionine uptake within the anaplastic component of a mixed-grade oligodendroglioma.

A 56-year-old woman presented with a mixed-grade oligodendroglioma. On 11C-methionine [MET]-positron-emission tomography images, heterogeneous uptake of MET was demonstrated in the mass lesion. The part of the lesion with higher MET uptake was identified as an ordinary oligodendroglioma, whereas the part of the lesion with lower MET uptake was an anaplastic component of oligodendroglioma. With oligodendrogliomas, we should be aware of the possibility that MET uptake decreases paradoxically with an increased anaplastic component of oligodendroglioma cells.

Biological activities of a recombinant adenovirus p53 (SCH 58500) administered by hepatic arterial infusion in a Phase 1 colorectal cancer trial.

The major focus of intrahepatic arterial (IHA) administration of adenoviruses (Ad) has been on safety. Currently, there is little published data on the biological responses to Ad when administered via this route. As part of a Phase I study, we evaluated biological responses to a replication-defective adenovirus encoding the p53 transgene (SCH 58500) when administered by hepatic arterial infusion to patients with primarily colorectal cancer metastatic to the liver. In analyzing biological responses to the Ad vector, we found that both total and neutralizing Ad antibodies increased weeks after SCH 58500 infusion. The fold increase in antibody titers was not dependent on SCH 58500 dosage. The proinflammatory cytokine interleukin-6 (IL-6) transiently peaked within 6 h of dosing. The cytokine sTNF-R2 showed elevation by 24 h post-treatment, and fold increases were directly related to SCH 58500 doses. Cytokines TNF-alpha, IL-1beta, and sTNF-R1 showed no increased levels over 24 h. Predose antibody levels did not appear to predict transduction, nor did serum Ad neutralizing factor (SNF). Delivery of SCH 58500 to tumor tissue occurred, though we found distribution more predominantly in liver tissues, as opposed to tumors. RT-PCR showed significantly higher expression levels (P<0.0001, ANOVA) for adenovirus type 2 and 5 receptor (CAR) in liver tissues, suggesting a correlation with transduction. Evidence of tumor-specific apoptotic activity was provided by laser scanning cytometry, which determined a coincidence of elevated nuclear p53 protein expression with apoptosis in patient tissue. IHA administration of a replication defective adenovirus is a feasible mode of delivery, allowing for exogenous transfer of the p53 gene into target tissues, with evidence of functional p53. Limited and transient inflammatory responses to the drug occurred, but pre-existing immunity to Ad did not preclude SCH 58500 delivery.

Relationship between regional cerebral metabolism and consciousness disturbance in traumatic diffuse brain injury without large focal lesions: an FDG-PET study with statistical parametric mapping analysis.

BACKGROUND: The cerebral metabolism of patients in the chronic stage of traumatic diffuse brain injury (TDBI) has not been fully investigated. AIM: To study the relationship between regional cerebral metabolism (rCM) and consciousness disturbance in patients with TDBI. METHODS: 52 patients with TDBI in the chronic stage without large focal lesions were enrolled, and rCM was evaluated by fluorine-18-fluorodeoxyglucose positron emission tomography (FDG-PET) with statistical parametric mapping (SPM). All the patients were found to have disturbed consciousness or cognitive function and were divided into the following three groups: group A (n = 22), patients in a state with higher brain dysfunction; group B (n = 13), patients in a minimally conscious state; and group C (n = 17), patients in a vegetative state. rCM patterns on FDG-PET among these groups were evaluated and compared with those of normal control subjects on statistical parametric maps. RESULTS: Hypometabolism was consistently indicated bilaterally in the medial prefrontal regions, the medial frontobasal regions, the cingulate gyrus and the thalamus. Hypometabolism in these regions was the most widespread and prominent in group C, and that in group B was more widespread and prominent than that in group A. CONCLUSIONS: Bilateral hypometabolism in the medial prefrontal regions, the medial frontobasal regions, the cingulate gyrus and the thalamus may reflect the clinical deterioration of TDBI, which is due to functional and structural disconnections of neural networks rather than due to direct cerebral focal contusion.

Evidence for white matter disruption in traumatic brain injury without macroscopic lesions.

BACKGROUND: Non-missile traumatic brain injury (nmTBI) without macroscopically detectable lesions often results in cognitive impairments that negatively affect daily life. AIM: To identify abnormal white matter projections in patients with nmTBI with cognitive impairments using diffusion tensor magnetic resonance imaging (DTI). METHODS: DTI scans of healthy controls were compared with those of 23 patients with nmTBI who manifested cognitive impairments but no obvious neuroradiological lesions. DTI was comprised of fractional anisotropy analysis, which included voxel-based analysis and confirmatory study using regions of interest (ROI) techniques, and magnetic resonance tractography of the corpus callosum and fornix. RESULTS: A decline in fractional anisotropy around the genu, stem and splenium of the corpus callosum was shown by voxel-based analysis. Fractional anisotropy values of the genu (0.47), stem (0.48), and splenium of the corpus callosum (0.52), and the column of the fornix (0.51) were lower in patients with nmTBI than in healthy controls (0.58, 0.61, 0.62 and 0.61, respectively) according to the confirmatory study of ROIs. The white matter architecture in the corpus callosum and fornix of patients with nmTBI were seen to be coarser than in the controls in the individual magnetic resonance tractography. CONCLUSIONS: Disruption of the corpus callosum and fornix in patients with nmTBI without macroscopically detectable lesions is shown. DTI is sensitive enough to detect abnormal neural fibres related to cognitive dysfunction after nmTBI.

p53 regulates ceramide formation by neutral sphingomyelinase through reactive oxygen species in human glioma cells.

The present study was designed to elucidate the relationship between p53 and ceramide, both of which are involved in apoptotic signaling. Treatment of human glioma cells with etoposide caused apoptosis only in cells expressing functional p53. p53 activation was followed by the formation of reactive oxygen species (ROS), superoxide anion (O2-*) measured by hydroethidium oxidation into ethidium and hydrogen peroxide (H2O2) measured by oxidation of 2',7'-dichlorofluorescin (DCFH) into 2',7'-dichlorofluorescein (DCF), which was accompanied with ceramide generation through the activation of neutral, but not acid, sphingomyelinase. Superoxide dismutase (SOD), a selective antioxidant for O2-*, had no effects on p53 expression but inhibited ceramide generation and apoptotic cell death caused by etoposide. However, catalase, a specific antioxidant for H2O2, only weakly inhibited and sodium formate, a hydroxyl radical (* OH) scavenger, unaffected etoposide-induced apoptosis. Like etoposide-induced cell death, treatment of glioma cells with the O2-*-releasing agent, pyrogallol, induced typical apoptosis and ceramide generation even in the presence of catalase. In contrast, human glioma cells lacking functional p53, either due to mutation or the expression of E6 protein of human papillomavirus, were highly resistant to etoposide and exhibited no significant change in the ceramide level. Moreover, expression of functional p53 protein in glioma cells expressing mutant p53 using a temperature-sensitive human p53(Val138) induced ceramide accumulation by the activation of neutral sphingomyelinase which was dependent on the generation of O2-*. Taken together, these results suggest that p53 may modulate ceramide generation by activation of neutral sphingomyelinase through the formation of O2-*, but not its downstream compounds H2O2 or * OH.

Influence of Bax or Bcl-2 overexpression on the ceramide-dependent apoptotic pathway in glioma cells.

Ceramide has recently been regarded as a potential mediator of apoptosis. In the present study, the effects of Bcl-2 and Bax on the ceramide-mediated apoptotic pathways were examined in glioma cells overexpressing Bcl-2 or Bax. Etoposide, cisplatin and tumor necrosis factor-alpha induced apoptosis of C6 rat glioma cells which was associated with ceramide formation due to activation of neutral sphingomyelinase, followed by release of mitochondrial cytochrome c into the cytosol and activation of caspases-9 and -3. The growth of C6 cells stably overexpressing either Bcl-2 or Bax was almost equal to that of the vector-transfected cells. Bax overexpression enhanced etoposide-induced apoptosis through acceleration of cytochrome c release and caspases activation. However, Bax had no effect on ceramide formation. Similar findings were obtained in C6 cells and U87-MG human glioblastoma cells which were transiently overexpressed with Bax. In contrast, Bcl-2 overexpression resulted in a retardation of the apoptotic process via prevention of cytochrome c release and caspases activation, and ceramide formation was also blocked when Bcl-2 was highly overexpressed in glioma cells. In addition, transient overexpression of Bcl-xL also exerted inhibitory effects on ceramide formation and apoptotic cell death induced by etoposide. These results indicate that Bax promotes apoptosis regardless of ceramide formation and that Bcl-2 or Bcl-xL prevents ceramide formation by repressing neutral sphingomyelinase as well as ceramide-induced cytochrome c release. Oncogene (2000) 19, 3508 - 3520

Molecular mechanisms of TNF-alpha-induced ceramide formation in human glioma cells: P53-mediated oxidant stress-dependent and -independent pathways.

The present study was designed to examine the roles of p53, reactive oxygen species (ROS), and ceramide, and to determine their mutual relationships during tumor necrosis factor (TNF)-alpha-induced apoptosis of human glioma cells. In cells possessing wild-type p53, TNF-alpha stimulated ceramide formation via the activation of both neutral and acid sphingomyelinases (SMases), accompanied by superoxide anion (O2-*) production, and induced mitochondrial depolarization and cytochrome c release, whereas p53-deficient cells were partially resistant to TNF-alpha and lacked O2-* generation and neutral SMase activation. Restoration of functional p53 sensitized glioma cells expressing mutant p53 to TNF-alpha by accumulation of O2-*. z-IETD-fmk (benzyloxycarbonyl-Ile-Glu-Thr-Asp fluoromethyl ketone), but not z-DEVD-fmk (benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone), blocked TNF-alpha-induced ceramide formation through both SMases as well as O2-* generation. Caspase-8 was processed by TNF-alpha regardless of p53 status of cells or the presence of antioxidants. Two separate signaling cascades, p53-mediated ROS-dependent and -independent pathways, both of which are initiated by caspase-8 activation, thus contribute to ceramide formation in TNF-alpha-induced apoptosis of human glioma cells.

p53-Independent ceramide formation in human glioma cells during gamma-radiation-induced apoptosis.

Although the p53 tumor-suppressor gene product plays a critical role in apoptotic cell death induced by DNA-damaging chemotherapeutic agents, human glioma cells with functional p53 were more resistant to gamma-radiation than those with mutant p53. U-87 MG cells with wild-type p53 were resistant to gamma-radiation. U87-W E6 cells that lost functional p53, by the expression of type 16 human papillomavirus E6 oncoprotein, became susceptible to radiation-induced apoptosis. The formation of ceramide by acid sphingomyelinase (A-SMase), but not by neutral sphingomyelinase, was associated with p53-independent apoptosis. SR33557 (2-isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxybphenethyl)amino]propyloxy)benzene-sulfonyl) indolizine, an inhibitor of A-SMase, suppressed radiation-induced apoptotic cell death. In contrast, radiation-induced A-SMase activation was blocked in glioma cells with endogenous functional p53. The expression of acid ceramidase was induced by gamma-radiation, and was more evident in cells with functional p53. N-oleoylethanolamine, which is known to inhibit ceramidase activity, unexpectedly downregulated acid ceramidase and accelerated radiation-induced apoptosis in U87-W E6 cells. Moreover, cells with functional p53 could be sensitized to gamma-radiation by N-oleoylethanolamine, which suppressed radiation-induced acid ceramidase expression and then enhanced ceramide formation. Sensitization to gamma-radiation was also observed in U87-MG cells depleted of functional p53 by retroviral expression of small interfering RNA. These results indicate that ceramide may function as a mediator of p53-independent apoptosis in human glioma cells in response to gamma-radiation, and suggest that p53-dependent expression of acid ceramidase and blockage of A-SMase activation play pivotal roles in protection from gamma-radiation of cells with endogenous functional p53.

Genistein inhibits potentiation by wortmannin of protein kinase C-activated phospholipase D in osteoblast-like cells.

We previously showed that protein kinase C (PKC) induces phosphatidylcholine-hydrolysing phospholipase D activation in osteoblast-like MC3T3-E1 cells and that tyrosine kinase is involved in this activation. Wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, markedly enhanced the formation of choline induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC in MC3T3-E1 cells. The effect of wortmannin was dose-dependent between 0.1 microM and 10 microM. ML-7, an inhibitor of myosin light chain kinase, had little effect on the TPA-induced formation of choline. Genistein, an inhibitor of protein tyrosine kinases, significantly suppressed the potentiation by wortmannin. These results strongly suggest that phosphatidylinositol 3-kinase is involved in the regulation of phospholipase D activation by PKC in osteoblast-like cells.

Effect of ceramide on interleukin-6 synthesis in osteoblast-like cells.

We previously showed that prostaglandin (PG) E1 stimulates the synthesis of interleukin-6 (IL-6) through activation of protein kinase (PK) A in osteoblast-like MC3T3-E1 cells and that PGF2alpha induces IL-6 synthesis through PKC activation. In other studies, we demonstrated that thrombin stimulates IL-6 synthesis, which depends on intracellular Ca2+ mobilisation in these cells and that tumour necrosis factor-alpha (TNF) induces IL-6 synthesis through sphingosine 1-phosphate, a product of sphingomyelin turnover. In the present study, among sphingomyelin metabolites, we examined the effect of ceramide on the IL-6 synthesis induced by various agonists in MC3T3-E1 cells. C2-ceramide, a cell-permeable ceramide analogue, suppressed the PGE1-induced IL-6 synthesis. C2-ceramide inhibited the IL-6 synthesis induced by PGF2alpha or 12-O-tetradecanoylphorbol-13-acetate, an activator of PKC. C2-ceramide reduced the IL-6 synthesis induced by cholera toxin, forskolin or dibutyryl cAMP. C2-ceramide inhibited the IL-6 synthesis induced by thrombin. The IL-6 synthesis stimulated by thapsigargin, which is known to stimulate Ca2+ mobilisation from intracellular Ca2+ stores, or A23187, a Ca-ionophore, was also inhibited by C2-ceramide. C2-ceramide did not affect the IL-6 synthesis induced by interleukin-1. On the contrary, C2-ceramide enhanced the TNF-induced IL-6 synthesis. D,L-threo-dihydrosphingosine, an inhibitor of sphingosine kinase, inhibited the enhancement by C2-ceramide as well as the TNF-effect. These results strongly suggest that ceramide modulates the IL-6 synthesis stimulated by various agonists in osteoblasts.

Effect of platelet-derived growth factor on phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like cells.

We examined the effect of platelet-derived growth factor (PDGF) on the activation of phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like MC3T3-E1 cells. PDGF-BB stimulated both the formation of choline (EC50 = 15 ng/ml) and inositol phosphates (EC50 = 5 ng/ml). However, PDGF-BB had little effect on the formation of phosphocholine. The formation of choline stimulated by a combination of PDGF-BB and 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C (PKC)-activating phorbol ester, was additive. H-7, an inhibitor of protein kinases, inhibited 12-O-tetradecanoylphorbol-13-acetate-induced choline formation, whereas HA1004, a control for H-7 as PKC inhibitor, had little effect. Neither H-7 nor HA1004 affected the PDGF-BB-induced formation of choline. Genistein and methyl 2,5-dihydroxycinnamate, inhibitors of protein tyrosine kinases, dose dependently inhibited the PDGF-BB-induced formation of choline. PDGF-BB stimulated Ca2+ influx from extracellular space. PDGF-BB-induced choline formation was significantly reduced by chelating extracellular Ca2+ with EGTA. PDGF-BB stimulated DNA synthesis of MC3YT3-E1 cells, and H-7 inhibited the DNA synthesis. These results strongly suggest that PDGF activates phosphatidyl-choline-hydrolyzing phospholipase D independently from PKC activated by phosphoinositide hydrolysis in osteoblast-like cells, and that both tyrosine kinase activation and Ca2+ influx are essential for this mechanism.

Interferon alpha2b gene delivery using adenoviral vector causes inhibition of tumor growth in xenograft models from a variety of cancers.

A recombinant adenovirus expressing human interferon alpha2b driven by the cytomegalovirus promoter, IACB, was shown to produce and secrete biologically active protein in vitro and in vivo. Intravenous administration of IACB in Buffalo rats resulted in circulating levels of biologically active human interferon at 70,000 international units/mL for up to 15 days. Distribution of interferon protein after IACB administration was different from that seen with the subcutaneous delivery of interferon protein. Higher levels of interferon protein were observed in liver and spleen after IACB delivery compared to protein delivery. The antitumor efficacy of IACB, as measured by suppression of tumor growth, was tested in athymic nude mice bearing established human tumor xenografts from different types of human cancer. Subcutaneous tumors most responsive to the intratumoral administration of IACB ranked as U87MG (glioblastoma) and K562 (chronic myelogenous leukemia), followed by Hep 3B (hepatocellular carcinoma) and LN229 cells (glioblastoma). Intravenous administration of IACB in animals bearing U87MG or Hep 3B xenografts was also effective in suppressing tumor growth, although to a lesser extent than the intratumoral administration. IACB was also tested in a metastatic model in beige/SCID mice generated with H69 (small cell lung carcinoma) cells and was found to prolong survival in tumor-bearing animals. This suggested that interferon gene delivery can be effective in suppressing tumor growth in a wide variety of cells.

ETA receptor mediates the signaling of endothelin-1 in osteoblast-like cells.

We previously reported that endothelin-1 (ET-1) stimulates phosphatidylcholine-hydrolyzing phospholipase D independently of phosphoinositide hydrolysis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the characteristics of the receptors mediating ET-1-induced intracellular signaling pathway in MC3T3-E1 cells. Cyclo-D-Trp-D-Asp-Pro-D-Val-Leu (BQ123), a selective ETA receptor antagonist, significantly inhibited the ET-1-induced formation of inositol phosphates in a dose-dependent manner in the range between 22 nmol/L (IC50) and 2.2 mumol/L (IC50 x 100). On the contrary, N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma MeLeu-D-Trp(COOMe)-D-Nle-ONa (BQ788), a selective ETB receptor antagonist, had no effect on the ET-1-induced formation of inositol phosphates in the range between 1.2 nmol/L (IC50) and 120 nmol/L (IC50 x 100). BQ123 significantly suppressed the ET-1-induced formation of choline dose-dependently, however, BQ788 did not affect the choline formation. BQ123 also inhibited the ET-1-induced release of arachidonic acid, but BQ788 had little effect. The results strongly suggest that ETA receptor mediates the three intracellular signaling pathways of ET-1: (1) phosphoinositide hydrolysis by phospholipase C; (2) phosphatidylcholine hydrolysis by phospholipase D; (3) arachidonic acid release in osteoblast-like cells.

Function of Ca2+ in phosphatidylcholine-hydrolyzing phospholipase D activation in osteoblast-like cells.

We investigated the function of Ca2+ in the activation of phosphatidylcholine (PC)-hydrolyzing phospholipase D (PLD) in osteoblast-like MC3T3-E1 cells. Fetal calf serum (FCS) stimulated the formation of choline in a dose-dependent manner in the range between 0.6% and 10%. The effect of a combination of FCS and 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C (PKC) activator, on the formation of choline was additive. Staurosporine, an inhibitor of protein kinases, enhanced the formation of choline induced by FCS. BAPTA/AM, a chelator of intracellular Ca2+, inhibited the formation of choline induced by FCS. The depletion of extracellular Ca2+ by EGTA markedly reduced the FCS-induced formation of choline. SK&F 96365, an inhibitor of receptor-operated Ca2+ entry, significantly inhibited the choline formation induced by FCS. On the other hand, nifedipine, an inhibitor of L-type voltage-dependent Ca2+ channels, had little effect on the choline formation. TMB-8, an inhibitor of Ca2+ mobilization from intracellular Ca2+ store, significantly inhibited FCS-induced choline formation. These results strongly suggest that Ca2+ mobilization, through both the influx via receptor-operated Ca2+ channel and the release from intracellular Ca2+ store, plays an important role in the activation of PLD in osteoblast-like cells.

Tyrosine kinase is involved in angiotensin II-stimulated phospholipase D activation in aortic smooth muscle cells: function of Ca2+ influx.

In the present study, we examined the effect of angiotensin II (Ang II) on phosphatidylcholine-hydrolyzing phospholipase D activity in subcultured rat aortic smooth muscle cells (SMC). Ang II dose-dependently stimulated the formation of choline and inositol phosphates. The effect of Ang II on the formation of inositol phosphates (EC50 was 0.249 +/- 0.091 nM) was more potent than that on the formation of choline (EC50 was 2.39 +/- 1.29 nM). A combination of Ang II and 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, additively stimulated the formation of choline. Staurosporine, an inhibitor of protein kinases, inhibited the TPA-induced formation of choline, but had little effect on the Ang II-induced choline formation. Ang II stimulated Ca2+ influx from extracellular space time- and dose-dependently. The depletion of extracellular Ca2+ by (ethylenebis(oxyethylenenitrilo)) tetraacetic acid (EGTA) significantly reduced the Ang II-induced formation of choline. Genistein and tyrphostin, protein tyrosine kinase inhibitors, significantly suppressed the Ang II-induced Ca2+ influx. Genistein and tyrphostin also suppressed the Ang II-induced formation of choline. These results suggest that Ang II stimulates phosphatidylcholine-hydrolyzing phospholipase D due to Ca2+ influx from the extracellular space in rat aortic SMC, and that protein tyrosine kinase is involved in the Ang II-induced Ca2+ influx, resulting in the promotion of phosphatidylcholine hydrolysis.